SYNTHESIS, CHARACTERISATION AND IN VITRO ...eprints.usm.my/30117/1/FULL_THESIS_VIVA_EDIT_LV.pdf1.7 Aim of the thesis 30 CHAPTER TWO : SYNTHESIS OF TRITHIOL MONOMER 2.1 Introduction

SYNTHESIS, CHARACTERISATION AND IN

VITRO EVALUATION OF DISULPHIDE CROSS-

LINKED POLYMERS AS POTENTIAL

CARRIERS FOR COLON SPECIFIC DRUG

DELIVERY SYSTEM

LAU YONG KHEE

UNIVERSITI SAINS MALAYSIA

2015

SYNTHESIS, CHARACTERISATION AND IN VITRO EVALUATION OF

DISULPHIDE CROSS-LINKED POLYMERS AS POTENTIAL CARRIERS

FOR COLON SPECIFIC DRUG DELIVERY SYSTEM

by

LAU YONG KHEE

Thesis submitted in fulfillment of the

requirements for the Degree

of Master of Science

FEBRUARY 2015

i

To my beloved parents, Lau Leong Bee and Lim Guat Hong,

and my brother, Lau Kian Khee

ii

ACKNOWLEDGEMENTS

I own my most sincere thanks to Dr. Lim Vuanghao, my wonderful and

helpful supervisor for giving me the opportunity to carry out this study. I have been

very fortunate to have Dr. Lim, a great mentor who gave me guidance during this

course of work and provided me with constructive comments and criticism. I am

deeply grateful to Dr. Nurzalina Karim Khan and Dr. Lee Hooi Ling, both my great

co-supervisors who guided me and unrelenting supported me throughout my research.

I would also express my special thanks to my wonderful friends and

colleagues, Siti Aishah, Masturah, Nurul Azieyan, Asila Dinie and Hui Wen for their

continuous support, understanding, encouragement and technical assistance. My

deepest appreciation also goes to the director and staffs of IPPT, USM for all their

support and assistance rendered during the course of my study.

Finally, I extend my deepest thanks to my family and my dearest girlfriend,

Renly who suffered when I should have been at home with them rather than working

on this research.

Lau Yong Khee

February 2015

iii

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ii

TABLE OF CONTENTS iii

LIST OF TABLES xi

LIST OF FIGURES xii

LIST OF SCHEMES xvii

LIST OF ABREVIATIONS xviii

ABSTRAK xx

ABSTRACT xxii

CHAPTER ONE : INTRODUCTION

1.1 Backgroud 1

1.2 Anatomy of the colon 2

1.3 Microflora of the gastrointestinal tract 4

1.4 Colon-specific drug targeting 7

1.5 Strategies for targeting drugs to the colon 8

1.5.1 pH responsive delivery 8

1.5.2 Time responsive delivery 10

1.5.3 Pressure responsive delivery 12

1.5.4 Bacteria responsive delivery 13

1.5.4.1 Azo-containing prodrugs 13

1.5.4.2 Azo polymers 16

1.5.4.3 Glycosidic prodrugs 17

iv

1.5.4.4 Polysaccharides 18

1.5.4.5 Disulphide polymers 21

1.6 Literature review 22

1.7 Aim of the thesis 30

CHAPTER TWO : SYNTHESIS OF TRITHIOL MONOMER

2.1 Introduction 31

2.2 Objective 32

2.3 Materials 32

2.4 Physical characterisation of synthesised compounds 33

2.4.1 Qualitative screening of synthesised compound 33

2.4.2 Spectroscopic analysis 33

2.4.3 Liquid Chromatography-Mass Spectrometry (LC-MS) 33

2.4.4 Elemental analysis and melting point tests 34

2.5 Experimental procedures 34

2.5.1 Synthesis of (triphenylmethyl) thioethylamine (3) 34

2.5.2 Synthesis of N,N',N''-tris[2-(tritylsulfanyl)ethyl]propane-1,2,3-

tricarboxamide (5) (tricarballylic acid based trityl monomer) 35

2.5.3 Deprotection of (5) to yield N,N',N''-tris(2-sulfanylethyl)

propane-1,2,3-tricarboxamide (6) (tricarballylic acid based

trithiol monomer) 36

2.5.4 Attempted synthesis of N,N',N''-tris(2-sulfanylethyl)prop-1-

ene-1,2,3-tricarboxamide (8) (aconitic acid based trityl

monomer) 37

2.6 Results and discussion 40

2.6.1 Elucidation of (triphenylmethyl) thioethylamine (3) 40

2.6.2 Elucidation of N,N',N''-tris[2-(tritylsulfanyl)ethyl]propane-

1,2,3-tricarboxamide (5) 42

v

2.6.3 Elucidation of N,N',N''-tris(2-sulfanylethyl)propane-1,2,3-

tricarboxamide (6) 44

2.6.4 General discussion 46

2.7 Conclusion 50

CHAPTER THREE : SYNTHESIS OF DISULPHIDE CROSS-

LINKED POLYMERS

3.1 Introduction 51

3.2 Objective 51

3.3 Materials and methods 52

3.3.1 Materials 52

3.3.2 Polymerisation 52

3.3.2.1 Oxidation of trithiol monomers 52

3.3.2.2 Detection of thiol with sodium nitroprusside reagent,

Na2Fe(CN)5NO 53

3.3.2.3 Physical characterisation of the synthesised polymers 53

3.3.2.4 Scanning Electron Microscope-Energy Dispersive

X-ray (SEM- EDX) 54

3.3.2.5 Raman spectroscopy 54

3.4 Results 54

3.4.1 Physical characterisations of disulphide cross-linked polymers 54

3.4.1.1 Solubility test for disulphide cross-linked polymers 55

3.4.1.2 Characterisation of disulphide cross-linked polymers 56

3.4.1.3 Raman spectroscopy 63

3.4.1.4 SEM for morphology aspects and EDX micrographs 63

3.4.1.5 Elemental mapping 70

3.5 Discussion 76

vi

3.6 Conclusion 81

CHAPTER FOUR : CHEMICAL REDUCTION STUDIES OF

DISULPHIDE CROSS-LINKED POLYMERS

4.1 Introduction 82

4.2 Objective 82

4.3 Materials and methods 83

4.3.1 Materials 83

4.3.1.1 Reagent for assay of thiols 83

4.3.1.2 Materials for chemical reduction 83

4.3.1.3 Polymer substrates 83

4.3.2 Assay for thiol 84

4.3.2.1 Preparation of Sørensen’s phosphate buffer 84

4.3.2.2 Determination of thiol content using Ellman’s reagent 84

4.3.2.3 Application of Beer-Lambert equation 85

4.4 Chemical reduction 85

4.4.1 Reduction of cystamine dihydrochloride by zinc/acetic acid 85

4.4.2 Reduction of disulphide cross-linked polymers by zinc/acetic 85

acid

4.5 Results 87

4.5.1 Chemical reduction of cystamine dihydrochloride 87

4.5.2 Chemical reduction of disulphide cross-linked polymers 89

4.5.2.1 Chemical reduction of polymer P10 89




vii



4.6 Discussion 96

4.6.1 Chemical reduction of cystamine dihydrochloride 96

4.6.2 Chemical reduction of disulphide cross-linked polymers 96

4.7 Conclusion 98

CHAPTER FIVE : DISSOLUTION STUDIES OF DISULPHIDE

CROSS-LINKED POLYMERS

5.1 Introduction 99

5.2 Objective 100

5.3 Materials 100

5.3.1 Pure culture bacteria 100

5.3.2 Incubation media 101

5.3.3 Other materials 101

5.3.4 Polymer substrates 101

5.4 Experimental procedures 102

5.4.1 Bacteriological techniques 102

5.4.2 Preparation of pre-reduced anaerobically sterile media 102

(PRAS-WCAB)

5.4.3 Preparation of Robertson’s cooked meat media (CMM) 103

5.4.4 Preparation of WCAB Bacteroides fragilis test culture 103

5.4.5 Preparation of bacterial pellets 104

5.4.6 Preparation of Bacteroides fragilis stock cultures 104

5.4.7 Preparation of Sørensen’s phosphate buffer pH 7.4 104

5.4.8 Polymer sample preparation 105

viii

5.4.9 Preparation of simulated gastric fluid 105

5.4.10 Preparation of simulated intestinal fluid 106

5.4.11 Degradation in simulated gastric fluid 106

5.4.12 Degradation in simulated intestinal fluid 107

5.4.13 Incubation of disulphide cross-linked polymers with

Bacteroides fragilis in Sørensen’s phosphate buffer 107

5.4.14 Control incubations 108

5.4.15 Detection thiol concentration using Ellman’s reagent 108

5.5 Results 108

5.5.1 Dissolutions of polymers in simulated gastric condition 109

5.5.1.1 Dissolution of polymer P10 in simulated gastric 109

condition


condition


condition


condition


condition


condition

5.5.1.7 Dissolution of polymer P10 in simulated intestine 115

condition


condition


condition


condition

ix


condition


condition

5.5.1.13 Incubation of polymer P10 in simulated colon 121

condition


condition


condition


condition


condition


condition

5.5.1.19 Summary of the thiol concentrations detected from 127

each formulated polymer after dissolution studies

5.5.2 Statistical analysis results 128

5.6 Discussion 132

5.7 Conclusion 135

CHAPTER SIX : GENERAL DISCUSSION 137

CHAPTER SEVEN : FURTHER STUDIES 141

7.1 Employ 2,2’-thiodiethanethiol as alternative dithiol monomer for 141

polymerisation

7.2 Use iodine as the oxidising agent for polymerisation 142

7.3 Coating of gelatine capsule by using disulphide cross-linked 142

polymers

x

REFERENCES 143

APPENDICES

Appendix A 158

Appendix B 158

Appendix C 159

Appendix D 159

Appendix E 160

Appendix F 160

Appendix G 161

Appendix H 161

Appendix I 162

Appendix J 162

Appendix K 163

Appendix L 163

xi

LIST OF TABLES

Table 2.1

Table 2.2

Table 2.3

Table 3.1

CHNS analysis of (3).



Solubility test of the synthesised polymers at

different molar ratios.

41

43

45

55

Table 3.2 Morphology of synthesised disulphide polymers. 56

Table 4.1 Thiol concentration of chemical reduction for six

formulated disulphide polymers.

95

Table 5.1 Dissolution studies of detected thiol concentrations

(x 10-6

mol L-1

) from each formulated polymer after

dissolution studies.

127

Table 5.2 The thiol concentration (x 10-6

mol L-1

) at hour 2 of

stimulated gastric condition, hour 3 of stimulated

intestine condition and hour 70 of stimulated colon

condition Mean ± SD, N = 3. Incubation medium of

s colon condition containing polymer + bacteria (3)

is controlled sample.

129

Table 5.3 The thiol concentration (x 10-6

mol L-1

) of different

incubation medium at hour 70 in simulated colon

condition. Mean ± SD, N = 3. Incubation medium

containing polymer + bacteria (3) is controlled

sample.

130

Table 5.4 The statistical analysis results of thiol concentration

(x 10-6

mol L-1

) of various molar combinations of

polymers at incubation time of 5, 30, 70 hours in

simulated colon condition containing Bacteroides

fragilis. Mean ± SD, N =3.

131

xii

LIST OF FIGURES

Figure 1.1 Structure of the colon (large intestine) (after

Wikipedia, 2014).

3

Figure 1.2

Concentration of microflora in different regions of

gastrointestinal tract. 5

Figure 1.3 Mechanism of enzymatic reduction of sulphasalazine

in the colon.

14

Figure 1.4 Molecular structure of new-generation prodrugs of 5-

ASA.

15

Figure 1.5 TFA deprotection of triphenylmethyl (a) and

TFA/TES scavenger deprotection of triphenylmethyl

(b).

23

Figure 1.6 Disulphide bond formation by oxidation using

DMSO (R2SO).

25

Figure 1.7 Proposed disulphide polymer network from

oxidation of solely trithiol monomers.

26

Figure 1.8 Proposed disulphide polymer network from

oxidation of trithiol and dithiol monomers.

27

Figure 2.1 FT-IR spectrum of (3). 41



Figure 3.1 FT-IR spectrum of polymer P10. 57





Figure 3.6

Figure 3.7

FT-IR spectrum of polymer P51.

Raman spectroscopy of polymer P15.

62

63

Figure 3.8 (a) Scanning electron micrograph of polymer P10 at

(i) 150X; (ii) 300X; (iii) 1000X; (b) scanning area

for EDX micrograph, (c) EDX micrograph of

polymer P10.

64

xiii




polymer P11.

65

Figure 3.10 (a) Scanning (a) Scanning electron micrograph of polymer P12 at



polymer P12.

66




polymer P15.

67




polymer P21.

68




polymer P51.

69

Figure 3.14 SEM micrograph at 300 X of polymer P10 and

elemental maps for carbon (C), oxygen (O), sulphur

(S) and nitrogen (N) for the same region obtained by

EDX mapping.

70




EDX mapping.

71




EDX mapping.

72




EDX mapping.

73




EDX mapping.

74




75

xiv

EDX mapping.

Figure 3.20 Disulphide polymer network from oxidation of

trithiol monomers.

78

Figure 3.21 Disulphide polymer network from oxidation of

trithiol and dithiol monomers.

78

Figure 4.1 Chemical reduction of cystamine by zinc/acetic acid

(mean, n = 3).

87

Figure 4.2 Standard calibration curve of cystamine. 88

Figure 4.3 Chemical reduction curves of polymer P10 using

zinc/acetic acid.

89


zinc/acetic acid.

90


zinc/acetic acid.

91


zinc/acetic acid.

92


zinc/acetic acid.

93


zinc/acetic acid.

94

Figure 5.1 Thiol concentration as a function of dissolution time

for polymer P10 over 120 mins. Dissolution was

carried out in 0.1 mol L-1 hydrochloric acid

containing NaCl and pepsin powder. Mean ± SD, n

= 3.

109



carried out in 0.1 mol L-1

hydrochloric acid


= 3.

110




hydrochloric acid


= 3.

111




hydrochloric acid


112

xv

= 3.




hydrochloric acid


= 3.

113




hydrochloric acid


= 3.

114



carried out in Sørensen’s phosphate buffer pH 6.8

containing pancreas powder. Mean ± SD, n = 3.

115





116





117





118





119

Figure 5.12 Thiol concentration as a function of incubation time




120


for polymer P10 over 70 hours period. Incubation

was carried out in Sørensen’s phosphate buffer pH

7.4 containing, ( ) Bacteroides fragilis and polymer

P10, ( ) polymer P10 only without bacteria, ( )

bacteria only without polymer P10 Mean ± SD, n =

3.

121

xvi







3.

122







3.

123







3.

124







3.

125







3.

126

Figure 7.1 Molecular structure of 2,2’-thiodiethanethiol. 141

xvii

LIST OF SCHEMES

Scheme 1.1 Reaction of Ellman’s reagent with thiol. 29

Scheme 1.2 Disulphide exchange of Ellman’s mixed

disulphide with thiol.

29

Scheme 2.1 Synthesis of (triphenylmethyl) thioethylamine (3). 35

Scheme 2.2 Synthesis of N,N',N''-tris[2-

(tritylsulfanyl)ethyl]propane-1,2,3-tricarboxamide

(5).

36

Scheme 2.3 Deprotection process to yield N,N',N''-tris(2-

sulfanylethyl)propane-1,2,3-tricarboxamide (6).

37

Scheme 2.4 Attempted synthesis of N,N',N''-tris(2-

sulfanylethyl)prop-1-ene-1,2,3-tricarboxamide

(8).

39

Scheme 2.5 Proposed mechanism for the formation of amide

bond using EDC.

48

Scheme 2.6 Synthetic routes for preparing (6). 49

xviii

LIST OF ABBREVIATIONS

GI Gastrointestinal

IBD Inflammatory bowel disease

5-ASA 5-aminosalicylic acid

HEMA 2-hydroxyethyl methacrylate

MMA Methylmethacrylate

BMAAB bis(methacryloylamino)azobenzene

SHIME Simulated human intestinal microbial ecosystem

TFA Trifluoroacetic acid

TES Triethylsilane

DTNB 5,5’-dithiobis-2-nitrobenzoic acid

TNB2-

5-thio-2-nitrobenzoic acid

SEM Scanning electron microscopy

EDX Energy dispersive X-ray

TLC Thin layer chromatography

FT-IR Fourier transform infrared

NMR Nuclear magnetic resonance

1H-NMR Proton nuclear magnetic resonance

13C-NMR Carbon nuclear magnetic resonance

LC-MS Liquid chromatography-Mass spectroscopy

EDC N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide

DMSO Dimethylsulphoxide

DCC N, N”-dicyclohexylcarbodiimide

DCM Dichloromethane

xix

DCU Dicyclohexylurea

HOBt 1-hydroxybenzotriazole hydrate

EDTA Ethylenediaminetetraacetic acid

WCAB Wilkens-Chalgren Anaerobic Broth

CMM Robertson’s cooked meat media

ANOVA One-way analysis of variance

HSD Honestly Significant Difference

s Singlet

d Doublet

t Triplet

m Multiplet

et al. et alii, others

h. hour

min minute

Rf Resolution factor

ml Milliliter (s)

MHz Mega Hertz

oC Degree Celsius

L Liter

mm Millimeter

xx

SINTESIS, PENCIRIAN DAN PENILAIAN SECARA IN VITRO BAGI

POLIMER DISULFIDA BERANGKAI-SILANG SEBAGAI

PENGANGKUTAN BERPOTENSI UNTUK SISTEM PENYAMPAIAN UBAT

KHAS KEPADA KOLON

ABSTRAK

Sistem penyampaian drug ke kolon adalah sangat berguna untuk menyampaikan drug

untuk rawatan penyakit kolon setempat seperti penyakit keradangan usus, ulser

kolitis dan penyakit Crohn. Polimer disulfida berangkai-silang telah mendapat

perhatian kerana keupayaannya untuk bertindak sebagai polimer sensitif redoks dan

ikatan disulfida hanya akan dilekangkan oleh persekitaran yang mempunyai potensi

redoks yang rendah di dalam kolon. Kebelakangan ini, polimer rantai bercabang

disulfida telah menerima perhatian yang lebih disebabkan oleh hakikat bahawa

polimer ini kurang terdedah untuk degradasi dalam keadaan pH rendah di perut

berbanding dengan polimer rantai linear disulfida. Oleh itu, tujuan kajian ini ialah

untuk mensintesiskan monomer trithiol berasaskan asid trikarbalilik untuk

pempolimeran ke polimer rantai bercabang disulfida. Polimer sintetik telah dicirikan,

dikaji secara kimia dan dinilai dengan menggunakan kajian disintegrasi secara in

vitro. Monomer ini telah disintesiskan melalui tindak balas gandingan amida antara

asid trikarbalilik dan (trifenilmetil) thioetilamina dengan menggunakan dua langkah

sintesis. Monomer tersebut dinyahlindungkan dengan menggunakan asid

trifluoroasetik dan trietilsilana untuk mendedahkan atom sulfur dalam persediaan

untuk pempolimeran selanjutnya. Serbuk pepejal putih diperolehi dengan hasil

xxi

lebih kurang 20-25%. Keputusan spektroskopi dan analisis unsur CHNS

mengesahkan monomer yang dikehendaki. FT-IR menunjukkan kehadiran puncak

amida dan disokong oleh analisis jisim menggunakan LC-MS. Pempolimeran secara

pengoksidaan N,N',N''-tris(2-sulfaniletil)propana-1,2,3-trikarboksamida (monomer

trithiol) dan 2,2'- (etilenadioksi)dietanathiol (monomer dithiol) dengan pelbagai

nisbah molar menghasilkan enam (6) polimer yang berbeza, dinamakan, P10, P11,

P12, P15, P21 dan P51. Spektroskopi Raman menunjukkan kemunculan ikatan

disulfida. Kajian reduksi kimia menunjukkan bahawa semua polimer disulfida telah

diturunkan dengan lengkapnya selepas 1 jam dengan kepekatan thiol yang berbeza.

Dalam keadaan simulasi gastrik dan usus kecil, semua polimer menunjukkan

kepekatan thiol yang rendah jika dibandingkan dengan kepekatan thiol yang dikesan

dalam keadaan simulasi kolon dengan kehadiran Bacteroides fragilis. Keputusan ini

membuktikan bahawa semua polimer disulfida dapat bertahan dalam persekitaran

gastrik dan usus kecil yang kasar dan degradasi hanya akan berlaku dengan

kehadiran bakteria anaerobik di kolon. Degradasi didapati lebih ketara dalam

polimer P15 menunjukkan kepekatan thiol yang tertinggi, iaitu 57 x 10-6

mol L-1

berbanding polimer yang lain. Keputusan ini memberi persetujuan umum bahawa

biodegradabiliti bergantung pada kebolehkembangan polimer-polimer ini dalam

keadaan akues. Penyelidikan ini telah mengidentifikasikan polimer berangkai silang

disulfida yang mungkin mempunyai potensi untuk sasaran ke kolon. Walau

bagaimanapun, kajian selanjutnya diperlukan bagi mencapai kestabilan dan

penggunaannya sebagai polimer dalam bentuk dosaj farmaseutikal.

xxii

SYNTHESIS, CHARACTERISATION AND IN VITRO EVALUATION OF

DISULPHIDE CROSS-LINKED POLYMERS AS POTENTIAL CARRIERS

FOR COLON SPECIFIC DRUG DELIVERY SYSTEM

ABSTRACT

Colon drug delivery system is very useful to deliver drugs for treatment of localised

colonic diseases such as inflammatory bowel disease, ulcerative colitis and Crohn’s

disease. Disulphide cross-linked polymer has received much attention because of its

ability to act as a redox sensitive polymer and will only be cleaved by the low redox

potential environment in the colon. Recently, branch-chained disulphide polymers

had received more attention due to the fact that it is less susceptible for degradation

in low pH condition of the stomach compared to linear-chained disulphide polymers.

Therefore, the aim of this work is to synthesise tricarballylic acid based trithiol

monomer for polymerisation into branch-chained disulphide polymers. The

synthesised polymers were characterised, studied chemically and evaluated using in

vitro disintegration studies. The monomer was synthesised by amide coupling

reaction between tricarballylic acid and (triphenylmethyl) thioethylamine by using

two synthetic steps. The monomer was deprotected using trifluoroacetic acid and

triethylsilane to expose the sulphur atoms in preparation for further polymerisation.

White powdery solid was obtained with yield around 20-25%. Spectroscopic and

CHNS elemental analysis results complemented with the desired monomer. FT-IR

showed the presence of amide peaks and supported by the mass analysis using LC-

MS. The oxidative polymerisation of N,N',N''-tris(2-sulfanylethyl)propane-1,2,3-

tricarboxamide (trithiol monomer) and 2,2’-(ethylenedioxy)diethanethiol (dithiol

xxiii

monomer) of various molar ratios resulted six (6) different polymers, namely, P10,

P11, P12, P15, P21 and P51. Raman spectroscopy demonstrated the emergence of

disulphide bond. Chemical reduction studies showed that all the disulphide polymers

were completely reduced after 1 hour of reduction with different thiol concentrations.

In simulated gastric and intestinal condition, all the polymers showed low thiol

concentrations when compared to the detected thiol concentrations in simulated

colon condition with the presence of Bacteroides fragilis. The results proved that all

the disulphide polymers were able to withstand the harsh environment of the gastric

and intestinal condition and degradation will only occur in the colon condition with

the presence of anaerobic colonic bacteria. Disintegration was found to be more

pronounced in polymer P15 as the highest thiol concentration of 57 x 10-6

mol L-1

among all the other polymers. This result correlated the general consensus that

biodegradability relies on the swelling ability of polymers in an aqueous environment.

This work has successfully identified a disulphide cross-linked polymer, which might

be potential for colon specific targeting. However, further study on these polymers is

required to establish the stability and use of the polymer within a pharmaceutical

dosage form.

1

CHAPTER ONE

INTRODUCTION

1.1 Background

To date, oral drug delivery is the most preferred, common, convenience and widely

accepted route among many other routes of drug administration (Rubinstein, 1995;

Laura et al., 2011). Upper gastrointestinal (GI) tract appeared to be the major region

for dissolution and absorption of orally administered drug. However, this approach

is not suitable for drugs to be absorbed in lower GI tract or advanced biotechnology

products such as peptides and proteins, whereby undesirable side effects and

treatment failure will occur. Therefore, studies had been done in developing specific

drug targeting especially to lower GI tract. Colon specific drug delivery had

received much interest with much work already been carried out in this area of drug

delivery (Yan et al., 2010; Kenawy et al., 2011; Ursekar et al., 2012; Lim et al.,

2013). The role of colon specific drug delivery is not only limited for localised

treatment but also crucial for systematic treatment (Tozer et al., 1995). Although

colon specific drug delivery can also be achieved via rectal route but this route

appeared to be less readily accepted and less appealing to patients. Moreover, study

showed that it is difficult to deliver drug to the proximal colon via the rectal route

(Ritschel, 1991).

Colon specific drug delivery is particularly important in localised treatment of colon

diseases such as inflammatory bowel disease (IBD), ulcerative colitis, Crohn’s

2

disease and colorectal cancer (Gupta et al., 2001; Rubinstein, 2005; Yan et al., 2010).

The nature of colon which is less hostile has a near neutral pH and lower activity of

peptidase enzyme favours the systemic absorption of protein and peptide drugs

(Maroni et al., 2012).

1.2 Anatomy of the colon

The GI digestive tract is a system of organs in human which responsible in taking

food, digesting it to extract energy and nutrients and eliminating waste. The normal

human adult male GI tract consists of the upper and lower GI tracts and is

approximately 6.5 meters long (Anthea et al., 1993).

The colon, or large intestine, is located at the distal end of the GI tract (Figure 1.1).

It is a muscular tube with a length of approximately 1.5 m long, has an average

diameter of approximately 6.5 cm, with a diameter that varies from 2 cm in the

sigmoid colon to 9 cm in the caecal region, running from the ileocaecal valve to the

anus (Cummings and MacFarlane, 1991). The human colon is composed of the

caecum, ascending segment, transverse segment, descending segment and sigmoid

region. Colon plays an important role in extracting water and salt from solid wastes

before they are eliminated from the body, storing waste, absorbing some vitamins

and providing a site for flora-aided fermentation. Unlike the small intestine, the

colon does not play a major role in absorption of foods and nutrients. Colon does not

have villi although it does demonstrate crescentric folds, which increase the internal

surface area of the colon to approximately of 1300 cm2 (Cummings and MacFarlane,

1991). Guinea pigs and rodents models were commonly used in the study of colonic

3

drug delivery (Friend, 1991). However, there are differences between the animal

models with normal human. The guinea pig has a very large caecum whereas

humans have a poorly defined caecal region continuous with the colon (Friend, 1991).

The transit time was found to be shorter in guinea pigs and slightly longer in rats if

compared to humans (Pettersson et al., 1976). The mucosa of the colon is lined

almost entirely by goblet mucus secreting cells, whereby the large amount of

bicarbonate ions from the mucus maintains the basic pH of the colonic region

(Binder and Sandle, 1994).

Figure 1.1 : Structure of the colon (large intestine) (after Wikipedia, 2014).

4

1.3 Microflora of the gastrointestinal tract

The upper GI tract is sparsely populated with micro-organisms. The acidic

environment of the stomach exhibits a strong bactericidal activity, whereby the

microbial concentration is usually less than 103 cfu/ml (Ilett et al., 1990). Acid-

tolerant micro-organisms such as gram-positive aerobic streptococci and lactobacilli

are the main inhabitants in the stomach (Hao and Lee, 2004). The upper two-thirds

of the small intestine which includes duodenum and jejunum contain similar bacterial

concentration to the stomach, between 103 to 10

4 cfu/ml. However, gram-negative

bacteria begin to outnumber gram-positive bacteria in the distal ileum. The reduction

in acidity and lower redox potentials play a major role in maintaining a more diverse

microflora and higher bacteria population (Tannock, 1983).

Colon is the primary site of microbial colonisation in human body. The colon

contains a very large quantity of bacteria from various species. The bacterial

concentration in the colon is around 1011

to 1012

cfu/ml and approximately one third

of the fecal dry weight consists of bacteria (Simon and Gorbach, 1984). The increase

in bacterial concentration in this region is due to a near neutral pH caused by

neutralisation of the bowel contents by intestinal juice and lower motility rate of the

colon. However, 99.9 % of colonic microflora is obligate anaerobes (Hao and Lee,

2004). There are over 400 different bacterial species found in the colonic region.

The predominant species isolated include Bacteroides, Eubacterium, Bifidobacterium

(Hill and Drasar, 1975). Anaerobic gram-positive Cocci, Enterococci, Clostridia and

other species of Enterobacteriaceae, Coliforms, Staphylococci, Lactobacillus,

Spirochetes, Pseudomonas, Proteus, Clostridium, Actinomyces, Borrelia,

5

Fusobacterium and Diphtheroides species are also found. However, Bacteroides

species account for nearly 32 % of all bacterial flora isolated from the GI tract

(Finegold et al., 1977). The comparison of microbial concentration in different

regions of GI tract is shown in Figure 1.2.

Figure 1.2 : Concentration of microflora in different regions of gastrointestinal tract

(taken with modification from Basit, 2005).

Certain diseases or antibiotic treatment will influence the microbial flora in the gut

(Friend and Tozer, 1992). The nature of certain disease, where reduction of gastric

acid secretion occurred, will lead to increase in bacterial growth in the stomach

(Rowland, 1988). The speed of peristalsis movement decreases from the jejunum to

upper ileum and the distal ileum. As it is approaching the ileo-caecal valve,

peristalsis slows down to an almost stagnant state and this has been the major factor

which cause the drastic rise in the bacterial population in this area. The bacteria

6

populations found in this area are mainly Bacteroides, Enterobacteria and

Bifidobacteria. Huge changes of some organisms in the region within the gut can

occur when antibiotic treatments are introduced where it has bactericidal effect and

suppresses the bacterial growth (Goldin and Grobach, 1977).

Colon is well known for its reductive environment, where the redox potential

becomes more negative from the small intestine to the large bowel. The redox

potential of proximal small bowel is -67 ± 90 mV, -196 ± 97 mV in the distal small

bowel and -415 ± 72 mV in the colon (Stirrup et al., 1990). The presence of the

bacterial microflora causes the changes in the redox potential along the GI tract. Due

to its low redox potential, the colonic region is very active in reductive and

degradative reactions (Rowland, 1986).

Bacterial microflora in the GI tract produce a wide range of reductive and hydrolytic

enzymes which include β-glucoronidase, β-galactosidase, β-xylosidase, azoreductase,

nitroreductase, urea hyroxlase and α-arabinosidase (Kinget et al., 1998). The ability

of colonic anaerobic bacteria to produce appropriate digestive enzyme in order to

digest the constantly changing carbohydrates complex was exploited for the

development of enzyme-sensitive prodrugs to achieve colonic drug targeting (Sinha

and Kumria, 2001a; Sinha and Kumria, 2001b).

7

1.4 Colon-specific drug targeting

The concept of delivering drug to the desired site is not new. All drugs exert their

beneficial effects along with undesirable side effects to varying degrees of severity.

Therefore, site-specific drug delivery is important by enhancing the beneficial effects

and limiting the side effects. The formulated dosage form for colon-specific drug

delivery must pass through the upper GI tract intact before delivering the drug to the

colonic region.

Physiological factors in the upper GI tract such as extreme acidity in stomach,

degradative enzyme activities and presence of bile salts will destroy the drug

molecules that are intended to colon, especially peptides and proteins. This will

result in poor bioavailability of drugs and treatment outcome. Due to the barriers of

the upper GI tract, colon has been focused as the targeting site for orally

administered drug. Numerous investigations have shown that some anti-

inflammatory (Wilson and Washington, 1989) and antidiabetic (Gleiter et al., 1985)

drugs are better absorbed from the colon rather than the upper GI tract.

The colon is found to have prolonged residence time for the colonic contents (Coupe

et al., 1991). The average transit time through the colon is 22-36 hours and it is

much higher if compared to the transit time in the stomach (0.5-3 hours) and the

small intestine (1-6 hours) (Coupe et al., 1991). On top of that, colonic transit time

has been shown to be independent of size and density of the dosage form (Parker et

al., 1988). The ideal property for a colon-specific drug delivery system is to spend

as little time as possible in the upper GI tract (Friend, 1991). Therefore, the

8

prolonged colonic transit time provides a longer time interval for drug absorption in

the colonic region. This will compensate the smaller surface area for absorption in

the colon compared to the small intestine.

1.5 Strategies for targeting drugs to the colon

The main responsibility of a colonic drug delivery system is to utilise the special

characterisitics of the colon in order to achieve localised drug release. The main

concept of orally administered colon-specific drug delivery is where the dosage form

must be able to withstand the harsh environment of the stomach and small intestine

but to achieve drug release in the colon. The major characteristics of the GI tract

include pH, transit time, enzymes, bacteria and pressure (Basit, 2005; Bhalerao and

Patel, 2012) were taken into considerations in order to develop a good formulation

for colonic drug delivery. These strategies are currently being studied and will be

further discussed below.

1.5.1 pH responsive delivery

pH in human GI tract changes when it shifts from one region to another. The

environment of the stomach is acidic (pH 1-2) and the pH increases in the small

intestine region (pH 6.5-7). The pH in the terminal ileum and colon (pH 7-8) is the

highest in the human GI tract. The concept of pH responsive delivery was designed

by using materials that are insoluble in acidic pH but dissolve in neutral to slightly

basic pH (Ibekwe et al., 2006). However, pH in the terminal ileum region is higher

than in the caecum. Thus, the dosage forms are often delayed at the ileocecal

9

junction. In short, the concept of using polymers that are sensitive to higher pH was

employed and adapted for colonic drug delivery instead of conventional enteric

coating (Edsbacker and Anderson, 2004).

Acrylic polymers from the Eudragit® range are commonly used for this purpose

because of its solubility when expose to pH above 6 (Basit, 2005). Eudragit-based

polymers can be made to dissolve at various basic pH by modifying the methylester

content (Peeters and Kinget, 1993). Eudragit® S and Eudragit® L were designed to

be specifically dissolved in pH 7 and pH 6 respectively. Dew et al. (1982) were the

first to employ Eudragit® S as a capsule coating by utilising gastrointestinal pH as a

trigger for drug release in the distal gut. The results were positive in most patients as

the capsule disintegrated in the distal gut. However, Ashford et al. (1993a, 1993b)

found that the drug release of Eudragit® S-coated tablets in the colon of healthy

volunteers was not adequately reproducible. The reason behind was due to the

decrease in pH after passage through the ileocaecal valve. Therefore, Eudragit® S

coating dissolves and releases its drug content in the ileum rather than colon.

Despite such evidence of premature release of drug contents in the ileum,

preparations include Asacol® (Eudragit® S) and Claveral

® (Eudragit® L) have been

formulated using which have approval for human use. The preparations were found

to be successful in treatment of IBD (Hardy et al., 1987). In order to compensate the

low release pH of Eudragit® L, a thicker layer was formulated. The release of drug

contents (5-aminosalycilic acid) from both preparations is reported to take place at

the distal ileum and right colon.

10

1.5.2 Time responsive delivery

The size, shape and density of the dosage form and the feed status of the individual

are the major factors which will affect the GI transit time (Davis et al., 1984;

Devereux et al., 1990). Residence time in the stomach varies from a few seconds to

few hours. However, small intestine transit time was found to be more consistent at

3-4 hours, without being affected by feed status of the individual (Davis et al., 1986).

The consistent transit time of small intestine has been exploited in developing colon

drug delivery system. Time responsive delivery is formulated to release their drug

contents after a predetermined lag time. Assumption was made by researchers,

whereby 5-6 hours are needed for the ingested dosage form to reach the colonic

region. Therefore, a nominal lag time of 5 hours is usually considered sufficient

(Davis et al., 1986).

The pioneer time responsive delivery system was developed by Wilding et al. (1992),

namely, PulsincapTM

device. The device consists of an impermeable hard gelatin

capsule sealed at one end with hydrogel plug. Upon contact with gastrointestinal

fluid, the plug hydrates and begins to swell and after a pre-set lag time, ejects from

the capsule body, enabling drug release to occur. The size and composition of the

plug are crucial in controlling the lag time of the entire device. Two different

PulsincapTM

devices with different pre-set lag time of 5 and 6 hours respectively

were studied in fasted human subjects using gamma scintigraphy (Hebden et al.,

1999). The authors found that the location of the device varied in human body at the

time of release. Further study reported that the device exhibits poor emptying feature

11

especially in distal bowel. Lack of fluid in the distal bowel appeared to be the major

factor which caused the drawback (Wilding, 2000).

Newer colon drug delivery system was described by merging of both pH and time

responsive mechanisms in order to increase the effectiveness (Ishibashi et al., 1998).

The system comprises of a conventional hard gelatin capsule which contains mixture

of drug content and organic acid. The outer surface of the capsule is coated with

three layers, including outer enteric layer, hydrophilic layer and acid-soluble layer.

The entire system was designed to survive in the harsh condition of the stomach.

Subsequently, the capsule will reach a higher pH condition in the small intestine,

whereby the outer enteric layer will dissolve followed by the intermediate

hydrophilic layer. The inner acid-soluble layer which dissolves at pH below 5 will

survive the condition of the intestine. However, fluid will enter the capsule and lead

to the dissolution of the organic acid. pH reduction occurs followed by the

dissolution of the acid-soluble layer, leading to drug release from the entire system.

Study was done on fasted and fed volunteers but the results showed variability for

the site of disintegration (Ishibashi et al., 1998).

From the report of several studies, individual variability is a major influence towards

the gastrointestinal transit time (Coupe et al., 1991). Moreover, transit was found to

be faster in the morning if compared to the evening (Hebden et al., 1999). Time

responsive delivery system was unable to detect individual’s gastrointestinal transit,

which limits the practicality of the formulation.

12

1.5.3 Pressure responsive delivery

Higher pressures are encountered in the colon as a result of peristalsis movement.

The grinding, propulsion and motility of the luminal contents were found to be

generating pressure due to the muscle contraction of the intestinal wall. However,

the intensity and duration of the generated pressure varies throughout the GI tract

(Takaya et al., 1995). The colon is believed to exert a higher effective luminal

pressure in a viscous environment. A capsule utilising pressure responsive system

was developed by Takaya et al. (1995), using a hydrophobic polymer ethycellulose.

Drug release can be achieved after the successful disintegration of the capsule due to

the luminal pressure of the colon. The thickness of the ethylcellulose layer plays a

crucial role for the disintegration of the dosage form.

Several works had been done on methods of manufacturing pressure responsive

delivery system. Takaya et al. (1995) had successfully coated the inner surface of

the gelatin capsule with hydrophobic ethylcellulose, Hu et al. (2000) reported a low

temperature coating of the capsule-shaped pieces of suppository base. The dipping

method into both ethanolic ethylcellulose and alkalised enteric polymer solution was

conducted by Jeong et al. (2001). Investigations were made for these formulations

which involved pharmacokinetic evaluation. Correlation between times for

appearance of drug in plasma with literature values for colon arrival times had been

done. However, the results did not conclusively show successful disintegration in

the colon due to gastric and intestine transit times are known to be variable.

13

1.5.4 Bacteria responsive delivery

Majority of the bacteria in the human colon are anaerobic in nature. These anaerobic

bacteria are responsible in producing various types of enzyme to metabolise

substrates, such as proteins and carbohydrates, which escape from the upper GI tract

(Cummings et al., 1989). Materials which are susceptible to the enzymatic

degradation in the colon but able to survive the condition of gastric and small

intestine, could be designed as carriers for colonic drug targeting. These enzyme

sensitive materials depend heavily on the enzyme-generating microflora in the colon.

The enzymes generated include azoreductase, peptidase, esterase and glycosidase.

To date, bacteria responsive colon drug delivery has received much attention

(Vandamme et al., 2002; Sinha and Kumria, 2003; Chourasia and Jain, 2004; Ibekwe

et al., 2008).

1.5.4.1Azo-containing prodrugs

Sulphasalazine appeared to be the first bacteria responsive colon drug delivery

system being invented. It is a prodrug consisting of the active drug content

(mesalazine) linked by an azo bond to a carrier molecule (sulphapyridine)

(Peppercorn and Goldman, 1972; Svartz, 1988). From the study done by Myers et al.

(1987), mesalazine was found to be rapidly absorbed from the small intestine.

However, absorption is inhibited in the gastric and small intestine until it reaches

colon when the prodrug is ingested. Bacteria in the colon will release enzyme such

as azo-reductase to cleave the azo bond of the prodrug, leading to release of the

active drug content of 5-aminosalicylic acid (5-ASA) at the inflammation region.

14

Klotz (1985) reported that approximately 85% of an orally administered

sulphasalazine survived unabsorbed into the colonic region.

Enzymatic reduction will break down sulphasalazine into 5-aminosalicylic acid (5-

ASA) and sulphapyridine as shown in Figure 1.3. 5-ASA is generally unabsorbed in

the colon where it is responsible in providing topical anti-inflammatory activity

(Peppercon, 1984). However, sulphapyridine is well absorbed in the colon (Klotz,

1985) and causes undesirable side effects such as allergic reactions, which occurs in

up to 50% of recipients.

Figure 1.3 : Mechanism of enzymatic reduction of sulphasalazine in the colon.

Sulphasalazine

Enzymatic reduction

5-aminosalicylic acid Sulphapyridine

15

In order to solve the toxicity issue of sulphapyridine, mesalazine has been azo

bonded to another mesalazine molecule, namely olsalazine (Campbell and Berlingdh,

1998) or inert carrier, namely balsalazide and ipsalazide (Chan et al., 1983).

Molecular structures of olsalazine, balsalazide and ipsalazide are shown in Figure 1.4.

These methods provide alternative prodrugs for use in IBD which can prevent the

toxicity problem of sulphapyridine.

Figure 1.4 : Molecular structure of new-generation prodrugs of 5-ASA.

Olsalazine

Balsalazide

Ipsalazide

16

1.5.4.2 Azo-polymers

Azo-reductase is a type of enzyme which is commonly found in human colon. The

use of bacterial azo-reductase to split prodrug sulphasalazine into sulphapyridine and

5-ASA was the first colon specific targeting system. Subsequently, several studies

were carried out by exploiting the azo-reductase enzyme as a release mechanism for

drugs within the colonic region. Coating of peptide insulin with polymers cross-

linked with azoaromatic group has been discovered to protect insulin from the harsh

environment of stomach and small intestine. Thereafter, azo bonds were reduced in

the colon, leading to the release of insulin (Saffran et al., 1986). Further study was

conducted to deliver similarly coated insulin dosage forms to the colon of diabetic

dogs (Saffran et al., 1990) and the release mechanism was concluded to be caused by

the enzymatic degradation of the azo-polymer coatings in the colon. The increase in

insulin level and reduction in glucose production and glucose level were found to be

statistically significant.

Copolymers were employed for potential colon drug targeting by Van den Mooter et

al. (1992, 1993 and 1995). Copolymers composing of 2-hydroxyethyl methacrylate

(HEMA) and methylmethacrylate (MMA) were prepared in the presence of a

bifunctional azo compound, which is bis(methacryloylamino)azobenzene (BMAAB).

Different molar ratios of HEMA and MMA were used in order to obtain different

copolymers. Copolymer comprised of 1:6 molar ratio of MMA/HEMA and polymer

without the addition of MMA showed significant bacterial reduction both in vitro

and in vivo. Both of the polymers demonstrated good resistant in simulated gastric

17

acid and intestinal fluid. Conclusions were made that the monomer composition has

a greater influence than the structure of the cross-linking agent.

Hydrophilic azo-polyamides were studied as a potential colonic drug targeting by

Schact et al. (1991). The films were found to be completely dissolved under

reductive medium (-430 mV) and simulated human intestinal microbial ecosystem

(SHIME). The degradation was detected by observing the colour changes from pink-

orange to white/colourless. Lloyd et al. (1994) suggested that complete reduction of

azo bonds to amine would be favorable for effective drug release. Hydrophilic

polymers appeared to meet the requirement because of reduction of such polymers

are reported to go toward amine formation. Meanwhile, hydrophobic polymers

reduction stops at the hydrazo stage which appeared to be undesirable (Schact et al.,

1996). Recent studies showed similar results in degradation of azo dyes by bacteria

(Pandey et al., 2007; Hong et al., 2008).

1.5.4.3 Glycosidic prodrugs

Colonic bacteria provide glycosidase activity by generating glycosidase enzyme

which forms the fundamental of a colon specific drug delivery system. Friend and

Chang (1984 and 1985) utilised corticosteroid prodrugs as glycosidic carrier by the

attachment of the active agent. Drug glycosides are poorly absorbed from small

intestine due to its hydrophilic nature. The drug glycosides can be cleaved by

glycosidase enzyme upon reaching colonic region, releasing the active drug contents

which will be absorb by the colonic mucosa. Several studies have reported that

18

absorption occur for corticosteroids obtained from hydrolysis of glycosidic prodrug

in the colonic region.

Strategy of glycosidic prodrug was evaluated in rats with two steroid prodrugs,

prednisolone 21-β-D-glucoside and dexamethasone 21-β-D-glucoside (Tozer et al.,

1991). The potential of glycosidic prodrug is assessed by determining the rates of

its hydrolysis down the alimentary canal of the guinea pig. It was found that the

caecum and colon contents showed greater hydrolytic activity than the stomach and

small intestine contents. On top of that, the movement and hydrolysis of the prodrug

down the GI tract of the guinea pig were also examined. The authors reported

approximately 20 to 30 % of an oral dose appeared to reach the caecum, whereby the

prodrug was rapidly hydrolysed to the active form. Greater site-specific delivery is

expected for human due to less glucosidase activity in the small intestine.

1.5.4.4 Polysaccharides

Natural or synthetic polysaccharides have received much attention for colon specific

drug delivery. The feature of natural polysaccharides which can be degraded by the

local bacterial enzyme upon entering colonic region was exploited for colon drug

delivery (MacFarlane and Cummings, 1991). These natural polysaccharides are

present in normal dietary food and usually are resistant to digestive enzyme in the

upper GI tract. Many of these polymers are already used as pharmaceutical

excipients in formulations. Therefore, problems associated with toxicity and

biocompatibility are much simplified. However, the hydrophilic nature of the

polysaccharides would cause swelling in the upper GI tract and eventually lead to

19

premature drug release. Thus, methods were developed to prevent premature drug

release by either chemically modified the polymers and mixed with hydrophobic

polymers. This will prevent the swelling in the upper GI tract, but still retaining the

solubilisation feature of the coating in the colon due to bacterial degradation.

Several studies have been done on the use of dextrans in colonic drug delivery

(Harboe et al., 1988; McLeod et al., 1993). Dextran has potential to act as a carrier

for drugs such as naproxen and dexamethasone. The authors reported that

degradation of dextran was significantly higher in colon if compared to upper GI

tract. Modification has been made for dextran by esterification with fatty acids in

order to reduce its solubility (Hirsch et al., 1995). In vitro degradation studies

showed that the modified dextran resisted the harsh condition of the upper GI tract,

but sensitive to degradation by colonic microflora. However, increment in degree of

crosslinking will render the reduction of colonic degradability of the polymer.

Pectinolytic enzymes found in the colonic region are responsible in the degradation

of pectin (Rubinstein and Sintov, 1992). This unique feature was exploited by the

researchers which pectin was used as a carrier for colonic drug delivery (Ashford et

al., 1994). Pectin Type 170 (30-40 % methoxylated) and Pectin USP (70 %

methoxlated) were used as a compression coating. A minimum thickness of 0.9 mm

(700 mg) of Pectin USP coat was found to be viable in resisting the intestinal

condition. However, a thicker coat of 1000 mg showed poor degradation in

pectinolytic enzyme medium due to poor penetration of fluid to hydrate the coating

for enzyme entry. From the scintigraphic result, pectin coating is able to protect the

20

drug until colonic region and majority of the release occurred in caecum and

ascending colonic region. Modification was made by cross-linking calcium with

pectin, yielding calcium pectinate (Adkin et al., 1997). The authors reported that

calcium pectinate tablets were degraded in rat caecum, whereby such modification

has potential for colonic drug delivery.

Guar gum is a galactomannan which has high viscosity. This polysaccharide has

potential to be used as a carrier, delivering drugs to the colon without undesirable

release in the upper GI tract. The potential of guar gum has been studied in the form

of a matrix and compression coat (Rubinstein and Gliko-Kabir, 1997; Krishnaiah et

al., 1998a and 1998b; Tugcu-Demiroz et al., 2004). A study utilising guar gum

compression coat of 400 mg that applied to tablet cores of 300 mg, containing 250

mg of ornidazole was found to be significantly delay the onset of drug release and

absorption (Krishnaiah et al., 2003). However, the drug from the coated formulation

was found to be absorbed in a slower rate from the colon.

Amylose is one of the major components of starch, apart from amylopectin

(Biliaderis, 1991). Amylose is susceptible to fermentation by a broad range of

colonic bacteria (Salyers et al., 1977), where more than half of the bacterial

population is able to digest amylose (MacFarlane and Englyst, 1986). The

polysaccharide amylose has been exploited as a carrier for colonic drug delivery in

the form of film coating (Siew et al., 2000; Leong et al., 2002). Addition of

hydrophobic ethylcellulose as a structuring agent is essential due to the fact that

amylose films tend to swell in aqueous media. Various ratios of amylose with

21

ethylcellulose were employed to investigate the applicability in colonic drug delivery.

Drug release from the coated formulations was reported in fermentation environment

of the colon. Therefore, amylose coated formulations have potential to become a

carrier for drug delivery to the colon.

1.5.4.5 Disulphide polymers

To date, disulphide polymers have received much interest due to its potential as

carrier for colonic drug delivery. Disulphide polymer was found to be resistant in the

upper GI tract but susceptible to degradation in the colonic region. Low redox

potential environment of the human colon is the key feature for the entire mechanism,

where disulphide bond will be reduced. It has been proposed that linear polymers

containing disulphide backbones could be degraded in the low redox potential

environment of the human colon (Schact and Wilding, 1991). Watts and Illum (1997)

reported that the tablets coated with polymer rapidly disintegrated, achieving

complete dissolution in the testing medium.

Recently, studies were focused on employing branch-chained disulphide polymers

instead of linear-chained. The major reason was due to the insolubility features of

the branch-chained polymers. Linear-chained polymers are easily degraded in low

pH condition and more soluble if compared to branch-chained polymers (Sahudin,

2001; Lim et al., 2013). The ability of disulphide polymers to degrade in low redox

potential of the colonic environment while sustaining the harsh environment of the

upper GI tract, have been exploited as a redox-sensitive polymers in colon-specific

drug delivery.

22

1.6 Literature review

Protection of the thiol group is crucial in many areas of organic research especially in

peptide synthesis. The thiol groups were found to be very reactive and therefore the

protection is necessary for the sulphur atom. A free thiol group can be protected as a

thioether or thioester. The protecting group can be easily cleaved via acidic

hydrolysis or by using sodium/ammonia (Wuts and Greene, 2006). Triphenylmethyl

thioether protecting group which is used in this synthesis can be remove by using

iodine, thallium trifluoroacetate and trifluoroacetic acid (TFA). Iodine and thallium

are not favorable as a deprotection agent because they will effect simultaneous

disulphide bond formation (Fields and Noble, 1990). Relatively, TFA is a more

favorable agent because it generates free thiol groups without forming disulphide

bond (Atherton and Sheppard, 1987). However, the using of TFA alone for the

deprotection procedure is not sufficient. The cleavage reaction is reversible due to

the high stability of the trityl cation and strong nucleophilic nature of the thiol group.

Hence, the use of triethylsilane (TES) scavengers appeared to be crucial in the

deprotection process. TES will quench the trityl carbocation to form

triphenylmethane and make the entire process irreversible (Figure 1.5) (Pearson et.al.

1989).

23

a)

b)

Figure 1.5 : TFA deprotection of triphenylmethyl (a) and TFA/TES scavenger

deprotection of triphenylmethyl (b).

TFA

Trityl protected thiol

Thiol

Trityl cation

Trityl protected thiol

Thiol

TFA/Et3SiH

Et3SiOCOCF3 +

Triphenylmethane

24

Linear disulphide polymers have been studied shown to be biodegradable in colonic

bacterial culture (Le, 1998). Such polymers have been suggested as the coating

materials for colon drug delivery system (Schacht and Wilding, 1991). However,

linear-chained disulphide polymers were found to be easily degraded in low pH

condition of the stomach (Sahudin, 2001). Therefore, branch-chained disulphide

polymers had received more attention due to less susceptible for degradation in low

pH condition of the stomach and more insoluble compared to linear-chained

disulphide polymers. Nevertheless, the insolubility feature of branch-chained

disulphide polymers would appear to be desirable in the stomach and small intestine

if the polymerisation of the polymers can be controlled. The important goal of the

polymer is to maintain the dosage form in an intact state and should be readily

degraded in low redox potential environment of the human colonic region.

Oxidative polymerisation method can be used for synthesis of disulphide cross-

linked polymer where the monomer is placed in a mild basic condition, exposed to

open air and with the presence of oxidising agent such as dimethyl sulphoxide

(DMSO) (Tan et al. 1991). DMSO is miscible with water at all concentrations, thus

it helps to improve the solubility of thiol monomers in aqueous solution. The

reaction mechanism of oxidation by DMSO was shown in Figure 1.6.

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SYNTHESIS, CHARACTERISATION AND IN VITRO ...eprints.usm.my/30117/1/FULL_THESIS_VIVA_EDIT_LV.pdf1.7 Aim of the thesis 30 CHAPTER TWO : SYNTHESIS OF TRITHIOL MONOMER 2.1 Introduction