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1
Formulation and investigation of specialised dosage forms for
the systemic delivery of the selective antineoplastic α-
tocopheryl succinate
Madhumathi Seshadri
BPharm, MTech Pharm Chem
School of Pharmacy
Faculty of Health Science
Griffith University, Gold Coast
Submitted in fulfilment of the requirements for the degree of Masters of Philosophy
13 August, 2010
i
STATEMENT OF ORGINALITY
I Madhumathi Seshadri hereby declare that this work has previously not been
submitted in whole or in part, for a degree or diploma in any university. To the best of
my knowledge and belief, the thesis contains no material previously published or
written by another person except where due reference is made in the thesis itself.
Madhumathi Seshadri
August 2010
ii
ACKNOWLEDGEMENT
I would like to extend my thanks and heartfelt gratitude to all those who helped make
the present study possible.
Dr. Gary Dean Grant, my supervisor for his patience, professionalism and belief in me
as a RHD student.
Assoc. Prof. Jiri Neuzil for providing us with the wonder drug and
RHD convenor Associate Prof. Michael Rathbone for his support
Dr. Jeffery Grice and Ms. Jenny Ordonez, PA hospital, Brisbane for assisting me with
the Human skin study.
Members and staff of the School of Pharmacy for their moral support and invaluable
guidance during my candidature, especially Ms. Gillian Salinos for her support and
tolerance in dealing with my emotional outbursts.
RHD colleagues specially Tanya Jones for being there always to listen to me when I
was angry or confused, Hsien-Kuo Sun for helping me with the analytical studies and
Ms. Tian Tian Zhang for sitting with me in the lab almost everyday.
My mother, father and my little sister for constantly supporting me emotionally,
financially and inspiring me in realising my dream to study an RHD program away
from home possible. Thank you for always being there.
My fiancé Saibal Roy for being who he is when I needed the most and listen to me talk
endlessly about my work whenever we had the chance to talk. I know it hasn’t been the
easy few years for him either.
My brother Anish Ramachandran for his undying effort to see me happy all the time.
Lastly I dedicate this work to Almighty God and my mother who always said “You haven’t
achieved anything great in life till it makes you feel content throughout life”. This is especially
for you both. Sai Ram.
iii
TABLE OF CONTENTS
LIST OF FIGURES VI
LIST OF TABLES VIII
LIST OF ABBREVIATIONS IX
ABSTRACT XII
CHAPTER 1. INTRODUCTION 1
1.1 BACKGROUND 1
2.1 NONMENCLATURE, STRUCTURAL FEATURES AND BIOLOGICAL ACTIVITIES OF VE 2
1.3 -TOCOPHERYL SUCCINATE 4
1.4 MECHANISM OF ACTION AND DEMONSTRATED EFFICACY OF -TOS 5
1.5 PHARMACOKINETICS OF Α-TOS 9
1.6 TRANSDERMAL DRUG DELIVERY OF -TOS 12
1.7 BARRIERS TO TDD 13
1.8 DRUG-RELATED FACTORS INFLUENCING TDD 15
1.9 TRANSDERMAL DELIVERY OF Α-TOS 15
CHAPTER 2. HYPOTHESIS AND AIMS 17
CHAPTER 3. DEVELOPMENT AND VALIDATION OF ANALYTICAL METHODS
FOR THE DETECTION AND QUANTIFICATION OF Α-TOS 19
3.1 BACKGROUND 19
3.2 MATERIALS AND METHODS 19
3.2.1 CHEMICAL CONFIRMATION OF SOURCED Α-TOS 20 3.2.2 DEVELOPMENT OF METHODS FOR THE QUANTIFICATION OF Α-TOS 22 3.2.3 DEVELOPMENT OF A UV SPECTRAL PROFILE OF Α-TOS 22
iv
3.2.4 DEVELOPMENT OF HPLC METHODS FOR THE DETECTION AND QUANTIFICATION OF Α-TOS
23 3.2.5 VALIDATION OF ANALYTICAL METHODS 24 3.2.6 DATA ANALYSIS 27
3.3 RESULTS AND DISCUSSION 27
3.3.2 RESULTS OF METHODS FOR THE QUANTIFICATION OF Α-TOS 33 3.3.3 RESULTS FOR THE VALIDATION OF QUANTIFICATION OF Α-TOS 36
CHAPTER 4. SAMPLE PREPARATION FOR ANALYTICAL PROCEDURES 45
4.1 BACKGROUND 45
4.2 MATERIALS AND METHODS 45
4.2.1 LIQUID-LIQUID EXTRACTION (LLE) 46 4.2.2 SOLID PHASE EXTRACTION (SPE) 46 4.2.4 DATA ANALYSIS 47
4.3 RESULTS AND DISCUSSION 48
CHAPTER 5. FORMULATION OF TRANSDERMAL DOSAGE FORMS
CONTAINING Α-TOS 53
5.1 BACKGROUND 53
5.2 METHODS AND MATERIALS 56
5.2.1 COMPOUNDING OF LIPODERM FORMULATIONS 56 5.2.2 COMPOUNDING OF DMSO-CONTAINING PATENT FORMULATION 57
5.3 RESULTS AND DISCUSSION 57
CHAPTER 6. IN-VITRO SKIN PERMEATION EXPERIMENTS 59
6.1 BACKGROUND 59
6.2 MATERIALS AND METHODS 61
6.2.1 SOLUBILITY STUDIES 62 6.2.2 SHAKE-FLASK DETERMINATION OF LOG P 62 6.2.3 IN-VITRO SKIN PERMEATION STUDY 63 6.2.5 DATA ANALYSIS 66
6.3 RESULTS AND DISCUSSION 66
CHAPTER 7. INFLUENCE OF ESTERASES ON THE SUCCESSFUL TRANSDERMAL
DELIVERY OF Α-TOS 72
v
7.1 BACKGROUND 72
7.1.1 ESTERASES 73
7.2 MATERIALS AND METHODS 74
7.2.1 COMPETITIVE ACHE AND BCHE ACTIVITY ASSAY 74 7.2.2 COMPARATIVE METABOLISM OF Α-TOS BY ACHE AND BCHE 75 7.2.3 EFFECT OF Α-TOS EXPOSURE ON ESTERASE ACTIVITY IN-VITRO 76 7.2.4 PLASMA INCUBATION STUDY 78 7.2.5 DATA ANALYSIS 78
7.3 RESULTS AND DISCUSSION 79
CHAPTER 8. CONCLUSIONS 89
REFERENCES 93
vi
LIST OF FIGURES
Figure 1. Structure and substituent (R) groups for the various forms of VE. ....... 2
Figure 2. Structure of α-TOS [27]. ............................................................................. 4
Figure 3. Evidenced -TOS mediated signalling pathways [44].............................. 8
Figure 4. Metabolic fate of orally and parenterally administered -TOS. ........... 10
Figure 5. Numbering used for spectral assignments of α-TOS. ............................. 20
Figure 6. Representative 1H-NMR spectrum obtained for donated -TOS. ........ 28
Figure 7. Representative ESI MS spectrum run in positive-ion obtained for
donated -TOS. The parent peak of 554.3amu corresponds to [M+Na]+
for -TOS. .................................................................................................. 30
Figure 8. Representative FTIR spectrum obtained for donated -TOS. ............. 31
Figure 9. Representative DSC curve for the donated -TOS. ............................... 32
Figure 10. Representative TGA thermogram of donated -TOS. .......................... 33 Figure 11. Absorbance-wavelength profile for α-TOS. Arrows indicated the
wavelengths of maximum absorbance potentially suitable for
quantitative calibration. ............................................................................ 34
Figure 12. Example chromatogram from the optimised isocratic HPLC method at
205nm. ......................................................................................................... 35 Figure 13. Example chromatogram from the optimised step-up gradient HPLC
method at 205 nm....................................................................................... 35
Figure 14. Standard curves for the isocratic HPLC (A) and gradient HPLC (B)
methods. ...................................................................................................... 37
Figure 15. Accuracy data for the isocratic (A) and gradient (B) HPLC methods at
205nm. Results represent the mean ± SD of replicate experiments (n =
3). ................................................................................................................. 38
Figure 16. Repeatability of the developed isocratic (A) and gradient (B) HPLC
method at 205nm. Results represent the mean ± SD of replicate
experiments (n = 3). ................................................................................... 40
Figure 17. Intermediate precision of the isocratic (A) and gradient (B) HPLC
method at 205nm. Results represent the mean ± SD of replicate
experiments (n = 3). ................................................................................... 41
Figure 18. Reproducibility of the isocratic (A) and gradient (B) HPLC method at
205nm. Results represent the mean ± SD of replicate experiments (n =
3). ................................................................................................................. 42
Figure 19. Representative isocratic chromatogram. The chromatograms shows 5,
10, 25µg/mL α-TOS, α-TOH and poorly resolving internal standard
extracted from spiked porcine plasma at 205nm. ................................... 48
Figure 20. Representative step-up gradient chromatogram. The chromatograms
shows 7.5µg/mL α-TOS and internal standard extracted from spiked
porcine plasma at 205nm. ......................................................................... 49
Figure 21. Spectral profiles obtained from the PDA during the isocratic (A) and
gradient (B) analysis of extracted plasma samples. ................................ 50
Figure 22. Efficiencies of LLE of plasma samples (A) and PBS containing 30%
ethanol (B). Samples were analysed by the step-up gradient method.
Data shown is the mean ± SD of triplicate values and is representative
of three independent experiments. ........................................................... 51
vii
Figure 23. Efficiencies of SPE of plasma samples (A) and PBS containing 30%
ethanol (B). Samples were analysed by the step-up gradient method.
Data shown is the mean ± SD of triplicate values and is representative
of three independent experiments. ........................................................... 52
Figure 24. Mechanisms of action liposomes as transdermal drug delivery systems.
(A) Free drug mechanism; (B) penetration enhancing effects of
liposome components; (C) vesicle absorption to and/or fusion with the
stratum corneum; and (D) illustrates intact vesicle penetration into or
into and through intact skin. [97] ............................................................. 54
Figure 25. Franz diffusion cell set up used for in-vitro skin permeability study ... 63
Figure 26. Solubility of α-TOS in tested co-solvent systems. ................................... 68
Figure 27. (A) The cumulative amount of α-TOS permeated per unit skin surface
area plotted against time for each of the transdermal formulations and
(B) the percentage α-TOS released from application. Data shown is the
mean ± SD of triplicate values and is representative of three
independent experiments. Data were analysed using one-way ANOVA
with Tukey-Kramer multiple comparisons post test. Significance levels
were defined as P < 0.05 (*) and P < 0.01 (**). ........................................ 70
Figure 28. Influence of -TOS on AChE (A) and BChE (B) activity. Data shown is
the mean ± SD of triplicate values and is representative of three
independent experiments. Data were analysed using one-way ANOVA
with Tukey-Kramer multiple comparisons post test. Significance levels
were defined as P < 0.05 (*). ..................................................................... 80
Figure 29. Metabolism of -TOS by AChE and BChE (1U/mL). Metabolism of 100
(A), 50 (B) and 25 M (C) -TOS is shown. Data shown is the mean ±
SD of triplicate values and is representative of three independent
experiments. Data were analysed using one-way ANOVA with Tukey-
Kramer multiple comparisons post test. No significant differences were
observed. ..................................................................................................... 82
Figure 30. Comparison of rates of -TOS metabolism by AChE and BChE over
the time-course of the experiment. Data shown is the mean of triplicate
values. .......................................................................................................... 83
Figure 31. Comparison of rates of -TOS metabolism by AChE and BChE relative
to the concentration of -TOS at 10 min. The relationship between
rates of metabolism and drug concentration were shown to be linear for
AChE (r2 = 0.99; y = 0.054 – 0.31) and BChE (r
2 = 0.97; y = 0.082 +
0.68). Data shown is the mean of triplicate values. ................................. 84
Figure 32. Cellular AChE activity after exposure to -TOS for 24h. Data shown is
the mean ± SD of triplicate values and is representative of three
independent experiments. Data were analysed using one-way ANOVA
with Tukey-Kramer multiple comparisons post test. No significant
differences were observed. ........................................................................ 85
Figure 33. Plasma stability of -TOS (A) and rate of metabolism relative to
concentration (B). Data shown is the mean ± SD of triplicate values and
is representative of three independent experiments. Data were analysed
using one-way ANOVA with Tukey-Kramer multiple comparisons post
test. Significance levels were defined as P < 0.01 (**). ........................... 87
viii
LIST OF TABLES
Table 1. Pro-apoptotic effects of 50 M -TOS on normal cells and cells with a
malignant or transformed phenotype after 12 h incubation [61]. ........... 6 Table 2. Literature and experimental
1H-NMR chemical shift assignments for α-
TOS. Crucial differences between α -TOH and α-TOS are shown and
highlighted by shaded cells. [83] ............................................................... 29
ix
LIST OF ABBREVIATIONS
%CV Percentage coefficient of variance
Α-TOS alpha-tocopheryl succinate
Α-TOA alpha-tocopheryl acetate
Α-TOH alpha-tocopherol
ACh Acetylcholine
AChE Acetylcholinesterase
Ag/ AgCl Silver/ Silver chloride
BChE Butyrylcholinesterase
CL Clearance rate
CPE Chemical permeation enhancer
DHE Dihydroergotamine
DMEM Dulbecco’s modification of Eagles minimal essential medium
DMSO Dimethyl sulphoxide
DSC Differential scanning calorimetry
E Epidermis
ESI Electrospray ionisation
FTIR Fourier transformation infra red
H2O2 Hydrogen peroxide
HPLC High performance Liquid Chromatography
HIF Hypoxia inducible factors
ICH International conference on Harmonisation
IVRT In vitro release testing
x
LOD Limit of Detection
LOQ Limit of Quantification
LLE Liquid-Liquid extraction
MOM Mitochondrial outer membrane
MS Mass spectroscopy
NMR Nuclear magnetic resonance
OE Oestradiol
OP Organophosphate
PA Princess Alexandra
PBS Phosphate buffered saline
PCCA Professional Compounding Centres of America
PDA Photodiode array
PDMS Polydimethyl siloxane
PEG Polyethylene glycol
PLO Pluronic lecithin organogel
PSA Prostate specific antigen
PTFE Polytetrafluoroethylene
Rt Retention time
RH Relative Humidity
ROS Reactive oxygen species
SC Stratum corneum
SD Standard deviation
SDS Sodium dodecyl sulphate
SPE Solid phase extraction
TDD Transdermal drug delivery
xi
TGA Thermo gravimetric analysis
TGF-β Transforming growth factor β
TMS Tetramethylsilane
UV Ultraviolet
VE Vitamin E
VEGF Vascular endothelial growth factor
xii
ABSTRACT
Background
-Tocopheryl succinate, a redox-silent analogue of vitamin E, has been shown to
selectively induce apoptosis in a variety of cancers. However, -tocopheryl succinate is
rendered ineffective when administered orally due to hepatic metabolism. Transdermal
delivery has been identified as an alternative approach for delivering in-tact -
tocopheryl succinate systemically.
Aims and objectives
The aim of this study was to compound a liposomal formulation of -tocopheryl
succinate and evaluate the transdermal diffusion in an in-vitro Franz diffusion cell
assay. The feasibility of transdermal delivery was further evaluated by studying the
potential metabolism of -tocopheryl succinate by esterases, which are commonly
located in the skin.
Methods
Large quantities of -tocopheryl succinate was sourced and characterised by nuclear
magnetic resonance, mass spectrometry, infrared spectroscopy and differential scanning
calorimetry for the compounding of transdermal dosage forms. Analytical high
performance liquid chromatography and extraction methods were developed and
validated to isolate, identify and quantify -tocopheryl succinate.
xiii
A commercially available pre-formed liposomal product, anhydrous Lipoderm®, was
identified as the most suitable product for compounding of transdermal dosage forms of
-tocopheryl succinate. Lipoderm® formulations, containing 5% pentylene glycol and
-tocopheryl succinate concentrations of 10, 15 and 30% w/w were compared to a
DMSO-containing patent formulation. In-vitro Franz diffusion cells, fitted with excised
human cadaveric skin as the barrier, were utilised for the comparison of lag-time,
transdermal flux and permeability coefficients of formulated dosage forms. A
competitive enzyme assay, utilising Amplex Red®, was used to assess and compare
affinity of -tocopheryl succinate for acetylcholinesterase and butyrylcholinesterase.
Modification of the competitive enzyme assay was used to assess the extent of -
tocopheryl succinate metabolism by these esterases. Finally, the extent of -tocopheryl
succinate metabolism was assessed in plasma.
Results
Gradient and isocratic high performance liquid chromatography assays were developed
for the identification and quantification of -tocopheryl succinate. The gradient elution
method was linear between 5 and 50 g/mL (y = 11.2x + 23.0; r2 = 0.997). The accuracy
and precision data was within 10% CV for the gradient elution method. In addition, this
method was shown to be selective for resolving -tocopheryl succinate in samples
extracted from plasma. A liquid-liquid extraction method, utilising ethanol and n-
hexane, was shown to be reliable for preparation of samples from plasma and receptor
phase solution. This method repeatedly produced extraction efficiencies greater than
95%.
xiv
Compounded Lipoderm® formulations containing 5, 15 and 30% w/w -tocopheryl
succinate were shown to successfully deliver drug across excised human skin. No lag-
time to diffusion was observed for compounded formulations. In addition, Lipoderm®
effectively released drug. The permeability coefficient values for Lipoderm®
formulations containing 5, 15 and 30% w/w -tocopheryl succinate were 0.50, 0.88 and
1.3 g/cm2/h. In comparison, the DMSO-containing formulation displayed superior
ability to flux -tocopheryl succinate across skin; however, this is most likely due to its
capacity to effectively solubilise higher quantities of drug.
-Tocopheryl succinate was shown to compete for the binding site of
acetylcholinesterase and butyrylcholinesterase. In addition, metabolism by these
enzymes was confirmed. Although results suggest that -tocopheryl succinate has
slightly higher affinity for butyrylcholinesterase, no statistical differences in the rates of
-tocopheryl succinate metabolism by these two enzymes was observed. Similar
profiles of -tocopheryl succinate metabolism were observed in plasma.
Conclusions
Lipoderm® formulations containing 5% pentylene glycol have been shown to
effectively delivery -tocopheryl succinate across the outer barriers of the skin. The
major limitation to enhanced flux appears to be solubilisation of the drug in the dosage
form. Further formulatory research is needed to optimise these transdermal dosage
forms prior to in-vivo studies. Despite evidence suggesting that -tocopheryl succinate
may effectively cross the skin, likelihood of maintaining systemic plasma
concentrations is going to be heavily dependent on ester metabolism in the skin and
plasma.
1
CHAPTER 1. INTRODUCTION
1.1 Background
In 2000, cardiovascular diseases (CVDs), diabetes, cancer, obesity, and respiratory
diseases accounted for about 60% of the 56.5 million deaths each year and almost half
of the global burden of disease [1]. In the developed world, cancer is exceeded only by
CVD [1]. In 2000, there were 10 million new cases and over 7 million deaths (13% total
mortality) worldwide from cancer [2]. From projections, the incidence rates for many
cancers was proposed to increase substantially in the near future, with up to 15 million
cases expected in 2020 [3]. By 2020, the total number of new cancer cases is expected
to increase by 29% and 73% in developed and developing countries, respectively [3].
Alarmingly, global cancer mortality is expected to be 5-fold greater in the developing
world, compared to established market economies [3]. This difference in projected
mortality is proposed to be due to late diagnosis, overburdened healthcare systems and
inadequate treatments for advanced stages of cancer [3]. In the developing world,
approximately 80% of patients had incurable disease when first diagnosed [3]. There
exist a definite need for the development of more effective cancer treatments that can be
made easily accessible to cancer sufferers, which have minimal impact on the already
overstretched healthcare resources of both developed and developing countries.
With improved understanding of cancer biology, novel agents are being discovered that
selectively target molecular pathways thought to be critical for tumour survival, growth
and metastases [4]. Current observations in the literature suggest that compounds
2
structurally derived from vitamin E (VE), the redox-silent analogues, may be suitable
candidates for development as treatments for various cancers [5-23].
2.1 Nonmenclature, structural features and biological activities of VE
VE refers to a family of eight compounds containing a chroman ring with an
alcoholic hydroxyl group (collectively termed the chromanol ring) and 12-carbon
aliphatic side chain with two central methyl groups and two additional terminal methyl
groups.
VE represents two homologous series, namely: tocopherols with a saturated side
chain; and tocotrienols with an unsaturated side chain. Tocopherol has three asymmetric
carbons and as a result eight diastereomers. All naturally occurring tocopherols have
three asymmetric carbons (2 , 4 and 8 of the ring and phytol tail) in the R-
configuration [7]. The four tocopherols have an alpha, beta, gamma and delta form,
named on the basis of number and position of the methyl groups on the chromanol ring
as shown in Figure 1. [24]
HO
R1
R2
R3
FORM R1 R2 R3
α CH3 CH3 CH3
β CH3 H CH3
γ H CH3 CH3
Figure 1. Structure and substituent (R) groups for the various forms of VE.
3
All forms of VE remain in the non-ionic, hydrophobic state [25]. The structure of
these compounds can be broadly defined by three distinct domains, namely: a
hydrophobic tail (phytyl chain) that mediates docking to membrane lipids or
lipoproteins; a chroman head that affects cell signaling; and a functional arm attached to
the chroman head that is considered responsible for redox or apoptotic activity. [26-27]
VE maintains the equilibrium between anti- and pro-oxidant reactions in tissues
[28]. Among the various forms, -tocopherol is considered the most biologically active
form of VE [18]. In comparison, - and -tocopherol have only ~30% and ~20% of the
activity of α-TOH, respectively [18]. Tocopherols, such as -TOH, possess radical
scavenging capacity and are therefore termed redox-active [27, 29-30]. This action of
VE reduces the rate of free radical attack on polyunsaturated fatty acids in membrane
phospholipids and in other important biological sites [28]. In addition to this antioxidant
action, the various forms of VE have been proposed to produce numerous beneficial
effects, including cancer prevention, anti-artherogenic properties, neuroprotective
effects and modulation of immune function. [18, 24, 30-34]. The cancer prevention
potential of supplemented VE is, however, limited by the hepatic system preventing
both hyper- and hypovitaminosis. The liver maintains circulating and tissue levels
within a narrow margin [35]. Despite the mechanistic potential, in the absence of
specific co-morbidity little association has been identified between VE supplementation
and incidence of neoplasia in large clinical investigations [36-38]. In addition, α-TOH
showed little to no activity against tumour cells in culture (in-vitro) or in-vivo [18].
Recent evidence suggests that analogues of α-TOH may hold greater potential
for the treatment of cancer [39-43]. Commercially available esterified forms, such as -
4
tocopheryl acetate (α-TOA), -tocopheryl nicotinamide (α-TN) and -tocopheryl
succinate (α-TOS), display distinctly different actions to natural VE [18]. Of these
esterified forms, -TOS epitomises the redox-silent analogues of VE, shown to
selectively induce apoptosis in a variety of cancers [44]. -TOS has been shown to
have distinctly different physicochemical and pharmacological properties to the
conventional tocopheryl analogues, such as -TOH [25].
1.3 -Tocopheryl Succinate
α-TOS is a naturally occurring compound first isolated from a green barley
extract [18]. This compound exhibits physicochemical and pharmacological properties
that differ from the conventional non-ionic forms of VE [25]. This VE analogue is an
amphiphilic succinyl ester of α-TOH with a molecular weight of 530.8 amu (Figure 2).
α-TOS exists as an odourless white powder with a melting point between 76-77 C [27,
29, 45] The succinyl moiety of α-TOS can exist in a charged (anionic) form at
physiological pH (pKa ~ 5.6), which increases its aqueous solubility compared to α-
TOH and may be a crucial pharmacophore for action [25, 27].
Succinyl moeity
Figure 2. Structure of α-TOS [27].
5
Despite this property, α-TOS remains a highly lipophilic compound, exhibiting
extremely poor aqueous solubility and limited solubility in vegetable oils [45-46]. In
contrast, α-TOS is freely soluble in alcohols, such as ethanol. In relation to chemical
instability, α-TOS degrades rapidly to α-TOH in alkali environments. [45] The powder
of α-TOS is also extremely hygroscopic and without appropriate storage can gain water
mass rapidly [45].
1.4 Mechanism of action and demonstrated efficacy of -TOS
The addition of the succinyl moiety at the C-6 position of the chromanol ring of
-TOH renders -TOS devoid of antioxidant action [26-27, 29]. This compound has
been shown to produce numerous biological effects distinct form VE precursors
including, namely: stimulation of prolactin and growth hormone; inactivation of
transcriptional factor nuclear factor B (NF- B); suppression of tumour cell growth;
induction of apoptosis; promotion of differentiation; and promotion of cell cycle arrest
[5, 7, 10, 12, 15-17, 20-21, 23, 47-59].
Importantly the activity of -TOS appears to be largely specific to malignant
cells having little effect on non-transformed cells [60]. -TOS at a concentration of
50 M has been shown to selectively kill cells with a malignant or transformed
phenotype. Although this research primarily focussed on the induction of early events of
apoptosis during relatively short incubation periods, the differences in activity towards
normal and malignant or transformed cells was marked (Table 1). Normal cells types,
such as haematopoietic cells, fibroblasts, endothelial cells, cardiomyocytes, hepatocytes,
6
and smooth muscle cells, were shown to be resistant to the pro-apoptotic effects of -
TOS. [61]
Table 1. Pro-apoptotic effects of 50 M -TOS on normal cells and cells with a
malignant or transformed phenotype after 12 h incubation [61].
Cell Type % Apoptosis
Control -TOS
Normal cells
Mouse peritoneal macrophages 2.1 ± 1.1 5.5 ± 3.4
Human peripheral monocytic cells 3.5 ± 2.8 6.2 ± 4.1
Human monocyte-derived macrophages 2.9 ± 1.5 5.2 ± 1.5
Human skin fibroblasts 3.5 ± 0.9 6.1 ± 2.5
Human umbilical vein endothelial cells 3.1 ± 1.2 6.5 ± 4.2
Rat intestine epithelial cells 1.8 ± 1.6 3.5 ± 4.8
Rat neonatal cardiomyocytes 1.1 ± 0.9 4.2 ± 3.1
Rat neonatal hepatocytes 2.5 ± 1.9 4.3 ± 3.2
Rat smooth muscle cells 2.5 ± 1.8 4.9 ± 3.2
Haematopoietic cell lines
Jurkat 8.9 ± 3.1 45.2 ± 6.8
HL-60 8.1 ± 2.6 52.2 ± 7.3
Adenocarcinoma lung cell line
A549 1.1 ± 0.5 28.1 ± 4.2
Breast carcinoma cell line
MCF-7 12.3 ± 1.5 36.3 ± 7.3
Bronchocarcinoma cell line
BEAS-2B 1.9 ± 1.1 36.2 ± 5.9
Colon carcinoma cell lines
CaCo-2 6.5 ± 2.2 63.1 ± 9.8
HCT-116 7.5 ± 1.1 37.3 ± 4.8
The proposed reasons for the aforementioned selectivity include the esterified
structure and the pro-oxidant action of -TOS [61]. Normal cells such as hepatocytes,
7
colonocytes, fibroblasts and cardiomyocytes generally express higher levels of
esterases, which hydrolyse -TOS to the antioxidant VE. In addition, in-tact -TOS
induces initial generation of reactive oxygen species (ROS), which then leads to a
cascade of events culminating in apoptosis. [44] The efficacy of -TOS to suppress
cancer growth has been demonstrated in-vitro, in-vivo and through preliminary clinical
case reports [5, 47, 62-65]. However, to date there are no large randomised controlled
trials which conclusively demonstrate the therapeutic potential of -TOS.
In general, -TOS has been shown to generate less than 5% apoptosis in normal
cells [44]. However, the presence and prevalence of adverse drug reactions associated
with the administration of -TOS still needs to be assessed clinically.
Both intrinsic and extrinsic pathways appear to contribute to the pro-apoptotic
action of -TOS in many tumour cell types [44]. Research suggests that -TOS
primarily initiates apoptotic signalling downstream of the mitochondria by causing
rapid ROS accumulation (within 1 h) by disrupting the electron flow within the
mitochondrial complex II in the respiratory chain [44] (Figure 3). Death receptor,
mitogen-activated protein kinase (MAPK), protein kinase C (PKC) and NF- ,
associated with the extrinsic signalling pathways, have also been linked to the pro-
apoptotic action of -TOS [23, 33, 44, 60, 66]. A detailed account of these molecular
mechanisms of -TOS action is, however, outside the scope of this study.
8
Figure 3. Evidenced -TOS mediated signalling pathways [44].
Of particular relevance to this study is the reported effective pro-apoptotic in-
vitro concentration. The reported pro-apoptotic concentration of -TOS ranges between
37 and 50 M for various malignant cell types. Approximately 50 to 90% apoptosis has
been reported for malignant cell types treated with these concentrations continuously for
48 h. [44, 57] In addition, tumour growth suppression (~50%) has been demonstrated
in-vivo at -TOS concentrations of 15 M [67]. Based on these findings it appears that
-TOS must be maintained at concentrations in excess of 15 M to exert its pro-
apoptotic effect. However, this must be confirmed by suitably designed in-vivo studies
and clinical investigations.
Various clinical case reports provide some level of anecdotal evidence in
support of the therapeutic potential of -TOS. These case reports demonstrate the
9
efficacy of -TOS in treating relatively progressed cancers which include lymphoma,
sarcoma, prostate, nasopharyngeal, basal cell skin, and small cell lung cancers. In the
case of small cell lung cancers, -TOS was effectively used as an adjuvant with
chemotherapy and irradiation therapy. In all other cases, -TOS was the sole agent
trialled after chemotherapy or irradiation therapy. Clinical response was demonstrated
in all reported cases. [65] It is, however, imperative that the clinical efficacy of -TOS
is assessed through more rigid randomised-controlled trials (RCTs).
The selective, potent pro-apoptotic action of -TOS together with the
preliminary clinical evidence of its efficacy in treating various cancers provides suitable
evidence of the therapeutic potential of -TOS and highlights its suitability for
formulation development.
1.5 Pharmacokinetics of α-TOS
Literature confirms that -TOS is ineffective when administered orally [44]
(Figure 4). The whole, intact -TOS compound is required for pro-apoptotic activity
[68]. Oral administration of -TOS merely liberates -TOH. The ester bond which links
the succinyl moiety to tocopherol is susceptible to hydrolysis by intestinal esterases.
[26, 69]
Oral pharmacokinetic studies have shown that -TOS is absorbed by epithelial
cells of the intestinal villi where it is completely hydrolysed. Liberated -TOH is
secreted in chylomicrons into mesenteric lymph and then enters bloodstream where it
10
distributes to other lipoproteins and to peripheral tissues. [25, 70] This metabolism
liberates free, redox-active -TOH which, as previously mentioned, is devoid of
significant pro-apoptotic action [27]. The liberation of -TOH may, however, contribute
to additional benefit by providing potent anti-oxidant effect [68].
Figure 4. Metabolic fate of orally and parenterally administered -TOS.
To retain the efficacy of -TOS alternate routes of administration have been
used during in-vivo studies and in clinical case reports. These include intravenous,
intraperitoneal, rectal and transdermal routes of administration. [25, 62, 65] However,
only the pharmacokinetics of the single bolus intravenous administration of -TOS
(100mg/kg) in Sprague-Dawley rats has been reported in literature [25]. This study
demonstrated the extended half-life of -TOS in-vivo (t1/2 ~ 10 h). -TOS was shown to
possess a relatively low apparent volume of distribution (Vd ~0.56mL/kg) coupled with
a low rate of clearance (Cl ~ 0.065 L/h/kg). The low Vd of -TOS was attributed, in
part, to serum lipoprotein or lipid binding. Interestingly, -TOS accumulated in liver
Esterified forms are absorbed
by the epithelial cells of villi
Taken orally
Hydrolysis
-TOH Succinic acid
Antineoplastic
activity
Anti-atherogenic
activity
Administered
Parenterally
Shift in activity
α-TOS -TOS
remains in
circulation
for sustained
period
11
and lung tissue. Distribution to these tissues appeared to be both rapid and sustained.
Relatively high concentrations were observed at both 24 and 48 h after single
intravenous bolus dosing in both these tissues. The liver/serum and lung/serum -TOS
concentration ratios were between 20-28 and 10-15 at 24-48 h, respectively. In contrast,
brain, kidney and heart concentrations of -TOS were shown to be considerably lower.
The in-vivo conversion of -TOS to -TOH was also demonstrated in this study. -
TOH levels slowly peaked 7-8 h after -TOS administration. -TOH levels were shown
to increase in brain, kidney, heart, lung and liver tissues. [25]
There are no published studies on the pharmacokinetics of -TOS in humans.
Nonetheless, these results suggest that the major limitation for the successful delivery of
in-tact -TOS is the pre-systemic metabolism by hepatic esterases. [25, 70] Further
evidence is provided by Neuzil and Massa [71]. Mice repeatedly injected with -TOS
were shown to accumulate the drug systemically. However, -TOS concentrations were
below limits of detection in blood drawn from the hepatic vein. In contrast, -TOH
concentrations were shown to increase to a limit in blood drawn from the hepatic vein at
similar time points. [71] Once -TOS reaches systemic circulation, it appears to
possess pharmacokinetic properties suitable for therapeutic treatment. Irrespective of
route of administration, the observed tissue distribution of -TOS suggests that this
agent may be more suitable for the treatment of cancers of the lung and liver. Of
particular interest would be the clinical efficacy of -TOS in treating either malignant
mesothelioma or small cell lung cancers.
12
Given these findings, it seems feasible to administer -TOS intravenously for
effective, rapid and accurate pharmacological action. [25] More importantly, these
findings provide evidence that other administration routes which similarly by-pass first-
pass metabolism may also maintain the antineoplastic properties of -TOS. Although
intravenous administration has been shown to deliver intact -TOS, this route is not
ideal for long-term self-treatment and therefore places added burden to health care
infrastructure. Transdermal delivery of -TOS is therefore proposed to be a suitable
treatment alternative. A single report provides evidence of successful transdermal
delivery of -TOS, however, scientific evidence supporting these claims is lacking [62,
65].
1.6 Transdermal drug delivery of -TOS
The skin represents the largest and most easily accessible organ of the body [72-
73]. The skin of an average adult body covers a surface area of approximately 2 m2 and
receives about one-third of the circulating blood [74]. Transdermal permeation or
percutaneous absorption can be defined as the passage of a substance, such as a drug,
from the outside of the skin through its various layers into the bloodstream [75].
Successful transdermal drug delivery (TDD) has several advantages over conventional
routes, such as oral or parenteral administrations. This route avoids factors that
influence drug absorption through the gastrointestinal tract, including: pH, food-intake
and intestinal motility. Successful TDD, like parenteral routes of administration, by-
passes first-pass metabolism and enhances the bioavailability of susceptible drugs [72].
Importantly, this route may eliminate pulsed drug delivery to the systemic circulation,
instead producing sustained, constant and controlled levels of drug in plasma. This route
13
also allows effective use of drugs with short-biological half-lives, increasing the
likelihood for patient compliance. [75] Importantly TDD offers a significant reduction
of costs because it can be self-administered.
Developed transdermal formulations must overcome a number of crucial
limiting factors to be feasible. Ideally, the drug must possess specific physicochemical
properties for it to be delivered successfully across the skin. Dosage forms must
facilitate transport of drug through the excellent barrier properties of the skin and must
ensure consistent drug delivery throughout the intended treatment period. It is important
that developed formulations reduce skin irritation rates and minimise alterations of
metabolism by microflora on the skin or enzymes in the skin. [72, 75-76]
The use of the skin for topical and systemic drug delivery has been well
documented and may be feasible for successful delivery of intact -TOS systemically,
however very little scientific evidence is available to support this concept. Systemic
delivery of drugs across the skin is determined by the anatomical properties of the skin,
drug features and formulation features [73].
1.7 Barriers to TDD
There are numerous processes that can interfere with the movement of drugs
across the skin and into systemic circulation. Drug delivery across the skin is primarily
limited by the extensive resistance offered by the non-viable layer of the epidermis, the
stratum corneum [73, 77-78]. Even prior to this, sweat, sebum and cellular debris on the
surface of the skin may also interfere with drug permeation of the stratum corneum. As
14
a drug partitions into and diffuses through the stratum corneum, it may interact with
many potential binding sites. Interaction with these binding sites may slow entry into
systemic circulation, by creating a drug reservoir that holds and delivers the agent over
an extended period of time. [75, 77]
In comparison to the stratum corneum, the viable epidermis offers minimal
resistance to drug passage across the skin. [77] There are, however, other potential
retardants to drug movement in these layers of skin. If a drug reaches the interface
between the stratum corneum and the viable epidermis, it must partition into water-rich
tissue. Although drugs with highly lipophilic properties cross the stratum corneum
easier, they may have greater difficulty leaving the stratum corneum to enter the dermis.
[75] It is apparent that a balance in aqueous and lipid solubility is important for
diffusion across the skin.
The skin is proposed to have enzymes with catalytic activity nearing 80-90% as
efficient as those in the liver. Possible enzymatic reactions in the skin may include:
hydrolytic, oxidative, reductive and conjugative processes. This enzyme activity may
significantly affect the pharmacokinetics of transdermally delivered drugs. Once a drug
moves into the dermis, additional receptors, metabolic processes and depot sites may
hinder its movement into systemic circulation. Drugs may also partition into
subcutaneous fat, underlying muscle tissue, and lymphatic drainage that delay drug
entry into systemic circulation. [75]
15
In order to produce systemic effects, drugs together with the excipients of the
formulation must overcome all the aforementioned barriers or obstacles of the skin.
1.8 Drug-related factors influencing TDD
There are two possible routes of permeation for small molecules through the
skin, namely the intercellular and intracellular pathways. The intercellular space
contains a mixture of lipids that produce both hydrophilic and hydrophobic domains. It
is the organisation of these lipids that dictate the required physicochemical properties of
a molecule to permeate the skin. [72] Generally, suitable candidates for transdermal
permeation are small molecules (< 1000 amu) with a balanced water and lipid solubility
(log P between 1 and 4). These characteristics are often indicated by the possession of a
low melting point, typically < 200 C. [72, 74, 79] Ideally, candidate drugs should
possess high potency and potential to exist in a non-ionic form. These required
characteristics match the physicochemical properties of α-TOS [45]. Importantly the
drug must be devoid of irritant or sensitizing action to the skin and should not stimulate
immunological reactions [74].
1.9 Transdermal delivery of α-TOS
Chan and Chow [65] provided the first report of successful transdermal delivery
of α-TOS. Various transdermal formulations containing dimethyl sulfoxide (DMSO),
almond oil, stearyl alcohol, petroleum jelly, mineral oil and distilled water were shown
to deliver α-TOS systemically. These transdermal formulations were shown to
16
successfully produce plasma α-TOS concentrations between 20 and 30 M in humans.
However, the relatively high content of DMSO (approximately 12 to 22%) in these
transdermal formulations limits its clinical usefulness. [65] DMSO has been shown to
cause marked swelling, distortion, and intercellular delamination of the stratum
corneum [80]. Although this has been shown to enhance skin penetration, the marked
inflammation caused by continued application of DMSO is restrictive. DMSO can
induce either a non-immunological immediate contact urticaria or an irritant reaction
depending on concentration and mode of application [81]. The composition of the
dermal cellular infiltrate was shown to be dependent on the concentration and number
of DMSO applications. The immediate reaction infiltrate, 3 h after application of 100%
DMSO, consisted of 50% granulocytes (mostly basophils). On the other hand, 12%
DMSO applied 3 x daily for 3 days (cumulative insult) caused a cellular reaction in
which 80% of the infiltrate consisted of mononuclear cells. [81]
Epicutaneous application of α-TOS was also shown to result in accumulation of
the drug in the skin [82]. This accumulation of α-TOS in skin may provide the reservoir
required for sustained (zero order) diffusion into systemic circulation. The study by
Gensler et al. [82] did, however, raise some concerns regarding the safety of topically
applied ester analogues of VE. The study identified that α-tocopherol acetate (α-TOA)
or α-TOS may enhance photocarcinogenesis induced by ultraviolet (UV)-B irradiation.
[82] These findings need to be investigation further.
Although the formulations proposed by Chan and Chow [65] had limited clinical
application, they provided the necessary proof of concept for this study.
17
CHAPTER 2. HYPOTHESIS AND AIMS
-TOS has been shown to be a potent inducer of apoptosis in a number of
malignant cells, while being largely non-toxic to normal cells. Given that oral
administration of -TOS leads to sub-therapeutic levels due its conversion to the non-
neoplastic agent -TOH, it is necessary to develop formulations to enhance its delivery.
Anecdotal evidence suggested that transdermal delivery of α-TOS may be a suitable
alternative to parenteral drug delivery. This research therefore investigated the
feasibility of delivering intact -TOS systemically using the transdermal route of
administration. Outcomes from this project provide guidance for optimising TDD for
future use in clinical studies.
The hypothesis of this study was that α-TOS could be successfully delivered in-
tact across the SC using various commercially available transdermal bases. An
optimized systemic dosage form would ensure sustained delivery of α-TOS across the
SC into the skin thereby increasing the likelihood of maintaining therapeutic plasma
concentrations. In addition, this study evaluated the impact that skin esterases may have
on the successful transdermal delivery of α-TOS.
The aims specific to this investigation were as follows:
Develop and validate analytical methods for the detection and quantification of α-
TOS. This aim was achieved by initially characterising sourced α-TOS raw
material utilising nuclear magnetic resonance (NMR), mass spectrometry (MS),
fourier transformation infra-red spectroscopy (FTIR), differential scanning
18
calorimetry (DSC) and thermogravimetric analysis (TGA). Characterised α-TOS
was further utilised in the development and validation of quantitative methods
which included UV spectroscopy, isocratic and step-up gradient high-performance
liquid chromatography (HPLC);
Validate and optimise methods for the extraction of α-TOS from various sample
matrices. This aim was achieved by employing liquid–liquid extraction (LLE) and
solid phase extraction (SPE) techniques;
Formulate various transdermal dosage forms using commercially available
products. This was achieved by initially assessing the physicochemical properties
and biopharmaceutical characteristics of α-TOS (log P and solubility). α-TOS was
subsequently formulated into various transdermal dosage forms;
Investigate the transdermal flux of formulated transdermal dosage forms
containing α-TOS through human cadaveric skin using in vitro Franz diffusion
cells;
Assess the potential metabolism of α-TOS by esterases. This aim was achieved by
assessing α-TOS substrate specificity for acetylcholinesterase (AChE) and
butyrylcholinesterase (BChE) using a competitive fluorometric Amplex Red
enzyme-based assay, quantifying the rate of α-TOS metabolism by AChE and
BChE utilising the validated HPLC assay, and evaluating the effect of α-TOS on
epithelial cell expression of esterases using a modified Amplex Red enzyme and
modified Ellman’s assay in an in-vitro cell-based assay.
19
CHAPTER 3. DEVELOPMENT AND VALIDATION OF
ANALYTICAL METHODS FOR THE DETECTION AND
QUANTIFICATION OF α-TOS
3.1 Background
Large quantities of α-TOS were required to formulate and evaluate transdermal
dosage forms. The large quantities required (>100g) restricted purchase of α-TOS from
a reputable scientific supplier. Instead food-grade α-TOS was sourced. The quality of
sourced α-TOS was therefore characterised prior to use in this study. The chemical
identity was confirmed by NMR, MS, FTIR and DSC. In addition, given the reported
hygroscopic properties of α-TOS powder, DSC and TGA were used to determine water
content.
Analytical methods for the detection and quantification of α-TOS were needed for the
completion of this study. Once characterised, α-TOS was used to develop and validate
analytical methods which were needed for the in-vitro experiments assessing
transdermal flux and potential metabolism by esterases.
3.2 Materials and Methods
A large quantity of α-TOS raw material, purchased from a non-scientific
commercial supplier, was kindly donated by Prof. Jiri Neuzil (School of Medical
20
Sciences, Griffith University, Gold Coast, Australia). d,l-α-TOH (labelled as approx.
95%) and d-α-tocopherol acetate (α-TOA) were obtained from Sigma-Aldrich (St.
Louis, MO). All chromatography grade organic solvents were sourced from Merck
(Darmstadt, Germany). The water used throughout this investigation was deionised and
filtered by a MilliQ-Academic system.
3.2.1 Chemical confirmation of sourced α-TOS
Chemical characterisation and structural confirmation of donated α-TOS, not
sourced from a scientific commercial supplier, was achieved by 1H-NMR, MS and
FTIR methods. In addition, melting point determination was performed by DSC and
water content of donated material evaluated by DSC and thermogravimetric analysis
(TGA).
1H-NMR spectra of donated α-TOS were acquired on a 400MHz Bruker Avance
spectrometer equipped with a cryoprobe at 298K, in DMSO-d6 as the solvent for
resolution and tetramethylsilane (TMS) as the internal standard. 1H-NMR chemical
shifts obtained were assigned (Figure 5) and cross-referenced to the findings of Baker
and Myers [83]. All resonances were reported in ppm (σ) downfield of TMS.
Figure 5. Numbering used for spectral assignments of α-TOS.
21
The MS method used was Electrospray Ionisation (ESI) performed on an
Applied Biosystems API 3200TM
MS/MS. Chromatography grade methanol was used
as the solvent for delivery and samples were directly introduced to the system by slow
injection. The parent peak [M+Na]+ was used to confirm the presence of -TOS by
identifying the molecular weight of the compound.
Infrared (IR) spectra were collected on a Bruker FTIR Tensor 27 Spectrometer
(GmbH, Germany) fitted with a general purpose PIKE (Madison, WI) attenuated total
reflection (ATR) cell. Donated material was placed in the ATR cell and spectra
recorded between 400 and 4000cm-1
by averaging 64 scans. α-TOS was confirmed by
the assignment of functional groups to absorption bands in the region of 1450 to
4000cm-1
and comparison of these and fingerprint region (600 to 4000cm-1
) to the
published IR spectra of Rosenkrantz [84]. Bands characteristic of the tocopherol
structure were also assigned. (3333, 1587, 1250, 1162 and 917cm-1
) [84].
DSC and TGA measurements were performed simultaneously using a Linseis
STA Platinum Series analyser under nitrogen purge (Linseis Messgeraete GmbH,
Germany). Approximately 5-10mg samples of donated α-TOS were placed into
aluminium crucibles fitted with perforated lids. Samples weights were accurately
determined by the analyser prior to each run. Samples were scanned over a temperature
range of 30-400 C at a heating rate of 5 C/min and the phase transition behaviour
recorded. The endotherm corresponding to the melting point was identified and
compared to reference literature for α-TOS (76-77 C) [45]. In addition, the mass of the
sample crucible was continuously recorded as a function of temperature. Determination
of water content was performed using the methods described by Khankari et al [85].
22
Tests were performed in triplicate on each day of analysis. Samples were re-
analysed at regular intervals throughout this investigation as part of the validation
process.
3.2.2 Development of methods for the quantification of α-TOS
A number of analytical HPLC methods exist for the quantification of VE
analogues [86-90]. These methods used both isocratic method and gradient methods for
the efficient separation and quantification of α-TOS. A comprehensive review and
preliminary trial of published methods highlighted some limitations and disadvantages,
including: excessively long run times, co-eluting peaks (especially when performed on
samples extracted from complex and biological matrices), majority of methods were not
based on C18 reverse-phase chromatography and relied on the use of columns which are
not readily available, and many methods were unable to resolve -TOS from other VE
analogues and other fat-soluble vitamins when trialled.
3.2.3 Development of a UV spectral profile of α-TOS
The UV spectral profile of α-TOS was obtained using a Shimadzu 1601 single
beam UV-visible spectrophotometer (Tokyo, Japan). Briefly, the wavelength-
absorbance profile of an appropriately diluted α-TOS standard solution was recorded in
a 1cm quartz cells between the wavelengths of 200–400nm. After baseline correction
with appropriate blanking solution, absorbances were recorded at 1nm intervals and the
wavelength-absorbance profile was plotted to determine the wavelengths of maximal
absorption (λmax) for α-TOS.
23
3.2.4 Development of HPLC methods for the detection and quantification of α-TOS
The HPLC system used throughout this investigation consisted of a Varian
Prostar 240 Solvent Delivery Module, Varian Prostar 430 Autosampler and Varian
Prostar 335 Photodiode Array Detector (PDA). The system was also equipped with a
solvent degasser and column thermostat. This investigation was adapted and validated
based on extensive reports of previously published methods [73-74]. Methods were
optimised by systematic modification of mobile phase composition, flow-rate,
temperature and column size. The optimised and validated isocratic and gradient
methods were then compared. The most appropriate method was then selected for
analysing samples obtained from experiments requiring the sensitive identification and
quantification of α-TOS from either simple or complex matrices.
Isocratic method for analysis
The isocratic method for analysis of α-TOS was adapted from Good et al, using
a Varian Pursuit reverse-phase C-18 column (3μm i.d., 3.3cm × 4.6mm) [86].
Chromatographic conditions include: Varian C-18 pursuit column (3 microns, 3cm x
4.6mm); column temperature of 25 ± 1°C; pump flow rate was 1.0mL/min; and a
detection wavelength of 205nm and 254nm. The mobile phase consisted of acetonitrile
and water (90:10 v/v), filtered and degassed under reduced pressure, prior to use.
Standards of the concentration 5-50µg/mL were used during method development.
Gradient method of analysis
A validated step up gradient HPLC method for the analysis of α-TOS from a
variety of sources (e.g. dosage forms or biological samples) was adapted from Siluk et
al [87].
24
The separation was achieved on a Phenomenex Gemini II reverse phase C-18
column (5μm i.d., 250mm × 4.6mm) connected to a C-18 Phenomenex cartridge guard
column. Stationary phase was thermostated at 30 ± 1°C throughout the run. VE
analogues were detected at dual wavelengths of 205 and 225nm. The spectral profiles
obtained by the photodiode array (PDA) detector were used to assess peak purity. By
matching the spectral profile obtained across the eluting peak to known reference
standards peak purity was effectively assessed. Chromatographic conditions include:
Varian C-18 pursuit column (5 microns, 250 x 4.6mm); column temperature of; pump
flow rate varying from 1.0mL/min to 1.2mL/min at different time intervals. The mobile
phase consisted of methanol acidified with 0.1% formic acid (Solvent A) and
acetonitrile (Solvent B) degassed under reduced pressure prior to use. In order to
achieve adequate resolution of α-TOS from other VE analogues and fat soluble
vitamins, the following optimized protocol was used during the run: 0-4min 30%A at a
flow rate of 1mL/min, 4 min step to 100%A at a flow rate of 1.2mL/min, 4-20min linear
gradient to 69%A, and 2-25 min step to 30%A at flow rate of 1mL/min. Standards of
the concentration range 5-50µg/mL were used during method development.
3.2.5 Validation of analytical methods
The objective of validating the developed analytical procedures was to
demonstrate their suitability for quantifying -TOS in various matrices. The validation
and performance of the developed UV, isocratic and gradient HPLC methods were
based on the International Conference on Harmonisation (ICH) guidelines for the
validation of analytical procedures [91]. The ICH parameters include measures of
specificity, linearity, range, accuracy, precision, limit of detection (LOD), limit of
25
quantitation (LOQ) and robustness. The definition and process for determining each of
these parameters that were followed in this investigation is defined below [91]:
Linearity
The linearity of an analytical procedure is its ability (within a given range) to
obtain test results which are directly proportional to the concentration (amount) of
analyte in the sample. Standards tested will be plotted for concentration versus
absorbance to calculate its correlation coefficient to determine the linearity through the
various concentration ranges tested during the method development. Linearity was
determined at five concentration levels with a minimum specified range from 80-120%
of the target concentration (i.e., 25 to 50 M) was selected.
Accuracy
The accuracy of an analytical procedure expresses the closeness of agreement
between the value which is accepted either as a conventional true value or an accepted
reference value and the value found (mean value within 15% not more than 20% at
lowest limit of quantification). For the assay of drug product, accuracy was evaluated by
analysing synthetic mixtures spiked with known quantities of components. Data was
collected from a minimum of nine determinations over a minimum of three
concentration levels covering the specified range (for example, three concentrations,
and three replicates each).
26
Precision
The precision of an analytical procedure expresses the closeness of agreement
between a series of measurements obtained from multiple sampling of the same
homogeneous sample under the prescribed conditions. Precision may be considered at
three levels: repeatability, intermediate precision and reproducibility. Precision should
be investigated using homogeneous, authentic samples. The precision of an analytical
procedure is usually expressed as the variance, standard deviation or coefficient of
variation of a series of measurements (coefficient of variance should not exceed 15%
and not more than 20% at the lowest limit of quantification). The precision was
determined from a minimum of nine determinations covering the specified range of the
procedure (three concentration levels, three repetitions each).
LOD
The LOD of an individual analytical procedure is the lowest amount of analyte
in a sample which can be detected but not necessarily quantified as an exact value. This
is based on signal-to-noise ratio of the developed method for analysis. A signal-to-noise
ratio of three-to-one was used for estimating the detection limit.
LOQ
The LOQ of an individual analytical procedure is the lowest amount of analyte
in a sample which can be quantitatively determined with suitable precision
(20%) and accuracy (80-120%). A signal-to-noise ratio of ten-to-one was used for the
estimation of LOQ.
27
Specificity
The method has to be specific, which means the ability to assess unequivocally
the analyte in the presence of components which may be expected to be present. The
peak purity was determined using the spectral profile obtained from the PDA to show
that no interfering peaks co-eluted with the compounds of interest.
3.2.6 Data analysis
Data was processed, graphed and analysed using GraphPad PRISM version 3
and GraphPad Instat version 3.10. Replicate data was expressed as the mean S.D of
replicate experiments. Coefficient of variance was calculated by dividing the standard
deviation obtained for replicates by the mean and expressing it as a percentage. Linear
equations and correlation coefficients were calculated using the GraphPad PRISM
software. Statistical comparisons were made by one way ANOVA and two tailed
student-t test where applicable.
3.3 Results and Discussion
3.3.1 Results of methods for the quantification of α-TOS
An example 1H-NMR spectrum is presented in Figure 6. The assignment of
protons, with exception of the aliphatics, is presented in Table 2.
The proton NMR data confirms the presence of the ester (CH2-C=O at 2.873
and 2.897ppm) providing evidence for the presence of the succinic acid derivative of -
28
TOH. In addition, absence of a chemical shift at 4.18ppm excludes the existence of the
phenolic hydroxyl group at the C-6 position, which would be expected if free -TOH
were present. [83]
Figure 6. Representative 1H-NMR spectrum obtained for donated -TOS.
29
Table 2. Literature and experimental 1H-NMR chemical shift assignments for α-TOS.
Crucial differences between α -TOH and α-TOS are shown and highlighted by
shaded cells. [83]
Superscripts a, b and c denote interchangeable chemical shift assignments.
An example spectra obtained from ESI-MS in the positive ion mode is presented
in Figure 7. The presence of m/z at 554.3amu corresponds to the monoisotopic mass of
Position α-TOH
Literature
α-TOS
Literature
α-TOS
Experimental
C2-H - - -
C3-H ~1.8 ~1.8 1.795-1.812
C4-H 2.60 2.58 2.591-2.613
C5-H - - -
C6-H - - -
C7-H - - -
C2-CH3 1.22 1.23 1.304
C5-CH3 2.11 1.96c 1.953
C7-CH3 2.15 2.01c 1.987
C8-CH3 2.11 2.08c 2.064
C4’-CH3 0.84a 0.84
a 0.861
C8’-CH3 0.83a 0.83
a 0.839
C12’-CH3 0.88b 0.88
b 0.883
C13’-H 0.85b 0.85
b 0.861
C6-OH 4.18 - -
CH2-COO - 2.8, 2.9 2.873, 2.897
30
the sodium ion adduct of -TOS [M+Na]+. Taking this into account, the desired mass of
-TOS was confirmed (Fw = 530.8) by the MS analysis.
Figure 7. Representative ESI MS spectrum run in positive-ion obtained for donated -
TOS. The parent peak of 554.3amu corresponds to [M+Na]+ for -TOS.
The absorption bands obtained from the FTIR analysis provides further
confirmation of -TOS structure (Figure 8). The absorption bands near 2924, 1405,
1263, 1156 and 934.63cm-1
are characteristic of the tocopherol structure [84]. In
addition, the presence of a strong absorption band at 1223cm-1
confirms the presence of
the phenolic C-O linkage which is present in the structure of -TOS (not -TOH) [84].
31
Similarities between the fingerprint region in spectra obtained in this study to those of
Rosenkrantz [84] provides further structural confirmation.
Figure 8. Representative FTIR spectrum obtained for donated -TOS.
32
Results obtained from the DSC curve in Figure 9 demonstrate a sharp
endothermic transition (77.1 C) consistent with the expected melting point of -TOS
(76-77 C). No other thermal transitions were observed. Both these observations suggest
a high degree of purity.
Figure 9. Representative DSC curve for the donated -TOS.
The TGA thermogram is presented in Figure 10. Weight loss of sample between
25 and 100 C was calculated as a percentage relative to sample weight. The sample
weight lost during heating was consistently shown to be less than 10% (approximately
9.8%). This weight loss corresponds to associated water content of the sample.
33
Figure 10. Representative TGA thermogram of donated -TOS.
The results from 1H-NMR, MS, FTIR and DSC/TGA provide excellent evidence
supporting the structure and purity of the donated -TOS. This evidence suggests that
the donated material can be used as a reliable reference material for the development of
analytical methods and is suitable for use in development and evaluation of transdermal
dosage forms.
3.3.2 Results of methods for the quantification of α-TOS
The absorbance-wavelength profile obtained for α-TOS by UV spectroscopy
identified three possible wavelengths suitable for use in the development of methods for
the quantification of α-TOS (205, 225 and 284nm) (Figure 11). Furthermore, the
spectral profile obtained highlights the unique pattern of this chemical which can be
34
cross-referenced to results obtained from the PDA during HPLC analysis and used to
help confirm identity and purity of eluting α-TOS peaks.
200 225 250 275 300 3250
1
2
3
Wavelength (nm)
Ab
so
rban
ce
Figure 11. Absorbance-wavelength profile for α-TOS. Arrows indicated the
wavelengths of maximum absorbance potentially suitable for quantitative
calibration.
Results for isocratic HPLC methods for quantification of α-TOS
An example chromatogram for the optimised isocratic method is presented in
Figure 12. This method provided adequate separation of α-TOS (Rt 4.10 0.1min), α-
TOH (not shown in the figure, Rt 6.08 0.1min) and α-TOA (Rt 7.130 0.1min). The
optimised method provided efficient resolution of α-TOS from other VE analogues.
35
Figure 12. Example chromatogram from the optimised isocratic HPLC method at
205nm.
Results for gradient HPLC methods for quantification of α-TOS
An example chromatogram representing the optimised step up gradient HPLC
method is presented in Figure 13. Under these optimised conditions, this method
provided adequate separation of α-TOS (Rt 12.7 0.2min), α-TOH (not shown in figure,
Rt 15.1 0.1min) and α-TOA (Rt16.861 0.1min).
Figure 13. Example chromatogram from the optimised step-up gradient HPLC
method at 205 nm.
36
Both isocratic and step up gradient HPLC methods, when optimised, produced
sharp symmetrical peaks with good baseline resolution and minimal tailing for all VE
analogues. A gradient of decreasing solvent strength (decreasing methanol, increasing
acetonitrile concentrations) was required to achieve adequate resolution of -TOS from
-TOH. The achieved adequate resolution of α-TOA, coupled with its physicochemical
similarities to α-TOS suggested that this agent is suitable for use as an internal standard
during experimentation.
3.3.3 Results for the validation of quantification of α-TOS
To increase sensitivity of the assay all validations were performed at 205nm.
Linearity
The standard curves, across the concentration range of 5-50µg/mL, for both
isocratic and gradient HPLC methods are shown in Figure 14. Both methods delivered
high correlation coefficients across the concentration range tested. Correlation
coefficients for isocratic and gradient methods were 0.998 and 0.997, respectively. The
linear equation for the isocratic and the gradient HPLC methods, across the
concentration range tested were 24.77x + 13.80 and 11.21x + 22.95, respectively.
Equations represent the averages of three replicate experiments (n= 3).
37
0 25 50 75 100 1250
1000
2000
3000
[ -TOS] ( g/mL)
Are
a (
mA
U*s
ec)
0 25 50 75 100 1250
500
1000
1500
[ -TOS] ( g/mL)
Are
a (
mA
U*s
ec)
A
B
Figure 14. Standard curves for the isocratic HPLC (A) and gradient HPLC (B)
methods.
Standard curves were for standard solutions of 5, 10, 25 and 50µg/mL α-TOS at 205nm.
Results represent the mean ± SD of replicate experiments (n = 3).
38
Accuracy
The accuracy data, determined across three concentrations in triplicates, for both
the isocratic and gradient HPLC methods are presented in Figure 15.
7.5 g/mL 15 g/mL 30 g/mL0
10
20
30
7.5 g/mL
15 g/mL
30 g/mL
Target Concentration ofVarification samples
[-T
OS
] (
g/m
L)
7.5 g/mL 15 g/mL 30 g/mL0
10
20
30
40
7.5 g/mL
15 g/mL
30 g/mL
Target Concentration ofVarification samples
[-T
OS
] (
g/m
L)
A
B
Figure 15. Accuracy data for the isocratic (A) and gradient (B) HPLC methods at
205nm. Results represent the mean ± SD of replicate experiments (n = 3).
Both HPLC methods were shown to have high accuracy in the determination of
verification samples within the concentration range of 7.5-30 µg/mL. Both methods
39
consistently determined the concentration of known verification samples within 5% of
target concentration.
Precision
The precision of both methods was assessed by looking at the repeatability (on
the same day), intermediate precision (on different days) and reproducibility (in an
adjacent laboratory)
The results for both methods are presented in Figure 16, 17 and 18. The isocratic
method proved to be highly sensitive with reliable intraday and interday repeatability (<
10%CV). The within- and between-run precision (%CV) of the gradient HPLC method
was <10% over the concentration range tested. The variability between the intra-day
and inter-day runs assessed during the triplicate assays for α-TOS was almost
negligible. This data indicates that both HPLC methods are extremely reproducible.
Both methods produced coefficients of variance less than 15%.
40
Area Height Rt
0.0
2.5
5.0
7.5
10.0Area
Height
Rt
Parameter
%C
V
Area Height Rt
0.0
2.5
5.0
7.5
10.0Area
Height
Rt
Parameter
%C
V
A
B
Figure 16. Repeatability of the developed isocratic (A) and gradient (B) HPLC
method at 205nm. Results represent the mean ± SD of replicate experiments (n = 3).
41
Area Height Rt
0.0
2.5
5.0
7.5
10.0Area
Height
Rt
Parameter
%C
V
Area Height Rt
0.0
2.5
5.0
7.5
10.0Area
Height
Rt
Parameter
%C
V
A
B
Figure 17. Intermediate precision of the isocratic (A) and gradient (B) HPLC
method at 205nm. Results represent the mean ± SD of replicate experiments (n = 3).
42
Area Height Rt
0.0
2.5
5.0
7.5
10.0Area
Height
Rt
Parameter
%C
V
Area Height Rt
0.0
2.5
5.0
7.5
10.0Area
Height
Rt
Parameter
%C
V
A
B
Figure 18. Reproducibility of the isocratic (A) and gradient (B) HPLC method at
205nm. Results represent the mean ± SD of replicate experiments (n = 3).
No statistical differences were observed for measures of precision of area
between either HPLC assays (P> 0.05, two tailed t-test). The isocratic method was more
precise when height was used as the unit of measure when compared to the gradient
43
elution method (P< 0.05, two tailed t-test). Shifts in retention however, were greater in
the isocratic method than the gradient HPLC method.
LOD & LOQ
The LOD and LOQ of both methods were determined by the sequential dilution
of standard solutions. The optimized isocratic and gradient HPLC methods produced a
LOD and LOQ of 1µg/mL and ≥ 2.5µg/mL, respectively. Importantly, the %CV at the
LOQ was less than 20% for both the isocratic and gradient HPLC methods.
Specificity
The specificity of both HPLC methods was assessed in the quantification of -
TOS extracted from spiked plasma samples (Refer to Chapter 4).
Both the developed methods have shown to be accurate and precise. In
comparison to the validated isocratic method published by Siluk et al [87] and Good et
al [86], both these methods performed with similar precision and accuracy. The
publication by Good et al. was the only study located that specifically defined the
validation of methods for the direct detection of -TOS. The published method of Good
et al [86] had run times in excess of 30 min for the resolution of all fat soluble vitamins,
including -TOS, -TOH and -TOA (Rt= 9.8, 18.66 and 28.03 min, respectively).
[86] Both the isocratic and gradient methods developed in this investigation achieved
comparable resolution of these analogues in shorter run times. Both methods produced
sharp peaks with good resolution and little tailing effect. These methods have proved to
be useful analytical tools for quantification of α-TOS. However, the developed gradient
elution method has the distinctive advantage of being able to be performed with
44
standard and readily available HPLC columns (i.e., C18 reverse phase HPLC column,
250 x 4.6mm, 5 m particle size).
45
CHAPTER 4. SAMPLE PREPARATION FOR
ANALYTICAL PROCEDURES
4.1 Background
To achieve the stated aims of this investigation, it was essential to
develop reliable methods for the extraction of α-TOS from two matrices. In addition,
extracted samples were used to assess the specificity of developed HPLC methods.
Methods for the extraction of α-TOS from various matrices was adapted and optimised
from previously published protocols [86-88, 90, 92-93]. Extraction efficiencies were
performed in plasma and phosphate buffered saline (PBS) containing 30% ethanol,
which is a common receptor solution for in-vitro transdermal experiments of highly
lipophilic drugs [94]. Porcine plasma, which is a complex matrix containing other fat
soluble vitamins, was used to test the selectivity of the developed analytical methods.
4.2 Materials and Methods
All chemicals used in the extraction procedure were analytical grade (AR) and
sourced from Sigma Aldrich (St. Louis, MO). Porcine plasma was purchased from
Sigma Aldrich (St. Louis, MO). The water used throughout this investigation was
deionised and filtered by a Milli Q-Academic system.
46
4.2.1 Liquid-Liquid extraction (LLE)
LLE was performed for spiked physiologically relevant concentrations of α-
TOS. 1000µL of plasma as verification sample was prepared by spiking with 7.5µg/mL
of α-TOS from a stock solution containing 10mg α-TOS in 10mL of methanol. 250µL
of internal standard (α-TOA10µg/mL of methanol) was added to the prepared plasma
sample. 200µL of water was added to 200µL of plasma from the plasma sample
prepared and vortexed for 10s. 3000µL of Sodium dodecyl sulphate (SDS) was added
and briefly vortexed for 10s to precipitate proteins. 400µL of ethanol (95%) acidified
with 0.1% ascorbic acid was added and the resultant solution vortexed for 10s. 800µL
of hexane acidified with 0.1% ascorbic acid was added and the solution was vortexed
for 3min and the resultant mixture was centrifuged at 3500g for 10 min in an Eppendorf
centrifuge 5810R. The hexane layer was collected and evaporated to dryness. The
resultant pellet was resuspended in 200 µL of methanol and centrifuged for 5 min on
14000rpm in Eppendorf centrifuge mini plus. The sample was then filtered through
0.45µm phenomenex syringe driven polytetrafluoroethylene (PTFE) filter and
transferred to HPLC standard vials fitted with 300µL glass inserts. A blank plasma
assay was also performed to prove the specificity of the method. It is analysed using the
validated analytical method to determine the extraction efficiency and to check the
percentage of sample extracted from biological samples. The aforementioned procedure
was applied to PBS containing 30% ethanol.
4.2.2 Solid Phase extraction (SPE)
SPE is used as an alternative method to extract physiologically relevant
concentrations α-TOS from plasma. A suitable and validated protocol was also
performed in a Varian ProStar Solid Phase Extractor with C-18 SPE cartridges used for
47
analysis. The SPE cartridge was washed with various organic solvents as shown in the
LLE protocol to remove proteins and unwanted materials. The final hexane layer
hexane layer was collected and evaporated to dryness. The resultant pellet was
resuspended in 200µL of methanol and centrifuged for 5 min on 14000rpm in
phenomenex mini centrifuge. The sample was then filtered through 0.45µm
phenomenex syringe filter and transferred to HPLC standard vials fitted with 300µL
glass inserts. A blank plasma assay was also performed to prove the specificity of the
method. It was analysed using the validated analytical method to determine the
extraction efficiency and to check the percentage of sample extracted from biological
samples. A comparison of the extraction efficiency was determined between the
conventional and SPE methods. The extraction efficiency was determined by comparing
the peak areas of known concentrations of α-TOS extracted from LLE and SPE to those
of α-TOS or solutions of corresponding concentration injected directly in the HPLC
system without extraction developed as a standard curve. The percentage recovery of
the known concentration was calculated using the value extrapolated from the slope of
the standard curve to the value obtained from the expected concentration of verification
samples. The aforementioned procedure was applied to PBS containing 30% ethanol.
4.2.4 Data analysis
Data was processed, graphed and analysed using GraphPad PRISM version 3
and GraphPad Instat version 3.10. Data was expressed as the mean S.D of replicate
experiments. The extraction efficiency was determined by dividing the concentration
obtained by the target concentration and expressing it as a percentage. Statistical
comparisons were made by one way ANOVA and two tailed student-t test where
applicable.
48
4.3 Results and Discussion
Spiked plasma and PBS containing 30% ethanol samples extracted by LLE and
SPE were analysed by the optimised isocratic and gradient methods. An example
isocratic HPLC chromatogram obtained from the analysis of extracted plasma sample is
presented in Figure 19. Despite this method producing adequate resolution of the α-
TOS, the calculated extraction efficiencies were consistently less than 80% of the
required target concentrations.
Figure 19. Representative isocratic chromatogram. The chromatograms shows 5,
10, 25µg/mL α-TOS, α-TOH and poorly resolving internal standard extracted from
spiked porcine plasma at 205nm.
This low extraction efficiency was due to the poorly resolving α-TOA peak.
Peaks were observed co-eluting with the internal standard (α-TOA) and as a result
affected the extraction efficiency calculation. Analysing the data at 225nm did not
remove the co-eluting peak with α-TOA. However, reanalysing the data at 284nm
removed the co-eluting peak but significantly reduced the sensitivity of the assay
(negatively affected both the LOD and LOQ). This poor selectivity precluded further
49
use of the developed isocratic method in this investigation. Future investigations may
focus on identifying an alternate internal standard which could be used more effectively
with this isocratic HPLC method.
Spiked plasma samples analysed by the gradient HPLC method, however,
produced chromatograms with no interfering peaks co-eluting with the compounds of
interest. An example chromatogram is shown in Figure 20.
Figure 20. Representative step-up gradient chromatogram. The chromatograms
shows 7.5µg/mL α-TOS and internal standard extracted from spiked porcine plasma at
205nm.
The gradient HPLC method presented here offered reliable separation of α-TOS
from spiked plasma samples and provided efficient resolution of α-TOS, α-TOH and
endogenous fat soluble vitamins. In addition, PDA analysis across the α-TOS peak
confirmed spectral purity (Figure 21), thereby providing evidence of the method’s
specificity.
50
A B
Figure 21. Spectral profiles obtained from the PDA during the isocratic (A) and
gradient (B) analysis of extracted plasma samples.
Using this method it became evident that LLE was far more efficient in the
extraction of α-TOS than the SPE method (Figures 22 and 23). The SPE method
consistently produced extraction efficiencies of less than 80% of the target
concentrations. In contrast, the LLE method consistently produced extraction
efficiencies greater than 95%. LLE efficiencies were within 5% for plasma samples
spiked at 7.5, 15 and 30µg/mL.
51
7.5 g/mL 15 g/mL 30 g/mL0
10
20
30
40
7.5 g/mL
15 g/mL
30 g/mL
Target concentration
[-T
OS
] (
g/m
L)
7.5 g/mL 15 g/mL 30 g/mL0
10
20
30
40
7.5 g/mL
15 g/mL
30 g/mL
Target concentration
[-T
OS
] (
g/m
L)
A
B
Figure 22. Efficiencies of LLE of plasma samples (A) and PBS containing 30%
ethanol (B). Samples were analysed by the step-up gradient method. Data shown is the
mean ± SD of triplicate values and is representative of three independent experiments.
52
7.5 g/mL 15 g/mL 30 g/mL0
10
20
30
7.5 g/mL
15 g/mL
30 g/mL
7.5 g/mL 15 g/mL 30 g/mL0
10
20
30
7.5 g/mL
15 g/mL
30 g/mL
A
B
Figure 23. Efficiencies of SPE of plasma samples (A) and PBS containing 30%
ethanol (B). Samples were analysed by the step-up gradient method. Data shown is the
mean ± SD of triplicate values and is representative of three independent experiments.
Based on these findings, the step-up gradient HPLC and the LLE protocols
proved to be the most accurate and reliable methods for the analysis of α-TOS from
complex biological matrices and as a result were chosen for further use throughout this
investigation.
53
CHAPTER 5. FORMULATION OF TRANSDERMAL
DOSAGE FORMS CONTAINING α-TOS
5.1 Background
Transdermal delivery systems are specifically designed to obtain systemic blood
levels of included drugs and involve continuous administration of therapeutic molecules
through the skin [75, 78]. Transdermal delivery has the attractive advantage of non-
invasivlryt maintaining constant plasma drug levels [72, 78]. A number of systems are
available to enhance transdermal drug delivery, however, utilisation of liposomes has
been extensively investigated due to their diverse composition, structure and
biocompatibility [95]. Research into topically applied liposomes initially focussed on
improving drug deposition into the skin and its appendages. Ganesan et al [96] were
among the first to report the enhanced systemic drug delivery of hydrophobic drugs
after topical application.
Liposomes are microscopic vesicles comprised of one or more membrane-like
phospholipid bilayers surrounding and aqueous medium. The lipid components are
predominantly phosphatidylcholine, derived from egg or soybean lechithins. Liposomes
can act as carriers for both hydrophilic and lipophilic drugs because of their biphasic
character. Lipophilic drugs are generally entrapped in the lipid bilayers of liposomes
and therefore aid in solubilisation. [95]
54
A number of mechanisms have been proposed to explain the skin permeating
effects of liposomes (figure 24).
Figure 24. Mechanisms of action liposomes as transdermal drug delivery systems.
(A) Free drug mechanism; (B) penetration enhancing effects of liposome
components; (C) vesicle absorption to and/or fusion with the stratum corneum;
and (D) illustrates intact vesicle penetration into or into and through intact skin.
[97]
Evidence suggests that liposomes can act as permeation enhancers.
Phospholipids of the liposome can diffuse into the stratum corneum and disrupt bilayer
fluidity, thereby decreasing the barrier properties of the skin. Phospholipids of liposome
potentially may mix with stratum corneum lipids thereby creating a lipid-enriched
environment, which enhances skin uptake of lipophilic drugs. Liposomal vesicles may
absorb to the stratum corneum surface and subsequently transfer the drug from the
vesicle to the skin. Liposomal vesicle can also potentially fuse and mix with the stratum
corneum lipid matrix thereby increasing drug partitioning into the skin. Intact liposomal
vesicles can potentially penetrate human skin and co-transport the drug with them. [97]
55
In order to further enhance transdermal drug delivery, enhancing methods have
been used to elicit synergistic effects with liposomes. Enhancers include iontophoresis,
ultrasound, electroporation and chemicals [98]. The inclusion of organic solvents
(chemical enhancers) in liposomes has been shown to enhance transdermal drug
delivery. More than 300 chemicals, termed permeation enhancers or penetration
enhancers, have been studied for their ability to increase transport of drugs across the
skin [99]. DMSO was the first chemical permeation enhancers studies, however, clinical
use is limited by skin irritation [99]. DMSO causes significant skin irritating effects at
concentrations used for skin penetration enhancement [73]. Contemporary permeation
enhancers which have been successfully included in liposomal formations, include
alcohols, such as pentylene glycol, propylene glycol and ethanol. The inclusion of these
permeation enhancers into liposomal formulations can significantly enhance skin
penetration. [95] Alcohol chemical enhancers have been shown to improve skin
permeation by a variety of mechanisms, which include: extraction of skin lipids and
proteins; swelling of the stratum corneum; improving drug partitioning into the skin;
and increasing the solubility of the drug in the formulation. [98]. In contrast to DMSO,
these agents have a good balance between safety and potency as chemical enhancers
[99].
Poorly formulated liposomal transdermal drug delivery systems can potentially
reduce the permeation of drugs. Phospholipids may form extra lipid barriers at the skin
surface which reduce permeation. Liposome structure may also slow the release rate of
drugs which negatively impact on flux and uptake in the skin. [95] It is therefore
imperative to systematically investigate formulated liposomal transdermal delivery
systems.
56
Lipoderm represents a commercially available pre-formed liposomal formulation for
the compounding and transdermal delivery of drugs. This patented formulation is
marketed by the Professional Compounding Chemists of Australia (PCCA). To date,
there are no peer-reviewed published studies on the skin permeation of drugs formulated
in Lipoderm . Annecdotal evidence suggests that Lipoderm effectively delivers
tramadol, ketoprofen, and promethazine across skin. Lipoderm , containing 5%
pentylene glycol as a chemical enhancer, reportedly outperforms other liposomal
transdermal dosage forms in delivering drugs such as ketoprofen and promethazine
across the skin. [100-102]
α-TOS was formulated in Lipoderm containing 5% pentylene glycol in order to assess
the extent of drug delivery across the outer layers of the skin and make comparisons to
the reported DMSO-containing patent formulation.
5.2 Methods and Materials
Lipoderm anhydrous and pentylene glycol were purchased from the PCCA
(NSW, Australia). All other chemicals used were analytical grade (AR) and sourced
from Sigma Aldrich (St. Louis, MO). The water used throughout this investigation was
deionised and filtered by a Milli Q-Academic system.
5.2.1 Compounding of Lipoderm formulations
α-TOS (5, 10, 15 and 25 g) was weighed on a Shimadzi analytical balance and
transferred to a glass slab. The volume of pentylene glycol required to produce a final
57
concentration of 5% w/w was measured and triturated with α-TOS on the glass slab
with a spatula. This mixture was further triturated in Lipoderm anhydrous by the
doubling method. 50g of 10, 15, 30, and 50% w/w α-TOS-containing Lipoderm
formulation were prepared.
5.2.2 Compounding of DMSO-containing patent formulation
For comparison of transdermal drug delivery, the DMSO-containing formulation
was prepared according to the methods outlined in the published patent [65]. Specified
weights of α-TOS, DMSO, almond oil, stearyl alcohol and distilled water were mixed
by Ultra-Turrax disperser (IKA, China). The patent formulation contained α-TOS at a
final concentration of 50% w/w.
5.3 Results and Discussion
Liposome anhydrous, containing 5% w/w pentylene glycol, was successfully
used to incorporate α-TOS at concentrations of 10, 15 and 30% w/w. At a concentration
of 50% w/w, significant amounts of α-TOS remained undissolved. Granular particles
remained visible despite extended trituration. As a consequence this formulation was
removed from further study. Pre-dissolving α-TOS in pentylene glycol proved to be a
crucial step, ensuring dissolution and even distribution of the drug throughout the
Lipoderm anhydrous base during trituration. Formulating α-TOS in Lipoderm
anhydrous containing 5% w/w pentylene glycol yielded a smooth, cream-like (semi-
solid) product with medium viscosity and pleasant odour. In contrast, the DMSO-
containing patent formulation had the consistency of a medium viscosity vegetable oil,
58
such as liquid paraffin. In addition, the DMSO-containing formulation had a distinct,
strong ‘garlic-like’ odour. Lipoderm formulations were prepared much quicker than the
DMSO-containing patent formulations and required less specialised equipment for
preparation.
Depending on the results obtained from in-vitro and in-vivo studies assessing skin
permeation, Lipoderm appears to be a relatively economical, easy and feasible way to
formulate α-TOS for transdermal delivery.
59
CHAPTER 6. IN-VITRO SKIN PERMEATION
EXPERIMENTS
6.1 Background
The purpose of this work was to evaluate the skin permeation of formulated -
TOS transdermal dosage forms through excised human skin using in-vitro Franz
diffusions cells.
The intrinsic ability of a drug to permeate across skin (i.e., percutaneous
permeation) has been linked to three key physicochemical properties, namely:
solubility, melting point and n-octanol-water partition coefficient (P and calculated log
P) [103-104]. High percutaneous permeation is associated with increased aqueous
solubility and low melting point. In contrast, low percutaneous permeation is associated
with low aqueous solubility and high melting point. [105] In the case of log P values,
drugs should preferably have a balanced lipophilic/hydrophilic to be potential
candidates for transdermal delivery [106]. Based on evidence, log P values of
approximately 2 are considered ideal for transdermal delivery [106]. The low melting
point of -TOS (76-77 C) suggests that this drug may possess suitable physicochemical
properties for permeation of the skin. However, details regarding the aqueous solubility
of -TOS at different pHs is still required and determination of log P needed.
60
Franz diffusion cells have been extensively used for studying drug release rates
from semi-solid dosage forms and drug diffusion through biological and synthetic
membranes [107-109]. Over the years, in-vitro Franz diffusion experiments have
evolved into one of the most important methods for researching transdermal drug
administration [107].
Franz diffusion cells, also termed vertical diffusion cells, have a static receptor
solution reservoir with a side-arm sampling port. These cells are comprised of two
compartments, one containing the active component (donor vehicle) and the other
containing a receptors solution. A thermal jacket is positioned around the receptor
compartment and is heated by external circulating bath. In order to create sink
conditions for highly lipophilic drugs in the receptor solution, ethanol can be included at
concentrations up to 35%. The inclusion of ethanol has little effect on the permeability
of the stratum corneum at these concentrations. [110-111] However, if ethanol is
included at these concentrations, it is necessary to assess the integrity of the excised
human skin for the duration of the experiment. Integrity of excised human skin can be
confirmed by assessing the rate and extent of caffeine flux. Excessive caffeine flux
indicates that the integrity of the excised human skin is compromised.
The two compartments are separated by a piece of excised skin or other suitable
membrane. Despite the reported use of numerous synthetic, substitute membranes in
skin permeation studies, evidence suggests that these are less reproducible and not
reliable predictors of in-vivo diffusion [107]. In contrast, excised skin is reported to
display very close correlation between in-vitro and in-situ permeation for approximately
4.5 h after commencement of experiments. After this period, permeation through
61
excised skin increases disproportionately due to proposed disruption of the stratum
corneum. [109] In the case of various skin types, all tested substitutes to excised human
skin demonstrate exceedingly high comparative flux (concentration per surface area per
unit time) [112]. Unless inaccessible, excised human skin should always be used for in-
vitro drug permeation studies.
During an experiment, small volumes are withdrawn from the stirred receptor
solution for analysis. Removed volumes are immediately replaced to maintain the
volume of the receptor solution. In-vitro Franz diffusion permeation experiments
provide important permeation data, such as lag time, permeability coefficients (Kp) and
flux. Variations in cutaneous blood flow, interfollicular transport and variable patterns
of lymphatic drainage from the skin are not accounted for in this investigative
technique. [113] It does, however, provide crucial evidence of local drug kinetics below
the immediate site of application, namely the stratum corneum.
The intrinsic ability of α-TOS to permeate across skin was firstly assessed by
determining its n-octanol-water partition coefficient (log P) and pH-related aqueous
solubility. Flux (cumulative amount of drug permeated through skin as a function of
time) and lag time to diffusion of α-TOS from formulated transdermal dosage forms
was subsequently assessed using Franz diffusion cells fitted with human excised skin as
the membrane.
6.2 Materials and Methods
62
All chemicals used were analytical grade (AR) and sourced from Sigma Aldrich
(St. Louis, MO). The water used throughout this investigation was deionised and
filtered by a Milli Q-Academic system.
6.2.1 Solubility studies
Excess α-TOS was added to a series of aqueous co-solvent systems to assess solubility
and determined suitable sink conditions in permeation studies. Co-solvent systems were
comprised of PBS and commonly used solvents, such as ethanol or polyethylene glycol
(PEG). The ratio of solvents to buffers ranged from a 0-100%. The mixture was agitated
in a water bath and maintained at 32°C (normal body temperature) for 24 h. Samples
were collected at the end of 24 h and centrifuged at 10,000 g. The supernatant was
collected and filtered through PTFE filters (0.45 µm pore diameter) for HPLC samples.
The samples collected were run on HPLC using the validated HPLC method for
analysis of α-TOS.
6.2.2 Shake-flask determination of log P
The n-octanol-water partition coefficient of α-TOS was measured using the traditional
shake-flask technique at 25ºC [114]. A known weight of α-TOS was allowed to
partition between equal volumes of n-octanol and water for 24 h on an invert shaker.
Mixtures were centrifuged at 4000 g for 10 min to separate immiscible phases. The
concentration in each phase was subsequently determined by the validated HPLC
method. The log P value was calculated using the following formula:
log P = log ([solute]n-octanol/[solute]water)
63
LogP determinations between 0 and 3 were considered to be low or moderately
lipophlilic. Log P determinations between 3 and 6 were considered to be highly
lipophilic. [115]
6.2.3 In-vitro skin permeation study
The Franz diffusion study, using excised human skin, was adapted from previously
published methods [116-117]. The procedure was performed under the supervision by
Dr Jeff Grice at the Therapeutics Research Unit (Princess Alexandra Hospital,
Brisbane). The apparatus consisted of 5 Franz diffusion cells, water bath, stirring
apparatus, battery operated multimeter, cork board and small stirring bars (Figure 25).
Figure 25. Franz diffusion cell set up used for in-vitro skin permeability study
64
1.33 cm2
surface areas of the human female stratum corneum and epidermal
membranes were used. The excised human skin was cut using a skin cutter to isolate the
stratum corneum and epidermis. This was placed on PTFE filter paper and cut into
circles with a skin cutter (approx. 25 mm diameter) on a cork board. The skin ID
number was recorded for future reference. High vacuum grease was applied evenly
between the surfaces of donor and receptor compartments. The membrane was mounted
between the cell halves (skin facing up) and the two compartments held together using
clamps. PBS was added to receiver compartment with a 5mL syringe, needle and
tubing. The level of the solution was marked on the sampling arm and the volumes
added into the receiver compartment were noted. The contents of the receiver solution
were stirred with the help of a magnetic bar at 100 rpm. Approximately 1mL of PBS
was added to the donor compartment. The donor compartment was capped with a glass
cap to prevent evaporation. The rack holding Franz diffusion cells was placed in the
water bath set at 35oC and left for 30 min. Prior to experimentation, silver/ silver
chloride (Ag/AgCl) electrodes were placed into both the receptor and donor
compartments (black AgCl electrode into donor compartment). The resistance was
recorded on a multimeter. A reading above 20 k was considered satisfactory, while a
reading below 20 k indicated that the integrity of the membrane surface was
compromised. The membrane was replaced wherever necessary. The PBS solution was
discarded from all cells and the skin was carefully blot dried. The required volume of
PBS containing 30% ethanol (to maintain sink conditions) was added to the receptor
compartment making sure all bubbles were eliminated. A sample (0.2 mL) was
withdrawn from the receiver chamber at 0 h for baseline correction.
65
The density of formulations, containing α-TOS, was determined in order to
allow for the calculation of sample weight applied. A chosen volume of known
concentration, based on density calculations, was applied onto the donor compartment
using a Gilson micropipette. The weight of the syringe plunger was recorded before and
after application of dosage form to the skin, in order to calculate the net weight of
formulation applied to the skin. Samples (0.2mL) were withdrawn from the receiver
chamber immediately after application of the formulation or solution (0 h) and at
intervals of 1, 2, 4 and 6 h. An equal volume of buffer was immediately replaced to
maintain constant volume in the receptor compartment. Each experiment was conducted
at least in triplicate. Permeation was determined by measuring the cumulative amount of
drug in the receiver chamber at collected intervals using the validated HPLC method for
analysis of α-TOS. Suitable negative controls were included in this analysis. The
cumulative amount of α-TOS permeated per unit skin surface area was plotted against
time for each of the transdermal formulations. The slope of the linear portion of the plot
was estimated as steady-state flux (Jss) and the Kp calculated as follows:
Kp = Jss/Cv
where Cv is the total donor concentration of α-TOS. [118]
6.2.4 Caffeine analysis
The concentration of caffeine in samples was assessed by HPLC using a method
adapted from Desbrow et al [119]. The HPLC system consisted of a Varian Prostar 240
Quaternary Solvent Delivery Module, a Varian Prostar Photodiode Array Detector, and
a Varian Prostar Autosampler. The column used in this study was a Phenomenex
Gemini C18 column (250 x 4.6 mm, 5- m particle size) and was thermostated at 30 C.
66
Eluted caffeine was detected at 273nm. Mobile phase consisted of 0.05M NaH2PO4
(adjusted to pH 5.5. using triethylamine) [solvent A] and methanol [solvent B].
Conditions used to elute caffeine (Rt = 8.65 min) were 75% solvent A and 25% solvent
B. This isocratic method detected caffeine with limits of quantification of ≥ 1.5 M
(signal-to-noise ratio of 10:1). The calculated standard curves for caffeine were linear in
the range of 2.5 to 50 M (y = 13.78x + 1.98; r2 = 0.999) and relative standard
deviations of < 5% were obtained for both repeatability and intermediate precision
studies.
6.2.5 Data analysis
Data was processed and graphed GraphPad PRISM version 3 (SanDiego, CA).
Data was expressed as the mean S.D of replicate experiments. Data were analysed
using a paired Student t-test or one-way ANOVA with Tukey-Kramer multiple
comparisons test, using Graphpad InStat3 software (SanDiego, CA). Significance levels
were defined as p<0.05 (*), p<0.01 (**) and p<0.001 (***).
6.3 Results and Discussion
The intrinsic ability of α-TOS to permeate across skin was assessed by
determining key descriptive parameters, namely: log P value; lag time to diffusion;
cumulative amount of drug permeated per unit skin surface area as a function of time;
and Kp.
Comment [GD1]: Figure legends refer
to Dunnetts post test. In the case of
Senescence data, I cannot do this test
because technically there were only 2
replicates. I can perform a Student-Neuwman-Keuls post test for this data for
which both the 25 and 50uM [ ] are
significantly different from vehicle control (0); p < 0.05 (*).
67
The traditional shake-flask method was used to determine the log P value for α-
TOS. The calculated log P value for α-TOS was 8.2 ± 0.5, confirming the highly
lipophilic nature of the drug. Chemicals with high log P values (> 4) are very
hydrophobic [120]. Based on previous evidence, this finding suggests that the imbalance
between water and lipid solubility of α-TOS may restrict skin permeation. A balanced
log P value, between 1 and 4, is generally considered ideal for effective transdermal
delivery. The dosage form, together with co-solvents, would have to maintain the
solubility of α-TOS to ensure adequate solution concentrations are available for diffusion
to occur. Hence α-TOS possesses low aqueous solubility and low melting point. The
consequence of these combined parameters on ability to permeate skin has not been
previously been reported.
Results obtained for the solubility determination of α-TOS are presented in
figure 26. The saturation aqueous solubility of α-TOS was 13.6 ± 1.3 g/mL, highlighting
its poor aqueous solubility. Addition of increasing concentrations of ethanol significantly
increased solubility when compared to control (P < 0.01). Solubility of α-TOS increased
in a linear fashion (y = 15.1x + 42.1; r2 = 0.992) with increasing concentrations of
ethanol. In contrast, PEG did not significantly improve α-TOS solubility, even at
concentrations of 50%.
68
0 10 20 30 40 50 600
250
500
750
1000Ethanol
PEG
[co-solvent] (% v/v)
[-T
OS
] (
g/m
L)
Figure 26. Solubility of α-TOS in tested co-solvent systems.
These findings confirm the suitability of ethanol as a co-solvent for the receptor
compartment solution. For highly lipophilic drugs, the inclusion of ethanol
concentrations up to 35% in receptor solutions are recommended [111]. α-TOS
concentrations of approximately 0.5mg/mL were observed at 30% ethanol
concentrations. The inclusion of 30% ethanol in the receptor solution would therefore
provide suitable sink conditions to maintain an adequate gradient for diffusion of α-TOS
across the membrane in Franz diffusion studies. In addition, the enhanced solubility of α-
TOS in 30% ethanol would prevent the highly lipophilic drug from floating at the
membrane surface due to immiscibility.
The skin permeation of α-TOS from the following formulations was assessed:
50% α-TOS in the DMSO-containing patent formulation (patent formulation); 10% α-
TOS in Lipoderm containing 5% pentylene glycol (formulation 1); 20% α-TOS in
Lipoderm containing 5% pentylene glycol (formulation 2); and 30% α-TOS in
69
Lipoderm containing 5% pentylene glycol (formulation 3). For each of the transdermal
dosage forms, results for both the cumulative amount of α-TOS permeated per unit skin
surface area plotted against time and the percentage α-TOS released from application are
presented in figure 27. Importantly, caffeine flux was not observed through membranes
throughout the experimental period. This result confirmed that the integrity of the
excised human skin was not compromised by the inclusion of 30% in the receptor
solution. There was no was observed lag time to α-TOS permeation of excised human
skin from any of the transdermal dosage forms tested, which could be attributed, in part,
to the highly lipophilic nature of α-TOS. The stratum corneum is mainly lipoidal and
high lipid solubility is required for maximal permeation [121]. The patent formulation,
which contained high concentrations of α-TOS and DMSO, produced significantly
higher flux of α-TOS across excised human skin when compared to Lipoderm
formulations (P < 0.01). Kp (permeability coefficient) values, calculated as the slope in
the first two hours, was 2.28, 0.50, 0.88 and 1.3 g/cm2/h for the DSMO-containing
patent formulation, formulation 1, formulation 2 and formulation 3, respectively.
Increasing the concentration of α-TOS in the formulation clearly increased the
permeability coefficient; however, values were still well below that of the DMSO-
containing patent formulation. Increasing α-TOS concentrations in Lipderm
formulations tended to increase the permeation profile of α-TOS across excised human
skin, however these trends were not statistically significant. In order to obtain high levels
of drug in the first layers of the stratum corneum it should also have a tendency to leave
the vehicle and migrate into the skin. The magnitude of partitioning is affected by the
composition of the vehicle (dosage form), and the chemical structure of the drug. [121]
Importantly, the percentage of α-TOS release from all applied formulations was rapid
and extensive (>50% in 6 h) in this study. These findings show that α-TOS does not
70
remain trapped within transdermal dosage forms and provides good evidence that α-TOS
is readily available to permeate the first layers of the stratum corneum. Despite the low
solubility and extremely high log P value of α-TOS, this study provides evidence that it
still can readily cross the stratum corneum, which is considered the rate limiting barrier
in transdermal permeation, and epidermis, [97].
0 1 2 3 4 5 6 70.0
2.5
5.0
7.5
10.0
12.5Patent Formulation
Formulation 1
Formulation 2
Formulation 3
**
**
**
**
*
Time (h)
Cu
mu
lati
ve
-TO
S d
iffu
sio
n p
er
un
it a
rea
(g
/cm
2)
0 1 2 3 4 5 6 70
50
100
150Patent Formulation
Formulation 1
Formulation 2
Formulation 3
Time (h)
% R
ele
ase
A
B
Figure 27. (A) The cumulative amount of α-TOS permeated per unit skin surface
area plotted against time for each of the transdermal formulations and (B) the
percentage α-TOS released from application. Data shown is the mean ± SD of
triplicate values and is representative of three independent experiments. Data were
71
analysed using one-way ANOVA with Tukey-Kramer multiple comparisons post
test. Significance levels were defined as P < 0.05 (*) and P < 0.01 (**).
Although the stratum corneum is the main barrier to skin permeation for most
drugs, once the drug has crossed the stratum corneum it must partition into the
underlying layers of the epidermis, dermis, lymphatic and circulatory systems. In
contrast to the stratum corneum, these other structures are more hydrophilic and can be a
barrier to extremely lipophilic drugs [121]. Although this study provides evidence that α-
TOS can cross the stratum corneum and epidermis, whether or not it can enter systemic
circulation must still be assessed by suitably designed in-vivo pharmacokinetic
experiments.
Lipoderm containing 5% pentylene glycol was shown to effectively deliver α-
TOS. Although it appeared less effective at delivering α-TOS across the skin than the
DMSO-containing patent formulation, the findings from this study are encouraging.
Further experimentation, which includes alteration of the type and concentration of
percutaneous enhancer, may improve the transdermal delivery of α-TOS.
Comment [GD2]: Figure legends refer
to Dunnetts post test. In the case of
Senescence data, I cannot do this test
because technically there were only 2
replicates. I can perform a Student-
Neuwman-Keuls post test for this data for which both the 25 and 50uM [ ] are
significantly different from vehicle control
(0); p < 0.05 (*).
72
CHAPTER 7. INFLUENCE OF ESTERASES ON THE
SUCCESSFUL TRANSDERMAL DELIVERY OF α-TOS
7.1 Background
There is growing evidence that the majority of common drug-metabolising
enzymes are expressed in the skin of humans [122]. There is good evidence that the skin
is capable of many metabolic functions common to visceral organs [123]. All major
enzymes important for systemic metabolism in the liver and other tissues have been
identified in the skin, albeit at lower levels [124]. Irrespective, the skin acts as an organ
of extrahepatic metabolism and has the capacity to biotransform foreign substances that
penetrate through the stratum corneum, the main physical barrier of the skin [125-126].
Skin biotransformation has the potential to decrease the therapeutic index of
transdermally delivered drugs [123, 126]. It is therefore important to know the types and
locations of skin enzymes, which could be involved in drug metabolism and the
potential influence they may have on transdermally delivered drugs. The viable
epidermis appears to be the major site of skin metabolism. Skin metabolising enzymes
reported have the capacity to catalyse phase I (oxidative, reductive and hydrolytic) and
phase II (conjugation) reactions [124, 127-130]. Chemical groups such as esters,
primary amines, alcohols, and acids are particularly susceptible to metabolism in the
skin. Of particular relevance to this study is that esters are hydrolysed by
caroxylesterases in the skin to their parent alcohol and acid molecules. [125, 127]
73
7.1.1 Esterases
Carboxylesterases, which include butyryl esterase, are members of serine
hydrolase superfamily [131]. These esterase enzymes are involved in the hydrolysis of a
wide range of ester-containing, xenobiotics [132]. In comparison to acetylcholinesterase
(AChE), carboxylesterases such as butyryl esterase (BChE) have lower substrate
specificity. Due to its wide range of substrates, BChE was termed the “nonspecific
cholinesterase”. Known substrates for BChE include acetylcholine, butyrylcholine,
succinylcholine, organophosphates and cocaine. In comparison to AChE, however,
wild-type BChE possess very low catalytic efficiency. [133]
Of particular interest is the finding that some carboxylesterases may be variably induced
by exogenous chemicals. For example, butylated hydroxyanisole, ticlopidine and
diclofenac have been shown to induce the production of carboxylesterase in human
hepatocytes through an unconfirmed mechanism. [131] In contrast, rifampcin and
omperazole had no effect on the expression of carboxylesterases [134]. Maruichi et al
provided preliminary evidence that increases in enzyme production may be linked to
nuclear factor-erythroid 2 related factor 2 (Nrf2), which is a transcriptional factor
activated by oxidative stress. [131] Given the reported generation of ROS by α-TOS, it
is possible that similar events could occur, which may ultimately lead to enhanced
conversion to α-TOH.
In this study we report the effects esterases may have on the delivery of α-TOS.
Although this was not intended to be an exhaustive enzyme kinetics study, it provides
important preliminary evidence. This investigation aimed to provide the following:
evidence on substrate specificity between AChE and BChE; potential for metabolism by
74
AChE and BChE; and a comparison of metabolism profiles between AChE and BChE.
AChE and BChE were chosen in this study because of their differences in reported
substrate specificities [133]. In addition, we investigated the potential for esterase
enzyme expression in an in-vitro cell-based assay, as well as the potential for
metabolism of α-TOS once it reaches the blood.
7.2 Materials and Methods
The Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit was
purchased from Invitrogen (Victoria, Australia). AChE and BChE, together with all
other chemicals and reagents, were purchased from Sigma Aldrich (St. Louis, MO).
Ellman’s reagent was sourced from Cayman Chemical (Michigan, USA). The water
used throughout this investigation was deionised and filtered by a Milli Q-Academic
system.
7.2.1 Competitive ACHE and BCHE activity assay
AChE activity was assessed by the relative extent of acetylcholine (ACh)
metabolism during a 60 min period in the presence or absence of different
concentrations of α-TOS. The AChE activity assay was performed according to
manufacturer’s instructions. Adaptations were made to study the competitive or
inhibitory effects of α-TOS on esterase activity. Briefly, the 100mM stock solution of
ACh was diluted with reaction buffer to produce final concentrations ranging from 0 to
100µM. The stock solutions of α-TOS were diluted with reaction buffer to produce final
concentrations ranging from 0.001µM to a 100µM. A volume of 100µL comprising of
75
75µL of α-TOS standards and 25µL of ACh standards were used for each reaction. A
positive control of 10µM H2O2 in reaction buffer was included. Reaction buffer was
used as a negative control. 100µL of samples and controls were pipetted into separate
wells of a 96 well microplate.
A reaction mixture was prepared by adding 200µL of 400µM Amplex red
reagent, 100µL of 2U/mL horseradish peroxidase, 100µL of 0.2U/mL choline oxidase
and 100µL of 1U/mL AChE. This reaction mixture was made up to 9.5mL with reaction
buffer. 100µL of this reaction mixture was added to each well containing samples and
controls. The plates were then incubated at RT protected from light for 60 min.
Fluorescence (excitation at 530nm, emission at 560nm) at 20, 30 and 60 min was
measured on a Fluoroskan Ascent microplate fluorometer (Thermo Scientific, Victoria,
Australia). Background fluorescence was accounted for and AChE activity determined
by extrapolation of observed relative fluorescence from the standard curve generated on
the day of experiment. Results are represented as percentage AChE activity relative
control wells (i.e., presence of ACh and absence of α-TOS). The experiment was
performed in triplicate.
The effect of α-TOS on BChE activity was assessed by repeating the above
protocol and substituting AChE with 1U/mL BChE.
7.2.2 Comparative metabolism of α-TOS by AChE and BChE
The metabolism of α-TOS by AChE and BChE was performed using the
developed and validated gradient HPLC method.
76
AChE enzyme stock solution was diluted with reaction buffer to produce a final
concentration of 1U/mL. The stock solution of α-TOS was diluted with reaction buffer
to produce final concentrations ranging from 0, 25, and 100µM. 10µL of these α-TOS
solutions was added to 96 well microtiter plates. 90µL of the enzyme (AChE) and 100
µL of the buffer were used to make up for the 200µL in each well. Wells containing 90
µL of the enzyme and 110µL of the reaction buffer were included for negative controls.
Sufficient wells were included for a sequential interval study. At intervals of 10, 20, 30
and 60 min, 200µL was withdrawn and analysed using the developed and validated
gradient HPLC method. α-TOS concentrations at each time point were determined by
extrapolation of observed peaks areas from standard curves generated on the day of
analysis. Experiments were performed in triplicate.
The metabolism of α-TOS by BChE was assessed by repeating the above
protocol and substituting AChE with 10U/mL BChE.
7.2.3 Effect of α-TOS exposure on esterase activity in-vitro
A549 and HepG2 cells were used to assess the effect α-TOS exposure may have
on esterase activity in-vitro. A549, an adenocarcinomic human alveolar basal epithelial
cell, is a squamous subdivision of epithelial cells. They grow adherently, as a
monolayer, in-vivo. HepG2, a human hepatocellular liver carcinoma cell line, is a
perpetual cell line which is epithelial in morphology. These two cell lines were chosen
because of their reported differences in basal esterase expression. HepG2 is known to
express significant quantities of carboxylesterases [131]. In contrast, A549 cells
reportedly produce significantly lower amounts of carboxylesterases [135]. Based on
77
these inherent differences, these two models were therefore considered suitable to study
the effects of α-TOS exposure on esterase expression in-vitro.
A549 cells were obtained from the ATCC (Manassas VA, USA). Cells were
grown and maintained at 37 C with 5% CO2 in complete Dulbecco’s Modified Eagle
Medium (DMEM)/F12 (Invitrogen Australia) containing high glucose, L-glutamine,
sodium pyruvate, Phenol Red, 10% fetal bovine serum (Invitrogen, Victoria, Australia)
and 500U/mL Penicillin-Streptomycin (Invitrogen, Victoria, Australia). All A549 cell
experiments were conducted with complete DMEM/F12.
HepG2 cells were obtained from the ATCC (Manassas VA, USA). Cells were
grown and maintained at 37 C with 5% CO2 in complete DMEM (Invitrogen Australia)
containing high glucose, L-glutamine, sodium pyruvate, Phenol Red, 10% fetal bovine
serum (Invitrogen, Victoria, Australia) and 500U/mL Penicillin-Streptomycin
(Invitrogen, Victoria, Australia). All HepG2 cell experiments were conducted with
complete DMEM.
A549 and HepG2 cells were seeded at a density of 1×104 cells/mL in 25cm
2
tissue culture flasks and allowed to recover for 24 h. Cells were subsequently treated
with 10 M α-TOS for a further 24 h at 37 C with 5% CO2. Cells were then washed
twice with PBS, resuspended in PBS, tubes immersed in ice and subjected to ultrasonic
cell lysis. Supernatant was then analysed using the Amplex Red
Acetylcholine/Acetylcholinesterase Assay Kit according to the protocol outlined above.
Appropriate negative controls were included for comparison. Protein concentration was
measured using Bradford protein assay (Bio-Rad, Munich, Germany) with bovine
78
serum albumin standards (Sigma Aldrich, St Louis, MO, USA). 10 L of supernatant
was reacted with 140 L of Bradford reagent and absorbance measured at 590nm.
Experiments were performed in triplicate and results represented as the mean AChE
activity per mg cell protein ± SD.
7.2.4 Plasma incubation study
25 M -TOS was incubated in porcine plasma at 37°C for 1, 6, 12 and 24 h,
and the level of the VE analog assessed by HPLC as described above. 25 M -TOS in
PBS containing 30% ethanol was included as a control. Esterase activity of porcine
plasma was confirmed by an adapted Ellman’s method [136]. 100 L of sample,
standard or control solution was transferred to a 96 well microtitre plate. 100 L of
reconstituted Ellman’s reagent was added to all wells. Suitable solvent controls were
included in the experiment. The plate was subsequently developed at 37 C for 30 min.
Absorbance was then measured at 450nm. Background absorbance was accounted for
and AChE activity determined by extrapolation of observed absorbances from the
standard curve generated on the day of experiment. Experiments were performed in
triplicate.
7.2.5 Data analysis
Unless otherwise outlined, results are presented as the means ± SD of replicate
experiments. Data were analysed using a paired Student t-test or one-way ANOVA with
Tukey-Kramer multiple comparisons test, using Graphpad InStat3 software (SanDiego,
CA). Significance levels were defined as p<0.05 (*), p<0.01 (**) and p<0.001 (***).
Comment [GD3]: Reference: Wilhelm, K., Determination of human plasma cholinesterase activity by adapted Ellman's method. Arh Hig Rada Toksikol, 1968. 19(2): p. 199-207.
Comment [GD4]: Figure legends refer
to Dunnetts post test. In the case of Senescence data, I cannot do this test
because technically there were only 2
replicates. I can perform a Student-
Neuwman-Keuls post test for this data for
which both the 25 and 50uM [ ] are
significantly different from vehicle control
(0); p < 0.05 (*).
79
7.3 Results and Discussion
Esterases have been shown to play an important role in the metabolism of drugs
in the skin [123, 127, 137-138]. Given the ester-based structure of α-TOS and the fact
that only the intact drug maintains antineoplastic activity, the potential role esterases
may have on successfully delivering α-TOS transdermally was investigated [27, 45].
Results for the effect α-TOS had on AChE and BChE are presented in Figure 28.
α-TOS appeared to produce a concentration-depended decrease in apparent AChE and
BChE, respectively. The greatest decrease in AChE and BChE activity was observed at
the highest concentration tested (100 M).
80
100.0 10.0 1.0 0.1 0.050
75
100
125
20min
30min
60min
** * *
[ -TOS] ( M)
% A
Ch
E A
cti
vit
y(r
ela
tive t
o c
on
tro
l)
100.00 10.00 1.00 0.10 0.0050
75
100
125
20min
30min
60min
**
[ -TOS] ( M)
% B
Ch
E A
cti
vit
y(r
ela
tive t
o c
on
tro
l)
*
A
B
Figure 28. Influence of -TOS on AChE (A) and BChE (B) activity. Data shown is
the mean ± SD of triplicate values and is representative of three independent
experiments. Data were analysed using one-way ANOVA with Tukey-Kramer
multiple comparisons post test. Significance levels were defined as P < 0.05 (*).
α-TOS produced a statistically significant decrease in AChE activity at both the
10 and 100 M concentrations after 60 min. 100 M α-TOS also produced significantly
decreased AChE activity at both the 20 and 30 min intervals, respectively. 100 M α-
Comment [GD5]: Figure legends refer
to Dunnetts post test. In the case of
Senescence data, I cannot do this test
because technically there were only 2
replicates. I can perform a Student-
Neuwman-Keuls post test for this data for
which both the 25 and 50uM [ ] are
significantly different from vehicle control
(0); p < 0.05 (*).
81
TOS produced a significant decrease in BChE activity at the 30 and 60 min intervals. In
contrast, 10 M α-TOS decreased BChE activity only at the 20min interval.
α-TOS, even at the highest concentration tested, produced only a modest
reduction in the activity of either AChE and BChE. AChE activity ranged from 89 to
92% in the presence of 100 M. BChE activity ranged from 67 to 88% in the presence of
100 M α-TOS. The effect of various α-TOS concentrations, ranging from 0.1 to 10 M,
on AChE and BChE, for the most part, was comparable. Although not statistically
significant, BChE activity was affected to a greater extent at the 100 M α-TOS
concentration. This result provides some level of evidence that the affinity of α-TOS for
either enzyme does not differ to greatly.
To complement these findings, the extent of α-TOS metabolism by either AChE
and BChE was assessed (Figure 29). Metabolism profiles produced for both AChE and
BChE were first-order in nature. Concentrations of α-TOS decreased more rapidly at
higher concentrations. As concentrations decreased, the decline in α-TOS concentration
slowed. In addition, there was a quantifiable increase in α-TOH (results not shown). No
significant differences were observed between the profiles obtains for AChE and BChE.
82
0 10 20 30 40 50 60 700
25
50
75
100
125
AChE
BChE
Time (min)
[-T
OS
] (
g/m
L)
0 10 20 30 40 50 60 700
10
20
30
40
50
60
70
AChE
BChE
Time (min)
[-T
OS
] (
g/m
L)
0 10 20 30 40 50 60 700
10
20
30
40
AChE
BChE
Time (min)
[-T
OS
] (
g/m
L)
A B
C
Figure 29. Metabolism of -TOS by AChE and BChE (1U/mL). Metabolism of
100 (A), 50 (B) and 25 M (C) -TOS is shown. Data shown is the mean ± SD of
triplicate values and is representative of three independent experiments. Data were
analysed using one-way ANOVA with Tukey-Kramer multiple comparisons post
test. No significant differences were observed.
Rates of metabolism were subsequently calculated and shown to decrease
throughout the time-course of the experiment (Figure 29). Rates of metabolism
confirmed the concentration-dependent nature of the reaction (Figures 30 and 31). As α-
TOS concentrations increased, the rates of metabolism increased. The linear regression
between rates of metabolism and -TOS concentrations were 0.99 (y = 0.054 – 0.31)
and 0.97 (y = 0.082 + 0.68) for AChE and BChE, respectively. At 100 M
concentrations of α-TOS, BChE (7.7 M/min) produced higher rates of metabolism
Comment [GD6]: Figure legends refer
to Dunnetts post test. In the case of Senescence data, I cannot do this test
because technically there were only 2
replicates. I can perform a Student-Neuwman-Keuls post test for this data for
which both the 25 and 50uM [ ] are
significantly different from vehicle control
(0); p < 0.05 (*).
83
when compared to AChE (5.7 M/min). These findings further suggest that α-TOS has
slightly higher affinity for BChE in comparison to AChE.
0 10 20 30 40 50 60 700.0
2.5
5.0
7.5
100 M
50 M
25 M
Time (h)
Rate
of
meta
bo
lism
(M
/min
)
0 10 20 30 40 50 60 700.0
2.5
5.0
7.5
10.0
100 M
50 M
25 M
Time (h)
Rate
of
meta
bo
lism
(M
/min
)
A
B
Figure 30. Comparison of rates of -TOS metabolism by AChE and BChE over
the time-course of the experiment. Data shown is the mean of triplicate values.
84
0 25 50 75 100 1250.0
2.5
5.0
7.5
-TOS concentration ( M)
Rate
of
meta
bo
lism
(M
/min
)
0 25 50 75 100 1250.0
2.5
5.0
7.5
10.0
-TOS concentration ( M)
Rate
of
meta
bo
lim
s
(M
/min
)
A
B
Figure 31. Comparison of rates of -TOS metabolism by AChE and BChE
relative to the concentration of -TOS at 10 min. The relationship between rates of
metabolism and drug concentration were shown to be linear for AChE (r2 = 0.99; y
= 0.054 – 0.31) and BChE (r2 = 0.97; y = 0.082 + 0.68). Data shown is the mean of
triplicate values.
85
These findings confirm that α-TOS is a substrate for AChE and BChE and is
rapidly metabolised by esterases which are commonly found in the epidermis.
Metabolism by esterases was shown to liberate free α-TOH. This, as described
previously, would accompany a decrease in desired antineoplastic activity [27].
The ability of α-TOS to influence esterase activity in both A549 and HepG2
cells after 24 h exposure is presented in Figure 32. 10 M α-TOS did not produce
statistically significant changes in esterase activity after 24 h exposure. The average
esterase activities of both HepG2 and A540 cells exposed to α-TOS, however, tended to
be slightly higher than control cells. Given this result, longer exposure to higher
concentrations of α-TOS may prove to cause significant changes in esterase activity.
These factors should be assessed in future studies. In addition, the effect of formulation
excipients on enzyme activity in the skin will have to be assessed.
HepG2 A5490
10
20Control
10 M -TOS
Cell line
[AC
HE
] m
U/m
g
Figure 32. Cellular AChE activity after exposure to -TOS for 24h. Data shown is
the mean ± SD of triplicate values and is representative of three independent
experiments. Data were analysed using one-way ANOVA with Tukey-Kramer
multiple comparisons post test. No significant differences were observed.
Comment [GD7]: Figure legends refer
to Dunnetts post test. In the case of Senescence data, I cannot do this test
because technically there were only 2
replicates. I can perform a Student-
Neuwman-Keuls post test for this data for
which both the 25 and 50uM [ ] are
significantly different from vehicle control
(0); p < 0.05 (*).
86
Once α-TOS reaches systemic circulation it will be exposed to plasma esterases.
The extent of this metabolism was assessed by incubating α-TOS in purchased porcine
plasma. The esterase activity of the purchased porcine plasma was confirmed by
Ellman’s assay. The plasma was shown to possess on average 1.3 ± 0.2U/mL (n=9)
esterase activity. The plasma stability of α-TOS is presented in Figure 33. The decline
in plasma α-TOS levels was consistent with the profiles observed for AChE and BChE
metabolism presented above (Figures 29 and 30). Metabolism appeared to be
concentration-dependent and first-order in nature. Plasma concentrations of -TOS
rapidly declined with the 24 h study period.
87
0 10 20 300
10
20
30Plasma
PBS
Time (h)
Co
ncen
trati
on
(M
)
**
**
**
0 500 1000 1500 20000.00
0.05
0.10
0.15
Time (h)
Rate
of
meta
bo
lism
(M
/min
)
A
B
Figure 33. Plasma stability of -TOS (A) and rate of metabolism relative to
concentration (B). Data shown is the mean ± SD of triplicate values and is
representative of three independent experiments. Data were analysed using one-way
ANOVA with Tukey-Kramer multiple comparisons post test. Significance levels were
defined as P < 0.01 (**).
Comment [GD8]: Figure legends refer
to Dunnetts post test. In the case of
Senescence data, I cannot do this test
because technically there were only 2
replicates. I can perform a Student-
Neuwman-Keuls post test for this data for
which both the 25 and 50uM [ ] are
significantly different from vehicle control
(0); p < 0.05 (*).
88
Despite the slightly higher AChE activity, rates of α-TOS metabolism were
shown to be considerably slower than the enzyme incubation study. The association of
α-TOS with plasma components may, in part, protect this drug from rapid esterase
metabolism. α-TOH has been shown to distribute to lipoproteins and it is possible that
systemic α-TOS behaves similarly [25]. This finding provides an additional explanation
as to why α-TOS was shown to have an unexpectedly long plasma half-life (~10 h) in
the pharmacokinetic study reported by Teng et al. [25].
It is clear from these findings that α-TOS, delivered transdermally, will not only
be subjected to metabolism by esterases in the skin, but also in plasma once it reaches
systemic circulation. Although α-TOS has been shown to successfully permeate the
stratum corneum in this study, metabolism by esterases, which are present in the skin
and plasma may still jeopardise the therapeutic action of the drug. These findings
suggest that strategies to reduce or avoid significant metabolism by esterases, such as
BChE and AChE, would need to be employed in the development of transdermal
dosage forms of α-TOS for them to be clinically useful. Unless a sufficient quantity of
α-TOS is consistently delivered across the stratum corneum into the epidermis, to
maintain high sink conditions, this agent may be significantly metabolised to an inactive
form prior to systemic absorption. The findings of this in-vitro study must still,
however, be confirmed in in-vivo models before conclusive statements can be made.
89
CHAPTER 8. CONCLUSIONS
α-TOS is a potent antineoplastic agent with strong pro-apoptotic potential. Due
to pharmacokinetic problems associated with the oral administration of α-TOS, it is
essential to develop reliable methods to deliver intact drug and ensure maintenance of
sustained plasma levels. An investigation was therefore undertaken to evaluate the
feasibility of delivering intact -TOS systemically using the transdermal route of
administration.
Large quantities of material and methods for the identification and quantification
of α-TOS were required to undertake this study. NMR, MS, FTIR, DSC and TGA were
used to characterise raw material sourced from a non-scientific commercial supplier.
The sourced material provided an economically feasible way to undertake this current
study. Structure and purity of source material was confirmed and provided evidence of
its suitability for further investigation.
HPLC was chosen as the technique for the development and validation of assays
for the detection and quantification of α-TOS. Although methods had previously been
published on the HPLC analysis of α-TOS, they failed to produce reliable and
reproducible results when attempts were made to replicate them. Gradient and isocratic
elution methods were therefore modified in order to elute and resolve α-TOS. Although
evidence from validation studies confirmed the accuracy, linearity and precision of both
methods, only the gradient elution method produced reliable selectivity. Analysis of
spiked plasma samples prepared by LLE using the isocratic HPLC method revealed co-
eluting peaks, which compromised quantification. In contrast, the developed gradient
90
method provided complete resolution of all eluting peaks. The accuracy, precision and
selectivity of this gradient HPLC method confirmed its suitability for identifying and
quantifying α-TOS from extracted samples. In addition, LLE using ethanol
saponification and hexane partitioning was shown to be a suitable method for the
extraction of α-TOS from plasma and aqueous reservoir solution. Using the protocols
outlined in this study, LLE methods were shown to be superior to SPE. The LLE
method used in this study consistently delivered high extraction efficiencies and was
therefore used to isolate and concentrate α-TOS from collected samples. Further
investigations should, however, validate the use of these methods for the identification
and quantification of α-TOS from other biological matrices, such as urine, faeces and
tissue. These validations will have to be completed prior to undertaking in-vivo
pharmacokinetic studies of α-TOS.
The transdermal delivery α-TOS in a formulation containing high concentrations
of DMSO has been reported. This formulation, which contained of 50% w/w α-TOS,
was reported to maintain therapeutic concentrations of intact drug. Due to concerns
relating to the irritant properties of DMSO, this formulation is considered unlikely to be
feasible for clinical investigations. Liposomal formulation was considered to be the
most suitable alternative method for the transdermal delivery of α-TOS. The ability of
liposomes to encapsulate and solubilise highly lipid soluble drugs, such as α-TOS, was
considered particularly important. In addition, the ability of liposomal formulations to
enhance percutaneous absorption of relative large drugs, such as α-TOS, was relevant to
the objectives of this study. The pre-formulated liposomal dosage form, anhydrous
Lipoderm , was selected for this study. The percutaneous enhancer, 5% pentylene
glycol, was included in the investigation due to its moderate efficacy and low irritant
91
properties when compared to DMSO. Using this formulation, α-TOS was successfully
compounded into Lipoderm at concentrations up to 30% w/w. The method utilised
for the preparation of the Lipoderm dosage form in this study was economical, easy
and did not require complex formulatory equipment. The compounded formulation was
aesthetically pleasing, without visible presence of undissolved drug or unpleasant
odour. Increasing the concentration of α-TOS above 30% w/w in the formulation
resulted in the appearance of undissolved material. The Lipoderm formulation
containing 5% pentylene glycol appeared to solubilise 20% less α-TOS than the DMSO-
containing formulation.
In-vitro Franz diffusion cells, fitted with excised human skin, were used to
compare the percutaneous delivery of α-TOS from compounded formulations. Given
that rates of drug diffusion through the skin are concentration-dependent, it was not
surprising that liposomal formulation delivered less α-TOS at a slower rate than DMSO-
containing formulations. If the solubility of α-TOS in anhydrous Lipoderm could be
increased to an equivalent concentration as that contained by the DMSO-containing
formulation, it is possible that percutaneous delivery profiles may be comparable.
Irrespective, the Lipoderm formulation containing 5% pentylene glycol was shown to
effectively deliver α-TOS. These results are promising; however, further formulatory
research will need to be undertaken for optimisation of the transdermal dosage form.
Results obtained from the esterase study, raises concerns about the metabolic
fate of delivering α-TOS transdermally. The reported presence of esterases in the skin
and the observed affinity of α-TOS for these enzymes suggest that considerable
amounts of drug would be converted to the inactive form. In addition, plasma
92
inactivation of α-TOS would further reduce circulating levels of α-TOS. Although the
transdermal route of administration by-passes first-pass hepatic metabolism,
extrahepatic metabolism could effectively neutralise the pro-apoptotic effect of α-TOS.
The findings of this in-vitro study provide the first report of successful delivery
of α-TOS across the outer barriers of the skin using a readily available commercial
Lipoderm base. The findings of this study suggest that further optimisation of
Lipoderm formulations could lead to the development of transdermal dosage form
suitable for in-vivo pharmacokinetic studies, wherein the concerns identified in this
study can be further investigated.
93
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