Embed Size (px)
Citation preview
Evaluation of transplacental pharmacology and toxicology from bench to bedside
by
Janine Rose Hutson
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy
Institute of Medical Science University of Toronto
© Copyright by Janine Rose Hutson 2014
ii
Evaluation of transplacental pharmacology and toxicology from bench to bedside
Janine Rose Hutson
Doctor of Philosophy
Institute of Medical Science University of Toronto
2014
Abstract
Many women require pharmacologic treatment during pregnancy. Clinical studies of drug
safety in human pregnancy are often limited because of ethical considerations and thus a
theoretical framework to evaluate drug safety in pregnancy is needed. Dual perfusion of a single
placental lobule is the only experimental model to study human placental transfer of substances
in organized placental tissue. The objectives of this thesis were to systematically evaluate the
perfusion model in predicting placental drug transfer and to develop a model to account for non-
placental pharmacokinetic parameters in the perfusion results. In general, the fetal-to-maternal
drug concentration ratios matched well between placental perfusion experiments and in vivo
samples taken at the time of delivery. After modeling for differences in maternal and
fetal/neonatal protein binding and blood pH, the perfusion results were able to accurately predict
in vivo transfer at steady state (R2 = 0.85, P < 0.0001). We then utilized the perfusion model to
evaluate the placental transfer of 6-mercaptopurine (6-MP), a drug commonly used in pregnancy
for the treatment of inflammatory bowel disease as well as the toxic effects of alcohol and formic
acid on the placenta. Placental transfer of 6-MP is limited and binding to placental tissue and
maternal pharmacokinetic parameters are the main factors that restrict placental transfer.
Evaluation of the placental perfusion results together with our meta-analysis of clinical studies
supported other evidence that the benefit of 6-MP outweighs any fetal risk when indicated. The
placental transfer of folic acid was decreased in pregnancies with heavy alcohol exposure and
iii
folic acid supplementation may decrease the toxic effects of formic acid on the placenta.
Furthermore, our validation of the ex vivo perfusion model showed that it is effective in
predicting fetal exposure to drugs and should have a place in clinical and regulatory
pharmacology and toxicology.
iv
Acknowledgments
I would like to thank the following individuals for their support and contributions during
the course of my training.
Dr. Gideon Koren for his exceptional mentorship, the many opportunities he has provided
me, and the encouragement and support to follow my ambitions.
Dr. Bhushan Kapur for his mentorship and support during my training and for the
opportunity to work on a completely new area of research! Drs Suzanne Schuh and Micheline
Piquette-Miller, my advisors, for their time and invaluable guidance that has facilitated my
degree completion. Drs. Yaron Finkelstein and Cindy Woodland for also being great mentors and
for giving me amazing opportunities.
I would also thank to express my gratitude to those who have collaborated with me on my
studies. Angelika Lubetsky for her expertise in the placental perfusion experiment and for never
saying no when I wanted to try numerous experiments in a day! I thank you for sharing your
expertise with me. To Myla Moretti, Jeremy Matlow, Dr. Facundo Bournissen, Amy Davis, Jeff
Eichhorst, Dr. Kathy Aleksa, Dr. Asnat Walfisch, Dr. Rinat Hackmon, Dr. Brenda Stade, Dr.
Denis Lehotay, and Dr. Christine Collier for your time and contributions to these studies. I could
not have completed this work without you. To Sarah MacDougall and Irene Zelner for their
friendship and support. The other members of the Koren Laboratory and the Motherisk Program
for their contribution and companionship through the course of this work. To the lab ladies who
lunch for the good times and great memories!
The generous support from the Canadian Institutes of Health Research Vanier
Scholarship Program, University of Toronto Open Fellowships, Canadian Foundation on Fetal
Alcohol Research, and the McLaughlin Centre for Molecular Medicine.
Finally, my parents, family and close friends for their encouragement and understanding.
You have instilled in me the courage to succeed. Lastly, to all of my friends from both the MD
and PhD programs for your support throughout the ride.
v
Contributions I am grateful to the following for the contributions they have made to the individual
studies as detailed below as well as their contributions to the preparation of the respective
manuscripts.
Amy Davis: A summer student who assisted me with the systematic searches for the in
vivo umbilical cord blood and maternal blood drug concentration values that are included in
Chapter 2.
Dr. Facundo Garcia-Bournissen who contributed to the study design and data
interpretation of Chapters 2 and 3.
Angelika Lubetsky for her technical assistance with the placental perfusion experiments
included in Chapters 3 and 6. Dr. Asnat Walfisch, Dr. Rinat Hackmon for recruitment of eligible
patients in Chapters 3 and 6, respectively. Dr. Brenda Stade and Dr. Christine Collier for
recruitment of patients and data collection in Chapter 5.
Jeremy Matlow who was the second reviewer to evaluate appropriateness of studies to
include for the meta-analysis in Chapter 4 and for data extraction from these studies. Myla
Moretti for data analysis in Chapter 4.
Jeff Eichhorst and Dr. Denis Lehotay for measurement of formic acid and folate in
Chapters 5 and 6.
Dr. Gideon Koren, my supervisor, for overseeing study design, data analysis,
interpretation of data, manuscript preparation, and obtaining funding. Dr. Bhushan Kapur for
obtaining funding for Chapters 5 & 6 and for overseeing these two studies as the principle
investigator.
vi
Table of Contents
Acknowledgments .......................................................................................................................... iv!
Contributions ................................................................................................................................... v
Table of Contents ........................................................................................................................... vi!
List of Tables ................................................................................................................................. ix!
List of Figures ................................................................................................................................. x!
List of Publications ....................................................................................................................... xii!
List of Abbreviations ................................................................................................................... xiii!
List of Appendices ....................................................................................................................... xvi!
Chapter 1 General Introduction ...................................................................................................... 1!
! Drug Exposure In Pregnancy ..................................................................................................... 1!1
1.1! Factors Affecting Maternal-Fetal Pharmacokinetics and Transplacental Drug Transfer ... 1!
1.1.1! Maternal pharmacokinetic considerations .............................................................. 2!
1.1.2! Fetal Pharmacokinetic Considerations .................................................................... 5!
1.1.3! Transplacental Drug Transfer ................................................................................. 6!
1.1.4! Methods for Studying Transplacental Drug Transfer ........................................... 12!
1.2! Therapeutic Drug Use during Pregnancy: Inflammatory Bowel Disease and Treatment with Thiopurines ............................................................................................................... 19!
1.3! Recreational Drug Use During Pregnancy: Alcohol and Fetal Alcohol Spectrum Disorder ............................................................................................................................. 23!
1.4! Overall Rational and Primary Objectives ......................................................................... 29!
Chapter 2 ....................................................................................................................................... 32!
! The human placental perfusion model: a systematic review and development of a model to 2predict in vivo transfer of therapeutic drugs ............................................................................ 33!
2.1! Abstract ............................................................................................................................. 33!
2.2! Introduction ....................................................................................................................... 34!
2.3! Methods ............................................................................................................................. 36!
vii
2.3.1! Literature Search ................................................................................................... 36!
2.3.2! Extraction of Data and Qualitative Comparisons ................................................. 36!
2.3.3! Quantitative Calculations ...................................................................................... 37!
2.3.4! Model Development .............................................................................................. 37!
2.4! Results ............................................................................................................................... 39!
2.4.1! Search Results ....................................................................................................... 39!
2.4.2! Qualitative Comparison of Perfusion Results to In Vivo Data ............................. 39!
2.4.3! Quantitative Comparison of Perfusion Results to In Vivo Data ........................... 42!
2.5! Discussion ......................................................................................................................... 45!
Chapter 3 ....................................................................................................................................... 52!
! The transfer of 6-mercaptopurine in the dually perfused human placenta ............................... 53!3
3.1! Abstract ............................................................................................................................. 53!
3.2! Introduction ....................................................................................................................... 54!
3.3! Methods ............................................................................................................................. 55!
3.4! Results ............................................................................................................................... 58!
3.5! Discussion ......................................................................................................................... 63!
3.6! Conclusions ....................................................................................................................... 65!
Chapter 4 ....................................................................................................................................... 67!
! The fetal safety of thiopurines for the treatment of inflammatory bowel disease in 4pregnancy: a meta-analysis ...................................................................................................... 68!
4.1! Abstract ............................................................................................................................. 68!
4.2! Introduction ....................................................................................................................... 69!
4.3! Methods ............................................................................................................................. 69!
4.4! Results ............................................................................................................................... 71!
4.5! Discussion ......................................................................................................................... 82!
Chapter 5 ....................................................................................................................................... 85!
viii
! Folic acid transport to the human fetus is decreased in pregnancies with chronic alcohol 5exposure ................................................................................................................................... 86!
5.1! Abstract ............................................................................................................................. 86!
5.2! Introduction ....................................................................................................................... 87!
5.3! Methods ............................................................................................................................. 89!
5.4! Results and Discussion ..................................................................................................... 90!
Chapter 6 ....................................................................................................................................... 98!
! Adverse placental effect of formic acid on hCG secretion is mitigated by folic acid ............. 99!6
6.1! Abstract ............................................................................................................................. 99!
6.2! Introduction ..................................................................................................................... 100!
6.3! Methods ........................................................................................................................... 101!
6.3.1! Materials ............................................................................................................. 101!
6.3.2! Placental perfusion .............................................................................................. 101!
6.3.3! Sample analysis ................................................................................................... 102!
6.4! Results ............................................................................................................................. 103!
6.5! Discussion ....................................................................................................................... 108!
6.6! Conclusions ..................................................................................................................... 110!
Chapter 7 ..................................................................................................................................... 111!
! General Discussion ................................................................................................................. 111!7
7.1! Summary of Research Findings and Future Directions .................................................. 112!
7.2! Summary ......................................................................................................................... 127!
! References .............................................................................................................................. 128!8
Appendices .................................................................................................................................. 161!
ix
List of Tables
Table 1-1. Advantages and disadvantages of different methods used to determine placental drug
transfer. ......................................................................................................................................... 15!
Table 1-2 The 1996 Institute of Medicine’s Diagnostic Criteria for FASD ................................. 24!
Table 1-3 Secondary Disabilities in Adolescents and Adults with FAS or FAE .......................... 25!
Table 2-1. Drugs that had the maternal-to-fetal transfer evaluated using the placental perfusion
model and where the fetal to maternal drug concentration ratios (F:M) from the perfusion agreed
with in vivo data from mother-infant pairs. .................................................................................. 41!
Table 2-2. Drugs that had the maternal-to-fetal transfer evaluated using the placental perfusion
model and where the fetal to maternal drug concentration ratios (F:M) from the perfusion
disagreed with in vivo data from mother-infant pairs. .................................................................. 41!
Table 2-3. The observed and calculated F:M ratios for data obtained from placental perfusion
experiments ................................................................................................................................... 43!
Table 3-1. Placental viability parameters and metabolic capacity throughout the perfusions ...... 59!
Table 4-1 Characteristics of studies included in the meta-analysis .............................................. 72!
Table 4-2 Pregnancy outcomes in thiopurine-exposed pregnancies versus controls .................... 74!
Table 4-3 Specific malformations and other drug use reported in the included studies ............... 75!
Table 5-1. Demographic information on alcohol-using women included in this study and fetal
parameters. .................................................................................................................................... 91!
Table 6-1 Placental viability parameters and metabolic capacity throughout the perfusion ...... 104!
x
List of Figures
Figure 1-1. ABC Transport proteins present on the human placental barrier. ................................ 9!
Figure 1-2. The transfer of the basic drug, bupivacaine, across the placental membrane at
equilibrium is determined by the acid-base equilibrium effect and protein binding.. .................. 13!
Figure 1-3. Schematic presentation of the placental perfusion experimental set-up. ................... 16!
Figure 1-4 Metabolic pathway for 6-mercaptopurine and its prodrug azathioprine. .................... 22!
Figure 2-1. Flow diagram of the search strategy and articles retrieved and included in the review.
....................................................................................................................................................... 40!
Figure 2-2. Agreement between perfusion fetal to maternal (F:M) concentration ratios and the in
vivo umbilical cord to maternal blood (C:M) drug concentration ratios ...................................... 44!
Figure 2-3. Comparison of F:M ratios for morphine obtained from placental perfusion
experiments versus maternal blood and umbilical cord blood ...................................................... 46!
Figure 3-1. Maternal-to-fetal transfer of 6-mercaptopurine after dual perfusion of a single
placental lobule ............................................................................................................................. 61!
Figure 3-2 Concentrations of 6-MP in the fetal and maternal circulations after perfusion of a
single placental lobule under equilibrative concentrations ........................................................... 62!
Figure 4-1 Overall effect of the incidence of congenital abnormalities after in utero exposure to
thiopurines………………………………………………………………………………………..76
Figure 4-2 Overal effect of the incidence of prematurity (<37 weeks gestation) after in utero
exposure to thiopurines…………………………………………………………………………..78
Figure 4-3 Overall effect of the incidence of low birth weight (< 2500 g) after in utero exposure
to thiopurines. ............................................................................................................................... 80!
Figure 4-4. Overall effect of in utero exposure to thiopurines on mean birth weight .................. 81!
xi
Figure 5-1. The transport proteins present on the syncytiotrophoblast that are involved in the
transfer of folate to the fetal circulation. ....................................................................................... 88!
Figure 5-2. Scatter-plot of the fetal to maternal (F:M) folate ratios as measured in cord blood and
maternal blood, respectively, at the time of delivery. ................................................................... 92!
Figure 5-3. Corresponding maternal and fetal folate concentrations at the time of delivery in
pregnancies with A) heavy alcohol exposure and B) in controls. ................................................. 93!
Figure 5-4. Mean (± SEM) cord and maternal plasma folate concentrations at the time of delivery
in alcohol-abusing women and controls ....................................................................................... 94!
Figure 6-1 Maternal-to-fetal transfer of formic acid after dual perfusion of a single placental
lobule in the presence or absence of folate ................................................................................. 105!
Figure 6-2 The rate of hCG secretion into the maternal circulation during the placental perfusion
was calculated in the pre-experimental control period (before addition of formic acid) and during
the experimental period in the presence and absenceof folate. ................................................... 106!
Figure 6-3 Tissue concentration of hCG in the perfused lobule at the end of the 180 minute
experimental period with formic acid expressed as a percentage of the initial hCG tissue
concentration in the same placenta in the presence and absence of folate. ................................ 107!
Figure 7-1 The interaction between alcohol use in pregnancy and the proposed mechanisms of
toxicity by reduced placental folate transfer to the fetus and by formic acid. ............................ 126!
xii
List of Publications
Etwel F, Hutson JR, Madadi P, Gareri J, Koren G. Fetal and Perinatal Exposure to Drugs and Chemicals; Novel Biomarkers of Risk Assessment. Ann Rev Pharmacol Tox 2014;54:295-315.
Hutson JR, Lubetsky A, Eichhorst J, Hackmon R, Koren G, Kapur BM. Adverse placental effect of formic acid on hCG secretion is mitigated by folic acid. Alcohol Alcohol 2013;48(3):283-7.
Hutson JR, Matlow JN, Moretti M, Koren G. The fetal safety of thiopurines for the treatment of inflammatory bowel disease in pregnancy: a meta-analysis. J Obstet Gynecol 2013;33(1):1-8.
Hutson JR, Stade B, Lehotay DC, Collier CP, Kapur B. Folic acid transport to the human fetus is decreased in pregnancies with chronic alcohol exposure. PLoS ONE 2012:7(5);e38057.
Hutson JR, Garcia-Bournissen F, Davis A, Koren G. The human placental perfusion model: a
systematic review and development of a model to predict in vivo transfer of therapeutic drugs. Clin Pharmacol Ther 2011;90(1):67-76.
Hutson JR, Lubetsky A, Walfisch A, Ballios BG, Garcia-Bournissen F, Koren G. The transfer
of 6-mercaptopurine in the dually perfused human placenta. Reprod Toxicol 2011;32:349–353.
Hutson JR. Prediction of Placental Drug Transfer Using the Human Placental Perfusion Model.
J Popul Ther Clin Pharmacol 2011;18(3):e533-e543.
Hutson JR, Koren G, Matthews SG. Placental P-glycoprotein and breast cancer resistance protein: influence of polymorphisms on fetal drug exposure and physiology. Placenta 2010;31(5):351-357.
xiii
List of Abbreviations
5-ASA 5-aminosalicylic acid
6-MMP 6-methylmercaptopurine
6-MP 6-mercaptopurine
6-TG 6-thioguanine
AAG α1-acid glycoprotein
ABC ATP-binding cassette
ADH alcohol dehydrogenase
AUC area under the curve
AZA azathioprine
B/F bound to free drug concentration ratio
BCRP breast cancer-resistance protein
C:M cord to maternal concentration ratio
CI confidence interval
CL clearance
Cmax peak serum concentration
CNT concentrative nucleoside transporter
Css steady state concentration
CYP 450 cytochrome P450
D dose
xiv
ENT equilibrative nucleoside transporters
F:M fetal to maternal concentration ratio
FAS fetal alcohol syndrome
FASD fetal alcohol spectrum disorder
FR-α folate receptor-α
GFR glomerular filtration rate
GMPS guanine monophosphate synthetase
hCG human chorionic gonadotropin
HPRT hypoxanthine–guanine phosphoribosyltransferase
IBD inflammatory bowel disease
IMPDH inosine monophosphate dehydrogenase
ip intraperitoneal
LBW low birth weight
MCT monocarboxylate transporters
MDMA 3,4-methylenedioxy-N-methylamphetamine
meTGMP methylated TGMP
meTIMP methylated TIMP
MRP multidrug resistance-associated proteins
NOAEL no observable adverse effect level
NSAID nonsteroidal anti-inflammatory drug
xv
OR odds ratio
PCFT proton-coupled folate transporter
PFR placental folate receptors
Pgp P-glycoprotein
RFC reduced folate carrier
RR relative risk
SA spontaneous abortion
SD standard deviation
SEM standard error of the mean
TGMP 6’thioguanosine 5’monophosphate
TGN thioguanine nucleotides
THC tetrahydrocannabinol
TIMP 6-thioinosine 5 ́ monophosphate
TPMT thiopurine S-methyltransferase
TXMP 6-thioxanthosine 5’monophosphate
UGT uridine diphosphate glucuronosyltransferase
Vd volume of distribution
WMD weighted mean difference
XO xanthine oxidase
xvi
List of Appendices
Appendix A: Placental Perfusion System Materials and Set-up……………………………162
Materials…………………………………………………………………………….162
Fetal Circuit Hardware Set-up………………………………………………………163
Maternal Circuit Hardware Setup…………………………………………………...165
Perfusion Chamber….………………………………………………………………167
Composition of Medium 199 Culture Medium in Fetal and Maternal Perfusate….. 168
Appendix B: Systematic Review of Perfusion Data - Supplementary Table (Chapter 2)…..169
Appendix C: Placental P-glycoprotein and breast cancer resistance protein: influence of
polymorphisms on fetal drug exposure and physiology…………………………………….202
1
Chapter 1 General Introduction
Drug Exposure In Pregnancy 1Historically, the placenta was believed to restrict transfer of xenobiotics from the
maternal to the fetal circulation, thereby protecting the fetus from any adverse effects of drugs
(Koren et al., 1998). The thalidomide disaster in the 1950s and early 1960s was the first large-
scale evidence that the fetus is not exempt from adverse drug effects as exposure during critical
periods during development resulted in severe limb defects and other organ dysgenesis (e.g.,
kidney and heart defects) (McBride, 1978). The thalidomide disaster prompted the belief that
every drug has a potential to be harmful to the fetus (Koren et al., 1998).
We now know that most small molecules cross the placenta in a measurable way,
sometimes achieving concentrations in fetal plasma as high as those in maternal plasma. Despite
most drugs reaching the fetal circulation, only a limited number of drugs have been proven to be
teratogenic (Koren et al., 1998). This is reassuring, as many women require pharmacologic
treatment during pregnancy for chronic medical conditions or illnesses that arise during
pregnancy. The benefit of drug treatment must be weighed against any potential risk to the
unborn child as well as risk of the untreated maternal disease. This potential maternal-fetal
conflict brings to light the need to identify drugs that can treat the mother without harming the
fetus. As the placenta is the interface between the maternal and fetal circulations, a thorough
understanding of the role of the placenta in maternal-fetal pharmacokinetics is vital to
understanding drug safety in pregnancy. Factors influencing placental drug transfer as well as
methods to study transfer will be reviewed. In addition, examples of therapeutic agents and
toxins that can be studied with respect to placental transfer will be reviewed.
1.1 Factors Affecting Maternal-Fetal Pharmacokinetics and Transplacental Drug Transfer
Dynamic changes in maternal, placental, and fetal pharmacokinetics occur throughout
pregnancy as a result of altered physiology during pregnancy and also by the growth and
2
development of the placenta and fetus. These changes can influence the pharmacokinetic
processes of drug absorption, distribution, metabolism and elimination. Three factors influence
maternal and fetal drug exposure and thus also influence drug response in pregnancy: 1)
maternal pharmacokinetics influenced by pregnancy-induced physiological changes 2)
distribution, metabolism, and elimination by the fetus and 3) the amount of drug that can cross
the placenta (Hutson et al, 2012).
1.1.1 Maternal pharmacokinetic considerations
During pregnancy, a variety of anatomical, physiological, hormonal and biochemical
changes occur, which affect virtually every aspect of the pharmacokinetics of drugs, including
absorption, distribution, metabolism, and elimination (Loebstein et al., 1997).
Absorption - Pregnancy can theoretically affect oral absorption of drugs, mainly in late
pregnancy when gastrointestinal transit time is increased by 30 to 50% (Chiloiro et al., 2001;
Loebstein et al., 1997). Opioids administered during labor can also result in delayed gastric
emptying, which can slow absorption of the drug and also the therapeutic effect (Porter et al.,
1997). During pregnancy, gastric acid secretions are reduced and there is an increase in mucous
secretions, resulting in an increase in gastric pH (Loebstein et al., 1997). A change in gastric pH
affects absorption of weak acids and bases by changing the proportion of ionized to unionized
drug: only the unionized form can be absorbed across biological membranes. For most drugs, the
change in total bioavailability during pregnancy is relatively small, however, the rate of
absorption may be altered and be clinically relevant when a rapid effect is desired. Nausea and
vomiting in pregnancy during the first trimester may also influence drug absorption. Absorption
of drugs across the lungs can be influenced by the increased cardiac output and tidal volumes
that occur in pregnancy (Hutson et al., 2012). Furthermore, increased pulmonary blood flow and
alveolar ventilation occur. At term, alveolar ventilation is 70% above normal in the supine
position. These physiological changes may favour alveolar uptake when administering drug
aerosols.
Distribution – Both total body water and fat content increase during pregnancy and
consequently often affect the volume of distribution (Vd) of drugs (Loebstein et al., 1997). There
3
is a mean increase of 8L in total body water, most (60%) of which is distributed to the fetus,
placenta, and amniotic fluid (Loebstein et al., 1997). Plasma volume during pregnancy expands
by 50% (Pirani et al., 1973) and can lead to a decrease in the peak serum concentration (Cmax)
of many drugs. For some drugs, a larger Vd could necessitate a higher initial dose to obtain
therapeutic plasma concentration. Adipose tissue content also increases by a mean of 19kg at the
end of pregnancy and can increase the Vd for lipophilic substances. For lipophilic drugs, such as
anesthetics, this can lead to persistence of a high drug concentration as the drug distributes back
out of the adipose tissue.
Albumin and α1-acid glycoprotein (AAG) are the two most important drug binding
plasma proteins. Albumin binds mainly weak acids and lipophilic drugs, whereas AAG binds
mainly basic drugs. Pregnant women may exhibit decreased protein binding because of
decreased levels of plasma albumin (Krauer et al., 1984). Throughout pregnancy, albumin
concentrations decrease, reaching approximately 70-80% of normal values at the time of
delivery. This decrease is a result of the plasma volume expanding at a greater rate than the
increase in albumin production. Furthermore, a 3-fold higher concentration of free fatty acids
towards term in the maternal circulation can displace drugs from binding to albumin (Nau et al.,
1984; Ridd et al., 1983). In contrast to albumin, plasma concentrations of AAG remain similar
throughout pregnancy. Since AAG is an acute phase protein that increases during trauma or
inflammation, it is expected to increase during labor; however, there was no increase in plasma
AAG concentrations during delivery when effective analgesia was provided using epidural
ropivacaine (Porter et al., 2001). Increases in placental and steroid hormones throughout
pregnancy can occupy and displace drugs from the protein binding sites. The clinical relevance
of this phenomenon is not clear. For example, protein binding of bupivacaine decreased during
maximum estrogen concentrations in patients undergoing in vitro fertilization (Tsen et al., 1997).
However, this decrease in protein binding is mainly attributed to estrogen decreasing the
concentration of plasma binding proteins rather than displacement of the drug from the binding
site. For highly protein-bound drugs, decreased protein binding has a variable effect on kinetics:
more drug is free and available for effect, yet more free drug is also available for metabolism
and/or elimination. Changes in protein binding during pregnancy can influence the transplacental
transfer of many drugs and will be further discussed in section 1.1.3.4.
4
Metabolism and Elimination - Although maternal physiologic changes begin early in
pregnancy, they are most pronounced in the third trimester. A rise in cardiac output begins in the
first trimester of pregnancy and can be as high as 50% above normal non-pregnant values. An
increase in hepatic clearance may result from the increase in hepatic blood flow as a result of
increased cardiac output. This may be especially critical for drugs with high extraction ratios,
such as lidocaine and morphine, and less significant for low hepatic extraction drugs such as
acetaminophen. Changes in hepatic enzyme activity can have an important impact on drug
clearance, mainly in the phase I cytochrome P450 (CYP450) pathway. As steady state
concentrations (Css) correlate with clearance rate (CL) (according to the equation: Css = D/CL),
a dose of drug (D) would have to be increased proportionally to increase clearance rate to
maintain a similar pharmacologic effect. Specifically, pregnancy increases the hepatic activity of
CYP3A4, CYP2D6, CYP2C9 and CYP2A6. Drugs predominately metabolized by these
enzymes, such as nonsteroidal anti-inflammatory drugs (NSAIDs), may require an increased
dose to avoid loss of efficacy (Anderson, 2005). In contrast, CYP1A2 and CYP2C19 activity is
decreased during pregnancy, suggesting that dosage reductions may be needed to minimize
potential toxicity of their substrates. Phase II enzymes can also be increased (ie. uridine
diphosphate glucuronosyltransferase (UGT) isoenzymes) or decreased (ie. N-acetyltransferase 2)
during pregnancy.
Blood flow to the kidneys increases 60-80% during pregnancy. As a result, glomerular
filtration rate (GFR) increases 50% by the first trimester and continues to increase throughout
pregnancy compared with postpartum values (Davison & Hytten, 1974). Only during the last
three weeks of pregnancy does GFR begin to decrease (Anderson, 2005). The increase in renal
clearance during pregnancy is likely to have notable effects on drugs that are eliminated
predominately unchanged by the kidneys (e.g. β-lactam antibiotics, lithium, digoxin) and their
dosage may need to be increased by 20-65% in order to maintain pre-pregnancy concentrations
(Anderson, 2005). The influence of pregnancy on drug transporters involved in tubular secretion
and reabsorption has not been extensively studied.
5
1.1.2 Fetal Pharmacokinetic Considerations
Distribution - Drugs that cross the placenta are carried to the fetus by the umbilical vein
and enter the unique fetal circulation. Drugs that readily transfer across the placenta often are
rapidly distributed to highly perfused fetal organs, such as anesthetics (Finster et al., 1972a).
Before entering the systemic circulation, the majority of blood from the umbilical vein flows
through the fetal liver. The fetal liver therefore has the potential to reduce the amount of drug
distributed to sensitive fetal organs. Lidocaine and thiopental were observed to bind and
accumulate in fetal liver (Finster et al., 1972a; Finster et al., 1972b). Drug distribution in the
fetus will also depend on plasma protein binding. Plasma concentrations of both albumin and
AAG in the fetus progressively increase throughout gestation. Between the 12th week of
gestation and term, albumin increases from 10.9 g/L to 34.2 g/L and AAG increases from 0.05
g/L to 0.21 g/L (Krauer et al., 1984). This dynamic process contributes to the change in the fetal
to maternal albumin/AAG concentration ratio at different gestational ages. The influence of this
change on drug transfer across the placenta is discussed in section 1.1.3.4. In fetuses with severe
cardiac insufficiency, elevated umbilical venous pressure may reduce placental drug transfer
(Schmolling et al., 2000).
Metabolism and Elimination - The fetal liver has generally less enzymatic activity for
drug metabolism compared to the adult. Throughout gestation, the amount and intra-lobular
distribution of drug metabolizing enzymes in the fetal liver undergo changes. Several drug-
metabolizing enzymes have been detected as early as 8 to 10 weeks of gestation (Hines, 2007).
However, CYP450 and some conjugating enzymes are located to the smooth endoplasmic
reticulum in the hepatocyte and a significant amount of smooth endoplasmic reticulum is not
present until mid-gestation (Myllynen et al., 2009). Activity of CYP1A1 has been detected in
first trimester fetal liver, but not in the second and third trimesters (Myllynen et al., 2009). No
significant activity of CYP1A2 has been detected (Sonnier & Cresteil, 1998). Activity of
CYP2C9 increases with gestational age, but CYP2C19 activity remains constant (Koukouritaki
et al., 2004). The expression of CYP2D6 is variable and activity has ranged from undetectable
to approximately 1% of adult activity (Myllynen et al., 2009). The major CYP3A isoform in the
fetal liver is CYP3A7 and it accounts for 50% of the total fetal hepatic CYP content (Ring et al.,
1999). After birth, CYP3A7 activity decreases and CYP3A4 becomes the predominant hepatic
CYP3A isoform. Hepatic activity of UGTs starts during the latter half of the second trimester
6
(Myllynen et al., 2009). Drug-metabolizing enzymes present in the fetal liver may contribute to
a first-pass effect. A study in the baboon demonstrated that the fetal liver had a high intrinsic
clearance toward morphine (a UGT substrate) and that fetal hepatic clearance of morphine was
flow limited (Garland et al., 2006). This study demonstrates that for specific drugs, there may be
significant placenta-fetal hepatic first-pass metabolism when drugs are administered to the
mother. Drugs entering the fetal circulation may also be excreted into the amniotic fluid through
the fetal kidneys. However, most drug elimination from the fetus is generally considered to be a
result of transfer back to the maternal circulation.
1.1.3 Transplacental Drug Transfer
Drug transfer across the placenta depends on the physicochemical properties of the drug
as well as the mechanism of transfer across the placental barrier, blood flow to the placenta,
metabolism and binding by the placenta, protein binding in both the mother and fetus, and the pH
of the maternal and fetal circulations. Furthermore, drug transfer will relate to the developmental
stage of the placenta as the thickness of the interface between the maternal and fetal circulations
changes as the placenta develops (Enders, 1981). The expression and function of drug transport
proteins and metabolic enzymes in the placenta also can change throughout pregnancy (Hutson
et al., 2010).
1.1.3.1 Placental Structure
The functional vascular unit of the placenta is the cotyledon. Each cotyledon contains
highly branched villi suspended in the intervillous space (Enders, 1981). This space is filled
with maternal blood supplied by spiral arteries and carried away by uterine veins. Each fetal
villous tree consists of capillary endothelium, villous stroma and a trophoblast layer. Throughout
gestation, the syncytium of cells separating the maternal and fetal circulations thins and the
surface area for transfer increases (Enders, 1981). The rate-limiting barrier for transfer across the
mature placenta is the single layer of polarized multinucleated cells called syncytiotrophoblasts
(Audus, 1999; Ceckova-Novotna et al., 2006). Consequently, drug transfer at term may represent
7
the highest fetal drug exposure compared to earlier gestational ages (Vahakangas & Myllynen,
2006).
1.1.3.2 Transfer Mechanisms
As in other lipid barriers, molecules that are relatively lipophilic, with molecular weights
< 600 Da, can readily cross the syncytiotrophoblast by diffusion in the unbound non-ionized
state. As a result, most drugs, being small molecules, cross the placenta by passive diffusion
(Ala-Kokko et al., 2000; Audus, 1999). Accordingly, the concentration gradient across the
placental barrier drives transfer for most drugs. A well-mixed double pool flow model best
describes transplacental kinetics where venous equilibrium exists between the maternal and fetal
compartments (Ala-Kokko et al., 2000).
Transfer of hydrophilic drugs is permeability-limited and transfer of lipophilic drugs is
usually considered flow-limited (Giroux et al., 1997; Syme et al., 2004). Blood flow to the
placenta increases during gestation from about 50 mL/min at 10 weeks to 600 mL/min at term
and factors that alter utero-placental blood flow may alter placental drug transfer (Syme et al.,
2004). These factors include anesthesia, nicotine, maternal position, and degenerative changes
within the placenta that are observed with conditions such as hypertension, diabetes, or renal
disease. During labour, uterine contractions can lead to intermittent decreases in uterine arterial
flow. These contractions can delay the transfer of drugs to the fetus and also delay the clearance
of drug from the fetal to the maternal circulation. For example, concentrations of diazepam were
higher in newborns born to mothers administered the drug at the onset of uterine contraction,
compared to those administered the drug during uterine diastole (Haram et al., 1978). The
steady-state fetal to maternal drug concentration ratio will not be affected by changes in blood
flow, however, it may accelerate or delay the rate of transfer.
A wide variety of drug transporters have been localized to the placenta. Although most
are found to have mainly physiological substrates, they can also transport several xenobiotics.
Active transport in the placenta is mediated by transport proteins expressed on both the
microvillous brush-border and basal membranes (maternal blood-facing and fetal endothelial
cell-facing, respectively) of the syncytiotrophoblast cells. Several members of the ATP-binding
8
cassette (ABC) drug efflux proteins have been localized to the placenta, including P-glycoprotein
(Pgp), breast cancer-resistance protein (BCRP), and multidrug resistance-associated proteins
(MRPs) (Figure 1-1) (Atkinson et al., 2003; Evseenko et al., 2007; MacFarland et al., 1994;
Maliepaard et al., 2001; Meyer Zu Schwabedissen et al., 2005; Meyer zu Schwabedissen et al.,
2006; Nagashige et al., 2003; St-Pierre et al., 2000; Sugawara et al., 1997; Sun et al., 2006;
Yeboah et al., 2006). Depending on the location of the protein, it can facilitate drug-uptake or
drug-efflux from the fetal or maternal circulations. The placement of Pgp and BCRP on the
large surface area of the syncytiotrophoblast brush-border is optimal to limit transfer across the
placental barrier to the fetal side. Pharmacologic blockade of efflux transporters can lead to
disruption of the placental barrier and increase the transfer of respective substrates to the fetal
side by several-fold, which may increase the fetal drug response or also be a mechanism for
teratogenicity. Conversely, transport proteins may also facilitate drug transfer across the
placenta. The monocarboxylate transporters (MCTs) are proton-dependent transporters, and
have been suggested to mediate the placental transfer of diclofenac and salicylic acid (Shintaku
et al., 2007; Shintaku et al., 2009). The influence of polymorphisms in placental drug transport
proteins with respect to fetal drugs exposure and physiology is described in Appendix C.
9
Figure 1-1. ABC Transport proteins present on the human placental barrier.
1.1.3.3 Metabolism and binding by the placenta
Both phase I and II drug metabolizing enzymes have been detected and characterized in
the placenta. For phase I enzymes, CYP1A1 is the major CYP isoform present in the placenta
and is induced by lifestyle factors such as smoking and medications such as glucocorticoids
(Myllynen et al., 2009). CYP1A1 is expressed throughout gestation, whereas CYP1A2 mRNA
has been detected in first trimester placenta but not in term placenta (Syme et al., 2004).
Functional activity of CYP2C, 2D6, and 3A has not been reported, whereas variable activity has
been reported for CYP2E1 (Myllynen et al., 2009; Syme et al., 2004). For phase II enzymes,
UGTs are suggested to have a significant role in placental metabolic activity and are present
throughout gestation (Syme et al., 2004). For example, morphine was observed to have a
placental clearance by UGTs slightly lower than that for total fetal clearance (Garland et al.,
10
2006). However, for most drugs, placental metabolism is a relatively minor and likely non-
significant factor in limiting placental drug transfer (Syme et al., 2004).
Drug transfer across the placenta can also be influenced by drug binding to placental
tissue. For example, bupivacaine disappeared from the maternal circulation more rapidly than
less lipophilic lidocaine during dual perfusion of a placental lobule ex vivo; however, because
bupivacaine was more extensively bound to the placental tissue, less drug appeared in the fetal
compartment compared to lidocaine (Ala-Kokko et al., 1995). Because of the substantial
placental tissue binding, the maternal clearance of bupivacaine exceeds the transplacental
clearance into the fetal circulation (Johnson et al., 1995). High binding to placental tissue can
lead to the placenta acting as a drug depot. In this case, after discontinuation of drug
administration to the maternal circulation, the drug can continue to wash out into the fetal
circulation and potentially prolong drug exposure. Less binding to the placenta can lead to a
more rapid drug equilibration with the fetus and thus a more rapid fetal drug response.
1.1.3.4 Maternal and fetal protein binding
The two most important drug binding plasma proteins are albumin and AAG. These two
proteins differ in concentration in the maternal and fetal circulations. Maternal plasma albumin
gradually decreases towards term while fetal albumin progressively increases. Compared to the
maternal circulation, the fetal circulation has an appreciably higher albumin concentration at
term, with the fetal-to-maternal (F:M) albumin concentration ratio increasing from 0.28 in the
first trimester to 1.20 at term (Krauer et al., 1984). Furthermore, a 3-fold higher concentration of
free fatty acids in the maternal circulation towards term can displace drugs from binding to
albumin in the maternal circulation (Nau et al., 1984; Ridd et al., 1983). Maternal protein binding
can also be reduced by therapeutic maneuvers such as aggressive fluid hydration prior to regional
anesthesia because it leads to a physiological protein dilution (Herman et al., 2000). Taken
together, there is increased binding to albumin in the fetal circulation compared to the maternal
circulation at term; however, the difference in binding between the two circulations is dynamic
throughout gestation. Because it is the free concentration of the drug that will equilibrate across
the placenta, increased fetal protein binding can lead to increased placental drug transfer. Alpha-
fetoprotein is considered to be the fetal analogue of albumin and differs slightly in structure.
11
However, the differences between maternal and fetal plasma protein concentrations influences
placental transfer to a greater degree than varying protein structures or competing endogenous
ligands (Hill & Abramson, 1988)
The other major plasma binding protein, AAG, stays fairly constant throughout gestation
in the maternal circulation while the fetal concentration gradually increases (Krauer et al., 1984).
The F:M AAG concentration ratio increases from 0.09 in the first trimester to 0.37 at term
(Krauer et al., 1984). Compared to maternal, there is lower binding to AAG in the fetal
circulation throughout pregnancy. Because of this, drugs bound primarily to AAG such as
bupivacaine and ropivacaine have a lower total concentration in the fetal circulation compared to
the maternal (Ala-Kokko et al., 1997; Datta et al., 1995). The F:M AAG concentration gradient
was found to be strongly associated with the distribution of ropivacaine and bupivacaine across
the placenta (Ala-Kokko et al., 1997) and is considered to be the main determinant in the steady-
state distribution across the maternal-placental-fetal unit for these agents (Figure 1-2).
1.1.3.5 Acid-Base Equilibrium Effect
Unionized lipid-soluble molecules are able to penetrate biological membranes more
quickly than ones that are ionized and less lipid-soluble. Maternal and fetal plasma pH are
important determinants in placental transfer for weak acid or basic drugs whose pKa is close to
physiologic pH. The fetal circulation is slightly more acidic than the maternal (7.35 vs 7.4
respectively) (Reynolds & Knott, 1989). Because of this difference, weak bases will become
more ionized in the fetal circulation and result in ion trapping (Figure 1-2). For the amide-type
anesthetics bupivacaine (pKa=8.1), lidocaine (pKa=7.9), ropivacaine (pKa=8.1), 2-
chloroprocaine (pKa=8.9), and mepivacaine (pKa=7.6), placental transfer to the fetal circulation
has been observed to increase with decreasing fetal pH (Johnson et al., 1996; Ueki et al., 2009).
For these agents, it is the basic uncharged drug concentration that determines placental transfer
(Ueki et al., 2009).
The difference between maternal and fetal pH is minimal (<0.1 pH units) under normal
circumstances. However, fetal pH may fall considerably in cases of fetal compromise.
Compared with general or epidural anesthesia, there is also a greater degree of fetal acidosis
12
observed during spinal anesthesia for Caesarean section (Reynolds & Seed, 2005). As fetal pH
decreases, the effect of ion trapping on basic drugs in the fetal circulation is accentuated and less
drug is able to cross back from the fetal to the maternal circulation. For amide-type anesthetics,
this leads to increased fetal concentrations and is expected to lead to increased fetal effect (Biehl
et al., 1978; Pickering et al., 1981). However, this phenomenon may be less pronounced for
agents that are rapidly metabolized and do not have the chance to accumulate. For example, at
the time of delivery, there was no association found between umbilical cord concentrations of 2-
chloroprocaine and a fetal pH ≤7.25 (Philipson et al., 1985).
In addition to influencing the level of drug ionization, plasma pH can also influence other
pharmacokinetic factors, such as protein binding. With decreasing pH, protein binding for
lidocaine was shown to decrease (Burney et al., 1978). Thus, the effect of decreased fetal pH on
placental drug transfer will be the net result of changes in both drug ionization and protein
binding. During fetal distress and fetal acidosis, there is increased perfusion to the heart and
brain, which may also alter fetal drug response. This may lead to increased drug delivery to
these important organs.
1.1.4 Methods for Studying Transplacental Drug Transfer
There is often limited information available on drug safety for new pharmacologics in
pregnancy and specifically on maternal-to-fetal pharmacokinetics. The full time-course of
pharmacokinetic changes during pregnancy is rarely studied. Obtaining data on placental and
fetal pharmacokinetics is also difficult since the feto-placental unit in situ is not easily accessible
until delivery. In vivo human studies evaluating the transplacental kinetics of drugs have
numerous ethical concerns regarding fetal and maternal safety. Because the placenta is the most
species-specific mammalian organ, animal studies cannot always be extrapolated to humans
(Ala-Kokko et al., 2000). Limited pharmacokinetic data can be obtained via cord blood
sampling in neonates whose mothers receive the agent to be studied. By measuring the difference
in drug concentrations between the umbilical vein and artery at term, fetal uptake and/or
metabolism of a drug can be estimated. For lidocaine, concentrations in the fetal umbilical vein
were 1.5 times higher than the umbilical artery, suggesting fetal uptake or metabolism of the
13
drug (De Barros Duarte et al., 2010). From these studies, it is difficult to characterize how the
drug distributes in the fetus and the amount/type of drug metabolism.
Figure 1-2. The transfer of the basic drug, bupivacaine, across the placental membrane at
equilibrium is determined by the acid-base equilibrium effect and protein binding. A)
Unbound bupivacaine (pKa=8.1) is more ionized in the more acidic fetal circulation as a result of
ion trapping. A fetal to maternal ratio of 1.08 for unbound bupivacaine results from ion trapping.
B) Bupivacaine is bound to α1-acid glycoprotein (AAG) and higher binding in the maternal
circulation results from the higher AAG concentration in plasma. As a result, the fetal to
maternal concentration ratio for total bupivacaine is 0.32.
14
Dual perfusion of a single placental lobule ex vivo is the only experimental model to
study human placental transfer of substances in organized placental tissue and theoretically may
be able to better predict fetal exposure compared to other experimental methods (Hutson et al.,
2011). In vitro models utilizing cell culture and membrane vesicles are mostly limited to the
investigation of specific mechanisms of transfer, such as active transport or passive diffusion, but
they lack anatomic integrity and blood flow. In vitro cellular models have focused on transport in
trophoblast cells and have largely neglected the potential role of the fetal endothelial cells to
influence transfer (Elad et al., 2014). The advantages and disadvantages of different methods
used to study placental drug transfer are provided in Table1-1.
1.1.4.1 Description of the Dual Perfusion of a Single Placental Lobule ex vivo
The first perfusion of an isolated human placental lobule was described by Panigel et al.
in 1967 (Panigel et al., 1967) and was subsequently modified by Schneider and Miller (Miller et
al., 1985; Schneider et al., 1972). Use of a placenta after delivery obviates ethical problems
because tissue collection is non-invasive and harmless to both the mother and newborn. Figure 1-
3 provides a schematic description of the placental perfusion system. Between laboratories,
experimental systems can vary with respect to cannulation method, isolation procedure,
perfusion fluid, oxygenation, perfusion flow, and control measurements (Mathiesen et al., 2010).
The perfusion model described here is routinely used in our laboratory in Toronto.
15
Table 1-1. Advantages and disadvantages of different methods used to determine placental
drug transfer.
Method Advantages Disadvantages
Placental Perfusion • Most closely resembles the
in vivo situation as structural integrity and cell-cell organization are maintained
• Can measure transfer over time
• Sampling is available from all compartments including placental tissue
• Often performed in term tissue and cannot be used to determine transfer in the first trimester
• No standardized criteria between laboratories
Measuring Umbilical
Cord Blood and
Maternal Blood
Drug Concentrations
at Delivery
• True in vivo measure in humans
• Sampling at delivery avoids ethical issues
• Timing of sampling usually restricted to delivery
• Represents only one point in time and large inter-individual variation
• New drugs or toxic substances can not be studied ethically
Animal Models • Transfer and toxicity at
different gestational ages can be studied
• Drug accumulation in fetal tissues can be studied
• Low inter-individual variation as a result of inbred animals
• Interspecies variability in placental structure and blood flood flow pattern
• Difficult to extrapolate findings to humans
Trophoblast Tissue
Preparations (Villous
Preparations, Membrane
Vesicles)
• Useful for studying transfer mechanisms (such as uptake, efflux and metabolism), even at earlier gestational ages
• Can study fetal and maternal plasma membrane fractions separately
• Not useful for studying trans-cellular transport.
• Transporter regulatory mechanisms may not be present in the preparation
• Does not incorporate the role of fetal endothelial cells
Trophoblast Cultures (primary cultures and
trophoblast cell lines)
• Useful for studying transfer mechanisms (such as uptake, efflux and metabolism)
• BeWo cell line available that forms cellular monolayers
• Transporter expression can vary between cultures
• Does not incorporate the role of fetal endothelial cells
16
Figure 1-3. Schematic presentation of the placental perfusion experimental set-up.
17
After tissue collection, a fetal vein-artery pair supplying a well-defined cotyledon is
identified. The corresponding maternal surface is also confirmed to have an intact basal plate and
no evident trauma. The fetal vein-artery pair is cannulated and flow of perfusate through the fetal
circulation is established. The lobule is clamped into a plexiglass chamber with the fetal side
downwards and the excess placental tissue is removed. Buffered saline in the chamber supports
the weight of the lobule and the chamber is placed in a water bath to maintain physiologic
temperature. Depending on the design of the perfusion chamber, saline solutions may not be
used and physiologic temperature may be maintained using a heated flowbench (Ala-Kokko et
al., 2000; Mathiesen et al., 2010).
Perfusion of the maternal side begins with insertion of blunt-tipped needles into the
intervillous space 2-3 mm below the decidual surface. Venous samples from the maternal
circulation are collected from multiple venous openings in the decidual plate. The fetal and
maternal circulations are independently controlled by roller pumps and are equilibrated with 95%
O2/5% CO2 in the maternal side and 95% N2/5% CO2 in the fetal side. Some groups utilize
atmospheric air instead of 95% O2/5% CO2 to avoid toxicity associated with hyperoxia
(Mathiesen et al., 2010). Flow rates are maintained in the fetal and maternal circulations at 3 and
12 mL/min, respectively. Flow rates used vary between laboratories (Mathiesen et al., 2010) and
can influence the time to achieve transplacental transfer and steady state between the fetal and
maternal circulations (Bassily et al., 1995; He et al., 2001).
The first portion of the experiment is usually a control period where the blood is washed
out and baseline parameters are established. After the control period, the compound of interest is
added either into the maternal or fetal circulation to investigate placental transfer in one
direction: maternal-to-fetal or fetal-to-maternal, respectively. The experiment is performed in
either a closed (recirculating) or open (single-pass or non-recirculating) configuration. In the
closed configuration, the perfusates are recycled to imitate physiological conditions. Drug
transfer, as well as the maternal-placental-fetal distribution, can be evaluated. Open
configuration allows for the calculation of drug clearance at steady-state concentrations. The
drug can also be added equally to both circulations in the closed configuration to assess any
accumulation in one direction (Gedeon et al., 2008; Kraemer et al., 2006; Pollex et al., 2008).
18
Placental viability and integrity are monitored by several parameters both during and
after the perfusion. However, there is no standard set of parameters that are required and these
vary between laboratories (Mathiesen et al., 2010). Common parameters measured during the
perfusion include pressure in the fetal circulation, pH, oxygen consumption, net fetal oxygen
transfer, glucose consumption, lactate production, and synthesis and secretion of proteins
including human chorionic gonadotropin (hCG), human placental lactogen, and leptin (Hutson et
al., 2011). The optimal measure of tissue viability and integrity is suggested to be measuring
fetal volume loss (Pienimaki et al., 1997). There is no generally accepted maximum fetal volume
loss, however, a loss of 2 to 4 mL/hour from the fetal reservoir is typically allowed (Mathiesen et
al., 2010). The addition of a flow-limited marker that undergoes only passive diffusion, such as
antipyrine, into the maternal circulation is also be used to measure tissue integrity. Antipyrine or
other reference markers are also used to standardize results to account for variability in flow or
lobule size among experiments.
Results obtained from placental perfusion experiments are often compared to in vivo
results (if available) that have been collected by measuring drug concentrations in maternal
blood and fetal/umbilical cord blood at the time of delivery. The calculation of cord to maternal
blood concentration ratio (C:M) can provide information on how much drug crosses the placenta
in vivo. Drugs are either measured after the mother has been taking the medication chronically
during pregnancy and is likely in steady state or after single bolus administration before delivery.
Venous blood is usually collected from the mother, while umbilical cord blood can be arterial,
venous, or mixed cord blood. Ideally, drug levels should be measured from the umbilical vein,
when evaluating placental drug transfer, since this receives blood directly from the placenta.
Drug concentrations are likely to be higher in the umbilical vein before reaching steady state
compared to mixed or arterial cord blood. Measuring the concentration difference between the
umbilical vein and artery can also provide information on whether there is drug distribution or
metabolism by the fetus. However, it is not possible from cord blood measurements to
determine if there is accumulation of the drug in fetal tissues.
It is difficult to accurately determine the transplacental kinetics of a particular drug by the
C:M concentration ratios because it is measured at only one time point and typically in a small
number of mother-infant pairs. Often there is large variability between pairs due to sample
contamination, timing of samples, sample site, inter-individual differences, and timing of
19
exposure. Maternal venous blood concentrations do not reflect those being presented to the
placenta and can depend on the tissues drained by the selected vein if an arteriovenous gradient
exists (Hermann et al., 1999). However, sampling at the time of delivery is easily accessible
using umbilical cord blood and is an ethically acceptable method. It is often the only source of in
vivo human data.
1.2 Therapeutic Drug Use during Pregnancy: Inflammatory Bowel Disease and Treatment with Thiopurines
Between 23 and 85% of women are estimated to require medication at some point during
their pregnancy (Gendron et al., 2009). Many maternal medical conditions such as diabetes,
epilepsy, and hypertension require drug therapy in order to ensure optimal health of both the
mother and fetus. A common medical condition encountered during pregnancy is inflammatory
bowel disease (IBD). The major types of IBD are Crohn's disease and ulcerative colitis and peak
incidence of IBD occurs in women of childbearing age (Cooper & Stroehla, 2003). During
pregnancy, maintaining disease remission is critical for positive fetal outcome (Coelho et al.,
2011; Kwan & Mahadevan, 2010) since disease activity is associated with adverse pregnancy
outcomes (Baiocco & Korelitz, 1984; Dominitz et al., 2002; Fedorkow et al., 1989; Woolfson et
al., 1990).
The thiopurines azathioprine (AZA) and 6-mercaptopurine (6-MP) are effective
treatments for IBD and are increasingly being used as treatment for this disease in pregnancy
(Goldstein et al., 2007). Thiopurines for the treatment of IBD can benefit patients by maintaining
remission during pregnancy, avoiding chronic use of steroids for disease control, and leading to
endoscopic remission (Ardizzone et al., 2006; Mowat et al., 2011; Timmer et al., 2007).
Avoiding the chronic use of oral corticosteroids in women of childbearing age is beneficial as it
has been associated with an increased risk of adverse perinatal outcomes including gestational
diabetes, preeclampsia, preterm delivery, oral cleft palate, premature rupture of membranes, and
impaired fetal growth (Jain & Gordon, 2011; Landy et al., 1988; Park-Wyllie et al., 2000; Schatz
et al., 2004).
20
Animal studies have investigated the potential teratogenic effect of the thiopurines. In
mice given AZA intraperitoneally (i.p.) at doses 4-13 times human therapeutic doses, increased
rates of cleft palate and skeletal anomalies were observed (Githens et al., 1965; Rosenkrantz et
al., 1967). A similar study at these high doses, however, observed no fetal anomalies in mice
(Tuchmann-Duplessis & Mercier-Parot, 1964). No malformations were observed in mice born to
mothers injected i.p. with therapeutic levels or twice therapeutic levels of AZA during the time
of organogenesis, however, there was an increased frequency of fetal loss and growth retardation
(Tuchmann-Duplessis & Mercier-Parot, 1964; Githens et al., 1965; Rosenkrantz et al., 1967). In
pregnant rabbits injected i.p. with AZA at a dose two to six times higher than that used in
humans, increased frequencies of limb malformations, ocular anomalies, and cleft palate were
observed in the offspring (Tuchmann-Duplessis & Mercier-Parot, 1964). In pregnant rats given
a single injection of 6-MP equivalent to 37.5 to 156 times the maximum human therapeutic dose
on the 11th or 12th day of gestation, an increased incidence of cleft palate, skeletal and urogenital
anomalies, and diaphragmatic hernia were observed in fetuses (Murphy, 1960; Bragonier et al.,
1964; Chaub and Murphy, 1968; Kury et al., 1968; Puget et al., 1975). However, no
malformations were induced in fetuses of pregnant rats treated orally at various times during
organogenesis with 6-MP at the equivalent of one to 12 times the maximum human dose
(Thiersch, 1954).
Several studies have investigated the safety of thiopurines in human pregnancy. A
review of 27 case series and three large retrospective and prospective cohort studies did not find
an increased risk for major malformations associated with the use of AZA in pregnancy (Cleary
& Kallen, 2009; Coelho et al., 2011; Francella et al., 2003; Goldstein et al., 2007; Polifka &
Friedman, 2002). These studies are discussed further in detail in Chapter 4. However, one recent
study reported a trend for increased malformations and an increased risk specifically for
ventricular/atrial septal defects after prenatal AZA exposure (Cleary & Kallen, 2009). An
increased risk for prematurity and lower birth weight has also been reported, but this may be
related to the underlying maternal disease (Cleary & Kallen, 2009; Goldstein et al., 2007).
Although evidence supports the relative safety of AZA in pregnancy compared to the untreated
disease (Coelho et al., 2011), safety concerns still arise from its mechanism of action and its
toxicity during pregnancy in animal studies (Polifka & Friedman, 2002). Because of these
remaining safety concerns, over eighty percent of women with IBD have concerns about
21
medications, including AZA, in pregnancy and fears surrounding medications are greater than
the negative effect of IBD exacerbation (Mountifield et al., 2010). Moreover, some physicians
still choose not to administer AZA during pregnancy because they consider it to be a potential
teratogen or it is discontinued in the third trimester (Mahey et al., 2013; Peyrin-Biroulet et al.,
2011).
After oral administration, AZA is extensively (>80%) cleaved into 6-MP in the blood
(Polifka & Friedman, 2002). 6-MP is a purine analogue, and after further conversion into 6-
thioguanine nucleotides (6-TGNs), the active metabolites (Figure 1-4), it can inhibit de novo
purine ribonucleotide synthesis leading to inhibition of cell proliferation. Furthermore, 6-MP
can be cytotoxic after incorporation of its metabolites into cellular DNA. Side effects include
bone marrow suppression, hepatotoxicity, and an increased risk of neoplasia (Nelson et al., 1975;
Van Scoik et al., 1985). Since 6-MP targets rapidly dividing cells, the developing fetus would
theoretically be expected to be sensitive to its cytotoxicity. However, even high dose 6-MP was
not found to be useful as a single-agent medical abortifacient in early pregnancy, leading the
authors to suggest that this drug does not alter cell division in trophoblastic tissues (Davis et al.,
1999).
The half-life of 6-MP is short at one hour (Chan et al., 1987; Ohlman et al., 1994). The
metabolite, 6-thioguanine (6-TG), is found intracellularly and the half-life is much longer at
three to 13 days (Lennard, 1992; Sandborn et al., 1995). The effect of pregnancy on the
pharmacokinetics of 6-MP has only recently been studied. Jharap et al. (2014) studied a small
cohort of 30 pregnant women with IBD receiving steady-state thiopurines. Median 6-TGN
concentrations decreased over the course of the pregnancy but returned to preconception baseline
after delivery. On the other hand, 6-methylmercaptopurine (6-MMP) concentrations increased
but returned to baseline after delivery. Although these changes were statistically significant, the
clinical significance is unknown as no woman in this study developed toxicity or therapeutic
failure. The authors also measured fetal concentrations of 6-TGN in umbilical cord blood
immediately after delivery and found fetal concentrations to correlate positively with maternal 6-
TGN levels.
22
Figure 1-4 Metabolic pathway for 6-mercaptopurine and its prodrug azathioprine.
GMPS, guanine monophosphate synthetase; HPRT, hypoxanthine–guanine phosphoribosyltransferase; IMPDH,
inosine monophosphate dehydrogenase; meTGMP, methylated TGMP; meTIMP, methylated TIMP; TGMP,
6’thioguanosine 5’monophosphate; TGN, thioguanine nucleotides; TIMP, 6-thioinosine 5 ́ monophosphate; TPMT,
thiopurine S-methyltransferase; TXMP 6-thioxanthosine 5’monophosphate; XO, xanthine oxidase;
23
1.3 Recreational Drug Use During Pregnancy: Alcohol and Fetal Alcohol Spectrum Disorder
In addition to therapeutic drugs, many women of childbearing age consume recreational
or illicit drugs. In a survey of Canadian women who had recently given birth, the self-reported
incidence of alcohol use in pregnancy was 5-15% and street drug use was 1-5% (Finnegan, 2013;
Zelner & Koren, 2013). However, because of the stigma associated with alcohol and recreational
drug use in pregnancy, self-reporting is likely to underestimate the true incidence (Ostrea et al.,
2001). In Canada, one standard drink contains 17.05 mL (13.45 g) of pure ethanol and in the US
it equals to 18 mL (14.00 g) of pure alcohol. This roughly corresponds to one 12 fl. oz. (341 mL)
bottle of beer, cider, or cooler (all 5% alcohol); one 5 fl. oz.(142 mL) glass of wine (12%
alcohol); or 1.5 fl.oz. (43 mL) shot of 80-proof spirits or liquor (40% alcohol) (Butt et al., 2011;
Centers for Disease Control and Prevention, 2012). Statistics Canada defines heavy drinking as
>5 drinks per occasion, >12 times over the past year (Dell & Roberts, 2006).
Prenatal alcohol exposure can lead to a wide range of deficits known as fetal alcohol
spectrum disorder (FASD). FASD is considered the leading preventable cause of intellectual
disability in the developed world (Abel & Sokol, 1986). The most severe form of FASD is fetal
alcohol syndrome (FAS) and is characterized by prenatal and postnatal growth restriction, facial
dysmorphology, and neurocognitive and behavioural dysfunction (Chudley et al., 2005). Fetal
alcohol syndrome is characteristic of heavy daily or weekend binge drinking (Abel, 1998). Also
under the umbrella term FASD are less severe outcomes such as partial FAS, alcohol-related
birth defects, and alcohol-related neurodevelopmental disorder. The less severe outcomes result
from differences in frequency and amount of alcohol consumed, timing of exposure during the
pregnancy, genetic factors, and maternal nutrition (Alati et al., 2006; Chudley et al., 2005;
Shankar et al., 2006; Warren & Li, 2005). Criteria for diagnosing FASD as set by the Institute of
Medicine (IOM) are detailed in Table 1-2.
24
Table 1-2 The 1996 Institute of Medicine’s Diagnostic Criteria for FASD (Stratton et al.,
1996)
Category Description 1. FAS with confirmed maternal alcohol exposure
• Confirmed maternal alcohol exposure • Facial anomalies including thin upper
lip, flattened philtrum flat midface, small palpebral fissures
• Growth retardation • Central nervous system
neurodevelopmental abnormalities 2. FAS without confirmed maternal alcohol exposure
• Same as above but without confirmed maternal alcohol consumption
3. Partial FAS with confirmed maternal alcohol exposure
• Confirmed maternal alcohol exposure • Facial anomalies (less than seen in 1 &
2) • Growth retardation • Central nervous system
neurodevelopmental abnormalities • Behavioural/cognitive abnormalities
4. Alcohol-related birth defects • Confirmed maternal alcohol exposure • Congenital anomalies such as cardiac
defects, skeletal defects, among many others
5. Alcohol-related neurodevelopmental disorder
• Confirmed maternal alcohol exposure • Central nervous system
neurodevelopmental abnormalities OR Behavioural/cognitive abnormalities
25
In addition to primary features associated with FASD, many children, adolescents, and
adults with FASD suffer from secondary disabilities (Table 1-3). The risk of developing
secondary disabilities associated with FASD is decreased 2- to 4-fold with an early diagnosis
and/or a stable, loving home environment (Streissguth et al., 2004). The large array of primary
and secondary deficits associated with FASD results in a large economic impact for affected
individuals, affected families, and society. In Canada, where the prevalence of FAS and FASD is
considered to be similar to USA general population estimates of 0.1% and 1.0% respectively
(May & Gossage, 2001), the associated annual economic loss is estimated at 4-6 billion dollars
(Stade et al., 2009). If extrapolated to include other countries, the global economic burden
would be enormous, exemplifying the need to establish FASD prevention strategies globally.
Table 1-3 Secondary Disabilities in Adolescents and Adults with FAS or FAE (O’Connor et
al., 2002; Streissguth et al., 2004)
Adverse Life Outcome Prevalence
Psychiatric Disorder 87% (n=23)
Disrupted School Experience 61% (n=415)
Trouble with the Law 60% (n=415)
Confinement 50% (n-415)
Inappropriate Sexual Behaviour 49% (n=415)
Alcohol/Drug Problems 35% (n=415)
26
Ethanol can freely cross the placenta and transfer is bidirectional between the maternal
and fetal circulations (Brien et al., 1983; Nava-Ocampo et al., 2004). Ethanol concentrations are
similar in the maternal and fetal circulations; however, amniotic fluid may act as a reservoir for
ethanol and prolong fetal exposure (Brien et al., 1983; Nava-Ocampo et al., 2004). Numerous
mechanisms have been proposed as contributing to alcohol-induced fetal damage. Ethanol itself
is generally considered to be the teratogenic substance, however, metabolites such as
acetaldehyde also may play a role (reviewed in Goodlett et al., 2005). Suggested mechanisms
include oxidative stress to both the fetus and placenta leading to excessive cell death,
interference with developmental regulatory proteins, interference with neurotransmitters, and
interference with trophic factors that regulate neurogenesis and cell survival (Bosco & Diaz,
2012; Goodlett et al., 2005). However, no single mechanism can account for all the phenotypes
observed in children with FASD and we still do not have a good understanding of the mechanism
of alcohol teratogenesis (Goodlett et al., 2005).
In addition to ethanol, alcoholic beverages may contain a small amount of methanol as a
congener (Lachenmeier et al., 2011). Furthermore, methanol is produced endogenously in the
pituitary from S-adenosylmethionine (Axelrod & Daly, 1965; Sarkola & Eriksson, 2001).
Methanol shares a metabolic pathway with ethanol in that it is first metabolized by alcohol
dehydrogenase (ADH) into formaldehyde or acetaldehyde, respectively (Kalant & Khanna,
2007). Second, it is further metabolized by acetaldehyde dehdydrogenase (ALDH) into formic
acid or acetic acid, respectively. In heavy drinkers, methanol may accumulate since ethanol has a
higher affinity for alcohol dehydrogenase (ADH) and consequently methanol may reach plasma
concentrations above 2 mM (Kapur et al., 2007; Majchrowicz & Mendelson, 1971). Formic acid,
the toxic metabolite of methanol, has been detected in both sera and cerebrospinal fluid of
alcoholics in concentrations that are neurotoxic (Kapur et al., 2007). There is no current
regulation of methanol content in Canada, however, the European Union has set a limit for
naturally occurring methanol at 10g methanol/ litre of ethanol which equates to 0.4% (v/v)
methanol at 40% alcohol (Paine & Davan, 2001).
Formic acid has also been recently detected in maternal blood and umbilical cord blood
of infants born to heavy drinkers (Kapur, et al., 2009). Furthermore, preliminary studies have
shown a negative correlation between formic acid concentrations in umbilical cord blood at the
time of delivery and cognitive function as measured using Bayley scores (r= -0.6154, p=0.025,
27
n=12 at 12 months and r=-0.6241, p=0.023, n=13 at 18 months) (Kapur, et al., 2009). Formic
acid has also been shown in animal studies to lead to growth restriction, physical malformations,
and depletion of glutathione in the embryo (Andrews et al., 1998; Brown-Woodman et al., 1995;
Hansen et al., 2005; Harris et al., 2004). Furthermore, human studies have reported fetal alcohol
syndrome-like facial features in infants born after solvent abuse (including methanol) by the
mother during pregnancy (Scheeres & Chudley, 2002). However, the role of methanol and
formic acid in the development of FASDs has not been investigated.
Formic acid is cleared by the body through metabolism into carbon dioxide and two non-
free radical pathways have been proposed for this step. First, oxidation through the catalase-
peroxidative system (Chance, 1950), and second by entering the one-carbon pool. Formate enters
the one carbon pool by combining with tetrahydrofolate (THF) to form 10-formyl-THF, a
reaction catalyzed by 10-formyl-THF synthetase (Johlin et al., 1987). This is followed by the
oxidation of 10-formyl-THF to carbon dioxide mediated by 10-formyl-THF dehydrogenase (10-
FTHFDH). Metabolism of formate through the one carbon pool has been shown to be the major
route (Johlin et al., 1987; Makar & Tephly, 1976; Chiao & Stokstad, 1977; Palese & Tephly,
1975) and also the predominant route in primates (McMartin et al., 1977). In humans, formate
oxidation to carbon dioxide is dependent upon folate (Johlin et al., 1987). Sokoro and
colleagues, using an animal model, showed that the formic acid half-life was significantly longer
in the folate deficient animals when compared with folate sufficient animals (Sokoro et al.,
2008). Studies on rat brain hippocampal slices showed that formic acid can cause neuronal cell
death at concentrations found in heavy alcohol users, and this toxicity can be mitigated by folate
(Kapur et al., 2007).
Folic acid deficiency is a common finding in chronic alcoholics (Herber et al., 1963;
Halsted et al., 2002a). Chronic alcohol ingestion reduces the intestinal absorption of dietary folic
acid leading to a decrease in the folate metabolic pool (Halsted et al., 2002b). A decrease in this
pool prolongs the formate blood levels by decreasing the rate at which formate combines with
tetrahydrofolate, the first step in its metabolism to carbon dioxide. Decreased detoxification of
formate may lead to formate-induced cytotoxicity mediated by oxidative stress (McMartin et al.,
1977). Since folic acid plays a major role as a coenzyme in one-carbon metabolism and is a key
participant in the biosynthesis of DNA, RNA and certain amino acids, it is vital to proper fetal
development. In an animal model, it has been suggested that ethanol can alter the normal transfer
28
of folate across the placenta and is described further in Section 5.2. Furthermore, Lin & Lester
(1985) showed that the folate distribution in the rat placenta was altered after ethanol exposure as
accumulation of methyltetrahydrofolate was increased compared to non-methyltetrahydrofolates.
Ethanol has been shown to decrease the activity of methionine synthase, which has been
suggested to decrease the regeneration of non-methyltetrahydrofolates (Barak et al., 1985;
Finkelstein et al., 1974). To our knowledge, there are no studies in humans to determine the
effect of folic acid on the toxicity of formic acid in pregnancy as this is a novel hypothesis to
explain the deficits observed in the FASDs.
29
1.4 Overall Rational and Primary Objectives
The human placenta performs many functions that support the maintenance of pregnancy
and the normal development of the fetus. This organ regulates the supply of oxygen and
nutrients to and removal of waste products from the fetus by bringing the maternal and fetal
circulations into close opposition. Because the human placenta separates the maternal and fetal
circulations, it can restrict fetal drug or toxin exposure from agents consumed by the pregnant
woman. Studies utilizing the human placenta are vital to understanding fetal drug exposure and
also drug safety in pregnancy. Dual perfusion of a single placental lobule is the only
experimental model to study human placental transfer of substances in organized placental
tissue. To date, there has not been any attempt at a systematic evaluation of this model. Careful
validation of the perfusion model is needed before it can be used routinely to predict placental
drug transfer during preclinical evaluation.
The first objective of this thesis was to systematically evaluate the placental perfusion
model in predicting placental drug transfer and to develop a pharmacokinetic model to
account for non-placental pharmacokinetic parameters in the perfusion results. Having a
validated and accurate model to predict drug transfer across the human placenta will be an
invaluable tool to help guide decisions regarding the benefits and risks of new medications
that may be required during pregnancy. We hypothesize that the placental perfusion model
will accurately predict in vivo placental drug transfer after adjusting results for non-placental
pharmacokinetic parameters.
The observed safety of AZA in pregnancy likely results, in part, from the placenta
limiting fetal exposure to the main metabolite, 6-MP. Indeed, only 1-2% of maternal 6-MP
concentrations are found in cord blood and the human placenta is considered to be a relative
barrier to 6-MP and its metabolites (de Boer et al., 2006; Saarikoski & Seppala, 1973).
However, the mechanism of how the placenta restricts 6-MP transfer to the fetus is unknown. 6-
MP is a substrate for several drug transport proteins, including breast cancer resistance protein
(BCRP) (de Wolf et al., 2008). BCRP is highly expressed in the placenta and is localized to the
brush border (maternal-facing) membrane of the syncytiotrophoblast (Maliepaard et al., 2001).
The importance of BCRP and other placental transporters in limiting fetal exposure to various
30
drugs has been demonstrated in both human and animal models (Jonker et al., 2000; Pollex et al.,
2008).
The second objective of this thesis was to further evaluate the safety of AZA and 6-MP
in pregnancy using two approaches. First, to systematically review the literature and perform
a meta-analysis of available studies in order to further quantify the safety of thiopurines in
pregnancy. Second, to determine the mechanisms by which the placenta restricts 6-MP
transfer using the validated perfusion model from objective one. Understanding these
mechanisms will allow for prediction of fetuses that may be at risk of higher exposure to 6-MP
as a result of drug interactions with, or polymorphisms in, placental drug efflux proteins. We
hypothesize that 6-MP does not increase the risk for fetal malformations when taken during
pregnancy and that 6-MP is a substrate for placental drug efflux transporters.
Heavy alcohol consumption during pregnancy is associated with numerous placental
alterations including placental dysfunction, decreased placental size, gene expression changes
and endocrine disruptions (Bosco & Diaz, 2012; Burd et al., 2007; Rosenberg et al., 2010).
Methanol and its toxic metabolite, formic acid, have been detected in high concentrations in sera
of heavy alcohol users and may contribute to the teratogenic nature of alcohol beverages (Kapur
et al., 2007). Since formic acid can produce oxidative stress (Harris et al., 2004), it may be
placentotoxic; however, this has not been studied to date. Since the placenta is a reservoir for
folate (Henderson et al., 1995) and folate is required for detoxification of formic acid, there may
also be a potential role for detoxification of formic acid within the placenta itself. In the human
placenta, short-term exposure to ethanol during a human placental perfusion had no effect on
either binding or transfer of 5-methyltetrahydrofolate (Henderson et al., 1995). However, animal
studies have suggested that alcohol exposure can decrease transfer to the fetus (Fisher et al.,
1985; Keating et al., 2009; Keating et al., 2008). The chronic effect of alcohol during pregnancy
to folate status in the human placenta has not been investigated. Decreased folic acid transport
may limit the detoxification of formic acid in the fetus in addition to the other negative
consequences of folate deficiency in the fetus.
The third objective of this thesis was to evaluate the potential toxicity of formic acid to
the placenta and if folate may be protective to this toxicity. To determine this, the effect of
alcohol consumption in human pregnancy on placental transfer of folate will first be
31
evaluated. We hypothesize that placental folate transfer to the fetus is decreased in
pregnancies with heavy alcohol exposure and that this is mediated by decreased expression of
placental folate transporters. Second, we will determine whether formic acid transfers across
the placenta and if it is toxic to the placenta. We hypothesize that formic acid can transfer
across the placenta because it is a small molecule and is toxic to the placenta but folate can
both decrease transplacental transfer of formic acid and mitigate toxicity.
32
Chapter 2
The human placental perfusion model: a systematic review and development of a model to predict in vivo transfer of therapeutic drugs Janine R Hutson1,2, Facundo Garcia-Bournissen1,2, Amy Davis1, Gideon Koren1,2
1Division of Clinical Pharmacology and Toxicology, Hospital for Sick Children, 555 University
Ave, Toronto, Ontario, M5G 1X8, Canada
2Institute of Medical Science, University of Toronto, 1 King’s College Circle, Toronto, Ontario,
M5S 1A8, Canada
This work has been published and reproduced with permissions: Hutson JR, Garcia-Bournissen F, Davis A, Koren G. The human placental perfusion model: a
systematic review and development of a model to predict in vivo transfer of therapeutic drugs. Clin Pharmacol Ther 2011;90(1):67-76.
33
The human placental perfusion model: a systematic 2review and development of a model to predict in vivo transfer of therapeutic drugs
2.1 Abstract
Dual perfusion of a single placental lobule is the only experimental model to study
human placental transfer of substances in organized placental tissue. To date, there has not been
any attempt at a systematic evaluation of this model. The aim of this study was to systematically
evaluate the perfusion model in predicting placental drug transfer and to develop a
pharmacokinetic-model to account for non-placental pharmacokinetic parameters in the
perfusion results. In general, the fetal-to-maternal drug concentration ratios matched well
between placental perfusion experiments and in vivo samples taken at the time of delivery. After
modeling for differences in maternal and fetal/neonatal protein binding and blood pH, the
perfusion results were able to accurately predict in vivo transfer at steady state (R2=0.85,
p<0.0001). Placental perfusion experiments can be used to predict placental drug transfer when
adjusting for extra parameters and can be useful for assessing drug therapy risks and benefits in
pregnancy.
34
2.2 Introduction
Studies have estimated that between 23-85% of women require medication at some point
during their pregnancy (Gendron et al., 2009). There is often limited information on the fetal
safety of medications in pregnancy and the decision to continue treatment relies on weighing the
benefit of the drug against the fetal risks. An important determinant in this risk assessment is
estimation of fetal exposure, based on quantifying the amount of drug that crosses the placenta.
The rate-determining step in placental drug transfer is the crossing of drug molecules across a
single layer of fetal-derived syncytiotrophoblasts (Ceckova-Novotna et al., 2006). In vivo studies
evaluating the transplacental kinetics of drugs in humans have numerous ethical concerns
regarding fetal and maternal safety. Furthermore, the feto-placental unit in situ is not easily, or
ethically, accessible until delivery. Animal studies cannot always be extrapolated to humans
because the placenta is the most species-specific mammalian organ (Ala-Kokko et al., 2000). In
vitro models utilizing cell culture and membrane vesicles are mostly limited to the investigation
of specific mechanisms of transfer, such as active transport or passive diffusion, but they lack
anatomic integrity and blood flow.
Dual perfusion of a single placental lobule ex vivo is the only experimental model to
study human placental transfer of substances in organized placental tissue and theoretically may
be able to predict fetal exposure compared to other experimental methods (Table 1.1). Although
tedious, the placental perfusion model has proven useful in studying both endogenous and
exogenous substrates, such as amino acids, hormones, electrolytes, viruses, therapeutics, and
illicit drugs (Ala-Kokko et al., 2000; Malek et al., 2009; Muhlemann et al., 1995; Omarini et al.,
1992). This model allows to investigate simultaneously many properties that can influence
placental drug transfer, such as physiochemical properties (size, pKa, and lipophilicity) and
pharmacokinetic factors (active transport, placental binding, and metabolism). A limitation of
this model is that it cannot fully incorporate non-placental pharmacokinetic factors such as
protein binding, elimination, and distribution in the maternal and fetal compartments. However,
these factors can be determined in vitro. While protein can be added to the perfusion medium
(Gavard et al., 2006; Hemauer et al., 2009; Johnson et al., 1999; Schenker et al., 1999), it is
difficult to mimic physiological conditions since there are numerous proteins and endogenous
factors that could influence binding in vivo. A description of the model is given in section
1.1.4.1.
35
Results obtained from placental perfusion experiments are often compared to in vivo
results (if available) that have been collected by measuring drug concentrations in maternal
blood and fetal/umbilical cord blood at the time of delivery. The calculation of cord to maternal
blood concentration ratio (C:M) can provide information on how much drug crosses the placenta
in vivo. Drugs are either measured after single bolus administration before delivery or after the
mother has been taking the medication chronically during pregnancy and is likely in steady state.
From the mother, venous blood is usually collected, while umbilical cord blood can be arterial,
venous, or mixed cord blood. Ideally, drug levels should be measured from the umbilical vein
for evaluating placental drug transfer, since this vessel receives blood directly from the placenta.
Drug concentrations are likely to be higher in the umbilical vein before reaching steady state
compared to mixed or arterial cord blood. Measuring the concentration difference between the
umbilical vein and artery can also provide information on whether there is drug distribution or
metabolism by the fetus. It is, however, not possible from cord blood measurements to
determine if there is accumulation of the drug in fetal tissues.
It is difficult to accurately determine the transplacental kinetics of a particular drug by
this in vivo method because it is measured at only one time point and typically in a small number
of mother-infant pairs. Often there is large variability between pairs due to sample
contamination, timing of samples, sample site, inter-individual differences, and timing of
exposure. Maternal venous blood concentrations do not reflect those being presented to the
placenta and can depend on the tissues drained by the selected vein if an arteriovenous gradient
exists (Hermann et al., 1999). However, sampling at the time of delivery is easily accessible
using umbilical cord blood and is an ethically acceptable method. It is often the only source of in
vivo human data.
Because in vivo data are not usually available, especially for new drugs, a method to
reliably estimate transfer across the placenta is needed. Careful validation of the perfusion
model is needed before it can be used routinely to predict placental drug transfer during
preclinical evaluation (Ala-Kokko et al., 2000). To date, no systematic evaluation of the
perfusion model has been conducted. This review aims to systematically evaluate the placental
perfusion model in predicting placental drug transfer by comparing it to in vivo data.
Subsequently, we constructed a pharmacokinetic model that best allows prediction of the in vivo
36
maternal-fetal drug distribution at steady state. Finally, we also provide recommendations to
improve the reliability of the predictions provided by the perfusion method.
2.3 Methods
2.3.1 Literature Search
The in vivo data available in the literature are mostly limited to maternal blood and
umbilical cord blood drug concentrations measured at the time of delivery. Since only one time
point is available from these studies, the best measure to compare in vivo to perfusion studies
was the fetal to maternal drug concentration ratio. A systematic search for papers that evaluated
placental transfer of a therapeutic drug was performed by searching Medline, EMBASE, and
EMBASE Classic from inception to August 31, 2010 using the search strategy, placenta AND
perfusion and limited to human studies (Omarini et al., 1992). Perfusion studies were included if
they 1) were published in English 2) investigated a therapeutic drug 3) completed dual perfusion
of a term human placenta 4) involved placing a compound in the maternal circulation and
measuring its appearance in the fetal circulation 5) the fetal to maternal ratio (F:M) at the end of
the experiment or during steady state could be extracted. Papers were excluded if they 1)
investigated an endogenous compound, illicit drug, or a natural health product 2) did not present
F:M or did not provide sufficient information to calculate it 3) the length of the perfusion was
not sufficient to provide useful information (<20 minutes, since this is the time it would take for
the maternal reservoir to be completely cycled through once, if it is in closed-configuration). For
each drug studied using the perfusion model, a subsequent search was performed to retrieve
papers that measured cord blood and maternal blood drug levels.
2.3.2 Extraction of Data and Qualitative Comparisons
For each drug, the following data were extracted from the perfusion studies: F:M ratio,
initial maternal drug concentration, whether the experiment reached steady state, whether protein
was added to the perfusate, whether the system was in open- or closed-configuration, and the
number of completed perfusion experiments. From the in vivo studies, cord blood to maternal
37
blood concentration ratio (C:M), time elapsed between drug administration and delivery (blood
sampling), and number of mother-child pairs included. The percentage of protein binding (in
non-pregnant adults) and clinical drug concentrations for each drug were obtained from
DRUGDEX® System [Internet database]. Greenwood Village, Colo: Thomson Reuters
(Healthcare) Inc. Updated periodically. Available www.micromedex.com). Data were
summarized and the placental transfer as determined by the perfusion and in vivo methods were
defined as limited transfer (F:M <0.1 ), transfer (F:M 0.1 to 1.0), or fetal accumulation (F:M >
1.0).
2.3.3 Quantitative Calculations
Quantitative comparisons between the perfusion and the in vivo data were performed on
data where a direct comparison was meaningful, ie. the perfusion and in vivo data were from
comparable conditions. The following inclusion criteria were developed to ensure comparable
conditions: 1) the perfusion was performed in closed-configuration 2) the perfusion and in vivo
experiments both reached steady state (the maternal and fetal compartments reached a fixed
proportionality resulting in a constant C:M or F:M; there was a constant ratio of at least three
data points and over at least 20 minutes) and 3) the placenta used in the perfusion and the cord
and maternal blood samples were both taken at the same gestational age (ie. term). The F:M and
C:M ratios were plotted and Pearson correlation was calculated using GraphPad Prism (version
4.0b for Macintosh, GraphPad Software, San Diego, California). Outliers were determined by
analysis of residuals.
2.3.4 Model Development
The perfusion experiments often did not take into account the physiological differences in
protein binding between fetal and maternal circulations. The physiological pH difference
between the circulations was also often not taken into consideration, where the fetal circulation is
slightly more acidic than maternal (7.35 vs 7.4 respectively) (Reynolds & Knott, 1989). To
account for these, the results obtained from the placental perfusion was adjusted using the
following F:M equation (Garland, 1998):
38
equation (1)
where %unboundM and %unboundF are the % of the free drug in the maternal and fetal
circulations, respectively; the pHM and pHF are the pH in the maternal and fetal circulations,
respectively; and the CLMF, CLFM, and CLF are the maternal-to-fetal, fetal-to-maternal and fetal
clearances, respectively. The CLMF/CLFM term was taken from the perfusion experiments as the
F:M ratio at steady state from a closed-configuration study, or from the clearance rates obtained
from an open-configuration study. Furthermore, to adjust the results, information regarding fetal
and maternal protein binding had to be available. Only drugs from studies providing this
information could be adjusted. To apply the F:M equation (equation 1), the following
assumptions were made: 1) only the free drug can cross the placenta passively 2) the free fraction
is not concentration dependent 3) only the un-ionized form of the drug can cross the placenta
passively and 4) the fetal clearance rate is negligible. If studies measuring the percentage of
protein binding in fetal and maternal blood were not available, binding could be estimated from
non-pregnant adult protein binding using the method by Hill and Abramson (Hill & Abramson,
1988) (Equations 2 to 4). This method requires that the drug be bound to only albumin or α1-
acid glycoprotein (AAG).
equation (2)
equation (3)
equation (4)
maternal
fetal
maternalfetal roteinP
roteinP
FB
FB
][][
×"#
$%&
'="#
$%&
'
adultpregnantnon
maternalorfetal
adultpregnantnonmaternalorfetal roteinP
roteinP
FB
FB
−−
×#$
%&'
(=#$
%&'
(][][
%unbound=100−100×B
FBF+1
€
F :M =%unboundM%unboundF
×1+10pKa − pHF
1+10pKa − pHM×
CLMFCLFM +CLF
39
where B/F is the ratio between bound and free drug in the fetal, maternal, or adult circulations;
[Protein] is the plasma protein concentration for either albumin or AAG. The following values
were used in the calculations (Hill & Abramson, 1988): for albumin, the plasma protein
concentration ratios between fetal:adult is 0.866, maternal:adult is 0.733, and fetal:maternal is
1.20; for AAG, the plasma protein concentration ratios between fetal:adult and fetal:maternal are
both 0.37 (Hill & Abramson, 1988).
2.4 Results
2.4.1 Search Results
A total of 1732 papers were retrieved in our systematic search for studies evaluating
placental drug transfer using the placental perfusion model. 1585 papers were excluded after
reviewing the title/abstract or by not being published in English. From the 147 full-text articles
that were assessed, 19 papers did not include sufficient information to extract the F:M ratio and
29 papers investigated a drug that had no corresponding in vivo data. Furthermore, 4 papers
were excluded since their perfusion lasted ≤ 5 minutes and 3 papers were excluded because the
experimental parameters fluctuated over the course of the experiment. Another 3 papers
described perfusion experiments after adding the drug to both maternal and fetal circulations
simultaneously. Of the 89 perfusion papers included, 70 drugs were compared. An additional 58
drugs have been investigated using the perfusion model, but were included in papers that were
excluded for the above reasons (Figure 2.1).
2.4.2 Qualitative Comparison of Perfusion Results to In Vivo Data
The agreement between perfusion and in vivo results for the drugs that met the inclusion
criteria are outlined in Table 2.1. The experimental parameters and results of the perfusion and
in vivo studies are detailed in Appendix B. Out of the 70 drugs compared, 49 showed placental
transfer (F:M 0.1 to 1.0) in both the placental perfusion model and in vivo. Another 9 drugs
showed limited transfer (F:M <0.1) in both models. The remaining 12 drugs had discrepancies
between the two methods (Table 2.2).
40
Figure 2-1. Flow diagram of the search strategy and articles retrieved and included in the
review.
41
Table 2-1. Drugs that had the maternal-to-fetal transfer evaluated using the placental
perfusion model and where the fetal to maternal drug concentration ratios (F:M) from the
perfusion agreed with in vivo data from mother-infant pairs.
Limited Transfer (F:M<0.1) Transfer (F:M 0.1 – 1.0) Azithromycin Enfuvirtide Fondaparinux Glyburide Heparin Indinavir Insulin Lispro Low Molecular Weight Heparins Saquinavir
Acetaminophen Fluoxetine Phenytoin Acyclovir Hydralazine Propofol Alfentanil Indomethacin Propranolol ASA Labetalol Pyrimethamine Atenolol Lamivudine Quetiapine Bupivacaine Lamotrigine Ranitidine Buprenorphine Lidocaine Ritonavir Carbamazepine Lopinavir Ritrodrine Celiprolol Methadone Ropivacaine Cimetidine Methimazole Salbutamol Citalopram Methohexital Salicylic Acid Clonidine Morphine Sufentanil Diclofenac Nelfinavir Sulindac Dideoxyinosine Nifedipine Theophylline Digoxin Nortriptyline Zidovudine Enalaprilat Olanzapine Erythromycin Phenobarbital
Table 2-2. Drugs that had the maternal-to-fetal transfer evaluated using the placental
perfusion model and where the fetal to maternal drug concentration ratios (F:M) from the
perfusion disagreed with in vivo data from mother-infant pairs.
42
2.4.3 Quantitative Comparison of Perfusion Results to In Vivo Data
Twenty-six drugs met the inclusion criteria for a quantitative comparison. Furthermore,
data were available for both total and free concentrations of bupivacaine and ropivacaine, so both
measures were included. There was a significant correlation between the F:M and C:M ratios
obtained by the perfusion and the in vivo measurements, respectively (R2=0.28, p=0.004) (2-2A).
Twenty-four drugs had sufficient data available to be adjusted using our model as per the
F:M equation (Table 2-3). After adjustment, there was a stronger correlation between the
perfusion and in vivo results (R2=0.54, p<0.001) compared to the unadjusted (Figure 2-2B).
There were 4 clear outliers in the adjusted comparison from digoxin, glyburide, metformin, and
oxcarbazepine. For digoxin, there was wide variability among the F:M ratio obtained from 4
different studies and among the C:M ratio obtained from 6 different studies. Since digoxin is a
substrate for P-glycoprotein and this efflux transporter decreases towards term, the concentration
ratio may be dependent on the gestational age of the placenta. Errors in digoxin measurements
from digoxin-like substances cross-reacting may further contribute to the variability in the C:M
ratios. For oxcarbazepine, the fetal and maternal protein binding were estimated from adult
binding to albumin only. This may not be accurate since it does not take into consideration
binding to other proteins or displacement by maternal endogenous substances. For metformin,
the in vivo studies may over estimate the C:M since in the study by Vanky et al. (Vanky et al.,
2005), the maternal blood sample was taken within an hour after birth and not necessarily at the
same time as fetal sampling. Other studies on metformin suggest a C:M closer to 1, but the exact
ratio could not be calculated from the information provided in the papers (Charles et al., 2006;
Eyal et al., 2010). For glyburide, both perfusion papers and an in vivo paper showed limited
transfer of glyburide at term. However, a recent study by Hebert et al. (Hebert et al., 2009)
observed a mean C:M of 0.7. Since many samples were below the limit of quantification, it is not
clear how these samples were used in the C:M calculation. Also, the gestational age at delivery
(mean = 34.6 weeks) was much shorter than that of the placentas routinely used for perfusion
experiments. After excluding the 4 outliers, the perfusion results avidly predicted the in vivo
results (R2=0.85, p<0.0001) (Figure 2-2C).
43
Table 2-3. The observed and calculated F:M ratios for data obtained from placental perfusion experiments
Drug In Vivo C:M*
Observed F:M*§
%unboundM %unboundF
pKa CLMF*** CLFM
Calculated F:M****
Protein Binding References
Alfentanil 0.30 - 0.36 6.5 1.02 0.37 (Meuldermans et al., 1986) Bupivacaine 0.30 0.69 0.20 8.1 - 0.15 (Mather et al. 1971) Carbamazepine 0.91 1.00 0.90 13.9 - 1.01 (Kuhnz et al., 1984) Diazepam 1.38 0.51 2.16 3.3 - 1.10 (Ridd et al., 1989) Dideoxyinosine 0.35 - - 9.13 0.27 0.30 - Digoxin 0.89 0.11 1.04** 13.5 - 0.13 (Gorodischer et al., 1974; Hill &
Abramson, 1988) Glyburide 0.59 0.03 - 5.3 - 0.03 - Insulin Lispro <LOD <LOD - - - <LOD - Lamotrigine 0.96 1.05 0.80 5.7 - 0.84 (Rambeck et al., 1997) Lidocaine 0.52 0.82 0.70 7.9 - 0.62 (Nau, 1985) Metformin 1.53 0.47 - 12.4 - 0.53 - Methadone 0.43 0.71 0.47** 8.25 - 0.37 (Hill & Abramson, 1988; Pond et al.,
1985) Methohexital 1.00 0.75 1.16** 7.9 - 1.27 (Hill & Abramson, 1988) Morphine 0.96 0.78 1.06** 8.0 - 0.91 (Hill & Abramson, 1988; Kurz et al., 1977) Oxcarbazepine 2.25 1.1 1.06** - - 1.17 (Hill & Abramson, 1988) Phenobarbital 0.96 0.99 1.02 7.4 - 1.07 (Nau et al., 1983) Phenytoin 0.95 0.77 0.99 8.3 - 0.84 (Hamar & Levy, 1980) Propranolol 0.26 1.00 0.54 9.0 - 0.60 (Belpaire et al., 1995) Propylthiouracil 1.51 1.00 1.15** 7.8 - 1.25 (Hill & Abramson, 1988) Ropivacaine 0.31 0.82 0.39 8.1 - 0.35 (Morton et al., 1997) Sufentanil 0.76 - 0.45 8.5 0.78 0.39 (Meuldermans et al., 1986) Theophylline 1.05 0.88 1.03** 8.7 - 1.01 (Frederiksen, et al., 1986; Hill &
Abramson, 1988) Valproic acid 1.51 0.88 1.65 4.8 - 1.48 (Froescher et al., 1984) Zidovudine 1.18 1.00 1.06** 9.68 - 1.12 (Hill & Abramson, 1988) § F:M ratio obtained from closed-configuration placental perfusions that obtained steady-state. *If more than one study available from (Appendix B), the weighted mean was calculated. **Fetal and/or maternal protein binding unavailable. Numbers were calculated using the method by Hill & Abramson (1988). ***Clearance ratios obtained from open-configuration placental perfusions. ****Calculated using equation
44
Figure 2-2. Agreement between perfusion fetal to maternal (F:M) concentration ratios and
the in vivo umbilical cord to maternal blood (C:M) drug concentration ratios A) with data
obtained from publications B) after adjusting the F:M ratios using the equation 1 and C)
after adjusting the F:M ratios using equation 1 and removing the 4 outliers where
experimental limitations preclude a direct comparison.
A)
B)
C)
45
2.5 Discussion
For drugs where there is limited fetal safety information from clinical studies, estimating
fetal drug exposure is critical in predicting whether a drug can be safely administered in
pregnancy. As studying human transplacental transfer in vivo is almost always unethical, other
experimental models are needed to estimate fetal exposure. Placental perfusion is the only
experimental model to study human placental transfer of substances in organized placental
tissue. In agreement with a previous study (Gavard et al., 2009), the placental perfusion model is
useful in predicting whether the drug will have limited placental transfer. When the drug does
transfer across the placenta, it has been suggested that in vivo data are required to adequately
quantify this transfer (Gavard et al., 2009).
By adjusting the perfusion results using the F:M equation, the transfer across the placenta
could be accurately predicted when the conditions from the placental perfusion experiment and
the in vivo data were comparable (ie. they were both from term placenta and they reached steady
state). Transfer was either underestimated or overestimated by the perfusion model mostly
because it did not mimic protein binding in the fetal circulations. Many groups added protein,
such as human or bovine serum albumin, to the perfusate in order to investigate the effects of
protein binding on transfer (Appendix B); however, addition of only albumin or AAG and in the
same concentration on both maternal and fetal sides, does not mimic physiological conditions.
Furthermore, use of human plasma in the perfusate is often not practical and also fails to mimic
physiological conditions since α-fetoprotein is present in fetal serum. Adding protein to the
perfusate is useful in evaluating the effects of protein on the rate of transfer, but theoretically
should not affect the F:M ratio of the free drug at steady state. By adjusting the perfusion results
using the F:M equation and addressing differences in protein binding, appreciably more accurate
predictions of in vivo placental transfer at steady state for was obtained (Figure 2-2). For
example, the adjusted perfusion data for bupivacaine, lidocaine and ropivacaine all resulted in
closer values to the in vivo situation compared to adding human plasma to the maternal circuit
during the perfusion (Table 2.3).
46
Figure 2-3. Comparison of F:M ratios for morphine obtained from placental perfusion
experiments versus maternal blood and umbilical cord blood (Kopecky et al., 2000;
Kopecky, 1999). Each in vivo data point represents a single patient’s F:M Ratio (n=8).
Each perfusion data point represents the mean ± SD value of 4 placental perfusions with
addition of morphine into the maternal compartment only.
Maternal and fetal plasma differ in both albumin and AAG concentrations, the two most
important drug binding plasma proteins. Towards term, albumin is appreciable higher in the fetal
circulation compared to the maternal, with the fetal to maternal albumin ratio increasing from
0.28 in the first trimester to 1.20 at term (Krauer et al., 1984). Furthermore, a 3-fold higher
concentration of free fatty acids towards term in the maternal circulation can displace drugs from
binding to albumin in the maternal circulation (Nau et al., 1984; Ridd et al., 1983). This leads to
increased binding to albumin in the fetal circulation compared to the maternal circulation at term.
Increased fetal binding can lead to increased placental drug transfer since it is the free
concentration of the drug that will equilibrate. Fetal accumulation of drugs bound to albumin at
term is expected if the drug is passively diffused. The perfusion model was not able to predict
the in vivo fetal accumulation observed with oxcarbazepine, diazepam, valproic acid, metformin,
and propylthiouracil (Table 2.2). With the exception of metformin, the other four drugs are all
47
highly bound to albumin and this is likely the reason that the perfusion model did not accurately
predict in vivo transfer. After adjusting for differences in fetal and maternal binding to albumin
using the F:M equation and the method by Hill & Abramson (Hill & Abramson, 1988), the fetal
accumulation observed in vivo could be predicted using the perfusion results (Table 2.3).
The other major plasma binding protein, AAG, gradually increases throughout gestation
in fetal plasma while maternal levels stay fairly constant (Krauer et al., 1984). The fetal to
maternal AAG ratio increases from 0.09 in the first trimester to 0.37 at term (Krauer et al., 1984).
At delivery, there is no apparent change in AAG levels compared to before induction of labour
(Porter et al., 2001). Compared to maternal, there is lower binding to AAG in the fetal
circulation throughout pregnancy. In contrast to drugs bound to albumin, the placental perfusion
model overestimated fetal exposure to drugs bound to AAG, including the anesthetics and
propranolol. After adjusting the perfusion results, there was a stronger correlation between the
perfusion F:M and the in vivo C:M for drugs bound to AAG (Table 2.3). Protein binding has
previously been suggested as the most significant factor in establishing the F:M ratio at steady
state (Hill & Abramson, 1988) and our results support this finding when it is the free
concentration of the drug that equilibrates across the placenta. Protein binding is not the most
significant factor if there is limited transfer of the free drug, as is the case with insulin lispro,
heparin, fondaparinux, and enoxaparin, where the large molecular size precludes placental
transfer. For drugs where efflux transporters limit transfer, such as indinavir, saquinavir,
enfuvirtide, azithromycin, and glyburide, protein binding is also not a significant factor. For
drugs that are effluxed back into the maternal circulation, the placental perfusion model was able
to show limited transfer. This suggests that the placental perfusion model is useful in identifying
drugs that are actively transported. The net transfer of these drugs differed from what would be
predicted based on the drug’s partition coefficient and passive diffusion alone. The perfusion
model can also be useful in identifying active transport when used under equilibrative conditions
(adding the drug to both the fetal and maternal circulations in equal concentrations). Drug
accumulation in one compartment represents drug transport against a concentration gradient.
The active transport of glyburide by placental breast cancer resistance protein (BCRP) was
established by this method (Kraemer et al., 2006; Pollex et al., 2008).
The placental perfusion model was able to accurately predict limited transfer of drugs
across the placenta at term. Thus, perfusion results that show no transfer to the fetal circulation
48
do not need to be adjusted using the F:M equation. Since the drug originates in the maternal
circulation, it will not even reach the fetal circulation if transfer is restricted by the placenta.
Thus, the term CLFM would not be reliably assessed by the perfusion model if the drug is placed
on the fetal side at much higher concentrations than would be reached in vivo. For example, if
CLMF and CLFM for indinavir from open-configuration placental perfusion (Sudhakaran et al.,
2005), the differential protein binding for maternal and fetal circulations (Sudhakaran et al.,
2007), and the pKa were put into the F:M equation, a F:M of 0.26 would result. This result
clearly overestimates placental transfer since in vivo data show very limited transfer (Appendix
B). Therefore, although adjusting the results using the F:M equation works well for drugs that
cross the placenta, it is not relevant to drugs which have limited transfer across the placenta.
Interpretation of placental perfusion results must take into consideration how the
experimental conditions relate to in vivo physiological conditions. If perfusion conditions do not
include protein in the perfusate, then the drug added into the system will theoretically be free
(unless it binds to tubing, dextran in the perfusate, or residual blood). When no protein is added
to the perfusate, then comparison to the in vivo results should be comparable to the free fraction
of the drug. For example, during a perfusion, alfentanil was added into the maternal circulation
in open-configuration (Zakowski et al., 1994). At steady state, the F:M ratio was 0.22 and the
authors commented that this value was similar to those obtained in vivo. However, in vivo, the
C:M ratio for free alfentanil is 0.97 (Gepts et al., 1986). A direct comparison of the free drug
would suggest that the perfusion underestimates exposure. However, since the perfusion was
performed in open-configuration, it underestimates the F:M ratio because the maternal circuit
remains at the initial concentration. In order to predict fetal exposure to the bound and free drug,
the perfusion results (clearances from open-configuration) can be adjusted using the F:M
equation. After adjustment, the calculated F:M ratio was 0.37 (Table 2.3), which is very similar
to the in vivo results for the free plus bound drug concentration.
Since protein binding is a critical factor in predicting the extent of placental transfer (Hill
& Abramson, 1988), experimental conditions must properly control for inadvertent binding.
Without addition of exogenous protein into the perfusion medium, protein resulting from
washout and placental secretion has been measured in both the fetal and maternal circulations.
Protein in the maternal circulation was found to be around 10-fold higher than the fetal
circulation (Shah & Miller, 1985). This likely results from the fetal side having a better washout
49
and the fact that some proteins (eg. hCG) are preferentially secreted into the maternal circulation
(Shah & Miller, 1985). Furthermore, haemoglobin content in the maternal circulation was found
to be 100-fold higher than in the fetal circulation (Mathiesen et al., 2010). If the drug is able to
bind to proteins that remain from washout or secreted placental proteins, then there may be an
experimental bias towards the drug being found in the maternal circulation.
The placental perfusion model offers a valid model to predict transplacental drug
passage. After careful interpretation of the results and adjustments to incorporate protein binding
and ion trapping, the perfusion model can predict the extent of placental transfer. A limitation of
the perfusion model is that it mostly uses term placentas and cannot necessarily be extrapolated
to earlier gestational ages. At term, the placental transfer layer is the thinnest and expression of
certain efflux transporters such as P-glycoprotein is substantially decreased (Gil et al., 2005;
Nanovskaya et al., 2008; Sun et al., 2006; Vahakangas & Myllynen, 2006). Therefore, drug
transfer at term may represent the highest exposure compared to earlier gestational ages
(Vahakangas & Myllynen, 2006). Other experimental methods may be useful to further
characterize transfer in first and second trimester tissue (Table 1.1). For example, knowing that
glyburide is a substrate for placental BCRP, kinetic parameters could be evaluated from
membrane vesicles prepared from early gestational tissue and compared to term tissue.
Comparing perfusion results to cord and maternal blood ratios at delivery also has
limitations. In vivo ratios obtained at delivery only represent one time point and it is not always
certain if the two circulations are at steady state (Chappuy et al., 2004). However, with a large
sample size and measurements at multiple times after the last dose, one can determine whether
the ratio is likely to be at steady state. A constant proportionality between the maternal and fetal
compartments occurs faster in vivo compared to the perfusion model. For example, for
bupivacaine, drug levels in maternal and cord blood equilibrate within minutes (Thomas et al.,
1976). In contrast, in the perfusion model, the F:M ratio equilibrates after 1 hour (Johnson et al.,
1995). For morphine, fetal and maternal concentrations in vivo were almost identical after 5 min
(Gerdin et al., 1990). In the perfusion model, equilibrium was not achieved until after 2 hours
(Figure 2-3) (Kopecky, 1999). The faster equilibrium obtained in vivo results from more rapid
distribution since the entire maternal blood volume is circulated by the heart in approximately
one minute. In the perfusion experiment, where the maternal reservoir is 250 mL, it would take
around 20 minutes to circulate this volume once. Furthermore, the villus surface area of the
50
whole placenta in vivo at term (12 m2 for the whole placenta) is much larger than that of a single
cotyledon (20 cotyledons per placenta on average) (Cabezon et al., 1985; Vermeulen et al.,
1982).
Recommendations for Future Standardization of Placental Perfusion Experiments
Only twenty percent of therapeutic drugs that have been studied using the perfusion
model were available for a quantitative comparison to the in vivo data. A major factor was that
in vivo data were not available for almost thirty percent of the drugs. However, the majority of
drugs could not be compared because of factors associated with study design or insufficient data
presented in the publication. Many perfusion studies did not achieve steady state as stated by the
authors or determined by inspection of the figures. In some publications, it was not possible to
determine whether steady state was achieved. Since the perfusion model equilibrates slower than
the maternal-fetal compartments in vivo, it is critical to ensure that the perfusion experiment lasts
a sufficient length of time. Many studies also reported only data that were standardized to a
reference marker and only final calculations. These standardized calculations do not provide
insight into the drug distribution and thus absolute values should always be reported (Ala-Kokko
et al., 2000). Reporting the absolute concentrations and a corresponding graph would add
valuable information to the literature and enable more secondary analyses to be performed. The
pH difference between the maternal and fetal compartments can influence the extent of drug
transfer, especially for weak bases (Ueki et al., 2009). Many studies did not report the actual pH
of the perfusates during the experiment or whether they controlled the pH during the course of
the experiment. This information should also be reported in publications. Mathiesen et al.
(2010) have also proposed criteria to be included in perfusion publications and have
recommended that each laboratory publish a detailed methods paper so that all experimental
parameters can easily be obtained (Mathiesen et al., 2010).
Summary
After systematically reviewing all drugs that have been evaluated by both the placental
perfusion model and in vivo, it is evident that the dual perfusion of a single placental lobule is a
valid method to predict placental drug transfer at term. Data obtained from perfusion
experiments must take into consideration many factors that are known to influence drug transfer
including placental pharmacokinetic factors (active transport, passive diffusion, and metabolism)
51
as well as physiochemical properties of the drug. Differential protein binding between the fetal
and maternal circulations is known to be a critical factor in the placental transfer of drugs (Hill &
Abramson, 1988). However, it is difficult and unpractical to exactly mimic physiological protein
binding during the perfusion experiment. Determining the protein binding in vitro using fetal
and maternal blood in parallel with perfusion experiments, and then modeling the results as
documented above would allow for more accurate predictions for placental drug transfer at
steady state. After modeling, results from placental perfusion experiments can be used to
accurately predict in vivo placental drug transfer and thus estimate fetal drug exposure. This will
allow conclusions to be reached regarding placental drug transfer for new drugs where there is
no in vivo fetal safety data available. When applied appropriately, the placental perfusion model
is an invaluable tool to help guide decisions regarding the benefits and risks of new medications
that may be required during pregnancy.
52
Chapter 3
The transfer of 6-mercaptopurine in the dually perfused human placenta Hutson JR1,2, Lubetsky A1, Walfisch A1, Ballios BG2, Garcia-Bournissen F1,2, Koren G1,2
1Division of Clinical Pharmacology and Toxicology, Hospital for Sick Children, 555 University
Ave, Toronto, Ontario, M5G 1X8, Canada
2Institute of Medical Science, University of Toronto, 1 King’s College Circle, Toronto, Ontario,
M5S 1A8, Canada
This work has been published and reproduced with permissions: Hutson JR, Lubetsky A, Walfisch A, Ballios BG, Garcia-Bournissen F, Koren G. The transfer
of 6-mercaptopurine in the dually perfused human placenta. Reprod Toxicol 2011;32:349–353.
53
The transfer of 6-mercaptopurine in the dually 3perfused human placenta
3.1 Abstract
The immunosuppressant azathioprine is increasingly being used in pregnancy. The
human placenta is considered a relative barrier to the major metabolite, 6-mercaptopurine (6-
MP), and likely explains the lack of proven teratogenicity in humans. The aim of this study was
to determine how the human placenta restricts 6-MP transfer using the human placental
perfusion model. After addition of 50ng/ml (n=4) and 500ng/ml (n=3) 6-MP into the maternal
circulation, there was a biphasic decline in its concentration and a delay in fetal circulation
appearance. Under equilibrative conditions, the fetal-to-maternal concentration ratio was >1.0 as
a result of ion trapping. Binding to placental tissue and maternal pharmacokinetic parameters are
the main factors that restrict placental transfer of 6-MP. Active transport is unlikely to play a
significant role and drug interactions involving, or polymorphisms in, placental drug efflux
transporters are not likely to put the fetus at risk of higher 6-MP exposure.
54
3.2 Introduction
The immunosuppressant azathioprine (AZA) is increasingly being used in pregnancy for
the treatment of autoimmune diseases or for organ transplant patients (Goldstein et al., 2007).
Peak incidence of autoimmune diseases, including inflammatory bowel disease (IBD) and
systemic lupus erythematosus, occurs in women during the childbearing years (Cooper &
Stroehla, 2003) and successful pharmacologic therapy in these women has improved the
feasibility of pregnancy (Polifka & Friedman, 2002). Treatment with AZA is required into
pregnancy to prevent relapse of the disease or organ rejection; thus, treatment can also minimize
adverse fetal effects associated with the underlying maternal disease (Polifka & Friedman, 2002).
Several studies have investigated the safety of AZA in pregnancy. A review of 27 case
series and three large retrospective and prospective studies did not find an increased risk for
major malformations associated with the use of AZA in pregnancy (Cleary & Kallen, 2009;
Coelho et al., 2011; Francella et al., 2003; Goldstein et al., 2007; Polifka & Friedman, 2002).
However, one recent study reported a trend for increased malformations and an increased risk
specifically for ventricular/atrial septal defects after prenatal AZA exposure (Cleary & Kallen,
2009). An increased risk for prematurity and lower birth weight has also been reported, but this
may be related to the underlying maternal disease (Cleary & Kallen, 2009; Goldstein et al.,
2007). Although evidence supports the relative safety of AZA in pregnancy compared to the
untreated disease (Coelho et al., 2011), safety concerns still arise from its mechanism of action
and its toxicity during pregnancy in animal studies (Polifka & Friedman, 2002). Over eighty
percent of women with IBD have unwarranted concerns about medications, including AZA, in
pregnancy and fears surrounding medications are greater than the effect of IBD exacerbation
(Mountifield et al., 2010). Moreover, some physicians still choose not to administer AZA during
pregnancy or to discontinue treatment in the third trimester (Peyrin-Biroulet et al., 2011).
After oral administration, AZA is extensively (>80%) cleaved into 6-mercaptopurine (6-
MP) in the blood (Polifka & Friedman, 2002). 6-MP is a purine analogue, and after further
conversion into active metabolites, it can inhibit de novo purine ribonucleotide synthesis leading
to inhibition of cell proliferation. Furthermore, 6-MP can be cytotoxic after incorporation of its
metabolites into cellular DNA. Since 6-MP targets rapidly dividing cells, the developing fetus
would theoretically be expected to be sensitive to its cytotoxicity. However, even high dose 6-
55
MP was not found to be useful as a single-agent medical abortifacient in early pregnancy,
leading the authors to suggest that this drug does not alter cell division in trophoblastic tissues
(Davis et al., 1999).
The observed safety of AZA in pregnancy likely results, in part, from the placenta
limiting fetal exposure to the main metabolite, 6-MP. Indeed, only 1-2% of maternal 6-MP
concentrations are found in cord blood and the human placenta is considered to be a relative
barrier to 6-MP and its metabolites (de Boer et al., 2006; Saarikoski & Seppala, 1973).
However, the mechanism of how the placenta restricts 6-MP transfer to the fetus is unknown. 6-
MP is a substrate for several drug transport proteins, including breast cancer resistance protein
(BCRP) (de Wolf et al., 2008). BCRP is highly expressed in the placenta and is localized to the
brush border (maternal-facing) membrane of the syncytiotrophoblast (Maliepaard et al., 2001).
The importance of BCRP and other placental transporters in limiting fetal exposure to various
drugs has been demonstrated in both human and animal models (Jonker et al., 2000; Pollex et al.,
2008). The objective of this study was to determine the mechanisms by which the placenta
restricts 6-MP transfer. Understanding these mechanisms will allow for prediction of fetuses that
may be at risk of higher exposure to 6-MP as a result of drug interactions with, or
polymorphisms in, placental drug efflux proteins.
3.3 Methods
Materials
6-methylmercaptopurine (6-MMP) and 6-thioxanthosine were obtained from MP
Biomedicals (Solon, Ohio). 6-thiouric acid was obtained from US Biological (Swampscott, MA)
and toluene from EMD Chemicals (Gibbstown, NJ). HPLC-grade methanol was ordered from
Fisher Scientific (Fair Lawn, NJ). All other chemicals were purchased from Sigma (St. Louis,
MO). The water used for all experimental procedures was obtained from a Milli-Q Advantage
A10 Ultrapure Water Purification System (Millipore, Billerica, MA).
56
Placental perfusion
The dual perfusion of a placental lobule was previously described by Miller et al. (Miller
et al., 1985) and adapted in our laboratory (Derewlany et al., 1991; Pollex et al., 2008). Term
placentas were obtained immediately after elective caesarean sections from healthy mothers with
uncomplicated pregnancies from Mount Sinai or St. Michael’s Hospitals in Toronto, Ontario.
Research ethics board approval at each hospital was obtained and maternal consent was obtained
prior to the surgery. The placentas were transferred to the lab in heparinised ice-cold phosphate-
buffered saline where a vein/artery pair supplying a clearly identifiable cotyledon were chosen
for cannulation (Derewlany et al., 1991). Maternal and fetal circulations were established within
30 minutes of delivery.
The perfusate consisted of M199 tissue culture medium (Sigma, St. Louis, MO)
containing heparin (2000 U/L), glucose (1.0 g/L), kanamycin (100 mg/L), and 40,000 molecular-
weight dextran (maternal 7.5 g/L; fetal 30 mg/L). Antipyrine (1 mM) was added to the maternal
circulation as a marker of passive diffusion. Flow rates were maintained at 2 to 3 and 13 to 14
ml/min in the fetal and maternal circuits, respectively. The maternal perfusate was equilibrated
with 95% O2, 5% CO2 and the fetal with 95% N2, 5% CO2. Maternal and fetal circuits were
maintained at pH 7.4 and 7.35, respectively, by the addition of small volumes of sodium
bicarbonate and hydrochloric acid. This pH mimics the slightly more acidic fetal circulation
observed in vivo (Reynolds & Knott, 1989). The temperature of the circuits and the perfusion
chamber was kept at 37°C.
Each perfusion consisted of a 1 h closed pre-experimental control period, followed by a 3
h closed experimental period, and a final 1 h closed post-control period. The post-control period
was omitted from the perfusions where 6-MP was added to only the maternal circulation since
this allowed measurement of drug binding to placental tissue. The perfusates in both circulations
were replaced with fresh media prior to each of the 3 periods. During the pre- and post-control
periods, samples were taken every 15 min to analyze glucose and oxygen consumption, and
human chorionic gonadotropin (hCG) secretion as measures of tissue viability. These measures
were taken every 30 minutes during the experimental period. Tissue viability measures were
calculated as previously described (Pollex et al., 2008). Fetal reservoir volume and fetal
perfusion pressure were monitored as an indicator of tissue integrity. pH, pO2, and pCO2 were
57
monitored using a blood gas analyzer (Radiometer ABL 725, Copenhagen, Denmark). During
the experimental period, 50 ng/ml or 500 ng/ml 6-MP was added to the maternal circulation only
or simultaneously to both the fetal and maternal circulations. 50 ng/ml is the maximum
concentration obtained after oral administration of AZA in adult renal transplant recipients
(Bergan et al., 1994; Zins et al., 1997) and similar doses are indicated for the treatment of IBD.
Samples were taken for measurement of 6-MP every 10 minutes for the first half-hour and every
half-hour following. The perfusion was terminated at any time if there was a >3 ml/hour loss in
fetal reservoir volume.
The expected fetal to maternal (F:M) ratio at steady state resulting from ion trapping –
given the change in pH between the fetal and maternal circulations - was calculated using a
modified form of the Henderson-Hasselbalch equation that incorporates the two pKa of 6-MP:
This equation was derived by assuming conservation of mass and rapid equilibration between the
neutral and protonated forms of 6-MP (6-MP, 6-MP+, and 6-MP++). Only the un-ionized form is
assumed to cross the membrane. Data are expressed as mean ± SEM unless otherwise indicated.
F:M ratios were compared using a two-tailed Student’s t-test.
Sample analysis
Perfusate samples were stored at -20°C until analysis. 6-MP and its metabolites (6-
MMP, 6-thioxanthosine, 6-thiouric acid, 6-thioguanine) were extracted using a method adapted
from Lennard and Singleton (Lennard & Singleton, 1992). 500 µl of perfusate was added to 500
µl of 6 mM DL-Dithiothreitol and 500 µl of 1.5 M H2SO4 in duplicate. Since 6-TG was not
detected in any of the perfusions, it was further used as an internal standard (100 ng/ml). One of
each sample was heated at 100°C for 1 hour for acid hydrolysis of the thionucleotide metabolites
back into the parent thiopurine and for extraction of 6-methylmercaptopurine (Lennard &
Singleton, 1992). Samples not being heated were kept on ice. After cooling on ice, 500 µl of 3.8
M NaOH was added followed by 6 ml of isoamyl alcohol (170 mM) and phenylmercury acetate
(1.3 mM) in toluene and placed in an automatic rotator for 15 min. The mixture was centrifuged
at 2500×g for 15 min at room temperature and the toluene layer was added to a new tube. 200 µl
€
FM
=1+10 pKa 2 − pHF( ) +10 pKa1 + pKa 2 −2pHF( )
1+10 pKa 2 − pHM( ) +10 pKa1 + pKa 2 −2pHM( )
58
of 0.1 M HCl was added and vortexed for 2 × 1 minute. 70 µl of the aqueous phase was injected
onto a Atlantis T3 Column (3µm, 4.6×150mm) protected with a guard cartidge (3 µm, 4.6×20
mm)(Waters, Milford, MA). The mobile phase consisted of acetonitrile, methanol, and KH2PO4
(0.02 M; pH 2.25) (3:1:96, v/v/v) (Hawwa, Millership, Collier, & McElnay, 2009) and was
filtered through a 0.45 µm filter. The flow rate was 1.0 ml/min.
The HPLC system (Shimadzu, Columbia, MD) consisted of a solvent delivery pump (LC-
10AT), auto injector (SIL-10A), degasser (DGU-14A), controller (CBM-20), and dual
wavelength UV detector (SPD-20A). 6-MP was monitored at 313 nm; 6-MMP at 303 nm; and
6-thioguanine, 6-thioxanthosine, and 6-thiouric acid at 342 nm. The chromatograms were
acquired and analyzed using Shimadzu Class-VP Software, Version 7.4 SP2. The limit of
quantification was 2 ng/ml for 6-MP and 6-thioguanine and 5 ng/ml for 6-MMP, 6-thiouric acid,
and 6-thioxanthosine.
3.4 Results
A total of 16 cotyledons from different placentae were perfused with 6-MP and the
physical parameters for the perfusions are given in Table 3-1. The mass of the perfused
cotyledons ranged from 10.22 to 32.0 g. Throughout the experiments, measures of placental
viability, integrity, and function remained within normal ranges and were not significantly
different between the control and experimental phases (Table 3-1). There was no significant
difference between experimental and control periods in the fetal arterial pressures. The rate of
antipyrine appearance in the fetal circulation during the experimental period was equal to the rate
of disappearance from the maternal circulation with values of 0.030 ± 0.002 and 0.032 ± 0.003
µmol/g per minute, respectively.
59
Table 3-1. Placental viability parameters and metabolic capacity throughout the perfusions
(n=16 cotyledons from independent placentae) (mean ± SEM)
Viability parameter Pre-control Experiment Post-control Fetal arterial inflow pressure (mm Hg) 40.36 ± 1.11 40.71 ± 1.08 42.91 ± 1.81 hCG production (mIU/g per min) 57.52 ± 10.12 39.62 ± 6.83 40.86 ± 13.07 Oxygen (µmol O2/g per min) Transfer 0.01 ± 0.00 0.01 ± 0.00 0.02 ± 0.00 Delivery 0.48 ± 0.05 0.48 ± 0.04 0.45 ± 0.07 Consumption 0.22 ± 0.02 0.22 ± 0.02 0.23 ± 0.04 Glucose consumption (µmol/g per min) 0.42 ± 0.05 0.36 ± 0.04 0.38 ± 0.06
After addition of 50 ng/ml 6-MP into the maternal circulation only, there was a biphasic
decline in its concentration from this compartment (Figure 3-1A); an initial rapid decline during
the first 30 minutes, followed by a slower decline in the final 150 minutes. There was a delay in
the transfer of 6-MP in the fetal circulation as measurable concentrations of 6-MP appeared only
after 30 minutes. This delay suggests uptake and retention of 6-MP by the placental tissue.
After 3 hours, the mean fetal concentration of 6-MP was 8.8 ± 1.71 ng/ml and the mean fetal to
maternal (F:M) concentration ratio was 0.44 ± 0.06. After addition of the higher 6-MP
concentration (500 ng/ml), there was a similar biphasic decline from the maternal circulation
(Figure 3-1B). 6-MP was detected in all three perfusions at 20 minutes, which is more rapid
compared to the perfusions at the lower concentration. After 3 hours, the mean fetal
concentration of 6-MP was 91.48 ± 7.50 ng/ml and the mean F:M ratio was 0.57 ± 0.04.
When equal concentrations (50 ng/ml or 500 ng/ml) were added to both fetal and
maternal circulations at the start of the experimental period, the fetal to maternal (F:M) ratio
increased over time (Figure 3-2). The F:M ratios of 6-MP at the end of the 180 minute
experimental period were 1.21 ± 0.08 and 1.19 ± 0.04 for 50 ng/ml and 500 ng/ml, respectively.
The F:M ratios increased significantly from time zero to 180 minutes at both the 50 and 500
ng/ml concentrations (p=0.007, p=0.02, respectively). 6-MP is a basic drug with two pKa’s
(pKa1=7.77, pKa2=11.17) (O'Neil et al., 2010), and basic drugs are more ionized in the fetal
circulation since it is slightly more acidic than the maternal (7.35 vs. 7.40, respectively)
(Reynolds & Knott, 1989). The expected F:M ratio at equilibrium based on ion trapping alone
was calculated to be 1.22.
60
After perfusion with 50 ng/ml 6-MP, the metabolites including 6-MMP, 6-thioguanine, 6-
thioxanthosine, 6-thioinosine, and 6-thiouric acid (and the related thionucleotide metabolites)
were not detected in the fetal nor the maternal circulations. At the ten-fold higher 6-MP
concentration of 500 ng/ml, only 6-MMP was detected with a mean concentration of 11.75 ±
2.02 and 8.65 ± 5.97 ng/ml in the fetal and maternal circulations, respectively, at 180 minutes.
Two of the seven perfusions at 500 ng/ml had 6-MMP below the limit of detection. The tissue-
to-maternal perfusate ratio was 0.49 ± 0.08 at the end of 180 minutes of perfusion (n=7). The
tissue-to-maternal perfusate ratio was not significantly different between the 50 ng/ml and the
500 ng/ml perfusions.
61
Figure 3-1. Maternal-to-fetal transfer of 6-mercaptopurine after dual perfusion of a single
placental lobule. 6-mercaptopurine was added to the maternal circulation at A) 50 ng/ml (n=4)
or B) 500 ng/ml (n=3) and transfer was determined for a period of 180 minutes. Data are shown
as mean values ± SEM at each time point.
0"50"100"150"200"250"300"350"400"450"
0" 50" 100" 150" 200"
6MP$(ng/ml)$
Time$(min)$
Fetal"Maternal"
0"5"10"15"20"25"30"35"40"45"50"
0" 50" 100" 150" 200"
6MP$(ng/ml)$
Time$(min)$
Fetal"Maternal"
A)
B)
62
Figure 3-2 Concentrations of 6-MP in the fetal and maternal circulations after perfusion of a
single placental lobule under equilibrative concentrations of A) 50 ng/ml (n=5) and B) 500 ng/ml
(n=4). C) The fetal to maternal (F:M) concentration ratios for the perfusion under equilibrative
conditions. Data are shown as mean values ± SEM at each time point.
A)
B)
C)
63
3.5 Discussion
The human placenta is considered a relative barrier to 6-MP and its metabolites (de Boer
et al., 2005; de Boer et al., 2006; Saarikoski & Seppala, 1973) and our study demonstrates that
placental binding and not active transport limits transfer. Limited fetal exposure to 6-MP may in
part explain why studies have not shown conclusive evidence that 6-MP is teratogenic (Cleary &
Kallen, 2009; Coelho et al., 2011; Goldstein et al., 2007). Understanding the mechanism of this
limited transfer will allow for prediction of fetuses that may be at risk for higher exposure or for
prediction of potential drug interactions.
After introduction of a clinically relevant concentration into the maternal circulation,
there was a rapid decline in maternal levels, however, there was a delay in appearance of 6-MP
in the fetal circulation. This delay can be attributed to the uptake and binding by the placental
tissue. After addition of a 10-fold higher concentration, there was also a delay in the appearance
of 6-MP in the fetal circulation. The appearance of 6-MP in the fetal circulation was more rapid
for 500 ng/ml, suggesting saturation of tissue binding sites. Although binding to the tissue
delayed placental transfer, the tissue to maternal perfusate ratio at the end of the perfusion was
low. This low ratio is similar to the placenta to maternal plasma ratio observed in vivo after
therapeutic abortions in the ninth to fifteenth week of gestation (Saarikoski & Seppala, 1973).
The human placenta does have a capacity to sequester 6-MP and thus decrease transfer to the
fetus (Saarikoski & Seppala, 1973).
To determine if there is active efflux of 6-MP into the maternal circulation, we
introduced the drug into both fetal and maternal circulations as this methodology has been used
previously to identify placental efflux (Kraemer et al., 2006; Pollex et al., 2008). After 180
minutes of perfusion, the F:M ratio was above 1.0. Under equilibrative conditions at 50 ng/ml
and 500 ng/ml, the term placenta was not able to concentrate 6-MP into the maternal circulation.
This suggests that efflux of 6-MP into the maternal circulation is not responsible for or plays a
very minor role in the limited placental transfer of 6-MP. The F:M ratio at the end of the 3 hour
experimental period at both 50 ng/ml and 500 ng/ml was not significantly different from the
calculated F:M ratio based on ion trapping. This supports ion trapping as the mechanism for the
observed F:M ratio at steady state.
64
During pregnancy, nucleoside transporters transfer nucleotides from the mother to the
fetus. Equilibrative nucleoside transporters (ENT) 1, 2 and concentrative nucleoside transporter
2 (CNT2) have been localized to the brush-border membrane of the syncytiotrophoblast
(Govindarajan et al., 2007). 6-MP is a substrate for the ENTs (Nagai et al., 2007), however,
these are energy-independent transporters and are only capable of equilibrative (not
concentrative) transport (Ganapathy et al., 2000) and are not likely to play a role in the transfer
under equilibrative conditions. Only mRNA expression (not protein) of CNT2 was demonstrated
in the human placenta (Govindarajan et al., 2007), thus it is unlikely that CNTs would play a
major role in the F:M distribution of 6-MP. 6-MP is also a substrate for multidrug resistance
protein 5 (MRP5) (Wielinga et al., 2002; Wijnholds et al., 2000). MRP5 is localized to the
basolateral membrane of the syncytiotrophoblast and also on fetal endothelial cells (Meyer Zu
Schwabedissen et al., 2005) and could thus transport 6-MP to the fetal circulation. However, the
expression of MRP5 decreases in term compared to pre-term placentas (Meyer Zu
Schwabedissen et al., 2005), thus it is unlikely that MRP5 plays an important role in our
perfusion model.
Our results show that active efflux of 6-MP is not the mechanism responsible for limited
placental transfer. After introducing 6-MP in the maternal circulation only, the fetal 6-MP
concentration was approximately half of the maternal. This is higher than that observed in vivo
where 6-MP transfer to the fetus is limited (de Boer et al., 2006; Saarikoski & Seppala, 1973).
The placental perfusion model is a useful model for determining transport mechanisms, but does
not always correlate well with in vivo data if non-placental pharmacokinetic variables are
important factors in fetal exposure (Hutson et al., 2011; Myllynen et al., 2005). Two non-
placental pharmacokinetic factors for 6-MP, namely distribution and clearance, are likely to be
important in the limited transfer in vivo. First, distribution of 6-MP includes binding to placental
tissue as demonstrated in our experimental model, but also 6-MP is rapidly converted into
intracellular metabolites. Because of the intracellular location, the metabolites are not free to
cross the placenta in vivo (de Boer et al., 2005). Second, maternal metabolism of 6-MP and its
corresponding short t1/2 of 1 hour (Chan et al., 1987; Ohlman et al., 1994) will limit fetal
exposure by decreasing the maternal area under the curve (AUC). Saarikoski & Seppala (1973)
suggested that maternal metabolism of 6-MP was important in limiting exposure as they
observed that 6-thiouric acid, an inactive metabolite, was the main metabolite transferred to the
65
fetus. Metabolism by the placenta does not likely play an important role in limited fetal 6-MP
exposure as in our perfusions 6-MMP was the only metabolite detected and only after perfusion
with 500 ng/ml of 6-MP. In vivo, 6-MMP was not detected in RBC from cord blood in 3 infants;
however, the limit of detection in this study was not provided (de Boer et al., 2006).
Furthermore, 6-MMP is not stable in blood samples and may have degraded before the samples
were analyzed (de Graaf et al., 2010).
A limitation of our placental perfusion model is that it uses term placentas.
Generalizations to earlier gestational ages are difficult, including extrapolations to the embryonic
period where the placental structure varies. Our results suggest that the distribution of 6-MP and
its short maternal t1/2 are the key factors limiting transfer. These factors are unlikely to change
in the different stages of pregnancy and monitoring maternal plasma for 6-MP and its
metabolites would prevent elevated drug concentrations during pregnancy (de Boer et al., 2006).
Another limitation to our study is that there is no information available regarding plasma
concentrations of 6-MP during pregnancy. The clinically relevant concentration used in our
study was determined in adults taking AZA at similar doses to those administered during
pregnancy. Although the Km for BCRP and 6-MP has not been published, a linear rate of
transfer was observed at higher concentrations than those utilized in our study (de Wolf et al.,
2008).
3.6 Conclusions
Pharmacological therapy with AZA during pregnancy is needed to prevent relapse of
autoimmune diseases or rejection of renal transplants. Studies looking at the safety of AZA and
its major metabolite, 6-MP, have demonstrated that the benefit of treating the disease outweighs
the risk of maintaining treatment during the pregnancy (Kwan & Mahadevan, 2010). Additional
large epidemiological studies and meta-analyses are needed to provide conclusive evidence
regarding the teratogenicity of 6-MP (Coelho et al., 2011), especially to account for the
pharmacogenetic variability in thiopurine S-methyltransferase (TPMT) (Sahasranaman, Howard,
& Roy, 2008). The placenta acts as a relative barrier to 6-MP and its metabolites (de Boer et al.,
2005; de Boer et al., 2006; Saarikoski & Seppala, 1973), and our results suggest that maternal
pharmacokinetic factors and placental binding limit transfer. These findings support monitoring
66
maternal plasma for elevated levels of 6-MP and metabolites is useful in preventing potential
fetal toxicity (de Boer et al., 2006). Our results also suggest that active transport of 6-MP into
the maternal circulation is not an important mechanism. Therefore, polymorphisms or drug
interactions involving active drug transport proteins in the placenta are unlikely to leave a fetus
more vulnerable to 6-MP exposure.
67
Chapter 4
The fetal safety of thiopurines for the treatment of inflammatory bowel disease in pregnancy: a meta-analysis
Janine R Hutson MSc1,2, Jeremy N Matlow BMSc1,3, Myla Moretti MSc1, Gideon Koren MD1,2,3
1Division of Clinical Pharmacology and Toxicology, Hospital for Sick Children, 555 University
Ave, Toronto, Ontario, M5G 1X8, Canada
2Institute of Medical Science, University of Toronto, 1 King’s College Circle, Toronto, Ontario,
M5S 1A8, Canada
3Department of Pharmacology, University of Toronto, 1 King’s College Circle, Toronto, Ontario,
M5S 1A8, Canada
This work has been published and reproduced with permissions: Hutson JR, Matlow JN, Moretti M, Koren G. The fetal safety of thiopurines for the treatment of
inflammatory bowel disease in pregnancy: a meta-analysis. J Obstet Gynecol 2013;33(1):1-8.
68
The fetal safety of thiopurines for the treatment of 4inflammatory bowel disease in pregnancy: a meta-analysis
4.1 Abstract
Maintaining remission of inflammatory bowel disease (IBD) during pregnancy is critical
for positive pregnancy outcomes. Conflicting data exist regarding the association between
thiopurine use for IBD treatment in pregnancy and adverse pregnancy outcomes and this meta-
analysis aims to clarify this association. A meta-analysis was performed of all original human
studies reporting outcomes in pregnancy in patients receiving thiopurines. Nine studies satisfied
the inclusion criteria and a total of 494 patients with IBD and 2,782 disease-matched controls
were reported. When compared to healthy women, those receiving thiopurines had an increased
risk for congenital malformations (RR 1.45; 95% CI 1.07 to 1.96; p = 0.02); however, when
compared to IBD controls, there was no increased risk (RR 1.37; 95% CI 0.92 to 2.05; p = 0.1).
These data provide support for thiopurines having a minimal risk, if any, to the fetus.
69
4.2 Introduction
The thiopurines azathioprine (AZA) and 6-mercaptopurine (6-MP) are effective
treatments for inflammatory bowel disease (IBD), leading to steroid-free, clinical and endoscopic
remission (Ardizzone et al., 2006; Mowat et al., 2011; Timmer et al., 2007). Peak incidence of
IBD occurs in women of childbearing age (Cooper & Stroehla, 2003) and thiopurines are
increasingly being used in pregnancy for their treatment (Goldstein et al., 2007). Maintaining
disease remission during pregnancy is critical for positive fetal outcome (Coelho et al., 2011;
Kwan & Mahadevan, 2010).
Animal studies have demonstrated that at high doses of thiopurines, there is an increased
risk of congenital malformations (reviewed in Polifka & Friedman, 2002). However, doses used
in human treatment regimens are below the no observable adverse effect level (NOAEL)
reported for mice/rats (Coelho et al., 2011; Platzek & Bochert, 1996). In humans, few studies
have evaluated the safety of thiopurines in pregnancy and are limited by their statistical power
(Coelho et al., 2011). Although there is evidence to support that the benefit of treatment with
thiopurines in IBD patients during pregnancy likely outweighs any fetal risk, safety concerns still
arise as a result of their cytotoxic mechanism of action and their toxicity during pregnancy in
animal studies. Over eighty percent of women with IBD have unwarranted concerns about
medications, including AZA, in pregnancy and fears surrounding medications are greater than
the effect of IBD exacerbation (Mountifield et al., 2010). Moreover, some physicians still
choose not to prescribe AZA during pregnancy or to discontinue treatment in the third trimester
(Peyrin-Biroulet et al., 2011). The present meta-analysis aims to clarify the potential effects of
thiopurines on fetal outcome in women with IBD and to compare outcomes from studies using a
general/healthy population compared to an IBD control group.
4.3 Methods
Study Selection
A literature search was performed using MEDLINE, EMBASE, and EMBASE Classic
for all articles from inception to March 2011 reporting pregnancy outcomes in women who took
thiopurines at any time during their pregnancy. The following search terms were used:
70
“azathioprine OR 6-mercaptopurine OR inflammatory bowel disease” AND “pregnancy” and the
results were limited to human studies. Web of Science Database of Abstracts, Cochrane Central
Register of Controlled Trials, Reprotox (an information system from the Reproductive
Toxicology Centre), International Pharmaceutical Abstracts, and articles from the references of
selected reviews were also consulted for additional articles.
Data Extraction
Data extraction was done independently by JRH and JNM. The following information
was extracted from each of the eligible articles: author, year of publication, journal, study design,
timeline and location of study, overlap with other studies, patient characteristics, definition of
malformations used in study, and data for study outcomes. In the case of a disagreement, a third
party (MM) served as a tie-breaker.
Inclusion and Exclusion Criteria
Studies were included if they met the following criteria: human subjects; either case-
controlled or cohort design with a control group; exposure to either AZA or 6-MP during any
stage of pregnancy; and at least one outcome of interest reported. Outcomes of interest included
incidence of congenital abnormalities, prematurity (<37 weeks gestation), birth weight, low birth
weight (LBW) (<2,500 g), and spontaneous abortion (SA). There were no language restrictions.
We excluded reviews, letters to the editor, animal studies, studies with no or inappropriate
control group, and studies with fewer than 10 subjects in either the control or experimental
group. If more than one paper had overlap in the study cohort, the study with the larger sample
size was chosen for inclusion.
Statistical Analysis
Data were combined using a random effects model. Analysis of dichotomous variables
(malformations, prematurity, and LBW) was performed by estimating relative risk (RR) values
and the continuous variable (birth weight) was analyzed using the weighted mean difference
(WMD). Both RR values and WMDs were reported with 95% confidence intervals (CIs) and
were calculated using Review Manager (version 5.0, Copenhagen: Cochrane Collaboration).
Heterogeneity was assessed by the χ2 and I2 tests. I2 refers to the percentage of variability that is
71
due to heterogeneity. While the Q test (χ2) determines the presence or absence of heterogeneity,
the I2 test allows for determination of the extent of heterogeneity.
4.4 Results
Eligible Studies
A total of 533 studies were identified by the initial search and another four unique articles
were identified using Reprotox, two abstracts through Web of Science Database of Abstracts,
and two articles from hand-searching reference lists. After inspection of the title and abstract, 45
papers were read in entirety. Eight peer-reviewed publications (Cleary & Kallen, 2009; Coelho
et al., 2011; Francella et al., 2003; Goldstein et al., 2007; Langagergaard et al., 2007; Norgard et
al., 2007a; Shim, Eslick, Simring, Murray, & Weltman, 2011; Tennenbaum et al., 1999) and one
abstract (Dejaco et al., 2005) met the inclusion criteria and were therefore included in the meta-
analysis (Table 4.1). Two additional studies that met the inclusion criteria were excluded since
their study cohort was believed to overlap with other included studies; Nørgård et al. (2003) had
overlap with both Nørgård et al. (2007) and Langagergaard et al. (2007); Angelberger et al.
(2011) had overlap with Dejaco et al. (2005). Five of the included studies compared women with
IBD receiving thiopurines to IBD controls not receiving thiopurines (Coelho et al., 2011;
Francella et al., 2003; Norgard et al., 2007a; Shim et al., 2011; Tennenbaum et al., 1999), where
IBD controls were those with any previous diagnosis of IBD. Two studies compared women
receiving thiopurines for any indication to healthy or general population controls (Goldstein et
al., 2007; Langagergaard et al., 2007). Two studies had both comparisons available (Cleary &
Kallen, 2009; Dejaco et al., 2005). Although Nørgård et al. (2007) and Langagergaard et al.
(2007) were believed to have overlap between their study cohorts, they were both included since
the former was used for the IBD group comparison and the latter was used for the any indication
group comparison. Independent analyses were performed comparing thiopurine-exposed to IBD
and healthy controls, thus both studies were included. Since disease-matched and healthy
controls were mostly from different studies and thus populations, there were not directly
compared.
72
Table 4-1 Characteristics of studies included in the meta-analysis
First Author Year Study Design
Thiopurine Thiopurine Exposed Patients
(n)
Controls (n)
IBD Any Indication*
IBD Disease-Matched
Healthy/ General
Population
Tennenbaum
1999
rc
AZA
12
-
137
-
Francella 2003 rc 6-MP 32 - 94 - Dejaco 2005 pc AZA or 6-MP 26 26 27 310,323 Norgard 2007 rc AZA or 6-MP 26 - 628 - Langagergaard 2007 rc AZA or 6-MP - 65 - 1,274 Goldstein 2007 pc AZA - 189 - 230 Cleary 2009 rc AZA 324 481 1,739 1,180,969 Coelho 2011 p/rc AZA or 6-MP 55 - 83 - Shim 2011 rc AZA or 6-MP 19 - 74 - Total 494 761 2,782 1,492,796
*Exposed patients in the IBD group are also included in the any indication group where applicable. rc, retrospective cohort; pc, prospective cohort; IBD, inflammatory bowel disease; AZA, azathioprine; 6-MP, 6-mercaptopurine
73
Malformation Rates
Six studies reported on the incidence of congenital malformations in 482 patients with
IBD taking a thiopurine and 2645 IBD controls (Cleary & Kallen, 2009; Coelho et al., 2011;
Dejaco et al., 2005; Francella et al., 2003; Norgard et al., 2007a; Shim et al., 2011) (Table 4.2).
No significant difference was observed (p = 0.12) (Table 4.2; Figure 4.1). A significant
difference was found in the incidence of congenital malformations in IBD patients receiving
thiopurines versus controls not receiving other medications for IBD (Coelho et al., 2011;
Norgard et al., 2007a) (RR 2.67; 95% CI 1.07 to 6.63; p = 0.03), but not versus controls
receiving other pharmacologic treatments for IBD (Cleary & Kallen, 2009; Coelho et al., 2011)
(p = 0.55). Other medications for treatment of IBD included systemic corticosteroids, 5-
aminosalicylic acid, antitumor necrosis factor alpha inhibitors, and other immunosuppressants
(Cleary & Kallen, 2009; Coelho et al., 2011). There was also a significant difference observed
between women receiving thiopurines for any indication versus healthy or general population
controls (Cleary & Kallen, 2009; Dejaco et al., 2005; Goldstein et al., 2007; Langagergaard et
al., 2007) (RR 1.45; 95% CI 1.07 to 1.96; p = 0.02). The specific malformations reported in the
exposed infants in seven of the nine included studies are reported in Table 4.3. The other two
studies did not report on specific malformations (Cleary & Kallen, 2009; Dejaco et al., 2005).
74
Table 4-2 Pregnancy outcomes in thiopurine-exposed pregnancies versus controls
Outcome of Interest No of Studies
Exposed Patients (n)
Controls (n)
RR (95% CI) p Value HG I2 Value
IBD vs Disease-Matched Controls*
Congenital Abnormalities 6 482 2,645 1.37 (0.92, 2.05) 0.12 0% Prematurity 6 470 2,589 1.67 (1.17, 2.37) 0.004 28% LBW 6 467 2,582 1.30 (0.92, 1.83) 0.13 0% SA 2 51 269 0.48 (0.21, 1.10) 0.08 0% IBD vs Disease-Matched Controls ON other medications
Congenital Abnormalities 2 379 1,795 1.15 (0.73, 1.81) 0.55 0% Prematurity 2 373 1739 1.49 (1.13, 1.96) 0.005 0% LBW 2 370 1,732 1.36 (0.95, 1.94) 0.09 0% IBD vs Disease-Matched Controls NOT on other medications
Congenital Abnormalities 2 81 655 2.67 (1.07, 6.63) 0.03 0% Prematurity 2 75 655 2.49 (0.92, 6.75) 0.07 57% LBW 2 75 655 2.06 (0.56, 7.52) 0.28 0% Exposed vs Healthy or General Population Controls**
Congenital Abnormalities 4 743 1,492,735 1.45 (1.07, 1.96) 0.02 0% Prematurity 3 688 1,146,056 3.97 (2.65, 5.94) <0.001 62% LBW 3 667 1,142,385 3.13 (2.44, 4.02) <0.001 0% *Disease-matched controls were on either other pharmacologic treatments for IBD, on no medication, or medication use was not specified **Exposed are women taking thiopurines for any indication (including IBD) during pregnancy HG, heterogeneity; LBW, low birth weight (<2500 g); RR, relative risk ratio; SA, spontaneous abortion; IBD, inflammatory bowel disease Significant results (p < 0.05) are shown in bold
75
Table 4-3 Specific malformations and other drug use reported in the included studies First Author Year Malformations from thiopurine-exposed
group Other drug use in thiopurine-exposed group
Tennenbaum 1999 Pyloric stenosis Hip dislocation
Not clearly reported
Francella 2003 Hydrocephalus
Not clearly reported
Dejaco 2005 Not specified Similar use of prednisolone /5-ASA in exposed and control groups
Norgard 2007 Cataracta congential Encephalocele occipitalis Malformation of sternocleidomastoideus Malformation congenitae aliae cutis
Other drugs used concurrently, but not specified
Langagergaard 2007 Stenosis of pulmonal valve Hydronephrosis Cyst at the ductus choledochus Occipital encephalocele Malformation of sternocleidomastoideus Asymmetrical face
All malformations in exposed-group were in women on multiple medications
Goldstein 2007 Hypospadias Port wine stain Cleft lip Dysmorphic features with ASD Hydrocephalus Hydrocephalus
39% of women taking AZA were also taking a potential teratogen
Cleary 2009 30 malformations, not all specified 9 VSD/ASD (all on many other meds) 2 hypospadias
Women in exposed group at higher risk for taking other medications including drugs for gastric ulcer, loperamide, antihypertensives, steroids,opioids, sedatives, antidepressants, immunosuppressants, heparins, antiplatelet agents
Coelho 2011 Bilateral cataract Cervical angioma
Exposed group also likely receiving 5-ASA, steroids, and anti-TNF.
Shim 2011 Congenital hyperplastic heart Exposed group more likely also receiving 5-ASA and corticosteroids
5-ASA, 5-aminosalicylic acid; anti-TNF, antitumor necrosis factor alpha
76
A)
B)
C)
D)
Figure 4-1 Overall effect of the incidence of congenital abnormalities after in utero
exposure to thiopurines A) In patients with IBD receiving thiopurines compared to disease-
matched controls B) In patients with IBD receiving thiopurines compared to disease-
matched controls on other medications only C) In patients with IBD receiving thiopurines
compared to disease-matched controls not receiving other medications D) In patients
receiving thiopurines for any indications compared to healthy or general population
controls.
77
Prematurity
Six studies reported on the incidence of premature birth (<37 weeks gestation) (Cleary &
Kallen, 2009; Coelho et al., 2011; Dejaco et al., 2005; Francella et al., 2003; Norgard et al.,
2007a; Shim et al., 2011). Patients with IBD receiving thiopurines were more likely to have
premature infants compared to IBD controls (RR 1.67; 95% CI 1.17 to 2.37; p = 0.004) and also
compared to IBD controls that received other IBD therapeutics (Cleary & Kallen, 2009; Coelho
et al., 2011) (RR 1.49; 95% CI 1.13 to 1.96; p = 0.005) (Figure 4-2). There was a trend towards
having an increased odds for prematurity in exposed IBD patients compared to IBD controls that
were not receiving other medications (Coelho et al., 2011; Norgard et al., 2007a) (RR 2.49; 95%
CI 0.92 to 6.75; p = 0.07). Women receiving thiopurines for any indication were also more
likely to have premature infants compared to healthy or general population controls (Cleary &
Kallen, 2009; Goldstein et al., 2007; Langagergaard et al., 2007) (RR 3.97; 95% CI 2.65 to 5.94;
p < 0.001).
78
Birth weight
A)
B)
Figure 4-2 Overall effect of the incidence of prematurity (< 37 weeks gestation) after in utero
exposure to thiopurines A) In patients with IBD receiving thiopurines compared to disease-matched
controls B) In patients receiving thiopurines for any indications compared to healthy or general
population controls.
79
The odds ratio for LBW (<2500 g) was not significant based on six studies (Cleary &
Kallen, 2009; Coelho et al., 2011; Dejaco et al., 2005; Francella et al., 2003; Norgard et al.,
2007a; Shim et al., 2011) comparing women with IBD receiving thiopurines and all IBD controls
(p = 0.13) (Figure 4-3). Likewise, the mean difference in birth weight was also not significant (p
= 0.07) based on three studies (Coelho et al., 2011; Norgard et al., 2007a; Shim et al., 2011) with
the same comparison group (Figure 4-4). Similar findings for LBW were observed when
comparing specifically to IBD controls either receiving (Cleary & Kallen, 2009; Coelho et al.,
2011) (p = 0.09) or not receiving (Coelho et al., 2011; Norgard et al., 2007a) (p = 0.28) other
therapeutics for IBD. There was, however, a lower mean birth weight in women with IBD
receiving thiopurines compared to IBD controls not receiving other medications (Coelho et al.,
2011; Norgard et al., 2007a) (-238.8 g; 95% CI -443.8 to -33.8; p = 0.02). A significant increase
was seen in the incidence of LBW in patients receiving thiopurines for any indication compared
to healthy or general population controls (Cleary & Kallen, 2009; Goldstein et al., 2007;
Langagergaard et al., 2007) (RR 3.13; 95% CI 2.44 to 4.02; p < 0.001). The mean difference in
birth weight for this comparison was significantly decreased (Goldstein et al., 2007;
Langagergaard et al., 2007) (-363.3 g; 95% CI -608.9 to -117.7; p = 0.004).
80
Figure 4-3 Overall effect of the incidence of low birth weight (< 2500 g) after in utero exposure
to thiopurines A) In patients with IBD receiving thiopurines compared to disease-matched
controls B) In patients receiving thiopurines for any indications compared to healthy or general
population controls.
A)
B)
81
Figure 4-4. Overall effect of in utero exposure to thiopurines on mean birth weight A) In
patients with IBD receiving thiopurines compared to disease-matched controls B) In patients
receiving thiopurines for any indications compared to healthy or general population controls.
Spontaneous Abortions
Only two studies reported on the incidence of SA in 51 women receiving thiopurines for
IBD compared to 269 IBD controls (Francella et al., 2003; Tennenbaum et al., 1999). No
significant difference was found, although there was a trend for a decreased rate of SA (p =
0.08). Shim et al. (2011) also reported on the incidence of SA in the same comparison group and
observed no SA in 19 thiopurine-exposed women or 74 controls.
B)
A)
82
4.5 Discussion
Compared to IBD controls, there was no increased risk for congenital malformations in
women receiving thiopurines for IBD. Subgroup analysis of the IBD control groups revealed
that no significant increase was observed when the comparison group comprised of women with
IBD receiving other medications. On the other hand there was a significant increase in risk when
the comparison group was made of women not receiving any other medication for their IBD or
taken from the healthy, general population. However, only two studies were included in each
IBD subgroup analysis. Medication use during pregnancy has been suggested to be a surrogate
for disease activity during pregnancy (Dominitz et al., 2002) and Shim et al. (2011) reported a
significantly higher rate of hospitalization for an IBD flare during pregnancy compared to
controls. The only two comparisons that showed a significant increase for abnormalities used
comparison groups that were either likely to be in remission since they were not receiving any
therapeutics for IBD, or were healthy controls. This supports other reports of disease activity
being associated with adverse pregnancy outcomes (Baiocco & Korelitz, 1984; Dominitz et al.,
2002; Fedorkow et al., 1989; Woolfson et al., 1990). Proper comparison groups and disease
activity will need to be maintained in future studies. Since women with IBD are considered
high-risk (Cornish et al., 2007), some malformations may be more likely to be detected
prenatally due to more intensive monitoring. Furthermore, the largest study in the pooled
analysis by Cleary and Källén (2009) included both major and minor abnormalities and this may
result in overestimation of the true risk (Cornish et al., 2007).
In some cases, studies also reported that women receiving thiopurines were more likely to
be receiving a potential teratogen compared to the control group, including systemic
corticosteroids (Cleary & Kallen, 2009; Goldstein et al., 2007; Shim et al., 2011). Women
receiving thiopurines for IBD treatment were often on other pharmacologic treatment, including
5-aminosalicylic acid (5-ASA), corticosteroids, and antitumor necrosis factor alpha (anti-TNF).
A meta-analysis evaluating the safety of 5-ASA drugs in pregnancy did not find a significant
association with congenital abnormalities (Rahimi et al., 2008). Systemic corticosteroid use in
pregnancy is associated with a small increased risk for oral clefts, growth restriction, and
prematurity (Park-Wyllie et al., 2000). To date, limited evidence suggests a low risk, if any, with
the use of anti-TNF in pregnancy, but further studies are needed (Osting & Carter, 2010).
83
Seven of the nine studies provided information on the specific malformations in the
thiopurine-exposed cohort (Table 4.3). Out of the 22 specific malformations reported in the
seven studies, there was no pattern observed. A previous review of 27 clinical series, mostly
with women receiving thiopurines for renal transplant, also did not identify a recurrent pattern of
congenital anomalies (Polifka & Friedman, 2002). Specifically, no pattern was identified from
six clinical series and reports in 40 IBD patients on thiopurines (Alstead et al., 1990; Campbell &
Ghosh, 2000; Hanauer, 1999; Khan et al., 2000; Levy et al., 1981; Present et al., 1989). Cleary
and Källén (2009) reported an increased risk for ASD/VSDs in women using AZA for any
indication compared to the general population (OR 3.18; 95% CI 1.45 to 6.04). However, this
association was not significant when comparing women using AZA for any indication to
unexposed women with IBD (OR 1.91; 95% 0.75 to 4.57). Three studies in addition to Cleary
and Källén (2009) reported the incidence of ASD/VSD malformations in exposed and unexposed
IBD patients (Coelho et al., 2011; Dejaco et al., 2005; Francella et al., 2003). However, these
three studies had no cases in both exposed and control groups and therefore the data could not be
pooled. In the other five studies (Goldstein et al., 2007; Langagergaard et al., 2007; Norgard et
al., 2007a; Shim et al., 2011; Tennenbaum et al., 1999), there were no cases of ASD/VSD in the
exposed group, however, no data were provided for the control group. Of the six clinical series
and reports reporting outcomes in 40 IBD patients, only one ASD/VSD was reported in the
exposed infants (Campbell & Ghosh, 2000).
The risk for preterm delivery was increased in women receiving thiopurines. Since use
of thiopurines in pregnancy may be related to active disease during conception or pregnancy
(Cleary & Kallen, 2009; Dominitz et al., 2002; Goldstein et al., 2007), this finding is not
surprising since active disease itself increases the risk for prematurity (Baird et al., 1990; Bortoli
et al., 2007; Ludvigsson & Ludvigsson, 2002). Furthermore, medical induction of preterm
delivery, rather than spontaneous, was observed to occur more in women receiving thiopurines
compared to healthy controls (Langagergaard et al., 2007). Compared to IBD controls, there was
no increased risk for LBW in women with IBD being treated with thiopurines. There was,
however, a decrease in the mean birth weight when comparing to IBD controls or the general
population. Similar to the risk of prematurity, active disease during pregnancy can also increase
the chances for LBW (Baird et al., 1990; Ludvigsson & Ludvigsson, 2002).
84
Limited data were provided regarding the risk of SA in women receiving thiopurines. A
decreased trend for SA in women with IBD on thiopurines was observed, however more studies
are needed. Cleary & Kallen (2009) reported on the incidence of SA in women on AZA for any
indication compared to the general population and did not observe a significant difference. Even
at high doses, 6-MP was not found to be useful as a single-agent medical abortifacient in early
pregnancy (Davis et al., 1999).
There are several limitations to our study. First, all nine of the studies included in the
meta-analysis were observational cohort studies, and only three were prospective. Since patients
were not randomized, the studies may be subject to selection bias and confounding variables.
However, studies evaluating the fetal safety of therapeutics in pregnancy are almost always
observational since there are many ethical concerns regarding randomization in this population.
Second, overall there was a low frequency of adverse outcomes, making statistical precision
difficult. Furthermore, subgroup analysis for IBD controls was difficult since only two studies
with relatively small sample sizes were available. Third, all studies except for Tennenbaum et al.
(1999) and Shim et al. (2011) did not report on disease activity during pregnancy. Since disease
activity may be related to adverse fetal outcomes, future studies should comment on this. Fourth,
there was limited information available regarding the concurrent medications that the exposed
and control groups were taking. Fifth, we included patients that received treatment with
thiopurines at any time during gestation and therefore were not able to specifically evaluate any
association with timing of exposure in pregnancy.
In conclusion, there is no significant difference in the risk for malformations or LBW in
women with IBD being treated with thiopurines compared to IBD patients not receiving
thiopurines. A significant risk for malformations and LBW was only observed when comparing
women receiving thiopurines to healthy or general population controls. Women receiving
thiopurines for IBD have an increased risk of having a premature infant, but this may be related
to disease activity. Future studies evaluating the safety of thiopurines in pregnancy should
include data on disease activity during pregnancy and use appropriate control groups.
85
Chapter 5
Folic acid transport to the human fetus is decreased in pregnancies with chronic alcohol exposure Janine R. Hutson1,2, Brenda Stade3, Denis C. Lehotay4, Christine P. Collier5,6, and Bhushan M.
Kapur1,7
1Division of Clinical Pharmacology and Toxicology, Hospital for Sick Children, Toronto,
Canada
2Institute of Medical Science, University of Toronto, Toronto, Canada
3Department of Paediatrics, Keenan Research Centre, St. Michael’s Hospital, Toronto, Canada
4College of Medicine, University of Saskatchewan, Regina, Canada
5Department of Pathology and Molecular Medicine, Queen's University, Kingston, Canada
6Kingston General Hospital, Kingston, Canada
7Department of Clinical Pathology, Sunnybrook Health Sciences Centre, Toronto, Canada
This work has been published and reproduced with permissions: Hutson JR, Stade B, Lehotay DC, Collier CP, Kapur B. Folic acid transport to the human fetus
is decreased in pregnancies with chronic alcohol exposure. PLoS ONE 2012:7(5);e38057.
86
Folic acid transport to the human fetus is decreased in 5pregnancies with chronic alcohol exposure
5.1 Abstract
Background: During pregnancy, the demand for folic acid increases since the fetus requires this
nutrient for its rapid growth and cell proliferation. The placenta concentrates folic acid into the
fetal circulation; as a result the fetal levels are 2 to 4 fold higher than maternal. Animal and in
vitro studies have suggested that alcohol may impair transport of folic acid across the placenta by
decreasing expression of transport proteins. We aim to determine if folate transfer to the fetus is
altered in human pregnancies with chronic alcohol consumption. Methodology/Principal
Findings: Serum folate was measured in maternal blood and umbilical cord blood at the time of
delivery in pregnancies with chronic and heavy alcohol exposure (n=23) and in non-drinking
controls (n=24). In the alcohol-exposed pairs, the fetal:maternal serum folate ratio was ≤1.0 in
over half (n=14), whereas all but one of the controls were >1.0. Mean folate in cord samples was
lower in the alcohol-exposed group than in the controls (33.15 ± 19.89 vs 45.91 ± 20.73,
p=0.04). Conclusions/Significance: Our results demonstrate that chronic and heavy alcohol use
in pregnancy impairs folate transport to the fetus. Altered folate concentrations within the
placenta and in the fetus may in part contribute to the deficits observed in the fetal alcohol
spectrum disorders.
87
5.2 Introduction
During pregnancy, the demand for folic acid increases since this nutrient is critically
important for DNA synthesis and cell proliferation. During pregnancy, the placenta concentrates
folic acid into the fetal circulation and as a result fetal levels are 2- to 4-fold higher than maternal
(Economides et al., 1992; Giugliani et al., 1985; Guerra-Shinohara et al., 2004; Molloy et al.,
2005; Navarro et al., 1984; Obeid et al., 2005; Relton et al., 2005; Stark et al., 2007; Thorand et
al., 1996). The fetal requirement for folate during pregnancy is paramount such that cord folate
status is maintained even when maternal status is low (Wallace et al., 2008). Transport of folates
across the placenta is mediated by placental folate receptors (PFRs) (Henderson et al., 1995),
namely folate receptor-α (FR-α) at the microvillous membrane of the syncytiotrophoblast
(Bisseling et al., 2004; Solanky et al., 2010). Folate in the maternal circulation binds to FR-α,
where it binds with high affinity and is internalized through receptor-mediated endocytosis
(Keating et al., 2009) (Figure 5-1). Other transporters including the reduced folate carrier (RFC)
and the proton-coupled folate transporter (PCFT) are also important for the placental uptake of
folate (Prasad et al., 1995; Yasuda et al., 2008). This results in a high intervillous blood
concentration of folate within the placenta where it can be transported to the fetus by passive
diffusion and by the RFC (Bisseling et al., 2004; Henderson et al., 1995). This mechanism of
transport is established early in pregnancy (within the first trimester (Solanky et al., 2010)) as
folate is vital to the proper development of the fetus.
88
Figure 5-1. The transport proteins present on the syncytiotrophoblast that are involved in
the transfer of folate to the fetal circulation.
Animal and in vitro studies have suggested that alcohol may impair transport of folic acid
across the placenta by decreasing expression of folate transport proteins (Fisher et al., 1985;
Keating et al., 2009; Keating et al., 2008). Indeed, there are similarities between deficits
observed in the fetal alcohol spectrum disorders (FASDs) and folate status during pregnancy.
These similarities include common physical malformations such as neural tube defects,
congenital heart defects, and limb defects (Chudley et al., 2005; Goh & Koren, 2008; Jones,
2006). Furthermore, lower folate status has been associated with hyperactivity, peer problems,
and lower cognitive function (Schlotz et al., 2010; Veena et al., 2010), which are all neuro-
developmental consequences of alcohol exposure during pregnancy (Chudley et al., 2005).
89
Folic acid is also important as an antioxidant during pregnancy. Alcohol consumption
during pregnancy creates oxidative stress to both the placenta and fetus and this stress can be
mitigated by folic acid (Cano et al., 2001; Gundogan et al., 2010). Formic acid, the toxic
metabolite of methanol, has been reported in the sera of alcohol abusing patients and has also
recently been detected in umbilical cord blood from pregnancies with heavy amounts of alcohol
consumption (Kapur, et al., 2009). Formic acid can lead to neurotoxicity and oxidative stress
(Kapur et al., 2007). Folic acid is required for the detoxification of formic acid. Folic acid is
critical to the rate of detoxification of formic acid (Sokoro et al., 2008). Furthermore, in folate
deficient animals, the adverse effects to the fetus after alcohol exposure are more severe
compared to controls (Gutierrez et al., 2007; Lin, 1991). Taken together, proper placental
transfer of folic acid is critical to proper fetal development and can influence the fetal effects of
alcohol.
To our knowledge, there are no studies that determine fetal folate levels from pregnancies
affected by heavy alcohol use. We hypothesize that folate transfer to the fetus is impaired in
alcohol-exposed pregnancies and that this may, in part, be responsible for the deficits associated
with the FASDs.
5.3 Methods
All procedures and protocols received prior approval from the Institutional Research
Ethics Board. The most severe diagnosis under the FASDs is fetal alcohol syndrome (FAS).
Children with FAS are usually born to mothers who consume large amounts of alcohol in the
pregnancy and for a long duration. However, there is large variation in the deficits produced with
alcohol consumption and likely results from differences in amount consumed, drinking patterns,
genetics, and nutrition. Only women consuming heavy amounts throughout the pregnancy were
included in this study and are thus the women most at risk of having a child affected by FAS.
This avoided any variability that may result from differences in amount of alcohol consumed and
timing of exposure. Healthy women with no known illicit drug or alcohol use during pregnancy
were also recruited to be representative of the general population to serve as our comparison
group. As part of this study, the infants were offered follow up care and diagnostic testing for
the FASDs.
90
Written informed consent was obtained from the alcohol abusing mothers included in this
study. Maternal and cord blood samples were collected at the time of delivery. These women
were identified at the time of delivery and questionnaires regarding drug use were administered
after delivery. For controls maternal and cord blood samples were collected as part of routine
clinical workup of women delivering at the hospital. Plasma folic acid was measured in our
routine clinical laboratory by chemiluminescent immunoassay using the Beckman Coulter
UniCel DxI 800 AccessH Immunoassay System (Beckman Coulter Canada, Inc., Mississauga,
Ont. Canada). Manufacturer recommended assay protocol was used. Besides folic acid results no
other information on these women was available to us.
Normally distributed data were compared as follows: maternal and fetal folate levels
were compared using a paired t-test; folate levels in the control group compared to the alcohol
group were compared using an independent t-test. Pearson correlation was used to compare
maternal and fetal folate levels. Fetal to Maternal folate ratios were compared using the Mann
Whitney U Test because they were not normally distributed. Significance was obtained if p<0.05
and all performed tests were two-tailed (where applicable).
5.4 Results and Discussion
Twenty-three women consuming heavy amounts throughout the pregnancy were included
in this study. Twenty-four women were recruited from the general population to serve as the
comparison group with no known illicit drug or alcohol use during pregnancy. Maternal
demographics and fetal information for the women drinking alcohol during pregnancy is given in
Table 5.1. Maternal age for this group ranged from 16 to 44 years. All women reported regular
alcohol consumption throughout the pregnancy and were considered to be dependent users. The
women were not recruited until later in their pregnancy and self-reported alcohol consumption
ranged from daily use to > 8 drinks per week. The women included in this cohort were being
followed by an FASD clinic and were therefore considered at high risk of having an affected
child because of their alcohol consumption. In addition to alcohol, fourteen women reported
occasional to frequent cocaine use, eight reported marijuana use, and two reported opiate use
during the pregnancy. Cigarette use was also common to this population.
91
Table 5-1. Demographic information on alcohol-using women included in this study and
fetal parameters. (n=23)
Mean (SD)
Maternal age 29.2 (6.9) years
Gravidity 4.8 (3.4)
Parity 2.4 (1.9)
Gestational age 36.6 (1.6) weeks
Length 42.3 (3.2) cm
Head Circumference 32.3 (1.4) cm
Birth weight 2830 (421) g
In order to determine whether the placenta was concentrating folate to the fetal
circulation, we calculated the fetal to maternal (F:M) folate ratio using the serum folate
measurements from the mother-cord pairs. The F:M folate ratio was ≤1.0 in over half (n=14) of
the alcohol-exposed pairs, whereas all but one of the controls were >1.0 (Figure 5.2). The F:M
folate ratios in the alcohol group were significantly lower than the control group (p=0.014). Also,
there was large variability observed in the F:M folate ratios in the alcohol group (range 0.24 to
7.65) and not in the control group (range 0.79 to 3.18), suggesting that the tight regulation of
folate transport to the fetus is deregulated (Figure 5.3).
As expected, the fetal folate levels in the control group were significantly higher than
maternal in controls (45.91nM ± 20.73nM vs 26.99nM ± 11.18nM, p<0.0001) (Figure 5.4). This
finding has been reported in all studies to date comparing maternal and fetal folate levels
(Economides et al., 1992; Giugliani et al., 1985; Guerra-Shinohara et al., 2004; Molloy et al.,
2005; Navarro et al., 1984; Obeid et al., 2005; Relton et al., 2005; Stark et al., 2007; Thorand et
al., 1996). However, this finding was not observed in the alcohol group and there was a trend for
lower fetal levels compared to maternal (33.15nM ± 19.89nM vs 38.13nM ± 29.55nM,
respectively, p=0.461). Furthermore, there was no correlation between the maternal and cord
folate levels in the alcohol group (R2=0.051, p=0.226), yet there was the expected correlation in
the control group (R2=0.550, p=0.018). Together, these data support folate transport being
impaired in pregnancies affected by chronic and heavy alcohol use. Because only half of mother-
fetal pairs had a F:M folate ratio of less than one, this suggests variability in the effect. This
92
could result from differences in drinking patterns, genetic susceptibility, or other drug use in
addition to alcohol (to be discussed below).
Figure 5-2. Scatter-plot of the fetal to maternal (F:M) folate ratios as measured in cord
blood and maternal blood, respectively, at the time of delivery.
93
Figure 5-3. Corresponding maternal and fetal folate concentrations at the time of delivery
in pregnancies with A) heavy alcohol exposure and B) in controls.
94
Figure 5-4. Mean (± SEM) cord and maternal plasma folate concentrations at the time of
delivery in alcohol-abusing women and controls. *p<0.05 for a two-tailed paired t-test.
**p<0.05 for a two-tailed independent t-test.
After observing an impaired ability to concentrate folic acid to the fetus, we next wanted
to see if the actual folate concentration in the fetal circulation was lower in the alcohol-exposed
group. Indeed, folate levels were significantly lower in cord blood in alcohol-exposed fetuses
compared to controls (p=0.04). Interestingly, there was no difference in folate levels between
the alcohol-using and control women. There was a trend for increased folate levels in the alcohol
group. This was not expected since folate deficiency is common with chronic alcohol
consumption (Halsted et al., 2002). Two studies however, have reported higher folate levels in
alcohol-using pregnant women compared to controls and may reflect women drinking beer,
which contains folates (Larroque et al., 1992; Stark et al., 2005). Despite having higher maternal
folate levels, fetal folate levels were still lower in the alcohol group – emphasizing that alcohol
likely deregulates placental folate transport.
95
It has been suggested that alcohol consumption during pregnancy may alter the placental
transport of folate in animal and in vitro studies; however, no conclusion has been reached. In
vitro studies using both a choriocarcinoma cell line (BeWo) and primary trophoblast cells from
human term placentas demonstrated decreased folic acid uptake after chronic exposure to ethanol
(Keating et al., 2008; Keating et al., 2009). Furthermore, after chronic exposure in rats, there
was a decrease in folic acid binding (Fisher et al., 1985). Conversely, other studies have shown
that alcohol may not alter folate transport. Dual-perfusion of a single lobule from a term human
placenta did not demonstrate altered folate binding or transfer after treatment with ethanol
(Henderson et al., 1995). Similarly, a single ethanol exposure to rats during pregnancy did not
alter transport of folate (Lin, 1991). The different findings likely result from the type of exposure
since only those studies where there was chronic alcohol exposure showed a decreased effect.
Our study population in the alcohol group was consuming alcohol continuously during
pregnancy and is in concordance with the chronic animal and in vitro studies.
Decreased folate transfer may be a result of altered expression of the folate binding and
transport proteins, including FR-α, RFC, and PCFT. After chronic alcohol exposure in vitro,
primary trophoblasts and BeWo cells had reduced mRNA expression of RFC and FR-α
respectively (Keating et al., 2008; Keating et al., 2009). However, there are no data available
looking at mRNA, protein, or folic acid uptake in human placenta from a population similar to
that in our study. Future studies should investigate this mechanism using human placentas from
alcohol-exposed pregnancies. Since this population is difficult and sensitive to recruit and has
many co-morbidities, animal studies may be crucial to a full mechanistic understanding.
A role for impaired folic acid transport in the development of FASDs is further supported
by animal studies. After alcohol exposure during pregnancy in mice and rats, more adverse fetal
effects were observed in those born to mothers receiving a folate free diet (Gutierrez et al., 2007;
Lin, 1988). Furthermore, several recent animal studies have reported that high dose folic acid
can mitigate the adverse fetal effects induced by alcohol, especially measures of oxidative stress
(Cano et al., 2001; Garcia-Rodriguez et al., 2003; Wang et al., 2009; Xu, Tang, & Li, 2008;
Yanaguita et al., 2008). A recent study has demonstrated that administration of folic acid by
injection decreases fetal resorption and malformations in mice consuming ethanol in pregnancy
(Wentzel & Eriksson, 2008). These findings suggests that it may be possible to overcome the
decreased placental transfer observed in this study if maternal levels are high enough.
96
The fetotoxic effects resulting from folate deficiency may result from different possible
pathways. Folic acid is critically important for DNA synthesis and cell proliferation as well as in
cellular protection through its antioxidant properties (Antony, 2007). Folate deficiency within
the placenta has also been shown to alter placental DNA methylation (Kim et al., 2009). Fetal
growth and development are closely linked to DNA methylation (Kim et al., 2009). Disruption
in placental DNA methylation may have a role in the Developmental Origins of Adult Health
and Disease hypothesis and alterations in the placenta have already been associated with the
future risk of cardiovascular disease and cancer (Gallou-Kabani et al., 2010).
An additional complication that may arise from folate deficiency is related to formic acid,
the toxic metabolite of methanol. Formic acid has recently been detected in maternal and cord
blood in pregnancies with chronic ethanol exposure (Kapur, et al., 2009). This formic acid is
endogenously produced from methanol naturally found in alcoholic beverages as well as
methanol produced as a by-product in the pituitary (Axelrod & Daly, 1965). Formic acid
requires folic acid for detoxification. A recent study by our group observed a negative
correlation between formic acid in cord blood and cognitive function (r=-0.6154, p=0.025, n=12
at 12 months and r=-0.6241, p=0.023, n=13 at 18 months) using Bayley scores (Kapur, BM et
al., 2009). Formic acid has been shown to cause neuronal cell death in the rat hippocampal
explants and this could be mitigated by the folic acid (Kapur et al., 2007). Thus, lower levels of
folate may leave the fetus more vulnerable to the potential toxic effects of formic acid.
The women included in our study in the alcohol group also reported use of other drugs of
abuse, including cocaine, tetrahydrocannabinol (THC in marijuana), and cigarettes. The
influence of these drugs on folate transfer cannot be ruled out. However, in vitro studies do not
support a role for cocaine in altering folate transport. Studies using primary trophoblasts showed
that acute or chronic exposure to cocaine did not alter folate uptake (Keating et al., 2009).
However, chronic exposure (but not acute) to THC, did decrease folate uptake in primary
trophoblasts (Keating et al., 2009). Furthermore, amphetamine, and ectasy (MDMA) decreased
folate uptake after both acute and chronic exposure. No women in our study reported use of
amphetamine or ecstasy, however, self-report may not be 100% accurate due to the stigma
associated with illicit drug use. Also, it is unknown whether the reported occasional use of THC
by the three women in this study would be enough to cause an effect on folate transport since
chronic exposure was needed in the in vitro studies to have an effect.
97
There are currently no in vivo data available that relate folate transport to drugs of abuse;
however, there are studies evaluating folate transport in pregnancy to cigarette smoking in
pregnancy. A small decrease in cord folate levels was determined in infants born to smoking
mothers compared to non-smoking mothers (Stark et al., 2007). However, the fetal folate levels
remained higher than maternal and the positive linear relationship was maintained in the
smoking group. The decrease in fetal folate levels and transfer was more profound in our study
population; thus the observed effect in our study is a result of more than simply maternal
cigarette use. An interaction or additive effect between smoking, alcohol, and other drugs of
abuse may be possible and should be further investigated.
To our knowledge, this is the first study to show that folic acid transport to the fetus is
compromised in pregnancies affected by heavy and chronic alcohol exposure. Decreased levels
of folate available to the fetus as well as bound to the placenta itself may in part be responsible
for the deficits observed in the FASDs. Although our study was focused on FASD, low fetal
folate may also be part of the aetiologies of diseases seen in children born to chronic alcohol
drinking women during pregnancy. Although future studies are needed to address the mechanism
for the decreased folate transfer, current practice should continue to properly counsel pregnant
women and women of childbearing age on proper folic acid supplementation as well as
abstinence from alcohol during pregnancy.
98
Chapter 6
Adverse placental effect of formic acid on hCG secretion is mitigated by folic acid Janine R Hutson1,2, Lubetsky, A1, Eichhorst, J3, Hackmon, R1, Koren, G1,2, Kapur, BM1,4
1Division of Clinical Pharmacology and Toxicology, Hospital for Sick Children, Toronto, ON
2Insitute of Medical Sciences, University of Toronto, Toronto, ON
3Saskatchewan Disease Control Laboratory, Regina, SK
4Dept of Clinical Pathology, Sunnybrook Health Sciences Center, Toronto, ON.
This work has been published and reproduced with permissions: Hutson JR, Lubetsky A, Eichhorst J, Hackmon R, Koren G, Kapur BM. Adverse placental
effect of formic acid on hCG secretion is mitigated by folic acid. Alcohol Alcohol 2013;48(3):283-7.
99
Adverse placental effect of formic acid on hCG 6secretion is mitigated by folic acid
6.1 Abstract
Formic acid has recently been detected in maternal blood and umbilical cord blood of
infants born to alcohol abusing mothers. This toxic metabolite of methanol requires folate for
detoxification. We hypothesized that formic acid produced in the maternal circulation will
transfer across the placenta and will be toxic to the placenta. Our objectives were first, to
determine whether formic acid transfers across the human placenta and whether it is toxic to the
placenta. Second, to determine whether folate can decrease transplacental transfer of formic acid
and mitigate toxicity. Dual perfusion of a single placental lobule ex vivo was used to
characterize the transfer of formic acid across the placenta. After a 1-hour control period, formic
acid (2mM) was introduced into the maternal circulation with (n=4) or without folate (1uM)
(n=4) and was allowed to equilibrate for 3-hours. Formic acid transferred rapidly from the
maternal to the fetal circulation and transfer was not altered with the addition of folate.
Compared to the control period, there was a significant decrease in hCG secretion (p=0.03) after
addition of formic acid. The addition of folic acid to the perfusate mitgiated the decrease in
hCG. Formic acid rapidly transfers across the placenta and thus has the potential to be toxic to
the developing fetus. Formic acid decreases hCG secretion in the placenta, which may alter
steroidogenesis and differentiation of the cytotrophoblasts, and this adverse effect can be
mitigated by folate.
100
6.2 Introduction
Alcoholic beverages may contain a small amount of methanol as a congener
(Lachenmeier et al., 2011). Furthermore, methanol is produced endogenously in the pituitary
from S-adenosylmethionine (Axelrod & Daly, 1965; Sarkola & Eriksson, 2001). In heavy
drinkers, methanol may accumulate since ethanol has a higher affinity for alcohol dehydrogenase
(ADH) and may reach plasma concentrations above 2 mM (Kapur et al., 2007; Majchrowicz &
Mendelson, 1971). Formic acid, the toxic metabolite of methanol, has been detected in both sera
and cerebral spinal fluid of alcoholics in concentrations that are neurotoxic (Kapur et al., 2007).
Formic acid has also been recently detected in maternal blood and umbilical cord blood of
infants born to heavy drinkers (Kapur et al., 2009).
Formic acid requires folate for detoxification and folate status can influence the clearance
rate (Johlin et al., 1987; Sokoro et al., 2008). Studies on rat brain hippocampal slices showed
that formic acid can cause neuronal cell death, and this toxicity can be mitigated by folate (Kapur
et al., 2007). Formic acid has also been shown in animal studies to lead to growth restriction,
physical malformations, and depletion of glutathione in the embryo (Andrews et al., 1998;
Brown-Woodman et al., 1995; Hansen et al., 2005; Harris et al., 2004). Furthermore, human
studies have reported fetal alcohol syndrome-like facial features in infants born after solvent
abuse (including methanol) by the mother during pregnancy (Scheeres & Chudley, 2002).
The placenta separates the maternal and fetal circulations throughout human pregnancy.
Heavy alcohol consumption during pregnancy is associated with numerous placental alterations
including placental dysfunction, decreased placental size, gene expression changes and endocrine
disruptions (Burd et al., 2007; Rosenberg et al., 2010). Since formic acid can produce oxidative
stress (Harris et al., 2004), it may be placentotoxic. Compromised placental function is
associated with growth restriction, a deficit associated with the fetal alcohol spectrum disorders
(FASDs). Since the placenta is a reservoir for folate (Henderson et al., 1995), there may also be
a potential role for detoxification of formic acid within the placenta itself. The objectives of the
study were to first determine whether formic acid transfers across the placenta and is toxic to the
placenta. Second, to determine whether folate can decrease transplacental transfer of formic acid
and mitigate potential toxicity.
101
6.3 Methods
6.3.1 Materials
Sodium formate was obtained from Sigma (St. Louis, MO). The water used for all
experimental procedures was obtained from a Milli-Q Advantage A10 Ultrapure Water
Purification System (Millipore, Billerica, MA).
6.3.2 Placental perfusion
The dual perfusion of a placental lobule was previously described by Miller et al. (1985)
and adapted in our laboratory (Derewlany et al., 1991; Pollex et al., 2008). Immediately after
elective caesarean sections, term placentas were obtained from healthy mothers with
uncomplicated pregnancies from St. Michael’s Hospital in Toronto, Ontario. Research ethics
board approval was obtained and maternal consent was attained prior to the surgery. For each
placenta, a vein/artery pair supplying a clearly identifiable cotyledon was chosen for cannulation
and maternal and fetal circulations were established within 30 minutes of delivery (Derewlany et
al., 1991).
The maternal perfusate was equilibrated with 95% O2, 5% CO2 and the fetal with 95%
N2, 5% CO2. The perfusate consisted of M199 tissue culture medium (Sigma, St. Louis, MO)
containing heparin (2000 U/L), glucose (1.0 g/L), kanamycin (100 mg/L), and 40,000 molecular-
weight dextran (maternal 7.5 g/L; fetal 30 mg/L). As a marker of passive diffusion, antipyrine (1
mM) was added to the maternal circulation. The fetal and maternal circuit flow rates were
maintained at 2 to 3 and 13 to 14 ml/min, respectively, and the temperature of both circuits and
the perfusion chamber was kept at 37°C. Small volumes of sodium bicarbonate and hydrochloric
acid were added to the perfusate to maintain the maternal and fetal circuits at pH 7.4 and 7.35,
respectively. This pH mimics the slightly more acidic fetal circulation observed in vivo
(Reynolds & Knott, 1989).
Each perfusion consisted of a 1 h pre-experimental control period, followed by a 3 h
experimental period, where both were in a recirculating (closed) configuration. The perfusates in
both circulations were replaced with fresh media prior to each of the 2 periods. During the pre-
102
experimental period, samples were taken every 15 min to analyze glucose and oxygen
consumption, and human chorionic gonadotropin (hCG) secretion as measures of tissue viability.
During the experimental period, these measures were taken every 30 minutes. Tissue viability
measures were calculated as previously described (Pollex et al., 2008). Fetal perfusion pressure
and fetal reservoir volume were monitored as an indicator of tissue integrity. The perfusion was
terminated at any time if there was a >3 ml/hour loss in fetal reservoir volume. pH, pO2, and
pCO2 were monitored using a blood gas analyzer (Radiometer ABL 725, Copenhagen,
Denmark).
During the experimental period, 2 mM sodium formate was added to the maternal
circulation. 2 mM was chosen since this is a clinically relevant concentration that was
previously observed in heavy drinkers (Kapur et al., 2007). For perfusions that included folate, 1
µM was added into both the fetal and maternal circuits at the beginning of both the pre-
experimental and experimental periods. Samples were taken for measurement of formate and
folic acid every 10 minutes for the first half-hour and every half-hour following.
6.3.3 Sample analysis
At the end of each perfusion, the perfused lobule was isolated. Perfused and unperfused
tissue from the same placenta was homogenized (1:5 (w/v) in phosphate buffered saline, pH 7.4)
using a Polytron (Brinkmann Instruments, Inc., Westbury, NY) for 2 minutes. The homogenate
was centrifuged at 5000 ×g for 15 minutes at 4 °C. The supernatant was removed and analyzed
for both hCG and formic acid. hCG levels were measured by enzyme-linked immunosorbent
assay (Alpha Diagnostic Intl. Inc., San Antonio, Texas) after a 1:80 dilution. The rate of hCG
secretion was determined by calculating the slope of the hCG concentration vs time curve during
the initial linear portion of the curve.
Perfusate samples were stored at -20°C until analysis. Formic acid was detected by gas
chromatography-flame ionization detection using a previously published method (Sokoro et al.,
2008). Area under the curve (AUC) was calculated using the trapezoidal rule. Tissue hCG levels
of the perfused tissue are expressed as a percentage of the initial tissue concentration
(Linnemann et al., 2000). Differences between the pre-experimental control and experimental
103
periods were compared using a paired t-test and differences between perfusion with and without
added folate were compared using an independent t-test. Differences were considered significant
if P < 0.05.
6.4 Results
A total of 8 lobules from different placentae were perfused with formic acid (4 with
folate added and 4 without folate added) and the physical parameters for the perfusions are given
in Table 6.1. The mass of the perfused cotyledons ranged from 8.53 to 17.12 g. Measures of
placental viability, integrity, and function throughout the experiments remained within normal
ranges and were not significantly different between the control and experimental phases (Table
6-1). Furthermore, the fetal arterial pressures remained similar between the control and
experimental periods. The rate of antipyrine disappearance from the maternal circulation was
equal to the rate of appearance in the fetal circulation during the experimental period with values
of 0.028 ± 0.004 and 0.029 ± 0.006 µmol/g per minute, respectively.
Formic acid transferred rapidly from the maternal to the fetal circulation (Figure 6-1). In
the presence or absence of folate to the perfusate, formic acid appeared in the fetal circulation
within 10 minutes in all eight perfusions. The addition of folate into the perfusate did not alter
the fetal AUC (1.30 ± 0.14 without folate; 1.23 ± 0.48 with folate; p = 0.79). Tissue
concentrations of formic acid measured in the perfused lobules at the completion of the
experiment were 425.83 ± 57.18 and 431.18 ± 133.07 nmol/g for perfusions without and with
folate added, respectively.
104
Table 6-1 Placental viability parameters and metabolic capacity throughout the perfusions
(mean ± SD)
Viability Parameter
Formic Acid without Folate (n=4) Formic Acid with Folate (n=4)
Pre-Experimental Control
Experiment
Pre-Experimental Control
Experiment
Fetal arterial inflow pressure (mm Hg)
43.65 ± 6.02 41.24 ± 2.15 41.50 ± 12.24 38.62 ± 9.05
Oxygen (µmol O2/g per min)
Transfer 0.02 ± 0.01 0.02 ± 0.01 0.02 ± 0.00 0.02 ± 0.00
Delivery 0.75 ± 0.33 0.72 ± 0.29 0.62 ± 0.09 0.64 ± 0.05
Consumption 0.35 ± 0.16 0.32 ± 0.13 0.31 ± 0.02 0.34 ± 0.03
Glucose consumption (µmol/g per min)
0.65 ± 0.58 0.42 ± 0.11 0.41 ± 0.09 0.32 ± 0.07
Lactate Production (µmol/g per min)
0.38 ± 0.11 0.25 ± 0.06 0.28 ± 0.17 0.22 ± 0.06
105
Figure 6-1 Maternal-to-fetal transfer of formic acid after dual perfusion of a single
placental lobule in the presence (n=4) or absence (n=4) of folate (1µM). Formic acid (2mM)
was added to the maternal circulation and transfer was determined for a period of 180 min.
Data are shown as mean values ± SEM at each time point.
106
Figure 6-2 The rate of hCG secretion into the maternal circulation during the placental
perfusion was calculated in the pre-experimental control period (before addition of formic
acid) and during the experimental period in the A) presence (n=4) and B) absence (n=4) of
folate. C) The rate of hCG secretion was compared between the experimental period in the
presence and absence of folate.
0"
2000"
4000"
6000"
0" 50" 100" 150" 200"
hCG$(m
IU/g)$
Time$(min)$
A)$
Experimental"
"Control""
0"
2000"
4000"
6000"
0" 50" 100" 150" 200"
hCG$(m
IU/g)$
Time$(min)$
B)$
Experimental"
Control"
0"1000"2000"3000"4000"5000"6000"
0" 50" 100" 150" 200"
hCG$(m
IU/g)$
Time$(min)$
C)$
Without"Folic"Acid"
With"Folic"Acid"
107
Figure 6-3 Tissue concentration of hCG in the perfused lobule at the end of the 180 minute
experimental period with formic acid expressed as a percentage of the initial hCG tissue
concentration in the same placenta in the presence (n=4) and absence (n=4) of folate.
Compared to the pre-experimental control period, there was a significant decrease the
rate of hCG secretion in the maternal circulation after addition of formic acid in the experimental
period (p = 0.03) (Figure 6-2). In contrast, there was no significant decrease when folate was
present in the perfusate. The percentage of initial placental tissue hCG was decreased in the
perfusions without folate compared to perfusions with folate (p = 0.04) (Figure 6-3).
108
6.5 Discussion
The small molecule, formic acid, crosses the placenta rapidly. Since formic acid can
reach high concentrations in heavy drinkers (Kapur et al., 2007; Kapur et al., 2009), our results
suggest that any formic acid produced by the mother is likely transferred to the fetus in utero.
Although there are a lack of in vivo human studies regarding the developmental toxicity of
formic acid (Ema et al., 2010), this molecule has the ability to cause neurotoxicity, growth
restriction, physical malformations, and depletion of fetal glutathione in animal studies (Andrews
et al., 1998; Brown-Woodman et al., 1995; Hansen et al., 2005; Harris et al., 2004; Kapur et al.,
2007).
Our current study demonstrates that formic acid is placentotoxic in that placental hCG
secretion was decreased. Other toxic compounds have been shown to decrease or inhibit
placental hCG secretion including cocaine, 2,3,7,8-tetrachlorodibenzo-p-dioxin, a
polychlorinated dibenzo-p-dioxin/polychlorinated dibenzo-P-furan mixture,
difluoromethylornithine, bisphenol A, benzo[a]pyrene, and high levels of zidovudine
(Augustowska et al., 2007; Boal et al., 1997; Moore et al., 1988; Morck et al., 2010; Simone et
al., 1996; Zhang et al., 1995). Since formic acid may be present in the maternal circulation at the
same time as ethanol, further studies should evaluate how these two agents interact to alter hCG
secretion since in vitro studies in human trophoblasts demonstrated that ethanol can increase
hCG secretion (Karl & Fisher, 1993; Karl et al., 1994; Karl et al., 1998; Wimalasena et al.,
1994).
Alterations in hCG secretion by the placenta may have consequential implications for the
pregnancy since hCG is vitally important in maintaining the pregnancy, regulating blood flow,
and delivering nutrients to the conceptus (Boal et al., 1997). In early pregnancy, hCG maintains
the corpus luteum for progesterone production. Even in later stages of pregnancy, hCG
continues to have numerous functions that have been recently reviewed by Cole (2010). Briefly,
hCG promotes cellular differentiation in the placenta to make syncytiotrophoblast cells (Cronier
et al., 1994); blocks the maternal immunological response to invading placental cells (Akoum et
al., 2005); promotes uterine and umbilical cord growth (Reshef et al., 1990); and increases blood
flow to the fetoplacental unit by promoting angiogenesis in the uterine vasculature and
maintaining umbilical cord elasticity (Berndt et al., 2009; Herr et al., 2007; Toth et al., 1994).
109
The addition of folate did not alter the transfer of formic acid, however, it did mitigate the
effects on hCG secretion. Since tissue concentrations of formic acid were similar in the presence
or absence of folate, this suggests that folate may mitigate toxicity to the placenta by acting as an
antioxidant to the oxidative stress caused from formic acid as opposed to increasing clearance of
formic acid. Our study shows that formic acid in the mother with heavy drinking during
pregnancy, at concentrations that are obtained through both endogenous production and
methanol found in alcoholic beverages, can be placentotoxic in addition to being neurotoxic as
shown in our previous study (Kapur et al., 2007). Using organotypic rat hippocampal brain slice
cultures, we previously showed that formic acid neurotoxicity is both dose and time-dependent:
cell death increased significantly between 1 to 5 mM formic acid and also between 24 and 72
hours (Kapur et al., 2007). Our current study evaluates the transfer and toxicity at 2 mM formic
acid as this is the maximal concentration observed in heavy drinkers (Kapur et al., 2007).
Studies evaluating fetal effects of formic acid using rodent whole embryo culture have
demonstrated concentration-dependent toxicity (Andrews et al., 1998; Brown-Woodman et al.,
1995; Hansen et al., 2005). After microinjection of sodium formate into the amniotic fluid of
pregnant mice and rats, incomplete fetal axial rotation was observed at concentrations as low as
0.029 mM (Hansen et al., 2005).
A limitation of our results is that we evaluated toxicity at one concentration of formic
acid, 2 mM. Since the placental perfusion model is very tedious, future in vitro studies are
needed to characterize the full dose-response relationship for placental toxicity. Formic acid in
heavy drinkers and in pregnant women has not been characterized in large populations. It is
possible that higher concentrations of formic acid may be obtained as methanol may accumulate
as long as ethanol is present in the blood (Kapur et al., 2007; Majchrowicz & Mendelson, 1971).
To properly characterize formic acid concentrations in pregnant alcohol-consuming women,
studies are needed that capture the full time-concentration curve and these patients will be
difficult to recruit. However, theoretically, we expect formic acid concentrations to reach similar
levels in pregnant women compared to non-pregnant adults. Preliminary results from our group
show formic acid concentrations as high as 140 and 533 µM in maternal blood and umbilical
cord blood, respectively, although these samples were not obtained at times where peak formic
acid concentrations are expected. In non-pregnant adults, a serum formic acid concentration of 2
mM was observed with a blood alcohol concentration of 150 mM (Kapur et al., 2007). This
110
would correspond to consuming over 12 standard drinks on one occasion. Binge drinking at and
exceeding this level has been reported in pregnant women (Kuehn et al., 2012; Malone et al.,
2010), thus it is likely that similar concentrations of formic acid are achieved in this population.
We have recently shown that folate transport to the fetus is compromised in pregnancies
affected by heavy and chronic alcohol exposure (Hutson et al., 2012). Although we did not
focus on FASD, one could speculate that with decreased folate transport there will be an increase
in formic acid which may play a significant role in the etiology of FASD.
6.6 Conclusions
In summary, formic acid at peak concentrations observed in heavy drinkers can rapidly
transfer across the placenta. Formic acid may be harmful to the developing fetus and our results
demonstrate that it is also directly placentotoxic. Formic acid decreases hCG secretion in the
placenta, which may alter steroidogenesis and differentiation of the cytotrophoblasts, and this
can be mitigated by folate.
111
Chapter 7
General Discussion 7Many women require pharmacologic treatment during pregnancy for chronic medical
conditions or illnesses that arise during pregnancy (Koren et al., 1998). Furthermore, many
women may be exposed to known teratogens inadvertently during pregnancy or because of
addiction issues. The benefit of drug treatment must be weighed against any potential risk to the
unborn child as well of risk of the untreated maternal disease. After the thalidomide disaster,
there is a current belief that every drug has the potential to be harmful to the fetus (Koren et al.,
1998) and thus every drug must be carefully evaluated for its safety in pregnancy before it can be
ethically administered to pregnant women. Clinical studies of drug safety in human pregnancy
are often limited because of ethical considerations, thus a validated model or framework for
predicting drug safety is needed. As the placenta is the interface between the maternal and fetal
circulations, a thorough understanding of the role of the placenta in maternal-fetal
pharmacokinetics is vital to understanding drug safety in pregnancy. Animal studies often do not
extrapolate well into humans because the placenta is the most species-specific mammalian organ
(Ala-Kokko et al., 2000). In vitro studies using trophoblast preparations are useful for studying
mechanisms of transfer but do not provide information on transfer in the intact organ, which
includes the often overlooked fetal endothelial cells that form part of the barrier (Elad et al.,
2014). Dual perfusion of a single placental lobule ex vivo is the only experimental model to
study human placental transfer of substances in organized placental tissue. Because of this
feature, it was thought that this experimental model was able to better predict fetal exposure
compared to other in vitro methods such as cell culture, placental explants, or membrane
vesicles. Validation of this placental perfusion model is needed before it can have a place in
clinical and regulatory pharmacology and toxicology. The studies conducted as part of this
doctoral thesis aimed to validate the placental perfusion model as an accurate source of
determining drug transfer across the human placenta. Furthermore, this thesis aimed to
subsequently use the perfusion model in conjunction with clinical studies to provide examples of
how the perfusion model can be used to draw conclusions that are clinically meaningful.
112
The present chapter deals with the significance of the specific research findings outlined
in the preceding chapters, and summarizes these findings in terms of clinical significance,
limitations of the conclusions, and future studies needed in each area.
7.1 Summary of Research Findings and Future Directions
I. To systematically evaluate the placental perfusion model in predicting placental
drug transfer and to develop a pharmacokinetic-model to account for non-
placental pharmacokinetic parameters in the perfusion results (Chapter 2).
A large number of studies have evaluated the transplacental transfer of therapeutic drugs
using the placental perfusion model. However, the accuracy of how well the results from the
perfusion model can predict the in vivo placental transfer in humans was unknown. This was
important to determine in order to better generalize results from perfusion studies into data that
can be used clinically. Knowledge translation is an important part of completing research and
finding ways to use basic science data in a clinically meaningful way. The results of this study
showed that:
• A systematic review of the literature determined that the fetal-to-maternal ratio in both
the perfusion model and in vivo was determined for 70 different drugs. The fetal-to-
maternal drug concentration ratios matched well between placental perfusion experiments
and in vivo samples taken at the time of delivery of the infant.
• The placental perfusion model was best able to predict drugs that have limited transfer
(F:M <0.1) across the term human placenta.
• After modeling for differences in maternal and fetal/neonatal protein binding and blood
pH, the perfusion results were able to accurately predict in vivo transfer at steady state
(R2 = 0.85, P < 0.0001).
• Determining the protein binding in vitro using fetal and maternal blood in parallel with
perfusion experiments, and then modeling the results as documented above, would allow
113
for more accurate predictions for placental drug transfer at steady state compared to
performing perfusion experiments alone.
• After modeling, the placental perfusion model can be used to accurately predict in vivo
placental drug transfer and thus estimate fetal drug exposure. This will allow conclusions
to be reached regarding placental drug transfer for new drugs for which no in vivo fetal
safety data are available.
Our systematic review and model development (Hutson et al., 2011, Chapter 2) determined
that after adjusting for maternal and fetal pharmacokinetic factors, the placental perfusion model
can be used to accurately predict in vivo placental drug transfer and thus estimate fetal drug
exposure. This will allow conclusions to be reached regarding placental drug transfer for new
drugs for which no in vivo fetal safety data are available. It is also the first study to show that
adjusting data from perfusion experiments mathematically for maternal and fetal protein binding
is more accurate than attempting to add protein into the maternal or fetal perfusate during the
experiment. This will prove to be cost effective in addition to improving the generalizability of
the results. Furthermore, drugs known to influence fetal growth or development that were studied
using our model showed good correlation including NSAIDs, antiepileptics, and endocrine
agents. In fact, Shintaku et al. were able to use data from perfusion experiments to predict fetal
toxicity of NSAIDs (detailed below) (Shintaku et al., 2012). Drugs where fetal exposure is
desired, for example antivirals, also showed good agreement between in vivo and perfusion
results.
A limitation of our study was that we compared a small proportion of drugs that have been
studied by the placental perfusion model. Out of 128 therapeutic drugs that have been
investigated by placental perfusion studies, only 26 drugs met inclusion criteria for a quantitative
comparison. Many of these drugs had no in vivo data available (there was no umbilical cord
blood to maternal blood drug concentration ratio available). Since many of these drugs are
considered safe in pregnancy and are commonly given to pregnant women, it may be useful to
measure these concentrations in order to increase the number of drugs compared quantitatively.
We also proposed that measuring protein binding in vitro in umbilical cord blood and maternal
blood would be a useful alternative to measuring in vivo protein binding for new drugs as new
medications can not be ethically administered to pregnant women without safety data. By
114
determining protein binding in vitro together with placental perfusion experiments would be a
useful framework for evaluating placental transfer of new medications that could be utilized by
regulatory agencies.
To apply this framework for the evaluation of new medications, a similar protocol for the
perfusion experiment must be performed by different laboratories. The basic experimental
perfusion model set-up as described in this thesis is performed by most laboratories utilizing this
experimental model, although slight variations do exist (Hutson et al., 2011; Mose et al., 2012).
It is recommended to transfuse a reference compound in all placental perfusions, such as
antipyrine (Mose et al., 2012). Antipyrine is a neutral molecule which undergoes passive
diffusion across the placenta in a flow dependent manner without binding to protein at
physiological pH (Schneider et al., 1972). Normalizing perfusion data of a specific drug to
antipyrine shows good agreement between laboratories who may use slightly different variations
of the placental perfusion setup (Mose et al., 2012). Published results from perfusion
experiments should therefore include enough both original data on the drug being studied as well
as data on antipyrine transfer. Furthermore, calculated normalized data should also be provided.
This will allow for secondary analyses of perfusion data to be performed (Hutson et al., 2011).
Mathiesen et al. (2010) have also proposed criteria to be included in perfusion publications and
have recommended that each laboratory publish a detailed methods paper so that all
experimental parameters can easily be obtained (Mathiesen et al., 2010).
Adjusting results from placental perfusion data for non-placental pharmacokinetic factors,
including protein binding and pH, showed that this experimental model was able to accurately
predict in vivo placental transfer at term. The main limitation of using this experimental model to
assess fetal drug exposure in pregnancy is the conclusions may not be generalizable to earlier
gestational ages. However, at term, drug transfer at term may represent the highest exposure
compared to earlier gestational ages (Vahakangas & Myllynen, 2006) since the placental transfer
layer is the thinnest and expression of certain efflux transporters such as P-glycoprotein is
substantially decreased (Gil et al., 2005; Nanovskaya et al., 2008; Sun et al., 2006; Vahakangas
& Myllynen, 2006). Using data from the perfusion experiments together with other in vitro
experimental methods using human placental tissue from earlier gestational ages, such as
placental explants, membrane vesicles, and primary cell culture, may be useful. For example,
knowing that a specific drug is a substrate for placental BCRP, kinetic parameters could be
115
evaluated from membrane vesicles prepared from early gestational tissue and compared to term
tissue to draw more generalizable conclusions.
Recently, a new in vitro model of placental transport was developed by Levkovitz et al.
(2013a). In customized wells, this model co-cultures human trophoblast cells with human
umbilical vein endolthelial cells on both sides of a denuded amniotic membrane resulting in a
monolayer mimicking the architecture of the human placenta. This is the first co-culture
technique reported to mimic the human placental barrier, however, other biological barriers have
been co-cultured successfully including the blood-brain barrier, the pulmonary blood-gas barrier,
and skin and eye (Dai et al., 2005; Hermanns et al., 2004; Levkovitz et al., 2013; Ma et al.,
2005). This model was shown to have good correlation with in vivo data for the transfer of
glucose across the placenta (Levkovitz et al. 2013b). This in vitro placental model more closely
resembles the in vivo architecture compared to other cellular monolayers that use only
trophoblast cells, however the amniotic membrane used to mimic the basement membrane is
thicker compared to in vivo which may influence transfer. Furthermore, it is unclear what
gestational age this placental model may best represent and whether drug transport proteins are
present consistently in these cellular preparations. Further studies are required to validate this
model, however, it may be an efficient model to for high throughput screening of drugs before
moving to the more tedious model of the placental perfusion. It may be yet another tool that
could be used in conjunction with the perfusion model to provide safety data for new
medications where no safety data in pregnancy is available.
One exciting new area of current research is the modeling of pharmacodynamic data together
with placental pharmacokinetic data. Shintaku et al. used transplacental pharmacokinetic
parameters obtained from placental perfusion studies and pharmacodynamic data obtained from
animal studies to quantitatively predict human fetal toxicity of NSAIDs (Shintaku et al., 2012).
Their model was able to predict the risk of constriction of the ductus arterioiusus and thus predict
fetal toxicity using maternal serum concentrations. Novel approaches, such as our model and the
integrated pharmacokinetic-pharmacodynamic modeling, are expected to be useful tools for
evaluation of drug safety in pregnancy. Developing models utilizing both pharmacodynamics
and pharmacokinetic data will allow better predictions of overall fetal drug safety and should be
validated in future studies. This will require the collaboration of basic science researchers,
clinical pharmacologists, and biomedical engineers to develop these complex models.
116
II. To further evaluate the safety of AZA and 6-MP in pregnancy by systematically
reviewing the literature and by determining the mechanisms by which the
placenta restricts 6-MP transfer (Chapters 3 & 4).
In addition to new medications where no clinical safety data is available, the perfusion model
is also useful to evaluate mechanisms of placental transfer, or lack of, for drugs where there may
still be concern regarding fetal safety. Another objective of this thesis was to utilize the validated
perfusion model in assessing drug safety in pregnancy in conjunction with available clinical
studies. An example of this is the use of thiopurines for IBD in pregnant women. Although there
is evidence to support that the benefit of treatment with thiopurines in IBD patients during
pregnancy likely outweighs any fetal risk, safety concerns still arise as a result of their cytotoxic
mechanism of action and their toxicity during pregnancy in animal studies (Hutson et al., 2013).
Despite the risks of untreated disease in pregnancy, some physicians still choose not to prescribe
AZA during pregnancy or to discontinue treatment in the third trimester (Peyrin-Biroulet et al.,
2011). By using our results from the placental perfusion experiments together with the results of
other pharmacokinetic parameters known about 6-MP and available human studies in pregnancy,
clinically relevant conclusions can be drawn to reassure women about the safety of thiopurines in
pregnancy.
• A meta-analysis was performed of all original human studies reporting outcomes in
pregnancy in patients receiving thiopurines and included nine studies and a total of 494
patients with IBD and 2,782 IBD controls.
• When compared with healthy women, those receiving thiopurines had an increased risk
for congenital malformations (RR 1.45; 95% CI 1.07–1.96; p = 0.02); however, when
compared with IBD controls, there was no increased risk (RR 1.37; 95% CI 0.92–2.05; p
= 0.1). These data provide support for thiopurines having a minimal risk, if any, to the
fetus.
• Patients with IBD receiving thiopurines were more likely to have premature infants (<37
weeks GA) compared with IBD controls (RR 1.67; 95% CI 1.17–2.37; p = 0.004) but this
may be related to disease activity. The risk ratio for LBW (<2,500 g) was not significant
when comparing women with IBD receiving thiopurines and all IBD controls (p = 0.13).
117
• In the dually perfused placental perfusion model, with 6MP being added to the maternal
circulation, there was a biphasic decline in its concentration and a delay in fetal
circulation appearance suggesting uptake and retention of 6MP by the placenta.
• In the dually perfused placental perfusion model, with equal concentrations of 6MP in
both the maternal and fetal circulations, the fetal-to-maternal concentration ratio was >1.0
as a result of ion trapping.
• In the dually perfused placental perfusion model, metabolites of 6MP were not found in
the fetal circulation at physiologic concentrations.
• Binding to placental tissue and maternal pharmacokinetic parameters are the main factors
that restrict placental transfer of 6-MP. Active transport is unlikely to play a significant
role and drug interactions involving, or polymorphisms in, placental drug efflux
transporters are not likely to put the fetus at risk of higher 6-MP exposure.
Our data from the meta-analysis and the perfusion experiments support the safety of using
azathioprine or 6-MP in pregnancy as the benefit of treating the disease likely outweighs the risk
of maintaining treatment during the pregnancy; however, this should be individualized to each
patient. Using the dually perfused placental perfusion model, we showed that binding to
placental tissue and maternal pharmacokinetic parameters are the main factors that restrict
placental transfer of 6-MP. Furthermore, we concluded that active transport is unlikely to play a
significant role and drug interactions involving, or polymorphisms in, placental drug efflux
transporters are not likely to put the fetus at risk of higher 6-MP exposure. This finding was not
in agreement with our hypothesis that BCRP would efflux 6-MP into the maternal circulation. It
is likely that placental expression of BCRP in normal physiological conditions does not
contribute significantly to the efflux of 6-MP and that other mechanisms of transfer are important
in achieving steady state concentrations. 6-MP was determined to be a substrate using an in vitro
model where MDCKII and HEK293 cells were transfected with human or mouse ABCG2 (de
Wolf et al., 2008). These cell lines consequently overexpressed BCRP and thus it is difficult to
extrapolate whether BCRP would play a rate-limiting role in placental drug transport in vivo.
Since expression of BCRP does not change throughout gestation (discussed in Appendix C), it is
unlikely that it would alter steady-state fetal to maternal concentrations in earlier gestations.
118
In addition to placental perfusion data and in vivo measurements of 6-MP fetal transfer,
several observational studies have been published regarding the safety of thiopurines in
pregnancy. Randomized controlled drug studies are usually not available because they most
always exclude pregnant women owing to fear of teratogenic risk. As a result, epidemiological
studies based on observational data constitute the main clinical data source (Schaefer et al.,
2008). Often, reporting of observational research is not sufficiently detailed to accurately assess
the potential strengths and weaknesses of these investigations (Etwel et al., 2014). Multiple
deficiencies such as heterogeneity, methodological quality, insufficient statistical methods, and
control of potential sources of bias have been widely recognized (Maguire et al., 2008;
Simunovic et al., 2009). The synthesis of these observational cohort studies into a systematic
review and meta-analysis yields an important signal for the safety/risk of drug use in pregnancy.
In fact, a recent systematic review determined that meta-analyses of earlier, albeit smaller, cohort
studies tend to generate an accurate overall teratogenic signal in estimating human teratogenicity
years before large and methodologically superior cohort studies are published (Etwel et al.,
2014).
We completed a systematic review of all original human observational studies reporting
outcomes in pregnancy in patients receiving thiopurines and included nine studies and a total of
494 patients with IBD and 2,782 IBD controls. When comparing women receiving thiopurines
for IBD compared to women with IBD not receiving thiopurines, there was no increased risk for
congenital malformations or LBW (<2,500 g). However, patients with IBD receiving thiopurines
were more likely to have premature infants (<37 weeks GA) but this may be related to disease
activity. A similar meta-analysis reporting on the safety of azathioprine and 6-MP was recently
published alongside our results (Akbari et al., 2013). They included only five studies (Cleary &
Kallen, 2009; Coelho et al., 2011; Francella et al., 2003; Norgard et al., 2007a; Shim et al., 2011)
and found results similar to ours: women with IBD exposed to thiopurines compared to IBD-
controls not receiving thiopurines, the pooled ORs for LBW, preterm birth, and congenital
abnormalities were 1.01 (95% CI 0.96, 1.06), 1.67 (95% CI 1.26, 2.20), and 1.45 (95% CI 0.99,
2.13), respectively. Akbari et al. (2013) limited their analysis to include only studies with an IBD
control group. However, compared to our analysis of studies with IBD control group
comparison, they did not include an article in French (Tennenbaum et al., 1999) and an abstract
(Dejaco et al., 2005). Results from our meta-analysis and that by Akbari et al. (2013) emphasize
119
the importance of study design when evaluating the safety of medications in pregnancy. An
increased risk for malformations was observed when thiopurine-exposed pregnancies were
compared to the general/healthy population, however, this association disappeared when a more
appropriate control group was used (IBD controls). As discussed in Chapter 4, disease activity
may be responsible for any increased fetal risk observed and thus it is important to have disease-
matched controls. Specifically for IBD, medication use during pregnancy has been suggested to
be a surrogate for disease activity during pregnancy and IBD flares during pregnancy are
associated with adverse pregnancy outcomes such as preterm birth and LBW (Norgard et al.,
2007b; Baiocco & Korelitz, 1984; Bush et al., 2004; Dominitz et al., 2002; Fedorkow et al.,
1989; Morales et al., 2000; Reddy et al., 2008; Woolfson et al., 1990).
A limitation of our meta-analysis was that there was not enough data provided by the studies
to subsequently look at the risk of cardiac malformations in thiopurine-exposed infants. Cleary
and Källén (2009) suggested an increased risk for ASD/VSD after fetal exposure to thiopurines;
however, three additional studies had no cases in both their exposed and control groups (Coelho
et al., 2011; Dejaco et al., 2005; Francella et al., 2003). Cleary and Källén (2009) used
retrospective study design using data from a national health registry. Since women with IBD are
considered high-risk (Cornish et al., 2007) and both women and physicians still perceive
thiopurines in pregnancy as unsafe (Mountifield et al., 2010; Peyrin-Biroulet et al., 2011), it is
likely that more postnatal tests and monitoring may be performed in order to ease any related
anxiety. Performing more postnatal tests may lead to an overestimate of the risk from
confounding by indication; a similar phenomena has been observed for the selective serotonin
reuptake inhibitors (SSRIs) in pregnancy (Jimenez-Solem et al., 2012). An increased risk for
VSD was originally associated with fetal SSRI exposure (Reis & Kallen, 2010); however, it was
also shown that in the first year of life, approximately twice as many echocardiograms were
performed in SSRI-exposed infants compared with infants of unexposed women (Bar-Oz et al.,
2007). This diagnostic bias may lead to more VSDs being detected since it is a congenital
malformation that often resolves on its own and may go undetected in otherwise healthy infants.
A large recent study was able to show that, in fact, the observed association with VSD and SSRIs
was a result of confounding by indication (Jimenez-Solem et al., 2012).
Since completion of our meta-analysis, three additional studies have recently been published.
First, a population-based study from the United Kingdom reviewed medical records of 1703
120
children born to mothers with IBD and 384,811 children of mothers without IBD (Ban et al.,
2014). Maternal exposure to azathioprine or 6MP was identified if a woman received a
prescription for these medications during the first trimester. Using logistic regression, the
adjusted odds ratios of a major congenital anomaly associated with azathioprine or 6MP
exposure was 1.27 (95% CI, 0.48–3.39). In early pregnancy, 24.6% of women did not receive
further prescriptions in pregnancy and were considered to have discontinued azathioprine/6MP
in pregnancy. There was no evident association between discontinuing these medications and
flares later in pregnancy, however, this study had limited power. Second, a retrospective,
multicenter study in IBD patients compared 187 thiopurine-exposed pregnancies compared to
318 non-exposed pregnancies (Casanova et al., 2013). The rate of unfavourable global pregnancy
outcome (those who developed obstetric complications including LBW, preterm labour,
spontaneous abortion, congenital malformations, or required ICU admission) was lower in the
thiopurine exposed pregnancies compared to the non-exposed controls (21.9% vs 31.8%,
p=0.01). Third, a prospective, multicenter study of children born to mothers with IBD assessed
the long-term effects of in utero thiopurine exposure (de Meij et al., 2013). Compared to a
control group (n=340), those children exposed to thiopurines (n=30) had no increased
susceptibility to infection or immunodeficiency and no difference in global medical and
psychosocial health status up to six years of age. Together, these studies alongside our results
support continuing AZA/6-MP therapy during pregnancy for IBD as the benefit of the treatment
outweighs the risk of untreated maternal disease (Ban et al., 2014).
A treatment algorithm for IBD in pregnancy has been recently proposed and recommends
AZA/6-MP as a safe pharmacologic choice in pregnancy (Habal & Huang, 2012). Considering
that maternal pharmacokinetic factors have the largest influence on fetal exposure to thiopurines,
our findings support the recommendations by other groups that maternal drug monitoring of
thiopurine levels may lead to avoidance of high levels of fetal drug exposure (de Boer et al.,
2006; Jharap et al., 2014). The effect of pregnancy on the pharmacokinetics of 6-MP has only
recently been studied. In a small cohort of 30 pregnant women with IBD receiving AZA or 6-MP
determined that 6-TGN levels may decrease throughout pregnancy but return to baseline after
delivery (Jharap et al., 2014). Future studies should determine whether there is a role for
therapeutic drug monitoring of 6-TGN levels in pregnancy or the role of monitoring maternal
CBC and liver enzymes (Jharap et al., 2014; Katz, 2013). Detecting any changes in maternal
121
CBC or liver enzymes may be a suitable surrogate marker for potential elevated 6-TGN levels as
toxic changes would start to be observed. Furthermore, monitoring maternal CBC and liver
enzymes would be available at any hospital laboratory and would not require specialized
equipment as would be required for therapeutic drug monitoring of 6-TGN.
III. To evaluate the effect of alcohol on folic acid transfer across the human placenta
and the potential toxicity of formic acid to the placenta (Chapters 5 & 6).
Compared to the study of therapeutic drug safety in pregnancy, similar experimental
approaches involving the placental perfusion model together with clinical studies can be utilized
to study known teratogens or toxins, such as alcohol. The placental perfusion model was
previously used to elucidate the mechanisms of folate transport across the placenta (Henderson et
al., 1995). Animal and in vitro studies were used to evaluate the mechanisms by which alcohol
can influence expression of folate transport proteins (Fisher et al., 1985; Keating et al., 2008;
Keating et al., 2009). Our cohort study comparing maternal blood and cord blood folate levels in
vivo demonstrated that chronic and heavy alcohol use in pregnancy impairs folate transport to
the fetus. When used together, these techniques are complimentary and allow us to better
understand how alcohol can lead to changes in folate transport across the placenta.
• Serum folate was measured in maternal blood and umbilical cord blood at the time of
delivery in pregnancies with chronic and heavy alcohol exposure (n=23) and in non-
drinking controls (n=24). In the alcohol-exposed pairs, the fetal:maternal serum folate
ratio was <1.0 in over half (n = 14), whereas all but one of the controls were >1.0. Mean
folate in cord samples was lower in the alcohol-exposed group than in the controls (33.15
± 19.89 vs 45.91 ± 20.73, p = 0.04) demonstrating that chronic and heavy alcohol use in
pregnancy impairs folate transport to the fetus.
• In the dually perfused placental perfusion model, formic acid at clinically relevant
concentrations transferred rapidly from the maternal to the fetal circulation, and transfer
was not altered with the addition of folate. The addition of folate did not alter fetal
exposure of formic acid originating from the maternal circulation, as measured by AUC
(1.30 ± 0.14 without folate; 1.23 ± 0.48 with folate; P = 0.79), or tissue concentration of
122
formic acid as measured in the perfused lobules (425.83 ± 57.18 and 431.18 ± 133.07
nmol/g for perfusions without and with folate added, respectively).
• In the dually perfused placental perfusion model, there was a significant decrease in hCG
secretion (p = 0.03) after addition of formic acid and this decrease was mitigated by the
addition of folic acid
• Formic acid decreases hCG secretion in the placenta, which may alter steroidogenesis
and differentiation of the cytotrophoblasts, and this adverse effect can be mitigated by
folate.
Our results from a small cohort of heavy drinking women showed that the majority of infants
have decreased serum folate concentrations compared to the maternal concentrations. Since it is
well known that the placenta concentrates folate into the fetal circulation and fetal concentrations
are normally 2- to 4-fold higher than maternal (Economides et al., 1992; Giugliani et al., 1985;
Guerra-Shinohara et al., 2004; Malloy et al., 2005; Navarro et al., 1984; Obeid et al., 2005;
Relton et al., 2005; Stark et al., 2007; Thorand et al., 1996), our data demonstrates that alcohol
alters normal placental folate transfer to the fetus. We hypothesize that the mechanism is
decreased expression of folate transport proteins in the placenta as this is observed in animal and
in vitro models after alcohol exposure as discussed in Chapter 5. Our results were the first to
show this finding in human pregnancies and were in agreement with our original hypothesis that
heavy alcohol exposure will decrease folate transport to the fetus. However, we were unable to
obtain the placentas from the deliveries in our cohort to look at protein expression of PFRs in
order to investigate the mechanism. Since many women who drink heavily in pregnancy receive
less prenatal care and many women do not admit to drinking in pregnancy because of the
associated social stigma, this is a difficult population to recruit and this was our experience in
our attempt to further this study. Furthermore, women can deliver at any time of the day or night
and at any institution and the placenta is often sent to pathology and is thus unavailable for
research purposes. Therefore, it is logistically difficult to collect tissue samples from this patient
population. A large collaborative effort with multiple institutions will be needed to recruit an
adequate number of human placentae from pregnancies with heavy alcohol exposure. If placental
samples are available, folate binding to FR-α can be determined by preparation of microvillous
membranes and binding can be measured by incubating the membranes with [3H]-folic acid after
123
an initial wash-out of endogenous folate using the method by Bisseling et al. (2004). Binding
characteristics should be compared to control placentae from births to women who do not use
alcohol during pregnancy. In addition to measuring binding, the amount of FR-α in the
microvillous membranes should be quantified using western blot. The amount of RFC in the
basolateral membrane vesicles should also be determined by western blot. Amounts of FR-α and
RFC should also be compared to control placentae.
The results of altering folic acid levels both within the placenta and to the fetus may have
many consequences since folic acid is critically important for DNA synthesis and cell
proliferation as well as in cellular protection through its antioxidant properties (Antony, 2007).
Our results showed that total folate concentrations are decreased in fetal umbilical cord blood in
those born to mothers drinking heavily compared to controls. The decrease in mean fetal folate
concentrations in the exposed group was 12 nM compared to controls. It is unknown whether this
decreased concentration is ultimately clinically significant on its effect on fetal development.
Lower folate status has been associated with hyperactivity, peer problems, and lower cognitive
function (Schlotz et al., 2010; Veena et al., 2010) but folate status in these studies was
determined by maternal folate concentrations and thus are not directly comparable.
Folate deficiency within the placenta has also been shown to alter placental DNA
methylation (Kim et al., 2009). Fetal growth and development are closely linked to DNA
methylation and disruption in placental and fetal DNA methylation may have a role in the
Developmental Origins of Adult Health and Disease hypothesis (Gallou-Kabani et al., 2010).
Alterations in the placenta have already been associated with the future risk of cardiovascular
disease and cancer (Gallou-Kabani et al., 2010). In fact, a recent study reported long-lasting
alterations in DNA methylation as a result of fetal alcohol exposure in a mouse model (Laufer et
al., 2013). Genomic imprinting by DNA methylation acts only on a select set of genes that are
important in early development, particularly neurodevelopment (Hager & Johnstone, 2003;
Laufer et al., 2013). Interestingly, in addition to folic acid supplementation in animal models,
treatments that contain other methyl group donors such as choline have been able to attenuate
some of the effects of alcohol on the fetus (Cano et al., 2001; Garcia-Rodriguez et al., 2003;
Wang et al., 2009; Wentzel & Eriksson, 2008; Xu et al., 2008; Yanaguita et al., 2008; Zeisel,
2011). Future studies should aim to elucidate the influence of altered folate transport both on
124
placental and fetal epigenetic patterns as this may shed light on the mechanism of alcohol
teratogenicity.
Folate is routinely given to pregnant women during pregnancy. High dose folate
supplementation (5mg/day) has been recommended by the Society of Obstetricians and
Gynaecologists of Canada to women with recreational drug use (including alcohol) because of
concerns surrounding adequate diet and compliance with prenatal vitamins (Wilson et al., 2007).
Since our results demonstrate that folate transport is decreased to the fetus after heavy alcohol
exposure, it would be beneficial to determine the optimal dose of folate supplementation that
may prevent any associated fetal effects. Animal studies have suggested that high dose folate
supplementation may mitigate adverse fetal effects induced by alcohol as discussed in Chapter 5
and thus it may be possible to overcome the decreased placental transfer. This would require a
large-scale clinical study to evaluate with adequate power to control for the numerous
confounding variables such as diet, socioeconomic status, other drug use, etc. This study would
also only be useful if the fetal effects directly resulting from alcohol altering placental transport
and binding are known as discussed above.
In addition to the direct toxic effects of alcohol on the placenta and fetus, our studies also
evaluated the role that formic acid, the toxic metabolite of methanol, may have in the
development of FASDs. Formic acid has recently been detected in maternal and cord blood in
pregnancies with chronic ethanol exposure (Kapur et al., 2009). Using the dually perfused
placental perfusion model, we determined that formic acid transferred rapidly from the maternal
to the fetal circulation, and transfer was not altered with the addition of folate. This was in
disagreement with our hypothesis. We hypothesized that folate would decrease transfer of formic
acid as it is known that folate can influence the rate of clearance of formic acid (Johlin et al.,
1989; Sokoro et al., 2008). This could be a limitation of the perfusion model as it cannot account
for potential changes on formic acid clearance after chronic exposure and accumulation of folate
reserves in the placenta (Henderson et al., 1995). Because the transfer of formic acid across the
placenta at clinically relevant concentrations is rapid, the developing fetus will be exposed to
formic acid from the maternal circulation. Since the perfusion model accurately predicts drug
transfer in term human placentas as determined in Chapter 2, it is likely that the fetus will rapidly
be exposed to formic acid from a heavy drinking mother.
125
We also determined the direct toxic effects of formic acid to the placenta during the placental
perfusion. Our results showed that there was a significant decrease in placental hCG secretion
after addition of formic acid and this decrease was mitigated by the addition of folic acid. This
was in agreement with our hypothesis. Alterations in hCG secretion by the placenta may have
consequential implications clinically since hCG is vitally important in maintaining the
pregnancy, regulating blood flow, and delivering nutrients to the conceptus (Boal et al., 1997).
The mechanism by which formic acid decreases hCG secretion should be determined in future
studies in addition to whether chronic exposure to methanol or formic acid has adverse placental
effects. The later will have to be completed in animal or in vitro studies since it is unethical to
study in humans.
Our research is the first to propose a role of methanol and formic acid in the development of
FASDs. The involvement of methanol in the toxic effect of alcoholic beverages is a novel
hypothesis proposed by Kapur et al. (2009). The proposed interaction of how alcohol and
methanol could lead to formic acid toxicity and also decreased placental folate is outlined in
Figure 7.1. Further studies are needed to fully understand how the toxicity of methanol/formic
acid plays a role together in the FASDs. Here, there is an important role for animal and in vitro
studies in addition to the validated placental perfusion model as it is difficult to recruit pregnant
women with chronic and heavy alcohol consumption.
126
Figure 7-1 The interaction between alcohol use in pregnancy and the proposed mechanisms
of toxicity by reduced placental folate transfer to the fetus and by formic acid.
127
7.2 Summary
In summary, our studies illustrate the benefits of using an integrative approach in the
desire to understand the safety of therapeutics or the toxicity of xenobiotics in pregnancy. When
used together, techniques including animal, in vitro, placental perfusion ex vivo, and clinical
studies can capture the dynamic nature of pregnancy and we can make generalized conclusions
and recommendations. Furthermore, meta-analyses offer clinicians, scientists, and regulators an
earlier signal for the presence or lack of teratogenic risk and hence can have an important impact
on clinical practice (Etwel et al., 2014). Since it is often unethical to study drug safety in
pregnant women, theoretical frameworks are needed on which to build the study of drugs in
human pregnancy. Our validation of the ex vivo perfusion model showed that it is effective in
predicting fetal exposure to drugs and should have a place in clinical and regulatory
pharmacology and toxicology. We then utilized the placental perfusion model in an integrative
approach to understand the safety of the therapeutic, 6-MP, and the toxicity of alcohol/methanol
in pregnancy. Our results from the placental perfusion model demonstrated that binding to
placental tissue and maternal pharmacokinetic parameters are the main factors that restrict
placental transfer of 6-MP. Active transport is unlikely to play a significant role and drug
interactions involving, or polymorphisms in, placental drug efflux transporters are not likely to
put the fetus at risk of higher 6-MP exposure. A meta-analysis showed that 6-MP does not
increase the risk of congential malformation when used during pregnancy and the benefit of
treating a woman with 6-MP and maintaining disease remission without the use of steroids
would outweigh any risk. Together, these findings support the safety of 6-MP in pregnancy and
woman and physicians can be reassured about its use in pregnancy. Using the validated perfusion
model, we also determined that formic acid transfers across the placenta rapidly and this transfer
is not influenced by folate. Pregnant women with heavy alcohol use are thus likely to exposure
the fetus to toxic levels of formic acid since we used clinically relevant concentrations of formic
acid that is found in heavy alcohol users. Formic acid was also found to be toxic to the placenta
at clinically relevant concentrations. Furthermore, we showed that placental transfer of folate is
decreased after heavy and chronic alcohol exposure. Since folate is required for formic
detoxification of formic acid, this may be a potential mechanism for the development of fetal
alcohol spectrum disorders.
128
References 8
Abel EL. (1998). Fetal alcohol syndrome: the 'American Paradox'. Alcohol Alcohol 33 (3), 195-
201.
Abel EL, & Sokol RJ. (1986). Fetal alcohol syndrome is now leading cause of mental
retardation. Lancet 2 (8517), 1222.
Akbari M, Shah S, Velayos FS, Mahadevan U, & Cheifetz AS. (2013). Systematic review and
meta-analysis on the effects of thiopurines on birth outcomes from female and male
patients with inflammatory bowel disease. Inflamm Bowel Dis 19 (1), 15-22.
Akoum A, Metz CN, & Morin M. (2005). Marked increase in macrophage migration inhibitory
factor synthesis and secretion in human endometrial cells in response to human chorionic
gonadotropin hormone. J Clin Endocrinol Metab 90 (5), 2904-2910.
Ala-Kokko TI, Myllynen P, & Vahakangas K. (2000). Ex vivo perfusion of the human placental
cotyledon: Implications for anesthetic pharmacology. Int J Obstet Anesth 9 (1), 26-38.
Ala-Kokko TI, Alahuhta S, Jouppila P, Korpi K, Westerling P, & Vahakangas K. (1997). Feto-
maternal distribution of ropivacaine and bupivacaine after epidural administration for
cesarean section. Int J Obstet Anesth 6 (3), 147-152.
Ala-Kokko TI, Pienimaki P, Herva R, Hollmen AI, Pelkonen O, & Vahakangas K. (1995).
Transfer of lidocaine and bupivacaine across the isolated perfused human placenta.
Pharmacol Toxicol 77 (2), 142-148.
Alati R, Al Mamun A, Williams GM, O'Callaghan M, Najman JM, & Bor W. (2006). In utero
alcohol exposure and prediction of alcohol disorders in early adulthood: a birth cohort
study. Arch Gen Psychiatry 63 (9), 1009-1016.
Alstead EM, Ritchie JK, Lennard-Jones JE, Farthing MJ, & Clark ML. (1990). Safety of
azathioprine in pregnancy in inflammatory bowel disease. Gastroenterology 99 (2), 443-
446.
129
Anderson GD. (2005). Pregnancy-induced changes in pharmacokinetics: a mechanistic-based
approach. Clin Pharmacokinet 44 (10), 989-1008.
Andrews JE, Ebron-Mccoy M, Schmid JE, & Svendsgaard D. (1998). Effects of combinations
of methanol and formic acid on rat embryos in culture. Teratology 58 (2), 54-61.
Angelberger S, Reinisch W, Messerschmidt A, Miehsler W, Novacek G, Vogelsang H et al.
(2011). Long-term follow-up of babies exposed to azathioprine in utero and via
breastfeeding. J Crohns Colitis 5 (2), 95-100.
Antony AC. (2007). In utero physiology: role of folic acid in nutrient delivery and fetal
development. Am J Clin Nutr 85 (2), 598S-603S.
Ardizzone S, Maconi G, Russo A, Imbesi V, Colombo E, & Bianchi Porro G. (2006).
Randomised controlled trial of azathioprine and 5-aminosalicylic acid for treatment of
steroid dependent ulcerative colitis. Gut 55 (1), 47-53.
Atkinson DE, Greenwood SL, Sibley CP, Glazier JD, & Fairbairn LJ. (2003). Role of MDR1
and MRP1 in trophoblast cells, elucidated using retroviral gene transfer. Am J Physiol Cell
Physiol 285 (3), C584-91.
Audus KL. (1999). Controlling drug delivery across the placenta. Eur J Pharm Sci 8 (3), 161-
165.
Augustowska K, Magnowska Z, Kapiszewska M, & Gregoraszczuk EL. (2007). Is the natural
PCDD/PCDF mixture toxic for human placental JEG-3 cell line? The action of the
toxicants on hormonal profile, CYP1A1 activity, DNA damage and cell apoptosis. Hum
Exp Toxicol 26 (5), 407-417.
Axelrod J, & Daly J. (1965). Pituitary gland: enzymic formation of methanol from S-
adenosylmethionine. Science 150 (698), 892-893.
Baiocco PJ, & Korelitz BI. (1984). The influence of inflammatory bowel disease and its
treatment on pregnancy and fetal outcome. J Clin Gastroenterol 6 (3), 211-216.
130
Baird DD, Narendranathan M, & Sandler RS. (1990). Increased risk of preterm birth for women
with inflammatory bowel disease. Gastroenterology 99 (4), 987-994.
Ban L, Tata LJ, Fiaschi L, & Card T. (2014). Limited Risks of Major Congenital Anomalies in
Children of Mothers With IBD and Effects of Medications. Gastroenterology 146 (1), 76-
84.
Barak AJ, Beckenhauer HC, Tuma DJ. (1985) Ethanol feeding inhibits the activity of hepatic
N5-methyltetrahydrofolate-homocysteine methyltransferase in the rat. IRCS Medical
Science 13 (8), 760-761.
Bar-Oz B, Einarson T, Einarson A, Boskovic R, O'Brien L, Malm H et al. (2007). Paroxetine and
congenital malformations: meta-Analysis and consideration of potential confounding
factors. Clin Ther 29 (5), 918-926.
Bassily M, Ghabrial H, Smallwood RA, & Morgan DJ. (1995). Determinants of placental drug
transfer: Studies in the isolated perfused human placenta. J Pharm Sci 84 (9), 1054-1060.
Belpaire FM, Wynant P, Van Trappen P, Dhont M, Verstraete A, & Bogaert MG. (1995).
Protein binding of propranolol and verapamil enantiomers in maternal and foetal serum. Br
J Clin Pharmacol 39 (2), 190-193.
Bergan S, Rugstad HE, Bentdal O, Endresen L, & Stokke O. (1994). Kinetics of mercaptopurine
and thioguanine nucleotides in renal transplant recipients during azathioprine treatment.
Ther Drug Monit 16 (1), 13-20.
Berndt S, Blacher S, Perrier d'Hauterive S, Thiry M, Tsampalas M, Cruz A et al. (2009).
Chorionic gonadotropin stimulation of angiogenesis and pericyte recruitment. J Clin
Endocrinol Metab 94 (11), 4567-4574.
Biehl D, Shnider SM, Levinson G, & Callender K. (1978). Placental transfer of lidocaine:
effects of fetal acidosis. Anesthesiology 48 (6), 409-412.
Bisseling TM, Steegers EA, van den Heuvel JJ, Siero HL, van de Water FM, Walker AJ et al.
(2004). Placental folate transport and binding are not impaired in pregnancies complicated
by fetal growth restriction. Placenta 25 (6), 588-593.
131
Boal JH, Plessinger MA, van den Reydt C, & Miller RK. (1997). Pharmacokinetic and toxicity
studies of AZT (zidovudine) following perfusion of human term placenta for 14 hours.
Toxicol Appl Pharmacol 143 (1), 13-21.
Bortoli A, Saibeni S, Tatarella M, Prada A, Beretta L, Rivolta R et al. (2007). Pregnancy before
and after the diagnosis of inflammatory bowel diseases: retrospective case-control study. J
Gastroenterol Hepatol 22 (4), 542-549.
Bosco C, & Diaz E. (2012). Placental hypoxia and foetal development versus alcohol exposure
in pregnancy. Alcohol Alcohol 47 (2), 109-117.
Bragonier JR, Roesky N, Carver MJ. (1964) Teratogenesis: Effects of substituted purines and the
influence of 4-hydroxypyrazolopyrimi- dine in the rat. Proc Soc Exp Biol Med 116, 685–
688.
Brien JF, Loomis CW, Tranmer J, & McGrath M. (1983). Disposition of ethanol in human
maternal venous blood and amniotic fluid. Am J Obstet Gynecol 146 (2), 181-186.
Brown-Woodman PD, Huq F, Hayes L, Herlihy C, Picker K, & Webster WS. (1995). In vitro
assessment of the effect of methanol and the metabolite, formic acid, on embryonic
development of the rat. Teratology 52 (4), 233-243.
Burd L, Roberts D, Olson M, & Odendaal H. (2007). Ethanol and the placenta: A review. J
Matern Fetal Neonatal Med 20 (5), 361-375.
Burney RG, DiFazio CA, & Foster JA. (1978). Effects of pH on protein binding of lidocaine.
Anesth Analg 57 (4), 478-480.
Bush MC, Patel S, Lapinski RH, & Stone JL. (2004). Perinatal outcomes in inflammatory bowel
disease. J Matern Fetal Neonatal Med 15 (4), 237-241.
Butt P, Beirness D, Gliksman L, Paradis C, and Stockwell T. (2011). Alcohol and health in
Canada: A summary of evidence and guidelines for low risk drinking (Ottawa, ON:
Canadian Centre on Substance Abuse).
132
Cabezon C, De la Fuente F, Jurado M, & Lopez G. (1985). Histometry of the placental structures
involved in the respiratory interchange. Acta Obstet Gynecol Scand 64 (5), 411-416.
Campbell S, & Ghosh S. (2000). Safety of azathioprine use in pregnancy. Gut 46, A13.
Cano MJ, Ayala A, Murillo ML, & Carreras O. (2001). Protective effect of folic acid against
oxidative stress produced in 21-day postpartum rats by maternal-ethanol chronic
consumption during pregnancy and lactation period. Free Radic Res 34 (1), 1-8.
Casanova MJ, Chaparro M, Domenech E, Barreiro-de Acosta M, Bermejo F, Iglesias E et al.
(2013). Safety of thiopurines and anti-TNF-alpha drugs during pregnancy in patients with
inflammatory bowel disease. Am J Gastroenterol 108 (3), 433-440.
Ceckova-Novotna M, Pavek P, & Staud F. (2006). P-glycoprotein in the placenta: expression,
localization, regulation and function. Reprod Toxicol 22 (3), 400-410.
Centers for Disease Control and Prevention (2012). Frequently Asked Questions - Alcohol.
Available at: http://www.cdc.gov/alcohol/faqs.htm#standDrink (accessed 2013 Sept. 6)
Chan GL, Canafax DM, & Johnson CA. (1987). The therapeutic use of azathioprine in renal
transplantation. Pharmacotherapy 7 (5), 165-177.
Chance B. (1950) On the Reaction of Catalase Peroxide with Acceptors. J Biol Chem 182, 649-
658.
Chappuy H, Treluyer JM, Jullien V, Dimet J, Rey E, Fouche M et al. (2004). Maternal-fetal
transfer and amniotic fluid accumulation of nucleoside analogue reverse transcriptase
inhibitors in human immunodeficiency virus-infected pregnant women. Antimicrob Agents
Chemother 48 (11), 4332-4336.
Charles B, Norris R, Xiao X, & Hague W. (2006). Population pharmacokinetics of metformin in
late pregnancy. Ther Drug Monit 28 (1), 67-72.
Chaub S, Murphy ML. (1968) The teratogenic effects of the recent drugs active in cancer
chemotherapy. Adv Teratol 3, 181–237.
133
Chiao F, Stokstad EL. (1977) Effect of methionine on the metabolism of formate and histidine
by rats fed folate/vitamin B-12-methionine-deficient diet. Biochimica et Biophysica Acta
497 (1), 225-33.
Chiloiro M, Darconza G, Piccioli E, De Carne M, Clemente C, & Riezzo G. (2001). Gastric
emptying and orocecal transit time in pregnancy. J Gastroenterol 36 (8), 538-543.
Chudley AE, Conry J, Cook JL, Loock C, Rosales T, & LeBlanc N. (2005). Fetal alcohol
spectrum disorder: Canadian guidelines for diagnosis. CMAJ 172 (5 Suppl), S1-S21.
Cleary BJ, & Kallen B. (2009). Early pregnancy azathioprine use and pregnancy outcomes.
Birth Defects Res A Clin Mol Teratol 85 (7), 647-654.
Coelho J, Beaugerie L, Colombel JF, Hebuterne X, Lerebours E, Lemann M et al. (2011).
Pregnancy outcome in patients with inflammatory bowel disease treated with thiopurines:
cohort from the CESAME Study. Gut 60 (2), 198-203.
Cole LA. (2010). Biological functions of hCG and hCG-related molecules. Reprod Biol
Endocrinol 8, 102.
Cooper GS, & Stroehla BC. (2003). The epidemiology of autoimmune diseases. Autoimmun Rev
2 (3), 119-125.
Cornish J, Tan E, Teare J, Teoh TG, Rai R, Clark SK et al. (2007). A meta-analysis on the
influence of inflammatory bowel disease on pregnancy. Gut 56 (6), 830-837.
Cronier L, Bastide B, Herve JC, Deleze J, & Malassine A. (1994). Gap junctional
communication during human trophoblast differentiation: influence of human chorionic
gonadotropin. Endocrinology 135 (1), 402-408.
Dai NT, Yeh MK, Liu DD, Adams EF, Chiang CH, Yen CY et al. (2005). A co-cultured skin
model based on cell support membranes. Biochem Biophys Res Commun 329 (3), 905-908.
Datta S, Camann W, Bader A, & VanderBurgh L. (1995). Clinical effects and maternal and fetal
plasma concentrations of epidural ropivacaine versus bupivacaine for cesarean section.
Anesthesiology 82 (6), 1346-1352.
134
Davis AR, Miller L, Tamimi H, & Gown A. (1999). Methotrexate compared with
mercaptopurine for early induced abortion. Obstet Gynecol 93 (6), 904-909.
Davison JM, & Hytten FE. (1974). Glomerular filtration during and after pregnancy. J Obstet
Gynaecol Br Commonw 81 (8), 588-595.
De Barros Duarte L, Moises EC, Cavalli RC, Lanchote VL, Duarte G, & Da Cunha SP. (2010).
Distribution of Bupivacaine Enantiomers and Lidocaine and its Metabolite in the Placental
Intervillous Space and in the Different Maternal and Fetal Compartments in Term Pregnant
Women. J Clin Pharmacol 51 (2), 212-217.
de Boer NK, Jarbandhan SV, de Graaf P, Mulder CJ, van Elburg RM, & van Bodegraven AA.
(2006). Azathioprine use during pregnancy: unexpected intrauterine exposure to
metabolites. Am J Gastroenterol 101 (6), 1390-1392.
de Boer NK, Van Elburg RM, Wilhelm AJ, Remmink AJ, Van Vugt JM, Mulder CJ et al.
(2005). 6-Thioguanine for Crohn's disease during pregnancy: thiopurine metabolite
measurements in both mother and child. Scand J Gastroenterol 40 (11), 1374-1377.
de Graaf P, Vos RM, de Boer NH, Sinjewel A, Jharap B, Mulder CJ et al. (2010). Limited
stability of thiopurine metabolites in blood samples: Relevant in research and clinical
practise. J Chromatogr B Analyt Technol Biomed Life Sci 878 (19), 1437-1442.
de Meij TG, Jharap B, Kneepkens CM, van Bodegraven AA, & de Boer NK. (2013). Long-term
follow-up of children exposed intrauterine to maternal thiopurine therapy during pregnancy
in females with inflammatory bowel disease. Aliment Pharmacol Ther 38 (1), 38-43.
de Wolf C, Jansen R, Yamaguchi H, de Haas M, van de Wetering K, Wijnholds J et al. (2008).
Contribution of the drug transporter ABCG2 (breast cancer resistance protein) to resistance
against anticancer nucleosides. Mol Cancer Ther 7 (9), 3092-3102.
Dell C, and Roberts G. (2006). Alcohol use and pregnancy: An important Canadian public health
and social issue (Ottawa, ON: Public Health Agency of Canada). Available at:
http://www.phac- aspc.gc.ca/publicat/fasd-ru-ectaf-pr-06/pdf/fasd-ru-ectaf-pr-06_e.pdf)
(accessed 2013 Sep. 10)
135
Dejaco C, Angelberger S, Waldhoer T, Meihsler W, Wenzl H, Haas T et al. (2005). Pregnancy
and birth outcome under thiopurine therapy for inflammatory bowel disease (IBD).
Gastroenterology 128 (4), A12.
Derewlany LO, Leeder JS, Kumar R, Radde IC, Knie B, & Koren G. (1991). The transport of
digoxin across the perfused human placental lobule. J Pharmacol Exp Ther 256 (3), 1107-
1111.
Dominitz JA, Young JC, & Boyko EJ. (2002). Outcomes of infants born to mothers with
inflammatory bowel disease: a population-based cohort study. Am J Gastroenterol 97 (3),
641-648.
Economides DL, Ferguson J, Mackenzie IZ, Darley J, Ware II, & Holmes-Siedle M. (1992).
Folate and vitamin B12 concentrations in maternal and fetal blood, and amniotic fluid in
second trimester pregnancies complicated by neural tube defects. Br J Obstet Gynaecol 99
(1), 23-25.
Elad D, Levkovitz R, Jaffa AJ, Desoye G, & Hod M. (2014). Have we neglected the role of fetal
endothelium in transplacental transport? Traffic 15 (1), 122-126.
Ema M, Naya M, Yoshida K, & Nagaosa R. (2010). Reproductive and developmental toxicity of
degradation products of refrigerants in experimental animals. Reprod Toxicol 29 (1), 1-9.
Enders AC. (1981). Anatomy of the placenta and its relationship to function. Mead Johnson
Symp Perinat Dev Med, 18, 3-7.
Etwel F, Hutson JR, Madadi P, Gareri J, & Koren G. (2014). Fetal and perinatal exposure to
drugs and chemicals: novel biomarkers of risk. Annu Rev Pharmacol Toxicol 54, 295-315.
Evseenko DA, Murthi P, Paxton JW, Reid G, Emerald BS, Mohankumar KM et al. (2007). The
ABC transporter BCRP/ABCG2 is a placental survival factor, and its expression is reduced
in idiopathic human fetal growth restriction. FASEB Journal 21 (13), 3592-3605.
Eyal S, Easterling TR, Carr D, Umans JG, Miodovnik M, Hankins GD et al. (2010).
Pharmacokinetics of metformin during pregnancy. Drug Metab Dispos 38 (5), 833-840.
136
Fedorkow DM, Persaud D, & Nimrod CA. (1989). Inflammatory bowel disease: a controlled
study of late pregnancy outcome. Am J Obstet Gynecol 160 (4), 998-1001.
Finkelstein JD, Cello JP, Kyle WE. (1974) Ethanol induced changes in methionine metabolism
in rat liver. Biochem Biophys Res Commun 61 (2), 525-531.
Finnegan L. (2013). Licit and illicit drug use during pregnancy: maternal, neonatal and early
childhood consequences. Available: www.ccsa.ca/./CCSA-Drug-Use-during-Pregnancy-
Report-2013-en.pdf .
Finster M, Morishima HO, Boyes RN, & Covino BG. (1972a). The placental transfer of
lidocaine and its uptake by fetal tissues. Anesthesiology 36 (2), 159-163.
Finster M, Morishima HO, Mark LC, Perel JM, Dayton PG, & James LS. (1972b). Tissue
thiopental concentrations in the fetus and newborn. Anesthesiology 36 (2), 155-158.
Fisher SE, Inselman LS, Duffy L, Atkinson M, Spencer H, & Chang B. (1985). Ethanol and
fetal nutrition: effect of chronic ethanol exposure on rat placental growth and membrane-
associated folic acid receptor binding activity. J Pediatr Gastroenterol Nutr 4 (4), 645-649.
Francella A, Dyan A, Bodian C, Rubin P, Chapman M, & Present DH. (2003). The safety of 6-
mercaptopurine for childbearing patients with inflammatory bowel disease: a retrospective
cohort study. Gastroenterology 124 (1), 9-17.
Frederiksen MC, Ruo TI, Chow MJ, & Atkinson AJJ. (1986). Theophylline pharmacokinetics in
pregnancy. Clin Pharmacol Ther 40 (3), 321-328.
Froescher W, Gugler R, Niesen M, & Hoffmann F. (1984). Protein binding of valproic acid in
maternal and umbilical cord serum. Epilepsia 25 (2), 244-249.
Gallou-Kabani C, Gabory A, Tost J, Karimi M, Mayeur S, Lesage J et al. (2010). Sex- and diet-
specific changes of imprinted gene expression and DNA methylation in mouse placenta
under a high-fat diet. PLoS ONE 5 (12), e14398.
137
Ganapathy V, Prasad PD, Ganapathy ME, & Leibach FH. (2000). Placental transporters relevant
to drug distribution across the maternal-fetal interface. J Pharmacol Exp Ther 294 (2), 413-
420.
Garcia-Rodriguez S, Arguelles S, Llopis R, Murillo ML, Machado A, Carreras O et al. (2003).
Effect of prenatal exposure to ethanol on hepatic elongation factor-2 and proteome in 21 d
old rats: protective effect of folic acid. Free Radic Biol Med 35 (4), 428-437.
Garland M. (1998). Pharmacology of drug transfer across the placenta. Obstet Gynecol Clin
North Am 25 (1), 21-42.
Garland M, Abildskov KM, Taylor S, Benzeroual K, Caspersen CS, Arroyo SE et al. (2006).
Fetal morphine metabolism and clearance are constant during late gestation. Drug Metab
Dispos 34 (4), 636-646.
Gavard L, Beghin D, Forestier F, Cayre Y, Peytavin G, Mandelbrot L et al. (2009). Contribution
and limit of the model of perfused cotyledon to the study of placental transfer of drugs.
Example of a protease inhibitor of HIV: nelfinavir. Eur J Obstet Gynecol Reprod Biol 147
(2), 157-160.
Gavard L, Gil S, Peytavin G, Ceccaldi PF, Ferreira C, Farinotti R et al. (2006). Placental transfer
of lopinavir/ritonavir in the ex vivo human cotyledon perfusion model. Am J Obstet
Gynecol 195 (1), 296-301.
Gedeon C, Anger G, Lubetsky A, Miller MP, & Koren G. (2008). Investigating the potential
role of multi-drug resistance protein (MRP) transporters in fetal to maternal glyburide
efflux in the human placenta. J Obstet Gynaecol 28 (5), 485-489.
Gendron MP, Martin B, Oraichi D, & Berard A. (2009). Health care providers' requests to
Teratogen Information Services on medication use during pregnancy and lactation. Eur J
Clin Pharmacol 65 (5), 523-531.
Gepts E, Heytens L, & Camu F. (1986). Pharmacokinetics and placental transfer of intravenous
and epidural alfentanil in parturient women. Anesth Analg 65 (11), 1155-1160.
138
Gerdin E, Rane A, & Lindberg B. (1990). Transplacental transfer of morphine in man. J Perinat
Med 18 (4), 305-312.
Gil S, Saura R, Forestier F, & Farinotti R. (2005). P-glycoprotein expression of the human
placenta during pregnancy. Placenta 26 (2-3), 268-270.
Giroux M, Teixera MG, Dumas JC, Desprats R, Grandjean H, & Houin G. (1997). Influence of
maternal blood flow on the placental transfer of three opioids--fentanyl, alfentanil,
sufentanil. Biol Neonate 72 (3), 133-141.
Githens JH, Rosenkrantz JG, Tunnock SM. (1965) Teratogenic effects of azathioprine (Imuran).
J Pediatr 66, 959–961.
Giugliani ER, Jorge SM, & Goncalves AL. (1985). Serum and red blood cell folate levels in
parturients, in the intervillous space of the placenta and in full-term newborns. J Perinat
Med 13 (2), 55-59.
Goh YI, & Koren G. (2008). Folic acid in pregnancy and fetal outcomes. J Obstet Gynaecol 28
(1), 3-13.
Goldstein LH, Dolinsky G, Greenberg R, Schaefer C, Cohen-Kerem R, Diav-Citrin O et al.
(2007). Pregnancy outcome of women exposed to azathioprine during pregnancy. Birth
Defects Res A Clin Mol Teratol 79 (10), 696-701.
Goodlett CR, Horn KH, & Zhou FC. (2005). Alcohol teratogenesis: mechanisms of damage and
strategies for intervention. Exp Biol Med 230 (6), 394-406.
Gorodischer R, Krasner J, & Yaffe SJ. (1974). Serum protein binding of digoxin in newborn
infants. Res Commun Chem Pathol Pharmacol 9 (2), 387-390.
Govindarajan R, Bakken AH, Hudkins KL, Lai Y, Casado FJ, Pastor-Anglada M et al. (2007).
In situ hybridization and immunolocalization of concentrative and equilibrative nucleoside
transporters in the human intestine, liver, kidneys, and placenta. Am J Physiol Regul Integr
Comp Physiol 293 (5), 1809-1822.
139
Guerra-Shinohara EM, Morita OE, Peres S, Pagliusi RA, Sampaio Neto LF, D'Almeida V et al.
(2004). Low ratio of S-adenosylmethionine to S-adenosylhomocysteine is associated with
vitamin deficiency in Brazilian pregnant women and newborns. Am J Clin Nutr 80 (5),
1312-1321.
Gundogan F, Elwood G, Mark P, Feijoo A, Longato L, Tong M et al. (2010). Ethanol-Induced
Oxidative Stress and Mitochondrial Dysfunction in Rat Placenta: Relevance to Pregnancy
Loss. Alcohol Clin Exp Res 34, 415-423.
Gutierrez CM, Ribeiro CN, de Lima GA, Yanaguita MY, & Peres LC. (2007). An experimental
study on the effects of ethanol and folic acid deficiency, alone or in combination, on
pregnant Swiss mice. Pathology 39 (5), 495-503.
Habal FM, & Huang VW. (2012). Review article: a decision-making algorithm for the
management of pregnancy in the inflammatory bowel disease patient. Aliment Pharmacol
Ther 35 (5), 501-515.
Hager R, & Johnstone RA. (2003). The genetic basis of family conflict resolution in mice.
Nature 421 (6922), 533-535.
Halsted CH, Villanueva JA, Devlin AM et al. (2002a) Folate deficiency disturbs hepatic
methionine metabolism and promotes liver injury in the ethanol-fed micropig. Proc Natl
Acad Sci U S A 99 (15), 10072-10077.
Halsted CH, Villanueva JA, Devlin AM, & Chandler CJ. (2002b). Metabolic interactions of
alcohol and folate. J Nutr 132 (8 Suppl), 2367S-2372S.
Hamar C, & Levy G. (1980). Factors affecting the serum protein binding of salicylic acid in
newborn infants and their mothers. Pediatr Pharmacol 1 (1), 31-43.
Hanauer SB. (1999). Updating the approach to Crohn's disease. Hosp Pract 34 (8), 77-78, 81-
83, 87-93.
Hansen JM, Contreras KM, & Harris C. (2005). Methanol, formaldehyde, and sodium formate
exposure in rat and mouse conceptuses: a potential role of the visceral yolk sac in
embryotoxicity. Birth Defects Res A Clin Mol Teratol 73 (2), 72-82.
140
Haram K, Bakke OM, Johannessen KH, & Lund T. (1978). Transplacental passage of diazepam
during labor: influence of uterine contractions. Clin Pharmacol Ther 24 (5), 590-599.
Harris C, Dixon M, & Hansen JM. (2004). Glutathione depletion modulates methanol,
formaldehyde and formate toxicity in cultured rat conceptuses. Cell Biol Toxicol 20 (3),
133-145.
Hawwa AF, Millership JS, Collier PS, & McElnay JC. (2009). Development and validation of
an HPLC method for the rapid and simultaneous determination of 6-mercaptopurine and
four of its metabolites in plasma and red blood cells. J Pharm Biomed Anal 49 (2), 401-
409.
He Y-L, Seno H, Tsujimoto S, & Tashiro C. (2001). The effects of uterine and umbilical blood
flows on the transfer of propofol across the human placenta during in vitro perfusion.
Anesth Analg 93 (1), 151-156.
Hebert MF, Ma X, Naraharisetti SB, Krudys KM, Umans JG, Hankins GD et al. (2009). Are we
optimizing gestational diabetes treatment with glyburide? The pharmacologic basis for
better clinical practice. Clin Pharmacol Ther 85 (6), 607-614.
Hemauer SJ, Patrikeeva SL, Nanovskaya TN, Hankins GD, & Ahmed MS. (2009). Opiates
inhibit paclitaxel uptake by P-glycoprotein in preparations of human placental inside-out
vesicles. Biochem Pharmacol 78 (9), 1272-1278.
Henderson GI, Perez T, Schenker S, Mackins J, & Antony AC. (1995). Maternal-to-fetal
transfer of 5-methyltetrahydrofolate by the perfused human placental cotyledon: evidence
for a concentrative role by placental folate receptors in fetal folate delivery. J Lab Clin Med
126 (2), 184-203.
Herbert V, Zalusky R, Davidson CS. (1963) Correlation of folate deficiency with alcoholism
and associated macrocytosis, anemia, and liver disease. Ann Intern Med 58, 977-988.
Herman NL, Li A-T, Van D,T.K., Johnson RF, Bjoraker RW, Downing JW et al. (2000).
Transfer of methohexital across the perfused human placenta. J Clin Anesth 12 (1), 25-30.
141
Hermann DJ, Egan TD, & Muir KT. (1999). Influence of arteriovenous sampling on
remifentanil pharmacokinetics and pharmacodynamics. Clin Pharmacol Ther 65 (5), 511-
518.
Hermanns MI, Unger RE, Kehe K, Peters K, & Kirkpatrick CJ. (2004). Lung epithelial cell lines
in coculture with human pulmonary microvascular endothelial cells: development of an
alveolo-capillary barrier in vitro. Lab Invest 84 (6), 736-752.
Herr F, Baal N, Reisinger K, Lorenz A, McKinnon T, Preissner KT et al. (2007). HCG in the
regulation of placental angiogenesis. Results of an in vitro study. Placenta 28 (Suppl A),
S85-S93.
Hill MD, & Abramson FP. (1988). The significance of plasma protein binding on the
fetal/maternal distribution of drugs at steady-state. Clin Pharmacokinet 14 (3), 156-170.
Hines RN. (2007). Ontogeny of human hepatic cytochromes P450. J Biochem Mol Toxicol 21
(4), 169-175.
Hutson JR, Klieger C, & Koren G. (2012). Pharmacokinetics in Pregnancy. In Ginosar Y,
Reynolds F, Halpern SH (Ed) Anesthesia and the Fetus (1st ed., p. 53-62). Oxford, UK:
Wiley-Blackwell.
Hutson JR, Garcia-Bournissen F, Davis A, & Koren G. (2011). The human placental perfusion
model: a systematic review and development of a model to predict in vivo transfer of
therapeutic drugs. Clin Pharmacol Ther 90 (1), 67-76.
Hutson JR, Koren G, & Matthews SG. (2010). Placental P-glycoprotein and breast cancer
resistance protein: influence of polymorphisms on fetal drug exposure and physiology.
Placenta 31 (5), 351-357.
Hutson JR, Matlow JN, Moretti ME, & Koren G. (2013). The fetal safety of thiopurines for the
treatment of inflammatory bowel disease in pregnancy. J Obstet Gynaecol 33 (1), 1-8.
Hutson JR, Stade B, Lehotay DC, Collier CP, & Kapur BM. (2012). Folic acid transport to the
human fetus is decreased in pregnancies with chronic alcohol exposure. PLoS ONE 7 (5),
e38057.
142
Jain V, & Gordon C. (2011). Managing pregnancy in inflammatory rheumatological diseases.
Arthritis Res Ther 13 (1), 206.
Jharap B, de Boer NK, Stokkers P, Hommes DW, Oldenburg B, Dijkstra G et al. (2014).
Intrauterine exposure and pharmacology of conventional thiopurine therapy in pregnant
patients with inflammatory bowel disease. Gut 63 (3), 451-457.
Jimenez-Solem E, Andersen JT, Petersen M, Broedbaek K, Jensen JK, Afzal S et al. (2012).
Exposure to selective serotonin reuptake inhibitors and the risk of congenital
malformations: a nationwide cohort study. BMJ Open 2 (3), e001148.
Johlin FC, Fortman CS, Nghiem DD, & Tephly TR. (1987). Studies on the role of folic acid and
folate-dependent enzymes in human methanol poisoning. Mol Pharmacol 31 (5), 557-561.
Johnson RF, Herman N, Arney TL, Gonzalez H, Johnson HV, & Downing JW. (1995).
Bupivacaine transfer across the human term placenta: A study using the dual perfused
human placental model. Anesthesiology 82 (2), 459-468.
Johnson RF, Herman N, Arney TL, Olenick M, Paschall RL, Johnson HV et al. (1999). Transfer
of lidocaine across the dual perfused human placental cotyledon. Int J Obstet Anesth 8 (1),
17-23.
Johnson RF, Herman NL, Johnson HV, Arney TL, Paschall RL, & Downing JW. (1996). Effects
of fetal pH on local anesthetic transfer across the human placenta. Anesthesiology 85 (3),
608-615.
Jones KL. (2006). Smith's recognizable patterns of human malformation. (6 ed., p. 704-705).
Philadelphia: WB Saunders.
Jonker JW, Smit JW, Brinkhuis RF, Maliepaard M, Beijnen JH, Schellens JH et al. (2000). Role
of breast cancer resistance protein in the bioavailability and fetal penetration of topotecan.
J Natl Cancer Inst 92 (20), 1651-1656.
Kalant H, and Khanna J. (2007). The Alcohols. In Principles of Medical Pharmacology,7th
edition. H Kalant, D Grant, and J Mitchell, eds. (Toronto, ON: Elsevier Canada), pp. 275–
288.
143
Kapur BM, Vandenbroucke AC, Adamchik Y, Lehotay DC, & Carlen PL. (2007). Formic acid, a
novel metabolite of chronic ethanol abuse, causes neurotoxicity, which is prevented by
folic acid. Alcohol Clin Exp Res 31 (12), 2114-2120.
Kapur, BM, Khuu M, Bennett D, Tran S, Lehotay D, Eichhorst JC et al. (2009). Endogenous
methanol derived formic acid correlates with cognitive dysfunction in children born to
drinking mothers. Alcohol Clin Exp Res 33 (s1), 134A.
Karl PI, Divald A, Diehl AM, & Fisher SE. (1998). Altered cyclic AMP-dependent human
chorionic gonadotropin production in cultured human placental trophoblasts exposed to
ethanol. Biochem Pharmacol 55 (1), 45-51.
Karl PI, Divald A, & Fisher SE. (1994). Ethanol enhancement of ligand-stimulated cAMP
production by cultured human placental trophoblasts. Biochem Pharmacol 48 (7), 1493-
1500.
Karl PI, & Fisher SE. (1993). Ethanol alters hormone production in cultured human placental
trophoblasts. Alcohol Clin Exp Res 17 (4), 816-821.
Katz S. (2013). My treatment approach to the management of ulcerative colitis. Mayo Clin Proc
88 (8), 841-853.
Keating E, Goncalves P, Campos I, Costa F, & Martel F. (2009). Folic acid uptake by the human
syncytiotrophoblast: interference by pharmacotherapy, drugs of abuse and pathological
conditions. Reprod Toxicol 28 (4), 511-520.
Keating E, Lemos C, Goncalves P, & Martel F. (2008). Acute and chronic effects of some
dietary bioactive compounds on folic acid uptake and on the expression of folic acid
transporters by the human trophoblast cell line BeWo. J Nutr Biochem 19 (2), 91-100.
Khan ZH, Mayberry JF, Spiers N, & Wicks AC. (2000). Retrospective case series analysis of
patients with inflammatory bowel disease on azathioprine. A district general hospital
experience. Digestion 62 (4), 249-254.
Kim JM, Hong K, Lee JH, Lee S, & Chang N. (2009). Effect of folate deficiency on placental
DNA methylation in hyperhomocysteinemic rats. J Nutr Biochem 20 (3), 172-176.
144
Kopecky EA. (1999). Transplacental pharmacology and toxicology of morphine in humans: in
vitro and in vivo studies. Dissertation, University of Toronto.
Kopecky EA, Ryan ML, Barrett JF, Seaward PG, Ryan G, Koren G et al. (2000). Fetal response
to maternally administered morphine. Am J Obstet Gynecol 183 (2), 424-430.
Koren G, Pastuszak A, & Ito S. (1998). Drugs in pregnancy. N Engl J Med 338 (16), 1128-1137.
Koukouritaki SB, Manro JR, Marsh SA, Stevens JC, Rettie AE, McCarver DG et al. (2004).
Developmental expression of human hepatic CYP2C9 and CYP2C19. J Pharmacol Exp
Ther 308 (3), 965-974.
Kraemer J, Klein J, Lubetsky A, & Koren G. (2006). Perfusion studies of glyburide transfer
across the human placenta: implications for fetal safety. Am J Obstet Gynecol 195 (1), 270-
274.
Krauer B, Dayer P, & Anner R. (1984). Changes in serum albumin and alpha 1-acid
glycoprotein concentrations during pregnancy: an analysis of fetal-maternal pairs. Br J
Obstet Gynaecol 91 (9), 875-881.
Kuehn D, Aros S, Cassorla F, Avaria M, Unanue N, Henriquez C et al. (2012). A prospective
cohort study of the prevalence of growth, facial, and central nervous system abnormalities
in children with heavy prenatal alcohol exposure. Alcohol Clin Exp Res 36 (10), 1811-
1819.
Kuhnz W, Steldinger R, & Nau H. (1984). Protein binding of carbamazepine and its epoxide in
maternal and fetal plasma at delivery: comparison to other anticonvulsants. Dev Pharmacol
Ther 7 (1), 61-72.
Kury G, Chaube S, Murphy ML. (1968) Teratogenic effects of some purine analogues on fetal
rat. Arch Pathol 86, 395–402.
Kurz H, Mauser-Ganshorn A, & Stickel HH. (1977). Differences in the binding of drugs to
plasma proteins from newborn and adult man. I. Eur J Clin Pharmacol 11 (6), 463-467.
145
Kwan LY, & Mahadevan U. (2010). Inflammatory bowel disease and pregnancy: An update.
Expert Rev Clin Immunol 6 (4), 643-657.
Lachenmeier DW, Schoeberl K, Kanteres F, Kuballa T, Sohnius EM, & Rehm J. (2011). Is
contaminated unrecorded alcohol a health problem in the European Union? A review of
existing and methodological outline for future studies. Addiction 106 (Suppl 1), 20-30.
Landy HJ, Isada NB, McGinnis J, Ratner R, & Grossman J. (1988). The effect of chronic steroid
therapy on glucose tolerance in pregnancy. Am J Obstet Gynecol 159 (3), 612-615.
Langagergaard V, Pedersen L, Gislum M, Norgard B, & Sorensen HT. (2007). Birth outcome in
women treated with azathioprine or mercaptopurine during pregnancy: A Danish
nationwide cohort study. Aliment Pharmacol Ther 25 (1), 73-81.
Larroque B, Kaminski M, Lelong N, d'Herbomez M, Dehaene P, Querleu D et al. (1992). Folate
status during pregnancy: relationship with alcohol consumption, other maternal risk factors
and pregnancy outcome. Eur J Obstet Gynecol Reprod Biol 43 (1), 19-27.
Laufer BI, Mantha K, Kleiber ML, Diehl EJ, Addison SM, & Singh SM. (2013). Long-lasting
alterations to DNA methylation and ncRNAs could underlie the effects of fetal alcohol
exposure in mice. Dis Model Mech 6 (4), 977-992.
Lennard L, & Singleton HJ. (1992). High-performance liquid chromatographic assay of the
methyl and nucleotide metabolites of 6-mercaptopurine: quantitation of red blood cell 6-
thioguanine nucleotide, 6-thioinosinic acid and 6-methylmercaptopurine metabolites in a
single sample. J Chromatogr 583 (1), 83-90.
Levkovitz R, Zaretsky U, Gordon Z, Jaffa AJ, & Elad D. (2013). In vitro simulation of placental
transport: part I. Biological model of the placental barrier. Placenta 34 (8), 699-707.
Levkovitz R, Zaretsky U, Jaffa AJ, Hod M, & Elad D. (2013). In vitro simulation of placental
transport: part II. Glucose transfer across the placental barrier model. Placenta 34 (8), 708-
715.
Levy N, Roisman I, & Teodor I. (1981). Ulcerative colitis in pregnancy in Israel. Dis Colon
Rectum 24 (5), 351-354.
146
Lin GWJ, Lester D. (1985) Altered placental folate coenzyme distribution by ethanol
consumption during pregnancy. Nutrition Reports Int 31 (6), 1375-1383.
Lin GW-J. (1988). Folate deficiency and acute ethanol treatment on pregnancy outcome in the
rat. Nutrition Res 8 (10), 1151-1160.
Lin GW. (1991). Maternal-fetal folate transfer: effect of ethanol and dietary folate deficiency.
Alcohol 8 (3), 169-172.
Linnemann K, Malek A, Sager R, Blum WF, Schneider H, & Fusch C. (2000). Leptin
production and release in the dually in vitro perfused human placenta. J Clin Endocrinol
Metab 85 (11), 4298-4301.
Loebstein R, Lalkin A, & Koren G. (1997). Pharmacokinetic changes during pregnancy and
their clinical relevance. Clin Pharmacokinet 33 (5), 328-343.
Ludvigsson JF, & Ludvigsson J. (2002). Inflammatory bowel disease in mother or father and
neonatal outcome. Acta Paediatr 91 (2), 145-151.
Ma SH, Lepak LA, Hussain RJ, Shain W, & Shuler ML. (2005). An endothelial and astrocyte
co-culture model of the blood-brain barrier utilizing an ultra-thin, nanofabricated silicon
nitride membrane. Lab Chip 5 (1), 74-85.
MacFarland A, Abramovich DR, Ewen SW, & Pearson CK. (1994). Stage-specific distribution
of P-glycoprotein in first-trimester and full-term human placenta. Histochem J 26 (5), 417-
423.
Maguire MJ, Hemming K, Hutton JL, & Marson AG. (2008). Overwhelming heterogeneity in
systematic reviews of observational anti-epileptic studies. Epilepsy Res 80 (2-3), 201-212.
Mahey R, Kaur SD, Chumber S, Kriplani A, & Bhatla N. (2013). Splenectomy during
pregnancy: treatment of refractory immune thrombocytopenic purpura. BMJ Case Rep In
Press.
Majchrowicz E, & Mendelson JH. (1971). Blood methanol concentrations during experimentally
induced ethanol intoxication in alcoholics. J Pharmacol Exp Ther 179 (2), 293-300.
147
Makar AB, Tephly TR. (1976) Methanol poisoning in the folate-deficient rat. Nature 261 (5562),
715-6.
Malek A, Obrist C, Wenzinger S, & von Mandach U. (2009). The impact of cocaine and heroin
on the placental transfer of methadone. Reprod Biol Endocrinol 7, 61.
Maliepaard M, Scheffer GL, Faneyte IF, van Gastelen MA, Pijnenborg AC, Schinkel AH et al.
(2001). Subcellular localization and distribution of the breast cancer resistance protein
transporter in normal human tissues. Cancer Res 61 (8), 3458-3464.
Malone SM, McGue M, & Iacono WG. (2010). Mothers' maximum drinks ever consumed in 24
hours predicts mental health problems in adolescent offspring. J Child Psychol Psychiatry
51 (9), 1067-1075.
Mather LE, Long GJ, & Thomas J. (1971). The binding of bupivacaine to maternal and foetal
plasma proteins. J Pharm Pharmacol 23 (5), 359-365.
Mathiesen L, Mose T, Morck TJ, Nielsen JK, Nielsen LK, Maroun LL et al. (2010). Quality
assessment of a placental perfusion protocol. Reprod Toxicol 30 (1), 138-146.
May PA, & Gossage JP. (2001). Estimating the prevalence of fetal alcohol syndrome. A
summary. Alcohol Res Health 25 (3), 159-167.
McBride WG. (1978). Teratogenic action of thalidomide. Lancet 1 (8078), 1362.
McMartin KE, Martin-Amat G, Makar AB, Tephly TR. (1977) Methanol poisoning. V. Role of
formate metabolism in the monkey. J Pharmacol Exp Ther 201 (3), 564-572.
Meuldermans W, Woestenborghs R, Noorduin H, Camu F, van Steenberge A, & Heykants J.
(1986). Protein binding of the analgesics alfentanil and sufentanil in maternal and neonatal
plasma. Eur J Clin Pharmacol 30 (2), 217-219.
Meyer zu Schwabedissen HE, Grube M, Dreisbach A, Jedlitschky G, Meissner K, Linnemann K
et al. (2006). Epidermal growth factor-mediated activation of the map kinase cascade
results in altered expression and function of ABCG2 (BCRP). Drug Metab Dispos 34 (4),
524-533.
148
Meyer Zu Schwabedissen HE, Grube M, Heydrich B, Linnemann K, Fusch C, Kroemer HK et
al. (2005). Expression, localization, and function of MRP5 (ABCC5), a transporter for
cyclic nucleotides, in human placenta and cultured human trophoblasts: effects of
gestational age and cellular differentiation. Am J Pathol 166 (1), 39-48.
Miller RK, Wier PJ, Maulik D, & di Sant'Agnese PA. (1985). Human placenta in vitro:
characterization during 12 h of dual perfusion. Contrib Gynecol Obstet 13, 77-84.
Molloy AM, Mills JL, Cox C, Daly SF, Conley M, Brody LC et al. (2005). Choline and
homocysteine interrelations in umbilical cord and maternal plasma at delivery. Am J Clin
Nutr 82 (4), 836-842.
Moore JJ, Lundgren DW, Moore RM, & Andersen B. (1988). Polyamines control human
chorionic gonadotropin production in the JEG-3 choriocarcinoma cell. J Biol Chem 263
(25), 12765-12769.
Morales M, Berney T, Jenny A, Morel P, & Extermann P. (2000). Crohn's disease as a risk
factor for the outcome of pregnancy. Hepatogastroenterology 47 (36), 1595-1598.
Morck TJ, Sorda G, Bechi N, Rasmussen BS, Nielsen JB, Ietta F et al. (2010). Placental
transport and in vitro effects of Bisphenol A. Reprod Toxicol 30 (1), 131-137.
Morton CP, Bloomfield S, Magnusson A, Jozwiak H, & McClure JH. (1997). Ropivacaine
0.75% for extradural anaesthesia in elective caesarean section: an open clinical and
pharmacokinetic study in mother and neonate. Br J Anaesth 79 (1), 3-8.
Mose T, Mathiesen L, Karttunen V, Nielsen JK, Sieppi E, Kummu M et al. (2012). Meta-
analysis of data from human ex vivo placental perfusion studies on genotoxic and
immunotoxic agents within the integrated European project NewGeneris. Placenta 33 (5),
433-439.
Mountifield RE, Prosser R, Bampton P, Muller K, & Andrews JM. (2010). Pregnancy and IBD
treatment: This challenging interplay from a patients' perspective. J Crohn's Colitis 4 (2),
176-182.
149
Mowat C, Cole A, Windsor A, Ahmad T, Arnott I, Driscoll R et al. (2011). Guidelines for the
management of inflammatory bowel disease in adults. Gut 60 (5), 571-607.
Muhlemann K, Menegus MA, & Miller RK. (1995). Cytomegalovirus in the perfused human
term placenta in vitro. Placenta 16 (4), 367-373.
Murphy ML. 1960. Teratogenic effects of tumor-inhibiting chemicals in the rat. In:
Wolstenholme GEW, O’Conner CM, editors. A CIBA Foundation Symposium on
congenital malformations. London: J & A Churchill Ltd. p 78.
Myllynen P, Immonen E, Kummu M, & Vahakangas K. (2009). Developmental expression of
drug metabolizing enzymes and transporter proteins in human placenta and fetal tissues.
Expert Opin Drug Metab Toxicol 5 (12), 1483-1499.
Myllynen P, Pienimaki P, & Vahakangas K. (2005). Human placental perfusion method in the
assessment of transplacental passage of antiepileptic drugs. Toxicol Appl Pharmacol 207 (2
Suppl), 489-494.
Nagai K, Nagasawa K, Kihara Y, Okuda H, & Fujimoto S. (2007). Anticancer nucleobase
analogues 6-mercaptopurine and 6-thioguanine are novel substrates for equilibrative
nucleoside transporter 2. Int J Pharm 333 (1-2), 56-61.
Nagashige M, Ushigome F, Koyabu N, Hirata K, Kawabuchi M, Hirakawa T et al. (2003). Basal
membrane localization of MRP1 in human placental trophoblast. Placenta 24 (10), 951-
958.
Nanovskaya TN, Nekhayeva IA, Hankins GDV, & Ahmed MS. (2008). Transfer of methadone
across the dually perfused preterm human placental lobule. Am J Obstet Gynecol 198 (1),
126e1-4.
Nau H. (1985). Clinical pharmacokinetics in pregnancy and perinatology. I. Placental transfer
and fetal side effects of local anaesthetic agents. Dev Pharmacol Ther 8 (3), 149-181.
Nau H, Helge H, & Luck W. (1984). Valproic acid in the perinatal period: decreased maternal
serum protein binding results in fetal accumulation and neonatal displacement of the drug
and some metabolites. J Pediatr 104 (4), 627-634.
150
Nau H, Luck W, Kuhnz W, & Wegener S. (1983). Serum protein binding of diazepam,
desmethyldiazepam, furosemide, indomethacin, warfarin, and phenobarbital in human
fetus, mother, and newborn infant. Pediatr Pharmacol 3 (3-4), 219-227.
Nava-Ocampo AA, Velazquez-Armenta Y, Brien JF, & Koren G. (2004). Elimination kinetics
of ethanol in pregnant women. Reprod Toxicol 18 (4), 613-617.
Navarro J, Causse MB, Desquilbet N, Herve F, & Lallemand D. (1984). The vitamin status of
low birth weight infants and their mothers. J Pediatr Gastroenterol Nutr 3 (5), 744-748.
Nelson JA, Carpenter JW, Rose LM, Adamson DJ. (1975) Mechanisms of action of 6-
thioguanine, 6-mercaptopurine, and 8-azaguanine. Cancer Res 35, 2872–2878.
Norgard B, Pedersen L, Christensen LA, & Sorensen HT. (2007a). Therapeutic drug use in
women with crohn's disease and birth outcomes: A danish nationwide cohort study. Am J
Gastroenterol 102 (7), 1406-1413.
Norgard B, Hundborg HH, Jacobsen BA, Nielsen GL, & Fonager K. (2007b). Disease activity in
pregnant women with Crohn’s disease and birth outcomes: a regional Danish cohort study.
Am J Gastroenterol 102 (9), 1947-1954.
O'Connor MJ, Shah B, Whaley S, Cronin P, Gunderson B, & Graham J. (2002). Psychiatric
illness in a clinical sample of children with prenatal alcohol exposure. Am J Drug Alcohol
Abuse 28 (4), 743-754.
O’Neil M, Heckelman P, Koch C, Roman K, editors. The Merck Index: An Encyclopedia of
Chemicals, Drugs, and Biologicals. 6-Mercaptopurine (Monograph #05871). Whitehouse
Station, NJ, USA: Merck & Co., Inc.; 2010.
Obeid R, Munz W, Jager M, Schmidt W, & Herrmann W. (2005). Biochemical indexes of the B
vitamins in cord serum are predicted by maternal B vitamin status. Am J Clin Nutr 82 (1),
133-139.
Ohlman S, Albertioni F, & Peterson C. (1994). Day-to-day variability in azathioprine
pharmacokinetics in renal transplant recipients. Clin Transplant 8 (3 Pt 1), 217-223.
151
Omarini D, Pistotti V, & Bonati M. (1992). Placental perfusion. An overview of the literature. J
Pharmacol Toxicol Methods 28 (2), 61-66.
Osting VC, & Carter JD. (2010). A safety assessment of tumor necrosis factor antagonists
during pregnancy. Expert Opin Drug Saf 9 (3), 421-429.
Ostrea EMJ, Knapp DK, Tannenbaum L, Ostrea AR, Romero A, Salari V et al. (2001).
Estimates of illicit drug use during pregnancy by maternal interview, hair analysis, and
meconium analysis. J Pediatr 138 (3), 344-348.
Paine A1, Davan AD. Defining a tolerable concentration of methanol in alcoholic drinks. (2001)
Hum Exp Toxicol 20 (11), 563-568.
Palese M, Tephly TR. (1975) Metabolism of formate in the rat. J Toxicol Environ Health 1 (1),
13-24.
Panigel M, Pascaud M, & Brun JL. (1967). [Radioangiographic study of circulation in the villi
and intervillous space of isolated human placental cotyledon kept viable by perfusion]. J
Physiol 59 (1 Suppl), 277.
Park-Wyllie L, Mazzotta P, Pastuszak A, Moretti ME, Beique L, Hunnisett L et al. (2000). Birth
defects after maternal exposure to corticosteroids: prospective cohort study and meta-
analysis of epidemiological studies. Teratology 62 (6), 385-392.
Peyrin-Biroulet L, Oussalah A, Roblin X, & Sparrow MP. (2011). The use of azathioprine in
Crohn's disease during pregnancy and in the post-operative setting: a worldwide survey of
experts. Aliment Pharmacol Ther 33 (6), 707-713.
Philipson EH, Kuhnert BR, & Syracuse CD. (1985). Fetal acidosis, 2-chloroprocaine, and
epidural anesthesia for cesarean section. Am J Obstet Gynecol 151 (3), 322-324.
Pickering B, Biehl D, & Meatherall R. (1981). The effect of foetal acidosis on bupivacaine
levels in utero. Can Anaesth Soc J 28 (6), 544-549.
Pienimaki P, Lampela E, Hakkola J, Arvela P, Raunio H, & kangas K. (1997). Pharmacokinetics
of oxcarbazepine and carbamazepine in human placenta. Epilepsia 38 (3), 309-316.
152
Pirani BB, Campbell DM, & MacGillivray I. (1973). Plasma volume in normal first pregnancy.
J Obstet Gynaecol Br Commonw 80 (10), 884-887.
Platzek T, & Bochert G. (1996). Dose-response relationship of teratogenicity and prenatal-toxic
risk estimation of 6-mercaptopurine riboside in mice. Teratog Carcinog Mutagen 16 (3),
169-181.
Polifka JE, & Friedman JM. (2002). Teratogen update: azathioprine and 6-mercaptopurine.
Teratology 65 (5), 240-261.
Pollex E, Lubetsky A, & Koren G. (2008). The role of placental breast cancer resistance protein
in the efflux of glyburide across the human placenta. Placenta 29 (8), 743-747.
Pond SM, Kreek MJ, Tong TG, Raghunath J, & Benowitz NL. (1985). Altered methadone
pharmacokinetics in methadone-maintained pregnant women. J Pharmacol Exp Ther 233
(1), 1-6.
Porter JM, Kelleher N, Flynn R, & Shorten GD. (2001). Epidural ropivacaine hydrochloride
during labour: protein binding, placental transfer and neonatal outcome. Anaesthesia 56
(5), 418-423.
Porter JS, Bonello E, & Reynolds F. (1997). The influence of epidural administration of fentanyl
infusion on gastric emptying in labour. Anaesthesia 52 (12), 1151-1156.
Prasad PD, Ramamoorthy S, Leibach FH, & Ganapathy V. (1995). Molecular cloning of the
human placental folate transporter. Biochem Biophys Res Commun 206 (2), 681-687.
Present DH, Meltzer SJ, Krumholz MP, Wolke A, & Korelitz BI. (1989). 6-Mercaptopurine in
the management of inflammatory bowel disease: short- and long-term toxicity. Ann Intern
Med 111 (8), 641-649.
Puget A, Cros S, Oreglia J, Tollon Y. (1975) E ́ tude de la sensibilite ́ embryonnaire de
l’ochotone afghan (Ochotona rufescens rufescens) a deux agents te ́ratoge`nes,
l’azathioprine et la 6 mercaptopurine. Zentralbl Veterinarmed [A] 22, 38–56.
153
Rahimi R, Nikfar S, Rezaie A, & Abdollahi M. (2008). Pregnancy outcome in women with
inflammatory bowel disease following exposure to 5-aminosalicylic acid drugs: a meta-
analysis. Reprod Toxicol 25 (2), 271-275.
Rambeck B, Kurlemann G, Stodieck SR, May TW, & Jurgens U. (1997). Concentrations of
lamotrigine in a mother on lamotrigine treatment and her newborn child. Eur J Clin
Pharmacol 51 (6), 481-484.
Reddy D, Murphy SJ, Kane SV, Present DH, & Kornbluth AA. (2008). Relapses of
inflammatory bowel disease during pregnancy: in-hospital management and birth
outcomes. Am J Gastroenterol 103 (5), 1203-1209.
Reis M, & Kallen B. (2010). Delivery outcome after maternal use of antidepressant drugs in
pregnancy: an update using Swedish data. Psychol Med 40 (10), 1723-1733.
Relton CL, Pearce MS, & Parker L. (2005). The influence of erythrocyte folate and serum
vitamin B12 status on birth weight. Br J Nutr 93 (5), 593-599.
Reshef E, Lei ZM, Rao CV, Pridham DD, Chegini N, & Luborsky JL. (1990). The presence of
gonadotropin receptors in nonpregnant human uterus, human placenta, fetal membranes,
and decidua. J Clin Endocrinol Metab 70 (2), 421-430.
Reynolds F, & Knott C. (1989). Pharmacokinetics in pregnancy and placental drug transfer. Oxf
Rev Reprod Biol 11, 389-449.
Reynolds F, & Seed PT. (2005). Anaesthesia for Caesarean section and neonatal acid-base
status: a meta-analysis. Anaesthesia 60 (7), 636-653.
Ridd MJ, Brown KF, Nation RL, & Collier CB. (1983). Differential transplacental binding of
diazepam: causes and implications. Eur J Clin Pharmacol 24 (5), 595-601.
Ridd MJ, Brown KF, Nation RL, & Collier CB. (1989). The disposition and placental transfer of
diazepam in cesarean section. Clin Pharmacol Ther 45 (5), 506-512.
Ring JA, Ghabrial H, Ching MS, Smallwood RA, & Morgan DJ. (1999). Fetal hepatic drug
elimination. Pharmacol Ther 84(3), 429-445.
154
Rosenberg MJ, Wolff CR, El-Emawy A, Staples MC, Perrone-Bizzozero NI, & Savage DD.
(2010). Effects of moderate drinking during pregnancy on placental gene expression.
Alcohol 44 (7-8), 673-690.
Rosenkrantz JG, Githens JH, Cox SM, Kellum DL. (1967) Azathioprine (Imuran) and
pregnancy. Am J Obstet Gynecol 97, 387–394.
Saarikoski S, & Seppala M. (1973). Immunosuppression during pregnancy: transmission of
azathioprine and its metabolites from the mother to the fetus. Am J Obstet Gynecol 115 (8),
1100-1106.
Sahasranaman S, Howard D, & Roy S. (2008). Clinical pharmacology and pharmacogenetics of
thiopurines. Eur J Clin Pharmacol 64 (8), 753-767.
Sandborn WJ, Van Os EC, Zins BJ, Tremaine WJ, Mays DC, Lipsky JJ. (1995) An intravenous
loading dose of azathioprine decreases the time to response in patients with Crohn disease.
Gastroenterology 109, 1808 –1817.
Sarkola T, & Eriksson CJ. (2001). Effect of 4-methylpyrazole on endogenous plasma ethanol and
methanol levels in humans. Alcohol Clin Exp Res 25 (4), 513-516.
Schaefer C, Ornoy A, Clementi M, Meister R, & Weber-Schoendorfer C. (2008). Using
observational cohort data for studying drug effects on pregnancy outcome--methodological
considerations. Reprod Toxicol 26 (1), 36-41.
Schatz M, Dombrowski MP, Wise R, Momirova V, Landon M, Mabie W et al. (2004). The
relationship of asthma medication use to perinatal outcomes. J Allergy Clin Immunol 113
(6), 1040-1045.
Scheeres JJ, & Chudley AE. (2002). Solvent abuse in pregnancy: a perinatal perspective. J
Obstet Gynaecol Can 24 (1), 22-26.
Schenker S, Yang Y, Mattiuz E, Tatum D, & Lee M. (1999). Olanzapine transfer by human
placenta. Clin Exp Pharmacol Physiol 26 (9), 691-697.
155
Schlotz W, Jones A, Phillips DI, Gale CR, Robinson SM, & Godfrey KM. (2010). Lower
maternal folate status in early pregnancy is associated with childhood hyperactivity and
peer problems in offspring. J Child Psychol Psychiatry 51 (5), 594-602.
Schmolling J, Renke K, Richter O, Pfeiffer K, Schlebusch H, & Holler T. (2000). Digoxin,
flecainide, and amiodarone transfer across the placenta and the effects of an elevated
umbilical venous pressure on the transfer rate. Ther Drug Monit 22 (5), 582-588.
Schneider H, Panigel M, & Dancis J. (1972). Transfer across the perfused human placenta of
antipyrine, sodium and leucine. Am J Obstet Gynecol 114 (6), 822-828.
Shah YG, & Miller RK. (1985). The pharmacokinetics of phenytoin in perfused human placenta.
Pediatr Pharmacol 5 (3), 165-179.
Shankar K, Hidestrand M, Liu X, Xiao R, Skinner CM, Simmen FA et al. (2006). Physiologic
and genomic analyses of nutrition-ethanol interactions during gestation: Implications for
fetal ethanol toxicity. Exp Biol Med 231 (8), 1379-1397.
Shim L, Eslick GD, Simring AA, Murray H, & Weltman MD. (2011). The effects of
azathioprine on birth outcomes in women with inflammatory bowel disease (IBD). J
Crohns Colitis 5 (3), 234-238.
Shintaku K, Hori S, Tsujimoto M, Nagata H, Satoh S, Tsukimori K et al. (2009). Transplacental
pharmacokinetics of diclofenac in perfused human placenta. Drug Metab Dispos 37 (5),
962-968.
Shintaku K, Hori S, Satoh H, Tsukimori K, Nakano H, Fujii T et al. (2012). Prediction and
evaluation of fetal toxicity induced by NSAIDs using transplacental kinetic parameters
obtained from human placental perfusion studies. Br J Clin Pharmacol 73 (2), 248-256.
Shintaku K, Arima Y, Dan Y, Takeda T, Kogushi K, Tsujimoto M et al. (2007). Kinetic analysis
of the transport of salicylic acid, a nonsteroidal anti-inflammatory drug, across human
placenta. Drug Metab Dispos 35 (5), 772-778.
Simone C, Byrne BM, Derewlany LO, & Koren G. (1996). Cocaine inhibits hCG secretion by
the human term placental cotyledon perfused in vitro. Life Sciences 58 (5), PL 63-66.
156
Simunovic N, Sprague S, & Bhandari M. (2009). Methodological issues in systematic reviews
and meta-analyses of observational studies in orthopaedic research. J Bone Joint Surg Am
91(Suppl 3), 87-94.
Sokoro AA, Zhang Z, Eichhorst JC, Zello GA, House JD, Alcorn J et al. (2008). Formate
pharmacokinetics during formate administration in folate-deficient young swine.
Metabolism 57 (7), 920-926.
Solanky N, Requena Jimenez A, D'Souza SW, Sibley CP, & Glazier JD. (2010). Expression of
folate transporters in human placenta and implications for homocysteine metabolism.
Placenta 31 (2), 134-143.
Sonnier M, & Cresteil T. (1998). Delayed ontogenesis of CYP1A2 in the human liver. Eur J
Biochem 251 (3), 893-898.
St-Pierre MV, Serrano MA, Macias RI, Dubs U, Hoechli M, Lauper U et al. (2000). Expression
of members of the multidrug resistance protein family in human term placenta. Am J
Physiol Regul Integr Comp Physiol 279 (4), R1495-1503.
Stade B, Ali A, Bennett D, Campbell D, Johnston M, Lens C et al. (2009). The burden of
prenatal exposure to alcohol: revised measurement of cost. Can J Clin Pharmacol 16 (1),
e91-102.
Stark KD, Pawlosky RJ, Beblo S, Murthy M, Flanagan VP, Janisse J et al. (2005). Status of
plasma folate after folic acid fortification of the food supply in pregnant African American
women and the influences of diet, smoking, and alcohol consumption. Am J Clin Nutr 81
(3), 669-677.
Stark KD, Pawlosky RJ, Sokol RJ, Hannigan JH, & Salem NJ. (2007). Maternal smoking is
associated with decreased 5-methyltetrahydrofolate in cord plasma. Am J Clin Nutr 85 (3),
796-802.
Stratton K, Howe C, Battaglia FC. (1996) Fetal alcohol syndrome: diagnosis, epidemiology,
prevention, and treatment. Institute of Medicine and National Academy Press, Washington,
USA.
157
Streissguth AP, Bookstein FL, Barr HM, Sampson PD, O'Malley K, & Young JK. (2004). Risk
factors for adverse life outcomes in fetal alcohol syndrome and fetal alcohol effects. J Dev
Behav Pediatr 25 (4), 228-238.
Sudhakaran S, Rayner CR, Li J, Kong DC, Gude NM, & Nation RL. (2007). Differential protein
binding of indinavir and saquinavir in matched maternal and umbilical cord plasma. Br J
Clin Pharmacol 63 (3), 315-321.
Sudhakaran S, Ghabrial H, Nation RL, Kong DCM, Gude NM, Angus PW et al. (2005).
Differential bidirectional transfer of indinavir in the isolated perfused human placenta.
Antimicrob Agents Chemother 49 (3), 1023-1028.
Sugawara I, Akiyama S, Scheper RJ, & Itoyama S. (1997). Lung resistance protein (LRP)
expression in human normal tissues in comparison with that of MDR1 and MRP. Cancer
Letters 112 (1), 23-31.
Sun M, Kingdom J, Baczyk D, Lye SJ, Matthews SG, & Gibb W. (2006). Expression of the
multidrug resistance P-glycoprotein, (ABCB1 glycoprotein) in the human placenta
decreases with advancing gestation. Placenta 27 (6-7), 602-609.
Syme MR, Paxton JW, & Keelan JA. (2004). Drug transfer and metabolism by the human
placenta. Clin Pharmacokinet 43 (8), 487-514.
Tennenbaum R, Marteau P, Elefant E, Rambaud JC, Modigliani R, Gendre JP et al. (1999).
[Pregnancy outcome in inflammatory bowel diseases]. e Gastroenterol Clin Biol 23 (5),
464-469.
Thiersch JB. (1954) The effect of 6-mercaptopurine on the rat fetus and on reproduction of the
rat. Ann NY Acad Sci 60, 220–227.
Thomas J, Long G, Moore G, & Morgan D. (1976). Plasma protein binding and placental
transfer of bupivacaine. Clin Pharmacol Ther 19 (4), 426-434.
Thorand B, Pietrzik K, Prinz-Langenohl R, Hages M, & Holzgreve W. (1996). Maternal and
fetal serum and red blood cell folate and vitamin B12 concentrations in pregnancies
affected by neural tube defects. Z Geburtshilfe Neonatol 200 (5), 176-180.
158
Timmer A, McDonald JW, & Macdonald JK. (2007). Azathioprine and 6-mercaptopurine for
maintenance of remission in ulcerative colitis. Cochrane Database Syst Rev 1, CD000478.
Toth P, Li X, Rao CV, Lincoln SR, Sanfilippo JS, Spinnato JAn et al. (1994). Expression of
functional human chorionic gonadotropin/human luteinizing hormone receptor gene in
human uterine arteries. J Clin Endocrinol Metab 79 (1), 307-315.
Tsen LC, Arthur GR, Datta S, Hornstein MD, & Bader AM. (1997). Estrogen-induced changes
in protein binding of bupivacaine during in vitro fertilization. Anesthesiology 87 (4), 879-
883.
Tuchmann-Duplessis H, Mercier-Parot L. (1964) Considerations sur les tests teratogenes.
Differences de reaction de trois especes animales a Iegard un antitumoral. CRSBA 158,
1984–1990.
Ueki R, Tatara T, Kariya N, Shimode N, & Tashiro C. (2009). Comparison of placental transfer
of local anesthetics in perfusates with different pH values in a human cotyledon model. J
Anesth 23 (4), 526-529.
Vahakangas K, & Myllynen P. (2006). Experimental methods to study human transplacental
exposure to genotoxic agents. Mutat Res 608 (2), 129-135.
Van Scoik KG, Johnson CA, Porter WR. 1985. The pharmacology and metabolism of the
thiopurine drugs 6-mercaptopurine and azathioprine. Drug Metab Rev 16, 157–174.
Vanky E, Zahlsen K, Spigset O, & Carlsen SM. (2005). Placental passage of metformin in
women with polycystic ovary syndrome. Fertil Steril 83 (5), 1575-1578.
Veena SR, Krishnaveni GV, Srinivasan K, Wills AK, Muthayya S, Kurpad AV et al. (2010).
Higher maternal plasma folate but not vitamin B-12 concentrations during pregnancy are
associated with better cognitive function scores in 9- to 10- year-old children in South
India. J Nutr 140 (5), 1014-1022.
Vermeulen RC, Kurver PH, Arts NF, Van Kessel H, Wilson GR, & Klopper A. (1982). The
relationship between the surface area of the trophoblast and some placental products.
Placenta 3 (4), 359-366.
159
Wallace JM, Bonham MP, Strain J, Duffy EM, Robson PJ, Ward M et al. (2008). Homocysteine
concentration, related B vitamins, and betaine in pregnant women recruited to the
Seychelles Child Development Study. Am J Clin Nutr 87 (2), 391-397.
Wang LL, Zhang Z, Li Q, Yang R, Pei X, Xu Y et al. (2009). Ethanol exposure induces
differential microRNA and target gene expression and teratogenic effects which can be
suppressed by folic acid supplementation. Hum Reprod 24 (3), 562-579.
Warren KR, & Li TK. (2005). Genetic polymorphisms: impact on the risk of fetal alcohol
spectrum disorders. Birth Defects Res A Clin Mol Teratol 73 (4), 195-203.
Wentzel P, & Eriksson UJ. (2008). Folic acid prevention of ethanol damage in rat offspring.
Alcohol Clin Exp Res 32 (s1), 48A.
Wielinga PR, Reid G, Challa EE, van der Heijden I, van Deemter L, de Haas M et al. (2002).
Thiopurine metabolism and identification of the thiopurine metabolites transported by
MRP4 and MRP5 overexpressed in human embryonic kidney cells. Mol Pharmacol 62 (6),
1321-1331.
Wijnholds J, Mol CA, van Deemter L, de Haas M, Scheffer GL, Baas F et al. (2000). Multidrug-
resistance protein 5 is a multispecific organic anion transporter able to transport nucleotide
analogs. Proc Natl Acad Sci U S A 97 (13), 7476-7481.
Wilson RD1, Johnson JA, Wyatt P, Allen V, Gagnon A, Langlois S, Blight C, Audibert F,
Désilets V, Brock JA, Koren G, Goh YI, Nguyen P, Kapur B; Genetics Committee of the
Society of Obstetricians and Gynaecologists of Canada and The Motherisk Program.
(2007) Pre-conceptional vitamin/folic acid supplementation 2007: the use of folic acid in
combination with a multivitamin supplement for the prevention of neural tube defects and
other congenital anomalies. J Obstet Gynaecol Can. 29 (12), 1003-1026.
Wimalasena J, Beams F, & Caudle MR. (1994). Ethanol modulates the hormone secretory
responses induced by epidermal growth factor in choriocarcinoma cells. Alcohol Clin Exp
Res 18 (6), 1448-1455.
160
Woolfson K, Cohen Z, & McLeod RS. (1990). Crohn's disease and pregnancy. Dis Colon
Rectum 33 (10), 869-873.
Xu Y, Tang Y, & Li Y. (2008). Effect of folic acid on prenatal alcohol-induced modification of
brain proteome in mice. Br J Nutr 99 (3), 455-461.
Yanaguita MY, Gutierrez CM, Ribeiro CN, Lima GA, Machado HR, & Peres LC. (2008).
Pregnancy outcome in ethanol-treated mice with folic acid supplementation in saccharose.
Childs Nerv Syst 24 (1), 99-104.
Yasuda S, Hasui S, Kobayashi M, Itagaki S, Hirano T, & Iseki K. (2008). The mechanism of
carrier-mediated transport of folates in BeWo cells: the involvement of heme carrier
protein 1 in placental folate transport. Biosci Biotechnol Biochem 72 (2), 329-334.
Yeboah D, Sun M, Kingdom J, Baczyk D, Lye SJ, Matthews SG et al. (2006). Expression of
breast cancer resistance protein (BCRP/ABCG2) in human placenta throughout gestation
and at term before and after labor. Can J Physiol Pharmacol 84 (12), 1251-1258.
Zakowski MI, Ham AA, & Grant GJ. (1994). Transfer and uptake of alfentanil in the human
placenta during in vitro perfusion. Anesth Analg 79 (6), 1089-1093.
Zeisel SH. (2011). What choline metabolism can tell us about the underlying mechanisms of
fetal alcohol spectrum disorders. Mol Neurobiol 44 (2), 185-191.
Zelner I, & Koren G. (2013). Alcohol consumption among women. J Popul Ther Clin
Pharmacol 20 (2), e201-206.
Zhang L, Connor EE, Chegini N, & Shiverick KT. (1995). Modulation by benzo[a]pyrene of
epidermal growth factor receptors, cell proliferation, and secretion of human chorionic
gonadotropin in human placental cell lines. Biochem Pharmacol 50 (8), 1171-1180.
Zins BJ, Sandborn WJ, McKinney JA, Mays DC, van Os EC, Tremaine WJ et al. (1997). A
dose-ranging study of azathioprine pharmacokinetics after single-dose administration of a
delayed-release oral formulation. J Clin Pharmacol 37 (1), 38-46.
161
Appendices
162
Appendix A: Placental Perfusion System Materials and Set-up
1. Materials 2. Maternal Ismatec Pump (Ismatec Instruments, Zurich Switzerland) 3. Fetal polystaltic SA 8031 pump (Buchler Instruments, (VWR Canlab) Missisauga
Canada) 4. Maternal Reservoir- 500 ml Flask 5. Fetal reservoir-chromatography Ampholine® column (type 8101) glass water jacket
(LKB Sweden) 6. Model 86 hot water bath (Precision Scientific, Chicago, USA) 7. Plexiglass Perfusion Chamber (Figure 5) 8. Buchi rotary evaporator- Brinkmann CH 9230 Rotavapor R (Laboratorium Technik AG,
Switzerland) 9. ABL 725 acid-base analysis laboratory (Radiometer A/S, Copenhagen Denmark) 10. Model 759 sphygmomanometer (Sunbeam Corp. (Canada) Ltd, Toronto, Canada) 11. Surgical instruments: fine and coarse scissors, 5.5" straight needle holder, 5.5"straight
Kelly clamps x 2, fine and coarse straight edge splinter forceps, German surgical 3.75" curved iris Castroviejo scissors (Irex, Toronto, Canada)
12. Three-way stop cocks x 13 13. Three hole rubber stopper x 1 14. # 5 French Catheter 15. # 8 French feeding tube 16. Blunt ended catheter 17. Narrow bore tubing (PE 280) 18. 19 gauge metal bulbous-tip cannula 19. 15 gauge metal bulbous-tip cannula 20. 1 cc and 3 cc syringes 21. 20 ml syringe 22. Oxygen- D tank (Praxair, Mississauga, Canada) with a pressure gauge, flow meter
regulator Model No ME1-540-FG (Medigas, Mississauga, Canada) 23. Carbon Dioxide- D tank (Praxair, Mississauga, Canada) with a pressure gauge, flow
meter regulator Model No ME1-540-FG (Medigas, Mississauga, Canada) 24. Nitrogen- C tank (Praxair, Mississauga, Canada) with a pressure gauge, flow meter
regulator Model No Ml-940 PG (Western Enterprises, Westlake, USA) 25. 4-0 black braided silk sutures with a 17 mm T-31 needles (Daves & Geek, Wayne, USA) 26. Catheter introducer (Becton-Dickinson, Rutherford, USA) 27. Gas dispersion tube 28. T-connector x 3 29. Y connector x 1 30. Ice-chest or cooler
163
2. Fetal Circuit Hardware Set-up
The fetal reservoir is a chromatography column glass water jacket, fitted with a three-
hole rubber stopper on the top and a three-way stopcock at the base as illustrated below in figure
1. A gas dispersion tube is inserted into the reservoir, through one of the three stopper holes, and
functions to both gas and mix the fetal perfusate. The fetal venous line is connected to a tube that
is inserted in a second of the three stopper holes. It returns perfusate to the reservoir for re-
circulation. Sampling the fetal perfusate from the reservoir is done through an umbilical catheter
(# 5 French) which is inserted in the third hole of the rubber stopper located at the top of the fetal
reservoir.
Tubing extending from the base of the reservoir leads to the fetal pump. From the pump,
the fetal circuit follows the following sequence: a flowmeter, then to a sampling port (three way
stopcock) from which fetal arterial (FA) samples are drawn. Distal to the sampling port, a
sphygmomanometer is connected to the fetal circuit tubing by a T-connector. From the
sphygmomanometer, the tubing leads to a connector located on the outside of the fetal perfusion
chamber. The FA cannula is connected to the inside of the chamber by a #5 French umbilical
vessel catheter with a short 19 gauge metal bulbous tip. The fetal vein (FV) cannula is also
connected to the inside of the chamber; it is a #8 French feeding tube with a short 15 gauge metal
bulbous tip. Tubing connected to the outside of the perfusion chamber at the venous port leads to
the FV sampling port (another three-way stopcock). From the FV sampling port, tubing extends
back to the fetal reservoir enabling the perfusate to be re-circulated or sampled (Figure 2).
164
Figure 1. Fetal reservoir setup
Figure 2. Configuration of fetal perfusion circuit.
165
3. Maternal Circuit Hardware Setup
The maternal reservoir is composed of a 500 ml flask attached to the steam duct of the
rotary evaporator (Figure 3). The maternal flask is rotated over the progress of the experiment
since this aids in the mixing and oxygenation of the maternal perfusate. There are three lines
inserted into the maternal reservoir: one line delivers oxygen into the maternal reservoir while
the other two lines deliver perfusate to and from the placenta. Perfusate samples from the
reservoir are withdrawn through a #5 French umbilical catheter that inserts into the maternal
reservoir. The maternal pump is a peristaltic pump that delivers perfusate to the placenta and
pumps maternal venous return from the placenta.
Prior to entering the placenta, the maternal perfusate enters a bubble trap consisting of a
20 ml syringe barrel fitted with a rubber stopper at the top (inflow). A small piece of gauze is put
inside the bubble trap to filter any tissue debris that could clog the maternal arterial cannulas.
From the bubble trap, the maternal artery (MA) sampling port (three way stopcock) branches off
the main line tubing by way of a T connector. Before reaching the perfusion chamber, the arterial
line branches at a Y-connector thereby creating two arterial inflows that enter the maternal
surface of the placenta. The MA cannulas are two #5 French umbilical catheters inserted into the
maternal surface of the placenta. The maternal venous (MV) return collects on the surface of the
lobule. The MV is suctioned off and returned to the maternal reservoir for re-circulation (Figure
4). The MV sampling port (three-way stopcock) branches off the main circuit tubing by way of
another T-connector.
166
Figure 3. Maternal reservoir Setup.
Figure 4. Configuration of maternal perfusion circuit.
167
4. Perfusion Chamber The plexiglass perfusion chamber consists of 3 individual sections (Figure 30):
1. The base of the perfusion chamber consists of three ports: FA port which leads perfusate to the reservoir and the FV leading perfusate away from the chamber. There is a third port used to fill the bottom section of the perfusion chamber with warmed phosphate buffered saline (amniotic fluid) once the placental tissue has been mounted.
2. The middle section of the chamber is a plexiglass collar that is used to firmly clamp the placental tissue to the bottom portion of the chamber. This causes the tissue surrounding the cotyledon of interest to be clamped into position within the chamber.
3. The top section of the perfusion chamber has three ports. Two of the ports support MA cannulas, which are attached to the inside of the chamber. The third port supports tubing that is attached on the inside of the chamber suctioning off maternal venous return to the maternal reservoir The top section of the perfusion chamber is securely clamped to the middle section enabling the maternal venous return to collect around the cotyledon without leaking out of the perfusion chamber.
Figure 5. Configuration of the perfusion chamber.
168
5. Composition of Medium 199 Culture Medium in Fetal and Maternal Perfusate
Compound mg/L Compound mg/L DL-Alanine 50.0 Thiamine HCl 0.01 L-Arginine ! HCl 70.0 Thymine 0.30 DL-Aspartic acid 60.0 Vitamin A acetate 0.14 L-Cysteine HCl ! H20 0.11 Xanthine ! Na 0.34 L-Cysteine ! 2HCl 26.0 Calcium chloride ! 2H2O 185.0 DL-Glutamic Acid 133.6 Ferric nitrate ! 9H2O 0.72 Glycine 50.0 Magnesium sulfate (anhydrous) 97.7 L-Histidine ! HCl ! H20 21.9 Potassium chloride 400.0 L-Hydroxyproline 10.0 Potassium phosphate 60.0 DL-Isoleucine 40.0 Sodium acetate (anhydrous) 50.0 DL-Leucine 120.0 Sodium chloride 8000.0 L-Lysine ! HCl 70.0 Sodium phosphate dibasic (anhydrous) 47.9 DL-Methionine 30.0 D-Glucose 1000.0 DL-Phylalanine 50.0 L-Proline 40.0 DL-Serine 50.0 DL-Threonine 60.0 DL-Tryptophan 20.0 L-Tyrosine ! 2Na 57.7 DL-Valine 50.0 Adenine sulfate 10.0 Adenosine triphosphate ! 2Na 1.0 Adenylic acid 0.20 α-Tocopherol phosphate ! 2Na 0.01 Ascorbic acid 0.05 Biotin 0.01 Calciferol 0.10 Cholestesterol 0.20 Choline chloride 0.50 Deoxyribose 0.50 Folic Acid 0.01 Glutathione (reduced) 0.50 Guanine ! HCl 0.30 Niacinamide 0.03 Nicotinic acid 0.03 PABA 0.05 D-Pantothenic acid ! Ca 0.01 Polyoxyethylene sorbitan 20.0 monooleate Pyridoxal HCl 0.03 Pyridoxine HCl 0.03 Riboflavin 0.01 Ribose 0.50
169
Appendix B: Systematic Review of Perfusion Data - Supplementary Table (Chapter 2) Supplementary Table. Therapeutics evaluated using the placental perfusion model and in vivo as identified through a systematic search. Data is presented as the mean fetal to maternal concentration ratio (F:M) in the placental perfusion model ex vivo and at the time of delivery in vivo as the mean umbilical cord blood to maternal blood concentration ratio (C:M). For the perfusion experiments, the amount of protein added to the perfusate is described. Analgesics
Perfusion In vivo Drug PB F:M [ ] on M SS Albumin Sys n Ref C:M Time after n Ref
last dose _____________________________________________________________________________________________________________________________________ ASA 50-80% 0.38 100µM Y 0.1% BSA C 11 (1) 0.39 12-123 min 9 (2) (SA) 50-80% 0.21 8µg/ml Y 2 g/l HSA O 3 (3) 0.75 12-123 min 9 (2)
1.6 8 hr 1 (4) >1 <20 hr 4 (5) >1 1-4 hr 115 (6)
~1.0*** 96 min 18 (7) _____________________________________________________________________________________________________________________________________ Diclofenac >99% ~0.6 20µg/ml Y 1 g/l BSA O 4 (8) 0.95*** 2 hr 30 (9)
0.12 100ng/ml Y 2 g/l HSA O 3 (10) _____________________________________________________________________________________________________________________________________ Buprenorphine 96% 0.12 10ng/ml Y No O 5 (11) 0.5 16.5 hr 1 (12)
0.48 10ng/ml Y No C 9 (11) <LOD 24 hr 1 (12) 0.59 10ng/ml Y 30g/l HSA C 5 (13) 0.47 10ng/ml Y M>F AAG C 4 (13) 0.43 10ng/ml Y HSA + AAG C 7 (13) 0.14 10ng/ml NR No O 4 (14)
_____________________________________________________________________________________________________________________________________ Methadone 71-88% 0.27 200ng/ml Y No O 8 (15) 0.21 SS 19 (16)
0.71 100ng/ml Y No C ≥5 (17) 0.58 SS 27 (18) 0.19*** 200ng/ml Y No O 8 (19)
<LOD 500ng/ml No 4% BSA C 6 (20) _____________________________________________________________________________________________________________________________________ Sulindac 93% 0.42 3.6µg/ml No 2-5g/dl HSA C 4 (21) 0.99 4.4-6.7 hr 9 (22)
0.34 64µg/ml No 2mg/ml HSA C 4 (23)
_____________________________________________________________________________________________________________________________________
170
Indomethacin 99% 0.45 32µg/ml No 2mg/ml HSA C 4 (23) 0.97 NR 26 (24) _____________________________________________________________________________________________________________________________________ Morphine 20-36% ~0.78 50ng/ml Y No C 4 (25) 0.96 5-74 min 5 (26)
~0.50 50ng/ml Y No O 2 (25) _____________________________________________________________________________________________________________________________________ Sufentanil 93% ~0.12 1ng/ml Y No O 10 (27) 0.38 ~45min 1 (28)
0.81 NR 8 (29)
_____________________________________________________________________________________________________________________________________ Alfentanil 92% 0.22 10ng/ml Y No O 10 (30) 0.29(0.97free) SS 10 (31) 0.29 6-10min PD 8 (28)
0.33 16-67 PI 6 (32) 0.35 9-25minPD 21 (33)
_____________________________________________________________________________________________________________________________________ Acetaminophen 10-30% 0.8 5-30µg/ml Y No C 4 (34) 0.78 overdose 1 (35)
>1 overdose 1 (36) _____________________________________________________________________________________________________________________________________ Anaesthetic
Perfusion In vivo Drug PB F:M [ ] on M SS Albumin Sys n Ref C:M Time after n Ref
last dose _____________________________________________________________________________________________________________________________________ Bupivacaine 95% 0.81 4µg/ml Y 2% HSA C 4 (37) 0.29 5-175 min 19 (38) 0.51 1µg/ml Y HP(M) / 4%HSA(F) C 6 (37) 0.32 28.5 min 10 (39) 0.73 4µg/ml Y 0.2% HSA O 4 (37) 0.35 0.58-3.5 hr 9 (40)
0.74 1µg/ml Y 2% HSA C 4 (41) 0.34 31 min 6 (42) 0.40 1µg/ml Y HP(M) / 4%HSA(F) C 4 (41) 0.24 3-135 min 31 (43)
0.56 2µg/ml Y 200mg/l HSA C 5 (44) 0.44 40-500 min 12 (45) 0.27 71 min 20 (46)
0.41 50 min 14 (47) 0.25 42 min 10 (48) 0.14 5-30 min 8 (49) 0.31 62 min 23 (50) 0.26(0.79free) 1-1.5 hr 10 (51) 0.25 NR 20 (52) 0.69(free) NR 29 (53) 0.29 38 min 28 (54) 0.37(0.77free) SS 12 (55)
171
_____________________________________________________________________________________________________________________________________ Lidocaine 33-80% 0.74 5µg/ml Y 2%HSA C 5 (56) 0.52 SS 1 (57)
1.10 5µg/ml Y HP(M) / 4%HSA(F) C 5 (56) 0.46 11 min 23 (58) 0.90 2µg/ml Y 200mg/l HSA C 5 (44) 0.48 10-71 min 18 (59) 0.55 7-39 min 9 (60) 0.54 40 min 11 (61)
0.69 NR 9 (62) 0.66 NR 19 (63) 0.48 30 min 29 (54) 0.37 15-18 min 10 (64)
_____________________________________________________________________________________________________________________________________ Ropivacaine 94% 0.82 1µg/ml Y 2%HSA C 4 (41) 0.28(0.81free) 77-103 min 11 (51)
0.42 1µg/ml Y HP(M) / 4%HSA(F) C 4 (41) 0.72(free) NR 31 (53) 0.30 1µg/ml Y 4%BSA O 6 (65) 0.31(0.74free) NR 9 (66)
0.33(0.66free) SS 12 (55) _____________________________________________________________________________________________________________________________________ Propofol 97-99% 0.51 15µg/ml Y 22g/l_M-44g/l_F O 6 (67) 0.65 5-14 min 20 (68)
0.13 15µg/ml Y 44g/l O 6 (67) 0.70 13-45 min 10 (69) 0.74 8.7-12.3min 8 (70) 0.85 4.0-7.4 min 10 (71) 0.72(0.2-1.7) 5-23 min 21 (72) 0.68 2-10min 13 (73) 0.22 4-10min 10 (74)
_____________________________________________________________________________________________________________________________________ Methohexital 80-85% 1.0 25µg/ml Y 2%HSA C 3 (75) -rapid transfer and approaches 1 1 (76)
0.66 25µg/ml Y M>F HSA C 9 (75) _____________________________________________________________________________________________________________________________________ Antidepressants
Perfusion In vivo Drug PB F:M [ ] on M SS Albumin Sys n Ref C:M Time after n Ref
last dose _____________________________________________________________________________________________________________________________________ Nortriptyline 86-95% 0.22 150 ng/ml Y 30 g/l BSA O 9 (77) 0.43 SS 1 (78)
0.68 SS 10 (79) _____________________________________________________________________________________________________________________________________ Citalopram 80% 0.30 1.23 µM Y 30g/l BSA O 8 (80) 0.71 SS 4 (81) 0.16 1.23 µM Y No O 5 (80) 0.83 SS 9 (82)
172
(Desmethylcitalopram) 0.19 0.60 µM Y 30g/l BSA O 8 (80) 0.54 SS 2 (81)
0.86 SS 9 (82) _____________________________________________________________________________________________________________________________________ Fluoxetine 95% 0.29 1.45 µM Y 30g/l BSA O 7 (80) 0.64 SS 15 (81) 0.04 1.45 µM Y No O 4 (80) 0.67 SS 4 (83) 0.91 SS 6 (84)
0.72 SS 2 (82) (Norfluoxetine) 0.30 1.23 µM Y 30g/l BSA O 7 (80) 0.65 SS 15 (81) 0.71 SS 4 (83)
1.04 SS 6 (84) 0.78 SS 2 (82)
_____________________________________________________________________________________________________________________________________ Antiepileptic
Perfusion In vivo Drug PB F:M [ ] on M SS Albumin Sys n Ref C:M Time after n Ref
last dose _____________________________________________________________________________________________________________________________________ Carbamazepine 76% 1.0 50µg/ml Y No C 6 (85) ~1 SS 16 (85)
~1 SS 5 (86) 0.73(1.42free) SS 7 (87) 0.78 SS 5 (88) 0.63*** 0.5->24hr 18 (89)
_____________________________________________________________________________________________________________________________________ Oxcarbazepine 40-60% ~1.1 50µg/ml Y No C 3 (90) ~1 SS 1 (91)
2.35 SS 3 (90) 2.40 SS 7 (92)
_____________________________________________________________________________________________________________________________________ Phenytoin 88-93% 0.73 0.16-0.5µg/ml Y No C 10 (93) 0.97 SS 3 (94)
0.94 0.7µg/ml Y No C 2 (95) 0.91 SS 3 (96) ~0.58 20µg/ml Y No O 4 (97) 0.91(1.1free) SS 7 (87)
~1 SS 8 (86) _____________________________________________________________________________________________________________________________________
173
Phenobarbital 20-60% 0.99 0.5µg/ml Y No C 2 (95) 1.05 SS 31 (98) ~1 SS 25 (99) 0.87 SS 23 (100) 0.95 SS 5 (94) 1.05 SS 25 (101) 0.86(1.13free) SS 13 (87) 0.84 SS 27 (102)
_____________________________________________________________________________________________________________________________________ Diazepam 94-99% 0.48 2µg/ml Y No C 4 (103) 1.31 0.1-9.5 hr 16 (104)
0.55 200ng/ml Y No C 3 (103) 1.73(0.92free) 1.5-3 hr 5 (105) 1.32 0.5-3.5 hr 7 (106)
1.46 ~15 hr 26 (107) 1.3 30-140 min 16 (108)
1.9 17-265 min 6 (108) 1.44 12 min 6 (109)
0.57 3-13 min 30 (110) 1.8 5-401min 37 (111) 1.69 NR 10 (112) >1*** 1-6 hr 8 (113) 0.84 3-13.5 min 30 (114) 2.4 0.6-10.9 hr 15 (115) 0.82 1-5 min 33 (116) 1.01 4-514 min 18 (117) >1 NR 20 (118)
_____________________________________________________________________________________________________________________________________ Valproic Acid 90% 0.90 80mg/l Y 0.2%BSA C 6 (119) 1.43 SS 1 (120) ~0.85 67µg/ml Y No C 4 (121) 1.7 SS 6 (122) ~0.50 20µg/ml Y No O 4 (121) 1.59 SS 8 (87)
1.71 SS 4 (94) 1.38 SS 18 (123)
_____________________________________________________________________________________________________________________________________ Lamotrigine 55% 0.83 2.5µg/ml Y No C 4 (124) 0.91 SS 51 (125)
1.26 10µg/ml Y No C 4 (124) 1.29 SS 2 (124) 0.9 SS 9 (126) 1.2 SS 1 (127) 0.90 SS 4 (128) 1.01 SS 6 (129)
_____________________________________________________________________________________________________________________________________
174
Antipsychotic Perfusion In vivo
Drug PB F:M [ ] on M SS Albumin Sys n Ref C:M Time after n Ref last dose
_____________________________________________________________________________________________________________________________________ Olanzapine 93% 0.20 10 ng/ml Y No C 3 (130) 0.32 SS 1 (131)
0.72 SS 13 (132) 0.3 SS 1 (133)
_____________________________________________________________________________________________________________________________________ Quetiapine 83% 0.12 75ng/ml Y 30g/l BSA O 6 (134) 0.24 SS 20 (132) _____________________________________________________________________________________________________________________________________ Antivirals
Perfusion In vivo Drug PB F:M [ ] on M SS Albumin Sys n Ref C:M Time after n Ref
last dose _____________________________________________________________________________________________________________________________________ Protease Inhibitors Indinavir 60% 0.06 7.6 mg/l Y No O 5 (135) <LOD 12h 21 (136)
0.04 7.6 mg/l Y No O 12 (137) 0.01 SS 4 (138) _____________________________________________________________________________________________________________________________________ Lopinavir 98-99% 0.30 1.1-10.6 mg/l NR 2g/l BSA O 6 (139) <250ng/ml SS 1 (140)
0.24 1.1-10.6 mg/L NR 10 g/l BSA O 3 (139) 0.22 SS 11 (141) 0.03 1.1-10.6 mg/L NR 40 g/l BSA O 3 (139) <LOD SS 11 (142)
0.23 SS 23 (143) 0.57 SS 6 (144) 0.2 SS 10 (145)
_____________________________________________________________________________________________________________________________________ Nelfinavir >98% 0.001 ~430 µg/l Y 2 g/l BSA O 3 (146) 0-0.3 SS 9 (140)
0.14 ~1.7 mg/l Y 2 g/l BSA O 6 (146) 0.24 SS 8 (141) 0.14 ~4.4 mg/ l Y 2 g/l BSA O 4 (146) 0.37 SS 7 (142)
0.49 SS 3 (144) 0.25 SS 73 (147) 0.24 SS 39 (138)
_____________________________________________________________________________________________________________________________________ Ritonavir 98-99% <LOD 1-2 µg/ml NR No C 2 (148) <LOD SS 2 (140)
0.12 0.3-1.1mg/l NR 2 g/l BSA O 3 (139) 0.31 SS 1 (141) 0.08 0.3-1.1mg/l NR 10 g/l BSA O 3 (139) 0.11 SS 15 (141) 0.00 0.3-1.1mg/l NR 40 g/l BSA O 3 (139) 0.55 SS 6 (144)
0.00 SS 10 (138)
175
_____________________________________________________________________________________________________________________________________ Saquinavir 97% 0.02 322-2197 ng/ml Y 2 g/l HSA O 10 (149) <LOD SS 1 (140)
0.04 SS 4 (141) <LOD SS 6 (138)
_____________________________________________________________________________________________________________________________________ Nucleoside Analogue Acyclovir 9-33% ~0.43 1µg/ml No 0.2g/L C 4 (150) 0.77 SS 10 (151)
0.16 1µg/ml NR 3%BSA C 4 (152) 0.74 SS 12 (153) 0.09 10µg/ml NR 3%BSA C 4 (152) 0.92 SS 5 (154) 0.89 100µg/ml NR 3%BSA C 2 (152) _____________________________________________________________________________________________________________________________________ Dideoxyinosine <5% ~0.35 1µM NR 0.1g/dl BSA O 5 (155) 0.38 SS 10 (156) (DDI) ~0.39 500µM NR 0.1g/dl BSA O 5 (155) 0.17 1 hr 2 (157)
~0.56 30µM No 2 g/l BSA C 5 (158)
_____________________________________________________________________________________________________________________________________ Lamivudine <36% 0.21 1.39µg/l NR 3% BSA C 3 (159) 1.12 SS 16 (160)
0.11 14.68µg/l NR 3% BSA C 3 (159) 0.96 SS 59 (156) 1.00 SS 10 (144) 1.31 2.3-35 hr 50 (161)
_____________________________________________________________________________________________________________________________________ Zidovudine <38% 0.90 1.0mg/ml No NR C 2 (162) 1.13-1.27 SS 2 (163)
~1.0 3.8mM Y N C 4 (164) 1.22 SS 75 (156) ~0.75 3µM No 0.2g/L HSA C 3 (165) 0.86 SS 10 (144) ~0.45 3µM No 0.1g/dl C 5 (166) 1.89*** 60 min 7 (167)
~1.0 10µM Y N C 5 (168) 3.55*** 2.5&3hr 2 (169) 1.25 0.25-4.5hr 7 (170)
_____________________________________________________________________________________________________________________________________ Antiretroviral Fusion Inhibitor Enfuvirtide 92% <LOD 12.4µg/ml** NR 2g/L HSA O 3 (171) <LOD SS 2 (172) _____________________________________________________________________________________________________________________________________
176
Asthma Perfusion In vivo
Drug PB F:M [ ] on M SS Albumin Sys n Ref C:M Time after n Ref last dose
_____________________________________________________________________________________________________________________________________ Theophylline 40% 0.33 15mg/l Y 0.2%BSA O 8 (173) 1.06 SS 9 (174)
0.88 15mg/l Y 0.2%BSA C 6 (175) 0.96 5&9 hr 2 (176) 1.10 SS 10 (177) 0.91 <6hr 3 (178)
_____________________________________________________________________________________________________________________________________ Salbutamol 10% 0.12 2µg/ml Y No O 7 (179) 0.70 27-105min 5 (180) _____________________________________________________________________________________________________________________________________ H-blockers
Perfusion In vivo Drug PB F:M [ ] on M SS Albumin Sys n Ref C:M Time after n Ref
last dose _____________________________________________________________________________________________________________________________________ Cimetidine 13-26% ~0.15 8µg/ml No 1 g/L BSA O 4 (8) 0.39 5-780min 17 (181) 0.46 4µg/ml No 0.1% BSA C 4 (182) 0.36 1-2hr 8 (183)
~0.5 1µg/ml No 2%HSA+13%HP C 6 (184) 0.6 1-1.5hr 16 (185) 0.8 1.5-2hr 16 (185)
_____________________________________________________________________________________________________________________________________ Ranitidine 15% ~0.4 60µg/ml No 2%HSA+13%HP C 3 (184) 0.9 0.5-8 hr (IV) 20 (186)
0.38 1-9 hr (oral) 45 (186) 0.95 5 hr 1 (187)
_____________________________________________________________________________________________________________________________________ Antimicrobials
Perfusion In vivo Drug PB F:M [ ] on M SS Albumin Sys n Ref C:M Time after n Ref
last dose _____________________________________________________________________________________________________________________________________ Pyrimethamine 87% 0.30 0.8mg/l NS 0-20g/l O 2 (188) 0.66 0.5-13 hr 10 (189)
0.30 5mg/l NS 0-20g/l O 11 (188) 0.5-1.0 SS 4 (190) 0.32 10mg/l NS 0-20g/l O 2 (188)
_____________________________________________________________________________________________________________________________________
177
Erythromycin 75-90% 0.10 2µg/ml Y N O 7 (191) 0.22 SS 12 (192) 0.06 NR 4 (193)
_____________________________________________________________________________________________________________________________________ Azithromycin 7-50% 0.09 0.3µg/ml Y N O 7 (191) limited transfer 6-168 hr 20 (194) _____________________________________________________________________________________________________________________________________ Ofloxacin 20-32% 0.07 10µg/ml No 3g/l C 6 (195) 0.8-0.9 3-4 hr 11 (196) 0.6-0.7 NR 8 (197) _____________________________________________________________________________________________________________________________________ Ticarcillin 45% 0.02 10-94µg/ml No 3g/dl BSA O 5 (198) 0.6 NR 5 (199) Clavulanic acid 25% <LOD 2-6µg/ml No 3g/dl BSA O 4 (198) 0.8 NR 5 (199)
0.05 10µg/ml No 3g/dl BSA O 4 (198) _____________________________________________________________________________________________________________________________________ Ceftizoxime 28-50% 0.03 56-166µg/ml No 3% BSA O 6 (200) 1.6 1-4 hr 20 (201) _____________________________________________________________________________________________________________________________________ Sulbactam 38% 0.03 40µg/ml N 3g/dl BSA O 4 (202) 1.3 NR 5 (199)
0.04 27-166µg/ml N 3g/dl BSA O 4 (202) _____________________________________________________________________________________________________________________________________ Diabetic Agents
Perfusion In vivo Drug PB F:M [ ] on M SS Albumin Sys n Ref C:M Time after n Ref
last dose _____________________________________________________________________________________________________________________________________ Insulin Lispro <LOD 100µU/ml N/A No O 4 (203) <LOD NR 4 (204)
<LOD 200µU/ml N/A No O 1 (203) <LOD 100µU/ml N/A NR C 4 (205) _____________________________________________________________________________________________________________________________________ Metformin 0.55 5µg/ml Y No C 12 (206) ~1.0?? 3.5-336 hr 7 (207)
0.16 5µg/ml Y No O 6 (206) 2.0 4-32 hr 8 (208) ~0.30 1µg/ml Y 3g/l C 6 (209) 0.11 1µg/ml NR 3g/l C 7 (210) 0.17 10mg/ml NR 3g/l C 2 (210)
_____________________________________________________________________________________________________________________________________
178
Glyburide 99% 0.006 1µg/ml NR 2g/dl C 3 (211) <LOD 8 hr 12 (212) 0.02 20µg/ml NR 2g/dl C 1 (211) 0.7 NR 66 (213)
0.26 150ng/ml NR No O 6 (214) 0.29 150ng/ml NR 42µg/mlHSA O 2 (214) 0.13 150ng/ml NR 210µg/mlHSA O 2 (214) 0.11 150ng/ml NR 630µg/mlHSA O 2 (214) 0.03 150ng/ml NR 2.1mg/mlHSA O 2 (214) 0.09 150ng/ml N No C 4 (215) 0.03 150ng/ml Y 30mg/mlHSA C 3 (215)
_____________________________________________________________________________________________________________________________________ Cardiac Drugs
Perfusion In vivo Drug PB F:M [ ] on M SS Albumin Sys n Ref C:M Time after n Ref
last dose _____________________________________________________________________________________________________________________________________ Digoxin 20-25% 0.39 5ng/ml No No C 4 (216) 0.26 SS 1 (217)
0.11 5.5ng/ml Y 3g/L BSA C 6 (218) 0.97 NR 3 (219) <LOD 5.5ng/ml Y 1mg/ml BSA C 5 (220) ~1 5-7 hr 7 (221) 0.11 5.5ng/ml Y 3mg/ml BSA C 4 (220) 0.27 SS/C 1 (222) 0.12 5.5ng/ml Y 5mg/ml BSA C 4 (220) 0.97*** 0.25-24 hr 6 (223) 0.70 5.7ng/ml No 21g/l BSA C 5 (224) 0.62 SS 1 (225) 0.61 5.7ng/ml No M>F BSA C 5 (224)
0.73 5.7ng/ml No 3g/l BSA C 5 (224) _____________________________________________________________________________________________________________________________________ Enalaprilat 50-60% ~0.7 150ng/ml No No C 5 (226) 0.59 NS 1 (226) _____________________________________________________________________________________________________________________________________ Clonidine 20-40% 0.85 4.4nM No 200mg/l HSA C 4 (227) ~1 SS 10 (228)
0.87 1-13.5 hr 5 (229) 1.0 SS 6 (230) 0.92 117 min 8 (231)
_____________________________________________________________________________________________________________________________________ Fondaparinux 94% <LOD 1.75 mg/l N/A 10g/l HSA O 6 (232) 0.1 22hrPD-SS 5 (233)
<LOD >24hr 1 (234) <0.07 >24hr 1 (235)
_____________________________________________________________________________________________________________________________________
179
Heparin 0.004 650IU/ml No No C 12 (236) NC 15-170 min 7 (237) _____________________________________________________________________________________________________________________________________ Low Molecular Weight Heparins Enoxaparin 80% <LOD 1 IU/mL N/A 10g/l HSA O 6 (232) NC*** 3 hr 14 (238) Other LMWHs NC*** 3 hr 5 (239)
NC*** 3 hr 7 (240) <LOD, NC*** 3&15 hr 15 (241) NC*** 3-7 hr 9 (242)
_____________________________________________________________________________________________________________________________________ Nitroglycerin 11-60% 0.19 100 nmol/l Y No O 6 (243) <0.01 1 min 32 (244) _____________________________________________________________________________________________________________________________________ Hydralazine 88-90% 0.58 15µg/ml NR No O 4 (245) ~1 SS 4 (246) 0.30 115µg/ml NR No O 4 (245) 0.40 750µg/ml NR No O 4 (245) _____________________________________________________________________________________________________________________________________ Atenolol <5% 0.09 5µg/ml Y 0.02&4g/dlHSA O 6 (247) 0.88 SS 6 (248)
~0.85 5µg/ml No 2g/dlHSA C 3 (247) _____________________________________________________________________________________________________________________________________ Celiprolol 22-24% 0.05 5µg/ml Y 0.02&4g/dlHSA O 2 (247) 0.28 SS 4 (249)
0.11 5µM NR No O 5 (250) _____________________________________________________________________________________________________________________________________ Labetalol 50% 0.27 10µg/ml Y 0.02g/dlHSA O 3 (247) ~0.5 NR 25 (251)
0.22 1µg/ml Y 4g/dl HSA O 2 (247) 0.10 6nmol/ml Y No NR 6 (252)
_____________________________________________________________________________________________________________________________________ Propranolol 93% 0.21 5µg/ml Y 0.02g/dl HSA O 5 (247) 0.26 3 hr 8 (253)
0.32 5µg/ml Y 4g/dl HSA O 5 (247) ~1 5µg/ml Y 2g/dl HSA C 3 (247) ~1.38 5µg/ml Y M<F HSA C 3 (247)
_____________________________________________________________________________________________________________________________________ Nifedipine 90-96% 0.18 0.2µM NS No O 5 (250) 0.76 2.5 hr 10 (254) _____________________________________________________________________________________________________________________________________
180
Immunological Agents Perfusion In vivo
Drug PB F:M [ ] on M SS Albumin Sys n Ref C:M Time after n Ref last dose
_____________________________________________________________________________________________________________________________________ Cyclosporin 90% <0.05 1.66µM NR No NR 6 (255) 0.56 8&10 hr 2 (256)
0.2 6 hr 1 (257) ~1 20 hr 1 (258)
Endocrine Agents
Perfusion In vivo Drug PB F:M [ ] on M SS Albumin Sys n Ref C:M Time after n Ref
last dose _____________________________________________________________________________________________________________________________________ Methimazole ~1 1.5µg/ml Y No C 3 (259) 0.77*** 2 hr 2 (260)
~1 15µg/ml Y No C 3 (259) ~1 1.5µg/ml Y 40g/l BSA C 3 (259)
_____________________________________________________________________________________________________________________________________ Propylthiouracil 80% ~1 4µg/ml Y No C 3 (259) 0.31*** 2 hr 2 (260)
~1 40µg/ml Y No C 3 (259) 1.99 1-9 hr 5 (261) ~1 4µg/ml Y 40g/l BSA C 3 (259)
_____________________________________________________________________________________________________________________________________ Tocolytic
Perfusion In vivo Drug PB F:M [ ] on M SS Albumin Sys n Ref C:M Time after n Ref
last dose _____________________________________________________________________________________________________________________________________ Ritodrine 32-38% 0.16 2µg/ml Y No O 4 (262) 0.58 NR 9 (263)
0.40 1.4-2.8µg/ml No 20g/L HSA C 8 (264) 1.17 10-990 min 28 (265) 0.30 120 min 8 (266) 0.70 SS 5 (267) 0.26 45-190 min 7 (268) 1.24 SS 19 (269)
_____________________________________________________________________________________________________________________________________ PB=protein binding [ ] on M= concentration of drug added into the maternal perfusate Sys=System configuration for perfusion experiment
181
BSA=bovine serum albumin HSA=human serum albumin HP=human plasma O=open C=closed NC=no change in anticoagulation activity compared to a control group No change in anti-Xa or anti-IIa activity ~ =calculated/estimated from data provided AAG=α1-acid glycoprotein NR=not reported SS=steady state ***placenta or in vivo results are from preterm pregnancies
182
Supplementary Table References
1. Jacobson, R.L., Brewer, A., Eis, A., Siddiqi, T.A., Myatt, L. Transfer of aspirin across the perfused human placental cotyledon. Am J Obstet Gynecol 165, 939-944 (1991).
2. Wolff, F., Berg, R., Bolte, A., Putter, J. Perinatal pharmacokinetics of acetylsalicylic acid. Arch Gynecol 233, 15-22 (1982).
3. Shintaku, K. et al. Kinetic analysis of the transport of salicylic acid, a nonsteroidal anti-inflammatory drug, across human placenta. Drug Metab Dispos 35, 772-778 (2007).
4. Garrettson, L.K., Procknal, J.A., Levy, G. Fetal acquisition and neonatal elimination of a large amount of salicylate. Study of a neonate whose mother regularly took therapeutic doses of aspirin during pregnancy. Clin Pharmacol Ther 17, 98-103 (1975).
5. Levy, G., Garrettson, L.K. Kinetics of salicylate elimination by newborn infants of mothers who ingested aspirin before delivery. Pediatrics 53, 201-210 (1974).
6. Noschel, H., Bonow, A., Moller, R., Estel, C., Muller, B. [Placental passage of sodium salicylate]. Zentralbl Gynakol 94, 437-442 (1972).
7. Elis, J., Sechserova, M., Stribrny, J., Drabkova, J. The distribution of sodium salicylate in the human fetus. Int J Clin Pharmacol Biopharm 16, 365-367 (1978).
8. Bassily, M., Ghabrial, H., Smallwood, R.A., Morgan, D.J. Determinants of placental drug transfer: Studies in the isolated perfused human placenta. J Pharm Sci 84, 1054-1060 (1995).
9. Siu, S.S., Yeung, J.H., Lau, T.K. A study on placental transfer of diclofenac in first trimester of human pregnancy. Hum Reprod 15, 2423-2425 (2000).
10. Shintaku, K. et al. Transplacental pharmacokinetics of diclofenac in perfused human placenta. Drug Metab Dispos 37, 962-968 (2009).
11. Nanovskaya, T., Deshmukh, S., Brooks, M., Ahmed, M.S. Transplacental transfer and metabolism of buprenorphine. J Pharmacol Exp Ther 300, 26-33 (2002).
12. Concheiro, M., Jones, H.E., Johnson, R.E., Choo, R., Shakleya, D.M., Huestis, M.A. Maternal buprenorphine dose, placenta buprenorphine, and metabolite concentrations and neonatal outcomes. Ther Drug Monit 32, 206-215 (2010).
13. Nanovskaya, T.N., Bowen, R.S., Patrikeeva, S.L., Hankins, G.D., Ahmed, M.S. Effect of plasma proteins on buprenorphine transfer across dually perfused placental lobule. J Matern Fetal Neonatal Med 22, 646-653 (2009).
14. Nekhayeva, I.A., Nanovskaya, T.N., Hankins, G.D.V., Ahmed, M.S. Role of human placental efflux transporter P-glycoprotein in the transfer of buprenorphine, levo-alpha-acetylmethadol, and paclitaxel. Am J Perinatol 23, 423-430 (2006).
183
15. Nanovskaya, T., Nekhayeva, I., Karunaratne, N., Audus, K., Hankins, G.D.V., Ahmed, M.S. Role of P-glycoprotein in transplacental transfer of methadone. Biochem Pharmacol 69, 1869-1878 (2005).
16. Harper, R.G., Solish, G., Feingold, E., Gersten-Woolf, N.B., Sokal, M.M. Maternal ingested methadone, body fluid methadone, and the neonatal withdrawal syndrome. Am J Obstet Gynecol 129, 417-424 (1977).
17. Nekhayeva, I.A., Nanovskaya, T.N., Deshmukh, S.V., Zharikova, O.L., Hankins, G.D., Ahmed, M.S. Bidirectional transfer of methadone across human placenta. Biochem Pharmacol 69, 187-197 (2005).
18. Rosen, T.S., Pippenger, C.E. Disposition of methadone and its relationship to severity of withdrawal in the newborn. Addict Dis 2, 169-178 (1975).
19. Nanovskaya, T.N., Nekhayeva, I.A., Hankins, G.D.V., Ahmed, M.S. Transfer of methadone across the dually perfused preterm human placental lobule. Am J Obstet Gynecol 198, (2008).
20. Malek, A., Obrist, C., Wenzinger, S., von Mandach, U. The impact of cocaine and heroin on the placental transfer of methadone. Reprod Biol Endocrinol 7, 61 (2009).
21. Higby, K., Elliott, B., King, T.S., Frasier, D., Langer, O. Human placental transfer of the prostaglandin inhibitor sulindac using an in vitro model. J Soc Gynecol Investig 2, 526-530 (1995).
22. Kramer, W.B. et al. Placental transfer of sulindac and its active sulfide metabolite in humans. Am J Obstet Gynecol 172, 886-890 (1995).
23. Lampela, E.S. et al. Placental transfer of sulindac, sulindac sulfide, and indomethacin in a human placental perfusion model. Am J Obstet Gynecol 180, 174-180 (1999).
24. Moise, K.J.J., Ou, C.N., Kirshon, B., Cano, L.E., Rognerud, C., Carpenter, R.J.J. Placental transfer of indomethacin in the human pregnancy. Am J Obstet Gynecol 162, 549-554 (1990).
25. Kopecky, E.A., Simone, C., Knie, B., Koren, G. Transfer of morphine across the human placenta and its interaction with naloxone. Life Sci 65, 2359-2371 (1999).
26. Gerdin, E., Rane, A., Lindberg, B. Transplacental transfer of morphine in man. J. Perinat. Med. 18, 305-312 (1990).
27. Krishna, B.R., Zakowski, M.I., Grant, G.J. Sufentanil transfer in the human placenta during in vitro perfusion. Can J Anaesth 44, 996-1001 (1997).
28. Meuldermans, W., Woestenborghs, R., Noorduin, H., Camu, F., van Steenberge, A., Heykants, J. Protein binding of the analgesics alfentanil and sufentanil in maternal and neonatal plasma. Eur J Clin Pharmacol 30, 217-219 (1986).
184
29. Loftus, J.R., Hill, H., Cohen, S.E. Placental transfer and neonatal effects of epidural sufentanil and fentanyl administered with bupivacaine during labor. Anesthesiology 83, 300-308 (1995).
30. Zakowski, M.I., Ham, A.A., Grant, G.J. Transfer and uptake of alfentanil in the human placenta during in vitro perfusion. Anesth Analg 79, 1089-1093 (1994).
31. Gepts, E., Heytens, L., Camu, F. Pharmacokinetics and placental transfer of intravenous and epidural alfentanil in parturient women. Anesth Analg 65, 1155-1160 (1986).
32. Heytens, L., Cammu, H., Camu, F. Extradural analgesia during labour using alfentanil. Br J Anaesth 59, 331-337 (1987).
33. Cartwright, D.P., Dann, W.L., Hutchinson, A. Placental transfer of alfentanil at caesarean section. Eur J Anaesthesiol 6, 103-109 (1989).
34. Weigand, U.W., Chou, R.C., Maulik, D., Levy, G. Assessment of biotransformation during transfer of propoxyphene and acetaminophen across the isolated perfused human placenta. Pediatr Pharmacol 4, 145-153 (1984).
35. Roberts, I., Robinson, M.J., Mughal, M.Z., Ratcliffe, J.G., Prescott, L.F. Paracetamol metabolites in the neonate following maternal overdose. Br J Clin Pharmacol 18, 201-206 (1984).
36. Sancewicz-Pach, K., Chmiest, W., Lichota, E. Suicidal paracetamol poisoning of a pregnant woman just before a delivery. Przegl Lek 56, 459-462 (1999).
37. Johnson, R.F., Herman, N., Arney, T.L., Gonzalez, H., Johnson, H.V., Downing, J.W. Bupivacaine transfer across the human term placenta: A study using the dual perfused human placental model. Anesthesiology 82, 459-468 (1995).
38. Decocq, G. et al. Serum bupivacaine concentrations and transplacental transfer following repeated epidural administrations in term parturients during labour. Fundam Clin Pharmacol 11, 365-370 (1997).
39. de Barros Duarte, L., Moises, E.C., Carvalho Cavalli, R., Lanchote, V.L., Duarte, G., Pereira da Cunha, S. Placental transfer of bupivacaine enantiomers in normal pregnant women receiving epidural anesthesia for cesarean section. Eur J Clin Pharmacol 63, 523-526 (2007).
40. Papini, O., Mathes, A.C., Cunha, S.P., Lanchote, V.L. Stereoselectivity in the placental transfer and kinetic disposition of racemic bupivacaine administered to parturients with or without a vasoconstrictor. Chirality 16, 65-71 (2004).
41. Johnson, R.F. et al. A comparison of the placental transfer of ropivacaine versus bupivacaine. Anesth Analg 89, 703-708 (1999).
185
42. Kuhnert, B.R., Zuspan, K.J., Kuhnert, P.M., Syracuse, C.D., Brown, D.E. Bupivacaine disposition in mother, fetus, and neonate after spinal anesthesia for cesarean section. Anesth Analg 66, 407-412 (1987).
43. Thomas, J., Long, G., Moore, G., Morgan, D. Plasma protein binding and placental transfer of bupivacaine. Clin Pharmacol Ther 19, 426-434 (1976).
44. Ala-Kokko, T.I., Pienimaki, P., Herva, R., Hollmen, A.I., Pelkonen, O., Vahakangas, K. Transfer of lidocaine and bupivacaine across the isolated perfused human placenta. Pharmacol Toxicol 77, 142-148 (1995).
45. Thomas, J., Climie, C.R., Mather, L.E. The maternal plasma levels and placental transfer of bupivacaine following epidural analgesia. Br J Anaesth 41, 1035-1040 (1969).
46. Scanlon, J.W., Ostheimer, G.W., Lurie, A.O., Brown wu, J.R., Weiss, J.B., Alper, M.H. Neurobehavioral responses and drug concentrations in newborns after maternal epidural anesthesia with bupivacaine. Anesthesiology 45, 400-405 (1976).
47. Magno, R., Berlin, A., Karlsson, K., Kjellmer, I. Anesthesia for cesarean section IV: placental transfer and neonatal elimination of bupivacaine following epidural analgesia for elective cesarean section. Acta Anaesthesiol Scand 20, 141-146 (1976).
48. McGuinness, G.A., Merkow, A.J., Kennedy, R.L., Erenberg, A. Epidural anesthesia with bupivacaine for Cesarean section: neonatal blood levels and neurobehavioral responses. Anesthesiology 49, 270-273 (1978).
49. Beazley, J.M., Taylor, G., Reynolds, F. Placental transfer of bupivacaine after paracervical block. Obstet. Gynecol. 39, 2-6 (1972).
50. Reynolds, F., Taylor, G. Maternal and neonatal blood concentrations of bupivacaine: a comparison with lignocaine during continuous extradural analgesia. Anaesthesia 25, 14-23 (1970).
51. Ala-Kokko, T.I., Alahuhta, S., Jouppila, P., Korpi, K., Westerling, P., Vahakangas, K. Feto-maternal distribution of ropivacaine and bupivacaine after epidural administration for cesarean section. Int J Obstet Anesth 6, 147-152 (1997).
52. Bader, A.M., Tsen, L.C., Camann, W.R., Nephew, E., Datta, S. Clinical effects and maternal and fetal plasma concentrations of 0.5% epidural levobupivacaine versus bupivacaine for cesarean delivery. Anesthesiology 90, 1596-1601 (1999).
53. Datta, S., Camann, W., Bader, A., VanderBurgh, L. Clinical effects and maternal and fetal plasma concentrations of epidural ropivacaine versus bupivacaine for cesarean section. Anesthesiology 82, 1346-1352 (1995).
54. Loftus, J.R., Holbrook, R.H., Cohen, S.E. Fetal heart rate after epidural lidocaine and bupivacaine for elective cesarean section. Anesthesiology 75, 406-412 (1991).
186
55. Irestedt, L., Ekblom, A., Olofsson, C., Dahlstrom, A.C., Emanuelsson, B.M. Pharmacokinetics and clinical effect during continuous epidural infusion with ropivacaine 2.5 mg/ml or bupivacaine 2.5 mg/ml for labour pain relief. Acta Anaesthesiol Scand 42, 890-896 (1998).
56. Johnson, R.F. et al. Transfer of lidocaine across the dual perfused human placental cotyledon. Int J Obstet Anesth 8, 17-23 (1999).
57. Banzai, M., Sato, S., Tezuka, N., Komiya, H., Chimura, T., Hiroi, M. Placental transfer of lidocaine hydrochloride after prolonged continuous maternal intravenous administration. Can J Anaesth 42, 338-340 (1995).
58. de Carvalho Cavalli, R. et al. Pharmacokinetics and transplacental transfer of lidocaine and its metabolite for perineal analgesic assistance to pregnant women. Eur J Clin Pharmacol 60, 569-574 (2004).
59. Thomas, J., Climie, C.R., Long, G., Nighjoy, L.E. The influence of adrenaline on the maternal plasma levels and placental transfer of lignocaine following lumbar epidural administration. Br J Anaesth 41, 1029-1034 (1969).
60. Shnider, S.M., Way, E.L. The kinetics of transfer of lidocaine (Xylocaine) across the human placenta. Anesthesiology 29, 944-950 (1968).
61. Guay, J., Gaudreault, P., Boulanger, A., Tang, A., Lortie, L., Dupuis, C. Lidocaine hydrocarbonate and lidocaine hydrochloride for cesarean section: transplacental passage and neonatal effects. Acta Anaesthesiol Scand 36, 722-727 (1992).
62. Kileff, M.E., James, F.M.r., Dewan, D.M., Floyd, H.M. Neonatal neurobehavioral responses after epidural anesthesia for cesarean section using lidocaine and bupivacaine. Anesth Analg 63, 413-417 (1984).
63. Abboud, T.K., Sarkis, F., Blikian, A., Varakian, L., Earl, S., Henriksen, E. Lack of adverse neonatal neurobehavioral effects of lidocaine. Anesth Analg 62, 473-475 (1983).
64. Kuhnert, B.R., Philipson, E.H., Pimental, R., Kuhnert, P.M., Zuspan, K.J., Syracuse, C.D. Lidocaine disposition in mother, fetus, and neonate after spinal anesthesia. Anesth Analg 65, 139-144 (1986).
65. Zuo, M., Duan, G.-L., Ge, Z.-G. Simultaneous determination of ropivacaine and antipyrine by high performance liquid chromatography and its application to the in vitro transplacental study. Biomed Chromatogr 18, 752-755 (2004).
66. Morton, C.P., Bloomfield, S., Magnusson, A., Jozwiak, H., McClure, J.H. Ropivacaine 0.75% for extradural anaesthesia in elective caesarean section: an open clinical and pharmacokinetic study in mother and neonate. Br J Anaesth 79, 3-8 (1997).
67. He, Y.L., Seno, H., Sasaki, K., Tashiro, C. The influences of maternal albumin concentrations on the placental transfer of propofol in human dually perfused cotyledon in vitro. Anesth Analg 94, 1312-1314 (2002).
187
68. Gin, T., Gregory, M.A., Chan, K., Oh, T.E. Maternal and fetal levels of propofol at caesarean section. Anaesth Intensive Care 18, 180-184 (1990).
69. Dailland, P. et al. Intravenous propofol during cesarean section: placental transfer, concentrations in breast milk, and neonatal effects. A preliminary study. Anesthesiology 71, 827-834 (1989).
70. Gin, T., Yau, G., Jong, W., Tan, P., Leung, R.K., Chan, K. Disposition of propofol at caesarean section and in the postpartum period. Br J Anaesth 67, 49-53 (1991).
71. Valtonen, M., Kanto, J., Rosenberg, P. Comparison of propofol and thiopentone for induction of anaesthesia for elective caesarean section. Anaesthesia 44, 758-762 (1989).
72. Moore, J., Bill, K.M., Flynn, R.J., McKeating, K.T., Howard, P.J. A comparison between propofol and thiopentone as induction agents in obstetric anaesthesia. Anaesthesia 44, 753-757 (1989).
73. Kanto, J., Rosenberg, P. Propofol in cesarean section. A pharmacokinetic and pharmacodynamic study. Methods Find Exp Clin Pharmacol 12, 707-711 (1990).
74. Sanchez-Alcaraz, A., Quintana, M.B., Laguarda, M. Placental transfer and neonatal effects of propofol in caesarean section. J Clin Pharm Ther 23, 19-23 (1998).
75. Herman, N.L. et al. Transfer of methohexital across the perfused human placenta. J Clin Anesth 12, 25-30 (2000).
76. Marshall, J.R. Human antepartum placental passage of methohexital sodium. Obtet Gynecol 23, 589-592 (1964).
77. Heikkinen, T., Ekblad, U., Laine, K. Transplacental transfer of amitriptyline and nortriptyline in isolated perfused human placenta. Psychopharmacology 153, 450-454 (2001).
78. Sjoqvist, F., Bergfors, P.G., Borga, O., Lind, M., Ygge, H. Plasma disappearance of nortriptyline in a newborn infant following placental transfer from an intoxicated mother: evidence for drug metabolism. J Pediatr 80, 496-500 (1972).
79. Loughhead, A.M., Stowe, Z.N., Newport, D.J., Ritchie, J.C., DeVane, C.L., Owens, M.J. Placental passage of tricyclic antidepressants. Biol Psychiatry 59, 287-290 (2006).
80. Heikkinen, T., Ekblad, U., Laine, K. Transplacental transfer of citalopram, fluoxetine and their primary demethylated metabolites in isolated perfused human placenta. BJOG 109, 1003-1008 (2002).
81. Hendrick, V., Stowe, Z.N., Altshuler, L.L., Hwang, S., Lee, E., Haynes, D. Placental passage of antidepressant medications. Am J Psychiatry 160, 993-996 (2003).
82. Rampono, J. et al. Placental transfer of SSRI and SNRI antidepressants and effects on the neonate. Pharmacopsychiatry 42, 95-100 (2009).
188
83. Rampono, J., Proud, S., Hackett, L.P., Kristensen, J.H., Ilett, K.F. A pilot study of newer antidepressant concentrations in cord and maternal serum and possible effects in the neonate. Int J Neuropsychopharmacol 7, 329-334 (2004).
84. Kim, J. et al. Stereoselective disposition of fluoxetine and norfluoxetine during pregnancy and breast-feeding. Br J Clin Pharmacol 61, 155-163 (2006).
85. Pienimaki, P. et al. Carbamazepine and its metabolites in human perfused placenta and in maternal and cord blood. Epilepsia 36, 241-248 (1995).
86. Bardy, A.H., Hiilesmaa, V.K., Teramo, K., Neuvonen, P.J. Protein binding of antiepileptic drugs during pregnancy, labor, and puerperium. Ther Drug Monit 12, 40-46 (1990).
87. Takeda, A., Okada, H., Tanaka, H., Izumi, M., Ishikawa, S., Noro, T. Protein binding of four antiepileptic drugs in maternal and umbilical cord serum. Epilepsy Res. 13, 147-151 (1992).
88. Kuhnz, W. et al. Carbamazepine and carbamazepine-10,11- epoxide during pregnancy and postnatal period in epileptic mother and their nursed infants: pharmacokinetics and clinical effects. Pediatr Pharmacol (New York) 3, 199-208 (1983).
89. Pynnonen, S., Kanto, J., Sillanpaa, M., Erkkola, R. Carbamazepine: placental transport, tissue concentrations in foetus and newborn, and level in milk. Acta Pharmacol Toxicol (Copenh) 41, 244-253 (1977).
90. Pienimaki, P., Lampela, E., Hakkola, J., Arvela, P., Raunio, H., kangas, K. Pharmacokinetics of oxcarbazepine and carbamazepine in human placenta. Epilepsia 38, 309-316 (1997).
91. Bulau, P., Paar, W.D., von Unruh, G.E. Pharmacokinetics of oxcarbazepine and 10-hydroxy-carbazepine in the newborn child of an oxcarbazepine-treated mother. Eur J Clin Pharmacol 34, 311-313 (1988).
92. Myllynen, P., ki, P., Jouppila, P., kangas, K. Transplacental passage of oxcarbazepine and its metabolites in vivo. Epilepsia 42, 1482-1485 (2001).
93. Shah, Y.G., Miller, R.K. The pharmacokinetics of phenytoin in perfused human placenta. Pediatr Pharmacol 5, 165-179 (1985).
94. Ishizaki, T., Yokochi, K., Chiba, K., Tabuchi, T., Wagatsuma, T. Placental transfer of anticonvulsants (phenobarbital, phenytoin, valproic acid) and the elimination from neonates. Pediatr Pharmacol (New York) 1, 291-303 (1981).
95. Kluck, R.M., Cannell, G.R., Hooper, W.D., Eadie, M.J., Dickinson, R.G. Disposition of phenytoin and phenobarbitone in the isolated perfused human placenta. Clin Exp Pharmacol Physiol 15, 827-836 (1988).
96. Mirkin, B.L. Diphenylhydantoin: placental transport, fetal localization, neonatal metabolism, and possible teratogenic effects. J Pediatr 78, 329-337 (1971).
189
97. Dickinson, R.G., Fowler, D.W., Kluck, R.M. Maternofetal transfer of phenytoin, p-hydroxy-phenytoin and p-hydroxy-phenytoin-glucuronide in the perfused human placenta. Clin Exp Pharmacol Physiol 16, 789-797 (1989).
98. Rayburn, W., Donn, S., Compton, A., Piehl, E. Oral phenobarbital given antenatally to reduce neonatal intraventricular hemorrhage. A comparison between maternal and umbilical cord serum levels at delivery. J Perinatol 9, 268-270 (1989).
99. Melchior, J.C., Svensmark, O., Trolle, D. Placental transfer of phenobarbitone in epileptic women, and elimination in newborns. Lancet 2, 860-861 (1967).
100. De Carolis, M.P. et al. Placental transfer of phenobarbital: what is new? Dev Pharmacol Ther 19, 19-26 (1992).
101. Shankaran, S., Cepeda, E.E., Ilagan, N., Kauffman, R.E. Pharmacokinetic basis for antenatal dosing of phenobarbital for the prevention of neonatal intracerebral hemorrhage. Dev Pharmacol Ther 9, 171-177 (1986).
102. Kuhnz, W., Koch, S., Helge, H., Nau, H. Primidone and phenobarbital during lactation period in epileptic women: total and free drug serum levels in the nursed infants and their effects on neonatal behavior. Dev Pharmacol Ther 11, 147-154 (1988).
103. Myllynen, P., Vahakangas, K. An examination of whether human placental perfusion allows accurate prediction of placental drug transport: studies with diazepam. J Pharmacol Toxicol Methods 48, 131-138 (2002).
104. Moore, R.G., McBride, W.G. The disposition kinetics of diazepam in pregnant women at parturition. Eur J Clin Pharmacol 13, 275-284 (1978).
105. Ridd, M.J., Brown, K.F., Nation, R.L., Collier, C.B. The disposition and placental transfer of diazepam in cesarean section. Clin Pharmacol Ther 45, 506-512 (1989).
106. Cavanah, D., Condo, C.S. Diazepam-- A pilot study of drug concentrations in maternal blood, amniotic fluid and cord blood. Curr Ther Res Clin Exp 6, 122-126 (1964).
107. Cree, J.E., Meyer, J., Hailey, D.M. Diazepam in labour: its metabolism and effect on the clinical condition and thermogenesis of the newborn. Br Med J 4, 251-255 (1973).
108. Kanto, J., Erkkola, R., Sellman, R. Accumulation of diazepam and N-demethyldiazepam in the fetal blood during the labour. Ann Clin Res 5, 375-379 (1973).
109. Mandelli, M. et al. Placental transfer to diazepam and its disposition in the newborn. Clin Pharmacol Ther 17, 564-572 (1975).
110. Bakke, O.M., Haram, K., Lygre, T., Wallem, G. Comparison of the placental transfer of thiopental and diazepam in caesarean section. Eur J Clin Pharmacol 21, 221-227 (1981).
111. Erkkola, R., Kangas, L., Pekkarinen, A. The transfer of diazepam across the placenta during labour. Acta Obstet Gynecol Scand 52, 167-170 (1973).
190
112. Owen, J.R., Irani, S.F., Blair, A.W. Effect of diazepam administered to mothers during labour on temperature regulation of neonate. Arch Dis Child 47, 107-110 (1972).
113. Idanpaan-Heikkila, J.E., Jouppila, P.I., Puolakka, J.O., Vorne, M.S. Placental transer and fetal metabolism of diazepam in early human pregnancy. Am J Obstet Gynecol 109, 1011-1016 (1971).
114. Haram, K., Bakke, O.M. Diazepam as an induction agent for caesarean section: a clinical and pharmacokinetic study of fetal drug exposure. Br J Obstet Gynaecol 87, 506-512 (1980).
115. Kanto, J., Scheinin, M. Placental and blood-CSF transfer of orally administered diazepam in the same person. Pharmacol Toxicol 61, 72-74 (1987).
116. Haram, K., Bakke, O.M., Johannessen, K.H., Lund, T. Transplacental passage of diazepam during labor: influence of uterine contractions. Clin Pharmacol Ther 24, 590-599 (1978).
117. Desilva, J.A., D'Arconte, L., Kaplan, J. The determination of blood levels and the placental transfer of diazepam in humans. Curr Ther Res Clin Exp 6, 115-121 (1964).
118. Eliot, B.W., Hill, J.G., Cole, A.P., Hailey, D.M. Continuous pethidine/diazepam infunsion during labour and its effects on the newborn. Br J Obstet Gynaecol 82, 126-131 (1975).
119. Barzago, M.M. et al. Placental transfer of valproic acid after liposome encapsulation during in vitro human placenta perfusion. J Pharmacol Exp Ther 277, 79-86 (1996).
120. Dickinson, R.G., Harland, R.C., Lynn, R.K., Smith, W.B., Gerber, N. Transmission of valproic acid (Depakene) across the placenta: half-life of the drug in mother and baby. J Pediatr 94, 832-835 (1979).
121. Fowler, D.W., Eadie, M.J., Dickinson, R.G. Transplacental transfer and biotransformation studies of valproic acid and its glucuronide(s) in the perfused human placenta. J Pharmacol Exp Ther 249, 318-323 (1989).
122. Nau, H., Rating, D., Koch, S., Hauser, I., Helge, H. Valproic acid and its metabolites: placental transfer, neonatal pharmacokinetics, transfer via mother's milk and clinical status in neonates of epileptic mothers. J Pharmacol Exp Ther 219, 768-777 (1981).
123. Froescher, W., Gugler, R., Niesen, M., Hoffmann, F. Protein binding of valproic acid in maternal and umbilical cord serum. Epilepsia 25, 244-249 (1984).
124. Myllynen, P.K., ki, P.K., kangas, K.H. Transplacental passage of lamotrigine in a human placental perfusion system in vitro and in maternal and cord blood in vivo. Eur J Clin Pharmacol 58, 677-682 (2003).
125. Kacirova, I., Grundmann, M., Brozmanova, H. Serum levels of lamotrigine during delivery in mothers and their infants. Epilepsy Res. 91, 161-165 (2010).
191
126. Ohman, I., Vitols, S., Tomson, T. Lamotrigine in pregnancy: pharmacokinetics during delivery, in the neonate, and during lactation. Epilepsia 41, 709-713 (2000).
127. Tomson, T., Ohman, I., Vitols, S. Lamotrigine in pregnancy and lactation: a case report. Epilepsia 38, 1039-1041 (1997).
128. de Haan, G.J. et al. Gestation-induced changes in lamotrigine pharmacokinetics: a monotherapy study. Neurology 63, 571-573 (2004).
129. Fotopoulou, C. et al. Prospectively assessed changes in lamotrigine-concentration in women with epilepsy during pregnancy, lactation and the neonatal period. Epilepsy Res. 85, 60-64 (2009).
130. Schenker, S., Yang, Y., Mattiuz, E., Tatum, D., Lee, M. Olanzapine transfer by human placenta. Clin Exp Pharmacol Physiol 26, 691-697 (1999).
131. Aichhorn, W., Yazdi, K., Kralovec, K., Steiner, H., Whitworth, S., Stuppaeck, C. Olanzapine plasma concentration in a newborn. J Psychopharmacol 22, 923-924 (2008).
132. Newport, D.J. et al. Atypical antipsychotic administration during late pregnancy: placental passage and obstetrical outcomes. Am J Psychiatry 164, 1214-1220 (2007).
133. Kirchheiner, J., Berghofer, A., Bolk-Weischedel, D. Healthy outcome under olanzapine treatment in a pregnant woman. Pharmacopsychiatry 33, 78-80 (2000).
134. Rahi, M. et al. Placental transfer of quetiapine in relation to P-glycoprotein activity. J Psychopharmacol 21, 751-756 (2007).
135. Sudhakaran, S. et al. Differential bidirectional transfer of indinavir in the isolated perfused human placenta. Antimicrob Agents Chemother 49, 1023-1028 (2005).
136. Mirochnick, M. et al. Concentrations of protease inhibitors in cord blood after in utero exposure. Pediatr Infect Dis J 21, 835-838 (2002).
137. Sudhakaran, S., Rayner, C.R., Li, J., Kong, D.C.M., Gude, N.M., Nation, R.L. Inhibition of placental P-glycoprotein: Impact on indinavir transfer to the foetus. Br J Clin Pharmacol 65, 667-673 (2008).
138. Chappuy, H. et al. Maternal-fetal transfer and amniotic fluid accumulation of protease inhibitors in pregnant women who are infected with human immunodeficiency virus. Am J Obstet Gynecol 191, 558-562 (2004).
139. Gavard, L. et al. Placental transfer of lopinavir/ritonavir in the ex vivo human cotyledon perfusion model. Am J Obstet Gynecol 195, 296-301 (2006).
140. Marzolini, C. et al. Transplacental passage of protease inhibitors at delivery. AIDS 16, 889-893 (2002).
192
141. Gingelmaier, A. et al. Placental transfer and pharmacokinetics of lopinavir and other protease inhibitors in combination with nevirapine at delivery. AIDS 20, 1737-1743 (2006).
142. Ivanovic, J. et al. Transplacental transfer of antiretroviral drugs and newborn birth weight in HIV-infected pregnant women. Curr HIV Res 7, 620-625 (2009).
143. Mirochnick, M. et al. Lopinavir exposure with an increased dose during pregnancy. J Acquir Immune Defic Syndr 49, 485-491 (2008).
144. Yeh, R.F. et al. Genital tract, cord blood, and amniotic fluid exposures of seven antiretroviral drugs during and after pregnancy in human immunodeficiency virus type 1-infected women. Antimicrob Agents Chemother 53, 2367-2374 (2009).
145. Stek, A.M. et al. Reduced lopinavir exposure during pregnancy. AIDS 20, 1931-1939 (2006).
146. Gavard, L. et al. Contribution and limit of the model of perfused cotyledon to the study of placental transfer of drugs. Example of a protease inhibitor of HIV: nelfinavir. Eur J Obstet Gynecol Reprod Biol 147, 157-160 (2009).
147. Hirt, D. et al. Pharmacokinetic modelling of the placental transfer of nelfinavir and its M8 metabolite: a population study using 75 maternal-cord plasma samples. Br J Clin Pharmacol 64, 634-644 (2007).
148. Casey, B.M., Bawdon, R.E. Placental transfer of ritonavir with zidovudine in the ex vivo placental perfusion model. Am J Obstet Gynecol 179, 758-761 (1998).
149. Forestier, F., de, R.,P., Peytavin, G., Dohin, E., Farinotti, R., Mandelbrot, L. Maternal-fetal transfer of saquinavir studied in the ex vivo placental perfusion model. Am J Obstet Gynecol 185, 178-181 (2001).
150. Henderson, G.I., Hu, Z.Q., Johnson, R.F., Perez, A.B., Yang, Y., Schenker, S. Acyclovir transport by the human placenta. J Lab Clin Med 120, 885-892 (1992).
151. Kimberlin, D.F. et al. Pharmacokinetics of oral valacyclovir and acyclovir in late pregnancy. Am J Obstet Gynecol 179, 846-851 (1998).
152. Gilstrap, L.C., Bawdon, R.E., Roberts, S.W., Sobhi, S. The transfer of the nucleoside analog ganciclovir across the perfused human placenta. Am J Obstet Gynecol 170, 967-972 (1994).
153. Frenkel, L.M. et al. Pharmacokinetics of acyclovir in the term human pregnancy and neonate. Am J Obstet Gynecol 164, 569-576 (1991).
154. Haddad, J., Langer, B., Astruc, D., Messer, J., Lokiec, F. Oral acyclovir and recurrent genital herpes during late pregnancy. Obstet Gynecol 82, 102-104 (1993).
193
155. Dancis, J., Lee, J.D., Mendoza, S., Liebes, L. Transfer and metabolism of dideoxyinosine by the perfused human placenta. J Acquir Immune Defic Syndr 6, 2-6 (1993).
156. Chappuy, H. et al. Maternal-fetal transfer and amniotic fluid accumulation of nucleoside analogue reverse transcriptase inhibitors in human immunodeficiency virus-infected pregnant women. Antimicrob Agents Chemother 48, 4332-4336 (2004).
157. Pons, J.C. et al. Fetoplacental passage of 2',3'-dideoxyinosine. Lancet 337, 732 (1991).
158. Henderson, G.I., Perez, A.B., Yang, Y., Hamby, R.L., Schenken, R.S., Schenker, S. Transfer of dideoxyinosine across the human isolated placenta. Br J Clin Pharmacol 38, 237-242 (1994).
159. Bloom, S.L., Dias, K.M., Bawdon, R.E., Gilstrap, I.I.I.,L.C. The maternal-fetal transfer of lamivudine in the ex vivo human placenta. Am J Obstet Gynecol 176, 291-293 (1997).
160. Moodley, D. et al. Pharmacokinetics of zidovudine and lamivudine in neonates following coadministration of oral doses every 12 hours. J Clin Pharmacol 41, 732-741 (2001).
161. Mandelbrot, L., Peytavin, G., Firtion, G., Farinotti, R. Maternal-fetal transfer and amniotic fluid accumulation of lamivudine in human immunodeficiency virus-infected pregnant women. Am J Obstet Gynecol 184, 153-158 (2001).
162. Olivero, O.A., Parikka, R., Poirier, M.C., Vahakangas, K. 3'-azido-3'-deoxythymidine (AZT) transplacental perfusion kinetics and DNA incorporation in normal human placentas perfused with AZT. Mutat Res 428, 41-47 (1999).
163. Watts, D.H. et al. Pharmacokinetic disposition of zidovudine during pregnancy. J Infect Dis 163, 226-232 (1991).
164. Boal, J.H., Plessinger, M.A., van, d.,Reydt C., Miller, R.K. Pharmacokinetic and toxicity studies of AZT (zidovudine) following perfusion of human term placenta for 14 hours. Toxicol Appl Pharmacol 143, 13-21 (1997).
165. Schenker, S., Johnson, R.F., King, T.S., Schenken, R.S., Henderson, G.I. Azidothymidine (Zidovudine) transport by the human placenta. Am J Med Sci 299, 16-20 (1990).
166. Liebes, L., Mendoza, S., Wilson, D., Dancis, J. Transfer of zidovudine (AZT) by human placenta. J Infect Dis 161, 203-207 (1990).
167. Gillet, J.Y., Garraffo, R., Abrar, D., Bongain, A., Lapalus, P., Dellamonica, P. Fetoplacental passage of zidovudine.[letter]. Lancet 1989;2(8657):269-270.
168. Collier, A.C., Keelan, J.A., Van, Z.,P.E., Paxton, J.W., Mitchell, M.D., Tingle, M.D. Human placental glucuronidation and transport of 3'azido-3'- deoxythymidine and uridine diphosphate glucuronic acid. Drug Metab Dispos 32, 813-820 (2004).
194
169. Pons, J.C., Taburet, A.M., Singlas, E., Delfraissy, J.F., Papiernik, E. Placental passage of azathiothymidine (AZT) during the second trimester of pregnancy: study by direct fetal blood sampling under ultrasound. Eur J Obstet Gynecol Reprod Biol 40, 229-231 (1991).
170. O'Sullivan, M.J. et al. The pharmacokinetics and safety of zidovudine in the third trimester of pregnancy for women infected with human immunodeficiency virus and their infants: phase I acquired immunodeficiency syndrome clinical trials group study (protocol 082). Zidovudine Collaborative Working Group. Am J Obstet Gynecol 168, 1510-1516 (1993).
171. Ceccaldi, P.-F., Ferreira, C., Gavard, L., Gil, S., Peytavin, G., Mandelbrot, L. Placental transfer of enfuvirtide in the ex vivo human placenta perfusion model. Am J Obstet Gynecol 198, 433.e1-433.e2 (2008).
172. Brennan-Benson, P. et al. Enfurvitide prevents vertical transmission of multidrug-resistant HIV-1 in pregnancy but does not cross the placenta. AIDS 20, 297-299 (2006).
173. Omarini, D., Barzago, M.M., Aramayona, J., Bortolotti, A., Lucchini, G., Bonati, M. Theophylline transfer across human placental cotyledon during in vitro dual perfusion. J Med 23, 101-116 (1992).
174. Labovitz, E., Spector, S. Placental theophylline transfer in pregnant asthmatics. JAMA 247, 786-788 (1982).
175. Omarini, D. et al. Placental transfer of theophylline in an in vitro closed perfusion system of human placenta isolated lobule. Eur J Drug Metab Pharmacokinet 18, 369-374 (1993).
176. Arwood, L.L., Dasta, J.F., Friedman, C. Placental transfer of theophylline: two case reports. Pediatrics 63, 844-846 (1979).
177. Ron, M., Hochner-Celnikier, D., Menczel, J., Palti, Z., Kidroni, G. Maternal-fetal transfer of aminophylline. Acta Obstet Gynecol Scand 63, 217-218 (1984).
178. Romero, R., Kadar, N., Gonzales Govea, F., Hobbins, J.C. Pharmacokinetics of intravenous theophylline in pregnant patients at term. Am J Perinatol 1, 31-35 (1983).
179. Nandakumaran, M., Gardey, C.L., Challier, J.C. Transfer of salbutamol in the human placenta in vitro. Dev Pharmacol Ther 3, 88-98 (1981).
180. Boulton, D.W., Fawcett, J.P., Fiddes, T.M. Transplacental distribution of salbutamol enantiomers at Caesarian section. Br J Clin Pharmacol 44, 587-590 (1997).
181. McGowan, W.A. Safety of cimetidine in obstetric patients. J R Soc Med 72, 902-907 (1979).
182. Ching, M.S., Mihaly, G.W., Morgan, D.J., Date, N.M., Hardy, K.J., Smallwood, R.A. Low clearance of cimetidine across the human placenta. J Pharmacol Exp Ther 241, 1006-1009 (1987).
195
183. Qvist, N., Storm, K., Holmskov, A. Cimetidine as pre-anesthetic agent for cesarean section: perinatal effects on the infant, the placental transfer of cimetidine and its elimination in the infants. J Perinat Med 13, 179-183 (1985).
184. Dicke, J.M., Johnson, R.F., Henderson, G.I., Kuehl, T.J., Schenker, S. A comparative evaluation of the transport of H2-receptor antagonists by the human and baboon placenta. Am J Med Sci 295, 198-206 (1988).
185. Howe, J.P., McGowan, W.A., Moore, J., McCaughey, W., Dundee, J.W. The placental transfer of cimetidine. Anaesthesia 36, 371-375 (1981).
186. McAuley, D.M., Moore, J., McCaughey, W., Donnelly, B.D., Dundee, J.W. Ranitidine as an antacid before elective Caesarean section. Anaesthesia 38, 108-114 (1983).
187. Armentano, G., Bracco, P.L., Di Silverio, C. Ranitidine in the treatment of reflux oesophagitis in pregnancy. Clin Exp Obstet Gynecol 16, 130-133 (1989).
188. Peytavin, G., Leng, J.J., Forestier, F., Saux, M., Hohlfeld, P., Farinotti, R. Placental transfer of pyrimethamine studied in an ex vivo placental perfusion model. Biol Neonate 78, 83-85 (2000).
189. Trenque, T. et al. Human maternofoetal distribution of pyrimethamine-sulphadoxine. Br J Clin Pharmacol 45, 179-180 (1998).
190. Dorangeon, P.H. et al. Transplacental passage of the pyrimethamine-sulfadoxine combination in the prenatal treatment of congenital toxoplasmosis. Presse Medicale 19, 2036 (1990).
191. Heikkinen, T., Laine, K., Neuvonen, P.J., Ekblad, U. The transplacental transfer of the macrolide antibiotics erythromycin, roxithromycin and azithromycin. BJOG 107, 770-775 (2000).
192. Kiefer, L., Rubin, A., McCoy, J.B., Foltz, E.L. The placental transfer of erythomycin. Am J Obstet Gynecol 69, 174-177 (1955).
193. Heilman, F.R., Herrell, W.E., Wellman, W.E., Geraci, J.E. Some laboratory and clinical observations on a new antibiotic, erythromycin (ilotycin). Proc Staff Meet Mayo Clin 27, 285-304 (1952).
194. Ramsey, P.S., Vaules, M.B., Vasdev, G.M., Andrews, W.W., Ramin, K.D. Maternal and transplacental pharmacokinetics of azithromycin. Am J Obstet Gynecol 188, 714-718 (2003).
195. Polachek, H. et al. Transfer of ciprofloxacin, ofloxacin and levofloxacin across the perfused human placenta in vitro. Eur J Obstet Gynecol Reprod Biol 122, 61-65 (2005).
196. Matsuda, S., Kashiwagura, T., Kokuho, K. Experimental and clinical studies of DL-8280 in the field of obstetrics and gynecology. Chemotherapy 32, 900-907 (1984).
196
197. Yamamoto, T., Yasuda, J., Kanao, M., Okada, H. Fundamental and clinical studies on DL-8280 in the field of obstetrics and gynecology. Chemotherapy 32, 944-949 (1984).
198. Fortunato, S.J., Bawdon, R.E., Swan, K.F., Bryant, E.C., Sobhi, S. Transfer of Timentin (ticarcillin and clavulanic acid) across the in vitro perfused human placenta: comparison with other agents. Am J Obstet Gynecol 167, 1595-1599 (1992).
199. Maberry, M.C., Trimmer, K.J., Bawdon, R.E., Sobhi, S., Dax, J.B., Gilstrap, L.C.r. Antibiotic concentration in maternal blood, cord blood and placental tissue in women with chorioamnionitis. Gynecol Obstet Invest 33, 185-186 (1992).
200. Fortunato, S.J., Bawdon, R.E., Maberry, M.C., Swan, K.F. Transfer of ceftizoxime surpasses that of cefoperazone by the isolated human placenta perfused in vitro. Obstet Gynecol 75, 830-833 (1990).
201. Fortunato, S.J., Bawdon, R.E., Welt, S.I., Swan, K.F. Steady-state cord and amniotic fluid ceftizoxime levels continuously surpass maternal levels. Am J Obstet Gynecol 159, 570-573 (1988).
202. Fortunato, S.J., Bawdon, R.E., Baum, M. Placental transfer of cefoperazone and sulbactam in the isolated in vitro perfused human placenta. Am J Obstet Gynecol 159, 1002-1006 (1988).
203. Boskovic, R., Feig, D.S., Derewlany, L., Knie, B., Portnoi, G., Koren, G. Transfer of insulin lispro across the human placenta: in vitro perfusion studies. Diabetes Care 26, 1390-1394 (2003).
204. Jovanovic, L. et al. Metabolic and immunologic effects of insulin lispro in gestational diabetes. Diabetes Care 22, 1422-1427 (1999).
205. Holcberg, G. et al. Transfer of insulin lispro across the human placenta. Eur J Obstet Gynecol Reprod Biol 115, 117-118 (2004).
206. Nanovskaya, T.N., Nekhayeva, I.A., Patrikeeva, S.L., Hankins, G.D.V., Ahmed, M.S. Transfer of metformin across the dually perfused human placental lobule. Am J Obstet Gynecol 195, 1081-1085 (2006).
207. Hague, W., Davoren, P., McIntyre, D., Norris, R., Xiaonian, X. Metformin crosses the placenta: A modulator for fetal insulin resistance? BMJ 327, 880-881 (2003).
208. Vanky, E., Zahlsen, K., Spigset, O., Carlsen, S.M. Placental passage of metformin in women with polycystic ovary syndrome. Fertil Steril 83, 1575-1578 (2005).
209. Kovo, M., Haroutiunian, S., Feldman, N., Hoffman, A., Glezerman, M. Determination of metformin transfer across the human placenta using a dually perfused ex vivo placental cotyledon model. Eur J Obstet Gynecol Reprod Biol 136, 29-33 (2008).
197
210. Kovo, M., Kogman, N., Ovadia, O., Nakash, I., Golan, A., Hoffman, A. Carrier-mediated transport of metformin across the human placenta determined by using the ex vivo perfusion of the placental cotyledon model. Prenat Diagn 28, 544-548 (2008).
211. Elliott, B.D., Langer, O., Schenker, S., Johnson, R.F. Insignificant transfer of glyburide occurs across the human placenta. Am J Obstet Gynecol 165, 807-812 (1991).
212. Langer, O., Conway, D.L., Berkus, M.D., Xenakis, E.M., Gonzales, O. A comparison of glyburide and insulin in women with gestational diabetes mellitus. N Engl J Med 343, 1134-1138 (2000).
213. Hebert, M.F. et al. Are we optimizing gestational diabetes treatment with glyburide? The pharmacologic basis for better clinical practice. Clin Pharmacol Ther 85, 607-614 (2009).
214. Nanovskaya, T.N., Nekhayeva, I., Hankins, G.D.V., Ahmed, M.S. Effect of human serum albumin on transplacental transfer of glyburide. Biochem Pharmacol 72, 632-639 (2006).
215. Nanovskaya, T.N. et al. Effect of albumin on transplacental transfer and distribution of rosiglitazone and glyburide. J Matern Fetal Neonatal Med 21, 197-207 (2008).
216. Derewlany, L.O., Leeder, J.S., Kumar, R., Radde, I.C., Knie, B., Koren, G. The transport of digoxin across the perfused human placental lobule. J Pharmacol Exp Ther 256, 1107-1111 (1991).
217. Kerenyi, T.D. et al. Transplancental cardioversion of intrauterine supraventricular tachycardia with digitalis. Lancet 2, 393-394 (1980).
218. Holcberg, G. et al. Lack of interaction of digoxin and P-glycoprotein inhibitors, quinidine and verapamil in human placenta in vitro. Eur J Obstet Gynecol Reprod Biol 109, 133-137 (2003).
219. Nagashima, M., Asai, T., Suzuki, C., Matsushima, M., Ogawa, A. Intrauterine supraventricular tachyarrhythmias and transplacental digitalisation. Arch Dis Child 61, 996-1000 (1986).
220. Tsadkin, M. et al. Albumin-dependent digoxin transfer in isolated perfused human placenta. Int J Clin Pharmacol Ther 39, 158-161 (2001).
221. Rogers, M.C., Willerson, J.T., Goldblatt, A., Smith, T.W. Serum digoxin concentrations in the human fetus, neonate and infant. N Engl J Med 287, 1010-1013 (1972).
222. Younis, J.S., Granat, M. Insufficient transplacental digoxin transfer in severe hydrops fetalis. Am J Obstet Gynecol 157, 1268-1269 (1987).
223. Saarikoski, S. Placental transfer and fetal uptake of 3H-digoxin in humans. Br J Obstet Gynaecol 83, 879-884 (1976).
224. Schmolling, J. Digoxin transfer across the isolated placenta is influenced by maternal and fetal albumin concentrations. Reprod Fertil Dev 8, 969-974 (1996).
198
225. Mimura, S., Suzuki, C., Yamazaki, T. Transplacental passage of digoxin in the case of nonimmune hydrops fetalis. Clin Cardiol 10, 63-65 (1987).
226. Miller, R.K., Jessee, L., Barrish, A., Gilbert, J., Manson, J.M. Pharmacokinetic studies of enalaprilat in the in vitro perfused human placental lobule system. Teratology 58, 76-81 (1998).
227. Ala-Kokko, T.I., Pienimaki, P., Lampela, E., Hollmen, A.I., Pelkonen, O., Vahakangas, K. Transfer of clonidine and dexmedetomidine across the isolated perfused human placenta. Acta Anaesthesiol Scand 41, 313-319 (1997).
228. Hartikainen-Sorri, A.L., Heikkinen, J.E., Koivisto, M. Pharmacokinetics of clonidine during pregnancy and nursing. Obstet Gynecol 69, 598-600 (1987).
229. Boutroy, M.J., Gisonna, C.R., Legagneur, M. Clonidine: placental transfer and neonatal adaption. Early Hum Dev 17, 275-286 (1988).
230. Buchanan, M.L. et al. Clonidine pharmacokinetics in pregnancy. Drug Metab Dispos 37, 702-705 (2009).
231. Cigarini, I. et al. Epidural clonidine combined with bupivacaine for analgesia in labor. Effects on mother and neonate. Reg Anesth 20, 113-120 (1995).
232. Lagrange, F. et al. Absence of placental transfer of pentasaccharide (fondaparinux, arixtra) in the dually perfused human cotyledon in vitro. Thromb. Haemost. 87, 831-835 (2002).
233. Dempfle, C.E. Minor transplacental passage of fondaparinux in vivo.[letter]. N Engl J Med 2004;350(18):1914-1915.
234. Mazzolai, L., Hohlfeld, P., Spertini, F., Hayoz, D., Schapira, M., Duchosal, M.A. Fondaparinux is a safe alternative in case of heparin intolerance during pregnancy. Blood 108, 1569-1570 (2006).
235. Harenberg, J. Treatment of a woman with lupus and thromboembolism and cutaneous intolerance to heparins using fondaparinux during pregnancy. Thromb Res 119, 385-388 (2007).
236. Bajoria, R., Contractor, S.F. Transfer of heparin across the human perfused placental lobule. J Pharm Pharmacol 44, 952-959 (1992).
237. Flessa, H.C., Kapstrom, A.B., Glueck, H.I., Will, J.J. Placental transport of heparin. Am J Obstet Gynecol 93, 570-573 (1965).
238. Dimitrakakis, C., Papageorgiou, P., Papageorgiou, I., Antzaklis, A., Sakarelou, N., Michalas, S. Absence of transplacental passage of the low molecular weight heparin enoxaparin. Haemostasis 30, 243-248 (2000).
199
239. Forestier, F., Daffos, F., Capella-Pavlovsky, M. Low molecular weight heparin (PK 10169) does not cross the placenta during the second trimester of pregnancy study by direct fetal blood sampling under ultrasound. Thromb Res 34, 557-560 (1984).
240. Forestier, F., Daffos, F., Rainaut, M., Toulemonde, F. Low molecular weight heparin (CY 216) does not cross the placenta during the third trimester of pregnancy.[letter]. Thromb Haemost 1987;57(2):234.
241. Melissari, E. et al. Use of low molecular weight heparin in pregnancy. Thromb Haemost 68, 652-656 (1992).
242. Omri, A., Delaloye, J.F., Andersen, H., Bachmann, F. Low molecular weight heparin Novo (LHN-1) does not cross the placenta during the second trimester of pregnancy. Thromb. Haemost. 61, 55-56 (1989).
243. Bustard, M.A., Farley, A.E., Smith, G.N. The pharmacokinetics of glyceryl trinitrate with the use of the in vitro term human placental perfusion setup. Am J Obstet Gynecol 187, 187-190 (2002).
244. David, M., Walka, M.M., Schmid, B., Sinha, P., Veit, S., Lichtenegger, W. Nitroglycerin application during cesarean delivery: plasma levels, fetal/maternal ratio of nitroglycerin, and effects in newborns. Am J Obstet Gynecol 182, 955-961 (2000).
245. Magee, K.P., Bawdon, R.E. Ex vivo human placental transfer and the vasoactive properties of hydralazine. Am J Obstet Gynecol 182, 167-169 (2000).
246. Liedholm, H., Wahlin-Boll, E., Hanson, A., Ingemarsson, I., Melander, A. Transplacental passage and breast milk concentrations of hydralazine. Eur J Clin Pharmacol 21, 417-419 (1982).
247. Schneider, H., Proegler, M. Placental transfer of beta-adrenergic antagonists studied in an in vitro perfusion system of human placental tissue. Am J Obstet Gynecol 159, 42-47 (1988).
248. Melander, A., Niklasson, B., Ingemarsson, I., Liedholm, H., Schersten, B., Sjoberg, N.O. Transplacental passage of atenolol in man. Eur J Clin Pharmacol 14, 93-94 (1978).
249. Kofahl, B., Henke, D., Hettenbach, A., Mutschler, E. Studies on placental transfer of celiprolol. Eur J Clin Pharmacol 44, 381-382 (1993).
250. Poranen, A.-K., Nurmi, H., Malminiemi, K., Ekblad, U. Vasoactive effects and placental transfer of nifedipine, celiprolol, and magnesium sulfate in the placenta perfused in vitro. Hypertension in Pregnancy 17, 93-102 (1998).
251. Michael, C.A. Use of labetalol in the treatment of severe hypertension during pregnancy. Br J Clin Pharmacol 8, 211S-215S (1979).
252. Nandakumaran, M., Angelo-Khattar, M., Ibrahim, M.E., Hathout, H. Transfer of labetalol in the perfused human placenta: An in vitro study. Med Princ Pract 1, 81-85 (1989).
200
253. Erkkola, R., Lammintausta, R., Liukko, P., Anttila, M. Transfer of propranolol and sotalol across the human placenta. Their effect on maternal and fetal plasma renin activity. Acta Obstet Gynecol Scand 61, 31-34 (1982).
254. Pirhonen, J.P., Erkkola, R.U., Ekblad, U.U., Nyman, L. Single dose of nifedipine in normotensive pregnancy: nifedipine concentrations, hemodynamic responses, and uterine and fetal flow velocity waveforms. Obstet Gynecol 76, 807-811 (1990).
255. Nandakumaran, M., Eldeen, A.S. Transfer of cyclosporine in the perfused human placenta. Dev Pharmacol Ther 15, 101-105 (1990).
256. Venkataramanan, R., Koneru, B., Wang, C.C., Burckart, G.J., Caritis, S.N., Starzl, T.E. Cyclosporine and its metabolites in mother and baby. Transplantation 46, 468-469 (1988).
257. Bourget, P., Fernandez, H., Bismuth, H., Papiernik, E. Transplacental passage of cyclosporine after liver transplantation. Transplantation 49, 663 (1990).
258. Flechner, S.M., Katz, A.R., Rogers, A.J., Van Buren, C., Kahan, B.D. The presence of cyclosporine in body tissues and fluids during pregnancy. Am J Kidney Dis 5, 60-63 (1985).
259. Mortimer, R.H., Cannell, G.R., Addison, R.S., Johnson, L.P., Roberts, M.S., Bernus, I. Methimazole and propylthiouracil equally cross the perfused human term placental lobule. J Clin Endocrinol Metab 82, 3099-3102 (1997).
260. Marchant, B., Brownlie, B.E., Hart, D.M., Horton, P.W., Alexander, W.D. The placental transfer of propylthiouracil, methimazole and carbimazole. J Clin Endocrinol Metab 45, 1187-1193 (1977).
261. Gardner, D.F., Cruikshank, D.P., Hays, P.M., Cooper, D.S. Pharmacology of propylthiouracil (PTU) in pregnant hyperthyroid women: correlation of maternal PTU concentrations with cord serum thyroid function tests. J Clin Endocrinol Metab 62, 217-220 (1986).
262. Nandakumaran, M., Gardey, C., Rey, E. Transfer of ritodrine and norepinephrine in human placenta: In vitro study. Dev Pharmacol Ther 4, 71-80 (1982).
263. Gandar, R., de Zoeten, L.W., van der Schoot, J.B. Serum level of ritodrine in man. Eur J Clin Pharmacol 17, 117-122 (1980).
264. Urbach, J., Mor, L., Fuchs, S., Brandes, J.M. Transplacental transfer of ritodrine and its effect on placental glucose and oxygen consumption in an in vitro human placental cotyledon perfusion. Gynecol Obstet Invest 32, 10-14 (1991).
265. Gross, T.L., Kuhnert, B.R., Kuhnert, P.M., Rosen, M.G., Kazzi, N.J. Maternal and fetal plasma concentrations of ritodrine. Obstet Gynecol 65, 793-797 (1985).
266. van Lierde, M., Thomas, K. Ritodrine concentrations in maternal and fetal serum and amniotic fluid. J Perinat Med 10, 119-124 (1982).
201
267. Fujimoto, S., Tanaka, T., Akahane, M. Levels of ritodrine hydrochloride in fetal blood and amniotic fluid following long-term continuous administration in late pregnancy. Eur J Obstet Gynecol Reprod Biol 38, 15-18 (1991).
268. Fujimoto, S., Akahane, M., Sakai, A. Concentrations of ritodrine hydrochloride in maternal and fetal serum and amniotic fluid following intravenous administration in late pregnancy. Eur J Obstet Gynecol Reprod Biol 23, 145-152 (1986).
269. Kuhnert, B.R., Gross, T.L., Kuhnert, P.M., Erhard, P., Brashar, W.T. Ritodrine pharmacokinetics. Clin Pharmacol Ther 40, 656-664 (1986).
202
Appendix C: Placental P-glycoprotein and breast cancer resistance protein: influence of
polymorphisms on fetal drug exposure and physiology
Janine R Hutsona,b, Gideon Korena,b, Stephen G Matthewsc
aInstitute of Medical Sciences, University of Toronto, 1 King’s College Circle, Toronto, Ontario M5S 1A8, Canada
bDivision of Clinical Pharmacology and Toxicology, Hospital for Sick Children, 555 University Ave, Toronto, Ontario M5G 1X8, Canada
cDepartments of Physiology, Obstetrics & Gynaecology and Medicine, University of Toronto, 1 King’s College Circle, Toronto, Ontario M5S 1A8, Canada
This work has been published and reproduced with permissions:
Hutson JR, Koren G, Matthews SG. Placental P-glycoprotein and breast cancer resistance protein: influence of polymorphisms on fetal drug exposure and physiology. Placenta 2010;31(5):351-357.
203
Abstract
Recent studies have illustrated the importance of placental drug transport proteins, such
as P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) in limiting fetal exposure to
drugs and toxins. Moreover, increasing evidence supports a role for Pgp and BCRP in the
normal development and physiological function of the placenta. Several single nucleotide
polymorphisms (SNPs) in the genes encoding Pgp and BCRP have been described and are
associated with altered protein expression, transporter activity, and clinical outcome in studies
focusing on tissues other than the placenta. This review aims to summarize current research
regarding the association between these polymorphisms and expression and function in the
placenta. The influence of these genotypes on fetal drug exposure and altered placental
physiology or development is also presented. To date, evidence suggests that SNPs in both
ABCB1 and ABCG1 can alter expression of their respective protein; however, the functional
significance of these polymorphisms is less clear. An understanding of this genotype-phenotype
relationship will allow for prediction of susceptible or favorable genotypes in order to
personalize medication choices to minimize fetal exposure to teratogens, or to maximize
pharmacological therapy to the fetus.
204
Introduction
Numerous factors are known to mediate transplacental transfer of substances, including
physiochemical and pharmacokinetic properties. Furthermore, transport proteins present on both
the microvillous brush-border and basal membranes (maternal blood-facing and fetal endothelial
cell-facing, respectively) of the syncytiotrophoblast cells have recently been identified as being
important for facilitated and active transport. Specifically, several members of the ATP-binding
cassette (ABC) drug efflux proteins are expressed, including P-glycoprotein (Pgp), breast cancer-
resistance protein (BCRP), and multidrug resistance-associated proteins (MRPs)(Table 1). Over
the last decade, the importance of Pgp and BCRP in mediating drug transfer across the placenta
and in the normal development and function of the placenta has been investigated.
Numerous polymorphisms have been reported in the genes encoding Pgp and BCRP
(ABCB1 and ABCG2, respectively). An understanding of these polymorphisms will allow
prediction of when normal placental physiology could be altered and lead to the development of
appropriate preventative therapies. Furthermore, knowledge of susceptible genotypes to fetal
drug exposure will influence medication choices during pregnancy, allowing minimized drug
exposure to potential teratogens or maximized drug exposure when pharmacological therapy to
the fetus is desired. This review summarizes current research regarding the association between
ABCB1 and ABCG2 polymorphisms and expression and function in the placenta and how this
knowledge will translate into personalized medicine.
205
Table 1. Selected substrates for Pgp and BCRP (Marzolini et al., 2004; Mao & Unadkat, 2005). Pgp Substrates BCRP Substrates Cimetidine Bisantrene Cyclosporin Diflomotecan Dexamethasone Cimetidine Digoxin Doxorubicin Doxorubicin Etoposide Erythromycin Flavonoids Indinavir Glyburide Levofloxacin Imatinib Morphine Lamivudine Phenobarbital Methotrexate Phenytoin Nitrofurantoin Quinidine Porphyrins Rifampin Sulfated estrogens Ritonavir Topotecan Saquinavir Zidovudine Talinolol Topotecan Verapamil Vinblastine
P-glycoprotein
Expression and function in the placenta
The expression of Pgp in the placenta throughout gestation has been localized to the
microvillous border of the syncytiotrophoblast[1-3]. Expression of Pgp is relatively high in the
placenta as ABCB1 mRNA levels are comparable to the intestine and liver[4]. However,
placental expression decreases throughout gestation[3;5]. This decrease in late gestation may be
important in allowing increased fetal exposure to endogenous factors in the maternal circulation;
including cortisol[3]. The decrease in Pgp may also increase susceptibility of the fetus to toxins
present in the maternal circulation. Many studies have shown close correlation between P-gp
expression and activity[6;7]. However, when P-gp levels are very low at term, this relationship is
not quite so clear[8].
Mechanisms regulating Pgp expression in the placenta are not fully understood as
regulation of Pgp appears tissue-specific[7;9]. In the trophoblast, ABCB1 transcription is
206
regulated by the Ap-2 and Sp families of transcription factors[10]. Hormonal regulation of Pgp
by progesterone has been proposed, however, administration of progesterone to pregnant mice
did not alter ABCB1 expression[7]. Placental expression of Pgp correlates with that of human
chorionic gonadotropin-β (hCG-β), suggesting that hCG-β may regulate Pgp expression or they
are both under the same control mechanisms[11]. Evidence suggests that cytokines may also be
important regulators of placental Pgp; in vitro incubation of primary term trophoblasts with
tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) decreased ABCB1 mRNA and
protein expression[12]. Furthermore, in rats, endotoxin-induced inflammation decreased
expression of placental Pgp[13].
Placental Pgp confers fetal protection to toxins in the maternal circulation. Deficiency of
mdr1a (knockout mice) or pharmacological inhibition of Pgp increased fetal exposure by up to
16-fold to digoxin, saquinavir, and paclitaxel after administration to pregnant dams[14]. Studies
using human placental tissue have also demonstrated altered drug transfer after inhibition of Pgp.
Using the dually perfused placenta model, increased drug transfer was seen for the protease
inhibitors saquinavir[15] and indinavir[16], and for methadone[17] after inhibition of Pgp.
Increased fetal exposure to protease inhibitors is warranted to prevent vertical transmission of
HIV, thus co-administration with other Pgp substrates or inhibitors will lead to the desired higher
fetal levels.
Polymorphisms in Pgp and effect on placental Pgp expression
The ABCB1 gene is over 100 kb in length and consists of a core promoter region and 28
exons located on the long arm of chromosome 7 at q21.1[18]. There is currently no evidence for
a spontaneous mutation in humans that would result in complete Pgp deficiency and may suggest
that mutations in ABCB1 may be embryolethal in humans[19]. There are, on the other hand,
over 50 SNPs and 3 insertion/deletion polymorphisms described[20]. Of these, specific
polymorphisms in an exon 26 wobble position (C3435T), an exon 21 coding region (G2677T/A),
an exon 12 coding region (C1236T), and the promoter region (T-129C) have been the focus of
studies in placental tissue. The C3435T SNP is synonymous, however, the G2677T/A SNP
results in an amino acid conversion (Ala to Thr/Ser) and the C1236T may influence RNA
stability[21;22].
207
Five studies have examined the relationship between placental Pgp expression and
ABCB1 polymorphisms and are summarized in Table 2. The first examined placentae from
Japanese women and detected nine SNPs. Compared to the -129TT genotype, -129CT exhibited
significantly reduced Pgp protein expression[21]. Interestingly, there were no subjects that were
homozygous with the mutation (-129CC). In a subsequent study, no placentae from Caucasian
subjects carried the T-129C SNP[23]. The maternal haplotype 2677T/3435T exhibited lower
mRNA and Pgp protein levels compared to other pooled haplotypes. Mothers that were
homozygous for both 2677T and 3435T (TT/TT) exhibited placental Pgp (protein) that was more
than 2-fold lower compared to GG/CC mothers. Interestingly, there was no significant
association between fetal genotype or haplotype and Pgp protein expression. When genotype of
both the mother and fetus was considered, there was a >30% reduction in Pgp protein expression
when both the mother and fetus were homozygous or heterozygous for the C3435T SNP (TT/tt
and CT/ct) compared to CC/cc. Limited sample size precluded full haplotype analysis. A
challenge with these correlation studies is that ABCB1 mRNA likely degrades rapidly after
delivery; rapid collection is critical. A more recent study, using brush border membrane
preparations, identified decreased protein expression in C1236T (11% decrease), C3435T (16%),
and G2677T/A (16%) variants compared to their homozygous wild-type control group[24]. A
fourth study contrasts to the previous three reports: a 29% higher Pgp protein expression for the
3435TT genotype was determined compared to 3435CC[25]. A major limitation of the above
studies is that most performed genotyping[21;24;25] and quantification of Pgp[23;25] using
whole placental tissue, which contains both maternal and fetal tissue. Since Pgp localized to the
syncytiotrophoblast will be of fetal origin, care must be taken to genotype only fetal tissue[23].
Furthermore, placental blood vessels of maternal origin have been shown to express Pgp, thus
fetal and maternal-derived Pgp should be separated in order to produce a clear genotype�
phenotype association[23].
Although most studies linking ABCB1 polymorphisms to protein expression have focused
on the coding region, Takane et al. focused on promoter variants[26]. Haplotypes that
simultaneously included T-1517aC, T-1017aC, and T-129C mutations in Japanese and Caucasian
subjects were associated with increased mRNA expression and altered protein-DNA binding,
independent of the C3435T mutation. These promoter polymorphisms also altered gene
208
transcription. In Japanese and Caucasian populations, the aforementioned haplotypes are found
in low frequency and thus their clinical relevance remains to be determined[26].
In addition to mutations in the ABCB1 gene, epigenetic regulation may control
transcription. Despite a CpG-rich promoter region found in the human ABCB1 gene, potential
transcription factor binding sites were found to be hypomethylated in full term-placenta
tissue[26]. On the other hand, hypermethylation of the ABCB1 promoter has been shown to
decrease Pgp expression in different types of cancer cells[27-29]. The small study in term
placenta (n=7), failed to identify an association between methylation status and mRNA
expression[26]. Further studies utilizing a larger sample size, expanded promoter mapping and
other promoter genotypes are clearly required.
In each study that examined placental ABCB1 polymorphisms, there is large variation in
Pgp expression found for each genotype. Given this large interindividual variation in expression,
the sample sizes of the five studies may be too small to provide adequate statistical power.
Furthermore, the large variability may result from other environmental factors that could also
influence expression. For example, common medications in pregnancy may influence Pgp
expression (such as morphine[30]). Only the study by Rahi et al.[25] required mothers to be
healthy and not taking any medications throughout the pregnancy. Once environmental factors
are controlled for, full haplotype analysis may result in less variability between genotypes and
stronger associations and should be included in future studies. Another limitation from the
studies linking ABCB1 genotype to Pgp expression is that only full term placentae were
analyzed. Since placental Pgp expression is at its lowest at term[3;5], any association with
genotype may be harder to establish and require a much larger sample size to obtain significance.
The influence of ABCB1 SNPs and Pgp expression outside of the placenta has also led to
conflicting results. A meta-analysis of three studies (total n=261) found no effect of the C3435T
SNP on intestinal ABCB1 mRNA expression[31]. On the other hand, a large systematic study
not included in the meta-analysis found that the 3435TT genotype had a 2-fold reduction in
intestinal Pgp protein expression compared to 3435CC[32]. Similar findings were observed in
lymphocytes[33;34] and renal epithelial tissue[35].
209
Table 2. Summary of the associations between A) ABCB1 genotype and placental Pgp expression B) ABCG2 genotype and placental BCRP expression. A) Study Population SNP Finding Placental Preparation Tanabe et al.(2001) Japanese (n=100) T-129C -129TC < -129TT protein from enriched trophoblasts G2677A/T NS C3435T NS Hitzl et al. (2004) Caucasian (n=73) T-129C N/A protein/mRNA from whole placental tissue G2677A/T NS C3435T NS Hemauer et al. (2009) Caucasian, C1236T 1236T < 1236CC protein from brush border membranes African-American G2677A/T 2677A/T < 2677GG Hispanic (n=199) C3435T 3435T < 3435CC Rahi et al. (2008) Caucasian (n=44) G2677A/T NS protein from whole placental tissue C3435T 3435TT > 3435CC Takane et al. (2004) Japanese (n=89) -1517aC, -1017aC, -129C mRNA from enriched trophoblasts
haplotype has > mRNA
B) Study Population SNP Finding Placental Preparation Kobayashi et al. (2005) Japanese (n=100) C376T Creates a stop codon protein from enriched trophoblasts
C421A 421AA < 421CC G34A NS
210
Pgp polymorphisms and altered placental transfer of drugs
The effect of ABCB1 polymorphisms on transplacental drug transfer has been
investigated for a limited number of substrates. The first series of studies investigated the
influence of 2 SNPs (C3435T and G2677A/T) on the transfer of the Pgp substrate saquinavir
using the dual perfusion of a single lobule in a human term placenta. There was no effect of the
C3435T or G2677A/T genotype on saquinavir transport[15;27]. On the basis of these studies,
the authors suggested that SNPs in ABCB1 do not have a significant impact on transfer.
However, these studies had small sample size and restricted the analysis to only two SNPs. A
more practical and rapid method for determining Pgp function is through preparation of inside
out vesicles from the brush border membrane[24]. Using vesicles from 105 term placentae,
Hemauer et al. identified a >40% increase in Pgp activity in the 1236TT genotype compared to
1236CC and the 3435TT genotype compared to 3435CC[24]. This increase in Pgp activity was
observed despite the decrease in protein expression for the 1236TT and 3435TT genotypes.
Further studies are clearly needed to elucidate the clinical relevance of these findings.
Pgp polymorphisms and influence on normal physiologic function of the placenta and
development
The importance of Pgp in normal physiology and development is still unclear. Mice
completely deficient in Pgp were initially thought to have little change in various physiological
parameters, including viability, fertility, multiple measures of serum clinical chemistry and
hematological parameters, and lymphocyte differentiation[36]. These initial physiological
observations led to the speculation that the role of Pgp was compensated by other
mechanisms[36]. We have shown that this is likely the case. Mice deficient in abcb1a and
abcb1b exhibit a substantial elevation of placental levels of BCRP protein (unpublished
observation).
Recently, an age-related phenotype has been identified in Pgp-deficient mice; mdr1a null
mice develop colitis[37]. This is likely due to inflammation caused by toxins that are normally
excluded from the intestinal epithelial cells by Pgp efflux[37] or from abnormalities in the
211
epithelial lining of the gut[38]. Pgp deficient mice also exhibit abnormal renal tubular function
as a result of altered intracellular ATP levels and altered mitochondrial morphology[39].
P-glycoprotein likely affects normal development and function of the placenta through
the apoptotic pathways. Pgp may counteract apoptosis by regulating intracellular concentrations
of intermediates in the apoptotic pathway, such as sphingomyelin[40]. Formation of the
syncytiotrophoblast layer involves apoptotic processes and increases in apoptosis are seen in
complications during pregnancy, such as pre-eclampsia and intra-uterine growth restriction (see
Heazell & Crocker[41] for a review). Interestingly, decreased expression of ABCB1 mRNA was
observed in placentas complicated by idiopathic fetal growth restriction (FGR) compared to
controls[42]. Any role of Pgp and its polymorphisms in these pathologies has yet to be
determined.
Breast Cancer Resistance Protein
Expression and function in the placenta
Similar to Pgp, placental BCRP expression has been localized to the microvillous border
of the syncytiotrophoblast[42-45]. BCRP is also expressed on the luminal membrane of fetal
endothelial cells in terminal and intermediate villi[42]. The number of fetal capillaries
expressing BCRP was higher in term tissue compared to that at 6-13 weeks of gestation[45].
Unlike Pgp, there is no clear consensus on how BCRP expression relates to gestational age since
three studies have obtained conflicting results.
The first study reported that ABCG2 mRNA levels and BCRP expression in microvillous
plasma membranes did not differ with gestational age[11]. The second study found no change in
ABCG2 mRNA levels with advancing gestational age, but did identify a significant increase in
whole placental BCRP protein levels at term[45]. The increase in protein but not mRNA levels
suggests that posttranscriptional regulation may be altered with advancing gestation.
Alternatively, the increase in BCRP protein levels may result from the increased BCRP
expression in fetal capillaries and not necessarily in the syncytiotrophoblast[45]. A third study
reported a 2-fold decrease in ABCG2 mRNA as well as BCRP protein from crude trophoblast
212
membranes at term compared to the preterm group (28 ± 1 week)[44]. All three studies
demonstrated large variability in BCRP expression within gestational age group. These studies
suggest that BCRP expression may be more dependent on genetic and environmental influences
than gestational age.
The regulation of human placental BCRP remains unclear; however, in vitro and animal
studies have brought forward possible mechanisms. The ABCG2 promoter region contains
estrogen and progesterone response elements[46;47]. Similar to Pgp, cytokines are also likely
important regulators of placental BCRP. Decreased expression of BCRP in trophoblasts isolated
from term placentae was observed after incubation with TNF-α and IL-1β[12]. Other factors
shown to influence BCRP expression include estriol, human placental lactogen, and prolactin in
BeWo cells[48]; and insulin-like growth factor II and epidermal growth factor in trophoblasts
isolated from term placentae[12;44].
Like Pgp, placental BCRP also confers fetal protection to toxins in the maternal
circulation. In Pgp-deficient mice, chemical inhibition of BCRP resulted in a 2-fold increase in
fetal exposure to maternally administered topotecan (a BCRP substrate) relative to wild-type[49].
Likewise, using a BCRP knockout model, fetal exposure to topotecan[50], genistein[51],
nitrofurantoin[52], and glyburide[53] was increased 2 to 5-fold compared to wild-type controls.
BCRP has also been demonstrated to limit fetal exposure to glyburide using the dually perfused
human placenta model: inhibition of BCRP resulted in an increase in the fetal-maternal ratio of
glyburide[54]. A recent meta-analysis examining the safety of the oral hypoglycemic, glyburide
in pregnancy compared to insulin treatment reported that there was no increased risk for fetal
hypoglycemia, macrosomia, and other adverse fetal effects[55]. Understanding the role of
BCRP in regulating glyburide transfer across the placenta will allow for identification of women
at risk of altered placental transfer due to drug interactions or polymorphisms in BCRP.
Polymorphisms in BCRP and effect on placental BCRP expression
The ABCG2 gene is located on 4q22 and contains 16 exons and 15 introns[56]. The
encoded protein, BCRP, has only one ATP-binding region and one transmembrane domain[56].
Unlike Pgp, BCRP is considered a half transporter and must homodimerize to become
213
functional[57]. In most ethnicities, the G34A and C421A SNPs occur at a relatively high
frequency[58-62], however, there are ethnic differences between SNP and haplotype
distributions[62;63]. The G34A and C421 SNPs result in amino acid changes (V12M and
Q141K respectively).
The relationship between ABCG2 polymorphisms and BCRP expression has only been
examined in one study (Table 2). In a Japanese cohort, 20 polymorphisms were detected in the
16 exons and 5’-flanking region: five SNPs resulted in amino acid substitutions[61]. A SNP
encoding a premature stop codon (C376T) was detected with an allelic frequency of 1%
(heterozygosity only). Due to the low frequency of the 376T allele, a statistical analysis could
not be undertaken. However, the two individuals with the 376T allele, exhibited low levels of
BCRP protein. There was also no association between ABCG2 mRNA and the C421A and G34A
SNPs, despite a difference in protein expression for the C421A SNP. The two SNPs, C421A and
G34A, did not result in an allelic expression imbalance. Interestingly, Kobayashi et al. attempted
to perform a haplotype analysis; however, the C421A variant did not coexist with G34A and
C376T in the Japanese population. Furthermore, the authors did not find any significant
haplotype-dependent changes in mRNA or protein expression when considering polymorphisms
in the 5’-flanking and 3’untranslated regions.
In tissues other than the placenta, the C421A SNP has been shown to decrease or not alter
BCRP expression[60]. There was no significant association between the C421A variant and
intestinal expression of BCRP protein or mRNA[62]. Functional changes in BCRP have also
been demonstrated in cancer patients, where patients with the 421A allele had 3-fold higher
plasma concentrations of diflomotecan compared to wild type controls[64]. The C421A SNP
results in an amino acid change that is differentially charged and is also located in the ATP-
binding region[61]. It has also been suggested that this C421A variant protein has altered
tertiary structure and is more easily degraded[60]. Thus, it is very likely that this SNP has
functional significance, even in the placenta.
Epigenetic modulation has also been shown to be an important regulator of BCRP
expression, particularly in cancer cells. The promoter of the ABCG2 gene contains a CpG island
and aberrant methylation has been shown to suppress transcription in renal carcinoma[65].
Moreover, in human multiple myeloma cell lines and patient plasma cells, expression of BCRP
214
was associated with promoter methylation[66]. Demethylation of the ABCG2 promoter
increased protein expression in lung cell carcinoma[67]. The role of epigenetic regulation of
BCRP has not been studied in the placenta.
BCRP polymorphisms and influence on normal physiologic function of the placenta,
development, and altered placental transfer of drugs
There are currently no studies available that have determined the effect of ABCG2
genotype on BCRP function in the placenta. As discussed previously, BCRP is important in
limiting fetal exposure to various drugs, therefore, altered function could lead to modified fetal
exposure to potential teratogens. Furthermore, new physiological roles for BCRP are currently
being identified and recent associations between BCRP and pregnancy complications involving
the placenta have been made.
Using a BeWo cell model, BCRP was shown to be important in trophoblast
differentiation. Silencing ABCG2 resulted in increases in phosphatidylserine externalization,
intracellular concentrations of ceramide, and apoptosis[68]. Additionally, there was a decrease
in markers of syncytial formation. Similar to Pgp, decreased placental expression of BCRP
(mRNA) was observed in pregnancies with idiopathic FGR[42]. It was suggested that BCRP is a
survival factor for trophoblasts, protecting them from cytokine- or ceramine-induced apoptosis.
Moreover, if reduced BCRP is associated with FGR or other placental complications, it could
render this vulnerable fetus more susceptible to exposure to drugs or toxins normally effluxed by
BCRP.
Another BCRP survival role has been indicated in myeloid progenitor cells from mice in
protecting against hypoxia. Hypoxia results in upregulation of BCRP by HIF-1 and in hypoxic
conditions, BCRP effluxes heme and porphyrins to protect against their toxic effects[69]. In fact,
mice deficient in BCRP develop a protoporphyria[50]. BCRP may also be important in
maintenance of amniotic stem cell differentiation, viability and pluripotency[70]. However, in
BeWo cells, silencing BCRP did not alter intracellular levels of protoporphyrin IX after exposure
to TNF-α and IFN-γ[42]. Thus, the significance of this protective role in the placenta requires
further investigation.
215
BCRP may also play a role in regulating placental estrogen synthesis by modulating
levels of precursor molecules, such as dehydroepiandrosterone-sulfate (DHEAS), or of metabolic
products, such as estrone-3-sulfate[71]. Regulation of transport of essential nutrients to the fetus,
such as folate and porphyrins also involves BCRP[50]. The localization of BCRP to the junction
between amnion epithelial cells and chorion nuclei suggest that the protein may also play a role
in intercellular communication and regulating access of substrates to the nuclear
compartment[72].
Summary and Implications
To date, there is evidence that the T-129C, C1236T, G2677T/A and C3435T SNPs in
ABCB1 alter expression of Pgp, and that the C376T and C421A SNPs in ABCG1 alter expression
of BCRP in the placenta. However, the functional significance of these polymorphisms in the
placenta is less clear. This lack of clarity likely partially results from methodological limitations
resulting in reduced statistical power. In addition, studies have focused on use of term placental
tissue (when P-gp levels are very low), thus the impact of polymorphisms in function of the
preterm tissue remain unknown. Animal studies will be crucial to investigate the functional
relevance of altered Pgp and BCRP expression earlier in gestation since normal human preterm
tissue will not be obtainable. Many of the human SNP studies have not included haplotype
analysis; investigation of one individual SNP may have poor predictive value[31]. More studies
are needed to determine the impact of polymorphisms in placental Pgp and BCRP on fetal drug
exposure and normal placental function and development.
To date, the role of polymorphisms in Pgp and BCRP in modulating drug-induced
malformations in humans has not been addressed[23]. However, there are many important
examples of how a better understanding of polymorphisms in Pgp and BCRP may lead to
personalized pharmacotherapy in pregnancy. First, the pharmacologic treatment of epilepsy is
often unavoidable in pregnancy because uncontrolled epilepsy carries risks for complications and
adverse fetal outcomes[73]. The antiepileptic drug phenytoin taken during pregnancy is
associated with a definite fetal malformation phenotype[74]. Phenytoin is a substrate for
Pgp[75] and variability in Pgp function due to genetic polymorphisms may explain why most
infants born to epileptic mothers are protected from the congenital malformations and cognitive
216
impairment while others are not[76]. Establishing a hierarchy of haplotypes could lead to genetic
counseling to better assess fetal risk to phenytoin exposure. This will influence drug choice and
give a better prediction of teratogenicity[76].
Other important examples include the use of glyburide and protease inhibitors during
pregnancy. Placental BCRP is important in limiting fetal exposure to glyburide. However, the
influence of BCRP genotype has not been specifically studied. Susceptible genotypes should be
investigated to avoid any potential increased fetal exposure to glyburide since it could lead to
fetal hypoglycemia. A safe orally administered hypoglycemic is desired for the treatment of
gestational diabetes since current treatment involves daily insulin injections. Daily injections
may lead to impaired adherence and the cost of insulin injections may not be affordable in many
parts of the world[55].
In contrast to glyburide where limited fetal exposure is desired, it may be favorable to
transfer protease inhibitors to the fetus to prevent vertical HIV transmission. Favorable
genotypes could be identified that will allow for optimal transfer of these drugs to the fetus and
for personalized medication selection. Furthermore, a complete understanding of the
relationship between placental drug transporter genotype and phenotype could result in better
drug design for safe and effective drugs in pregnancy.
In summary, a full understanding of which SNPs in Pgp and BCRP affect placental
expression and function would be useful for predicting altered fetal exposure to drugs. This is
clinically relevant when limiting fetal exposure to potential teratogens is desired or when drug
delivery to the fetus is warranted for therapeutic effects. The importance of Pgp and BCRP in
normal placental physiology and development is emerging and susceptible genotypes to
complications such as idiopathic FGR may be identified. Future studies must give consideration
to haplotypes, environmental factors, sample size, gestational age of the placenta, comorbidities
and concurrent medications in order to avoid spurious associations.
217
Reference List
[1] Macfarland A, Abramovich DR, Ewen SWB, Pearson CK. Stage-specific distribution
of P-glycoprotein in first-trimester and full-term human placenta. Histochem J 1994;
26:417-23.
[2] Sugawara I, Akiyama S, Scheper RJ, Itoyama S. Lung resistance protein (LRP)
expression in human normal tissues in comparison with that of MDR1 and MRP.
Cancer Lett 1997; 112:23-31.
[3] Sun M, Kingdom J, Baczyk D, Lye SJ, Matthews SG, Gibb W. Expression of the
Multidrug Resistance P-Glycoprotein, (ABCB1 glycoprotein) in the Human Placenta
Decreases with Advancing Gestation. Placenta 2006; 27:602-9.
[4] Bremer S, Hoof T, Wilke M, Busche R, Scholte B, Riordan JR et al. Quantitative
expression patterns of multidrug-resistance P-glycoprotein (MDR1) and differentially
spliced cystic-fibrosis transmembrane-conductance regulatory mRNA transcripts in
human epithelia. Eur J Biochem 1992; 206:137-49.
[5] Gil S, Saura R, Forestier F, Farinotti R. P-glycoprotein expression of the human
placenta during pregnancy. Placenta 2005; 26:268-70.
[6] Atkinson DE, Greenwood SL, Sibley CP, Glazier JD, Fairbairn LJ. Role of MDR1 and
MRP1 in trophoblast cells, elucidated using retroviral gene transfer. Am J Physiol Cell
Physiol 2003; 285:C584-91.
[7] Petropoulos S, Kalabis GM, Gibb W, Matthews SG. Functional changes of mouse
placental multidrug resistance phosphoglycoprotein (ABCB1) with advancing gestation
and regulation by progesterone. Reprod Sci 2007; 14:321-8.
[8] Hemauer SJ, Patrikeeva SL, Nanovskaya TN, Hankins GDV, Ahmed MS. Opiates
inhibit paclitaxel uptake by P-glycoprotein in preparations of human placental inside-
out vesicles. Biochem Pharmacol 2009; 78:1272-8.
[9] Kohno K, Sato S, Uchiumi T, Takano H, Kato S, Kuwano M. Tissue-specific enhancer
of the human multidrug-resistance (MDR1) gene. J Biol Chem 1990; 265:19690-6.
218
[10] Loregger T, Pollheimer J, Knofler M. Regulatory transcription factors controlling
function and differentiation of human trophoblast - A review. Placenta 2003; 24:S104-
S110.
[11] Mathias AA, Hitti J, Unadkat JD. P-glycoprotein and breast cancer resistance protein
expression in human placentae of various gestational ages. Am J Physiol Regul Integr
Comp Physiol 2005; 289:R963-R969.
[12] Evseenko DA, Paxton JW, Keelan JA. Independent regulation of apical and basolateral
drug transporter expression and function in placental trophoblasts by cytokines,
steroids, and growth factors. Drug Metab Dispos 2007; 35:595-601.
[13] Petrovic V, Wang JH, Piquette-Miller M. Effect of endotoxin on the expression of
placental drug transporters and glyburide disposition in pregnant rats. Drug Metab
Dispos 2008; 36:1944-50.
[14] Smit JW, Huisman MT, Van Tellingen O, Wiltshire HR, Schinkel AH. Absence or
pharmacological blocking of placental P-glycoprotein profoundly increases fetal drug
exposure. J Clin Invest 1999; 104:1441-7.
[15] Molsa M, Heikkinen T, Hakkola J, Hakala K, Wallerman O, Wadelius M et al.
Functional role of P-glycoprotein in the human blood-placental barrier. Clin Pharmacol
Ther 2005; 78:123-31.
[16] Sudhakaran S, Rayner CR, Li J, Kong DCM, Gude NM, Nation RL. Inhibition of
placental P-glycoprotein: Impact on indinavir transfer to the foetus. Br J Clin
Pharmacol 2008; 65:667-73.
[17] Nanovskaya T, Nekhayeva I, Karunaratne N, Audus K, Hankins GDV, Ahmed MS.
Role of P-glycoprotein in transplacental transfer of methadone. Biochem Pharmacol
2005; 69:1869-78.
[18] Callen DF, Baker E, Simmers RN, Seshadri R, Roninson IB. Localization of the human
multiple drug resistance gene, MDR1, to 7q21.1. Hum Genet 1987; 77:142-4.
219
[19] Lankas GR, Wise LD, Cartwright ME, Pippert T, Umbenhauer DR. Placental P-
glycoprotein deficiency enhances susceptibility to chemically induced birth defects in
mice. Reprod Toxicol 1998; 12:457-63.
[20] Li YH, Wang YH, Li Y, Yang L. MDR1 gene polymorphisms and clinical relevance.
Acta Genetica Sinica 2006; 33:93-104.
[21] Tanabe M, Ieiri I, Nagata N, Inoue K, Ito S, Kanamori Y et al. Expression of P-
glycoprotein in human placenta: Relation to genetic polymorphism of the multidrug
resistance (MDR)-1 gene. J Pharmacol Exp Ther 2001; 297:1137-43.
[22] Mathijssen RHJ, Marsh S, Karlsson MO, Xie R, Baker SD, Verweij J et al. Irinotecan
pathway genotype analysis to predict pharmacokinetics. Clin Cancer Res 2003; 9:3246-
53.
[23] Hitzl M, Schaeffeler E, Hocher B, Slowinski T, Halle H, Eichelbaum M et al. Variable
expression of P-glycoprotein in the human placenta and its association with mutations
of the multidrug resistance 1 gene (MDR1, ABCB1). Pharmacogenetics 2004; 14:309-
18.
[24] Hemauer SJ, Nanovskaya TN, Abdel-Rahman SZ, Patrikeeva SL, Hankins GDV,
Ahmed MS. Modulation of human placental P-glycoprotein expression and activity by
MDR1 gene polymorphisms. Biochem Pharmacol 2009; In Press.
[25] Rahi M, Heikkinen T, Hakkola J, Hakala K, Wallerman O, Wadelius M et al. Influence
of adenosine triphosphate and ABCB1 (MDR1) genotype on the P-glycoprotein-
dependent transfer of saquinavir in the dually perfused human placenta. Hum Exp
Toxicol 2008; 27:65-71.
[26] Takane H, Kobayashi D, Hirota T, Kigawa J, Terakawa N, Otsubo K et al. Haplotype-
oriented genetic analysis and functional assessment of promoter variants in the MDR1
(ABCB1) gene. J Pharmacol Exp Ther 2004; 311:1179-87.
[27] Baker EK, Johnstone RW, Zalcberg JR, El Osta A. Epigenetic changes to the MDR1
locus in response to chemotherapeutic drugs. Oncogene 2005; 24:8061-75.
220
[28] Fryxell KB, McGee SB, Simoneaux DK, Willman CL, Cornwell MM. Methylation
analysis of the human multidrug resistance 1 gene in normal and leukemic
hematopoietic cells. Leukemia 1999; 13:910-7.
[29] Kusaba H, Nakayama M, Harada T, Nomoto M, Kohno K, Kuwano M et al.
Association of 5' CpG demethylation and altered chromatin structure in the promoter
region with transcriptional activation of the multidrug resistance gene in human cancer
cells. Eur J Biochem 1999; 262:924-32.
[30] Aquilante CL, Letrent SP, Pollack GM, Brouwer KLR. Increased brain P-glycoprotein
in morphine tolerant rats. Life Sci 1999; 66:PL47-51.
[31] Chowbay B, Li H, David M, Cheung YB, Lee EJD. Meta-analysis of the influence of
MDR1 C3435T polymorphism on digoxin pharmacokinetics and MDR1 gene
expression. Br J Clin Pharmacol 2005; 60:159-71.
[32] Hoffmeyer S, Burk O, Von Richter O, Arnold HP, Brockm+¦ller J, Johne A et al.
Functional polymorphisms of the human multidrug-resistance gene: Multiple sequence
variations and correlation of one allele with P-glycoprotein expression and activity in
vivo. Proc Natl Acad Sci USA 2000; 97:3473-8.
[33] Fellay J, Marzolini C, Meaden ER, Back DJ, Buclin T, Chave JP et al. Response to
antiretroviral treatment in HIV-1-infected individuals with allelic variants of the
multidrug resistance transporter 1: A pharmacogenetics study. Lancet 2002; 359:30-6.
[34] Hitzl M, Drescher S, Van der Kuip H, Scha¦êffeler E, Fischer J, Schwab M et al. The
C3435T mutation in the human MDR1 gene is associated with altered efflux of the P-
glycoprotein substrate rhodamine 123 from CD56+ natural killer cells.
Pharmacogenetics 2001; 11:293-8.
[35] Siegsmund M, Brinkmann U, Scha¦êffeler E, Weirich G, Schwab M, Eichelbaum M et
al. Association of the P-glycoprotein transporter MDR1C3435T polymorphism with the
susceptibility to renal epithelial tumors. J Am Soc Nephrol 2002; 13:1847-54.
221
[36] Schinkel AH, Mayer U, Wagenaar E, Mol CAAM, Van Deemter L, Smit JJM et al.
Normal viability and altered pharmacokinetics in mice lacking mdr1-type (drug-
transporting) P-glycoproteins. Proc Natl Acad Sci USA 1997; 94:4028-33.
[37] Panwala CM, Jones JC, Viney JL. A novel model of inflammatory bowel disease: Mice
deficient for the multiple drug resistance gene, mdr1a, spontaneously develop colitis. J
Immunol 1998; 161:5733-44.
[38] Resta-Lenert S, Smitham J, Barrett KE. Epithelial dysfunction associated with the
development of colitis in conventionally housed mdr1a-/- mice. Am J Physiol
Gastrointest Liver Physiol 2005; 289:G153-G162.
[39] Huls M, Kramers C, Levtchenko EN, Wilmer MJG, Dijkman HBPM, Kluijtmans LAJ
et al. P-glycoprotein-deficient mice have proximal tubule dysfunction but are protected
against ischemic renal injury. Kidney Int 2007; 72:1233-41.
[40] Smyth MJ, Krasovskis E, Sutton VR, Johnstone RW. The drug efflux protein, P-
glycoprotein, additionally protects drug-resistant tumor cells from multiple forms of
caspase-dependent apoptosis. Proc Natl Acad Sci USA 1998; 95:7024-9.
[41] Heazell AEP, Crocker IP. Live and Let Die - Regulation of Villous Trophoblast
Apoptosis in Normal and Abnormal Pregnancies. Placenta 2008; 29:772-83.
[42] Evseenko DA, Murthi P, Paxton JW, Reid G, Emerald BS, Mohankumar KM et al. The
ABC transporter BCRP/ABCG2 is a placental survival factor, and its expression is
reduced in idiopathic human fetal growth restriction. FASEB J 2007; 21:3592-605.
[43] Maliepaard M, Scheffer GL, Faneyte IF, Van Gastelen MA, Pijnenborg ACLM,
Schinkel AH et al. Subcellular localization and distribution of the Breast Resistance
Protein Transporter in normal human tissues. Cancer Res 2001; 61:3458-64.
[44] Meyer Zu Schwabedissen HE, Grube M, Dreisbach A, Jedlitschky G, Meissner K,
Linnemann K et al. Epidermal growth factor-mediated activation of the MAP kinase
cascade results in altered expression and function of ABCG2 (BCRP). Drug Metab
Dispos 2006; 34:524-33.
222
[45] Yeboah D, Sun M, Kingdom J, Baczyk D, Lye SJ, Matthews SG et al. Expression of
breast cancer resistance protein (BCRP/ABCG2) in human placenta throughout
gestation and at term before and after labor. Can J Physiol Pharmacol 2006; 84:1251-8.
[46] Ee PLR, Kamalakaran S, Tonetti D, He X, Ross DD, Beck WT. Identification of a
Novel Estrogen Response Element in the Breast Cancer Resistance Protein (ABCG2)
Gene. Cancer Res 2004; 64:1247-51.
[47] Wang H, Lee EW, Zhou L, Leung PCK, Ross DD, Unadkat JD et al. Progesterone
receptor (PR) isoforms PRA and PRB differentially regulate expression of the breast
cancer resistance protein in human placental choriocarcinoma BeWo cells. Mol
Pharmacol 2008; 73:845-54.
[48] Wang H, Unadkat JD, Mao Q. Hormonal regulation of BCRP expression in human
placental BeWo cells. Pharm Res 2008; 25:444-52.
[49] Jonker JW, Smit JW, Brinkhuis RF, Maliepaard M, Beijnen JH, Schellens JHM et al.
Role of breast cancer resistance protein in the bioavailability and fetal penetration of
topotecan. J Natl Cancer Inst 2000; 92:1651-6.
[50] Jonker JW, Buitelaar M, Wagenaar E, Van der Valk MA, Scheffer GL, Scheper RJ et
al. The breast cancer resistance protein protects against a major chlorophyll-derived
dietary phototoxin and protoporphyria. Proc Natl Acad Sci USA 2002; 99:15649-54.
[51] Enokizono J, Kusuhara H, Sugiyama Y. Effect of breast cancer resistance protein
(Bcrp/Abcg2) on the disposition of phytoestrogens. Mol Pharmacol 2007; 72:967-75.
[52] Zhang Y, Wang H, Unadkat JD, Mao Q. Breast cancer resistance protein 1 limits fetal
distribution of nitrofurantoin in the pregnant mouse. Drug Metab Dispos 2007;
35:2154-8.
[53] Zhou L, Naraharisetti SB, Wang H, Unadkat JD, Hebert MF, Mao Q. The breast cancer
resistance protein (Bcrp1/Abcg2) limits fetal distribution of glyburide in the pregnant
mouse: An Obstetric-Fetal Pharmacology Research Unit Network and University of
Washington Specialized Center of Research Study. Mol Pharmacol 2008; 73:949-59.
223
[54] Pollex E, Lubetsky A, Koren G. The Role of Placental Breast Cancer Resistance
Protein in the Efflux of Glyburide across the Human Placenta. Placenta 2008; 29:743-7.
[55] Moretti ME, Rezvani M, Koren G. Safety of glyburide for gestational diabetes: A meta-
analysis of pregnancy outcomes. Ann Pharmacother 2008; 42:483-90.
[56] Allikmets R, Schriml LM, Hutchinson A, Romano-Spica V, Dean M. A human
placenta-specific ATP-binding cassette gene (ABCP) on chromosome 4q22 that is
involved in multidrug resistance. Cancer Res 1998; 58:5337-9.
[57] Kage K, Tsukahara S, Sugiyama T, Asada S, Ishikawa E, Tsuruo T et al. Dominant-
negative inhibition of breast cancer resistance protein as drug efflux pump through the
inhibition of S-S dependent homodimerization. Int J Cancer 2002; 97:626-30.
[58] Backstrom G, Taipalensuu J, Melhus H, Brandstrom H, Svensson AC, Artursson P et
al. Genetic variation in the ATP-binding Cassette Transporter gene ABCG2 (BCRP) in
a Swedish population. Eur J Pharm Sci 2003; 18:359-64.
[59] Bosch TM, Kjellberg LM, Bouwers A, Koeleman BPC, Schellens JHM, Beijnen JH et
al. Detection of single nucleotide polymorphisms in the ABCG2 gene in a Dutch
population. Am J Pharmacogenomics 2005; 5:123-31.
[60] Imai Y, Nakane M, Kage K, Tsukahara S, Ishikawa E, Tsuruo T et al. C421A
polymorphism in the human breast cancer resistance protein gene is associated with
low expression of Q141K protein and low-level drug resistance. Mol Cancer Ther
2002; 1:611-6.
[61] Kobayashi D, Ieiri I, Hirota T, Takane H, Maegawa S, Kigawa J et al. Functional
assessment of ABCG2 (BRCP) gene polymorphisms to protein expression in human
placenta. Drug Metab Dispos 2005; 33:94-101.
[62] Zamber CP, Lamba JK, Yasuda K, Farnum J, Thummel K, Schuetz JD et al. Natural
allelic variants of breast cancer resistance protein (BCRP) and their relationship to
BCRP expression in human intestine. Pharmacogenetics 2003; 13:19-28.
224
[63] Maekawa K, Itoda M, Sai K, Saito Y, Kaniwa N, Shirao K et al. Genetic variation and
haplotype structure of the ABC transporter gene ABCG2 in a Japanese population.
Drug metab pharmacokinet 2006; 21:109-21.
[64] Sparreboom A, Gelderblom H, Marsh S, Ahluwalia R, Obach R, Principe P et al.
Diflomotecan pharmacokinetics in relation to ABCG2 421C>A genotype. Clin
Pharmacol Ther 2004; 76:38-44.
[65] To KKW, Zhan Z, Bates SE. Aberrant promoter methylation of the ABCG2 gene in
renal carcinoma. Mol Cell Biol 2006; 26:8572-85.
[66] Turner JG, Gump JL, Zhang C, Cook JM, Marchion D, Hazlehurst L et al. ABCG2
expression, function, and promoter methylation in human multiple myeloma. Blood
2006; 108:3881-9.
[67] Nakano H, Nakamura Y, Soda H, Kamikatahira M, Uchida K, Takasu M et al.
Methylation status of breast cancer resistance protein detected by methylation-specific
polymerase chain reaction analysis is correlated inversely with its expression in drug-
resistant lung cancer cells. Cancer 2008; 112:1122-30.
[68] Evseenko DA, Paxton JW, Keelan JA. The Xenobiotic Transporter ABCG2 Plays a
Novel Role in Differentiation of Trophoblast-like BeWo Cells. Placenta 2007; 28:S116-
20.
[69] Krishnamurthy P, Ross DD, Nakanishi T, Bailey-Dell K, Zhou S, Mercer KE et al. The
stem cell marker Bcrp/ABCG2 enhances hypoxic cell survival through interactions
with heme. J Biol Chem 2004; 279:24218-25.
[70] Aye IL, Paxton JW, Evseenko DA, Keelan JA. Expression, Localisation and Activity of
ATP Binding Cassette (ABC) Family of Drug Transporters in Human Amnion
Membranes. Placenta 2007; 28:868-77.
[71] Grube M, Reuther S, Meyer Zu Schwabedissen H, Köck K, Draber K, Ritter CA et al.
Organic anion transporting polypeptide 2B1 and breast cancer resistance protein
225
interact in the transepithelial transport of steroid sulfates in human placenta. Drug
Metab Dispos 2007; 35:30-5.
[72] Yeboah D, Kalabis GM, Sun M, Ou RC, Matthews SG, Gibb W. Expression and
localisation of breast cancer resistance protein (BCRP) in human fetal membranes and
decidua and the influence of labour at term. Reprod Fertil Dev 2008; 20:328-34.
[73] Viinikainen K, Heinonen S, Eriksson K, Kälviäinen R. Community-based, prospective,
controlled study of obstetric and neonatal outcome of 179 pregnancies in women with
epilepsy. Epilepsia 2006; 47:186-92.
[74] Nulman I, Laslo D, Koren G. Treatment of epilepsy in pregnancy. Drugs 1999; 57:535-
44.
[75] Luna-Tortos C, Fedrowitz M, Loscher W. Several major antiepileptic drugs are
substrates for human P-glycoprotein. Neuropharmacology 2008; 55:1364-75.
[76] Atkinson DE, Brice-Bennett S, D'Souza SW. Antiepileptic medication during
pregnancy: Does fetal genotype affect outcome? Pediatr Res 2007; 62:120-7.
[77]Marzolini C, Paus E, Buclin T, Kim RB. Polymorphisms in human MDR1 (P-
glycoprotein): recent advances and clinical relevance. Clin Pharmacol Ther 2004
;75:13-33
[78] Mao Q, Unadkat JD. Role of the breast cancer resistance protein (ABCG2) in drug
transport. AAPS J 2005 ;7:E118-33.
[79] St-Pierre MV, Serrano MA, Macias RI, Dubs U, Hoechli M, Lauper U et al.
Expression of members of the multidrug resistance protein family in human term
placenta. Am J Physiol Regul Integr Comp Physiol 2000; 279:R1495-503.
[80] Meyer Zu Schwabedissen HE, Grube M, Heydrich B, Linnemann K, Fusch C, Kroemer
HK, Jedlitschky G. Expression, localization, and function of MRP5 (ABCC5), a
transporter for cyclic nucleotides, in human placenta and cultured human trophoblasts:
effects of gestational age and cellular differentiation. Am J Pathol 2005;166:39-48.
226
[81] Nagashige M, Ushigome F, Koyabu N, Hirata K, Kawabuchi M, Hirakawa T et al.
Basal membrane localization of MRP1 in human placental trophoblast. Placenta.
2003;24:951-8.
