DOXORUBICIN CONJUGATED TO D-α-TOCOPHERYL

POLYETHYLENE GLYCOL 1000 SUCCINATE (TPGS):

IN VITRO CYTOTOXICITY, IN VIVO

PHARMACOKINETICS AND BIODISTRIBUTION

CAO NA

(B.ENG., XI’AN JIAOTONG UNIVERSITY)

A THESIS SUBMITTED FOR THE DEGREE OF MASTER

OF ENGINEERING

DEPARTMENT OF CHEMICAL AND BIOMOLECULAR

ENGINEERING

NATIONAL UNIVERSITY OF SINGAPORE

2007

ACKNOWLEDGEMENTS

First of all, I would like to take this opportunity to express my deepest gratitude and

appreciation to the following people:

My supervisor, Associate Professor Feng Si-Shen, for his invaluable advice, guidance,

unconditional support and encouragement during the period of my research in Chemical &

Biomolecular Engineering. I have learnt how to carry out research work, how to overcome

the difficulty in research.

My senior, Zhang Zhiping, for his unconditional support and invaluable advice in the

study. His sharing on research experience as well as his help in training is greatly

appreciated.

My Laboratory colleagues, Dr. Dong Yuancai, Miss Lee Siehuey, Miss Chen Shilin, Dr.

Mei Lin, Mr. Pan Jie, Ms Sun Bingfeng and others, for their kind support.

Lab officers, Ms. Tan Mei Yee Dinah, Mr. Beoy Kok Hong, Ms. Chai Keng, Mr. Chia Pai

Ann, Ms. Sandy Koh, Dr. Yuan Zeliang and others I may neglect to mention here, for

their kind assistance.

The financial support provided by National University of Singapore in the form of GST

stipend is greatly acknowledged.

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TABLE OF CONTENT

ACKNOWLEDGEMENTS ................................................................................................. i

TABLE OF CONTENT...................................................................................................... ii

SUMMARY...................................................................................................................... vii

NOMENCLATURE .......................................................................................................... ix

LIST OF TABLES............................................................................................................. xi

LIST OF FIGURES .......................................................................................................... xii

LIST OF SCHEMES........................................................................................................ xiv

1 INTRODUCTION ....................................................................................................... 1

1.1 General Background ...................................................................................... 1

1.2 Objective and Thesis Organization................................................................ 4

2 LITERATURE SURVEY............................................................................................ 6

2.1 Cancer and Cancer Chemotherapy................................................................. 6

2.1.1 Cancer ..................................................................................................... 6

2.1.2 Cancer Treatment.................................................................................... 7

2.1.3 Cancer Chemotherapy............................................................................. 8

2.1.4 Problems in Chemotherapy..................................................................... 9

2.1.5 Chemotherapeutic Engineering............................................................. 10

2.2 Polymeric Drug Carrier................................................................................ 11

2.2.1 Polymers as Drug Carrier...................................................................... 11

2.2.1.1 Biodegradable Polymers................................................................ 11

2.2.1.2 Polyethylene Glycol ...................................................................... 15

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2.2.2 Polymeric Drug Formulations............................................................... 16

2.2.2.1 Paste............................................................................................... 16

2.2.2.2 Micelles ......................................................................................... 17

2.2.2.3 Liposomes ..................................................................................... 19

2.2.2.4 Microspheres ................................................................................. 21

2.2.2.5 Nanoparticles................................................................................. 22

2.3 Prodrug......................................................................................................... 23

2.3.1 Design and Synthesis of Polymeric Prodrugs....................................... 24

2.3.1.1 N-hydroxysuccinimide (NHS) Ester Coupling Method................ 25

2.3.1.2 Carbodiimide Coupling Method.................................................... 26

2.3.1.3 Dextran-prodrug ............................................................................ 28

2.3.1.4 N-(2-hydroxypropyl)methacrylamide (HPMA)-prodrug .............. 30

2.3.1.5 Dendrimer Conjugates................................................................... 31

2.3.2 PEGylated Drug Conjugation ............................................................... 34

2.3.2.1 PEGylation of Small Organic Molecules ...................................... 34

2.3.2.2 PEGylation of Polypeptide (Peptides and Proteins)...................... 36

2.3.2.3 Targeting PEGylation.................................................................... 37

2.4 Vitamin E TPGS .......................................................................................... 38

2.4.1 Chemistry of Vitamin E TPGS ............................................................. 38

2.4.2 Solubilizer for Water-insoluble Compounds ........................................ 40

2.4.3 Absorption Enhancer ............................................................................ 41

2.4.4 Bioavailability Enhancer....................................................................... 41

2.4.5 Anticancer Enhancer ............................................................................. 44

2.4.6 Drug Delivery Applications.................................................................. 45

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2.5 Doxorubicin ................................................................................................. 46

2.5.1 History................................................................................................... 46

2.5.2 Mechanism of Action............................................................................ 47

2.5.3 Side Effects and Limitations................................................................. 49

2.5.4 Formulations ......................................................................................... 50

3 SYNTHESIS AND CHARACTERIZATION OF THE TPGS-DOX CONJUGATE52

3.1 Introduction.................................................................................................. 52

3.2 Experiment Section...................................................................................... 52

3.2.1 Materials ............................................................................................... 52

3.2.2 Synthesis of TPGS-DOX ...................................................................... 53

3.2.2.1 TPGS Succinoylation .................................................................... 53

3.2.2.2 TPGS-DOX Conjugation .............................................................. 54

3.2.3 Characterization of TPGS-DOX Conjugate.......................................... 55

3.2.3.1 FT-IR ............................................................................................. 55

3.2.3.2 1H NMR......................................................................................... 56

3.2.3.3 GPC ............................................................................................... 56

3.2.3.4 Drug Conjugate Efficiency............................................................ 56

3.2.3.5 Stability of the Conjugate.............................................................. 57

3.3 Results and Discussion ................................................................................ 57

3.3.1 FT-IR Spectra........................................................................................ 57

3.3.2 1H NMR Spectra ................................................................................... 58

3.3.3 GPC Results .......................................................................................... 59

3.3.4 Drug Loading Capacity......................................................................... 61

3.3.5 In Vitro Stability ................................................................................... 62

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3.4 Conclusions.................................................................................................. 62

4 IN VITRO STUDIES ON DURG RELEASE KINETICS, CELLULAR UPTAKE

AND CELL CYTOTOXICITY OF THE TPGS-DOX CONJUGATE............................ 63

4.1 Introduction.................................................................................................. 63

4.2 Materials and Methods................................................................................. 63

4.2.1 Materials ............................................................................................... 63

4.2.2 In Vitro Drug Release ........................................................................... 64

4.2.3 Cell Culture........................................................................................... 64

4.2.4 In Vitro Cell Uptake Efficiency ............................................................ 64

4.2.5 Confocal Laser Scanning Microscopy .................................................. 65

4.2.6 In Vitro Cytotoxicity ............................................................................. 65

4.2.7 Statistics ................................................................................................ 66

4.3 Results and Discussion ................................................................................ 66

4.3.1 In Vitro Drug Release ........................................................................... 66

4.3.2 In Vitro Cellular Uptake........................................................................ 68

4.3.3 Confocal Laser Scanning Microscopy .................................................. 72

4.3.4 In Vitro Cytotoxicity ............................................................................. 74

4.4 Conclusions.................................................................................................. 79

5 IN VIVO INVESTIGATION ON PHARMACOKINETICS AND

BIODISTRIBUTION OF THE TPGS-DOX CONJUGATE ........................................... 81

5.1 Introduction.................................................................................................. 81

5.2 Materials and Methods................................................................................. 81

5.2.1 Animal................................................................................................... 81

5.2.2 In Vivo Pharmacokinetics ..................................................................... 82

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5.2.2.1 Drug Administration...................................................................... 82

5.2.2.2 Blood Collection and Sample Analysis......................................... 82

5.2.2.3 Pharmacokinetic Parameters ......................................................... 83

5.2.3 In Vivo Biodistribution.......................................................................... 84

5.2.3.1 Drug Administration...................................................................... 84

5.2.3.2 Tissues Collection and Samples Analysis ..................................... 84

5.2.3.3 Statistics......................................................................................... 85

5.3 Results and Discussion ................................................................................ 85

5.3.1 Pharmacokinetics .................................................................................. 85

5.3.2 Biodistribution ...................................................................................... 88

5.4 Conclusions.................................................................................................. 91

6 CONCLUSIONS AND RECOMMENDATIONS .................................................... 93

6.1 Conclusions.................................................................................................. 93

6.2 Recommendations........................................................................................ 95

7 REFERENCE............................................................................................................. 96

vi

SUMMARY

Polymer-drug conjugation is one of the major strategies for drug modifications, which

manipulate therapeutic agents at molecular level to increase their solubility, permeability

and stability, and thus biological activity. Polymer-drug conjugation can significantly

change biodistribution of the therapeutic agent, thus improving its pharmacokinetics (PK)

and pharmacodynamics (PD), increasing their therapeutic effects and reducing their side

effects, as well as provide a means to circumvent the multi-drug resistance (MDR). D-α-

tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS or simply, TPGS), a

water-soluble derivative of natural Vitamin E, is a PEGylated vitamin E, which is formed

by esterification of vitamin E succinate with PEG 1000. Its amphiphilic structure,

comprising lipophilic alkyl tail and hydrophilic polar head, is bulky and has large surface

areas, which enables it to be an effective emulsifier and solubilizer. TPGS has been

intensively used in our research either as an effective macromolecular emulsifier or as a

component for a novel biodegradable copolymer PLA-TPGS for nanoparticle formulation

of therapeutic agents, which resulted in high drug encapsulation efficiency, high cellular

uptake and high in vitro cytotoxicity and in vivo therapeutic effects. Some reports

demonstrated that TPGS can increase the oral bioavailability and enhance cytotoxicity of

drugs. TPGS-drug conjugation should thus be an ideal solution for the drugs that have

problems in their adsorption, distribution, metabolism and excretion (ADME).

The aim of this study was to develop a novel TPGS-DOX conjugate to enhance the

therapeutic potential and reduce the systemic side effects of the drug, doxorubicin. In this

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research a novel prodrug, TPGS-doxorubicin conjugate, was successfully developed. The

hydroxyl terminal group of TPGS was activated by succinic anhydride (SA) and interacted

with the primary amine group of doxorubicin. The polymer-drug conjugation was

confirmed by 1H NMR, FT-IR and GPC to characterize the molecular structure and

molecular weight. The efficiency was determined to be 8% and stability of the conjugate

was also favorable for required storage period. The drug release from the conjugate was

pH dependent without significant initial burst. The cellular uptake, intracellular

distribution, and cytotoxicity of the polymer-drug conjugation were accessed with MCF-7

breast cancer cells and C6 glioma cells as in vitro model. The conjugate showed higher

cellular uptake and broader distribution within the cells. Judged by IC50, the conjugate was

found 31.8, 69.6, 84.1% more effective with MCF-7 cells and 43.9, 87.7, 42.2% more

effective with C6 cells than the pristine drug in vitro after 24, 48, 72 h culture,

respectively. In vivo pharmacokinetics confirmed the advantages of the prodrug. The area-

under-the-curve (AUC) was found to be 6,810 h·ng/mL for the prodrug but 289 h·ng/mL

for the doxorubicin, which implied 23.6 times more effective, and the half-life of the drug

is 9.65±0.94 h for the TPGS-DOX conjugation but 2.53±0.26 h for the original DOX,

which implied 3.81 times longer (p<0.01), at the 5 mg/kg DOX dose i.v. injection. Effects

of the conjugation on biodistribution showed the impaired systemic toxicity, especially for

heart, stomach and intestine.

The TPGS-DOX prodrug showed great potential to become a novel dosage form of

doxorubicin and may also be applied to other anticancer drugs.

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NOMENCLATURE

ACN Acetonitrile

AUC The area under the curve

BBB Blood brain barrier

BD Biodistribution

CL Clearance

CLSM Confocal laser scanning microscopy

CMC Critical micelle concentration

CyA Cyclosporine A

DCC N,N'-dicyclohexylcarbodiimide

DCM Dichloromethane

DMAP Dimethylaminopyridine

DMEM Dulbecco’s modified eagle medium

DMSO Dimethyl sulfoxide

DOX Doxorubicin

FBS Fetal bovine serum

FT-IR Fourier transform infrared spectroscopy

GI Gastrointestinal

GPC Gel permeation chromatography

HPLC High performance liquid chromatography

HPMA N-(2-hydroxypropyl)methacrylamine

IC50 The drug concentration at which 50% of the cell growth is inhibited

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MDR Multi-drug resistance

MRT Mean residence time

MTD Maximum tolerated dose

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

NHS N-hydroxysuccinimide

NMR Nuclear magnetic resonance spectroscopy

PBS Phosphate-buffered saline

PEG Poly(ethylene glycol)

PGA Poly(L-glutamic acid)

P-gp P-glycoprotein

PK Pharmacokinetics

SA Succinic anhydride

t1/2 Half-life time

TEA Triethylanmine

teffect Therapeutic effective period

THF Tetrahydrofuran

TPGS Vitamin E TPGS, d-α-tocopheryl polyethylene glycol 1000

succinate

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LIST OF TABLES

Table 4-1 IC50 values (in equivalent µM DOX) of MCF-7 and C6 cancer cells cultured with the TPGS-DOX conjugate vs the pristine DOX in 24, 48, 72 h ............................... 77 Table 5-1 Pharmacokinetic parameters of the TPGS-DOX conjugate vs the pristine DOX i.v. injected in the SD rats at the equivalent 5 mg/kg dose ............................................... 86 Table 5-2 AUC values (µg·h/g) of biodistribution in various organs after intravenous injection of the free DOX and the TPGS-DOX conjugate to rats at 5 mg/kg equivalent dose ................................................................................................................................... 91

xi

LIST OF FIGURES

Fig 2-1 Schematic of a micelle ......................................................................................... 18

Fig 2-2 Liposomes for drug delivery ................................................................................ 20

Fig 2-3 Schematic presentation of (a) polymeric conjugate with targeting moiety and (b) hyperbranched polymeric conjugate with targeting and imaging components................. 25

Fig 3-1 FT-IR spectra of DOX (red line), TPGS (purple line) and TPGS-DOX (blue line)........................................................................................................................................... 58

Fig 3-2 1H NMR spectra of (a) TPGS, (b) TPGS-SA, (c) DOX, (d) TPGS-DOX with the insert for a magnification of the region between 6 and 10 ppm ....................................... 60

Fig 3-3 Gel permeation chromatogram (GPC) of the TPGS-DOX conjugate and the unconjugated TPGS .......................................................................................................... 61

Fig 4-1 Release of DOX from TPGS-DOX conjugate incubated in phosphate buffer at 37 oC (mean ± SD and n=3)................................................................................................... 68

Fig 4-2 (a) MCF-7 and (b) C6 cell uptake efficiency cultured with the pristine DOX or the TPGS-DOX for 0.5, 1, 4, 6 h respectively at equivalent drug concentration 1 µg/mL (mean ± SD and n=6)........................................................................................................ 70

Fig 4-3 Cell uptake of the TPGS-DOX and DOX in (a) MCF-7 and (b) C6 cells after 4 h incubation (mean ± SD and n=6) ...................................................................................... 71

Fig 4-4 Confocal laser scanning microscopy (CLSM) of MCF-7 cells after 4 h incubation with (a) the pristine drug DOX and (b) with the TPGS-DOX conjugate, and that of C6 cells with (c) the DOX and (d) the TPGS-DOX conjugate at the equivalent 1 µg/mL DOX concentration..................................................................................................................... 74

Fig 4-5 Cellular viability of (a) MCF-7 breast cancer cells and (b) C6 glioma cells after 24, 48, 72 h culture with the DOX-TPGS conjugate respectively in comparison with that of the pristine DOX at various equivalent DOX concentrations (mean ± SD and n=6) ....... 76

xii

Fig 4-6 Cellular viability of MCF-7 breast cancer cells and C6 glioma cells after 24, 48, 72 h culture with TPGS respectively at various equivalent concentrations in the conjugate (mean ± SD and n=6)........................................................................................................ 78

Fig 5-1 Pharmacokinetic profile of the pristine DOX and the DOX-TPGS conjugate after intravenous injection in rats at a single equivalent dose of 5 mg/kg (mean ± SD and n=4)........................................................................................................................................... 85

Fig 5-2 The DOX levels (µg/g) in heart, lung, spleen, liver, stomach, intestine and kidney after i.v. administration at 5 mg/kg equivalent drug of (a) the free DOX, (b) the TPGS-DOX conjugate (mean ± SD and n=3).............................................................................. 90

xiii

LIST OF SCHEMES

Scheme 2-1 PEO families ................................................................................................. 13

Scheme 2-2 Chemical structure of polyanhydride............................................................ 14

Scheme 2-3 Chemical structure of some polyesters ........................................................ 15

Scheme 2-4 Chemical structure of PEG ........................................................................... 16

Scheme 2-5 NHS esters compounds react with nucleophiles to release NHS leaving group........................................................................................................................................... 26

Scheme 2-6 Carbodiimide amide coupling scheme.......................................................... 27

Scheme 2-7 Chemical structure of DCC, DIC and EDC, respectively............................. 27

Scheme 2-8 Chemical structure of dextran....................................................................... 28

Scheme 2-9 Chemical structure of dextran-methylprednisolone hemisuccinate.............. 29

Scheme 2-10 Synthesis of FITC-labeled CM dextran ...................................................... 30

Scheme 2-11 Examples of synthesis of HPMA-drug antibody conjugates ...................... 32

Scheme 2-12 Typical architecture of a third generation dendrimer ................................. 33

Scheme 2-13 Chemical Structure of (a) monomethoxy-PEG and (b) di-hydroxyl PEG.. 34

Scheme 2-14 Synthetic schemes of (a) PEG-Ara-C4 and (b) PEG-Ara-C8 conjugates .... 36

Scheme 2-15 Architecture of multi-block PEG-Dox with targeting antibody conjugated38

Scheme 2-16 Chemical structure of TPGS: red = hydrophobic vitamin E, green = hydrophilic PEG chain, black = succinate linker.............................................................. 39

Scheme 2-17 Chemical structure of doxorubicin.............................................................. 47

xiv

Scheme 3-1 Scheme of TPGS succinoylation .................................................................. 54

Scheme 3-2 Scheme of TPGS-DOX conjugation............................................................. 55

xv

1 INTRODUCTION

1.1 General Background

Prodrug is a pharmacological substance in an inactive (or significantly less active) form

that is formulated through transient modification of a given drug. In other words, a

prodrug is an inactive precursor of a drug. The involved temporary chemical modification

in prodrug can be metabolized in the body in vivo and leave the inherent pharmacological

properties of the parent drug intact (Saltzman 2001). A conjugation of a drug to a polymer

was called “polymeric prodrug”, which has led a new era of polymer drug delivery system

(Pasut and Veronese 2007). Polymeric prodrug has quite a few merits over the precursor

drug, such as increased solubility, enhanced bioavailability, improved pharmacokinetics,

ability of targeting and protected activity of protein drug (Khandare and Minko 2006). In

particular, Polymer-drug conjugation can significantly change biodistribution of the

therapeutic agent, thus improving its pharmacokinetics (PK) and pharmacodynamics (PD),

increasing their therapeutic effects and reducing their side effects, as well as provide a

means to circumvent the multi-drug resistance (MDR), which is caused by overexpression

of MDR transporter proteins such as p-glycoproteins (p-gp) in the cell membrane that

mediate unidirectional energy-dependent drug efflux and thus reduce intracellular drug

levels. The MDR transporter proteins are rich in the liver, kidney, and colon cells. Tumors

also acquire drug resistance through induction of MDR transport proteins during treatment

(Harris and Hochhauser 1992; Borst and Schinkel 1996; Schinkel 1997; Gottesman, Fojo

et al. 2002; Liscovitch and Lavie 2002; Müller, Keck et al. 2003). The medical solution

1

for MDR is to apply inhibitors of MDR transporters such as cyclosporine A, which may

also suppress the body immune system and thus cause medical complications. Moreover,

the inhibitors themselves may have problems in formulation and delivery. The engineering

solution is thus preferred, which does not use any p-glycoprotein inhibitor. Instead, it

applies high technologies such as nanotechnology and polymer-drug conjugation, to

engineering the drugs to avoid being recognized by the p-glycoproteins (Feng and Chien

2003; Feng 2004; Feng 2006).

Various architectures of polymers have been utilized as carrier to deliver drugs, some of

which have stepped into clinical development and some have shown promise (Kopeček,

Kopečková et al. 2001; Duncan 2003). In synthetic polymers, N-(2-

hydroxypropyl)methacrylamine (HPMA) copolymers (Kopeček, Kopečková et al. 2000;

Chytil, Etrych et al. 2006), Poly(ethylene glycol) (PEG) (Greenwald, Choe et al. 2003;

Harris and Chess 2003), and poly(L-glutamic acid) (PGA) (Li, Price et al. 1999) have

been predominantly utilized as the carriers of anticancer drugs such as doxorubicin,

campothecin and platinates. In particular, PEG is water soluble, biocompatible and

nontoxic, facilitating its application for conjugation with paclitaxel (Feng, Yuan et al.

2002), camptothecin (Conover, Greenwald et al. 1998), methotrexate (Riebeseel,

Biedermann et al. 2002) and doxorubicin (Veronese, Schiavon et al. 2005) to improve

their water solubility, plasma clearance and biodistribution.

D-α-Tocopheryl Polyethylene glycol 1000 succinate (vitamin E TPGS or simply, TPGS),

a water-soluble derivative of natural vitamin E, is formed by esterification of vitamin E

succinate with PEG 1000. Its amphiphilic structure enables it to be an effective emulsifier

2

and solubilizer (Fischer, Harkin et al. 2002). In our lab, TPGS has been successfully

applied as a novel emulsifier in preparation of PLGA nanoparticles and as a component of

a novel biodegradable polymer, PLA-TPGS for nano-carrier of anticancer agents, which

exhibited high emulsification efficiency, high drug encapsulation efficiency and improved

cellular uptake, cytotoxicity and therapeutic effects (Feng 2004; Feng 2006). TPGS was

also demonstrated possessing the ability to enhance the oral bioavailability of

cyclosporine A, vancomycin hydrochloride and talinolo in animals (Prasad, Puthli et al.

2003; Bogman, Zysset et al. 2005). Besides, co-administration of TPGS could enhance the

cytotoxicity of doxorubicin, vinblastine and paclitaxel by the inhibition on p-glycoprotein

mediated MDR (Dintaman and Silverman 1999).

Doxorubicin, an anthracyclinic antibiotic, is a DNA-interacting chemotherapeutic drug

(http://en.wikipedia.org/wiki/Doxorubicin; Takakura and Hashida 1995), which is

effective in treating breast cancer (Bonfante, Ferrari et al. 1986) as well as ovarian (ten

Bokkel Huinink, van der Burg et al. 1988), prostate (Raghavan, Koczwara et al. 1997),

cervix (Hoffman, Roberts et al. 1988) and lung cancers (Schuette 2001). However, clinical

use of doxorubicin is limited because of the short half-life (Al-Shabanah, El-Kashef et al.

2000) and acute systemic toxicity (Blum and Carter 1974). Additionally, the intracellular

level of doxorubicin can be reduced by the MDR effects (Krishna and Mayer 2000). This

triggered us to take the advantages of TPGS and involve it as a carrier by conjugation with

doxorubicin to enhance the drug’s therapeutic potential in vitro and in vivo as well as to

reduce systemic toxicity.

3

1.2 Objective and Thesis Organization

This thesis tells a story of TPGS-DOX conjugation for chemotherapy. The novel prodrug,

TPGS-DOX conjugate, was investigated on drug loading efficiency, conjugate stability,

drug release property, intracellular uptake, in vitro cytotoxicity and in vivo

pharmacokinetics and biodistribution. This project provides a novel dose form of

doxorubicin with improved therapeutic effects, whose methodology can also be utilized in

other anti-cancer drugs.

The thesis consists of six chapters. First, an introduction with general background and

thesis organization are included in chapter 1. Second, chapter 2 gives a comprehensive

literature review on polymeric-prodrug, highlighting the TPGS advantages in

chemotherapy. Then chapter 3 is dedicated to the synthesis and characterization of TPGS-

DOX conjugate. TPGS was first activated by succinic anhydride through ring opening

reaction. Then it was covalently attached to the primary amine group in doxorubicin. The

resultant product was characterized by FT-IR and NMR for the chemical structure, GPC

for the molecular weight and the distribution. The doxorubicin content conjugated to

TPGS was determined by fluorescence detection using microplate reader. The stability of

the prodrug was investigated in PBS at 4°C. Chapter 4 shows the in vitro results including

drug release from the conjugate, the intracellular uptake efficiency of the conjugate using

MCF-7 and C6 cancer cells as in vitro model with comparison of free DOX, and

cytotoxicity at various drug concentrations. At last, in chapter 5, pharmacokinetics and

biodistribution via intravenous administration of the TPGS-DOX and free DOX are

4

included, followed by final conclusions and some recommendations for future work in

chapter 6.

5

2 LITERATURE SURVEY

2.1 Cancer and Cancer Chemotherapy

2.1.1 Cancer

Cancer, caused by disordered division of cells, has been seriously threatening human

health. It is always combined with malignant behavior of these cells, which tend to spread

by direct invasion into adjacent tissue or metastasis to distant sites

(http://en.wikipedia.org/wiki/Cancer). Although rapider diagnosis of cancer has been

achieved than before, many forms are still incurable. According to BBC news, cancer has

become NO. 1 killer with 135,000 death per year compared to 110,000 from heart disease

(http://news.bbc.co.uk/1/hi/health/1015657.stm).

So far, the specific mechanisms of formation and spreading of cancer have not been well

explained. The external effects and internal factors are the most important two

contributors to the cause of cancer. Radiation, overexposure to certain chemical

substances, infectious agents, diet and lifestyle can initiate and promote carcinogenesis. In

particular, the tobacco products can even cause about 80% of all cancers, especially in

high risk of larynx, oesophagus, pancreas, bladder, kidney, cervix and lung cancers.

Regarding the internal factors, many theories have been demonstrated. The most important

seven contributors conclude failure of apoptosis, overactivation of oncogenes, inactivation

of tumor suppressor genes, cell cycle activation of quiescent cells, acquisition of

metastatic behavior by malignant cells, disordered responses to cellular growth factors,

6

and immune system surveillance failure (Hanahan and Weinberg 2000). These factors

may act together or sequentially, no matter the external or internal ones.

2.1.2 Cancer Treatment

Cancer cells may break away and grow into a distant point of a normal tissue, which is

called metastasize. Therefore, tumors are classified to two types, benign and malignant.

Benign tumors do not spread, which are localized in one part of a body without lethal

threaten. In contrast, malignant tumors can spread from the original location to other parts

via the blood or/and the lymphatics, which usually happens in late stage of cancer.

Different cancer cells have specific propensity in metastasis. Prostate cancer intends to

spread into bones, while colon cancer prefers liver (Fausto).

Prevention is more active measure than cure to defense against cancer, which is classified

into primary and secondary type for a patient without and with a history of disease,

respectively. It was reported that low meat intake and certain coffee consumption are

associated with the reduced risk of cancer (Ward, Sinha et al. 1997; Sinha, Peters et al.

2005; Larsson and Wolk 2007). Moreover, some vitamins (Lieberman, Prindiville et al.

2003), β-carotene (http://www.cancer.gov/cancertopics/factsheet/Prevention/betacarotene)

and other chemoprevention agents, such as tamoxiten (Vogel, Costantino et al. 2006) and

finasteride (Baron, Sandler et al. 2006), have been demonstrated to be protective against

cancer. When prevention fails, cure is necessary.

Cancer can be treated through several methods, the effective ones among which are

surgery, radiotherapy, chemotherapy, hormone therapy and immunotherapy, although they

7

possess their own advantages and disadvantages. According to the stage of the cancer and

the location and grade of the tumor, as well as the condition of the patients, different

therapy or a combination will be employed. Removing the cancer completely without

damage to the normal tissue is the ideal solution of the treatment. It can be achieved

sometimes by surgery, but this is not always feasible, especially when the cancer can

metastasize to other sites in body. Besides, patients have to suffer from physical pain and

even the danger of infections. What is worse, it may speed up the growth rate of the

remaining cancer cells and even cause death by metastatic cancer. The larger excised

tumor causes the greater possibility of death. Radiation therapy utilizes ironing radiation

to kill cancer cells and shrink tumors, which can be almost employed into every type of

solid tumors. It can damage not only the cancer cells but also normal cells. However, the

normal cells can recover itself after the treatment. As radiotherapy is a localized strategy

like surgery, it is not effective to the patients suffering metastasis. Hormonal therapy

employs or blocks certain hormones to inhibit the cancer cell growing, which may cause

quite a few side effects such as nausea, swelling of limbs and weight gain. Immunotherapy

is a therapeutic strategy, which induces the patient’s own immune system to fight against

cancer. Cancer chemotherapy, using chemotherapeutical agents to killing or suppressing

cancer cells, first succeeded in 1950s and was widely employed in 1960s. However, most

chemotherapeutic agents cause serious side effects, which limits its application (Feng and

Chien 2003). Combination of chemotherapy with other strategies is a prominent treatment

recently in cancer cure.

2.1.3 Cancer Chemotherapy

8

Chemotherapy sometimes is defined as the use of chemical substances to treat disease

(http://en.wikipedia.org/wiki/Chemotherapy), which, however, is often toxic and even

life-threatening. Chemotherapy of cancer utilizes chemotherapeutic agents to control or

kill cancer cells, usually, via damaging to DNA or RNA of cancer cells

(http://www.chemocare.com/whatis/cancer_cells_and_chemotherapy.asp). So far,

hundreds of anti-cancer drugs have been found for clinical application, some of which are

natural extracts while others are synthetic or semi-synthetic agents. The majority of the

drugs can be classified into: alkylating agents, antimetabolites, anthracyclines, plant

alkaloids, topoisomerase inhibitors, monoclonal antibodies, etc. Some drugs perform

better while working together with other anti-cancer drugs, which is called combination

chemotherapy. Nanotechnology has been designed and investigated to achieve cancer

chemotherapy at home (Feng 2004).

2.1.4 Problems in Chemotherapy

Chemotherapy is an effective and complicated procedure for treating cancer. However,

anti-cancer drugs not only damage cancer cells, but also affect normal cells, which usually

leads to serious side effects including hair loss, nausea and vomiting, diarrhea or

constipation, anemia, malnutrition, memory loss, depression of the immune system,

hemorrhage, secondary neoplasms, cardiotoxicity, hepatotoxicity, hephrotoxocity,

ototoxicity and even death (http://en.wikipedia.org/wiki/Chemotherapy). The specific side

effects are up to what and how much of the chemotherapeutic agent is administrated and

how the body may response. One or more of the above effects may be observed after the

treatment.

9

Most of anticancer drugs are hydrophobic agents, thus adjuvants are required to make

them available in clinical use. For example, paclitaxel, a diterpenoid extracted from the

bark of Pacific yew tree, exhibits a wide anti-cancer spectrum. However, it is too

hydrophobic with a water solubility ≤0.5 mg/l to be directly administrated into body

(Mathew, Mejillano et al. 1992). Therefore, Cremophor EL was used as an adjuvant in

dose form but it causes serious side effects, such as hypersensitivity reactions,

nephrotoxicity, neurotoxicity and cardiotoxicity (Weiss, Donehower et al. 1990; Li, Price

et al. 1999).

Moreover, although chemotherapy increasingly succeeds, especially in initial stage, it

usually becomes less effective in the long-term therapy, which is associated with drug

resistance. The drug resistance can be due to three mechanisms: pharmacokinetic

resistance, kinetic resistance and genetic resistance. In a combinational chemotherapy,

multidrug resistance (MDR) may develop, which is found to be relevant with an

overexpression of P-glycoprotein (P-gp). P-gp is well known for removal of toxic

substance in normal function. It effluxes the drugs out of cells by an energy mediated

unidirectional process. These MDR transporter proteins are rich in the liver, kidney, and

colon cells and also involved in the various physiological drug barriers such as the

gastrointestinal (GI) drug barrier for oral chemotherapy, the blood-brain barrier (BBB) for

brain tumor treatment and the intratumoral barrier for efficient drug delivery in the tumor

(Harris and Hochhauser 1992; Schinkel 1997; Krishan 2000; Liscovitch and Lavie 2002;

Müller, Keck et al. 2003).

2.1.5 Chemotherapeutic Engineering

10

Chemotherapeutic engineering refers to the application and development of chemical

engineering principles and methodologies for chemotherapy so that better efficacy can be

achieved with fewer side effects. In order to solve the above problems, effective drug

delivery system seems as important as the discovery of new drugs, which makes the

development of chemotherapeutic engineering emergent. In resent decades, the

development of diversity drug delivery system with improved efficacy but reduced side

effects became a hot spot in chemotherapy. Numerous drug delivery strategies have been

intensively investigated, among which microspheres (Couvreur and Puisieux 1993),

nanoparticles (Couvreur, Roblot-Treupel et al. 1990; Brigger, Dubernet et al. 2002),

liposomes (Daoud, Hume et al. 1989; Pinto-Alphandary, Andremont et al. 2000), micelles

(Jones and Leroux 1999), cyclodextrins (Utsuki, Brem et al. 1996), pastes (Li, Price et al.

1999) and prodrug (Senter, Svensson et al. 1995; Soyez, Schacht et al. 1996; Springer and

Niculescu-Duvaz 1996; Khandare and Minko 2006) have attracted much attention.

2.2 Polymeric Drug Carrier

2.2.1 Polymers as Drug Carrier

In 1974, the discovery of the ability of macromolecules to localize to lysosomes started a

new era for macromolecules used as drug carriers (De Duve, De Barsy et al. 1974). Since

1975, when the rationale of polymeric targeted drug delivery was formulated on the basis

of previous investigations (Ringsdorf 1975), polymers, either natural or synthetic origin,

have been widely used in drug delivery system.

2.2.1.1 Biodegradable Polymers

11

The natural polymers, such as collagen, fibrin, alginate, silk, hyaluronic acid and chitosan,

are abundant and have good biodegradability and biocompatibility, which counteract their

drawbacks such as immunogenicity, instability and lack of chemical group for

modification (Duncan and Kopeček 1984). Meanwhile, perhaps synthetic polymers are the

most widely used materials as growth factor delivery carries, especially for biodegradable

synthetic polymers, which provide excellent chemical and mechanical properties that

natural polymers often fail to possess. The frequently used biodegradable polymers

include poly(ortho esters) (POE), polyanhydride and polyesters.

POE was developed in early 1970s, since when, four families of POE have been indicated

as shown in Scheme 2-1 (Tomlinson, Heller et al. 2003). The polymer can degrade to a

diol and a lactone, which will further produce γ-hydroxybutyric acid. The high

hydrophobic property of the polymer results in the limitation of the amount that water can

penetrate the polymer. As the erosion rate of the polymers is extremely slow, they can be

as a compression molded disk in an aqueous environment. More importantly, POE II and

POE IV have been found to have excellent potential as a delivery system in ocular

application (Tomlinson, Heller et al. 2003).

12

Scheme 2-1 PEO families

Polyanhydrides are a kind of biodegradable polymers in which anhydride bonds connect

monomer units of the chain. The chemical structure of a polyanhydride molecule with n

repeating units is shown in scheme 2-2. Water-labile anhydride bonds in polyanhydrides

give two carboxylic acid groups, which results in easy metabolization and

biocompatibility. Polyanhydrides consist of three main classes, which are aliphatic,

unsaturated and aromatic polyanhydrides (Hazra, Golenser et al. 2002). Polyanhydrides

can degrade uniformly into non-toxic metabolites and thus become useful for controlled

drug delivery (Hazra, Golenser et al. 2002). Gliade®, made of a polyanhydride wafer

containing a chemotherapeutic agent, can deliver carmustine to the malignant glioma

tumor, telling a successful story of polyanhydrides (Mishra and Jain 2000). However, as

polyanhydrides are quite sensitive to water, some hydrophobic monomers are employed to

resist the water penetration and increase the stability of polyanhydrides in water (Saltzman

2001).

13

Scheme 2-2 Chemical structure of polyanhydride

Polyesters, which contain the ester functional group in the main chain, have been

approved by FDA and are widely used as biodegradable polymers. The homo or

copolymers based on glycolic and lactic acid are significantly common, which have been

studied for more than 50 years (Lowe 1954; Schneider 1955). Polyglycolide (PGA) is the

simplest linear, aliphatic polyester. It exhibits somewhat unique solubility, in that the high

molecular weight form is insoluble in almost all the organic solvents while the low

molecular weight form is more soluble. On the other hand, PGA is soluble in fluorinated

solvents, which can be utilized for melt spinning and film preparation (Schmitt 1973). The

initial application of PGA was limited because of its hydrolytic instability. Recently, PGA

and its copolymers with lactic acid, ε-caprolacone or trimethylene carbonate are in wide

use as materials for absorbable sutures and being investigated in biomedical area (Gilding

and Reed 1979; Middleton and Tipton 1998). Polylactide is thermoplastic, aliphatic

polyester synthesized from lactide by ring opening polymerization. Due to the chiral

nature of lactide, there are two optical stereoisomers, poly(L-lactide) (PLLA), poly(D-

lactide) (PDLA) and poly(D,L-lactide) (PDLLA). As PDLLA degrades faster than others

do, it attracts more attention as a drug delivery system (Conti, Pavanetto et al. 1992).

Poly(lactic-co-glycolic acid) (PLGA), also a FDA approved copolymer, can be

14

synthesized by random ring-opening co-polymerization of glycolic acid and lactic acid,

producing different forms depending on the ratio of two monomers. PLGA can be

degraded by hydrolysis in water. Besides, PLGA can be dissolved in a wide range of

common solvents not as the poor solubility of PLA and PGA in organic phase. So far,

PLA and PLGA are in a dramatically wide application among a variety of biomedical

devices because of their satisfactory biodegradability, biocompatibility and restorability

(Holland, Tighe et al. 1986; Van Rensburg, Jooné et al. 1998; Riebeseel, Biedermann et al.

2002). In addition, poly (ε-caprolactone) (PCL) is another biodegradable polyester, which

degrades by hydrolysis quite slow in physiological conditions. Thus it is more attractive

for the preparation of implantable devices and used in controlled release and targeted drug

delivery (Sinha, Bansal et al. 2004; Zhang, Lee et al. 2007).

Scheme 2-3 Chemical structure of some polyesters

2.2.1.2 Polyethylene Glycol

Poly (ethylene glycol) (PEG, Scheme 2-4) is a unique polyether diol, which has been

approved by FDA for human intravenous, oral and dermal application. It can be

manufactured by interaction of ethylene oxide with water, ethylene glycol or ethylene

glycol oligomers. The ratio of reagents determines the chain length, producing a variety of

molecular weights of polymers (http://chemindustry.ru/Polyethylene_Glycol.php).

Initiation of ethylene oxide polymerization using anhydrous alkanols results in a

15

monoalkyl capped PEG such as methoxy PEG, which is amphiphilic and can dissolve in

both organic solvents and water. In addition to linear PEG chains, there are also some

branched PEGs in which two or more PEG chains may be joined through linkers such as

lysine (Monfardini, Schiavon et al. 1995) and triazine (Abuchowski, van Es et al. 1977).

Similarly, amphiphilic copolymers have been developed such as PLA-PEG (Ramaswamy,

Zhang et al. 1997; Nguyen, Allemann et al. 2003; Youk, Lee et al. 2005), PCL-PEG

(Gyun Shin, Yeon Kim et al. 1998; Kim and Lee 2001; Luo, Tam et al. 2002), PVL-PEG

(Tomlinson, Heller et al. 2003), PGA-PEG (Smith, Schimpf et al. 1990; Kim, Shin et al.

1999), etc., which contains both hydrophilic portion and hydrophobic part. Although PEG

is known to be non-degradable, the non-toxic property (Pang 1993), excellent solubility in

aqueous solution (Powell 1980), low level of protein or cellular absorption, extremely low

immunogenicity and antigenicity (Dreborg and Akerblom 1990), and satisfactory

pharmacokinetic and biodistribution behavior (Yamaoka, Tabata et al. 1994) enable it to

be an ideal material in pharmaceutical applications (Hooftman, Herman et al. 1996).

Scheme 2-4 Chemical structure of PEG

2.2.2 Polymeric Drug Formulations

2.2.2.1 Paste

Local administration or direct tumor injection of chemotherapeutic agents has been

expected by a method that can maximize local drug level in tumor environment but

16

minimize systemic exposure to normal organs. Chemotherapeutic polymer-paste is one of

the strategies to reduce local recurrence of disease at tumor site in the surgery. To date,

paclitaxel-loaded polymeric surgical paste has been designed and well developed. PCL,

which has low melting range (50-60 °C) and biodegradation lifetime of 6-9 months in vivo

(Pitt, Gratzl et al. 1981), was employed as the base component. Paclitaxel was added into

melt PCL and then poured into a prepared mould. The polymer with paclitaxel would

solidify after cooling down to obtain the paste. In surgical application, paste was melt and

delivered via injection directly to the tumor resection as a liquid which formed a solid

conform at surgical wound under body temperature (Winternitz, Jackson et al. 1996).

Adding mPEG was reported to reduce the onset of the melting temperature by 5-10 °C

(Winternitz, Jackson et al. 1996), while gelatin, albumin or methylcellulose can speed up

the release of paclitaxel from the matrix (Dordunoo, Oktaba et al. 1997). Therapeutic drug

level can be maintained in the region of implanted site but reduced at non-targeting distant

site. Optimally, all paste formulations showed no great effect on body weight of the

treated animals, revealing the well tolerated drug dose (Zhang, Jackson et al. 1996).

However, the brittle and inflexible properties and difficult manipulation limit the further

development of the paste as the optimal formulations of chemotherapeutic agents.

2.2.2.2 Micelles

A micelle is an aggregate of surfactant molecules dispersed in a liquid colloid, which

contains normal phase micelles (oil-phase-water) and inverse phase micelles (water-in-oil).

When the concentration is higher than the Critical Micelle Concentration (CMC), which is

the concentration of a monomeric amphiphile the micelle appears (Charman 1992), almost

17

all of the polymers can formulate to be micelles as a core-shell structure (Fig 2-1) with a

small particle size (less than 100 nm). Polymeric micelles are manufactured from

copolymers comprising both hydrophilic and hydrophobic polymers as the core and

hydrophobic drug can be mainly entrapped into core. For the hydrophobic part, a

biodegradable polymer such as PLA, PCL, PGA and PLGA is used and for the

hydrophilic part, PEG is the most used polymer to yield high in vitro stability (Yokoyama,

Sugiyama et al. 1993). The hydrophobic drug can be loaded in the amphiphiles by dialysis,

salting out, emulsion and solvent evaporation method.

Fig 2-1 Schematic of a micelle

As drug carriers, micelles can provide quite a few obvious advantages. They can solubilize

poorly soluble drugs and increase drugs’ bioavailability. The size of micelles permits them

to accumulate in body regions through leaky vasculature. The unique structure makes

them hardly reactive to blood or tissue components. By attachment via a certain ligand to

the outer shell, they can result in targeted efficacy. Last but not least, micelles can be

prepared in large quantities easily and reproducibly (Torchilin 2002). So far, some

chemotherapeutic agents have been formulated in micelles. Paclitaxel-loaded micelles of

PEtOx-PCL copolymers was fabricated by dialysis method, which had a size of only 20

18

nm with a drug loading efficiency of 0.5-7.6%. The micelles showed comparable

therapeutic efficacy to the commercial paclitaxel in vitro (Cheon Lee, Kim et al. 2003). By

conjugating folate to PCL-mPEG copolymer, Park et al (Park, Lee et al. 2006) formulated

the targeted micelles. Doxorubicin-loaded micelles of PEO-P(Asp) (Yokoyama, Kwon et

al. 1992), PEO-PBLA (Kwon, Naito et al. 1995), PEO-PDLLA (Pişkin, Kaitian et al.

1995), PAA-PMMA (Inoue, Chen et al. 1998), etc. have been investigated. Although

micelles can give some improvement in chemotherapy, the instable problems for storage

became a restriction in further application (Tomlinson, Heller et al. 2003).

2.2.2.3 Liposomes

Liposomes is a phospholipids spherical vesicle, consisting of an aqueous core surrounded

by a lipid bilayer or multilayer (Fig 2-2) (http://en.wikipedia.org/wiki/Liposome), usually

range in size from 0.05 to 5.0 µm. Phospholipids possess a polar head and two

hydrophobic tails, which is significant to the formulation of bilayer in aqueous solution.

Due to the unique structure, liposomes can protect and carry hydrophilic drug in aqueous

core or hydrophobic drug in the lipid layers. The preparation methods may varies

according to different size and characteristic liposomes, generally including thin-film

hydration, freeze-drying, detergent dialysis, calcium induced fusion, reverse-phase

evaporation, sonication and extrusion (Sharma and Sharma 1997).

19

Fig 2-2 Liposomes for drug delivery

Liposomes have attracted much attention in medical application, which is attributed to

their biocompatibility with cell membranes, capability to protect drug from degradation,

especially for protein drugs, and ability to add specific targeting ligands on surface (Lee

and Yuk). As liposomes are manufactured from lipids which are relatively non-toxic, non-

immunogenic, biocompatible and biodegradable, a broad range of water-insoluble drugs

such as cyclosporine and paclitaxel can be encapsulated. Sharma et al. (Sharma, Mayhew

et al. 1997) reported that paclitaxel liposomes could deliver drug effectively in body

system and improve therapeutic index in in vivo model. Traditional liposomes are taken up

by RES and cleared quickly from blood circulation (Poste, Bucana et al. 1982).

Cholesterol and PEG modified liposomes significantly impaired such limitations (Wheeler,

Wong et al. 1994). Moreover, PEGylated liposomes tagged with transferring exhibited 2-

to 3-fold higher targeting effect than the plain liposomes (Visser, Stevanovic et al. 2005)

and folate-targeted liposomes showed much higher affinity to tumor cells (Goren,

Horowitz et al. 2000).

20

Although liposomes have achieved some improvement in drug delivery system, issues like

stability, reproducibility, sterilization method, low drug loading efficiency, particle size

control and short circulation half-life in body are the remaining problems need to be

solved (Sharma and Sharma 1997).

2.2.2.4 Microspheres

Microspheres, especially biodegradable polymeric microspheres, have been studied

extensively. Solvent evaporation and spray drying are two commonly used methods for

microspheres preparation, as well as hot melt microencapsulation, solvent removal, phase

inversion microencapsulation (Vasir, Tambwekar et al. 2003). In solvent evaporation

method, spherical droplets can be formed by dispersing oil soluble monomers in aqueous

solution (oil in water, O/W) or water soluble monomers in an organic phase (water in oil,

W/O). Often, a double emulsion is employed, which means the first W/O emulsion in

which drug is loaded in aqueous phase is then dispersed in another aqueous medium to get

the final O/W emulsion. Microspheres are able to protect the drug molecules against

degradation, control their release after administration and facilitate their passage across

biological barriers. Some researchers have achieved a constant release of drug from the

polymeric microspheres via a W/O/W double emulsion solvent evaporation method after

the initial burst (Yang, Chung et al. 2000). Liggins et al (Liggins, D'Amours et al. 2000)

indicated that microparticles with less than 8 µm may be cleared from the peritoneum into

the lymph nodes. Recently, poly(ortho-ester) microspheres was made for delivering DNA

vaccines and tested in vivo (Wang, Ge et al. 2004). However, the relatively large size of

microspheres may limit its application in some cases such as intravenous delivery.

21

2.2.2.5 Nanoparticles

Polymer nanoparticles are microscopic particles with particles size less than 1 µm

diameter, which have became an attractive area for drug delivery application, especially

for biodegradable polymeric nanoparticles. Nanoparticles can be used to deliver

hydrophilic or hydrophobic drugs, proteins, vaccines, biological macromolecules, etc..

Nanoparticles can be fabricated by dispersion of polymers and polymerization of

monomers, which involves solvent extraction/evaporation method, salting-out method,

dialysis method, supercritical fluid spray technique and nanoprecipitation method (Feng

and Chien 2003). The most commonly used method is solvent extraction/evaporation,

which can encapsulate hydrophobic drugs by single emulsion and hydrophilic drugs via

double emulsion method.

Polymeric nanoparticles have been investigated as potential drug carriers because of their

unique advantages including providing a controlled release of drugs, targeting drugs to

tumors, showing available size for intravenous injection, reducing the uptake of drugs to

RES, improving biodistribution of drugs in body (Kim, Lee et al. 2003). It has been

indicated that the new-concept chemotherapy by nanoparticles of biodegradable polymers

can realize personalized chemotherapy with controlled dosage and duration, localized

chemotherapy by targeting, sustained and controlled chemotherapy with favorable release

profile of drugs, chemotherapy across biological barriers like GI and BBB, chemotherapy

at home via oral, nasal or ocular administration (Feng 2004). Innovatively fabricated PLA-

TPGS, PLGA-MMT and PLA-mPEG nanoparticles for paclitaxel formulation exhibited

great superiority over Taxol and even the PVA-emulsified PLGA nanoparticles (Li, Price

et al. 1999; Feng and Huang 2001; Feng, Yuan et al. 2002; Riebeseel, Biedermann et al.

22

2002; Mu and Feng 2003; Mu and Feng 2003; Yin Win and Feng 2005; Dong and Feng

2006; Win and Feng 2006; Zhang and Feng 2006). Besides, targeting (Song, Labhasetwar

et al. 1998; Nishioka and Yoshino 2001) and multifunctional (Kopelman, Lee Koo et al.

2005) nanoparticles also become attractive and have been investigated recently.

Nanoparticles, indeed, showed a promising approach, however, the initial burst and

incomplete release of the encapsulated molecules in microspheres and nanoparticles,

particularly for protein, may influence their application (Hong Kee Kim 1999; Kwon,

Baudys et al. 2001).

2.3 Prodrug

Polymer-drug conjugation is a major strategy for drug modifications, which manipulates

therapeutic agents in molecular level in order to increase their biological activity. Such a

strategy is based on a central assumption that the molecular structure of drugs can be

modified to make analogous agents, which are chemically distinct from the original

compound, but produce a similar or even better biological effect. Drug modifications are

frequently directed to alter the properties of the drug that influence its concentration

(solubility), its duration of action (stability), or its ability to move between compartments

in tissues (permeability). Polymer-drug conjugation can modify biodistribution of

therapeutic agents, thus improving their pharmacokinetics (PK) and pharmacodynamics

(PD), increasing their therapeutic effects and reducing their side effects, as well as provide

a means to circumvent the multi-drug resistance (MDR). Prodrug, as the pharmacological

substance in an inactive form, can be metabolized in the body in vivo into the active

compound once administered (Saltzman 2001). Polymer-anticancer drug conjugation has

been intensively investigated in the literature (Khandare and Minko 2006). Some

23

polymeric prodrugs have stepped into clinical development and several conjugates have

shown promise (Kopeček, Kopečková et al. 2001; Duncan 2003).

2.3.1 Design and Synthesis of Polymeric Prodrugs

Polymers have been a hot spot as carriers of drugs over the latest decades, especially

designed to be polymeric prodrugs. A number of polymeric conjugation methods have

been investigated since 1955, when peptamin-polyvinylpyrrolidone conjugates with

improved efficacy was reported (Jactzkewits 1955). In present, there are three major types

of polymeric prodrugs in use: (a) prodrugs which can be broken down in cells to release

active agents, (b) prodrugs in combination of more than one substance, (c) prodrugs with

targeting ability. Generally, an ideal polymeric prodrug was revealed to possess one or

more of the following components: (a) a polymeric backbone as a carrier, (b) active

therapeutic agents, (c) appropriate spacers, (d) an imaging substance and (e) a targeting

molecule (Fig 2-3) (Khandare and Minko 2006). The choice of an appropriate polymer

and a targeting agent is crucial for the success of a prodrug. The selection of a polymer

should meet the following criteria: (a) available chemical functional groups to permit

covalent linkage with drugs or targeting agents, (b) hydrophilic property to ensure water

solubility, (c) degradability to ensure excretion from the body, (d) biocompatibility to

avoid immunogenic response, (e) availability in reproduction and administration (Soyez,

Schacht et al. 1996). The selected polymers can be classified by the origin, chemical

structure, biodegradability and molecular weight. In addition, modification of a polymer is

significant, which depends upon the reactive chemical groups in polymer and the

functional group of the drug. Most of the biomolecules like ligands, peptides, proteins,

carbohydrates, lipids, polymers, nucleic acid and oligonucleotide possess functional

24

groups for conjugation or have potential to be activated for further conjugation. According

to different molecules, a suitable method, process and reagents are, indeed, vital to the

successful conjugation. The following are some strategies commonly used to obtain a

polymeric prodrug, mainly regarding the active group and polymer backbone.

Fig 2-3 Schematic presentation of (a) polymeric conjugate with targeting moiety and (b) hyperbranched polymeric conjugate with targeting and imaging components

2.3.1.1 N-hydroxysuccinimide (NHS) Ester Coupling Method

An N-hydroxysuccinimide (NHS) ester is perhaps the most common activation chemical

method for creating reactive acylating agents (Scheme 2-5). Carboxyl groups activated by

NHS esters show highly reactive to amine nucleophiles. NHS ester derivatives can be

prepared in nonaqueous condition employing water-insoluble carbodiimides or

25

condensing agents, or in aqueous media using the water-soluble carbodiimide EDC in situ

(Hermanson 1996). Due to the high reactivity at physiological pH, NHS is widely utilized

as an acylating agent and preferred for conjugation with amine terminal compounds (Cruz,

Iglesias et al. 2001; Luis J. Cruz 2001; Chockalingam, Gadgil et al. 2002; Kajiyama,

Kobayashi et al. 2004; Cabrita, Abrantes et al. 2005; Natarajan, Xiong et al. 2005).

However, both hydrolysis and amine reactivity would increase with the increase of pH.

Thus maintaining high concentrations of protein or other target molecules in the reaction

is quite necessary.

Scheme 2-5 NHS esters compounds react with nucleophiles to release NHS leaving group

2.3.1.2 Carbodiimide Coupling Method

A carbodiimide is a functional group comprising the formula N=C=N, which is used to

mediate the formation of an amide or phosphoramidate linkage between a carboxylate and

an amine or a phosphate and an amine, respectively (Scheme 2-6). The carbodiimides are

either water-soluble such as EDC or water-insoluble such as DCC and DIC. Water soluble

carbodiimides can be used for biomacromolecular conjugation in a variety of buffer

solutions. On the other hand, water-insoluble carbodiimides are preferred for conjugation

26

of polymers with drugs, imaging agents, targeting agents or other polymers in organic

solvents (Hermanson 1996).

Scheme 2-6 Carbodiimide amide coupling scheme

The most commonly used carbodiimides are N,N'-dicyclohexylcarbodiimide (DCC), N,N'-

diisopropylcarbodiimide (DIC) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide

hydrochloride (EDC) (Scheme 2-7). DCC can bring high yielding amide coupling reaction

but lead dangerous allergic reaction contacted with skin and the insoluble byproduct N,

N’-dicyclohexylurea is difficult to be removed completely, which limits its application in

certain reactions. DIC, as a liquid, can be easily removed by extraction and does not lead

an allergic reaction, which has been developed as an alternative to DCC. With a favorable

solubility in water, EDC was widely used in peptide synthesis, protein crosslinking with

nucleic acids and immunoconjugation (Nakajima and Ikada 1995).

Scheme 2-7 Chemical structure of DCC, DIC and EDC, respectively

27

2.3.1.3 Dextran-prodrug

Dextran is a complex, branched, natural polysaccharide, which possesses a huge number

of primary and secondary carbohydrate hydroxyl groups (Scheme 2-8). Thus it can be

conjugated to drugs and proteins which have active chemical groups either by direct

conjugation or incorporation of a spacer. Dextran is manufactured from sucrose by certain

lactic-acid bacteria, the best-known as Leuconostoc Mesenteroides and Streptococcus

mutans. The homogeneous structure of dextran makes the synthesized conjugates easy to

characterize and the high water solubility can be restored (Larsen 1989).

Scheme 2-8 Chemical structure of dextran

Dextran has been extensively investigated as a polymeric carrier to deliver anticancer

drugs through conjugation. Mehtyprednisolone and dexamethasome were covalently

attached to dextran using succinate linker, which showed potential of the dextran prodrug

in colon-specific delivery of glucocorticoides (McLeod, Friend et al. 1993).

Methylprednisolone-dextran prodrug (Scheme 2-9) also employed succinic acid as a linker

and 1,1’-Carbonyldidmidaxole as coupling agent, which achieved controlled hydrolysis

(Mehvar, Dann et al. 2000). The multiple hydroxyl groups on dextran provide efficient

functional sites for conjugation. In camptothecin (CPT) analogue-carboxymethyl (CM)

28

dextran conjugate via Gly-Gly-Gly linker, fluorescein isothiocyanate (FITC) was also

conjugated to dextran, producing a prodrug with imaging moiety as shown in Scheme 2-

10 (Harada, Murata et al. 2001). A dextran-peptide-methotrexate conjugate with tumor

targeting capability was reported (Chau, Tan et al. 2004). Due to the conjugated linker,

Pro-Val-Gly-Leu-Ile-Gly peptide cleavable by matrix metalloproteinase II (MMP-2) and

IX (MMP-9) that are over expressed in tumors, the conjugate exhibited satisfactory

stability and high selectivity uptake.

Scheme 2-9 Chemical structure of dextran-methylprednisolone hemisuccinate

29

Scheme 2-10 Synthesis of FITC-labeled CM dextran

2.3.1.4 N-(2-hydroxypropyl)methacrylamide (HPMA)-prodrug

N-(2-hydroxypropyl)methacrylamide (HPMA) is a water-soluble, nonimmunogenic,

synthetic polymer, which has suitability as carriers for drug delivery (Kopecek 1990).

HPMA copolymers are not biodegradable but are highly biocompatible (Kopeček,

Kopečková et al. 2000). HPMA homopolymer was designed and manufactured in

Czechoslovakia as a plasma expander (Kopecek and Bazilova 1973). Many functional

groups such as charged groups, targeting moieties and drugs can be incorporated into the

polymer backbone. The spacers in this connection can be either nondegradable or

degradable. The dipeptide glycylglycin (GG) is always utilized as nondegradable spacer

(Drobník, Kopeček et al. 1976) while the acid sensitive cis-aconityl bond and the

30

lysosomal enzyme sensitive tetrapeptide GFLG are employed as degradable spacers

(Rejmanova, Kopecek et al. 1985).

HPMA copolymers and their conjugates with drugs have become one of the most

extensively investigated systems. An HPMA copolymer with doxorubicin conjugated with

the peptidyl linker GFLG exhibited superior cytotoxicity in vitro and successfully

overcame P-gp mediated drug resistance (Minko, Kopečková et al. 1999; Kopeček,

Kopečková et al. 2001), which also displayed significant effectiveness in reducing both

sensitive and resistant tumors in vivo as compared to free DOX (Kopeček, Kopečková et

al. 2000). Various targeting moieties, such as carbohydrates and antibodies, were attached

to HPMA copolymer conjugate, which achieved the biorecognizability at the plasma

membrane (Chytry, Letourneur et al. 1998; Chytil, Etrych et al. 2006). A targeted

galactosamine containing HPMA copolymer-GFLG-DOX was found to induce dramatic

tumor associated toxicity (Duncan 1992). These preclinical studies prepared the clinical

trials of the conjugate, which is available currently. A number of synthetic methods have

been developed (Scheme 2-11) for antibodies attached to HPMA copolymer conjugates

via amino groups and oxidized saccharide (Omelyanenko, Kopečková et al. 1996; Chytil,

Etrych et al. 2006).

2.3.1.5 Dendrimer Conjugates

Dendrimer, which was defined in the late 1970s and early 1980s, is a new class of

macromolecules with highly branched, three-dimensional features like the architecture of

a tree. A typical dendrimer (Scheme 2-12) consists of a multifunctional central core (C),

branched units (B) and surface groups (S). The branched units represent the repeating

31

monomer unit which is also called “generations” (Tomalia, Naylor et al. 1990).

Dendrimers are synthesized from monomers by a step-growth polymerization process,

including mainly two approaches: divergent (Tomalia 1996) and convergent (Hawker and

Frechet 1990). The gradual stepwise process of synthesis determines a well defined size

and structure. The surface characteristics facilitate conjugation of a broad range of

molecules with different functionality, no matter with drugs, imaging agents or targeting

moieties (Dufes, Uchegbu et al. 2005).

Scheme 2-11 Examples of synthesis of HPMA-drug antibody conjugates

The highly branched structure, the low polydispersity and modifiable end groups assure

that dendrimer can be used to develop novel drug conjugates. Polyamidoamine (PAMAM)

dendrimers were the first complete dendrimer family (G=0-7), which are prevalent

32

nanovehicles for delivery of bioactives. 5-fluorouracil (5FU)-PAMAM conjugates with

cyclic cores was synthesized using the “time-sequenced propagation” technique, which

exhibited slow release of the drug (Zhuo, Du et al. 1999). Conjugation of the famous

anticancer drug cisplatin with PAMAM dendrimers showed slower release, higher

accumulation in tumors and lower systemic toxicity than free cisplatin (Malik, Evagorou

et al. 1999). The use of dendrimers as vectors for tumor targeting was also investigated.

Fréchet et al. fabricated ester-terminated dendrimers containing folic acid, or methotrexate

(MTX) as drug delivery with tumor cell specificity (Kono, Liu et al. 1999). The conjugate

of folic acid to the dendrimer utilized coupling of γ-carboxyl group in folic acid to

hydrazide groups in dendrimers. Dendrimer-peptide conjugates were also reported for the

development of synthetic vaccines, which had multiple copies of the peptide so that small

concentrations could result in the required immune response (Tam 1988; McGeary,

Jablonkai et al. 2001).

Scheme 2-12 Typical architecture of a third generation dendrimer

33

2.3.2 PEGylated Drug Conjugation

PEG possesses an ideal array of properties such as very low toxicity, excellent solubility

in aqueous media, availability in various molecular weights, extremely immunogenicity

and antigenicity, ready excretion after administration although non-biodegradable,

favorable pharmacokinetics and biodistribution, which enable PEG conjugates to become

classical prodrugs with approval for human use (Goren, Horowitz et al. 2000). Most

commonly used PEGs for prodrug modification are monomethoxy PEG and di-hydroxyl

PEG (Scheme 2-13). PEG has limited conjugation capacity since only two terminal

functional groups can be conjugated to biocomponents. Recently, this limitation has been

overcome by coupling amino acid to double the number of active groups (Greenwald,

Choe et al. 2003). To date, a number of strategies have been developed in PEGylated

conjugates. Anticancer agents have particularly benefited from this technology as well as

applications to larger proteins.

Scheme 2-13 Chemical Structure of (a) monomethoxy-PEG and (b) di-hydroxyl PEG

2.3.2.1 PEGylation of Small Organic Molecules

34

Water-soluble PEG-drug conjugates are considered to be most promising in drug delivery

system, especially for delivering hydrophobic drugs with low molecular weight in cancer

therapy. The most straightforward strategy towards drug-PEG conjugates is relying on

involvement of the terminal primary hydroxyl groups of PEG to affect the conjugate

reaction. Direct conjugation of drugs to hydroxyl groups of PEG can be achieved by

forming ether, carbonate or urethane bonds. With the use of certain spacers like succinate,

PEG can be used in a larger range of drugs (Goren, Horowitz et al. 2000).

A prodrug strategy using PEG as solubilizing agent has been reported in the case of

paclitaxel (Greenwald, Gilbert et al. 1996), in which PEGs with different molecular

weight were employed and compared. Esters with PEG in the α-position were found quite

effective as linking groups in the design of the prodrug. In vivo test suggested that

molecular weight play a significant role in solubilizing effect. PEG polymers can be

modified by small amino acids or other aliphatic chains molecules. For example, The

antitumor agent 1-β-D arabinofuranosilcytosyne (Ara-C) was covalently attached to linear

or branched PEG through amino acid spacer for controlled release (Schiavon, Pasut et al.

2004). Furthermore, the hydroxyl group of PEG was functionalized with a bicarboxylic

amino acid to form a tetrafunctional derivative (Scheme 2-14), resulting in the conjugates

with four or eight Ara-C molecules in one PEG chain. Besides, PEG-doxorubicin

conjugate using amino acid as spacer was investigated (Veronese, Schiavon et al. 2005).

Although less cytotoxicity in vitro was observed, the prolonged plasma circulation time

and greater tumor targeting showed its promise compared to free drug.

35

Scheme 2-14 Synthetic schemes of (a) PEG-Ara-C4 and (b) PEG-Ara-C8 conjugates

2.3.2.2 PEGylation of Polypeptide (Peptides and Proteins)

The conjugation of PEG with the polypeptide (peptides and proteins) has been

accomplished mainly via reaction with available amino groups, while other reactive sites

such as histidine and cysteine can be utilized. So as to achieve successful coupling, PEG

36

must be activated first, which may be done using active ester, active carbonates,

maleimides or thiazolidine (Greenwald, Choe et al. 2003).

In the pioneering work, PEG-proteins were prepared by conjugation of bovine serum

albumin (BSA) with mPEG-dichlorotriazine, which exhibited significantly reduced

immunogenicity and antigenicity (Abuchowski, van Es et al. 1977). R-hirudin, an

effective antithrombotic peptide, can be modified with two strands of PEG through

urethane bonds to achieve a dramatically prolonged half-life time with enhanced

antithrombotic activity and no immunogenicity (Esslinger, Haas et al. 1997). PEGylation

of granulocyte-colony stimulating factor (G-CSF) contained a single strand of PEG-20000

covalently bound with the N-terminal amino group of the methionyl moiety of G-CSF

(Holmes, O'Shaughnessy et al. 2002). The resultant high molecular weight of the prodrug

significantly reduced renal clearance. Clinical investigation of several other PEGylated

protein drugs have also been reported, such as IL-2 (Yang, Topalian et al. 1995), growth

hormone antagonist (Trainer, Drake et al. 2000), and α-interferon (Wang, Youngster et al.

2000).

2.3.2.3 Targeting PEGylation

Targeting of the anticancer agents is crucial to the specific sites as it can maximize cancer

cell death effect and minimize normal cell survive effect. The prodrug that antibody-

targeted biodegradable PEG multi-block attached to N2,N5-diglutamyllysine tripeptide

with doxorubicin conjugated via acid-sensitive hydrazone bond has been designed and

synthesized (Ulbrich, Etrych et al. 2002). PEG was first activated with phosgene and N-

hydroxysuccinimide and then interacted with amine group of triethyl ester of tripeptide

37

N2,N5-diglutamyllysine to get a degradable multi-block polymer, which, later, was

converted to the polyhydrazide by hydrazinolysis of the ethyl ester with hydrazine hydrate.

A part of the hydrazide group in polymer was modified with succinimidyl 3-(2-

pyridyldisulfanyl) porpanoate to introduce a pyridyldisulfanyl group for conjugation with

an activated antibody. DOX was coupled to the remaining hydrazide groups by acid-labile

hydrazone bonds to the polymer (Scheme 2-15). This prodrug was found having very high

antitumor activity and significant decrease of non-specific accumulation in the heart, liver

and lungs. Several other targeting prodrugs, such as DU-257-PEG-KM231 mAb (Suzawa,

Nagamura et al. 2000) and luteinizing hormone-releasing-hormone-PEG-camptothecin

(Dharap, Qiu et al. 2003) conjugate, have also been investigated recently.

Scheme 2-15 Architecture of multi-block PEG-DOX with targeting antibody conjugated

2.4 Vitamin E TPGS

2.4.1 Chemistry of Vitamin E TPGS

D-α-tocopheryl polyethylene glycol 1000 succinate (Vitamin E TPGS or TPGS) was

developed in the 1950s as a water-soluble derivative of lipid-soluble natural vitamin E,

which is prepared by esterification of d-α-tocopheryl succinate (TOS) with polyethylene

38

glycol 1000. TPGS appears a waxy solid at room temperature and stable in air. The

molecular weight is around 1542 Da. The thermal degradation and melt temperature are

199 °C and 38-41 °C, respectively (http://www.eastman.com). TPGS possesses a very low

critical micelle concentration (CMC) that is 0.02% and can form low viscosity solutions in

water until the concentration at 20 wt%, beyond which lipid crystalline phases may form

(Bogman, Zysset et al. 2005).

Scheme 2-16 Chemical structure of TPGS: red = hydrophobic vitamin E, green = hydrophilic PEG chain, black = succinate linker

TPGS is an amphiphile consisting of lipophilic alkyl tail (TOS) and hydrophilic polar

head portion (PEG) as shown in Scheme 2-16, which results in the bulky structure and

large surface area. The hydrophile-lipophile balance (HLB) of TPGS is about 13.2. The

unique chemical properties of TPGS have suggested its application as a solubilizer, an

emulsifier, an absorption enhancer, a plasticizer, a water-soluble source of vitamin E, and

a carrier of hydrophobic drugs. TPGS shows no human toxicity data to date. National

cancer institute reported that the safety oral dosage of TPGS can be up to 1 g/kg/day

39

(http://www.eastman.com). The acute oral LD50 of adults is larger than 7 g/kg for either

TPGS or its moieties, PEG 1000 and TOS (Krasavage and Terhaar 1977). The Japanese

government’s Ministry of Health, Labour and Welfare approved the application of TPGS

as a pharmaceutical excipient for oral drug formulations in 2005

(http://www.inpharmatechnologist.com).

2.4.2 Solubilizer for Water-insoluble Compounds

It is well known that quite a few drugs, especially anticancer drugs, are poorly water

soluble. There are many strategies improving solubility and TPGS is a powerful tool to

solubilize many hydrophobic agents. TPGS was used as solubilizing adjuvant to formulate

emulsion of an oily composition of several antitumor drugs (Kawata, Ohmura et al. 1986).

Another early step on the solubilizing effect of TPGS is the work by Ismailos et

al.(Ismailos, Reppas et al. 1994). The aqueous solubility of cyclosporine A (CyA) at three

different temperatures (5, 20 and 37 °C) showed 2- to 16-fold increase in the presence of

various concentrations of TPGS ranging from 0.01 to 0.05 mM. Later, Yu, L. et al (Li,

Price et al. 1999) reported that TPGS significantly improved solubility of the poorly water

soluble drug amprenavir, which is a potent HIV protease inhibitor. The solubility linearly

increased with increasing TPGS concentration through micelle solubilization since no

increase showed when the concentration of TPGS was below CMC. TPGS was also found

enhancing the solubility of another selective HIV-1 non-nucleoside reverse transcriptase

inhibitor, thiocarboxanilide UC-781 (Deferme, Van Gelder et al. 2002). Moreover, a

dramatic increase in estradiol solubility was found in the aqueous solvent as well as in

various concentration of ethanol with TPGS (Sheu, Chen et al. 2003). Recently, a new

40

successful story was documented involving anticancer drug paclitaxel (Varma and

Panchagnula 2005). Aqueous solubility of paclitaxel was significantly enhanced up to 38

times by TPGS, revealing TPGS is one of the best semi-solid oral dosage forms for

taxoids.

2.4.3 Absorption Enhancer

TPGS has caused much attention on its ability to enhance the absorption of several drugs

that, otherwise, have poor bioavailability. Sokol et al. (Sokol, Narkewicz et al. 1991),

Boudreaux et al. (Boudreaux, Hayes et al. 1993) and Chang et al. (Youk, Lee et al. 2005)

demonstrated that TPGS could enhance absorption of the lipophilic drug cyclosporine,

which is an immunosuppressive agent to prevent the rejection of transplanted organs. As a

consequence, the required daily dosage of cyclosporine was significantly reduced to

maintain therapeutic blood plasma concentration of the drug. TPGS also displayed well as

an absorption enhancer in amprenavir (Li, Price et al. 1999), which has been already used

in commercial amprenavir capsules, an agenerase that contains approximately 36 IU of

vitamin E in 50 mg capsule. Besides many examples of TPGS used for poorly water

soluble drugs, examples of TPGS used in poorly permeable drugs that are water soluble

were also reported. Vancomycin hydrochloride, a glycopeptide antibiotic, is water soluble

but poorly absorbed from the gastrointestinal tract. By introducing TPGS, the Cmax value

of vancomycin hydrochloride in plasma of rats increased from negligible to over 4.98

µg/mL (Prasad, Puthli et al. 2003).

2.4.4 Bioavailability Enhancer

41

As a water-soluble derivative of natural vitamin E, TPGS alone or TPGS/d-α-tocopheryl

acetate blend provides enhanced bioavailability in not only animals but also some

populations of human. Quite a few studies supported the use of TPGS to deliver vitamin E

in human exhibiting fat malabsorption (Traber, Kayden et al. 1986; Sokol, Heubi et al.

1987; Sokol, Butler-Simon et al. 1993). In the trial, Solol et al. dosed TPGS to 60 children

with chronic cholestasis, who were unresponsive even to 70-212 IU/kg/day of other oral

vitamin E forms. All children responded to TPGS at only 25 IU/kg/day oral dose of TPGS

and neurological function was improved or stabilized in most of patients. The vitamin E

trial was performed using an emulsified form of tocopheryl acetate, showing the

concentrations of tocopherols in plasma, erythrocytes and adipose tissues after

administration were high with TPGS. Thus TPGS provided better bioavailability than the

other forms. The proposed mechanism explained that the formed TPGS micelles could

cross from the intestinal lumen to enterocytes. TPGS may be hydrolyzed to release the

tocopherol which may be transferred into the enterocyte or the whole TPGS micelle could

be taken up by the enterocyte. Besides, TPGS can also improve the bioavailability of other

fat soluble vitamins, such as vitamin D (Argao, Heubi et al. 1992).

Not only can TPGS improve the bioavailability of vitamins, it also can enhance the

bioavailability of pharmacological compounds. Khoo found that by employing TPGS the

solid dispersion of halofantrine, an important anitmalarial agent, could result in 5-fold

improvement in absolute oral bioavailability (Khoo, Porter et al. 2000). TPGS enhanced

bioavailability of cyclosporine A in liver transplant patients and reduced daily dosage to

40-70% (Sokol, Butler-Simon et al. 1993). Paclitaxel, an antimicrotubule anticancer drug,

exhibits very poor absorption because of both poor solubility and permeability and thus it

42

is usually administered via intravenous injection. TPGS enhanced the bioavailability of

paclitaxel in the in vivo studies due to the increased solubility and permeability. It was

able to improve not only the bioavailability of oral paclitaxel but also that of other

biopharmaceutical classification system class II-IV drugs (Varma and Panchagnula 2005).

Regarding the mechanism of TPGS enhancing bioavailability, while Sokol originally

attributed the reason to micelles formation enhancing solubility, others later provided

evidence suggesting it could be due to P-glycoprotein (P-gp) inhibition resulting in

enhanced permeability (Dintaman and Silverman 1999; Wacher, Wong et al. 2002). P-gp

is primarily expressed on the luminal surface of epithelial cells from certain tissues such

as intestine, liver, kidney (Thiebaut, Tsuruo et al. 1987). TPGS can play a role as reversal

agent of P-gp mediated multidrug resistance (MDR) and inhibit P-gp substrate drugs

transporting. For example, TPGS significantly increased the bioavailability of P-gp

substrate talinolol in coadministration with talinolol form in vitro cell permeability on

Caco-2 cells and in vivo intraduodenal perfusion study (Bogman, Zysset et al. 2005). It

has also been reported that vitamin E was able to protect the human hepatocellular

carcinoma cells from free-radical induced lipid peroxidation and oxidative DNA damage

so as to inhibit the MDR phenotype (Fantappiè, Lodovici et al. 2004). Moreover, TPGS

was found to enhance the cytotoxicity of doxorubicin, vinblastine, paclitaxel and colchine

that are P-gp substrate agents in G185 cells but not for that of 5-FU which is not P-gp

substrate (Dintaman and Silverman 1999). TPGS can improve the permeability of a drug

across cell membranes by inhibiting P-gp, and thus enhance the bioavailability and

absorption of a drug.

43

2.4.5 Anticancer Enhancer

TPGS, a PEG-conjugated derivative of TOS, has been found possessing potent anticancer

activity since TOS has anticancer properties against wide spectra of cancers such as colon,

melanomas, breast, brain, lung and prostate cancers (Malafa and Neitzel 2000; Neuzil,

Weber et al. 2001; Barnett, Fokum et al. 2002; Zhang, Ni et al. 2002; Swettenham,

Witting et al. 2005). As a comprising moiety, α-tocopherol had the multidrug resistance

(MDR)-neutralizing activity of the chemosensitizers cyclosporine A, verapamil GF120918,

clofazimine and a derivative of clofazimine, B669 (Van Rensburg, Jooné et al. 1998). The

anticancer activity of TOS is related to its apoptosis-inducing characteristics, which

seemed to be mediated involving the generation of reactive oxygen species (ROS) which

can damage DNA, proteins and fatty acids in cells leading to apoptotic cell death (Ottino

and Duncan 1997; Wang, Witting et al. 2005). In xenograft studies, TOS was able to

suppress tumor growth either alone or in combination with other anticancer agents but

showed less antiproliferative potent in normal cell types including intestinal epithelial

cells, prostate cells and hepatocytes than that in tumor cells (Neuzil, Weber et al. 2001;

Barnett, Fokum et al. 2002; Weber, Lu et al. 2002; Zhang, Ni et al. 2002). However, the

poor water solubility of TOS limits its therapeutic efficacy. Through the conjugation to

PEG, the solubility was significantly increased, which was expected to show better

efficacy. Youk et al. have confirmed that TPGS possessed more effective inhibition on the

growth of human lung carcinoma cells in in vitro cells and in nude mice than that of TOS

(Youk, Lee et al. 2005). The superiority of TPGS over TOS in anticancer efficiency was

attributed to the increased apoptosis rather than increased cell uptake.

44

2.4.6 Drug Delivery Applications

Polymeric drug carriers, by which the drug can be entrapped, encapsulated, adsorbed,

attached or coupled, open a new era of drug delivery to provide sustained, controlled and

targeted effects. In drug delivery, TPGS can be used to improve the dissolution rate and

bioavailability by forming a solid dispersion or a solid solution, controlling or modifying

the release of the drug, and masking the bitter taste of a drug. Due to the bulky

amphiphilic structure and large surface area, TPGS is expected to act as a good emulsifier

or surfactant in drug delivery system, especially in fabricating micro/nanoparticles. As Ke

et al (Bogman, Zysset et al. 2005) indicated, TPGS can be a good surfactant in oral

delivery of protein drugs and in synthesis of biodegradable magnetic microspheres. TPGS

can also be surfactant coating material in fabrication of PLGA nanoparticles by modified

solvent extraction/evaporation method (Mu, Seow et al. 2004). Mu and Feng demonstrated

that TPGS acted as a more effective and safer emulsifier for fabrication and

characterization of polymeric nanoparticles in comparison with traditional PVA

(Riebeseel, Biedermann et al. 2002). TPGS can achieve far higher emulsification

efficiency with the amount of 0.015% (w/w) in fabrication whereas 1% PVA is required in

similar process. Co-polymerizing TPGS with a polymer such as PLA, PLGA or PCL can

enhance the emulsification efficiency and drug loading capability as demonstrated in the

studies on microencapsulated paclitaxel (Mu and Feng 2003; Zhang and Feng 2006). The

in vitro drug release was increased by 2-fold after one day and 3-fold after 31 days for the

PLGA and PLA/TPGS nanoparticles, respectively. It also showed to tailor the release rate

via increasing the entrapped drug release (Xie and Wang 2005). Besides, TPGS can be

used as a good additive in fabricating PLGA nanoparticles and discs for paclitaxel

45

formulation with up to 80% drug loading efficiency (Wang, Ng et al. 2003; Mu, Teo et al.

2005).

The further in vitro and in vivo investigation confirmed the great potential of TPGS in

drug delivery. Feng et al (Feng 2004) demonstrated that TPGS emulsified nanoparticles

could increase cancer cell mortality and enhance the cellular uptake of Caco-2 cells that

used as a model of the GI barrier. Nanoparticles coated by TPGS eliminated the acute side

effects caused by Cremophor EL which is an adjuvant in commercial Taxol. In in vivo PK

investigation of PLA-TPGS nanoparticles prepared by the dialysis method, 1.8-times

larger AUC and 11.6-times longer half-life were observed than that of Taxol after i.v.

administration (Feng 2006). As it is for TPGS emulsified PLGA nanoparticles, 4.9-times

increased AUC and 2.4-times prolonged therapeutic time in comparison of Taxol after i. v.

injection were reported (Win and Feng 2006).

Moreover, TPGS displayed promise as a matrix material in nasal delivery. Prepared by the

combination of double emulsion method and spray drying method, diphtheria taxoid

loaded PCL/TPGS microspheres exhibited high immune response than that achieved using

PCL microspheres alone, which showed favorable potential of TPGS as a novel adjuvant

either alone or with other appropriate delivery materials (Somavarapu, Pandit et al. 2005).

2.5 Doxorubicin

2.5.1 History

Doxorubicin, (8s, 10s)-10-(40amino-5hydroxy-6-methyl-tetrahydro-2H-pyran-2-yloxy)-6,

8, 11-trihydroxy-8-(2-hydroxyacetyl)-1-methoxy-7, 8, 9, 10-tetrahydrotetracene-5, 12-

46

dione, is an anthracycline antibiotic. The history of its discovery can trace back to the

1950s, when a bright red pigment, later named daunorubicin, was isolated from

Streptomyces peucetius. The clinical use began in 1960s for treating acute leukemia and

lymphoma, although fatal cardiac toxicity was recognized in 1967 (Tan, Tasaka et al.

1967). In 1969, a strain of Streptomyces mutated by N-nitroso-N-methyl urethane

produced a different, red-colored antibiotic, doxorubicin (Scheme 2-17), which exhibited

better anticancer activity than daunorubicin but the cardiotoxicity remained (Arcamone,

Cassinelli et al. 1969). Doxorubicin is one of the most powerful chemotherapeutic drugs

for the treatment of some leukemias, hodgkin’s lymphoma, as well as cancers of the

bladder, breast, stomach, lung, ovaries, thyroid, soft tissue sarcoma, multiple myeloma,

etc.

Scheme 2-17 Chemical structure of doxorubicin

2.5.2 Mechanism of Action

47

Despite doxorubicin has been frequently used in the treatment of numerous human

malignancies, the exact mechanism of the action is still somewhat unclear. Several

mechanisms have been proposed and often subject to controversy.

One of the most popular explanations is the capability of doxorubicin to inhibit DNA

synthesis which may be due to (a) DNA intercalation or inhibition of DNA polymerase

activity such as Topoisomerase II (Tanaka and Yoshida 1980; Gewirtz 1999), (b) effects

on signaling issues of growth arrest and p53 function (Kastan, Onyekwere et al. 1991), (c)

induction of enzymatic or chemically activated DNA adducts (Cullinane, Cutts et al. 1994)

and DNA cross-linking (Skladanowski and Konopa 1994), (d) interference with DNA

strand separation and helicase activity (Fornari, Randolph et al. 1994).

Another widely accepted theory is that oxidative stress and the resultant free radicals that

can lead to direct oxidative injure to DNA are related to doxorubicin action (Gewirtz 1999)

through two different ways. As for the first way, a doxorubicin semiquinone free radical

were formed involving some NADPH-dependent reductases and the oxygen, redox

cycling of adriamycin-derived quinine-semiquinone produced superoxide radicals (Olson

and Mushlin 1990; Singal, Li et al. 2000). In the other way, doxorubicin free radicals may

be from the reaction with iron which is non-enzymatic. A Fe2+ doxorubicin free radical

complex could be produced after a redox reaction of doxorubicin and Fe3+ (De Beer,

Bottone et al. 2001). Besides, several other mechanisms have also been suggested such as

alterations in calcium metabolism (Singal and Panagia 1984), and non-physiological

mechanisms (Gewirtz 1999).

48

2.5.3 Side Effects and Limitations

Although doxorubicin is one of most effective chemotherapeutic agents with a wide

anticancer spectrum, its clinical use is still limited by the serious side effects including

nausea, vomiting, mild alopecia, palmarplantar erythrodysesthesia (Hand-Foot Syndrome),

neutropenia, and the most serious one, cardiotoxicity. Doxorubicin can not only cause

acute cardiovascular chances including hypotension, tachycardia and various arrhythmias

after minutes intravenous administration but also lead to life-threatening chronic effects

such as hypotension, tachycardia, cardiac dilation and ventricular failure after several

weeks or months and even years (Edward, Lefrak et al. 1973; Edward A. Lefrak, tcaron et

al. 1973; Singal, Deally et al. 1987). The cardiotoxicity confines the cumulative dose of

DOX to 500-600 mg/m2, which still can be increased for tumor but not for heart (Petit

2004). On the basis of mechanisms of the action, the cardiotoxicity mechanisms of

doxorubicin can be attributed to free radical induction, Ca2+ overload accumulation, toxic

doxorubicin metabolites, production of prostaglandins and platelet activating factor,

simulation of histamine release, and direct interaction with the actin-myosin contractile

system (De Beer, Bottone et al. 2001).

Besides these serious irreversible side effects, other drawbacks also limit the clinical use

of doxorubicin. The multi-drug resistance (MDR) is one of the serious limitations in the

treatment of cancers through overexpression of MDR transporter proteins such as P-

glycoproteins (P-gp) and multidrug resistance associated protein (MRP). P-gp and MRP

can be overexpressed in malignant cells, as well as liver, kidney, and colon cells, to pump

anticancer drugs out of the cancer cells, significantly restricting the intracellular level of

the drug for effective therapy. Doxorubicin is the very substrate of P-gp and MRP, which

49

results in its shot half-life in circulation and low therapeutic efficacy (Krishna and Mayer

2000).

2.5.4 Formulations

In order to impair the side effects and other drawbacks of doxorubicin in chemotherapy, a

number of studies on the drug formulations have been undertaken. Liposome entrapped

doxorubicin achieved nigericin-induced “remote release” which resulted in a subsequent 8

times more effective cytotoxicity than the free drug (Goren, Horowitz et al. 2000). Recent

report showed that doxorubicin-loaded heparin-liposomes possessed high drug levels for

up to 64 h after i.v. administration and the half-life was approximately 8.4-fold increased

than the control liposomes (Han, Lee et al. 2006). Jane et al. (Janes, Fresneau et al. 2001)

fabricated chitosan nanoparticles as colloidal carriers for doxorubicin, achieving a

minimal burst release and retained cytotoxicity but induced anti-proliferative activity.

Later, dextran-doxorubicin conjugate loaded chitosan nanoparticles exhibited enhanced

therapeutic effect and survival rate in vivo (Mitra, Gaur et al. 2001). With in vitro and in

vivo work, doxorubicin-loaded polyisohexylcyanoacrylate nanoparticles exhibited

superiority to overcome the MDR phenotype and lead to a higher anti-tumor efficacy on

hepatocellular carcinoma that is well known as chemoresistant (Barraud, Merle et al.

2005).

Numerous investigations involving polymeric prodrug strategy in delivery of doxorubicin

were also demonstrated and showed favorable results. In early 1995,

monomethoxypoly(ethylene glycol)-propionic acid N-hydroxysuccinimide ester

conjugated doxorubicin was designed and studied, which had controlled release via

50

enzyme dependent hydrolysis, less cytotoxicity in vitro, longer circulation time, greater

extent in tumor xenografts (Senter, Svensson et al. 1995). Influence of polymer structure

on drug release, in vitro cytotoxicity, biodistribution and antitumor activity in PEG-

doxorubicin conjugate were extensively investigated by Veronese et al. (Veronese,

Schiavon et al. 2005). The highest MW PEG showed the longest plasma residence time

and the greatest targeting but all conjugates were 10-fold less toxic. The doxorubicin

prodrug N-[4doxorubicin-N-carbonyl (oxymethyl) phenyl] O-β-glucuronyl carbonate

could achieve completely parent drug activation, 5-fold decreased peek concentration of

doxorubicin in heart and 10-fold increased AUC in normal tissues than those in tumor

tissue (Houba, Boven et al. 2001). In the HPMA-based prodrug with doxorubicin attached

to the carrier via oligopeptide spacer or amide bonds, some of “classic” prodrugs have

been developed to clinical test (Kopeček, Kopečková et al. 2001; Chytil, Etrych et al.

2006). Water soluble HPMA copolymer-doxorubicin prodrug with pH-controlled

activation promisingly displayed high cytostatic activity in vitro and superior anti-tumor

activity in vivo in comparison of free drug (Chytil, Etrych et al. 2006).

Although hundreds of studies have been reported in formulations of doxorubicin, the

problems in stability of liposomes, incomplete release of nanoparticles and reduced

cytotoxicity in vitro of most prodrugs require new better drug delivery systems emerge.

51

3 SYNTHESIS AND CHARACTERIZATION OF THE TPGS-DOX

CONJUGATE

3.1 Introduction

TPGS is the derivative of PEG 1000 by conjugation to α-tocopheryl succinate and thus the

strategy of TPGS-DOX conjugate was similar as PEGylation (Hazra, Golenser et al. 2002).

As to get the TPGS-DOX conjugate, the terminal hydroxyl group in TPGS was first

reacted with succinic anhydride through ring-opening polymerization in the presence of

DMAP as base. Then the carboxyl group needed to be activated by NHS using DCC as

catalyst. At last, the amine group in DOX interacted with the activated carboxyl group in

TPGS-SA to get the conjugate, TPGS-DOX. The mechanism for the reaction was shown

in scheme 3-1 and scheme 3-2. The synthesized conjugate was characterized by fourier

transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR) for the

molecular structure, gel permeation chromatography (GPC) for the weight-averaged

molecular weight and polydispersity, microplate reader for drug loading efficiency. The

stability of the conjugate was studied in PBS at 4 °C.

3.2 Experiment Section

3.2.1 Materials

Vitamin E TPGS was obtained from Eastman Chemical Company (TN, USA).

Doxorubicin hydrochloride, N,N’-Dicyclohexylcarbodiimide (DCC),

dimethylaminopyridine (DMAP), N-hydroxysuccinimide (NHS), succinic anhydride (SA),

triethylanmine (TEA), diethyl ether and tetrahydrofuran (THF) were received from Sigma-

Aldrich (St. Louris, MO, USA). All solvents used are HPLC grade, which include

52

dichloromethane (DCM), dimethyl sulfoxide (DMSO, anhydrous) from Aldrich, and ethyl

acetate from Merck. All reagent water used in the laboratory was pretreated with Milli-Q

Plus System (Millipore Corporation, Bredford, USA).

3.2.2 Synthesis of TPGS-DOX

3.2.2.1 TPGS Succinoylation

TPGS was activated by succinic anhydride through ring-opening reaction mechanism in

the presence of DMAP (Sheme 3-1). In brief, 0.77 g (0.5 mmol) TPGS, 0.10 g (1mmol)

succinic anhydride and 0.12 g (1mmol) DMAP were mixed and heated at 100 °C under

nitrogen atmosphere for 24 h (Hazra, Golenser et al. 2002). The mixture was cooled to

room temperature, taken up in 5.0 mL cold DCM, filtered to remove excessive succinic

anhydride and then precipitated in 100 mL diethyl ether at -10 °C overnight. The white

precipitate was filtered and dried in vacuum to obtain TPGS succinate reagent with a yield

of 90%. To synthesize TPGS-SA as a precursor polymer for DOX conjugation, TPGS was

succinoylated with an excess of succinic anhydride because complete succinoylation is

essential to avoid polymer cross-linking during DOX conjugation (Tomlinson, Heller et al.

2003).

53

O

O

O n

COC

OO

O

O

+

Succinic AnhydrideVintamin E TPGS

CH2CH2O H

O

O

O n

COC

O

OHO

OCH2CH2O

TPGS-SA

100 OC

DCC, DMAP

Scheme 3-1 Scheme of TPGS succinoylation

3.2.2.2 TPGS-DOX Conjugation

The succinoylated TPGS (191.3 mg, 0.12 mmol) was reacted with DOX·HCl (102.5 mg,

0.18 mmol) with the presence of DCC (74.2 mg, 0.36 mmol), NHS (41.4 mg, 0.36 mmol)

and TEA (50 µL, 0.36 mmol ) in DMSO at room temperature under nitrogen atmosphere

for 24 h. In this process, the reaction can be separated into two steps. First, TPGS-SA was

esterized with NHS using DCC as coupling agent. Then the TPGS-NHS was interacted

with DOX to get the conjugate. The product was filtered to remove N,N-dicyclohexylurea

(DCU) and then dialyzed in DMSO for 24 h to remove excess reagents and unconjugated

DOX, and further dialyzed against Millipore water for 24 h to remove DMSO. The

resultant solution was freeze-dried to get the red powder with a yield of 83% and a purity

of 98%. The conjugate scheme is shown in Scheme 3-2.

54

O

O

O n

COC

O

OHO

OCH2CH2O

TPGS-SA

+ NOH

O

O

DCCO

O

O n

COC

O

OO

OCH2CH2O N

O

O

TPGS-NHS

NHS

O

O

OH

OH

O OH

OH

OO

O

NH2OH

Doxorubicin

O

O

O n

COC

O

O

O

O

O

OH

OH

O OH

OH

OO

O

HO NHCH2CH2O

TPGS-DOX

DMSO TEA DMSO

Scheme 3-2 Scheme of TPGS-DOX conjugation

3.2.3 Characterization of TPGS-DOX Conjugate

3.2.3.1 FT-IR

55

The chemical structure of TPGS-DOX was investigated by Fourier Transform Infrared

Spectroscopy (FT-IR) (Shimadzu, Japan). The FT-IR samples were prepared by mixing

99% KBr with 1% conjugate and then pressing to be a transparent tablet.

3.2.3.2 1H NMR

The molecular structure of TPGS-SA and TPGS-DOX were further confirmed by 1H

Nuclear Magnetic Resonance Spectroscopy (NMR) in DMSO-d6 at 300 MHz (Bruker

ACF300).

3.2.3.3 GPC

The molecular weight and the polydispersity of the conjugate were determined using gel

permeation chromatography (GPC, Aglient 1000 series GPC analysis system with RI-

G1362A refractive index detector). Agilent PL gel 5 µm mixed-C 300×7.5 mm column

was employed, which was eluted by THF as a mobile phase at a flow rate of 1mL/min.

The injection volume was 100 µL of sample solution with a concentration at 1 mg/mL in

mobile phase. Molecular weight of the conjugate was calculated using a series of

Polystyrene standards (Mw: 640, 7150, 111400, 1997000).

3.2.3.4 Drug Conjugate Efficiency

The DOX content conjugated to TPGS was measured using microplate reader (GENios,

Tean, Switzerland) in DMSO with fluorescence detection at λex=480 nm and λem=560nm.

A standard curve was generated by using free DOX at concentration range of 100 ng/mL -

500 µg/mL in DMSO.

56

3.2.3.5 Stability of the Conjugate

The stability of the conjugate was determined as follows. Five mg prodrug was suspended

in 5 mL PBS buffer (10 mM, pH 7.8) and sealed in a dialysis bag (M.W. cutoff: 1,000).

The dialysis bag was incubated in 20 mL PBS buffer at 4oC. The released DOX in the

incubation medium was collected at the designated time intervals and analyzed at 480 nm

fluorescence.

3.3 Results and Discussion

3.3.1 FT-IR Spectra

The spectra of DOX, TPGS and TPGS-DOX are shown in Fig 3-1. The FT-IR spectrum of

doxorubicin in Fig 3-1 exhibits multiple peaks at 3346, 2940, 2354, 1617, 1416 and 1072

cm-1, which corresponded to the different quinine and ketone carbonyls in DOX. However,

it cannot be delineated the specific bonds between specific quinine and ketone because

they both have carbonyl groups. The peaks at 872 and 806 cm-1 corresponded to the wag

of primary amine NH2 and deformation of N-H bonds, respectively. As for the spectrum

of TPGS, the peak at 1740 cm-1 was due to the carbonyl group. The peak region of 2870-

3000 cm-1 was generated by the aliphatic (C-H) functional group. From the spectrum of

TPGS-DOX conjugate, we can find that the peak at 872 and 806 cm-1 had disappeared and

instead, a peak at 1430 cm-1 turned up standing for N-H deformation in secondary amine

structure, which confirmed the secondary amine linkage between –COOH group in TPGS

and –NH2 group in doxorubicin. The conjugate showed significant signal from TPGS but

relative weak one of DOX, which was due to the low drug content in the conjugate.

57

Fig 3-1 FT-IR spectra of DOX (red line), TPGS (purple line) and TPGS-DOX (blue line)

3.3.2 1H NMR Spectra

1H NMR employing DMSO-d6 as solvent was used to confirm the product identity. The

spectrum of TPGS-SA demonstrated that the succinoyl methylene (CH2) gave signals at

2.3-2.4 ppm in comparison with that of TPGS as shown in Fig 3-2a, b. The typical 1H

NMR spectra of DOX and TPGS-DOX conjugate are shown in Fig 3-2c, d. The spectrum

of DOX exhibited typical peaks at 3.99 ppm (H3C-O-C-1), 5.45 ppm (HC-1’), 7.69 ppm

(HC-3) and 7.91 ppm (HC-2 and HC-4). The spectrum of TPGS showed an intense peak

at 3.51 ppm, which was the characteristic of methylene protons of the PEG part of TPGS.

The spectrum of TPGS-DOX contained both signals originating from DOX and TPGS,

58

but with much weaker intensity than the signals of DOX. This may be due to the small

molecular weight of DOX compared with that of TPGS.

3.3.3 GPC Results

The TPGS-DOX conjugate was analyzed by gel permeation chromatography (GPC) as

shown in Fig 3-3. The prodrug was eluted earlier than the unconjugated TPGS, which

demonstrated that DOX was conjugated to TPGS. The molecular weight of the conjugate

and the TPGS was 2,655 and 2,221, respectively. The ratio of the weight-averaged over

the-number molecular weight of the conjugate (Mw/Mn) was 1.16. The slight increase was

due to the DOX conjugation and the loss of the low molecular weight TPGS fraction in

the purification process. By GPC analysis, it can be confirmed that the TPGS-DOX

conjugate was synthesized successfully and the product was not a physical mixture of

TPGS and DOX.

59

60

Fig 3-2 1H NMR spectra of (a) TPGS, (b) TPGS-SA, (c) DOX, (d) TPGS-DOX with the insert for a magnification of the region between 6 and 10 ppm

-350

-300

-250

-200

-150

-100

-50

0

50

100

150

200

15.5 16.5 17.5 18.5 19.5 20.5 21.5 22.5

Retention time (min)

MV

TPGS

TPGS-DOX

Fig 3-3 Gel permeation chromatogram (GPC) of the TPGS-DOX conjugate and the unconjugated TPGS

3.3.4 Drug Loading Capacity

The DOX content in the TPGS-DOX conjugate was determined to be 8.0% by the

microplate reader using fluorescence at 480 nm. It is important to note that the DOX

loading capacity (8.0 wt%) of our TPGS-DOX conjugate is as favorable as that for other

polymer–DOX conjugates being tested clinically, which is, for example, 8.5 wt% for

HPMA copolymer-DOX conjugate (Vasey, Kaye et al. 1999), 2.5-5 wt% for PEG-DOX

conjugate (Rodrigues, Beyer et al. 1999), and 5-16 wt% for PGA-DOX conjugate (Hoes,

Grootoonk et al. 1993). The satisfactory drug loading capacity of TPGS-DOX conjugate

facilitates its clinical test in future.

61

3.3.5 In Vitro Stability

As the stability regards, the result is also satisfactory. It was found that after 24 h in PBS

buffer (pH 7.8) at 4 oC, the conjugate underwent only a small amount of hydrolysis

resulting in the release of approximately 2% of the bound DOX. Even after 5 months, only

12% of the conjugated drug was released from the conjugate. Therefore, most of the DOX

can be stably attached to the polymer under the test conditions, which enables its

relatively long time storage.

3.4 Conclusions

TPGS-DOX conjugate were synthesized via the interaction between the carboxyl group in

TPGS-SA and the primary amine group in DOX in the presence of the coupling agent.

The conjugate was characterized by FT-IR, 1H NMR and GPC to detect the molecular

structure, molecular weight and polydispersity, in which results confirmed the successful

synthesis of the conjugate. The TPGS-DOX also exhibited favorable drug loading

capacity in comparison with the clinical prodrug candidate. Besides, the in vitro stability

result showed the stable attachment between TPGS and DOX, which made the regular

storage feasible.

62

4 IN VITRO STUDIES ON DRUG RELEASE KINETICS,

CELLULAR UPTAKE AND CELL CYTOTOXICITY OF THE

TPGS-DOX CONJUGATE

4.1 Introduction

The release of the drug from the polymeric carrier is important for its further therapeutic

function. Usually, the release of drug can be mediated by simple hydrolysis. In this study,

the drug release was studied in PBS at 37 oC with different pH values. It is demonstrated

that TPGS can enhance the intracellular cell uptake of human colon carcinoma cells (Win

and Feng 2006). As for the in vitro study, MCF-7 breast adenocarcinoma cells and C6

glioma cells were utilized as in vitro model. The TPGS-DOX may also enhance the

cellular uptake of the doxorubicin through escaping from P-gp drug efflux pump, which

was investigated in this chapter. Cellular uptake of the conjugate was further visualized by

confocal laser scanning microscopy (CLSM) to indicate the intracellular distribution.

Cancer cell viability of TPGS-DOX conjugate was quantitatively determined by MTT

method. All the experiments were conducted in close comparison with the pristine DOX.

4.2 Materials and Methods

4.2.1 Materials

3-(4,5-cimethylthiazol-z-yl)-2,5-diphenyl chloroformate (MTT), phosphate buffered

saline (PBS), Dulbecco’s Modified Eagel Medium (DMEM), penicillin-streptomycin

solution, Trypsin-EDTA and Triton® X-100 were obtained from Sigma-Aldrich (St.

63

Louris, MO, USA). Fetal bovine serum (FBS) was received from Gibco (Life

Technologies, AG, Switzerland).

4.2.2 In Vitro Drug Release

The rate of DOX release from the conjugate was investigated in 10 mM PBS at pH 3.0,

5.0 and 7.4 at 37 oC, respectively. The conjugate solution of 200 µg/mL equivalent DOX

concentration was placed in a dialysis bag (MW cutoff: 1,000) and incubated in 20 mL of

the PBS solution with gentle shaking. The incubated solution was collected at designated

time points and equal volume of fresh medium was compensated. The released DOX

amount was determined by fluorescence detection at 480 nm.

4.2.3 Cell Culture

MCF-7 breast adenocarcinoma cells and C6 glioma cells (American Type Culture

Collection, VA) were employed as in vitro models. The cells were cultured in the DMEM

medium supplemented with 10% FBS, 1% penicillin-streptomycin solution, and incubated

in SANYO CO2 incubator at 37oC in humidified environment of 5.0% CO2. The medium

was replenished every other day until confluence was achieved. The cells were then

washed with PBS and harvested with 0.125% Trypsin-EDTA solution.

4.2.4 In Vitro Cell Uptake Efficiency

In the quantitative investigation, MCF-7 and C6 cells were seeded in 96-well black plates

(Costar, IL, USA) at a density of 3×104 cells/well. When the cells reached about 80%

confluence, as for the investigation of comparison of the prodrug and the free drug, the

medium was replaced by 100 µL TPGS-DOX or free DOX solution in medium at 1

64

µg/mL DOX concentration for 0.5, 1.5, 4, 6 h, respectively. Regarding the concentration

related effects in cell uptake, the medium was replaced by 100 µL TPGS-DOX or free

DOX at 1, 5, 25 µg/mL DOX for 4 h. For each sample, six wells were seeded for positive

control and six wells for sample wells. At the designated time interval, the sample wells

were washed three times with 50 µL PBS and then added 100 µL culture medium. All the

wells were then lysed by 50 µL 0.5% Triton 100 in 0.2 M NaOH. The fluorescence

intensity of each sample was detected by the microplate reader (Tecan, Männedorf,

Switzerland, λex = 480 nm, λem = 560 nm). Cellular uptake efficiency was expressed as the

percentage of the fluorescence associated with the cell vs that present in the positive

control (Zhang, Lee et al. 2007).

4.2.5 Confocal Laser Scanning Microscopy

As for the qualitative investigation, MCF-7 and C6 cells were incubated with TPGS-DOX

conjugate or free DOX medium solution at 1 µg/mL DOX concentration under 37oC for 4

h. Then the cells were rinsed with cold PBS for three times, fixed by 75% ethanol for 20

min, and then washed twice by PBS. Finally, the cells were mounted by the mounting

medium (DAKO® Fluorescent Mounting Medium) and observed under confocal laser

scanning microscopy (Zeiss LSM 510, Germany).

4.2.6 In Vitro Cytotoxicity

MCF-7 and C6 cells were seeded at a density of 5×103 cells/well in 96-well plates (Costar,

IL, USA) and incubated for 24 h. The medium was then replaced by the free DOX or

TPGS-DOX conjugate at various drug concentrations from 0.002 to 100 µM in the

medium. The cell viability was determined by the MTT assay. At the designated time

65

intervals 24, 48, 72 h, the medium was removed and the wells were washed twice with

PBS. Ten percent MTT (5 mg/mL in PBS) in medium was added and the cells were

incubated for 3-4 h. After that, the precipitant was dissolved in 100 µL isopropanol and

each well was finally analyzed by the microplate reader with absorbance detection at 570

nm. The cell viability was figured out using the following formula,

Cell viability (%) = (Abss / Absc) × 100

where Abss stands for the fluorescent absorbance of the wells of the drug samples and

Absc is that of the wells of the culture medium used as positive control.

The MCF-7 and C6 cell viability of TPGS was also investigated at various concentrations

equivalent to those from 0.008 to 400 µM for the conjugate experiment using the same

method.

4.2.7 Statistics

Statistical analysis was conducted by using the student’s t-test with p<0.05 as significant

difference.

4.3 Results and Discussion

4.3.1 In Vitro Drug Release

In vitro release of the drug from TPGS-DOX conjugate was measured at various pH

conditions. As shown in Fig 4-1, the DOX release showed no dramatic initial burst. It was,

however, significantly pH-dependent. The lower the pH value was, the faster the drug

released. Specifically, the drug released rapidly from the conjugate at pH 3.0, reaching

66

52.3±2.4% after 48 h and almost 100% within 10 days, whereas the DOX release at pH

5.0 was much slower, implying 27.1±1.4% and 43.6±2.8% in the same period,

respectively (p<0.01). As desired, only a small amount of drug (12.6±0.2%) was released

at pH 7.4 over the observed period.

The release of the free active drug from the carrier may play an important role, which is

considered as a prerequisite for the conjugate bearing drugs bound to the polymer via

degradable bonds. In most cases, release of anticancer drugs from the polymer carrier is

mediated by simple hydrolysis (Maeda, Seymore et al. 1992; Takakura and Hashida 1995).

The free DOX is known to be stable in the pH range 3.0-6.5 (Vigevani and Williamson

1980). In the TPGS-DOX conjugate, DOX might be released from the conjugate by

hydrolysis, which was sensitive to pH value. This effect may be involved to speed up the

drug release inside cancer cells as the proton concentration in lysosomes is usually at least

100-fold higher (<pH 5.0) than the outside of the cells (pH 7.4). The release profile

confirmed that the linkage was labile under mild acidic conditions anticipating endosomal

environment and sufficiently stable in blood circulation (pH 7.4) to allow the transport of

the prodrug to tumor cells.

67

0

20

40

60

80

100

0 50 100 150 200 250

Time (h)

Cum

ulat

ive

rele

ased

DO

X (%

)

pH 3.0

pH 5.0

pH 7.4

Fig 4-1 Release of DOX from TPGS-DOX conjugate incubated in phosphate buffer at 37 oC (mean ± SD and n=3)

4.3.2 In Vitro Cellular Uptake

It is known that the cellular internalization and sustained retention of the drug play

significant role in therapeutic effects. The in vitro investigation can give preliminary

information of the priority of the prodrug over the parent drug despite it cannot make sure

the definitely same results in vivo.

Fig 4-2 shows (a) MCF-7 and (b) C6 cellular uptake efficiency of the TPGS-DOX

conjugate compared to the pristine DOX after 0.5, 1.5, 4, 6 h cell culture. The same 1

µg/mL DOX concentration in the culture medium was applied in the experiment. It can be

seen from Fig 4-2 that the TPGS-DOX conjugate exhibited enhanced cellular uptake of

1.7- (69.8±8.8% for TPGS-DOX vs 39.9±2.4% for DOX), 1.3- (75.5±6.0% for TPGS-

68

DOX vs 56.7±3.4% for DOX), 1.2- (78.6±6.8% for TPGS-DOX vs 64.2±6.4% for DOX),

1.2-fold (86.0±5.1% for TPGS-DOX vs 72.5±2.0% for DOX) (p<0.05) for the MCF-7

cells and that of 5.4- (27.0±1.7% for TPGS-DOX vs 4.93±0.1% for DOX) , 5.9-

(34.0±2.7% for TPGS-DOX vs 5.70±0.2% for DOX), 1.3- (48.7±7.8% for TPGS-DOX vs

37.4±5.4% for DOX), 1.1-fold (73.6±1.8% for TPGS-DOX vs 69.7±3.5% for DOX)

(p<0.05) for the C6 cells after 0.5, 1.5, 4, 6 h cell culture, respectively, in comparison with

pristine DOX. Besides, the absolute cellular uptake efficiency for the MCF-7 cells is

generally higher than that for the C6 cells. Almost all of the TPGS-DOX samples in MCF-

7 cells displayed the cell uptake efficiency above 70%, whereas most of the TPGS-DOX

cell uptake efficiency in C6 cells was below 70%.

a

0

10

20

30

40

50

60

70

80

90

100

0.5 1.5 4 6

Time (h)

MC

F-7

cell

upta

ke e

ffic

ienc

y (%

)

DOX

DOX-TPGS

69

b

0

10

20

30

40

50

60

70

80

90

100

0.5 1.5 4 6

Time (h)

C6

cell

upta

ke e

ffic

ienc

y (%

)

DOX

DOX-TPGS

Fig 4-2 (a) MCF-7 and (b) C6 cell uptake efficiency cultured with the pristine DOX or the TPGS-DOX for 0.5, 1, 4, 6 h respectively at equivalent drug concentration 1 µg/mL (mean ± SD and n=6)

Fig 4-3 exhibits the cellular uptake efficiency of TPGS-DOX and free DOX in (a) MCF-7

and (b) C6 cells, respectively, which was assayed upon 4 h culture with different drug

concentration. It is obvious that both prodrug and free drug showed decreased cellular

uptake efficiency when the equivalent drug concentration was increased from 1 µg/mL to

25 µg/mL. The trend was found in both MCF-7 and C6 cancer cells, which indicated a

saturated and limited property of cellular uptake of the prodrug and free drug. Similarly as

the above results, the cell uptake of TPGS-DOX was higher than that of DOX, no mater in

which cell line and at which drug concentration. The cell uptake was enhanced to 1.2-

(78.6±6.8% vs 64.2±6.4%), 1.1- (30.6±1.2% vs 27.5±0.3%), 1.1-fold (26.4±0.6% vs

24.0±2.0%) (p<0.05) in MCF-7 cells and 1.3- (48.7±7.8% vs 37.4±5.4%), 1.4- (39.5±8.0%

70

vs 28.9±4.9%), 1.1-fold (25.4±6.9% vs 323.5±4.2%) (p<0.05) in C6 cells by TPGS-DOX

at 1, 5, 25 µg/mL, respectively.

a

0

10

20

30

40

50

60

70

80

90

100

0.5 1.5 4 6

Time (h)

MC

F-7

cell

upta

ke e

ffic

ienc

y (%

)

DOX

DOX-TPGS

b

0

10

20

30

40

50

60

70

80

90

100

0.5 1.5 4 6

Time (h)

C6

cell

upta

ke e

ffic

ienc

y (%

)

DOX

DOX-TPGS

Fig 4-3 Cell uptake of the TPGS-DOX and DOX in (a) MCF-7 and (b) C6 cells after 4 h incubation (mean ± SD and n=6)

71

The difference of the cell uptake between the conjugate and the free drug may be due to

the MDR effect, which is a major cause for the failure of anti-cancer chemotherapy

because of the overexpression of MDR transporter proteins such as P-glycoprotein (P-gp)

that mediate unidirectional energy-dependent drug efflux and thus reduce intracellular

drug levels. As the free DOX regards, most of the molecules would be effluxed out by P-

gp pump proteins except those that could bind to DNA after entering the cells (Zhang and

Feng 2006). In contrast, the TPGS has inhibitory effect on the P-gp, the multidrug

resistance transporter, so that less drug would thus be pumped out of the cells, resulting in

a higher cell uptake efficiency (Dintaman and Silverman 1999).

4.3.3 Confocal Laser Scanning Microscopy

The cellular uptake of TPGS-DOX conjugate was further investigated by confocal laser

scanning microscope (CLSM). The four pictures in Fig 4-4 show respectively the

confocal laser scanning microscopy of MCF-7 cancer cells after 4 h incubation with (a)

the pristine drug DOX and (b) with the TPGS-DOX conjugate, and that of C6 cancer cells

with (c) the DOX and (d) the TPGS-DOX conjugate at the same 1 µg/mL DOX

concentration. The cells in (b) and (d) had become unhealthy, which was agreeable with

the cellular uptake results as shown in Fig 4-3 and thus confirmed the advantages of the

TPGS-DOX conjugate over the original DOX. Moreover, it can be seen from Fig 4-4 that

the free DOX was mainly distributed within the nucleus (Fig 4-4 a, c) in both cancer cells,

while the conjugate could be observed around the nucleus resulting a broad intracellular

distribution in the cytoplasm (Fig 4-4 b, d). The change of intracellular distribution of the

72

drug by TPGS conjugation may play an important role, which is similar to that reported in

the literature for the PEG-DOX conjugate (Rodrigues, Beyer et al. 1999). This result

reveals that the conjugate may deliver the drug into the cells in a different way from the

simple diffusion of the free drug in the cells. Besides intercalation with DNA, the

conjugate might exert its cytotoxicity by a different mode of action.

(a)

(b)

73

(c)

(d)

Fig 4-4 Confocal laser scanning microscopy (CLSM) of MCF-7 cells after 4 h incubation with (a) the pristine drug DOX and (b) with the TPGS-DOX conjugate, and that of C6 cells with (c) the DOX and (d) the TPGS-DOX conjugate at the equivalent 1 µg/mL DOX concentration

4.3.4 In Vitro Cytotoxicity

The MCF-7 and the C6 cell line were employed as in vitro models to investigate the

cytotoxicity of the TPGS-DOX conjugate in close comparison with the pristine DOX as a

positive control. Fig 4-5 shows the cellular viability of (a) MCF-7 cells and (b) C6 cells

after 24, 48, 72 h culture respectively with the TPGS-DOX conjugate in comparison with

that of the pristine DOX at various DOX concentrations. It can be seen from the two

graphs that the TPGS-DOX conjugate exhibited significantly higher cytotoxicity

74

(equivalent lower cell viability) at low drug concentration (p<0.05) or at least, comparable

cytotoxicity at high drug concentration in comparison with the pristine DOX. This

advantage became more significant for longer time cell culture.

As the cytotoxicity of TPGS regards, no significant cell killing ability was found for C6

cancer cells and only slight cytotoxicity was observed for MCF-7 cancer cells (Fig 4-6).

However, the cell viability of TPGS in each sampling point is above 50%. This means that

the enhanced cytotoxicity of the TPGS-DOX came from the conjugation strategy but not

from the TPGS carrier.

a

0

20

40

60

80

100

120

0.002 0.02 0.2 2 20 100

Concentration (µM)

MC

F-7

cell

viab

ility

(%)

DOX-1DAY DOX-TPGS-1DAY DOX-2DAYSDOX-TPGS-2DAYS DOX-3DAYS DOX-TPGS-3DAYS

75

b

0

20

40

60

80

100

120

0.002 0.02 0.2 2 20 100

Concentration (µM)

C6

cell

viab

ility

(%)

DOX-1DAY DOX-TPGS-1DAY DOX-2DAYSDOX-TPGS-2DAYS DOX-3DAYS DOX-TPGS-3DAYS

Fig 4-5 Cellular viability of (a) MCF-7 breast cancer cells and (b) C6 glioma cells after 24, 48, 72 h culture with the DOX-TPGS conjugate respectively in comparison with that of the pristine DOX at various equivalent DOX concentrations (mean ± SD and n=6)

It is obvious that the cell viability decreased with increase of the drug concentration. The

IC50 value, i.e. the drug concentration at which 50% cells have been killed in a given

period, can thus be used as an quantitative index to evaluate in vitro the therapeutic

effects of a drug in that given period. Table 4-1 lists the IC50 values of the TPGS-DOX

conjugate and the free drug DOX after 24, 48, 72 h cell culture, respectively. It can be

concluded from this Table that the TPGS-DOX conjugate achieved much lower IC50

values than the free drug DOX in all the cases for MCF-7 and C6 cells at various periods.

The trends became more significant for longer time incubation. The IC50 of the MCF-7

cells was found to be 52.3, 0.357, 0.00916 µM for the TPGS-DOX conjugate vs 76.7,

0.1173, 0.0577 µM for the pristine drug DOX, which implied 31.8, 69.6, 84.1% more

76

effective in vitro, after 24, 48, 72 h culture, respectively. The IC50 of C6 cells was found to

be 20.8, 0.0712, 0.00468 µM for the TPGS-DOX conjugate vs 37.1, 0.568, 0.00810 µM

for the pristine drug DOX, which implied 43.9, 87.7, 42.2% more effective in vitro, after

24, 48, 72 h culture, respectively.

Table 4-1 IC50 values (in equivalent µM DOX level) of MCF-7 and C6 cancer cells cultured with the TPGS-DOX conjugate vs the pristine DOX in 24, 48, 72 h

IC50 (µM)

C6 cells MCF-7 cells Incubation time (h) DOX TPGS-DOX DOX TPGS-DOX

24 37.1 20.8 76.7 52.3

48 0.568 0.0712 0.1173 0.0357

72 0.00810 0.00468 0.0577 0.00916

It is thus evidenced at least in vitro that the TPGS conjugation greatly enhanced the

therapeutic effects of doxorubicin. What’s more, the IC50 values of TPGS in both cell lines

were larger than 400 µM. It is thus evidenced, once again, that the enhanced cytotoxicity

of the TPGS-DOX came from the conjugation strategy but not from the TPGS carrier.

77

0

20

40

60

80

100

120

0.008 0.08 0.8 8 80 800

TPGS concentration (µM)

Cell

viab

ility

(%)

C6-1DAY C6-2DAYS C6-3DAYSMCF7-1DAY MCF7-2DAYS MCF7-3DAYS

Fig 4-6 Cellular viability of MCF-7 breast cancer cells and C6 glioma cells after 24, 48, 72 h culture with TPGS respectively at various equivalent concentrations in the conjugate (mean ± SD and n=6)

It is well known that polymer-drug conjugation usually results in higher IC50 values in

vitro than the parent drug due to changes caused in cellular pharmacokinetics such as

slower endocytic capture compared with rapid transmembrane passage of the parent drug

(Duncan 1992). The DBM2-PEG4000-S-PEG3000-GFLG-DOX conjugate was found 10-20-

fold less toxic than free DOX against B16F10 murine melanoma in vitro (Andersson,

Davies et al. 2005). The PEG-DOX conjugates were demonstrated over 40-fold less toxic

than the free DOX toward H2981 human lung adenocarcinoma cells (Senter, Svensson et

al. 1995). On the contrary, our TPGS-DOX conjugate showed much lower IC50 values in

comparison with the parent drug, which indicated that TPGS-DOX is promising in

78

enhancing the therapeutic effects of the conjugated drug. Although TPGS itself showed

slight cytotoxicity for MCF-7 cells, its IC50 is much larger than that of the DOX and the

TPGS-DOX. This proves that the enhanced therapeutic effect mainly comes from the

conjugation strategy but not from the carrier itself. This great advantage may be attributed

to the unique pharmaceutical properties of TPGS, which is also involved after the

conjugation. First, it can induce apoptosis, a cell death mechanism shared by the majority

of anticancer drug, to exert anti-tumor effect (Porta 2004; Youk, Lee et al. 2005). Second,

TPGS is able to inhibit the MDR effect caused by P-gp efflux pump. The mechanism of

TPGS-DOX internalization may deliver DOX to intracellular compartments that are less

accessible to the P-gp. In this respect, the result of the cytotoxicity experiment was

consistent with that of the cellular uptake. Last but not least, as TPGS possesses great

ROS (reactive oxygen species)-generating ability (Youk, Lee et al. 2005), it is plausible

that TPGS shows selective cytotoxicity to facilitate cytotoxicity of the conjugated drug

toward cancer cells due to the disparities between normal and cancer cells (Renschler

2004).

4.4 Conclusions

In vitro release of DOX from the conjugate was found pH dependent with no significant

initial burst. TPGS-DOX conjugate displayed higher cellular uptake efficiency than that of

free DOX, no matter the drug concentration, incubation time nor MCF-7 or C6 cancer

cells. The in vitro confocal laser scanning microscope imaging confirmed that the TPGS-

DOX conjugate was found distributed around the nucleus and cytoplasm while the DOX

was mainly entrapped in the nucleus. The prodrug showed higher cytotoxicity and

achieved much lower IC50 values in comparison with the pristine DOX, especially for

79

MCF-7 cancer cells. Longer time treatment resulted in better cytotoxicity. Further in vivo

investigation is then required to confirm the preliminary and satisfactory results of in vitro

studies.

80

5 IN VIVO INVESTIGATION ON PHARMACOKINETICS AND

BIODISTRIBUTION OF THE TPGS-DOX CONJUGATE

5.1 Introduction

The in vivo pharmacokinetics (PK) and biodistribution (BD) of the TPGS-DOX conjugate

were investigated on rats in comparison with commercial DOX formulation. The

noncompartmental PK analysis, which estimates the exposure to a drug by the

determining the area under the curve of a concentration-time graph, was employed to test

the in vivo therapeutic effects of the TPGS-DOX conjugate. Quite a few methods have

been developed to determine the drug level in blood and tissues. Here HPLC detection

method was utilized due to its easy handling with acceptable sensitivity and selectivity.

The blood and organs collected from the rats after drug administration needed to be

treated through liquid-liquid extraction and no internal standard was needed in assay. And

then the samples were measured by HPLC using fluorescence detection.

5.2 Materials and Methods

5.2.1 Animal

Male Sprague-Dawley (SD) rate, which were 150-200 g and 4-5 weeks old, were provided

by the Laboratory Animals Centre of Singapore and maintained at the Animal Holding

Unit (AHU) of National University of Singapore. They were kept in well-ventilated rooms

at a temperature of 25±2 °C and a humidity of 50-60% under nature lighting conditions.

All rats caring and handling procedures and protocols were approved by Institutional

81

Animal Care and Use Committee (IACUC), Office of Life Science, National University of

Singapore under the authority of Animal Welfare Act (AWA).

5.2.2 In Vivo Pharmacokinetics

5.2.2.1 Drug Administration

The SD rats were randomly assigned to two groups with each of four rats; one group for

i.v. administration of free DOX and the other for that of TPGS-DOX. Before drug

administration, commercial DOX or TPGS-DOX conjugate were diluted in normal saline

containing 1.9% w/v NaCl to obtain an estimated injection volume of 1-1.5 ml.

Intravenous injection was given via the tail vein at a 5 mg/kg equivalent drug dosage. All

animals were observed for mortality, general condition and potential clinical signs.

5.2.2.2 Blood Collection and Sample Analysis

The blood samples were collected by heparinized tube at 0 (pre-dose), 10, 30 min, 1, 2, 4,

8, 12, 24, 48, 72 h post-treatment. Plasma samples were harvested by centrifugation at

1500×g for 10 min and stored at -20 oC until analysis. Liquid-liquid extraction was

performed prior to the HPLC analysis. Briefly, the plasma (100 µL) was mixed with 100

µL of 10 mM phosphate-buffered saline (pH 7.8). The drug was extracted by

dichloromethane-isopropanol (4:1, v/v) on a vortex-mixer for 90 s. Upon centrifugation at

2000×g for 15 min, the upper aqueous layer was removed by aspiration and the organic

layer was transferred to a glass tube and evaporated under nitrogen at room temperature

overnight. The residue was dissolved in 100 µL of the HPLC mobile phase (1/15 M

KH2PO4/CH3CN=75:25 v/v, pH 4.16, adjusted with H3PO4) by vortex and transferred to

auto sampler vials containing limited-volume inserts (100 µL). The standards needed to be

82

prepared using blank plasma with a series concentration of commercial DOX (0.05 µg/ml-

100µg/ml), followed by the same treatment as the samples done. The drug concentration

in samples was calculated using the standard calibration curve.

For the HPLC analysis, the drug concentration in plasma was determined using Agilent®

1100 Series installed with Agilent® Eclipse XDB-C18 column with 5 µm pore size. The

mobile phase was delivered at a rate of 1 mL/min. Twenty µL of sample were injected and

the column effluent was detected with a fluorescence detector (Ex 470 nm, Em 585 nm)

(Watson, Stewart et al. 1985; Park, Lee et al. 2006).

5.2.2.3 Pharmacokinetic Parameters

Non-compartmental Analysis (NCA), done by Kinetica Software (Thermo Electron

Corporation, USA), provides an estimate of the kinetic parameters of a drug based on

statistical moment theory. As for the specific parameters, the maximum drug

concentration (Cmax) and the corresponding time (tmax) can be observed from the plasma

concentration vs time curve. The elimination half-life (t1/2), an important index, can be

calculated as㏑ 2/λn, in which λn is the elimination constant obtained via log-linear

regression analysis of the terminal phase of the profile. The area under the curve (AUC)

and area under the first moment (AUMC) can be figured out using log-linear trapezoid

rule. The mean residence time (MRT) is calculated as AUMC/AUC. Apparent volume of

distribution at steady state (Vss) and plasma clearance (CL) were obtained as Dosage ×

AUMCinf/(AUCinf)2 and Dosage/AUCinf, respectively, in which AUMCinf and AUCinf

mean the corresponding value from 0 to infinity.

83

5.2.3 In Vivo Biodistribution

5.2.3.1 Drug Administration

The SD rats were randomly assigned to two groups, i.e. Group A with i.v. injection of the

pristine DOX suspension and Group B of the TPGS-DOX conjugation at the equivalent 5

mg/kg, respectively. Each group has 4 sets corresponding to 4 time points, and each set

having 3 rats. Before drug administration, commercial DOX or TPGS-DOX conjugate

were diluted in normal saline containing 1.9% w/v NaCl to obtain an estimated injection

volume of 1-1.5 ml. Intravenous injection was given via the tail vein at a 5 mg/kg

equivalent drug dosage. All animals were observed for mortality, general condition and

potential clinical signs.

5.2.3.2 Tissues Collection and Samples Analysis

Animals in each set were sacrificed by cardiac stick exsanguinations at 0.5, 2, 8 and 24 h

respectively after the injection and tissues (heart, spleen, stomach, lung, intestine, kidney

and liver) were collected. The tissues were then washed with saline and stored at -80oC

prior to analysis.

For the analysis, the tissues were freeze-dried, homogenized. After that, 30 mg organ for

each was mixed with 300 µL PBS, followed by extraction and HPLC analysis as the blood

samples done. The standards of different organs needed to be prepared using the blank

tissues collected from the rats without any drug administration. A series of commercial

DOX was added in the different blank organs respectively and then the standards were

84

treated in the same way as the samples done. The drug level in organs was figured out

using the standard calibration curve.

5.2.3.3 Statistics

Statistical analysis was conducted by using the student’s t-test with p<0.05 as significant

difference.

5.3 Results and Discussion

5.3.1 Pharmacokinetics

1

10

100

1000

10000

100000

0 10 20 30 40 50 60 70 80

Time (h)

DO

X in

pla

sma

(ng/

ml)

DOX-TPGS DOX LOWEST EFFECTIVE LEVEL PEAK LEVE OF MTD

Fig 5-1 Pharmacokinetic profile of the pristine DOX and the DOX-TPGS conjugate after intravenous injection in rats at a single equivalent dose of 5 mg/kg (mean ± SD and n=4)

85

The plasma concentration level of DOX was determined after a single intravenous

injection of the commercial DOX or the TPGS-DOX conjugate at a dose of 5 mg/kg DOX

equivalent in male SD rats, which is shown in Fig 5-1 in a period up to 72 h. The free

DOX rapidly disappeared from the circulation due to its short half-life. In contrast, the

TPGS-DOX conjugate showed a much longer circulation time. Both formulations reached

the highest level 10 min after the injection, which is 199.5±38.0 ng/mL for the pristine

DOX and 4,110±1,419 ng/mL (p<0.01) for the TPGS-DOX conjugate. The drug

concentration of the TPGS-DOX conjugate achieved much higher drug concentration in

the plasma than the pristine DOX did all the time in the experiment (p<0.05), but lower

than the peak concentration (16.4 µM) with the MTD (maximum tolerated dose)

administration (8mg/kg) of free DOX (Houba, Boven et al. 2001).

Table 5-1 Pharmacokinetic parameters of the TPGS-DOX conjugate vs the pristine DOX i.v. injected in the SD rats at the equivalent 5 mg/kg dose

Parameter DOX TPGS-DOX

t1/2 a (h) 2.53±0.26 9.65±0.94

teffectb (h) 9.90±0.51 62.1±2.53

MRTc (h) 2.86±0.39 10.9±1.87

AUC0-∞d (h·ng/mL) 288±54 6812±2149

CLtote (L/h/kg) 17.3±3.08 0.734±0.164

Vdssf (L/kg) 49.6±6.13 8.02±0.91

a half-life time; b therapeutic effective period; c mean residence time; d area under the curve; e total clearance; f volume of distribution at steady state.

86

The pharmacokinetic parameters are summarized in Table 5-1. It can be seen from the

Table 5-1 that the half-life of the drug was 9.65±0.94 h for the TPGS-DOX conjugate and

2.53±0.26 h for the original DOX, 3.81 times longer (p<0.01). Our measurement of the

half-life of the doxorubicin at the 5 mg/kg dose was in good agreement with that found

from the literature (Gao, Lee et al. 2005). The mean residence time of the drug in the

plasma was 10.9±1.87 h for the TPGS-DOX conjugate vs 2.86±0.39 h for the pristine

DOX, also 3.81 times longer (p<0.05) since it has the same physical meaning as the half-

life. The therapeutic effective period was 62.1±2.53 h for the TPGS-DOX conjugation and

9.90±0.51 h for the DOX alone, 6.27 times longer (p<0.05) (the minimum effective level

10 nM was found from the literature (Gavenda, Ševčík et al. 2001)). This may be due to

the enhanced permeability and retention effect (ERF) by the TPGS conjugation. As

prolonged plasma circulation is the driving force for increased tumor targeting (Seymour,

Miyamoto et al. 1995), the conjugate was supposed to show improved therapeutic efficacy.

Indeed, the area-under-the-curve (AUC), which is a key therapeutic index of a drug, was

6,810±2149 h·ng/mL for the TPGS-DOX conjugate and 288±54 h·ng/mL for the DOX

alone, 23.6 times larger (p<0.05). The volume distribution at steady state was 8.02±0.91

L/kg for the TPGS-DOX conjugate and 49.6±6.13 L/kg for the DOX alone, which was

6.18 times smaller (p<0.05), and the total clearance of the drug from the plasma was

0.763±0.164 L/h/kg for the TPGS-DOX conjugate vs 17.3±3.08 L/h/kg for the DOX alone,

22.7 times longer (p<0.05). All these results showed the significant enhancement of the

drug’s pharmacokinetics through TPGS conjugation. It should be emphasized that,

although polymer-drug conjugation enhanced the pharmacokinetics of the drug in general,

our TPGS-DOX conjugate showed the most significant effects compared with other

polymer-DOX conjugates in the literature (Senter, Svensson et al. 1995; Veronese,

87

Schiavon et al. 2005). Although both increased AUC and retention time were

demonstrated but they were not as significant as the TPGS-DOX had achieved, compared

to the pristine DOX.

5.3.2 Biodistribution

The distribution of DOX in solid tissues such as heart, lung, spleen, liver, gastric, intestine

and kidney was investigated at four time points: 0.5, 2, 8 and 24 h. The results are shown

in Fig 5-2 with graph (a) for pristine DOX and graph (b) for the TPGS-DOX

administration of 5 mg/kg doxorubicin dose. It can be seen that TPGS conjugation

significantly changed the biodistribution of the drug. The peak concentrations were

detected at 0.5 or 2 h for DOX administration while most peak concentrations were found

at 2 or 8 h for the TPGS-DOX conjugate. TPGS conjugation delayed the time of the peak

concentrations and reduced their magnitude, which may imply reduced side effects and

thus have clinical significance. Such an advantage of TPGS conjugation may be attributed

to the slower penetration of the prodrug into the tissues. For DOX, the highest

concentration was found in heart (50.08±5.27 µg/g at 2h) followed by stomach

(46.97±12.61 µg/g at 2 h), lung (44.44±12.85 µg/g at 2 h), kidney (42.63±9.27 µg/g at 0.5

h) and intestine (41.71±4.92 µg/g at 2 h). On the contrary, the peak concentration in these

tissues for the TPGS-DOX conjugate dropped by 5.4-, 7.3-, 2.2-, 2.4- and 5.3-fold, which

was 9.22±6.69 µg/g at 2 h, 6.44±5.93 µg/g at 0.5 h, 20.19±11.2 µg/g at 8 h, 17.66±9.56

µg/g at 2 h and 7.83±4.66 µg/g at 2 h, respectively (p<0.05). The conjugate presented a

little higher DOX peak levels than the free drug in spleen (22.52±8.50 vs 12.68±0.74 µg/g)

and liver (17.57±3.24 vs 14.70±6.13 µg/g) at 2 and 8 h, respectively (p>0.05).

88

Table 5-2 summarizes the AUC values of DOX in various tissues after intravenous

injection of the TPGS-DOX conjugate vs the free DOX. In comparison with that of the

DOX, the AUC of the TPGS-DOX conjugate was a bit higher in liver (262±47 vs 228± 20

µg·h/g, p=0.31) and spleen (335±43 vs 127.0±32.6 µg·h/g, p<0.05), comparable in lung

(313±50 vs 304±86 µg·h/g, p=0.88) and kidney (169.4±39.6 vs 166.3±21.6 µg·h/g,

p=0.91), and lower in heart (155.2±33.1 vs 307±64 µg·h/g, p<0.05), stomach (114.3±38.6

vs 313±14 µg·h/g, p<0.01) and intestine (86.7±20.1 vs 246±51 µg·h/g, p<0.01). The

TPGS-DOX showed great superiority in decreasing the AUC in heart by over 5 times,

which was indicated only 3 times decline for prodrug DOX-GA3 (Houba, Boven et al.

2001) and other polymer-DOX conjugate (Tomlinson, Heller et al. 2003; Veronese,

Schiavon et al. 2005).

a

0

10

20

30

40

50

60

heart lung spleen liver stomach intestine kidney

Tissues

µg D

OX/

g or

gan

for

DO

X

0.5h2h8h24h

89

b

0

10

20

30

40

50

60

heart lung spleen liver stomach intestine kidney

Tissues

µg D

OX/

g or

gan

for

DO

X-T

PG

S0.5h2h8h24h

Fig 5-2 The DOX levels (µg/g) in heart, lung, spleen, liver, stomach, intestine and kidney after i.v. administration at 5 mg/kg equivalent drug of (a) the free DOX, (b) the TPGS-DOX conjugate (mean ± SD and n=3) DOX is a lipophilic molecule and can rapidly penetrate into normal tissues (Houba, Boven

et al. 2001), which may account for unfavorable side-effects. The hydrophilic TPGS

moiety of the TPGS-DOX would prevent the rapid diffusion of the prodrug into tissue

cells. This is consistent with the delayed time and decreased magnitude of the peak drug

concentration in normal tissues of the TPGS-DOX conjugate. Significantly higher kidney

level of the free drug was observed at 0.5 and 2 h due to its on-going renal elimination,

which was also consistent with the faster clearance of the free drug. High accumulation of

the prodrug in liver and spleen may suggest that these two organs be the major ones for

the clearance, in which the prodrug was preferentially uptaken by the reticuloendothelial

90

system. More importantly, in contrast with the increased level of DOX in liver and spleen

after TPGS-DOX conjugate administration, the concentrations of the drug in heart,

stomach and intestine for the TPGS-DOX administration were reduced much more. As the

major side effect of DOX is considered as serious cardiac and gastrointestinal toxicity

(Mishra and Jain 2000), the TPGS-DOX prodrug significantly impaired such limitations

and showed great superiority over the free DOX.

Table 5-2 AUC values (µg·h/g) of biodistribution in various organs after intravenous injection of the free DOX and the TPGS-DOX conjugate to rats at 5 mg/kg equivalent dose

heart lung spleen liver stomach intestine kidney

DOX 307±64 304±86

127.0±32.6

228±20 313±14 246±51 166.3±21.

6

TPGS-DOX

155.2±33.1

313±50 335±43 262±4

7 114.3±38.6

86.7±20.1

169.4±39.6

5.4 Conclusions

The in vivo pharmacokinetics investigation showed that the TPGS-DOX resulted in much

higher AUC and much longer half-life than the original DOX, which implied enhanced

therapeutic effects. The peak value of the drug concentration in the tissues was reduced

and the time at which the peak concentration was delayed, which implied reduced side

effects. The biodistribution investigation showed that the drug accumulation for the

TPGS-DOX administration was higher in liver and spleen, and lower in heart, stomach

91

and intestine than that for the pristine DOX administration, which indicated the reduced

systemic toxicity, especially impaired the cardiotoxicity.

92

6 CONCLUSIONS AND RECOMMENDATIONS

6.1 Conclusions

The main objective of this project is to develop a novel polymeric anti-cancer drug

conjugate, TPGS-Doxorubicin, for cancer chemotherapy, which comprises prodrug

synthesis and confirmation, in vitro characterization of release, cytotoxicity and cellular

uptake, in vivo investigations in pharmacokinetics and biodistribution. There has been a

lot of work on polymer-drug conjugation, specifically on PEG-DOX conjugation. To our

knowledge, however, TPGS conjugation represents a novel idea in polymer-drug

conjugation strategy since TPGS is a PEGylated vitamin E, which can further improve the

cellular uptake and half-life and thus the therapeutic effects of the drug.

As chapter 3 indicated, TPGS-DOX conjugate was successfully synthesized by chemical

attachment between the carboxyl group in TPGS-SA and the primary amine group in

DOX in the presence of the coupling agent. 1H NMR, FT-IR and GPC were employed to

determine the chemical structure and molecular weight, which confirmed the success of

the DOX conjugation onto TPGS. The drug loading efficiency of the conjugate was

detected using microplate reader, implying 8%. Moreover, the stability study showed that

only 12% drug was released after 5 months as stored at 4 oC, indicating DOX could be

stably attached to TPGS. In chapter 4, in vitro investigation demonstrated that the prodrug

could release DOX for more than 10 days without initial burst in a controlled way under

different pH values. Compared to free DOX, the conjugate displayed higher cellular

uptake and better cytotoxicity in both C6 and MCF-7 cell lines. The lower IC50 values of

93

the TPGS-DOX showed its great superiority over the existing polymeric DOX conjugates.

Besides, the conjugate was found to be distributed differently in cancer cells from the free

DOX, which may imply the different mechanism of drug delivery. At last, the in vivo

pharmacokinetics and biodistribution were further demonstrated in chapter 5. The TPGS-

DOX conjugate resulted in controlled release in blood and tissues as well as less toxicity

in comparison with pristine DOX. On one hand, the TPGS-DOX conjugate resulted in

much higher AUC and much longer half-life in plasma than the original DOX, which

implied greatly enhanced therapeutic effects. On the other hand, the peak value of the drug

concentration in normal tissues after TPGS-DOX administration was reduced and the time

of the peak concentration was delayed, which implied reduced side effects.

All in all, the work above demonstrated that TPGS could carry doxorubicin by

conjugation and realize controlled and sustained release under mild acidic conditions that

allowed the transport of the prodrug to tumor cells and facilitate drug release to exert its

therapeutic function there. The higher in vitro cellular uptake efficiency, cytotoxicity and

much lower IC50 values of the conjugate in comparison with the pristine DOX or other

reported polymer-DOX conjugate approved the great clinical promise of TPGS for drug

conjugation. The in vivo pharmacokinetics index further revealed that the significant

enhancement of the drug’s pharmacokinetics by TPGS conjugation. As for the

biodistribution, the drug accumulation for the TPGS-DOX administration was much lower

in heart, gastric and intestine than that for the pristine DOX administration, which greatly

impaired the limitation of commercial doxorubicin. The TPGS-DOX prodrug showed

great potential to become a novel dosage form of doxorubicin and the methodology can

also be applied to other anticancer drugs.

94

6.2 Recommendations

To further improve the TPGS-drug conjugate in cancer chemotherapy, the following work

is recommended:

To develop TPGS prodrug for other hydrophobic drug such as Docetaxel, hydrophilic

drug or protein drug;

To transplant different tumor models in animals and evaluate the inhibition effect of

TPGS-drug conjugate (Xenograft);

To apply the TPGS-drug conjugate in clinic phase I test for further investigation in

therapeutic and side effects;

To conjugate targeting agent on to the prodrug for targeted delivery of the drug, for

example folate targeted TPGS-DOX conjugate.

95

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