STUDY OF GENE POLYMORPHISMS RELATED TO DRUG LEVEL AND CLINICAL BEHAVIOR IN KIDNEY TRANSPLANT PATIENTS: AN INDIAN

SCENARIO

A Thesis submitted to the

University of Mumbai for the

M.Sc. (by research) Degree In Applied Biology

Submitted By

Himanshu S. Raje

Under The Guidance Of

Dr. T. F. Ashavaid. Ph. D., FACB Consultant Biochemist

Head Dept. of Lab Medicine Jt. Director Research

RESEARCH LABORATORIES P.D. HINDUJA NATIONAL HOSPITAL AND MEDICAL RESEARCH CENTRE

MUMBAI - 400 016

SEPTEMBER 2008

STATEMENT BY THE CANDIDATE As required by the University Regulation No. R.2190, I wish to state that the work embodied in this thesis titled “Study of gene polymorphisms related to drug level and clinical behavior in kidney transplant patients: an Indian scenario” forms my own contribution to the research work carried out under the guidance of “Dr. T. F. Ashavaid” at the “P.D. Hinduja National Hospital And Medical Research Center.” This work has not been submitted for any other degree of this or any other university. Whenever references have been made to previous works of others, it has been clearly indicated as such and included in the Bibliography. Certified By Dr. T.F. Ashavaid. Ph.D., FACB Himanshu S. Raje Consultant Biochemist, Research Student Head Laboratory Medicine, Joint Director, Research. P.D. Hinduja National Hospital And Medical Research Centre.

CONTENTS No. CHAPTERS PAGE No. 1. INTRODUCTION 1 2. REVIEW OF LITERATURE 5 3. AIMS AND OBJECTIVES 39 4. MATERIALS AND METHODS 40 5. RESULTS AND DISCUSSION 52 6. CONCLUSION 67 7. BIBLIOGRAPHY 70 8. APPENDIX 9. SYNOPSIS

CHAPTER 1

INTRODUCTION

1

Chapter 1 - Introduction

The transplantation of organs to replace those lost or damaged through injury,

disease or deliberate mutilation has been practiced longer than most people realize,

but not successfully achieved until the later half of the twentieth century. Legend

has it that saints Cosmas and Damian, in the third century, replaced an entire

gangrenous leg removed from Justinian, a church elder in Constantinople, with one

taken from a corpse. While it is highly unlikely that this actually happened, records

dating back many centuries show that physicians on the Indian subcontinent

developed techniques for moving flaps of skin from an individual’s forehead

downward onto their face to reconstruct noses lost through injury, infection or legal

penalties involving mutilation. The first scientifically documented successful case in

western medical literature was described in 1596 by Gaspari Toggliacozzi at the

University of Bologna who, knowing that transplants between individuals had little or

no hope of success and deciding against using a wooden or metal substitute,

transferred a piece of skin from a man’s arm onto his face to reconstruct the

patient’s nose. The paramount importance of the genetic relationship between the

host and the donor to the success of a transplant was not definitively recognized

until the late 1920s when Little and Tyzzer noted a pattern in the rejection of

transplanted tumours in mice. Grafts exchanged between closely related donors

and hosts had a far greater chance of success than those done between unrelated

individuals (Auchimloss et al 1998, Charlton B et al 1994).

Because of tremendous development in the field of medicine, it is now possible to

transplant several organs like heart, liver, pancreas etc. However, the procedure to

transplant failed kidneys has significantly developed. The number of patients

undergoing kidney transplantation as well as the survival rate of organ and hence

the success rate of the procedure exceeds that of any other organ transplantation.

2

Since 1950s, renal transplantation has been increasingly used and the total number

of transplants performed on a worldwide basis, now exceeds half a million. The

increased reliance on transplantation has been possible because of constant

improvements in optimizing the matching of donors and recipients, and in the ability

to manage the host immune system to permit graft survival.

Now a days the concept of renal transplantation has become relatively

straightforward and sufficient technical skills have become available for the transfer

of grafts. However the attempts have been defeated by the recipient’s own immune

system. The immune system is unable to distinguish between the presence of “good

foreign” and “bad foreign” cells. Essentially the immune system of the recipient

views the transfer of even beneficial foreign cells as threats and attempt to destroy

the transplant. The increase in the success of renal transplantation outcome is

mainly attributed to two general discoveries 1) the role of genetics in determining

what the immune system views as foreign, leading to an ability to optimally match

donors and recipients, and 2) the development of techniques to inhibit the ability of

the immune response to damage or destroy transplanted grafts.

Most available grafts are allografts (grafts obtained from genetically non-identical

person) because of genetic diversity among humans, therefore; the alternative

solution to an attack by the immune system upon the graft is to employ some

method of minimizing the capacity of the immune system to do so, that is

immunosuppression. It is defined as the generalized inhibition of the immune

system without reference to the specificity of the rejection response.

Generalized immunosuppression is usually achieved by the application of chemical

agents or antibodies directed against cells of the immune system, particularly

lymphocytes. Drugs such as cyclosporine (CsA) and tacrolimus (FK506) (commonly

called as calcineurin inhibitors or CNIs) suppress the transcription of Interleukin-2

(IL-2) gene and target their effect on those T lymphocytes that are most actively

proliferating and responding at the time of application (presumably those that are

3

attacking the graft) leaving the remaining quiescent T and B lymphocytes relatively

unaffected and available for use in the future if needed (Humar A et al 2001).

The worldwide use of immunosuppressive drugs especially the calcineurin inhibitors

(CNIs) eg. CsA and tacrolimus have enabled this strategy to obtain its current place.

However it is well recognized that responses to calcineurin inhibitors have

significant inter- and intra-individual variation in transplant patients, which have

considerable influence on the drug effects and human bodies. Over-dose of these

drugs has been proved to influence the lifespan of the allograft, they also have their

side effects; most serious of which is nephrotoxicity. The narrow therapeutic indices

for the currently used immunosuppressive drugs owing to their poor bioavailability

and large individual variability in drug administration make the posttransplant drug

therapeutic strategy difficult. Therefore empirical dose has lost its value in the

posttransplant therapy and an individualized dosage regimen must be established

to achieve the optimal immunosuppressive effect (Wu J et al 2004, Bradley J et al

2003).

Many genetic and nongenetic factors such as organ function, drug interactions and

the nature of the disease may influence the effects of these drugs. Genetic factors

are estimated to contribute much to the interindividual variations in drug

administration; it is estimated that genetics can account for 20 to 95 percent of

variability in drug disposition and effects (Kalow W et al 1998, Evans W et al 2003).

Although many nongenetic factors influence the effects of medications, including

age, organ function, concomitant therapy, drug interactions and the nature of the

disease, there are now numerous examples of cases in which interindividual

differences in drug response are due to sequence variants in the genes encoding

drug-metabolizing enzymes, drug transporters or drug-targets. Unlike other factors

influencing drug response, inherited determinants generally remain stable

throughout a person’s lifetime. Pharmacogenetics is such a subject to determine the

genetic factors describing the inherited nature of individual variations, thereby

providing a strong scientific basis for optimizing drug therapy on the basis of each

4

patient’s genetic constitution. In recent years, extensive studies on

pharmacogenetics of immunosuppressive drugs have been focused on the

contribution of drug-metabolizing enzyme cytochrome P450 (CYP)3A and the drug

transporter P-glycoprotein (P-gp). Their contribution has been studied with respect

to the individual administration of CNIs in organ transplantation for they are thought

to be the main determinant of the pharmacokinetics of calcineurin inhibitors (Kalow

W et al 1998, Evans W et al 1999).

Thus, there are two factors responsible for accumulation of drug metabolites in the

body, first is defective metabolism and the other is decreased efflux of the formed

drug metabolites. The cause of this defective metabolism as well as decreased

efflux is mainly attributed to the gene polymorphisms in the responsible genes

respectively.

The study of single nucleotide polymorphisms in the genes involved in CNI

metabolism and CNI-drug target genes is therefore essential to develop an

individualized postoperative therapy so as to avoid the incidences of nephrotoxicity.

CHAPTER 2

REVIEW OF LITERATURE

5

Chapter 2 – Review Of Literature

Structure of kidney

The kidneys are two bean shaped organs lying in the retroperitoneal space, each

weighing 150g. The kidney is an anatomically complex organ, consisting of many

different types of highly specialized cells, arranged in a highly organized three-

dimensional pattern. The functional unit of the kidney is called a nephron (there are

approximately 1 million nephrons in one human kidney); each consists of a

glomerulus and a long tubule, which is composed of a single layer of epithelial cells.

The nephron is segmented into distinct parts e.g. proximal tubule, loop of Henle,

distal tubule, collecting duct- each with a typical cellular appearance and special

functional characteristics.

The nephrons are packed together tightly to make up the kidney parenchyma, which

can be divided into regions. The outer layer of kidney is called as cortex. It consists

of glomeruli, much of the proximal tubule, and some of the more distal portions as

well. The inner section called the medulla, consists largely of the parallel arrays of

the loops of Henle and collecting ducts. The medulla is formed into cone-shaped

regions, called pyramids (the human kidney typically has seven to nine), which

extend into the renal pelvis. The tips of the medullary pyramids are called papillae.

The medulla is important for concentration of the urine; the extracellular fluid in this

region of the kidney has much higher solute concentration than plasma- as much as

four times higher- with highest solute concentration reached at the papillary tips.

6

The renal artery, which enters the kidney at the renal hilum, carries about one fifth

of the cardiac output; this represents the highest tissue-specific blood flow of all

larger organs in the body (about 350ml/min per 100g of tissue). As a consequence

of this generous perfusion, the renal arteriovenous O2 difference is much lower than

that of most other tissues (and blood in the renal vein is noticeably redder in colour

than that in the other veins). The renal artery bifurcates several times after it enters

the kidney and then breaks into arcuate arteries, which run in an archlike fashion,

along the border between the cortex and the outer medulla. Kidney is the organ,

which purifies blood and maintains the body fluid volume (Figure 1 and 2).

Functions of kidney:

The main functions of the kidneys are:1) maintenance of body composition 2)

Excretion of metabolic end products and foreign substances 3) Production and

secretion of enzymes and hormones like rennin, erythropoetin etc. Thus the

principal job of the kidneys is the correction of perturbations in the composition and

volume of body fluids that occur as a consequence of food intake, metabolism,

environmental factors, and exercise. Typically, in healthy people, such perturbations

are corrected within a matter of hours so that in the long term, body fluid volume

and the concentration of most ions do not deviate much from normal set points. In

many disease states, however, these regulatory processes are disturbed, resulting

in persistent deviations in body fluid volumes or ionic concentrations. In case of

severe disease state like ESRD (End Stage Renal Disease) however there is

absolute dysfunction of kidney and hence the permanent solution is kidney

transplantation (Giebisch G et al 2003, Guyton A et al 1991, Koeppen B et al 1992).

7

Renal transplantation

The process of transplantation is the most effective and economical form of renal

replacement treatment. In this process the foreign kidney grafts are obtained from

cadaveric organ donors, living related donors (usually siblings or parents), living

unrelated altruistic donors etc. once the potential recipient has been shown to be fit

for surgery the operation can proceed.

The donor renal vessels are anastomosed to the iliac vessels in the recipient’s

pelvis and the ureter, joined directly to the bladder. Allograft survival is dependant

on the degree of HLA (Human Leukocyte Antigen) matching between the donor and

recipient (the greater the better) and the condition of the kidney at the time of

transplantation. Immunosuppression with combination of agents is started

immediately before operation and continued for as long as the transplant is

functioning, or has the potential to do so and the risks are acceptable. The

commonly used immunosuppressants, CNIs (cyclosporine A and tacrolimus) block

T cell proliferation by affecting cytokine production (mainly IL-2). These agents help

in maintaining allograft and preventing rejection. The causes of early allograft

failures are irreversible acute rejection, vascular thrombosis, and patient death from

infection or co-morbid conditions such as ischemic heart disease. Late transplant

failure results from chronic rejection (a poorly understood process which is in part

immunologically mediated), stopping or inconsistent taking of immunosuppressive

drugs and occasionally recurrence of the original cause of the renal failure, in the

graft. The recipients have an increased risk of death from complications of

immunosuppression such as infection and malignant disease such as lymphoma

and from cardiovascular disease.

Episodes of acute rejection are diagnosed by reduction in allograft function, and

usually confirmed by a biopsy revealing injury and invasion of tubules and vascular

endothelium by lymphocytes. These are usually treated with higher doses of

immunosuppressants. The side effects of these immunosuppressive agents are

8

largely the consequence of reduced immunity but cyclosporine and tacrolimus are

themselves nephrotoxic and may cause hirsutism, gum hypertrophy and tremor.

CsA and tacrolimus are chemically distinct molecules that bind to the intracellular

immunophillins, cyclophillin and FKBP (FK506 Binding Protein) respectively. When

bound, both the molecules inhibit the phosphatase; calcineurin, which is required for

the movement of nuclear factors from cytoplasm to nucleus. Decreased secretion of

IL-2 prevents proliferation of inflammatory responses via B cells and T cells. The

attenuated inflammatory response greatly reduces overall function of the immune

system (Kasiske B 2006).

The bioavailability and metabolism of cyclosporine and tacrolimus are primarily

controlled by the members of the cytochrome P450 isoenzyme system found in the

liver and gastro-intestinal tract. The main efflux pump involved in transporting them

is p-glycoprotein encoded by MDR-1 gene. PGP is a transmembrane transporter

capable of transporting numerous endogenous substances from the cytoplasm to

the exterior of the cell. In the intestines, PGP limits oral bioavailability of drugs by

expelling them from the interior of enterocytes into the gut lumen. After tacrolimus

and cyclosporine reach the blood stream, they are subject to metabolism and

systemic clearance by CYP enzymes, specifically CYP3A4 and CYP3A5. Many

clinical drugs are substrates of the CYP3A4 and CYP3A5 enzyme found in high

concentrations in the cells of the intestine and along with PGP, may reduce the

bioavailability of many drugs. CYP3A4 and CYP3A5 are also concentrated in the

liver and account for much of the first pass clearance from the hepatic portal vein

and systemic clearance of tacrolimus and cyclosporine (Hesselink D et al 2003,

Zheng H et al 2004, Yates C et al 2003, Haufroid V et al 2004, Anglicheu D et al

2003, Macphee I et al 2005, Uesugi M et al 2006, Formea C et al 2004).

9

Cyclosporine A (CsA)

Cyclosporine A (CSA), the first calcineurin inhibitor available for clinical use, was

launched in 1980s and radically changed the field of organ transplantation. The

incidence of acute rejection in solid organ transplantation decreased significantly,

making this event an unusual cause of graft failure nowadays. Early (1-2 years)

graft and patient survival has increased to unparalleled levels. CSA inhibits

interleukin-2 gene transcription and the transition of T lymphocytes from the G0 to

G1 phase of the cell cycle. It binds to a cytoplasmic immunophillin cyclophillin. The

complex CSA-cyclophillin decreases calcium signaling and blocks calcineurin, a

calcium dependant enzyme responsible for the nuclear translocation and

dephosphorylation of the cytosolic activating nuclear factor of T cells which initiates

the transcription of several cytokines mainly interleukin-2 (Allen R et al 1985).

Discovery of cyclosporine in 1971 began a new era in immunopharmacology. It was

the first immunosuppressive drug that allowed selective immunoregulation of T cells

without excessive toxicity. Cyclosporine was isolated from the fungus

Tolypocladium inflatum. It was first investigated as an anti-fungal antibiotic but it’s

spectrum was too narrow to be of any clinical use. J. F. Borel (Figure 3a)

discovered it’s immunosuppressive activity in 1976. This led to further investigations

into it’s properties involving further immunological tests and investigations into it’s

structure and synthesis (Borel J 1976, 1982). Cyclosporine has unwanted side

effects, notably nephrotoxicity. Animal testing showed cyclosporine to be sufficiently

non-toxic to begin clinical trials. These studies initially failed due to poor absorption

of the drug. Once this had been overcome, results were encouraging enough for

cyclosporine to be licensed for use in clinical practice. Cyclosporine changed the

face of renal transplantation. It decreased morbidity and enabled the routine

transplantation of organs that until then had only been done experimentally (Borel J

1981, 1982).

10

CsA is a cyclic polypeptide consisting of 11 amino acids and possess potent

immunosuppressive properties (Figure 3b). The introduction of CsA in the early

1980s dramatically altered the outcomes of renal transplantation. With CsA, five

year patient survival rate after cadaveric transplant is about 90% and five-year graft

survival rate is 75-80%. Thus CsA continued to play a central role in most

immunosuppressive protocols in spite of the introduction of the other newer

immunosuppressants.

The chemical structure of cyclosporine A is shown in the adjoining figure. It belongs

to a family of cyclic polypeptides and includes a unique 9-carbon acid in position 1.

All amide nitrogens are either hydrogen bonded or methylated. It contains a single

D- amino acid residue in position 8 and the methyl amide between residues 9 and

10 in cis configuration; all other methyl amides are in the trans form. Cyclosporine A

is lipophilic and very hydrophobic therefore for clinical administration it must be

solubilized (Cockburn I 1986).

After CsA has been internalized by lymphocytes, it binds to cyclophillin and inhibits

IL-2 by the mechanism described later in this chapter. It is cleared from the body

largely in the form of monohydroxylated (M1 and M17) and N-demethylated (M21)

metabolites. The major site of CsA metabolism is the endoplasmic reticulum of

hepatocytes where the number of cytochrome P450 3A proteins is more. Although

there is considerable interindividual variation in it’s metabolism, the cyclic peptide

structure of cyclosporine is relatively resistant to metabolism, but the side chains

are extensively metabolized. While metabolism is thought to result in the

inactivation of the immunosuppressive properties of the drug, some of the

metabolites are found to contribute to immunosuppression or toxicity (M1 and M9).

CsA and it’s metabolites are excreted mainly through the bile into the feces,

approximately 6% is excreted through the urine. In the presence of hepatic

dysfunction, adjustment in the dose may be necessary (Aoyama T et al 1989, Dai Y

et al 2004).

11

Adverse effects of CsA can be grouped into renal and non-renal side effects. It has

a vasoconstrictor effect on renal vasculature. This (probably a transient, reversible

and dose dependant phenomenon) may cause early post transplant graft

dysfunction.

CsA has been monitored by using 12-hour trough level (C0) with relative success in

the past 2 decades. However data have shown that this technique does not always

correlate with area under the curve or drug exposure, particularly in poor absorbers

of CsA. As stated earlier the microemulsion formulation of cyclosporine, Neoral,

offers more complete and reliable absorption from the gastrointestinal tract than

sandimmune. Recent studies showed that measuring the CsA blood level at 2 hours

(C2) correlated better with the area under the curve (Total drug exposure to the

body from first dose to the next dose) compared with C0. Data suggests that C2

level upto 1500 ng/ml for the first month is a therapeutic range. A C2 level of

approximately 800ng/ml at 1 year may be associated with improved long-term

outcome and less nephrotoxicity (Burdmann E et al 2003).

12

Tacrolimus (FK506)

Tacrolimus (Figure 4a) was discovered in 1987 by a Japanese team headed by T.

Goto, T. Kino and H. Hatanaka; it is among the first macrolide immunosuppressants

discovered, preceded by the discovery of rapamycin (sirolimus) in 1975. It is a

metabolite of an actinomycete Streptomyces tsukubaensis, was first demonstrated

to be immunologically effective in vivo in rat heart allograft recipients in 1987. It was

soon found to be a potent alternative to CsA in several experimental models. The

name ‘tacrolimus’ is reportedly derived from “Tsukuba macrol ide

immunosuppressant (Kino T et al 1987, Pritchard D et al 2005).

Pharmacokinetics: Because tacrolimus is minimally soluble in aqueous solvents, it

is formulated in alcohol and a surfactant for continuous intravenous administration.

The oral formulation is composed of capsules of a solid dispersion of tacrolimus in

hydroxypropyl methylcellulose. Absorption of tacrolimus is incomplete after oral

administration. Its bioavailability ranges from 10-60%, with peak blood level after 1

to 2 hours and half-life of 8 to 24 hours. The oral dose of tacrolimus needs to be

higher than intravenous doses. Administration of tacrolimus by the intravenous

route leads to a rapid distribution of the drug reflected as a rapid decline of the initial

peak concentration, followed by a slower decline over next 24 hours. Tacrolimus is

highly bound to plasma proteins eg. Albumin, and to red blood cells and

lymphocytes. Most of the solid organs exhibit a high concentration of tacrolimus

particularly in the lungs, heart, kidney, pancreas, spleen, and liver. The major part of

metabolism takes place in the intestinal wall and in the liver by the cytochrome

P450 system. At least 15 metabolites have been detected, and some of them show

pharmacological activity. Drug level monitoring is required, because tacrolimus has

high inter and intra- individual variability and a narrow therapeutic index. Drug levels

can be monitored by an enzyme linked immunosorbent assay (ELISA), RIA or by

FPIA from whole blood.

13

Pharmacodynamics: The mechanism of action is similar for tacrolimus and CsA.

The process is initiated by binding of the tacrolimus molecule to cytoplasmic

immunophillins, FKBP (FK506 binding protein), of which the isoform FKBP12 seems

to be involved in the immunosuppressive effect caused by tacrolimus. The

tacrolimus-FKBP complex inhibits the activity of calcineurin, a serine-threonine

phosphatase that regulates IL-2 promoter induction after T cell activation. Inhibition

of calcineurin impedes calcium dependant signal transduction, and inactivates

transcription factors (NFAT) that promote cytokine gene activation, because they

are direct or indirect substrates of calcineurin’s serine-threonine phosphatase

activity. As a consequence, the transcription of cytokines IL-2, IL-3, IL-4, IL-5,

interferon-γ, tumor necrosis factor-α, granulocyte-macrophage colony stimulating

factor, IL-2 and IL-7 receptors, is suppressed by tacrolimus (Liu J 1993, Rao A

1994).

Metabolism: Tacrolimus is metabolized to oxidized metabolites in liver by CYP3A

enzymes. Three mono-demethylated metabolites (M-I, M-II and M-III), three di-

demethylated metabolites (M-V, M-VI and M-VII), one mono-hydroxylated

metabolite (M-IV) and one metabolite modified by multiple reactions (M-VIII) are the

products of metabolism (Figure 4b, c) (Iwasaki K et al 1995).

14

Tacrolimus inhibits lymphocyte activation in vitro 10 to 100 times more potently that

CsA. One explanation might be the higher binding affinity of tacrolimus to FKBP

compared to the binding of cyclosporine to it’s immunophillin called cyclophillin.

Other immunosuppressive effects of tacrolimus include the inhibition of T cell

proliferation and the inhibition of primary or secondary T cytotoxic cell proliferation

in vitro, whereas direct cytotoxicity and calcium dependant T cell stimulation are not

affected. Tacrolimus also suppresses B cell activation in vitro: both induced Ig

production by B cells and the proliferation of stimulated B cells. In vivo, tacrolimus

inhibits proliferative and cytotoxic responses to alloantigens and suppresses

primary antibody responses to T cell dependant antigens, whereas secondary

antibody responses, IL-2 stimulated cell proliferation and natural killer or antibody

dependant cytotoxic cell function are not inhibited (Luke R et al 1994, Manez R et al

1995).

Tacrolimus has been investigated in clinical transplantation of all solid organs, and it

has been approved as an immunosuppressant agent for primary therapy in patients

with liver and kidney transplants. In renal transplantation, tacrolimus was used first

in 1989 in Pittsburgh. Many clinical trials and reports in renal allograft recipients

have been published. Tacrolimus has been proved effective in the patients with

steroid- resistant rejection episodes. In the most recent randomized, comparative

multicenter trial including 412 patients, tacrolimus was equivalent to CsA in 1-year

graft and patient survival. The number and severity of biopsy-proven acute rejection

episodes were significantly lower in the tacrolimus group. After 3 years, patient and

graft survival was still equivalent for both groups, but the number of graft failures

defined, as loss of graft excluding death was significantly lower in the tacrolimus

group. A higher overall incidence of post-transplant diabetes mellitus was observed

in the same group.

Significant nephro and neuro-toxicity have been reported in patients receiving

tacrolimus treatment. One possible mechanism for the neurotoxicity is the inhibition

of calcineurin phosphatase, but the etiology of it’s renal vasculopathic effects is

15

unclear. Reduced renal glomerular and cortical blood flow and increased renal

vascular resistance are generally associated with increased thromboxane A2,

endothelin production or stimulated intra-renal rennin production. Cardiomyopathy,

anemia, chronic diarrhea, onset of diabetes and allergies have been reported in the

patients receiving tacrolimus. Lymphoproliferative diseases and infections are

associated with tacrolimus based immunosuppressive protocols.

In the cytoplasm, cyclosporine A (CsA) binds to it’s immunophillin, cyclophillin

(CpN), forming a complex between cyclosporine and CpN. The cyclosporine-CpN

complex binds and blocks the function of the nuclear factor of activated T cells

(NFATc), and thereby the transport of NFATc to the nucleus and the binding of

NFATc to the nuclear component of the nuclear factor of activated T cells (NFATn).

The NFATc-NFATn complex binds to the promoter of the interleukin 2 (IL-2) gene

and initiates IL-2 production. Consequently, T cells do not produce IL-2, which is

necessary for full T-cell activation. Tacrolimus (FK506) binds to FK506-binding

protein (FKBP), forming a FK506-FKBP complex, which binds to and blocks CaN.

The FK506-FKBP-CaN complex inhibits the activation of NFATc, thus preventing it’s

entrance into the nucleus. Although the pre-drugs cyclosporine and FK506 bind to

different target molecules, both drugs inhibit T-cell activation in the same fashion

(Figure 5) (Rao A 1994, Andrus L et al 1981).

16

Target of CNIs : IL-2 gene

Cytokines are potent immunomodulatory molecules that act as mediators of

inflammation and the immune response. Primarily secreted by T cells and

macrophages, they influence cellular activation, differentiation and function.

Cytokine production is under genetic control. This is evidenced by the identification

of polymorphisms in cytokine gene regulatory regions that correlate with intra-

individual variations in actual cytokine production. As these polymorphisms

segregate independently, each person is a mosaic of high-, intermediate- and low

producing phenotypes. Cytokine polymorphisms have been implicated in a number

of diseases, disorders and toxicities (Cox E et al 2001).

The activation of cytokine genes is dependant on the binding of proteins, so called

transcription factors, to the DNA flanking the genes. In particular, the portion of DNA

immediately upstream of the cytokine gene, known as the promoter region,

influences the expression of cytokine genes. The promoter region of IL-2 gene is

now very well characterized and contains binding sites for many different

transcription factors. Some of these protein factors exist in the cytoplasm and, after

they are modified by phosphorylation or dephosphorylation, translocate to the

nucleus where they bind to the DNA. Alternatively, they are already present in the

nucleus where they are activated, again by the addition or removal of phosphate

groups. The binding of protein factors influences the conformation of DNA and

allows the access and assembly of the complex of RNA polymerase enzymes

required to make an RNA copy of the gene, the process of transcription (Grunfeld J

et al 1998).

DNA binding sites for certain factors appear quite frequently in cytokine gene

promoters. For example, the factor NFAT (nuclear factor of activated T cells) is

important for the regulation of IL-2, TNF, Interferon gamma, IL-4 and others.

Cyclosporine and Tacrolimus act to prevent the translocation of the cytoplasmic

NFAT molecule to the nucleus, thereby suppressing the activation of cytokine gene

17

expression. Other factors include NF-kB, CERB (cyclic adenosine monophosphate

(cAMP) response element binding and AP-1 (activation protein 1). The transcription

factor binding sites present in IL-2 gene are shown in the adjoining figure. One

notable point is that coordinately regulated cytokines (eg. The proinflammatory or

inflammatory cytokines) share the same pattern of transcription factor binding sites

(Figure 6).

The transcription factors are activated as a final event in the different second

messenger pathways that exist in T cells. Flux of calcium leads to NFAT activation,

whereas protein kinase C (PKC) is responsible for the translocation of NFkB.

Elevation of cAMP levels activates protein kinase A (PKA), which phosphorylates

and activates CERB, whereas many cell surface cytokine receptors activate factor

AP-1. Therefore T cell responds differentially to stimuli that selectively activate

second messenger pathways.

This concept is extremely important because we now realize that

immunosuppressive agents can be used to modify second messenger pathways

and cause the T cell to behave differently. Thus, certain drugs or combinations of

drugs can selectively inhibit the production of targeted cytokines.

It is now clear that there are polymorphisms in cytokine genes that predispose to

higher or lower production of cytokines. The DNA sequence in the promoter region

of cytokine genes in different individuals can vary so that although the cytokine itself

is the same, the amount produced can differ 4- to 50-fold. It is because of this

reason the study of promoter or regulatory region gene polymorphisms of cytokine

genes assumes significance (Turner D et al 1997, Grunfeld J et al 1998).

Morgun et al (2003) have recently shown that T-330G regulatory region

polymorphism of IL-2 gene affects allograft outcome after renal transplantation.

They have observed that this polymorphism is related to in vitro IL-2 levels with the

T/T and T/G genotypes being associated with low production and the G/G genotype

18

associated with high production, however a higher frequency of T/T genotype was

observed in renal transplant patients who experienced at least one acute rejection

in the first 3 months after transplantation than in those without rejection. Whereas

Cox et al have observed G/G genotype to be associated with high in vitro IL-2

production in peripheral blood lymphocytes. Effects of this polymorphism on the

incidences of CNI induced nephrotoxicity as well as on IL-2 levels need to be further

studied. Cox et al have also observed that the carriers of G/G genotype have shown

high in vitro IL-2 production. It is also observed that an increase in IL-2 production

has been associated with acute rejection events in renal transplant recipients

(Morgun et al 2003).

As stated earlier, nephrotoxicity is the most worrying side effect of CsA and

Tacrolimus. It is of particular concern in the case of renal transplantation, in which it

has to be distinguished from rejection as a cause of deteriorating renal function. In

the early rat and dog models of transplantation, nephrotoxicity was not noted. It

became evident soon after the initial clinical use of CsA. It was then subsequently

shown in the animal models using higher doses and more sophisticated evaluation

of renal function and some of the morphological changes attributed to nephrotoxicity

were observed in humans (Burdmann E et al 2003).

Nephrotoxicity

Drug induced nephrotoxicity may be caused due to patient related or medication

related risk factors. While patient related factors include age, sex, weight, height

etc. medication related factors are Their inherent nephrotoxic potential, Increased

dose, Their duration, frequency and form of administration, Repeated exposure,

Impairment in the metabolism of these drugs, leading to accumulation of their

metabolites in the body.

19

Because decreased renal function is often unsuspected in elderly or asymptomatic

patients, overdosing with nephrotoxic agents may occur resulting renal injury. When

the frequency of nephrotoxicity is assessed, it has become clear that most renal

injuries tend to cluster around certain patients and specific clinical situations that

represent risk factors. Because nephrotoxicity may limit the clinical usefulness of

many diagnostic and therapeutic agents, recognition of factors associated with

higher risk for renal injury becomes important.

To evaluate nephrotoxicity, it is necessary to define exactly the increase in serum

creatinine concentration that will be used clinically to signal it’s presence. The

common wisdom is to accept increases of 0.5 mg/dL if the baseline level is normal,

or increases of 1 mg/dL or more if the baseline concentration is already elevated.

However, serum creatinine levels should always be evaluated (Thatte L et al 1996).

Three clinical types of nephrotoxicity were observed with CsA and tacrolimus. The

first occurs immediately after transplantation, usually in the kidney already damaged

by ischemia and perhaps associated with the use of intravenous CsA. In humans,

the incidence of delayed function after renal transplantation has tended to be higher

in the patients treated with CsA than in those treated with azathioprine and steroids

(Burdmann E et al 2003)

The second type of nephrotoxicity is seen after the first two or three weeks and is

associated with deteriorating renal function, usually but not always associated with

high blood levels of CNIs, and responds to a reduction in CNI dosage. This type of

nephrotoxicity has to be differentiated from the episodes of acute rejection.

The mechanism of CNI induced nephrotoxicity has not been clarified, but it would

seem that a direct toxic action on proximal tubular cells is not a significant factor. A

decrease in renal blood flow with an increased renal vascular resistance at the level

of the afferent arteriole of the glomerulus and a decreased glomerular filtration rate

20

is the primary cause of nephrotoxicity. The metabolites of CNIs also have a similar

effect (Campistol J et al 2000).

The third type of CNI induced nephrotoxicity is a chronic condition in which there is

a slow and steady deterioration in the renal function and the histology of the kidney

may reveal several interstitial fibrosis. This type of nephrotoxicity shows some

improvement in renal function with a decrease in CNI dosage but this improvement

tends to be relatively short lived. It is likely that many of the changes observed

result from chronic immunological damage on which is superimposed some element

of nephrotoxicity.

Acute nephrotoxicity is the predominant kidney abnormality, seen within the first 6 to

12 months after initiating the treatment and is characterized by an acute or

subacute reduction in kidney function that is often dose-dependant. It is a functional

and reversible abnormality related to a renal imbalance of vasoconstrictor and

vasodilators mediators. The main feature of this form of nephrotoxicity is an intense

internal vasoconstriction reflected by increased renal vascular resistance and

reciprocal renal blood flow decrease, followed by variable degrees of glomerular

filtration rate (GFR) impairment. This vasoconstriction occurs preferentially in the

afferent arterioles but also in the adjacent small arteries, including the glomerular

tuft.

The clinical manifestations of nephrotoxicity are similar for CsA and tacrolimus,

consisting of acute reversible kidney dysfunction and chronic interstitial

nephropathy. Generally kidney dysfunction is nonprogressive and remits when the

dose is lowered or the drug is discontinued. Virtually every patient who receives

therapeutic doses of a CNI will experience a component of persistent, reversible

reduction in glomerular filtration rate (GFR) and renal blood flow. The mechanism of

this acute nephrotoxicity is homodynamic and results from the ability of CNIs to

induce intense renal vasoconstriction. CNIs do not cause vasoconstriction directly

but may act by stimulating production of other vasoconstrictor compound such as

21

thromboxane A2, endothelin and leukotrienes (English J et al 1987, Rosen S et al

1990).

In kidney transplant recipients, within the first year after transplant, it is often difficult

to distinguish acute nephrotoxicity from acute rejection. A stable or slowly

progressive increase in serum creatinine that reverses when the CsA or tacrolimus

dose is reduced suggests nephrotoxicity. Kidney biopsy can be helpful in this

setting, since aggressive inflammatory cell infiltrates are usually absent in acute

nephrotoxicity and their presence in a biopsy specimen would suggest ongoing

rejection. Isometric tubular vacuolization may be observed with calcineurin inhibitor

induced toxicity. Although serum drug levels are frequently used to monitor

therapeutic efficacy and to prevent toxicity, there is only a rough correlation

between serum levels and clinical events.

Chronic nephrotoxicity is defined by the development of interstitial fibrosis with

reduced levels of GFR in the patients receiving long-term treatment with CNIs. The

clinical features of chronic CsA nephrotoxicity are better characterized. Although

chronic nephrotoxicity also occurs with tacrolimus, generally 6 to 12 months of

treatment are required before signs of chronic nephrotoxicity become apparent.

Because of the irreversible nature of the morphologic abnormalities, this form of

toxicity is more ominous than the acute form. Histologically, chronic nephrotoxicity is

characterized by focal or striped medullary interstitial fibrosis. Often, these changes

are accompanied by tubular atrophy and obliterative arteriolar changes. In more

advanced cases, diffuse interstitial fibrosis with focal and segmental glomerular

sclerosis can be seen.. Although the mechanism of chronic nephrotoxicity is not

known, it is likely that cumulative dose, arterial hypertension and immunologic injury

contribute to the development of the lesion (Rosen S et al 1990).

Nephrotoxicity is an example of adverse drug reaction (ADR) caused because of

inappropriate dose of CNIs. There exist interindividual differences in the metabolism

of CNIs and therefore it is well recognized that different patients respond in different

22

ways to the same medication. Individual genetic status has been shown to

contribute much to these variations.

Interpatient variability in response to drug therapy is the rule and not the exception,

for almost all medications. It is well recognized that individuals may respond quite

differently to the same dose or even the same plasma concentration of a drug.

Potential causes for such variability in drug effects include the pathogenesis and

severity of the disease being treated, drug interactions and the individual’s age,

nutritional status, organ function and concomitant diseases. In many cases,

however, genetic factors may have an even greater influence on drug efficacy and

toxicity.

With the completion of Human Genome Project (HGP) and the ongoing annotation

of it’s data, the time is rapidly approaching when the sequences of virtually all genes

encoding enzymes that catalyze phase I and phase II drug metabolism will be

known. The same will be true for genes that encode drug transporters, drug

receptors and other drug targets. As a result, the traditional phenotype-genotype

pharmacogenetic and pharmacogenomic research paradigm is reversing the

direction to create a complementary genotype to phenotype flow of information.

The convergence of advances in pharmacogenetics and human genetics means

that physicians can now individualize the therapy for few drugs. As our knowledge

of genetic variations in the genes involved in the uptake, distribution and

metabolism, and action of various drugs improves, our ability to test for that

variation and, as a result to select the best drug and their optimal dose for each

patient should also increase.

23

Drug metabolism

Human body continuously remains in homeostasis; everything that comes in, must

go out. If the balance is disturbed, the organism accumulates the compound and the

concentration will rise, eventually reaching toxic levels (eg. nephrotoxicity in case of

the accumulation of CsA or tacrolimus). If not eliminated, these compounds would

stay in the body forever and with each repeated dose their concentration would rise

(Meyer U et al 2004).

Once the drug is administered, the drug is absorbed and distributed to it’s site of

action, where it’s interaction with targets (such as receptors and enzymes),

undergoes metabolism, and is then excreted. Each of these processes could

potentially involve clinically significant genetic variations in drug metabolism and

drug transporter genes (Meyer U et al 2004, Cascorbi I et al 2006).

There are more than 30 families of drug-metabolizing enzymes in humans, and

essentially all have genetic variants, many of which translate into functional

changes in the proteins encoded. There are monogenic as well as multigenic effects

on the metabolism due to these variations. There is an example of multigenic effect

involving the CYP3A family of P450 enzymes. About three quarters of whites and

half of blacks have a genetic inability to express functional CYP3A5. The lack of

functional CYP3A5 may not be readily evident, because many medications

metabolized by CYP3A5 are also metabolized by the universally expressed

CYP3A4. For medications that are equally metabolized by both the enzymes, the

net rate of metabolism is the sum of that due to CYP3A4 and CYP3A5; the

existence of this dual pathway partially obscures the clinical effects of genetic

polymorphism of CYP3A5 but contributes to the large range of total CYP3A activity

in humans. The CYP3A pathway of drug elimination is further confounded by the

presence of SNPs in the CYP3A4 gene that alter the activity of this enzyme for

some substrates but not for others. The genetic basis of CYP3A5 deficiency is

predominantly a SNP in intron 3 that creates a cryptic splice site causing 131

24

nucleotides of the intronic sequence to be inserted into the RNA, introducing a

termination codon that prematurely truncates the CYP3A5 protein. Although it is

now possible to determine which patients express both functional enzymes (ie.

CYP3A4 and CYP3A5), the clinical importance of these variants remains unclear

(Evans W et al 1999, Morisseau C et al 2005).

R. T. Wiiliams first defined the field of biotransformation in 1947. He proposed that

drugs or compounds could be biotransformed in two phases: functionalization,

which uses oxygen to form a reactive site and conjugation, which results in addition

of a water-soluble group to the reactive site. These two steps, functionalization and

conjugation, are termed phase I and phase II detoxification, respectively. The result

is the biotransformation of a lipophilic compound, not able to be excreted in urine, to

a water-soluble compound able to be removed in urine. Therefore detoxification is

not one reaction, but rather a process that involves multiple reactions and multiple

players (Estabrook R et al 1996, Liska D et al 1998, Satoh T et al 2002).

Currently over 10 families of phase I enzymes have been described, which include

at least 35 different genes. Phase II reactions are equally complex, and involve

multiple gene families as well (Meyer U et al 2004, Zanger U et al 2004).

The phase I system

The phase I detoxification system, composed mainly of the cytochrome P450

supergene family of enzymes, is generally the first enzymatic defense against

foreign compounds. Most pharmaceuticals are metabolized through phase I

biotransformation. In a typical phase I reaction, a cytochrome P450 enzyme

(Cyp450) uses oxygen and, as a cofactor, NADH, to add a reactive group, such as

hydroxyl radical. As a consequence of this step in detoxification, reactive molecules,

which may be more toxic than the parent molecule, are produced. If these reactive

molecules are not further metabolized by phase II conjugation, they may cause

damage to proteins, RNA and DNA within the cell. Several studies have shown the

25

evidence of association between induced phase I and/or decreased phase II

activities and an increased risk of disease, such as cancer, systemic lupus

erythromatosus and Parkinson’s disease. Compromised phase I and/or phase II

activity has also been implicated in adverse drug responses.

There are at least 10 families of phase I activities described in humans. The major

P450 enzymes involved in metabolism of drugs or endogenous toxins are the

Cyp3A4, Cyp1A1, Cyp1A2, Cyp2D6 and the Cyp2C enzymes. The amount of each

of these enzymes present in the liver reflects their importance in drug metabolism.

Most information on the phase I activities has been derived from studies with drug

metabolism; however, phase I activities are also involved in detoxifying endogenous

molecules, such as steroids.

CYP catalytic cycle is shown in the adjoining figure. In this cycle, CYP molecule

converts a substrate to it’s oxidized product. Several molecules are required for this

reaction. These include the addition of two hydrogen ions, oxygen and transfer of

two electrons, the accessory proteins NADPH and cytochrome b5 allow the electron

transfer (Meyer U et al 1990, Ingelman S et al 2004, Guengerich F et al 1999).

The phase II system

Phase II conjugation reactions generally follow phase I activation, resulting in a

compound that has been transformed into a water-soluble compound which can

then be excreted through urine or bile. Several types of conjugation reactions are

present in the body, including glucuronidation, sulfation and glutathione and amino

acid conjugation. These reactions require cofactors, which must be replenished

through dietary sources (Liska D 1998).

26

The phase III system

Drug transporters

Transport proteins have an important role in regulating the absorption, distribution

and excretion of many medications. Members of the adenosine triphosphate (ATP)-

binding cassette family of membrane transporters are among the most extensively

studied transporters involved in drug-disposition and effects. A member of the ATP

binding cassette family (ABC family), p-glycoprotein (pgp) is the energy dependant

cellular efflux of substrates, including bilirubin, several anticancer drugs, cardiac

glycosides, immunosuppressive agents, glucocorticoids and many other

medications. The expression of p-glycoprotein in many normal tissues suggests that

it has a role in the excretion of drug metabolites into urine, bile and the intestinal

lumen. At the blood-brain barrier, p-glycoprotein in the choroid plexus limits the

accumulation of many drugs in the brain including cyclosporine and tacrolimus.

Structure: All ABC transporters are transmembrane proteins having their core

functional unit as two polytropic membrane spanning domains (MSDs) typically

consisting 6 transmembrane (TM) helices (in some proteins may range from 5 to

10) and two nucleotide binding domains (NBDs). All these domains are encoded in

a single polypeptide in the order NH2 – MSD – NBD – MSD – NBD – COOH. The

NBDs of all ABC proteins contain walker A and walker B motifs that are essential for

ATP binding and hydrolysis. In addition they also contain a motif known as “C

signature” that is characteristic of ABC ATPases. C signature motif has a core

sequence LSGGQ. It appears in general that the role of this motif is not essential as

far as the transport of substrate is concerned (Figure 9) (Higgins C et al 1992, Doyle

L et al 1998).

Mechanism: The mechanism of substrate transport by ABC protein (eg. pgp) is

called as it’s transport cycle. The cyclic events that occur in the mechanism of

transport are as follows (Figure 10)

27

Step 1) When substrate binds to high affinity site of ABC protein it induces

conformational changes that enhance the ATP binding to NBD1.

Step 2) The initial binding of ATP to NBD1 stabilizes the interaction between NBDs

by establishing contact with the C signature motif of NBD2, facilitating the binding of

second molecule of ATP.

Step 3) Binding of the second ATP molecule completes the formation of closed

NBD dimmer and also causes conformational changes in NBD2. The combined

positional and conformational changes resulting from ATP binding are transmitted to

MSDs resulting in a decrease in the affinity for the substrate.

Step 4) The protein maintains a low affinity state following hydrolysis of ATP at

NBD2, as long as NBD1 is occupied by ATP and ADP has not been released by

NBD2. The mechanism by which the protein returns to it’s original conformation

remains unclear, however the proposed mechanism depending on the available

experimental data is described below (Chang X et al 1997, Gao M et al 1996,

Payen L et al 2003, Zhao Q et al 2004).

Step 5) If NBD1 lacks ATPase activity, resetting of protein might occur after the

release of only ADP from NBD2 or may require the release of ADP from NBD2 and

ATP from NBD1.

Step 6) It is also proposed that hydrolysis of ATP by NBD1 is required to reset the

protein (Jones P et al 2002, Koike K et al 2004, Loo T et al 2002, Situ D et al 2004,

Zhang D et al 2004).

Recently, antiporter activity (p-glycoprotein or multi-drug resistance) has been

defined as the phase III detoxification system. Antiporter activity is an important

factor in the first pass metabolism of pharmaceuticals and other xenobiotics. The SUB

28

antiporter is an energy dependant efflux pump, which pumps toxic material out of a

cell, thereby decreasing their intracellular concentration.

Antiporter activity in the intestine appears to be co-regulated with intestinal phase I

Cyp3A4 enzyme. This observation suggests the antiporter may support and

promote detoxification. Possibly, it’s function of pumping non-metabolized toxic

compounds out of the cell and back into intestinal lumen may allow more

opportunities for phase I activity to metabolize the toxic compound before it is taken

into circulation (Figure 11).

The two genes encoding antiporter activity have been described: the multi-drug

resistance gene 1 (MDR-1) and multi-drug resistance gene 2 (MDR-2). The MDR-1

gene product is responsible for drug resistance of many cells and is normally found

in epithelial cells in the liver, kidney, pancreas, small and large intestine, brain and

testes. MDR-2 activity is expressed primarily in liver, and may play a role similar to

that of intestinal MDR-1 for liver detoxification enzymes; however, it’s function is

currently undefined.

Genetic variation in drug-metabolizing enzymes and drug transporters is an

important contributor to interindividual differences in drug disposition and is

associated with significant clinical consequences. Many commonly used drugs are

dependant on cytochrome P450 monooxygenase enzymes (CYP450s) for their

metabolism and for elimination on ATP binding cassette family protein (p-gp) coded

by MDR-1 gene. More than 80% of all phase I-dependent metabolism of clinically

used drugs is carried out by the CYP450s, and these enzymes also metabolize a

large number of chemicals. The polymorphic forms of CYP450s are responsible for

the development of a significant number of adverse drug reactions and may also

contribute to nonresponsiveness to drug therapy (Chin K et al1993, Wacher V et al

1995, Deeley R et al 2006).

29

Adverse reaction to medication is a major cause of death and is partly ascribed to

inconsistent drug response displayed by different patients. Polymorphisms in

cytochrome P450 and multidrug resistance genes have the capacity to change the

expression of genes, leading to altered drug metabolism and disposition, which in

due course may result in adverse drug reactions.

The construction of single nucleotide polymorphism (SNP) profiles constitutes an

approach to differentiate between patients who respond favorably to medication and

those who experience undesirable side effects. The field pharmacogenomics aims

to permit the physician to improve medication response by way of individualized

prescriptions. This procedure will result in safer, more effective and cost-effective

medicine.

As mentioned above, increased dose for a particular individual leading to increased

levels of CNIs in the body may lead to nephrotoxicity; it is also true that their faster

metabolism causing their decreased levels leads to inadequate

immunosuppression. Because of this there are chances of strong host immune

response against graft causing nephrotoxicity or graft versus host disease (GVHD)

and in severe cases graft rejection. Hence monitoring levels of CNIs and correlating

them with the polymorphisms present in the above-mentioned genes assumes

significance.

In recent years, research has focused on the possible causes of inter and intra-

individual differences in the pharmacokinetics of CNIs. Genetic polymorphisms have

been identified in these genes that are associated with the expression of the CYP3A

enzymes and P-gp. It has become clear that the genetic variations of CYP3A and P-

gp may play an important role in the individual administration of immunosuppressive

drugs.

30

CYP3A4

In 1985 Watkins et.al. identified a glucocorticoid inducible cytochrome P450 in

human liver. Molowa et.al (1986) reported the complete cDNA sequence of this

P450. Wrighton and Vandenbranden isolated a CYP3-type cytochrome P450 from

human fetal liver. By somatic cell hybridization and in situ hybridization, Riddell et.al

(1987) assigned it to chromosome 7 and identified it as a cytochrome P450 enzyme

family member that encodes the enzyme nifedipine oxidase (CYP3). Brooks B et.al

(1988) concluded that the most likely location of CYP3 is 7q21-q22.1. Inoue et.al

(1992) mapped CYP3A4 to chromosome 7q22.1 by fluorescence in situ

hybridization.

CYP3A4 is one of the most abundant P450s in human liver. It is inducible by a

variety of agents including glucocorticoids and Phenobarbital and plays a central

role in the metabolism of immunosuppressive cyclic polypeptides i.e. CsA and

tacrolimus. It is responsible for oxidative metabolism of wide variety of xenobiotics,

including an estimated 60% of all clinically used drugs. Although the expression of

CYP3A4 gene is known to be induced in response of variety of compounds the

mechanism underlying this induction which represents a basis of drug interactions

in the patients was not clear (Ando Y et al 1999). Lehman et.al (1998) identified a

human orphan nuclear receptor that binds to a response element in CYP3A4

promoter and is activated by a range of drugs known to induce CYP3A4 expression.

Rebbeck et al (1998) have observed a single nucleotide polymorphism (SNP) in the

nifedipine specific response element (NFSE) of CYP3A4 promoter. They termed the

SNP as CYP3A4-V. It is an A to G transition at -290bp that alters 10bp.

(AGGGCAAGAG) to (AGGGCAGGAG) located -287 to -296bp from the

transcription start site of CYP3A4 gene. Unlike other human P450s there is no

evidence of null allele in case of CYP3A4. Genetic variations found in the flanking,

intronic and exonic regions of the gene may influence the level or function of

CYP3A4 protein, but full-length m-RNA has been detected in all adults studied so

31

far. Five different SNPs have been observed in the 5’-flanking region of the human

CYP3A4 gene. By far the most common is the one described above (A-290G or

CYP3A4-V or CYP3A4*1B). It’s mutant allele frequency varies among different

ethnic groups: 0% in Taiwanese, Chinese, Chinese Americans and Japanese

Americans; 2 – 9.6% in Caucasians; 9.3 – 11% in Hispanic Americans and 35 –

67% in African Americans. The other allelic variants in the 5’-flanking region appear

to be of much lower frequencies.

Of the five promoter SNPs identified, only CYP3A4-V has been studied to ascertain

the effect of the mutation on transcriptional activity and in vivo catalytic activity.

Amiramani et al (1999) examined the effect of this polymorphism on leuciferase

reporter transcription in HepG2 hepatoma and MCF7 breast cancer cell lines. They

observed that luciferase expression from the mutant (G) promoter construct

occurred at a rate that was 1.4 to 1.9 fold higher than that observed for wild type (A)

construct. This finding was supported by an observation of 1.6- and 2.1 fold higher

level of nifedipine oxidation and CYP3A4 protein in human livers carrying at least

one G-290 allele, although the difference was not statistically significant. However,

results from studies with a larger sample size demonstrate no clear association

between this polymorphism and CYP3A4 specific content or catalytic activity among

tissue banks from predominantly Caucasian donors.

The functional significance of this polymorphism has also been evaluated in vivo,

but with populations of limited size. Ball S et al (1999) studied the metabolic fate of

CYP3A probe substrates in healthy African American volunteers. They found no

difference in mean demethylation rate or mean oral clearance measured in

individuals with a homozygous A-290 (n=8) or homozygous G-290 (n=23) CYP3A4

genotype. Wandel et al (2000) have observed no no relationship between genotype

and oral midazolam clearance for this polymorphism however they found 30% lower

systemic midazolam clearance for subjects homozygous for the variant G allele

compared to that observed in subjects with A allele.

32

CYP3A5

The CYP3A5 cDNA sequence was first described independently by Aoyama et al

(1989) and Schuetz et al (1996). The allele corresponding to this cDNA and the

respective expressed protein was designated wild type, CYP3A5*1A. Subsequent

work described 5’-flanking sequence of two distinctly different genomic clones from

a human liver containing CYP3A5 protein. The clones contain identical sequences

for exon 1 but differed slightly in the 5’-flanking sequence. The flanking sequence

corresponding to CYP3A5*1A was notable for minor differences compared to the

CYP3A4 gene. More recent work revealed that the putative CYP3A5*1A promoter

sequence described by Jaunaidi et al (1994) actually corresponds to that of a

closely related CYP3A pseudogene, CYP3API.

In 2000, Paulussen et al (2000) described two linked mutations in the proximal 5’-

flanking region of CYP3A5 gene that were in complete concordance with

polymorphic hepatic CYP3A5 protein content. The first report of a potentially

important CYP3A5 coding SNP came from Jaunaidi et al (1994) in their study of

livers obtained from Caucasian donors. These investigators described a

transversion in exon 11 of CYP3A5 gene. The most frequent and functionally

important SNP in the CYP3A5 gene consists of an A6986G transition within intron 3

(CYP3A5*3) this polymorphism creates an alternative splice site within the pre-

mRNA and production of aberrant mRNA (SV1-mRNA) that contains 131 bp. Of

intron 3 sequence (exon 3B) inserted between exon 3 and exon 4. The exon 3B

insertion results in a frameshift and predicted truncation of the translated protein at

amino acid 102. Among Caucasians, a CYP3A5*3/*3 genotype was perfectly

concordant with very low or undetectable hepatic CYP3A5 protein content, a finding

that has been confirmed by other investigators. In addition, homozygosity for the

CYP3A5*3 allele was also strongly associated with low CYP3A5 protein content in

African-American livers and Caucasian intestinal mucosa (Kuehl et al 2001).

33

Focusing on the functional wild type allele (A6986), the frequency is reported as 5-

15% for Caucasians and 45-73% for African Americans. Hustert et al (2001) also

reported an A6986 allele frequency of 27% in Chinese and 30% in Koreans.

Sequence information from more limited data sets suggested a A6986 allele

frequency of 35% in Chinese, 15% in Japanese, 25% in Mexicans and 60% in

Southwestern American Indians (Kuehl et al 2001, Hustert et al 2001, Lamba J et al

2002).

There are mutations within intron 4 and intron 5 of the CYP3A5 gene which also

create cryptic splice sites that can affect the production of functional mRNA (SV2

and SV3). However, these rarer mutations have, to date always been seen in

combination with the intron 3 SNP and thus, represent CYP3A5*3 haplotypes (*3A,

*3B and *3C). Additional rare mutations that also give rise to splicing defects have

been reported. Several other intronic and flanking CYP3A5 SNPs have also been

described, although the haplotype for these mutations has not yet been determined.

In their publication Kuehl et al reported on the close association between high

hepatic CYP3A5-specific content and the presence of the CYP3A5*1A allele.

Moreover, they also demonstrated that Caucasians and African American livers with

at least one copy of the CYP3A5*1A allele exhibited a mean midazolam

hydroxylation activity that was approximately 2-fold higher than that observed for

the corresponding CYP3A5*3/*3 livers. While there was considerable phenotypic

variability within different CYP3A5 genotype groups, presumably as a result of

variable CYP3A4 expression, livers carrying a functional CYP3A5 allele were more

likely to exhibit more catalytic activity than those that did not. Hustert et al, reported

a similar concordance between hepatic CYP3A5 protein content and the

CYP3A5*1A genotype, however, the presence of CYP3A5 protein was less

prevalent than that seen in other Caucasian liver banks.

The impact of CYP3A5 splice variants on the production of mRNA is quite

interesting. Although livers with a homozygous CYP3A5*1 genotype produce only

34

properly spliced, wild type (wt) mRNA, those homozygous for the CYP3A5*3 variant

allele produce both wt and variant (SV1) mRNA. Utilizing a quantitative Taqman

assay, Lin et al (2002) found that the mean wt-CYP3A5 mRNA level was 4-fold

higher in CYP3A5*1/*3 livers compared to CYP3A5*3/*3 livers. SV1-CYP3A5

mRNA was absent from CYP3A5*1/*1 tissue, and present in heterozygous livers at

levels that were approximately 10-fold lower than wt-CYP3A5 mRNA. Moreover,

inter-individual differences in wt-CYP3A5 mRNA content could explain 53% of the

variability in CYP3A5 protein content. A similar correlation was seen between

hepatic CYP3A4 mRNA and protein content, demonstrating the variable

transcriptional control is the dominant source of inter-individual differences in

hepatic CYP3A levels. Interestingly, the relative amount of wt- and SV1-CYP3A5

mRNA were highly correlated with each other, for both CYP3A5*1/*3 and

CYP3A5*3/*3 sub-populations, suggesting that the probability of proper and

improper splicing at the exon 3/exon 4 boundry is pre-determined by the interaction

of the pre-mRNA with the spliceosome.

Because the substrate specificity and product regioselectivity of CYP3A5 can differ

from that of CYP3A4, it is expected that the impact of CYP3A5 genetic

polymorphism on drug disposition will be drug dependent. There are substrates,

which are common for CYP3A4 and CYP3A5 however for which the catalytic

activity of CYP3A5 is comparatively much lower than that of CYP3A4, whereas for

few substrates the catalytic activity is comparable.

Even when CYP3A5 is shown to be as efficient a catalyst of drug metabolism as

CYP3A4, one can still identify significant differences in product regioselectivity.

CYP3A5 catalyzes the formation of the M1 metabolite of cyclosporine, but it does

not make AM9 and AM4N. CYP3A4 produces all the three metabolites. There is

little information in the literature pertaining to the in vivo significance of newly

identified CYP3A5 polymorphisms with respect to metabolic drug clearance

(Aoyama T et al 1989). Undoubtfully, many pharmacogenetic studies are currently

underway and results can be expected to appear in the literature.

35

Aoyama et al (1989) screened a liver cDNA library with CYP3A4 as probe and

isolated a cDNA encoding CYP3A5, which they termed PCN3. Immunoblot analysis

of liver microsomes showed that CYP3A5 is expressed as a 52.5KD protein. The

deduced 502 amino acid CYP3A5 protein shares 85% sequence similarity with

CYP3A4. Analysis of enzymatic activity established that CYP3A4 and CYP3A5

have overlapping substrate specificity with minor differences in the metabolism of

steroids and cyclosporine, suggesting that CYP3A4 may bind these drugs at more

than 1 site.

Jaunaidi et al (1994) isolated the 5-prime flanking region of CYP3A5 from a

genomic clone on chromosome 7. Promoter analysis determined that CYP3A5 uses

a CATAA, rather than a TATA box at positions -23 to -28 and has a basic

transcription element from -35 to -50.

Yamakoshi et al (1999) isolated a cDNA clone for CYP3A5 from a prostate library

and determined that it has a unique 5-prime untranslated sequence, suggesting that

CYP3A5 is differentially regulated in liver and prostate. Enzymatic analysis showed

that the prostate form of the enzyme could metabolize sex hormones.

Variation in CYP3A enzymes, which act in drug metabolism, influences circulating

steroid levels and responses to half of all oxidatively metabolized drugs. CYP3A

activity is the sum activity of the family CYP3A genes, including CYP3A5, which is

polymorphically expressed at high levels in a minority of Caucasians. Previous

studies on CYP3A5 showed that a greater degree of heterogeneity seems to be

conferred by differences in the expression of CYP3A5. Genetic polymorphisms of

CYP3A5 have been found to be associated with more significant pharmacokinetic

effects on immunosuppressive drugs than those of CYP3A4.

36

MDR-1

In 1976, a 170KD glycosylated membrane protein was isolated from colchicine-

resistant Chinese hamster ovary cells. Since this glycoprotein appeared unique to

sublines displaying altered drug permeability, it was named para-glycoprotein.

About 10 years later, the human MDR1 gene was isolated from multidrug-resistant

KB carcinoma cells and was demonstrated to encode human p-glycoprotein. To

date, several isoforms of p-glycoprotein have been identified from humans, mice,

hamsters and rats and these are encoded by a family of closely related genes.

Based on their 3’-untranslated regions they are classified into 3 subclasses: class 1

containing human MDR1, mouse MDR1a or (mdr-3), hamster Pgp1 and rat Pgp1,

class 2 containing of mouse MDR-1b or (mdr-1), hamster Pgp-2 and rat pgp-2 or

(mdr-1b) and class 3 containing of human MDR3 or (MDR2), mouse mdr2, hamster

Pgp3 and rat pgp3.

It has been elucidated that human para-glycoprotein is a phosphorylated and

glycosylated protein with 1280 amino acid residues and consists of 2 homologous

halves containing 6 putative hydrophobic Transmembrane segments and an

intracellular binding site for ATP. The halves are separated by a flexible linker

polypeptide. Structural analysis by electron microscopy and image analysis using

Chinese hamster ovary cells suggested that P-glycoprotein approximates a cylinder

of about 10nm in diameter and a maximum height of 8nm, thus about one half of the

molecule is in the membrane. A large aqueous chamber within the membrane is

open at the cytoplasmic face. Extensive mutational analysis revealed that the two

halves of human para-glycoprotein interact to form a single transporter and the

major drug binding domains reside in or near transmembrane domains 5, 6 and 11,

12. P- glycoprotein is understood to act as a pump which detects and removes it’s

substrates into and out of cells, however another model which has been proposed

states that P-glycoprotein acts as a flippase carrying it’s substrate from the inner

leaflet of lipid bilayer to outer leaflet. P-gp belongs to a large group of functional

proteins that share common functional and structural properties. This superfamily is

37

known as ATP-binding cassette superfamily. To date more than 40 human ABC

transporter genes have been identified and sequenced. Pgp is defined as ABCB1.

Recently the term MDR1 has come to be used instead of Pgp.

Multidrug resistance-1 gene codes for para glycoprotein (pgp) a 170kDa

transmembrane glycoprotein. It is a member of the adenosine triphosphate (ATP)

binding cassette family of membrane transporters and thus contributes to phase III

metabolism of drugs. It functions as an energy dependant transmembrane efflux

pump that exports a number of substrates from inside to outside of cells to reduce

the accumulation of toxic substances and metabolites within the cells. Pgp is

reportedly expressed in various human tissues, including the intestine, adrenal,

kidney, liver and capillary endothelial cells of brain and testes. The disposition of

pgp expression suggests that pgp may play a role in the absorption and excretion of

it’s substrates and protect organism from toxic xenobiotics.

Great interindividual variations in the expression and function of pgp are due to

genetic factors. About 15 mutations and 28 SNPs have been identified so far in the

MDR-1 gene. Out of these SNPs the one in exon 21 (G2677T) and the other in

exon 26 (C3435T) are of particular interest because they have significant

pharmacological effects and are associated with alteration of pgp expression and/or

function. The SNP in exon 26 (C3435T) has been studied extensively. Hoffmeyer et

al (2000) first showed that SNP was associated with variation in intestinal

expression and function of pgp. The 3435C/C allele was more strongly associated

with pgp expression than the 3435T/T allele in enterocytes, and the circulating

concentrations of orally administered digoxin were higher for the 3435T/T allele than

for the 3435C/C allele. Several other groups also reported that SNP is associated

with pgp expression in CD56+ NK cells and placental tissues. SNP in exon 26 is a

silent one that does not result in any amino acid changes, while the heterozygosity

for the 3435T allele in exon 26 results in a 2-fold reduction in intestinal pgp

expression (Ameyaw et al 2001). This may be interpreted by the tight linkage with

the recently reported exon 21 G2677T SNP which alters the protein sequence

38

resulting in an A893S amino acid change. A noncoding SNP located in the promoter

of MDR-1 (T-129C) is also associated with weaker expression of pgp in human

placenta.

Investigation of the frequency of polymorphisms of MDR-1 found that there is

significant variability among various ethnic groups. In the African-American group

there is a 68% to 83% frequency of the MDR-1 3435CC genotype in contrast to

21% to 25% in Caucasian subjects, and 29% to 38% in Asians.

Relationship between CYP3A and MDR-1

It is known that there is a close relationship between CYP3A and PGP, which

overlap in substrates, and both contribute significantly to absorption and phase I

metabolism and share localization in small intestinal enterocytes. In the gut, PGP is

present on the apical membrane of the cells of the brush border, whereas the

CYP3A enzyme is located deeper in the cell, within the endoplasmic reticulum. This

arrangement limits or regulates access of drugs to CYP enzymes and prevents CYP

enzymes from being overwhelmed by the high drug concentration in the intestine.

Functional interactions between PGP and CYP3A have been suggested, though not

yet completely understood, at 3 levels 1) co-regulation of gene expression; 2)

functional interaction and 3) active transport of drug metabolites. One study showed

that the C3435T polymorphism in the MDR1 gene affects the enterocyte expression

level of CYP3A4 rather than PGP. Moreover, Anglicheu et al (2003) found in

Caucasians that CYP3A5*3 allele carriers were more likely to possess the MDR1

3435T allele.

CHAPTER 3

AIMS AND OBJECTIVES

39

Chapter 3 – Aims and Objectives

Drug induced nephrotoxicity is the most common phenomenon occurring in the

case of renal transplant recipients. It is the major factor affecting the outcome of

renal transplantation. As described earlier, gene polymorphisms in drug

metabolizing genes, drug transporter genes and drug targets has been shown to be

a contributing factor in influencing the activities of these enzymes. Therefore, SNP

profiling of patients undergoing kidney transplant may help to decide how each

patient will behave clinically to a given drug dose. Depending on the genetic status

of the individual, his/her metabolic status could be decided.

Depending on the metabolic status of individual, clinicians would be able to decide

the individualized dosage regimen, which in turn would help in reducing the chance

of occurrence of drug induced nephrotoxicity.

Keeping this hypothesis, we decided to study,

A-290G polymorphism of CYP3A4 gene,

A6986G polymorphism of CYP3A5 gene,

C3435T polymorphism of MDR-1 gene and

T-330G polymorphism of IL-2 gene

in 100 healthy controls and 100 renal transplant recipients so as to obtain a

comparative data between diseased and general population. We further aimed at

correlating the occurrence of these SNPs with the blood levels of CNIs and

subsequent incidences of nephrotoxicity. IL-2 SNP data as well as the incidences of

nephrotoxicity and rejection will be correlated with IL-2 plasma levels to observe the

role of IL-2 gene SNP in influencing it’s levels. As per the hypothesis, this could

guide clinicians to establish individualized dosage regimen for CNIs in order to

minimize the incidences of drug induced nephrotoxicity.

CHAPTER 4

MATERIALS AND METHODS

40

Chapter 4 – Materials and Methods

Subjects

Consecutive 100 renal transplant recipients receiving either CsA or tacrolimus were

included in patient group. Their clinical details like renal profile, liver profile, past

history of diseases or disorders were noted down as per the proforma. Patient

samples were collected from P. D. Hinduja National Hospital (N=12) as well as from

Muljibhai Patel Urological Hospital (MPUH), Nadiad, Gujrat (N=88). Plasma

separation and sample storage was done by laboratory staff of MPUH and samples

were collected from them in periodic visits to the hospital (by the gap of 1 to 11/2

month). Transportation of samples was done in a sealed container having ice pack.

Control group comprised of consecutive 100 subjects having normal renal profile.

Controls were selected in order to compare the genotype and allele frequencies of

the polymorphisms included in the study for the patients with general healthy

population. This group was chosen from the subjects attending regular health check

program at our hospital. After sample collection blood samples of patients as well as

controls were stored at 4°C and separated plasma samples of patients were stored

at -70°C.

Sample collection

Blood sample collection was done in EDTA tube.

For patients –

One 5cc EDTA pretransplant before starting CNIs and

One 5cc EDTA on 6th day after transplant

For controls –

One 5cc EDTA

Pre- as well as posttransplant blood sample collection was done for patients to use

the separated plasma for IL-2 ELISA.

Collected blood samples were used for genomic DNA extraction.

41

Genomic DNA extraction

Mammalian DNA is usually isolated by treatment of cells with proteinase K in the

presence of EDTA and SDS followed by extractions with phenol and isoamyl

chloroform. However this method is cumbersome especially when large number of

samples are involved. This method also involves risk to perform because of

involvement of hazardous solvents. Therefore simple salting out procedure

described by Miller et al was preferred for all the extractions done. The procedure

used is as follows.

1. 5 ml of anticoagulated blood (heparinized) was overlaid on equal amount of

'Histopaque' (Sigma) in 15 ml polypropylene tubes and centrifuged at 2,500 rpm

x 30 mins. Buffy coats of nucleated cells were obtained.

2. The cells were washed thrice with phosphate buffered saline (PBS) [See

appendix].

3. After washing, 3 ml of nuclei lysis buffer (See appendix) was added to the cells.

4. The cell lysate was digested overnight at 37°C with 0.2 ml of 10% SDS (See

appendix) and 0.5 ml of Proteinase K Solution (1 mg/ml Proteinase K in 1% SDS

and 2mM Na2EDTA).

5. After digestion the solution obtained was clear and very viscous. It was treated

with 6M sodium chloride solution [See appendix] (such that the final molarity of

sodium chloride is around 1.5 M (1 ml 6M NaCl per 3.5 ml lysis solution). Most of

the proteins precipitate out at this concentration.

6. The suspension was mixed vigorously and centrifuged at 2500 rpm x 20 mins.

The precipitated protein settled at the bottom of the tube and formed a pellet.

The viscous supernatant containing the DNA was transferred to another 15 ml

polypropylene tube.

7. Exactly 2 volumes of RT absolute ethanol (Hayman Ltd.) were added and the

tubes inverted several times until the DNA precipitated.

8. The precipitated DNA strands were removed with a plastic spatula or pipette and

transferred to a fresh 1.5 ml micro-centrifuge tube, washed with 70% ethanol

42

and allowed to dry. (Total drying is avoided, as it becomes difficult to redissolve

the DNA). After partial drying, DNA was redissolved in 100 µl of TE buffer (See

appendix).

9. The DNA was allowed to dissolve for 2 hrs at 37°C or overnight at 4°C before

quantitating.

10. The absorbance of the DNA was measured at 260 nm and 280 nm and the yield

obtained from this technique was 0.5-1.0 µg/µl. The ratio of A260 to A280 was

consistently between 1.7-2.0, demonstrating good deproteinization.

11. The DNA samples were electrophoresed on 0.8% agarose gel (See appendix) in

1X TAE buffer (See Appendix) as described later. Lambda DNA/Hind III digest

was used as a marker. The DNA was found to be of high molecular weight. DNA

samples extracted from the whole blood of all the subjects were preserved at -

20°C until use for PCR.

Genomic DNA amplification

In 1983, Dr. Kary Mullis from Cetus conceived a novel concept, which ultimately

earned Dr. Mullis the Nobel prize for Chemistry in 1993. This concept involves

utilizing an enzyme that normally is responsible for DNA synthesis (a DNA

polymerase), specific sequences (DNA primers) and other reagents such as

magnesium which is a co-factor for the activity of DNA polymerase, dNTPs etc. He

invented a test tube process of repetitive DNA synthesis. The process is termed as

Polymerase Chain Reaction (PCR) amplification. Application of this powerful

technology has touched every aspect of medical science and the practice of

medicine, from the study of gene regulation to the diagnosis of infectious, neoplastic

and hereditary diseases. The invention of PCR has upgraded recombinant DNA

technology, to a new level: it allows the scientist and the clinician to directly amplify

a defined stretch of DNA sequence in an exponential fashion, in short span of

several hours. The simplicity and sensitivity of this process makes it possible to

utilize this technology for the analysis of a minute amount of a specific genetic

material.

43

Principle –

DNA is made from four nucleotides adenine, guanine, thymine and cytosine. The

DNA molecule normally is present inside the cell as a complex of two polynucleotide

strands which, at the molecular level, complement each other so that adenine (a

purine) on one strand always corresponds to thymidine (a pyrimidine) on the other

strand, and a guanine (a purine) on one strand always corresponds to cytosine (a

pyrimidine) on the opposite strand. This complementation allows maximum

hydrogen bond formation between the purine and pyrimidine bases and at the same

time provides for the same spacing for each of the three base pairs [always a

purine, a fused 5- and 6- member ring, with a pyrimidine, a 6- member ring]. The

two DNA strands are oriented in an antiparallel direction (5’ to 3’) orientation

according to the linkage of phosphodiester backbone and twist around each other,

creating a double helix.

DNA polymerase is capable of synthesizing a second strand of DNA always in a

5’to 3’orientation, using the four nucleotide triphosphates as substrates, one DNA

strand as a template and a short piece of complementary DNA as primer. To initiate

DNA synthesis in vitro, the double stranded DNA is first heated to separate the two

strands, (denaturation), It is then cooled, thus allowing the annealing of primers

(which are provided in an excess amount) to the complementary DNA fragments. If

two specific DNA primers are provided that are complementary to the DNA

sequences on the opposite strand of parental DNA molecule, DNA synthesis

mediated by the DNA polymerase (extension) will yield two double stranded

molecules from the parental DNA duplex. If this process is repeated a second time,

the result would be four DNA molecules. In other words, the DNA multiplies

exponentially (doubling each time) as the same cycle of reaction is repeated. This is

the basis of PCR. PCR amplification

44

theoretically can amplify a short stretch of DNA fragment (upto several thousand

base-pair long), from a single molecule to 1,000 copies (210 = 1,000), if the process

is repeated 10 times, to one million copies (220 = 1,000,000) if the process is

repeated twenty times, and to one billion copies (230 = 1,000,000,000) if the process

is repeated 30 times. This process can be carried out automatically in a machine

called as thermal cycler that repeats this cycle of denaturation (94 to 96°C)

annealing of primers (40 to 60°C) and DNA synthesis (65 to 75°C), using a

thermostable DNA polymerase. A billion fold amplification, can be accomplished in

a span of 3 to 4 hours, yielding microgram quantity of DNA for direct analysis or

further procedures.

Amplification of CYP3A4 gene

Genomic DNA was amplified by PCR for the desired region of CYP3A4 gene by

using the primers mentioned in the table. PCR reactions were carried out in 0.5ml

microcentrifuge tubes with following conditions

Initial denaturation for 5min at 94°C followed by denaturation at 94°C for 1min,

primer annealing temperature at 60°C for 1min and amplification at 72°C for 1min.

These steps were repeated for 38 cycles.

A 50µl reaction contained following reagents

1X PCR buffer (see appendix for details)

2mM MgCl2

200µM dNTP

5pmol of both forward and reverse primers and

1 unit of the enzyme Taq polymerase (Fermentas)

500 ng DNA

45

Amplification of CYP3A5 gene

Genomic DNA was amplified by PCR for the desired region of CYP3A5 gene by

using the primers mentioned in the table. PCR reaction was carried out in 0.5ml

microcentrifuge tube with following conditions

Initial denaturation of 94°C for 5min followed by the denaturation step of 1min,

annealing of the primers at 55°C for 1min and extension of 72°C for 1min. These

steps were repeated for 35 cycles with final extension of 10min at 72°C.

A 25µl reaction contained following reagents

1X PCR buffer (see appendix for details)

1.5mM MgCl2

200µM dNTP

50pmol of both forward and reverse primers and

1.5 units of the enzyme Taq polymerase (Fermentas)

500 ng DNA

Amplification of MDR-1 gene

Genomic DNA was amplified by PCR for the desired region of MDR-1 gene by

using the primers mentioned in the table. PCR reaction was carried out in 0.5ml

microcentrifuge tube with following conditions

Initial denaturation of 94°C for 5min followed by the denaturation step of 1min,

annealing of primers at 55°C for 1min and extension of 72°C for 1min. These steps

were repeated for 35 cycles followed by final extension of 10min at 72°C.

46

A 25µl reaction contained following reagents

1X PCR buffer (see appendix for details)

1.5mM MgCl2

200µM dNTP

25 pmol of both forward and reverse primers and

1 unit of the enzyme Taq polymerase (Fermentas)

500 ng DNA

Amplification of IL-2 gene

Genomic DNA was amplified by PCR for the desired region of IL-2 gene by using

the primers mentioned in the table. PCR reaction was carried out in 0.5ml

microcentrifuge tube with following conditions

Initial denaturation of 94°C for 5mni followed by a denaturation step of 1min,

annealing of primers at 59°C for 30 seconds and extension of 72°C for 1min. These

steps were repeated for 30 cycles followed by final extension of 72°C for 10min.

A 25µl reaction contained following reagents

1X PCR buffer (see appendix for details)

1.0mM MgCl2

200µM dNTP

5 pmol of both forward and reverse primers and

1 unit of the enzyme Taq polymerase (Fermentas)

500 ng DNA

47

Detection of the amplified product

Principle

Detection of the amplified products was done using agarose gel electrophoresis.

Agarose gels are cast by melting the agarose in the desired buffer until a clear,

transparent solution is obtained. The melted solution is then poured into a mould

and allowed to harden. Upon hardening of the agarose it forms a matrix, the density

of which is determined by the concentration of agarose. When an electric field is

applied across the gel, DNA, which is negatively charged at neutral pH, migrates

towards anode. The rate of migration depends on

1) Molecular size of DNA – linear or double stranded DNA migrate through gel

matrices at rates that are inversely proportional to log10 of number of base

pairs.

2) Agarose concentration – There is a linear relationship between log of

electrophoretic mobility of DNA and gel concentration.

3) Conformation of DNA – Superhelical circular, nicked circular and linear DNAs

migrate at different rates through agarose gels.

4) Applied voltage – To obtain maximum resolution of DNA fragments greater

than 2kb in size, gel should be run at no more than 5V/cm.

5) Direction of electric field.

6) Base composition and temperature.

7) Presence of intercalating dyes – EtBr reduces electrophoretic mobility of

DNA by 15%. The dye intercalates between the stacked base pairs and

increases length of linear and nicked DNA making them more rigid.

48

Method

1) Edges of clean dry glass tray were sealed with tapes. Mould was set on

horizontal section of bench.

2) Sufficient electrophoresis buffer was prepared 1X TAE (see appendix for

details) and appropriate quantity of powdered agarose was weighed and

added to buffer in a conical flask. Care was taken that the buffer did not

occupy more than 50% of the conical flask.

3) The slurry was heated in microwave oven till all the grains of agarose are

dissolved.

4) The solution was cooled to 60°C and EtBr was added from a stock solution of

10mg/ml in water to final concentration of 0.5µg/ml and mixed thoroughly.

5) Remaining agarose was poured into the mould

6) After the gel is completely set the combs were removed.

7) Enough electrophoresis buffer was added in the running tank so as to cover

the gel properly to a depth of about 1mm.

8) DNA samples or amplified products were mixed with gel loading dye (see

appendix for details) and loaded into the wells formed in the gel.

9) Tank was closed with lid and connected with wires to electrode so that DNA

can migrate towards anode.

10) After the desired separation of the tracking dye Bromophenol blue, the gel

was observed under UV light in the gel documentation system.

49

Genotyping of samples

1) CYP3A4 A-290G polymorphism – The samples were genotyped for this SNP

by using 10 units of restriction enzyme MboII per reaction with 1X buffer (see

appendix) and 10µl of amplified product. The digested products were

separated on 3.5% high resolution agarose gel (Sigma).

2) CYP3A5 A6986G polymorphism – The samples were genotyped for this SNP

by using 1 unit of restriction enzyme SspI per reaction with 1X buffer (see

appendix) and 10µl of amplified product. The digested products were

separated on 3% agarose gel (Himedia).

3) MDR-1 C3435T polymorphism – The samples were genotyped for this SNP by

using 5 units of restriction enzyme DpnII per reaction with 1X buffer (see

appendix) and 10µl of amplified product. The digested products were

separated on 3% agarose gel (Himedia).

4) IL-2 T-330G polymorphism – The samples were genotyped for this SNP by

using 10 units of restriction enzyme FspBI per reaction with 1X buffer (see

appendix) and 5µl of amplified product. The digested products were separated

on 3.5% high resolution agarose gel (Sigma).

Quality control and confirmation of results

In order to check the handling of reagents and method of work, a negative control

involving sterile distilled water instead of DNA sample was incorporated in each

batch of genomic DNA amplification reaction.

For confirmation of results obtained by restriction enzyme digestion method,

representative samples for all the four polymorphisms (12 bidirectional reactions)

were sent for automated sequencing (by ABI Prism) to MWG Biotech, Banglore

Genei and GenOm Bio.

50

Estimation of human plasma Interleukin-2 levels by ELISA

Principle

The ELISA of IL-2 plasma levels was performed by the method provided in the kit

(Immunotech, Beckman Coulter). It is a two immunological step sandwich type

assay. The first step leads to the capture of IL-2 by a monoclonal antibody bound to

the wells of a microtiter plate and the binding of the second monoclonal antibody,

which is biotinylated, to the solid phase antigen complex. In the second step, the

streptavidine peroxidase binds to the biotinylated antibody. After incubation, the

wells are washed and the antigen complex bound to the well detected by addition of

a chromogenic substrate. The intensity of the coloration is proportional to the IL-2

concentration in the sample.

Method

1) All the components of the kit were equilibrated at room temperature for

30min. before starting the assay.

2) Serial dilutions of the standards were made as per the instructions given in

the kit insert (see appendix for details).

3) Wash buffer was diluted 1X from 20X.

4) 50µl of standard or sample and 50µl of biotinylated antibody was added into

wells

5) Plate was incubated for 2hrs at room temperature on microplate shaker at

350RPM

6) After incubation wells were washed by the wash buffer 5 times. Washing was

done by adding 300µl of wash buffer per well and inverting the plate over

discard. The plate was tapped several times on absorbent paper so as to

remove the wash buffer completely.

7) 100µl of streptavidin-HRP conjugate was added to the wells.

51

8) Plate was incubated for 30min at room temperature on microplate shaker at

350RPM.

9) Wells were washed with wash buffer 5 times

10) 100µl of substrate was added to all the wells.

11) Plate was incubated for 20min at room temperature on microplate shaker.

at 350RPM

12) 50µl of stop solution (see appendix) was added to all the wells in order to

stop the reaction.

13) Absorbance was taken at 450nm on a microplate reader capable of

plotting graph and determining the unknown concentrations as per the

curve fit equation in quadratic mode given in the kit insert.

Statistical analysis

Pearson’s chi-square test is applied to test the relationship of categorized

dependant and independent variables. If expected number in a table is below 5 in

any 2xn table then Fisher’s exact test (one sided) was used as the test of

significance. To study the effect on value, when classifying variable had greater

than 2 subgroups then Kruskal-Wallis ANOVA (Analysis of Variance) was used as

the test of significance. For all the tests p value < 0.05 was considered to be

statistically significant. SPSS 15.0, STATA 10.0 and XLSTATS packages were used

to perform all the statistical analysis.

CHAPTER 5

RESULTS AND DISCUSSION

52

Chapter 5 – Results and Discussion

Calcineurin inhibitors have significantly improved the outcome of renal

transplantation. However their inherent nephrotoxic potential limits the survival of

the graft. This CNI induced nephrotoxicity is the result of longer retention of

unmetabolized drug in the circulation. Single nucleotide polymorphisms in the genes

responsible for drug metabolism, drug efflux and drug targets have been shown to

be a major contributing factor towards drug retention in the body and hence

nephrotoxicity. Therefore we hypothesized that the study of these SNPs could

generate data, which would be helpful to clinicians in order to establish

individualized dosage regimen depending on patient’s SNP profile.

In the present study we decided to include 100 consecutive renal transplant

recipients who received either CsA or tacrolimus and 100 healthy controls in order

to compare the genotype as well as allele frequencies in diseased and general

population.

Total 200 subjects including 100 patients and 100 controls were analyzed for the

mentioned SNPs of CYP3A4, CYP3A5, MDR-1 and IL-2 genes. Patient samples

were collected from P. D. Hinduja National Hospital (n=12) and Muljubhai Patel

Urological Hospital (MPUH), Nadiad, Gujrat (n=88). 100 control samples were

collected from the subjects attending regular health check-up program at our

hospital. As the control group included in this study is not related to or a subgroup of

the patient population, the subjects comprising control group were not receiving

CNIs.

Among the consecutive 100 renal transplant patients included in the study, the

incidence of kidney failure was observed more in the age group of 30-50 years.

Gender wise distribution of patients shows that the prevalence of kidney failure was

found more in males (80%) than in females (20%).

53

In order to segregate the patients who achieved higher 6th day CNI levels, both the

categories of patients (ie. Those on CsA and those on tacrolimus) were divided as

per the therapeutic range of the respective drug levels.

In case of the patients receiving CsA (n=56), the division was made as those who

achieved <1500 ng/ml 6th day C2 level and those who achieved >1500 ng/ml 6th day

C2 level likewise in the case of tacrolimus receiving patients, the division was made

as those who achieved <10 ng/ml 6th day trough level and those who achieved >10

ng/ml 6th day trough level.

Out of the 44 patients receiving tacrolimus, 27 were receiving Azathiopurine along

with tacrolimus and 17 were receiving Mycophenolate mofetil along with it.

From the 56 patients receiving CsA 5 patients developed nephrotoxicity (mean 6th

day C2 level 1699 ng/ml) within one month of transplantation which was confirmed

by tissue biopsy. Out of the 44 patients receiving tacrolimus, 3 patients developed

biopsy proven nephrotoxicity (mean 6th day trough level 17.43 ng/ml) within the

same period of time.

54

CYP3A4 A-290G polymorphism

The A-290G SNP of CYP3A4 gene also called CYP3A4-V is the most common SNP

found in it’s 5’-flanking domain. The change lies in the putative nifedipine response

element (NFSE). Despite the expanding knowledge about CYP3A4 gene mutations,

the impact of variant alleles on enzyme activity is still under-recognized because the

frequency reported for many CYP3A4 variant alleles including G-290 is extremely

low (Ball S et al 1999, Felix C et al 1998. Rebbeck T et al 1998, Rivory L et al

2000).

G-290 allele frequency varies among different ethnic groups, 0% in Taiwanese,

Chinese, Chinese Americans and Japanese Americans, 2 to 9.6% in Caucasians,

9.3 to 11% in Hispanic Americans and 35 to 67% in African Americans (Gonzalez F

et al 1988, Rebbeck T et al 1998, Walker A et al, Hsich K et al 2001, Ball S et al

1999, Garcia-Martin E et al 2002, Paris P et al 1999).

As CYP3A4 substantially contributes to the metabolism of many clinically important

drugs including CNIs, it may be speculated that the observed interindividual

difference in their metabolism is likely attributed to the polymorphic expression of

this enzyme. However the attempts to link SNPs in CYP3A4 gene with functional

effects on drug pharmacokinetics have mostly shown negative results. Results from

many studies have showed no significant pharmacological impact of this

polymorphism on CsA pharmacokinetics. Von Ahsen et al (2001) have reported that

this SNP had no significant effect on trough blood concentrations of CsA in 124

stable Caucasian renal transplant recipients. They also found no significant

difference in the CsA doses needed to maintain similar trough concentrations in the

patients with or without G-290 allele. Similar conclusions have been drawn in many

other studies. It was thought that CYP3A4-V genotype in the transplant population

lacked an association with CsA clearance. Though some results showed a positive

correlation with tacrolimus and or sirolimus, it was associated with drug exposure

and functional influence of CYP3A5. the study of Hesselink et al (2003) showed that

G-290 allele carriers have lower tacrolimus dose-adjusted trough levels than

55

patients with the wild-type genotype. This may be explained by an unlikely

mechanism. The polymorphism is located in the 5’- transcriptional regulatory

element of the CYP3A4 coding region, more likely causing changes in protein

expression rather than activity. Pregnane X receptor and retinoid X receptor are

shown to be transcription initiation factors of CYP3A genes. Thus this polymorphism

may affect the binding of these factors to the regulatory element of CYP3A4 thereby

affecting the gene expression rather than affecting protein per se. On the other

hand, the variability in the pharmacokinetics of orally administered CNIs is largely

determined by interindividual differences in phase I elimination, whereas the C0 of

tacrolimus correlates better with total drug exposure than the results were

essentially influenced by CYP3A5 SNP. The exact contribution still needs to be

elucidated (Ball S et al 1999, Felix C et al 1998, Rebbeck T et al 1998, Von Ahsen

et al 2001, Rivory L et al 2000, Anglicheu D et al 2005, Hesselink D et al 2003,

Kuehl P et al 2001).

Table 2 compares the genotype frequencies of CYP3A4 A-290G SNP in 100

healthy controls and 100 patients. The distribution indicates that there is no

significant difference between genotype frequencies and number of alleles in both

the groups.

Table 3 shows number of genotypes in two subgroups of cyclosporine receiving

patients. It can be observed that these subgroups showed the prevalence of AA

(wild type) genotype and consequently A allele. The five patients who developed

nephrotoxicity were homozygous wild type for this polymorphism.

Table 4 shows number of genotypes in the two subgroups of tacrolimus receiving

patients. In this case also it can be observed that both these subgroups showed the

prevalence of AA (wild type) genotype and consequently A allele. The three patients

who developed nephrotoxicity were homozygous wild type for this polymorphism.

56

Table 5 shows the distribution of genotypes of CYP3A4 A-290G SNP in case of the

patients receiving tacrolimus and azathiopurine. All the patients in this case also

were found A allele carriers and most of them are homozygous wild type (AA n =

26).

Table 6 shows the distribution of genotypes of CYP3A4 A-290G SNP in case of the

patients receiving tacrolimus and Mycophenolate mofetil. All the patients in this

case also were found homozygous wild type (AA, n = 17).

57

CYP3A5 A6986G polymorphism

The 6986 A/G variation within intron 3 seems to be the most important functional

polymorphism in the CYP3A5 gene. CYP3A5*3 (G at position 6986) creates an

aberrantly spliced site in the pre- mRNA with a stop codon and leads to a truncated

CYP3A5 protein. Among CYP3A5*3/*3 subjects, CYP3A5 expression comprises

only 4.2% of total CYP3A in the jejunum. Among heterozygous CYP3A5*1/*3

subjects, however, CYP3A5 expression is appreciable, with 50% of total CYP3A in

the liver and 61% of CYP3A in the jejunum (Lin Y et al 2002). It has been reported

that CYP3A5*1 (A at position 6986) allele is related to the increased expression of

CYP3A5 enzyme and only people with at least one CYP3A5*1 allele actually

express CYP3A5 protein (Kuehl P et al 2001). In some patients, the coexistence of

the other variant alleles CYP3A5*2, CYP3A5*6 and CYP3A5*7 with CYP3A5*1

allele may prevent the expression of CYP3A5 (Hustert E et al 2001). As a result of

racial differences in the frequency of the most prevalent CYP3A5 alleles, about one

quarter of Caucasians and half of African Americans generally have the ability to

express functional CYP3A5. Japanese and Chinese people express CYP3A5

approximately three times as frequently as Caucasian subjects (Kuehl et al 2001,

Chou F et al 2001, Hiratsuka M et al 2002).

Several studies have shown that the dose of tacrolimus is associated with CYP3A5

SNPs. Patients with CYP3A5*1 allele require a larger dose of tacrolimus (kg/day) to

achieve the same blood concentration than the homozygous patients with

CYP3A5*3 in pediatric heart and adult lung transplantation (Zheng H et al 2003,

Zheng H et al 2004). The result was also shown in renal transplant recipients

(Thevert E et al 2003, Zhang X et al 2005). Moreover, others studied SNPs in a

pseudogene CYP3AP1 (-44A�G) and CYP3A5*3 and found that determination of

the CYP3A5*3 genotype could be used to predict the dose of tacrolimus (Macphee I

et al 2005, Macphee I et al 2002) . A study shows that polymorphisms of the donor

CYP3A5 gene seemd to contribute more to the individual variation of the dose of

tacrolimus. In case of CsA, however few studies have shown the role of this SNP on

CsA pharmacokinetic characteristics. Hesselink et al (2003) found no association

58

between CYP3A5 genetic polymorphism and dose adjusted predose concentration.

Interestingly unexpected result was reported in a recent study demonstrating that

CsA oral clearance was significantly lower in CYP3A5 expressers than in the

nonexpressers (Yates C et al 2003). They suggested that this observation was

artefact attributed to a linkage in their study population between CYP3A5

nonexpressers and MDR-1 3435T allele carriers. They failed to independently

assess whether CYP3A5 correlated with any CsA pharmacokinetic parameter since

all CYP3A5 nonexpressers were also MDR-1 3435T allele carriers. Another report

demonstrated that an effect of CYP3A5 intron 3 polymorphism was associated with

CsA dosage to a lesser extent other than tacrolimus in stable renal transplant

recipients (Haufroid V et al 2004). Dose adjusted trough concentrations were 3 fold

and 1.6 fold higher in CYP3A5*3/*3 patients than in CYP3A5*1/*3 patients for

tacrolimus and CsA, respectively (Hesselink D et al 2003, Evans W et al 1999,

Kamdem et al 2005). Although the polymorphism of CYP3A5 is thought to be

associated with the tacrolimus requirement but not associated with CsA

pharmacokinetics (von Ahsen et al 2001), the exact mechanism of CYP3A5 on CsA

pharmacokinetics is still controversial and needs to be further clarified.

Table 7 compares the genotype frequencies of CYP3A5 A6986G SNP in 100

healthy controls and 100 patients. The distribution indicates that there is no

significant difference between genotype frequencies and number of alleles in both

the groups.

Table 8 shows number of genotypes in the two subgroups of cyclosporine receiving

patients. It can be observed that both these subgroups showed the prevalence of G

allele. On comparing the A allele carriers (AA+AG) and homozygous mutants (GG)

from <1500ng/ml group with those of >1500ng/ml group we did not find significant

difference. All the five patients who developed nephrotoxicity were heterozygous for

this polymorphism.

59

Table 9 shows number of genotypes in the two subgroups of tacrolimus receiving

patients. It can be observed that all the 8 patients who achieved greater than

10ng/ml 6th day trough level of tacrolimus were homozygous mutants (GG). As per

the observations of Hesselink et al (2003), the patients having GG genotype for this

SNP require lower tacrolimus dose to achieve target drug level, which means they

show higher trough levels if dose adjustments are not done. Consequently, the

observations from above table suggest that a significant difference (p<0.05) exists

between A allele carriers and homozygous mutants (GG) in <10ng/ml and >10ng/ml

groups and the group achieving higher levels is prevalent in GG genotype. This

indicates the risk of nephrotoxicity. The three patients who developed biopsy proven

nephrotoxicity were all mutants for this polymorphism.

In order to observe the effect of CYP3A5 A6986G polymorphism on the

pharmacokinetics of tacrolimus we tabulated the median of level/dose (L/D) ratio

against the three genotypes. Observation from the following table shows that the

median L/D ratio was found to be significantly higher in the patients having GG

genotype (n = 24) by Kruskal-Wallis test (table 10).

Table 11 shows the genotype distribution of CYP3A5 A6986G SNP in the patients

receiving tacrolimus and azathiopurine. All the patients who achieved >10ng/ml 6th

day trough level of tacrolimus showed homozygosity for mutant G allele.

Table 12 shows the genotype distribution of CYP3A5 A6986G SNP in the patients

receiving tacrolimus and mycophenolate mofetil. In this case also all the patients

who achieved >10ng/ml 6th day trough level of tacrolimus showed homozygosity for

mutant G allele.

60

MDR-1 C3435T polymorphism

Both CsA and tacrolimus are the substrates for P-gp. Numerous studies have

associated SNPs in the MDR-1 gene with dosage of CsA and tacrolimus in organ

transplant recipients. Yates et al (2003) investigated 10 renal transplant recipients

and concluded that MDR-1 3435T allele carriers had enhanced CsA oral clearance

compared with individuals with the CC genotype. A recent study also shows that the

donor’s MDR-1 C3435T genotype was associated with the development of CsA

nephrotoxicity in renal transplant patients (Anglicheau D et al 2003). P-gp is also an

important factor in CNI induced nephrotoxicity. Recently the donor's MDR1 C3435T

genotype was associated with the development of CsA nephrotoxicity (Hauser I et

al 2005).

However, conflicting results have been reported. Von Ahsen et al (2001) found no

significant difference in CsA doses needed to maintain similar CsA trough

concentrations in 124 stable Caucasian renal transplant recipients. Goto et al

(2002) found no association between 10 SNPs including C3435T of exon 26 and

the tacrolimus concentration/dose ratio during the first postoperative days after liver

transplantation. Others found no evidence supporting a role of MDR-1 C3435T in

tacrolimus dose or CsA dose adjusted predose concentrations (Hesselink D et al

2003). Further Macphee I et al (2002) reported only a weak association of MDR-1

C3435T with tacrolimus concentrations and hence dose. These conflicting results

might be explained by the delay after transplantation. Several months after

transplantation, side effects of immunosuppressive treatment frequently require the

addition of concomitant medications (antihypertensive, antilipemic and uricosuric)

that might interact with CNI absorption and excretion (Bonhomme-Faivre L et al

2004).

Haplotype analysis of MDR-1 gene exon 12, 21 and 26 SNPs have attracted more

attention from researchers. Anglicheau et al performed a haplotype analysis on

exon 12, 26 and 21 in 81 renal transplant recipients. They showed a 61% increase

in tacrolimus dose regimen for patients who were homozygous for the wild type

61

allele in SNP of all three exons. Bernal M et al (2003) have reported the frequency

of C3435T polymorphism to be 26% (C/C), 22% (T/T), 52% (C/T) in a Spanish

population. In 461 White subjects Cascorbi I et al (2004) have reported the

frequency of mutants to be 28.6% and that of heterozygots to be 53.9%.

A study by Li-Yan-Miao et al (2007) on comparing allele frequencies of MDR-1

C3435T SNP in 105 healthy Chinese volunteers and 50 renal transplant recipients

shows that the frequencies do not differ in both these groups (p>0.05). Zheng H et

al (2003) have concluded in their study on pediatric heart transplant recipients that

the T allele of C3435T SNP of MDR-1 has been associated with increased blood

concentration of tacrolimus and homozygous mutants of this polymorphism require

larger tacrolimus dose to maintain their tacrolimus blood concentration. However a

study done by the same authors Zheng et al (2004) on adult lung transplant

recipients from USA shows no association between the homozygocity for the

mutant allele of this polymorphism and tacrolimus blood concentration. Whereas

Wang J et al (2006) state in their study on adult lung transplant recipients that

haplotypes are predictive of tacrolimus dose requirements. Research by Zhang X et

al (2005) on 118 renal transplant recipients from China does not show any

association between MDR-1 C3435T genotypes and tacrolimus trough levels at 1

week, 1 month or 3 months. Hu YF et al (2006) have studied 106 Chinese renal

transplant recipients receiving cyclosporine and concluded that MDR-1 C3435T

SNP is not a determinant of CsA trough levels and the median C0 level of all the

recipients did not differ in the wild type, heterozygous and mutant genotypes

(p>0.05). Whereas a study on 88 Iranian renal transplant recipients receiving CsA

by Azarpira N et al (2006) has demonstrated that level/dose (L/D) ratio was

significantly higher in the subjects homozygous mutant for MDR-1 C3435T SNP i.e.

(TT). These authors have also stated that pharmacogenetics methods could be

used to help select the initial dosage within few days after transplantation and

individualize the immunosuppressive therapy. A small study on 19 Caucasian renal

transplant recipients by Yates et al shows that MDR-1 3435T allele carriers have

enhanced oral clearance of CsA, however they fail to assess it’s independent role

62

because MDR-1 3435T allele carriers were also CYP3A5 nonexpressors. A study

by Hesselink et al (2003) on renal transplant recipients from Netherlands (on CsA n

= 110, on tacrolimus n = 64) however shows no correlation between MDR-1

C3435T genotypes and tacrolimus dose requirements. Roy JN et al (2006), in their

study on 44 renal transplant recipients from Canada concludes that the complete

absence of CYP3A5*3 allele and the accumulation of less than 3 copies of MDR-1

T-129C, C3435T and G2677T SNPs are associated with lower tacrolimus blood

levels. However Tsuchiya N et al (2004) state in their study on 30 consecutive renal

transplant recipients from Japan that MDR-1 C3435T SNP is not associated with

tacrolimus pharmacokinetics. Study by Haufroid et al (2004) on 50 renal transplant

recipients receiving tacrolimus showed no association between trough blood

concentration of tacrolimus dose requirement and MDR-1 C3435T genotypes. Wei-

Lin W et al (2006) have shown that MDR-1 C3435T genotypes influence tacrolimus

trough levels and hence dose requirements in 50 Chinese liver transplant recipients.

Foote CJ (2006) et al have shown that MDR-1 C3435T polymorphism is an

independent predictor of CsA dose within few days after renal transplant. Kotrych K

et al (2007) have studied 116 renal transplant recipients from Poland receiving CsA

and concluded that this SNP is not a CsA dose predictor. Utecht KN et al (2006)

state that the influences of CYP3A5 and MDR-1 alleles on CsA metabolism and

MDR-1 alleles on tacrolimus metabolism remain controversial. However Akbas SH

et al (2006) in their study on 92 Turkish renal transplant recipients state that

tacrolimus daily doses were significantly lower in the patients with 3435TT genotype

at 1 and 6 month after transplant. Therefore knowledge of MDR-1 genotypes may

be useful to adjust the optimal dose of tacrolimus in transplant patients thereby

rapidly achieving target blood concentrations.

Table 13 compares the genotype frequencies of MDR-1 C3435T SNP in 100

healthy controls and 100 patients. The distribution indicates that there is no

significant difference between genotype frequencies and number of alleles in both

the groups.

63

Table 14 shows number of genotypes in the two subgroups of cyclosporine

receiving patients. It can be observed that both these subgroups showed the

prevalence of T allele hence the difference was insignificant. Of the five patients

who developed nephrotoxicity one patient with the level 1830 ng/ml was

homozygous mutant whereas rest all were heterozygous.

Table 15 shows number of genotypes and alleles in the two subgroups of tacrolimus

receiving patients. It can be observed that all the 8 patients who showed >10ng/ml

6th day trough level of tacrolimus showed the prevalence of mutant (TT) genotype (n

= 7), which suggests the risk for developing nephrotoxicity. All the three patients

who developed nephrotoxicity were homozygous mutants for this polymorphism.

In order to observe the effect of genotypes of MDR-1 C3435T polymorphism on the

pharmacokinetics of tacrolimus, we tabulated the median L/D ratios of tacrolimus

against the genotypes. The observation from the following table shows that the

median L/D ratio was significantly higher in the subjects having TT genotype (n =

18) by Kruskal-Wallis test (table 16).

Table 17 shows the genotype distribution of MDR-1 C3435T SNP in the patients

receiving tacrolimus and azathiopurine. All the patients who achieved >10ng/ml 6th

day trough level of tacrolimus showed homozygosity for mutant T allele.

Table 18 shows the genotype distribution of MDR-1 C3435T SNP in the patients

receiving tacrolimus and mycophenolate mofetil. No significant difference was

observed in the patients with respect to the prevalence of the homozygous mutant

genotype.

64

IL-2 T-330G polymorphism

Cyclosporine and tacrolimus exhibit their immunosuppressive action by inhibiting

the transcription of interleukin-2. Though the molecular mechanisms of

immunosuppression for both these drugs are different, they have a common target.

T-330G polymorphism in the promoter region of IL-2 gene is relatively recent SNP

and hence not much literature is available about it's association studies (Morgun A

et al 2003).

Polymorphisms in the promoter region of cytokine genes have been shown to

influence their levels as well as some of them have been associated with toxicity or

rejection episodes in case of solid organ transplant patients. Therefore the study of

this SNP with respect to CNI blood levels and the incidences of nephrotoxicity with

IL-2 plasma level assumes significance.

Morgun et al (2003) have studied 67 heart and 63 renal transplant recipients for this

SNP and found that wild type genotype (TT) is associated with kidney rejection.

However in the same study, no association was observed for heart transplant

recipients. Holweg et al (2003) have also found no association of this SNP with

rejection episodes in 301 heart transplant recipients.

A study by Satoh S et al (2007) on 50 recipients with stable renal graft function

shows that the frequencies of the IL-2 T-330G TT genotype were higher in the

patients with progressive CAN (Chronic/sclerosing Allograft Nephropathy)

(p=0.046). The study also concludes that the presence of IL-2 T-330G TT genotype

may be a risk factor for CAN, however further studies with a large number of

subjects would contribute to the ability to make progressive determinations or tailor

immunomodulatory regimens after renal transplantation. Cox ED et al (2001) from

Washington, U.S.A. Have studied 160 (102 whites, 43 African-Americans) renal

transplant recipients and concluded that renal transplant recipients differ from the

general population with regard to IL-2, IL-6 and Interferon gamma gene

65

polymorphisms. These findings again suggest the relevance in making prognostic

determinations or tailoring immunomodulatory regimens after renal transplantation.

Jordan S et al (1993) have shown that IL-2 plasma levels were not predictive of

allograft rejection in the case of Caucasian cardiac transplant recipients. Studies by

Chang DM et al (1996) and Grant SC et al (1996) also concludes the same in

cardiac transplant patients from Taiwan and United Kingdom respectively.

MacMillan M et al (2003) have stated in their study on 95 consecutive donor and

recipient that the high producer G allele of IL-2 T-330G increases risk for acute graft

versus host disease in case of unrelated donor bone marrow transplantation.

A study by Kutukcular et al (1995) on 16 renal transplant recipients from England

shows that out of the 16 patients 7 showed clinical evidence of acute allograft

rejection and 5 showed good allograft function with no signs of rejection. Primary

nonfunction was observed in 4 patients and the IL-2 plasma levels whenever

detectable were found to be predictive of allograft rejection.

The observation from our study shows that out of the 100 patients studied, 8

patients showed detectable plasma posttransplant IL-2 levels (pg/ml) (13.656 on

tac, 3.930 on CsA, 5.779 on CsA, 9.706 on tac, 0.748 on CsA, 11.786 on tac,

30.077 on tac and 2.521 pg/ml on tac) withe their respective IL-2 T-330G genotypes

as (TT, TT, GG, TT, TG, TG, TG, TT). As shown in the bracket above, 5 patients

out of them were on tacrolimus and remaining 3 were on cyclosporine. Out of the 5

patients on tacrolimus, 3 developed nephrotoxicity within one month of

transplantation. Their IL-2 levels were 13.656 pg/ml (TT for IL-2 T-330G), 11.786

pg/ml (TG for IL-2 T-330G) and 30.077 pg/ml (TG for IL-2 T-330G).

Table 19 compares the genotype and allele frequencies of IL-2 T-330G SNP in 100

healthy controls and 100 patients. The distribution indicates that there is no

significant difference between genotype frequencies and number of alleles in both

the groups.

66

Table 20 shows number of genotypes in the two subgroups of cyclosporine

receiving patients. It can be observed that both these subgroups showed the

prevalence of wild type T allele. Of the five patients who developed nephrotoxicity 3

were heterozygous and 2 were wild type for this polymorphism.

Table 21 shows number of genotypes in the two subgroups of tacrolimus receiving

patients. In this case also it can be observed that both these subgroups showed the

prevalence of wild type T allele. Of the three patients who developed nephrotoxicity

2 were heterozygous and 1 was wild type for this polymorphism.

Table 22 shows the distribution of genotypes of IL-2 T-330G SNP in the patients

who were receiving tacrolimus and azathiopurine. No significant difference was

observed in the patients having <10 and >10ng/ml tacrolimus drug levels with

respect to homozygous mutant genotype.

The genotype distribution of the patients receiving tacrolimus and mycophenolate

mofetil shows a significant difference between the two groups with respect to the

number of homozygous mutants (table 23).

CHAPTER 6

CONCLUSION

67

Chapter 6 – Conclusion The success of kidney transplantation gets affected by drug induced nephrotoxicity.

Calcineurin inhibitors are the commonly used immunosuppressants worldwide.

These drugs have shown significant improvement in the outcome of kidney

transplantation; however limiting it’s success by their side effect i.e. nephrotoxicity.

This condition occurs because of improper metabolism of these drugs.

Pharmacogenomics emphasizes that every individual metabolizes drugs

differentially. This difference, in case of CNIs has been attributed to polymorphisms

in the genes involved in their metabolism (CYP3A4, CYP3A5), efflux (MDR1) and

those genes which are their targets (IL-2). Therefore we aimed at studying A-290G

(CYP3A4), A6986G (CYP3A5), C3435T (MDR1) and T-330G (IL-2) polymorphisms

in Indian de novo renal transplant recipients. This data was then correlated with the

blood levels of cyclosporine and tacrolimus. The hypothesis in this study was that if

either of these polymorphisms was found to be associated with higher levels of

either CsA or tacrolimus then it could guide physicians to predict the likelihood of

development of nephrotoxicity early and thereby to adjust the dose.

Upon studying 100 consecutive de novo renal transplant recipients receiving either

CsA or tacrolimus and 100 controls showing normal renal profile, we found that the

genotype frequencies of all the polymorphisms studied did not differ significantly

between controls and patients (p>0.05). In the case of CYP3A4 (A-290G) SNP the

mutant allele frequencies in our healthy subjects (0.050; n=100) lies between that in

the Australians (0.031, Amanda S et al 2002) and Caucasians (0.09, Von-Ahsen et

al 2001). The mutant G allele frequency of CYP3A5 A6986G polymorphism from

our study (0.705; n=100) is at par with that in Chinese (0.76, Nie-xin-min et al 2005)

and higher than that found in Indian immigrants in Singapore (0.59, Balram C et al

2003). In case of the mutant allele frequency of the MDR-1 C3435T polymorphism

we found that the frequency from our study (0.610; n=100) is at par with that found

in Indian immigrants in Singapore (0.63, Balram C et al 2003). However the mutant

68

G allele frequency of IL-2 T-330G polymorphism in our study (0.495; n=100) is

higher than that found in Whites (0.26) and in African- Americans (0.06).

In the patient group, 56 patients who received cyclosporinedid not show significant

difference for genotype frequencies of the polymorphisms studied in the patients

who developed <1500ng/ml and >1500ng/ml 6th day C2 levels. 44 patients who

were receiving tacrolimus also did not show significant difference with respect to

their genotype frequencies of CYP3A4 and IL-2 gene polymorphisms in those who

achieved <10ng/ml and >10ng/ml 6th day trough levels.

However for tacrolimus treated patients CYP3A5 6986G and MDR1 3435T

polymorphism frequencies were significantly different in those who achieved

<10ng/ml and >10ng/ml 6th day trough levels (p=0.010, CYP3A5 and p=0.015,

MDR1 by Fisher’s exact test). Significantly higher tacrolimus levels were observed

in the homozygous mutant cases of CYP3A5 and MDR1 gene polymorphisms

(p=0.011, CYP3A5 and p=0.0122, MDR-1 by Kruskal- Wallis test).

IL-2 ELISA data shows that detectable levels were obtained in the 5 patients who

received tacrolimus. Out of the 5 patients on tacrolimus who developed detectable

IL-2 levels 3 were wild type and 2 were heterozygous. Out of the three patients who

developed nephrotoxicity 1 was wild type and 2 were heterozygous for IL-2 T-330G

SNP.

Thus in the present study on 100 patients we have observed significant association

of CYP3A5 A6986G and MDR1 C3435T polymorphisms with increased levels of

tacrolimus. Larger sample size can only strengthen this association. This data

would be helpful to physicians so that by knowing the genotype of the patient before

undergoing transplantation they would be able to decide upon the starting dose of

tacrolimus so as to avoid high trough levels and consequently nephrotoxicity.

69

Future directions of work

Pharmacogenomics is an upcoming field. Individualized medicine concept is

gradually gaining place in regular clinical practice. Genetic polymorphisms as well

as mutations have now been identified in all the main genes in cytochrome P450

family, however their effect on translation, respective protein levels or on the protein

conformation is not known for all. Therefore proteomic studies could develop

simultaneously with genomics. There is lack of large, conclusive, prospective

studies showing improvement of drug efficacy following genotyping; this is the major

reason why the incorporation of the knowledge gained from pharmacogenetics into

routine medical practice is lacking. The cost of genotyping is decreasing rapidly,

cost effective high throughput genotyping techniques are becoming available and

hence our knowledge about the benefits of predictive genotyping is increasing, this

could be used in designing large association studies. In December 2004 the FDA

(Food and Drug Administration) has approved a microarray chip designed to

routinely identify polymorphisms of drug transporters and cytochrome P450s. This

technique along with the findings from proteomic studies could provide physicians

with valuable information to individualize drug treatment.

70

Figure 1 – Structure of kidney

Source - http://www.sci.sdsu.edu/classes/bio100/Lectures/Lec t16/Image271.gif Figure 2 - Functions of kidney

Source - http://www.engin.umich.edu/~CRE/web_mod/viper/pics/ nephron.gif

71

Figure 3 – a) Dr. J. F. Borel, inventor of cyclospo rine, b) Chemical structure of

cyclosporine

a)

Source –

www.worldoffungi.org/Mostly_Medical/Harriet_Upton/H arriet_Upton.htm

b)

Source- http://dailymed.nlm.nih.gov/dailymed/image.cfm?id=2 988&name=gengraf-capsules-

structure.jpg&CFID=426818&CFTOKEN=98e97d54d4965467- ABA54B97-CA47-C63D-

03D3681BAC969B62&jsessionid=ca305bfcd7d516306626

Cyclic peptide

Side chain

72

Figure 4 – a) Chemical structure of tacrolimus b) M etabolites of tacrolimus c)

Metabolism of tacrolimus

c)

a)

b)

Source - Iwasaki K (2007). Metabolism of tacrolimus (FK506) and recent topics

in clinical pharmacokinetics. Drug Metab. Pharmacok inet., 22 (5), 328-355.

73

Figure 5 - Mechanism of action of cyclosporine or t acrolimus (FK506)

Source – Expert reviews in Molecular Medicine, Camb ridge university press,

2000.

74

Figure 6 – Promoter region of IL-2 gene and the fac tors binding at various

positions.

Source - Grunfeld J, Bach J, Kreis H et al (1998). Advances in nephrology. St.

Lois: Mosby.

75

Figure 7 – ADME process (Absorption Distribution Me tabolism Excretion)

Source - Waterbeemd H, Gifford E (2003). ADMET in silico m odelling: towards

prediction paradise?. Nature Reviews Drug Discovery , 2, 192-204.

Figure 8 – The phase I system, mechanism of action of cytochrome p450

Source - http://www.reactome.org/figures/p450_cat_c ycle.JPG

76

Figure 9 – Topology of p-glycoprotein

Figure 10 – Mechanism of substrate efflux by p-glyc oprotein

Source (Fig. 9 & 10) - Deeley R, Westlake C, Cole S (2006). Transmembrane

transport of endo- and xenobiotics by mammalian ATP -binding cassette

multidrug resistance proteins. Physiol. Rev., 80, 849-899.

SUB SUB

SUB

SUB

77

Figure 11 – The phase III system

Source - Liska D (1998). The detoxification enzyme systems. Altern. Med.

Rev., 3(3), 187-198.

78

Figure 12 – Structure of promoter region of CYP3A4 gene

Figure 13 – Relative position of CYP3A4 and CYP3A5 genes on chromosome

Figure 14 – Phenotypic effect of CYP3A5 A6986G poly morphism

Source (Fig 12, 13, 14) - Lamba JK, Lin YS, Schuetz EG et al (2002). Genetic

contribution to variable human CYP3A-mediated metab olism. Advanced Drug

Delivery Reviews, 54, 1271-1294.

79

Figure 15 – Positions of various SNPs in MDR-1 gene with their positions in

protein

Source - Anglicheu D, Verstuyft C, Laurent-Pung P e t al (2003). Association of

the multidrug resistance-1 gene single-nucleotide p olymorphisms with the

tacrolimus dose requirements in renal transplant re cipients. J. Am. Soc.

Nephrol., 14, 1889-1896.

80

Figure 16 – Steps in Polymerase Chain Reaction

Source - http://employees.csbsju.edu/hjakubowski/cl asses/ch331/dna/pcr.gif

Figure 17 – Approximate temperatures used in DNA am plification

Source - http://www.mun.ca/biology/scarr/PCR_sketch _3.gif

81

Table 1 – Details of the genotyping of the polymorp hisms included in the

study

Gene

Primers

Adapted

from

Amplified

product

length

Restriction

enzyme

used

Fragment

lengths

after

digestion

CYP3A4

Forward

5’gaatgaggacagccatagagacaagggga3’

Reverse

5’cctttcagctctgtgttgctctttgctg3’

Cavalli et

al (2001)

385 bp.

Mbo II

Wt - 175, 169

Ht - 210, 175,169

Mt - 210, 175

CYP3A5

Forward

5’catcagttagtagacagatga3’

Reverse

5’ggtccaaacagggaagaaata3’

Ron HN et

al

(2002)

293 bp.

SspI

Wt - 148, 125

Ht - 168, 148,125

Mt - 168, 125

MDR-1

Forward

5’gatctgtgaactcttgttttca3’

Reverse

5’gaagagagacttacattaggc3’

Calado RT

et al

(2002)

244 bp.

Dpn II

Wt - 172, 72

Ht - 244, 172,72

Mt - 244

IL-2

Forward

5’tattcacatgttcagtgtagttct3’

Reverse

5’agactgactgaatggatgtaggtg3’

Cox ED et

al (2001)

188 bp.

Fsp BI

Wt - 188

Ht - 188, 164,24

Mt - 164, 24

82

Figure 18 – Photograph of IL-2 ELISA plate Figure 19 – OD versus concentration graph of the st andards

Standards

83

Figure 20 – Genotyping of CYP3A4 A-290G polymorphis m

W W H W W Ma

Wild type - 175, 169 Heterozygous - 210, 175, 169 Mutant - 210, 175 Ma – pBR322 MspI digest

84

Figure 21 – Genotyping of CYP3A5 A6986G polymorphis m

Wild type - 148 and 125 Heterozygous - 168, 148, 125 Mutant - 168, 125

Ma – pBR322 MspI digest

M H M H H Ma M H H H M

85

Figure 22 – Genotyping of MDR1 C3435T polymorphism

Wild type - 172, 72 Heterozygous - 244, 172, 72 Mutant – 244 Ma – 50bp DNA ladder

M W W H Ma M H H

86

Figure 23 – Genotyping of IL-2 T-330G polymorphism

H M M H H W H H H W W Ma

Wild type - 188 Heterozygous - 188, 164, 22 Mutant – 164, 22 Ma – 50bp DNA ladder

CYP3A4 A-290G SNP

Table 2 – Comparison of genotype frequencies between controls and patients

Table 3 – Comparison of the genotype frequencies between the two groups of cyclosporine treated patients

Table 4 – Comparison of the genotype frequencies between the two groups of tacrolimus treated patients

CYP3A4Level AA AG GG Total

Controls 92 6 2 100Patients 96 3 1 100

Total 188 9 3 200Fisher's exact = 1.000, NS

CYP3A4Level AA AG GG Total

40 2 1 4313 0 0 13

Total 53 2 1 56Fisher's exact = 1.000, NS

<1500 ng/ml CsA> 1500 ng/ml CsA

CYP3A4Level AA AG GG Total

35 1 0 368 0 0 8

Total 43 1 0 44Fisher's exact = 0.818, NS

<10 ng/ml tac> 10 ng/ml tac

Table 5

Tac, AZA AA AG GG A allele carriers

Homozygous mutants

<10 ng/ml 22 1 0 23 0

>10 ng/ml 4 0 0 4 0

Table 6

Tac, MMF AA AG GG A allele carriers

Homozygous mutants

<10 ng/ml 13 0 0 13 0

>10 ng/ml 4 0 0 4 0

CYP3A5 A6986G SNP

Table 7 – Comparison of genotype frequencies between controls and patients

Table 8 – Comparison of the genotype frequencies between the two groups of cyclosporine treated patients

Table 9 – Comparison of the genotype frequencies between the two groups of tacrolimus treated patients

CYP3A5Level AA AG GG Total

Controls 6 47 47 100Patients 3 62 35 100

Total 9 109 82 200Fisher's exact = 0.0989, NS

CYP3A5Level AA AG GG Total

2 31 10 430 12 1 13

Total 2 43 11 56Fisher's exact = 0.357, NS

<1500 ng/ml CsA> 1500 ng/ml CsA

CYP3A5Level AA AG GG Total

<10 ng/ml tac 1 19 16 360 0 8 8

Total 1 19 24 44> 10 ng/ml tac

Fisher's exact = 0.010

Table 10

Table 11

Tac, AZA AA AG GG A allele carriers

Homozygous mutants

<10 ng/ml 1 11 11 12 11

>10 ng/ml 0 0 4 0 4

Table 12

Tac, MMF AA AG GG A allele carriers

Homozygous mutants

<10 ng/ml 0 8 5 8 5

>10 ng/ml 0 0 4 0 4

CYP3A5L/D ratio AA AG GG

2.66 6.44 8.11Median (ng/ml/mg/kg/day)K-W ANOVA = 0.011

MDR1 C3435T SNP

Table 13 – Comparison of genotype frequencies between controls and patients

Table 14 – Comparison of the genotype frequencies between the two groups of cyclosporine treated patients

Table 15 – Comparison of the genotype frequencies between the two groups of tacrolimus treated patients

MDR-1Level CC CT TT Total

Controls 12 54 34 100Patients 14 54 32 100

Total 26 108 66 200Chi square = 0.8462, NS

MDR-1Level CC CT TT Total

12 20 11 430 10 3 13

Total 12 30 14 56Fisher's exact = 0.107, NS

<1500 ng/ml CsA> 1500 ng/ml CsA

MDR-1Level CC CT TT Total

<10 ng/ml tac 2 23 11 360 1 7 8

Total 2 24 18 44> 10 ng/ml tac

Fisher's exact = 0.015

Table 16

Table 17

Tac, AZA CC CT TT C allele carriers

Homozygous mutants

<10 ng/ml 1 16 6 17 6

>10 ng/ml 0 0 4 0 4

Table 18

Tac, MMF CC CT TT C allele carriers

Homozygous mutants

<10 ng/ml 1 7 5 8 5

>10 ng/ml 0 1 3 1 3

MDR-1L/D ratio CC CT TT

6.22 6.22 8.83Median (ng/ml/mg/kg/day)K-W ANOVA = 0.0122

IL2 T-330G SNP

Table 19 – Comparison of genotype frequencies between controls and patients

Table 20 – Comparison of the genotype frequencies between the two groups of cyclosporine treated patients

Table 21 – Comparison of the genotype frequencies between the two groups of tacrolimus treated patients

IL-2Level TT TG GG Total

Controls 24 53 23 100Patients 33 54 13 100

Total 57 107 56 200Chi square = 0.1354, NS

IL-2Level TT TG GG Total

12 25 6 435 7 1 13

Total 17 32 7 56Fisher's exact = 1.000, NS

<1500 ng/ml CsA> 1500 ng/ml CsA

IL-2Level TT TG GG Total

13 19 4 363 3 2 8

Total 16 22 6 44

<10 ng/ml tac> 10 ng/ml tac

Fisher's exact = 0.427, NS

Table 22

Tac, AZA TT TG GG T allele carriers

Homozygous mutants

<10 ng/ml 6 13 4 19 4

>10 ng/ml 2 2 0 4 0

Table 23

Tac, MMF TT TG GG T allele carriers

Homozygous mutants

<10 ng/ml 7 6 0 13 0

>10 ng/ml 1 1 2 2 2

87

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