Formulation and investigation of specialised dosage forms … · Formulation and investigation of...

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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

Transcript of Formulation and investigation of specialised dosage forms … · Formulation and investigation of...

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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

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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

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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.

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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

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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

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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

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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

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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

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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

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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

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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

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TGA Thermo gravimetric analysis

TGF-β Transforming growth factor β

TMS Tetramethylsilane

UV Ultraviolet

VE Vitamin E

VEGF Vascular endothelial growth factor

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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.

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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%.

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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.

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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

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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.

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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 -

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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].

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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,

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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,

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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.

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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

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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

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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

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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.

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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

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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

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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]

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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

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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.

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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

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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.

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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

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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.

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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].

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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.

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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].

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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

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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).

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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.

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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 -

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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.

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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

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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].

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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.

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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.

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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

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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.

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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.

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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).

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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).

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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

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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%.

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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).

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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).

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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

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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

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standard and readily available HPLC columns (i.e., C18 reverse phase HPLC column,

250 x 4.6mm, 5 m particle size).

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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.

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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

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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.

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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

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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.

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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.

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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.

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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.

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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]

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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]

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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.

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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

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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,

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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.

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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.

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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

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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

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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)

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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

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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.

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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.

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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 (*).

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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%.

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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

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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

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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

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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 (*).

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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]

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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

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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

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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.

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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

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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

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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 (*).

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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).

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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 (*).

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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.

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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 (*).

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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.

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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.

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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 (*).

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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.

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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 (*).

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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.

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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

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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

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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

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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.

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