Role of multidrug resistance associated protein 4 (MRP4 ABCC4) in the resistance and toxicity of oxazaphosphorines

ROLE OF MULTIDRUG RESISTANCE-ASSOCIATED PROTEIN 4 MRP4/ABCC4 IN THE IN VITRO ACTIVITY OF CYCLOPHOSPHAMIDE AND IFOSFAMIDE... The aim of this study was to investigate the resistance profi

ROLE OF MULTIDRUG RESISTANCE-ASSOCIATED

PROTEIN 4 (MRP4/ABCC4) IN THE IN VITRO ACTIVITY

OF CYCLOPHOSPHAMIDE AND IFOSFAMIDE

ƒ ACKNOWLEDGEMENTS

I like to thank my former supervisor, Dr Shufeng Zhou and my supervisor Prof Ho Chi Lui, Paul and co-supervisor Prof Ng Ka-Yun, Lawrence, for their great support, guidance and encouragement, for their invaluable assistance in the planning and conducting of the project, and for their advice when difficulties were encountered

I also like to thank Prof Tan May Chin, Theresa of the Department of Biochemistry, National University of Singapore for providing the MRP4

transfected HepG2 cell line, which was the focus of this project

I like to acknowledge the technical assistance given by all laboratory officers and students in my department and acknowledge the scholarship from the National University of Singapore and the generous support of the National

University of Singapore Academic Research Funds

Finally, I want to make a special acknowledgement to my family for their great moral support

ƒ PUBLICATIONS ARISING FROM THIS THESIS

Referred Journal Papers

1 Zhang J, Ng LK, and Ho PC Interaction of Oxazaphosphorine Anticancer

Agents with Multidrug Resistance Associated Protein 4 Biochemical

Pharmacology (under revision)

2 Tian Q, Zhang J, Chan E, Dun W, and Zhou SF Multidrug resistance proteins

(MRPs) and implication in drug development Drug Development Research 2005;

64(1):1-18

3 Tian Q, Zhang J, Tan TM, Chan E, Duan W, Chan SY, Boelsterli UA, Ho PC,

Yang H, Bian JS, Huang M, Zhu YZ, Xiong WP, Li XT and Zhou SF Human Multidrug Resistance Associated Protein 4 Confers Resistance to Camptothecin

Analogs Pharmaceutical Research 2005; 22(11): 1837-1853

4 Zhang J, Tian Q, Chan SY and Duan W, and Zhou SF Insights into

oxazaphosphorine resistance and possible approaches to its circumvention Drug Resistance Updates 2005; 8(5): 271-297

5 Zhang J, Tian Q, Chan SY, Duan W, Li SC, Zhu YZ, and Zhou SF

Metabolism and Transport of Oxazaphosphorines and the Clinical Implications

Drug Metabolism Reviews 2005; 37 (4):611-703

6 Tian Q, Zhang J, Chan SY, Tan TM, Duan W, Huang M, Zhu YZ, Chan E, Yu

Q, Nie YQ, Ho PC, Li Q, Ng LK, Yang HY, Hong W, Bian JS, and Zhou SF

Topotecan is a Substrate for Multidrug Resistance Associated Protein 4 Current Drug Metabolism 2006; 7(1): 105-118

7 Zhang J, Tian Q, and Zhou SF Clinical pharmacology of cyclophosphamide

and ifosfamide Current Drug Therapy 2006; 1(1):55-84

8 Zhang J, Tian Q, and Zhou SF Reversers for oxazaphosphorine resistance

Current Cancer Drug Targets 2006; 6(5): 385-407

Published Conference Abstracts

1 Zhang J, Tian Q, and Zhou SF Resistance profiles of multidrug resistance

associated protein 4 to anticancer drugs 17th Singapore Pharmacy Congress, 30 June-3 July 2005, Singapore

2 Tian Q, Zhang J, Tan MQ, Chan E, Chan SY, and Zhou SF Resistance

profiles of camptothecins in HepG2 cells with overexpression of MRP4 17thSingapore Pharmacy Congress, 30 June-3 July 2005, Singapore

3 Suhaiemi TN, Zhang J, and Zhou SF Multidrug resistance associated protein

4 (MRP4) confers resistance to cyclophosphamide 17th Singapore Pharmacy Congress, 30 June-3 July 2005, Singapore

4 Zhang J, Tian Q, and Zhou SF Resistance profiles of multidrug resistance

associated protein 4 to anticancer drugs Inaugural AAPS-NUS Student Chapter Symposium, 16 September 2005, Singapore

5 Tian Q, Zhang J, and Zhou SF Multidrug resistance associated protein 4

confers resistance to camptothecins Inaugural AAPS-NUS Student Chapter Symposium, 16 September 2005, Singapore

6 Tian Q, Zhang J, Tan MQ, Chan E, Chan SY, Duan W, and Zhou SF Human

multidrug resistance associated protein 4 confers resistance to camptothecins 1st

Postgraduate Congress of Faculty of Science of NUS, 21-22 September 2005, Singapore

ƒ TABLE OF CONTENTS

ƒ  ACKNOWLEDGEMENTS I 

ƒ  PUBLICATIONS ARISING FROM THIS THESIS II 

ƒ  TABLE OF CONTENTS V 

ƒ  SUMMARY IX 

ƒ  LIST OF TABLES XII 

ƒ  LIST OF FIGURES XIII 

ƒ  LIST OF ABBREVIATIONS XV 

CHAPTER 1  INTRODUCTION 1 

1.1   MULTIDRUG RESISTANT ASSOCIATED PROTEINS (MRP S ) 2  

1.1.1  An overview of MRPs 2 

1.1.2  The specific role of MRP4/ABCC4 11 

1.2   CYCLOPHOSPHAMIDE AND IFOSFAMIDE 17  

1.2.1  Clinical activity and mechanism of action of cyclophosphamide and ifosfamide 17 

1.2.2  Pharmacokinetics and pharmacodynamics of cyclophosphamide and ifosfamide 20 

1.2.3  Drug resistance to cyclophosphamide and ifosfamide 29 

1.3   OBJECTIVES 33  

CHAPTER 2  CONFIRMATION OF THE EXPRESSION AND FUNCTION OF MRP4/ABCC4 TRANSFECTED HEPG2 CELLS 35 

2.1   I NTRODUCTION 35  

2.2   M ATERIALS AND METHODS 36  

2.2.1  Chemicals 36 

2.2.2  Cell Culture 37 

2.2.3  Cytotoxicity assay in V/HepG2 and MRP4/HepG2 cells 37 

2.2.4  Western blot analysis 38 

2.2.5  Quantitative analysis of MRP4/ABCC4 expression by immunostaining 39 

2.2.6  Quantitative Real-time Polymerase Chain Reaction (PCR) 39 

2.2.7  Statistical analysis 40 

2.3   R ESULTS 40  

2.3.1  Cytotoxicity assay 40 

2.3.2  Western blot analysis 43 

2.3.3  Quantitative analysis of MRP4/ABCC4 expression by immunostaining 44 

2.3.4  Quantitative Real-time PCR 45 

2.4   D ISCUSSION 46  

CHAPTER 3  THE ROLE OF MRP4/ABCC4 ON THE CYTOTOXICITY OF CYCLOPHOSPHAMIDE AND IFOSFAMIDE IN HEPG2 CELLS 48 

3.1   I NTRODUCTION 48  

3.2   MATERIALS AND METHODS 49  

3.2.1  Chemicals 49 

3.2.2  Cell Culture 50 

3.2.3  Cytotoxicity assay in V/HepG2 and MRP4/HepG2 cells 50 

3.2.4  Cytotoxicities of cyclophosphamide and ifosfamide in V/HepG2 and MRP4/HepG2 cells with different MRP4/ABCC4 inhibitors or GSH synthesis inhibitor 50 

3.2.5  Cytotoxicities of cyclophosphamide and ifosfamide in V/HepG2 and MRP4/HepG2 cells with MRP4/ABCC4 inducer 51 

3.2.6  Western blot analysis 51 

3.2.7  Quantitatove analysis of MRP4/ABCC4 expression by immunostaining 51 

3.2.8  Statistical analysis 51 

3.3   R ESULTS 52  

3.3.1  Cytotoxicity assay 52 

3.4   D ISCUSSION 72  

CHAPTER 4  THE EFFECT OF MUTANT MRP4/ABCC4 ON THE CYTOTOXICITY OF CYCLOPHOSPHAMIDE AND IFOSFAMIDE IN HEPG2 CELLS 76 

4.1   I NTRODUCTION 76  

4.2   M ATERIALS AND M ETHODS 78  

4.2.1  Chemicals 78 

4.2.2  Cell culture 78 

4.2.3  Cytotoxicity assay in V/HepG2 and MRP4/HepG2 cells 79 

4.2.4  Statistical analysis 79 

4.3   R ESULTS 79  

4.3.1  Cytotoxicity assay 79 

4.4   D ISCUSSION 86  

CHAPTER 5  INDUCING POTENCY OF CYCLOPHOSPHAMIDE AND IFOSFAMIDE ON THE MRP4/ABCC4 EXPRESSION 88 

5.1   I NTRODUCTION 88  

5.2   M ATERIALS AND M ETHODS 89  

5.2.1  Chemicals 89 

5.2.2  Cell culture 89 

5.2.3  Western blot analysis 89 

5.2.4  Quantitatove analysis of MRP4/ABCC4 expression by immunostaining 90 

5.2.5  Real-time PCR 90 

5.2.6  Statistical analysis 90 

5.3   R ESULTS 90  

5.3.1  Western blot analysis 90 

5.3.2  Quantitative analysis of MRP4/ABCC4 expression by immunostaining 94 

5.3.3  Real-time PCR 96 

5.4   D ISCUSSION 98  

CHAPTER 6  CONCLUSIONS AND FUTURE DIRECTIONS 102 

6.1   C ONCLUSIONS 102  

6.2   T HESIS ACHIEVEMENTS 105  

6.3   F UTURE DIRECTIONS 106  

ƒ  BIBLIOGRAPHY 108 

ƒ SUMMARY

Multidrug resistance associated protein 4 (MRP4/ABCC4) is an organic anion pump capable of transporting nucleoside, nucleotide analogs and cyclic nucleotide Increased expression of MRP4/ABCC4 in tumor cells is associated with resistance to various chemotherapeutic agents such as methotrexate (MTX), topotecan and others MRP4/ABCC4 is identified as the contributor of multidrug resistance (MDR) The oxazaphosphorines including cyclophosphamide (CP) and ifosfamide (IF), represent an important group of therapeutic agents due to their substantial antitumor and immuno-modulating activity Resistance to

oxazaphosphorines is a major clinical problem often resulting in therapeutic failure Detailed investigations aimed at identification of resistant proteins and circumventing approaches of intrinsic drug resistance are thus warranted

The aim of this study was to investigate the resistance profiles of

MRP4/ABCC4 to oxazaphosphorines including CP and IF in the absence and presence of various MRP4/ABCC4 inhibitors or MRP4/ABCC4 inducers by using the MRP4/ABCC4 overexpressing HepG2 cells Overexpression of

MRP4/ABCC4 conferred significant resistance to CP and IF in the 48-hr exposure assays In MRP4/ABCC4 overexpressing HepG2 cells, the presence of the MRP4/ABCC4 inhibitors including diclofenac, MK571, and celecoxib

drug-decreased the cytotoxicity of CP and IF in 48-hr exposure assay In addition, the presence of DL-buthionine-(S,R)-sulphoximine (BSO), the glutathione (GSH) synthesis inhibitor, partially reversed the resistance to CP and IF in

MRP4/ABCC4 overexpressing HepG2 cells Furthermore, clofibrate (CFB), which was reported to be a MRP4/ABCC4 inducer in mice, was found to enhance MRP4/ABCC4-mediated resistance to CP and IF Moreover, this resistance to CP and IF was enhanced in mutant MRP4/ABCC4 (F324A or F324W)

overexpressing HepG2 cells when compared with MRP4/ABCC4 overexpressing HepG2 cells This demonstrates that CP and IF are highly possible substrates of MRP4/ABCC4 and GSH may play an important role in the resistance to CP and

IF mediated by MRP4/ABCC4

Oxazaphosphorines may also have effects on the expression of

MRP4/ABCC4 This was investigated by detecting MRP4/ABCC4 expression in HEK293 cells and HepG2 cells after these cells were incubated with media

containing different concentration of oxazaphosphorines including CP and IF The MRP4/ABCC4 inducer CFB was used as a positive control The present study showed that the positive control CFB can up-regulate MRP4/ABCC4 expression

at protein level in HEK293 cells In addition, CP significantly increased the MRP4/ABCC4 expression at both protein level and mRNA level in HEK293 cells

at higher concentration, while IF significantly decreased the MRP4/ABCC4 expression at mRNA level at lower concentration only It indicates that CFB can modulate MRP4/ABCC4 in vitro; whereas CP is a MRP4/ABCC4 inducer at higher concentration Furthermore, Overexpression of MRP4/ABCC4 conferred significant resistance to CFB in the 48-hr drug-exposure assays The

MRP4/ABCC4 inhibitors MK571 and celecoxib partially reversed the resistance caused by CFB It suggests that CFB is a potential substrate of MRP4/ABCC4

In summary, the present study demonstrated that MRP4/ABCC4 plays an

important role in the in vitro activity of oxazaphosphorines In addition, this study

suggests that CFB is a potential substrate of MRP4/ABCC4 and a modulator of

MRP4/ABCC4 in vitro Further studies are needed to explore the effect of

MRP4/ABCC4 on the transport of oxazaphosphorines and CFB It would also be interesting to investigate the effect of the combination of oxazaphosphorines and

MRP4/ABCC4 inhibitors on cancer treatment, and to investigate the effect of MRP4/ABCC4 on the toxicity of CFB

ƒ LIST OF TABLES

Table 1-1 Reported Substrates and Inhibitors for MRPs 9 

Table 2-1 Drug sensitivity of HepG2 cells expressing MRP4/ABCC4 or vector

only to some MRP4/ABCC4 substrates 41 

Table 3-1 Drug sensitivity of HepG2 cells expressing MRP4/ABCC4 or vector

only to cyclophosphamide (CP) and ifosfamide (IF) with or without BSO and some known MRP4/ABCC4 inhibitors The cells were preincubated with BSO (200 µM) for 24 h, and celecoxib (50 µM), diclofenac (200 µM), or MK571 (100 µM) for 2 h 55 

Table 3-2 Drug sensitivity of HepG2 cells expressing MRP4/ABCC4 or vector

only to cyclophosphamide (CP) and ifosfamide (IF) with the presence of MRP4/ABCC4 inducer 61 

Table 3-3 Drug sensitivity of HepG2 cells expressing MRP4/ABCC4 or vector

only to clofibrate without or with the presence of some known MRP4/ABCC4 inhibitors 67 

Table 4-1 Drug sensitivity of HepG2 cells expressing mutant MRP4/ABCC4,

MRP4/ABCC4 or vector only to MRP4/ABCC4 substrate (MTX) after drug exposure time of 4 hr 80 

Table 4-2 Drug sensitivity of HepG2 cells expressing mutant MRP4/ABCC4,

MRP4/ABCC4 or vector only to cyclophosphamide (CP) and ifosfamide (IF) after drug exposure time of 48 hr 83 

ƒ LIST OF FIGURES

Figure 1-1 Metabolism of Cyclophosphamide 22 

Figure 1-2 Metabolism of Ifosfamide 24 

Figure 2-1 Cytotoxicity of MTX and bis-POM-PMEA in V/HepG2 (□) and

MRP4/HepG2 (■) cells when the drugs were incubated for 4 hr (MTX) or 48 hr (bis-POM-PMEA) 42 

Figure 2-2 Western blot analysis of MRP4/ABCC4 expression in Hep G2 cells

Lane 1 shows the V/HepG2 cells and lane 2 shows the MRP4/HepG2 cells The molecular mass (kDa) is indicated 43 

Figure 2-3 Quantitative analysis of MRP4/ABCC4 expression by

immunostaining in Hep G2 cells Column 1 shows the V/HepG2 cells and column

2 shows the MRP4/HepG2 cells 44 

Figure 2-4 Quantitative real time PCR analysis of MRP4/ABCC4 mRNA in

V/HepG2 cells and MRP4/HepG2 cells MRP4/ABCC4 mRNA was quantified using real time PCR analysis (SYBR green) standardizing against the endogenous control GAPDH Data were normalized to controls and expressed as fold change ratio to control levels 45 

Figure 3-1 Cytotoxicity of cyclophosphamide (CP) and ifosfamide (IF) in

V/HepG2 (□) and MRP4/HepG2 (■) cells when the drugs were incubated for 48

hr 53 

Figure 3-2 Cytotoxicity of cyclophosphamide (CP) incubated for 48 hr in

V/HepG2 (□) and MRP4/HepG2 (■) cells with the presence of BSO (200 µM), diclofenac (200 µM), celecoxib (50 µM), and MK571 (100 µM) 57 

Figure 3-3 Cytotoxicity of ifosfamide (IF) incubated for 48 hr in V/HepG2 (□)

and MRP4/HepG2 (■) cells with the presence of BSO (200 µM), diclofenac (200 µM), celecoxib (50 µM), and MK571 (100 µM) 59 

Figure 3-4 Cytotoxicity of cyclophosphamide (CP) incubated for 48 hr in

V/HepG2 (□) and MRP4/HepG2 (■) cells with the presence of clofibrate (CFB) (200 µM and 400 µM) 62 

Figure 3-5 Cytotoxicity of ifosfamide (IF) incubated for 48 hr in V/HepG2 (□)

and MRP4/HepG2 (■) cells with the presence of clofibrate (CFB) (200 µM and

400 µM) 63 

Figure 3-6 MRP4/ABCC4 protein expression by western blot in V/HepG2 and

MRP4/HepG2 cells exposed to different concentration of clofibrate (CFB) for 6 days The bar graph shows the quantification of band intensity *p<0.05, **p<0.01, significantly different from the control group 65 

Figure 3-7 Quantitative analysis of MRP4/ABCC4 protein expression by

immunostaining in V/HepG2 and MRP4/HepG2 cells exposed to different

concentration of clofibrate (CFB) for 6 days **p<0.01 significantly different from the control group 65 

Figure 3-8 Cytotoxicity of clofibrate (CFB) incubated for 48 hr in V/HepG2 (□)

and MRP4/HepG2 (■) cells without or with the presence of celecoxib (50 µM) and MK571 (100 µM) 68 

Figure 3-9 Cytotoxicity of some alkylating agents including melphalan, busulfan,

nimustine hydrochloride and mechlorethamine hydrochloride incubated for 48 hr

in V/HepG2 (□) and MRP4/HepG2 (■) cells 71 

Figure 4-1 Cytotoxicity of methotrexate (MTX) incubated for 4 hr in V/HepG2

(□), MRP4/HepG2 (■), Mutant (F324A) (░) and Mutant (F324W) (▓) cells 81 

Figure 4-2 Cytotoxicity of cyclophosphamide (CP) and ifosfamide (IF) incubated

for 4 hr in V/HepG2 (□), MRP4/HepG2 (■), Mutant (F324A) (░) and Mutant (F324W) (▓) cells 85 

Figure 5-1 MRP4/ABCC4 protein expression by western blot in HepG2 cells

exposed to different concentration of cyclophosphamide (CP), ifosfamide (IF) or clofibrate (CFB) for 6 days Contro 1 and control 2 are the controls for cyclophosphamide, ifosfamide (or clofibrate) respectively The bar graph shows the quantification of band intensity 92 

Figure 5-2 MRP4/ABCC4 protein expression by western blot in HEK293 cells

exposed to different concentration of cyclophosphamide (CP), ifosfamide (IF) or clofibrate (CFB) for 6 days Contro 1 and control 2 are the controls for cyclophosphamide, ifosfamide (or clofibrate) respectively The bar graph shows the quantification of band intensity *p<0.05, **p<0.01, significantly different from the control group 93 

Figure 5-3 Quantitative analysis of MRP4/ABCC4 protein expression by

immunostaining in HEK293 cells exposed to different concentration of cyclophosphamide, ifosfamide or clofibrate for 6 days *p<0.05, **p<0.01 significantly different from the control group 95 

Figure 5-4 Quantitative real-time PCR analysis of MRP4/ABCC4 mRNA in

HEK293 cells exposed to different concentration of cyclophosphamide, ifosfamide or clofibrate for 6 days MRP4/ABCC4 mRNA was quantified using quantitative real-time PCR (SYBR green method) standardizing against the endogeneous control GAPDH Data were normalized to GAPDH and expressed as ratio to GAPDH levels (n=3) *p<0.05, **p<0.01 significantly different from the control group 97 

ANOVA One-way analysis of variance

AUC Area under the plasma concentration-time curve

AZT Azidothymidine

BCRP Breast cancer resistance protein

Bis-POM-PMEA Bis(pivaloxymethyl)-9-(2-phosphonylmethoxyethyl)adenine

BSEP Bile salt export pump

BSO DL-buthionine-(S,R)-sulphoximine

cAMP Cyclic adenosine monophosphate

cGMP Cyclic guanosine monophosphate

cMOAT Canalicular multispecific organic anion transporter

CYP Cytochrome P-450

DHEAS Dehydroepiandrosterone-3-sulfate

DMEM Dulbecco’s modified Eagle’s medium

MRP4/HepG2 HepG2 cells with stably transfected MRP4

MTT 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazonium bromide MTX Methotrexate

- xvii

Nrf2 Nuclear factor-E2-related factor 2

NSAIDs Nonsteroidal antiinflammatory drugs

OAT Organic anion transporter

PAH p-Aminohippurate

PBS Phosphate buffered saline

PgP P-glycoprotein

PG Prostaglandin

PMEA 9-(2-phosphonylmethoxyethyl)adenine PMEDAP 9-(2-phosphonomethoxyethyl)-2,6-diaminopurine PMEG 9-(2-phosphonomethoxyethyl)guanine PPARα Peroxisome proliferator-activated receptor α

CHAPTER 1 INTRODUCTION

Conventional chemotherapy aims to kill or disable tumor cells by direct or indirect mechanisms, while preserving the normal cells [1] Many chemotherapeutic

agents (e.g Vinca alkaloids, oxazaphosphorines, camptothecins, etc) are developed to

treat many different malignancies However, conventional chemotherapy is only successful when the cancer is detected at its early stage, or is limited to certain types

of cancer (e.g leukemia) Most chemotherapeutic agents can cause dose-limiting toxicities which may be transient toxicity, or have long-term effects on the lungs, heart and reproductive organs causing permanent organ damage, secondary tumor (e.g leukemia and brain tumor), or death Increasing the selectivity of chemotherapeutic agents may reduce dose-limiting toxicities, and coadministration of multiple

chemotherapeutic agents has become standard regimen for the treatment of nearly all carcinomas and haematological malignancies In addition, the major cause of failure

of antitumor chemotherapy is the development of multidrug resistance (MDR) Tumor cells may have intrinsic resistance to current chemotherapeutic agents or develop resistance after exposure Cellular mechanisms of MDR include defective drug

transport (reduced drug transport or increased drug efflux), altered drug activation or inactivation, and/or enhanced repair or tolerance to DNA damage

Multidrug resistance associated proteins (MRPs) are believed to contribute to the development of MDR Therefore, knowledge on structures and functions of MRPs

is important to develop successful approaches on reversion of MDR The rest of this chapter will present an overview on structures and functions of MRPs, the specific role of MRP4/ABCC4 in chemotherapy, mechanism of action of oxazaphosphorines, pharmacokinetics and pharmacodynamics of oxazaphosphorines, drug resistance to oxazaphosphorines and concludes with objectives of this research

1.1 MULTIDRUG RESISTANT ASSOCIATED PROTEINS (MRPs)

MDR is the phenomenon in which cells show simultaneous resistance to

several different structurally and functionally unrelated drugs that do not have the same mechanism of actions P-glycoprotein (PgP/ABCB1) is the first drug efflux pump identified as the contributor of MDR [2] It is a 170 kDa plasma glycoprotein encoded byhuman MDR1 gene, which belongs to the ATP-binding cassette (ABC)family of transporters Such protein is expressed constitutively in a number of normal tissues with high levels on the apical surfaces of epithelial cells in the liver (bile

canaliculi), kidney (proximal tubule), and small and large intestine (columnar mucosal cell) [3] Furthermore, PgP/ABCB1 can cause considerable resistance to a number of chemotherapeutic agents, including anthracyclines (e.g daunorubicin and doxorubicin)

[4], Vinca alkaloids (vincristine and vinblastine) [5], paclitaxel [6], and camptothecins

[7] In addition to PgP/ABCB1, the mitoxantrone resistance gene, MXR, also known

as the breast cancer resistance protein (BCRP/ABCG2) is a potent contribtor of

MDR[8] BCRP/ABCG2 has the ability to confer high levels of resistance to

anthracyclines (e.g daunorubicin and doxorubicin) [9], mitoxantrone [9], bisantrene [8], and the camptothecins (e.g topotecan and SN-38) [10, 11] Other than

PgP/ABCB1 and BCRP/ABCG2, many different transporters can confer the

resistance to clinically important anticancer agents Almost all transporters belong to MRP family, which is a subfamily of ABC transporters

1.1.1 An overview of MRPs

All MRP members have hydrophobic transmembrane domains and

cytoplasmic nucleotide binding domains (NBDs) [12] The NBDs are responsible for the ATP binding/hydrolysis that drives drug transport [13] The transmembrane

domains contain the drug-binding sites, which are likely to be located in a flexible internal chamber that is sufficiently large to accommodate different drugs MRPs can

be categorized according to the presence or absence of a third (NH2-terminal)

membrane-spanning domain in their structure This topological feature can be found

in MRP1/ABCC1, MRP2/ABCC2, MRP3/ABCC3, MRP6/ABCC6 and

MRP7/ABCC10, while it is not found in MRP4/ABCC4, MRP5/ABCC5,

MRP8/ABCC11 and MRP9/ABCC12 MRPs with this structural feature have the ability to transport conjugates, while MRPs without it are able to transport cyclic nucleotides Various MRPs show considerable differencesin their tissue distribution, substrate specificities, and proposedphysiological and pharmacological functions

MRP1/ABCC1 was found in 1992 in lung cancer H69AR cell line conferring resistance to many chemotherapeutic agents, which had been demonstrated not to overexpress PgP/ABCB1 previously [14, 15] MRP1/ABCC1 is nearly present in all major tissues and in all peripheral blood cell types MRP1/ABCC1 is capable of conferring resistance to several families of natural product drugs like PgP/ABCB1,

including Vinca alkaloids (vincristine and vinblastine), anthracyclines (e.g

daunorubicin and doxorubicin), epipodophyllotoxins (etoposide) [16], and

camptothecins (irinotecan (CPT-11) and SN-38) [17] In addition, the fluorescent probe calcein [18, 19], the human immunodeficiency virus (HIV) protease inhibitors (saquinavir and ritonavir) [20], the antiandrogen flutamide and its active metabolite hydroxyflutamide [21], and the organic arsenic as a triglutathione conjugate [22] are transported by MRP1/ABCC1 Furthermore, MRP1/ABCC1 is able to transport different structural glutathione (GSH), glucuronate and sulfate conjugates, including estradiol 17-β-D-glucuronide (E217βG), the cysteinyl leukotriene C4 (LTC4), and sulfated bile acids [23, 24] Substrates of MRP1/ABCC1 also include neutral and

basic cytotoxic compounds without conjugation with GSH or other anionic drugs [25, 26] However, intracellular GSH is needed when MRP1/ABCC1 transports these chemicals [27, 28] Moreover, various inhibitors that inhibit MRP1/ABCC1 transport activity have been described Some inhibitors such as probenecid, sulfinpyrazone, indomethacin and benzbromarone can modulate the activity of many different

transporters including organic anion transporters(OATs) that do not belong to the ABC family [29] Some inhibitors that are relatively specific to the MRP-related transporters are sulphonylurea, glibenclamide [30], ONO-1078, and MK571 [31] Many inhibitors are dual inhibitors of MRP1/ABCC1 and PgP/ABCB1 These include PAK-104P [32], verapamil and cyclosporin A [25], and several bioflavonoids

(genistein and quercetin) [19] In addition, LY475776 is a highly specific and potent MRP1/ABCC1 inhibitor in a GSH-dependent manner [33] Recently, the flavonoid myricetin showed the ability to modulate MRP1/ABCC1 mediated resistance to the anticancer drug vincristine in MRP1/ABCC1 transfected MDCKII cells [34]

MRP2/ABCC2 is also known as the canalicular multispecific organic anion transporter (cMOAT) Mutations of the MRP2/ABCC2 gene result in the Dubin-Johnson syndrome which is characterized by conjugated hyperbilirubinemia and impaired hepatobiliary secretion of a wide range of amphipathic compounds [35] MRP2/ABCC2 has shown resistance to a broad range of natural product drugs,

including etoposide, Vinca alkaloids (vincristine and vinblastine), anthracyclines (e.g

daunorubicin and doxorubicin) [36], and camptothecins [7] It seems that

MRP1/ABCC1 and MRP2/ABCC2 have some substrates in common However, there are some important differences With respect to anticancer agents, MRP2/ABCC2 functions as a cellular cisplatin [37] and taxanes (paclitaxel and docetaxel) [38]

transporter In addition, MRP2/ABCC2 efficiently transports the HIV protease

inhibitors including saquinavir, indinavir and ritonavir [39] Both MRP1/ABCC1 and MRP2/ABCC2 transport many of the glutathione, glucuronate, and sulfate conjugates, but the affinities of the two transporters can differ significantly For example,

bilirubin mono- and bis-glucuronides are better substrates for MRP2/ABCC2,

whereas MRP1/ABCC1 has higher affinity for LTC4 [40].Inhibitors of

MRP2/ABCC2 have been described Recently, in vitro studies showed that the

flavonoids including robinetin and myricetin appeared to be good inhibitors for

MRP2/ABCC2 [41] In addition, the non-nucleoside reverse transcriptase inhibitors including delavirdine and efavirenz, and the nucleoside reverse transcriptase inhibitor emtricitabine showed a significant and concentration-dependent inhibition of

MRP1/ABCC1, MRP2/ABCC2 and MRP3/ABCC3 [42]

Among the MRP family, MRP3/ABCC3 has 58% and 48% amino acid

sequence resemblance with MRP1/ABCC1 and MRP2/ABCC2 [43] MRP3/ABCC3

is mainly detected in the liver, colon, intestine, and prostate at the mRNA level and in adrenal gland, kidney, colon, pancreas, gallbladder, and liver at the protein level [44, 45] As an OAT like MRP1/ABCC1 and MRP2/ABCC2, MRP3/ABCC3 confers resistance to some anticancer drugs However, the substrate profile of MRP3/ABCC3

is narrow and limited to vincristine, methotrexate (MTX), and epipodophyllotoxins (teniposide and etoposide) [46, 47] Overexpression of MRP3/ABCC3 is specifically associated with increased ATP-dependent transport of E217βG, 2,4-dinitrophenyl S-glucuronide (DNP-SG), and LTC4 The transport of E217βG by MRP3/ABCC3 was inhibited by both etoposide and methotrexate in a concentration-dependent manner [48] In addition, MRP3/ABCC3 may function as a backup detoxification system for liver cells if normal canalicular route is damaged by cholestatic diseases

MRP3/ABCC3 can recognize the monoanionic bile acids such as glycocholate and

taurocholate, which is distinguished from other MRP family members [49, 50]

Recently, Zamek-Gliszczynski MJ et al reported that MRP3/ABCC3 mediates the hepatic basolateral excretion of diverse glucuronide conjugates in MRP3/ABCC3 knock out mice [51]

MRP5/ABCC5 is able to transport cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), but with differential affinity [52] MRP4/ABCC4 has a higher affinity for cAMP than that of MRP5/ABCC5, whereas MRP5/ABCC5 has a higher affinity for cGMP than that of MRP4/ABCC4 [53] The transport of cyclic nucleotides by MRP4/ABCC4 and MRP5/ABCC5 has led to the speculation that they might be involved in the regulation of the intracellular

concentration of these important second messengers MRP5/ABCC5 has also shown resistance to 9-(2-phosphonylmethoxyethyl)adenine (PMEA), 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) [54] In addition, MRP5/ABCC5 confers resistance to

5-fluorouracil and transports its monophosphorylated metabolites in vitro This

resistance can be reversed by several inhibitors including probenecid, MK571,

sildenafil and trequinsin [55] However, MRP5/ABCC5 shows no resistance to natural anticancer compounds or MTX

MRP6/ABCC6 is an amphipathic anion transporter able to transport

glutathione conjugates, such as LTC4 and N-ethylmaleimide S-glutathione (NEM-GS), and the cyclopentapeptide BQ123 which has a low affinity to E217βG Effective inhibitors of MRP1/ABCC1 and MRP2/ABCC2, including indomethacin, probenecid, and benzbromarone, can inhibit the MRP6/ABCC6-dependent NEM-GS transport [56] Additionally, MRP6/ABCC6 shows low level of resistance to several natural product agents, including etoposide, teniposide, doxorubicin, and daunorubicin [57] MRP6/ABCC6 is mainly present in the liver and kidney, besides in several other

organs with a low expression level [58] MRP7/ABCC10 has the lowest amino acid sequence identity with other MRP family members [59] It is able to mediate the transport of amphipathic anions, such as E217βG [60] In addition, MRP7/ABCC10 is

a resistance factor to several natural product anticancer agents including vincristine, vinblastine, and taxanes, especially docetaxel [61] However, MRP7/ABCC10 mRNA

is present in normal organs at very low levels

MRP8/ABCC11 and MRP/ABCC12 have high amino acid sequence

resemblance with MRP4/ABCC4 and MRP5/ABCC5 MRP8/ABCC11 is mainly present in normal breast and testis, while little is in the liver, brain, and placenta MRP8/ABCC11 has been characterized as an amphipathic anion transporter It

participates in physiological processes involving the monoanionic bile acids

glycocholate and taurocholate, conjugated steroids such as LTC4, DNP-SG, E217βG, and dehydroepiandrosterone-3-sulfate (DHEAS), and cyclic nucleotides [62] With the ability to efflux cAMP and cGMP, MRP8/ABCC11 confers resistance to purine and pyrimidine nucleotide derivatives [63] MRP9/ABCC12 is a specific member of MRP family, with two major transcripts of 4.5 and 1.3 kb Because the larger 4.5-kb

is mainly expressed in breast tumor and seldom in normal breast, MRP9/ABCC12 may represent a novel target in the treatment of breast cancer [64] However, the functional characteristics of MRP9/ABCC12 are still unclear

MRPs are capable of transporting a structurally diverse array of endo- and xenobiotics and their metabolites across cell membranes They play an important role

in the absorption, disposition and elimination of many therapeutic agents in the body

In particular, increased expression of these drug transporters in tumor cells is

associated with resistance to a number of important chemotherapeutic agents With the accumulation of information on drug resistance profile and physiological function

of MRP family, the relationship between drug selectivity and specific transporter level will be more significant and helpful in clinical cancer treatment

Table 1-1 Reported Substrates and Inhibitors for MRPs

MRP1(ABCC1) Vincristine, vinblastine, doxorubicin, daunorubicin,

etoposide (VP16), epirubicin, CPT-11, SN-38, methotrexate, ritonavir, sequinovar, flutamide, hydroxyflutamide, GSH conjugates, glucuronide conjugates, sulfate conjugates, GSH, GSSG, calcein, arsenic

Probenecid, sulfinpyrazone, indomethacin, benzbromarone MK571, ONO-1078, sulphonylurea, glibenclamide, verapamil, genistein, cyclosporine A, PAK-104P, quercetin, myricetin, LY475776, delavirdine, efavirenz, emtricitabine

[16-22, 24, 25,

29-34, 42, 65]

MRP2(ABCC2) Cisplatin, etoposide (VP16) , epirubicin, vincristine,

vinblastine, doxorubicin, daunorubicin, methotrexate, camptothecins, paclitaxel, docetaxel, saquinavir, ritonavir, indinavir, GSH, GSSG, GSH conjugates, glucuronide conjugates, sulfate conjugates

MK571, robinetin, myricetin, delavirdine, efavirenz,

MRP3(ABCC3) Vincristine, methotrexate, etoposide (VP16), teniposide,

glycocholate, glucuronide conjugates

Etoposide (VP16), methotrexate, delavirdine, efavirenz,

MRP4(ABCC4) AZT, methotrexate, PMEA, bis-POM-PMEA, ganciclovir,

6-mercaptopurine, 6-thioguanine, topotecan, CPT-11,

cefmetazole, cefotaxime

MK571, genistein, probenecid, sulfinpyrazone, dilazep,

MRP5(ABCC5) PMEA, heavy metals, 6-mercaptopurine, 6-thioguanine,

MRP6(ABCC6) BQ123, LTC 4 , N-ethylmaleimide S-glutathione,

Low-level resistance to doxorubicin, teniposide, daunorubicin, cisplatin and etoposide (VP-16)

MRP7(ABCC10) Taxanes, E 2 17βG unknown [60, 61]

MRP8(ABCC11) PMEA, methotrexate, cGMP, cAMP, LTC4, DNP-SG,

taurocholate

TXB2 = thromboxane B2

1.1.2 The specific role of MRP4/ABCC4

The chromosomal location of MRP4/ABCC4 gene was mapped to the long

arm of chromosome 13 at 13q32 by fluorescence in situ hybridization

MRP4/ABCC4 gene contains 31 exon encoding 1325 amino acids [84]

MRP4/ABCC4 has particular tissue expression profile, drug resistance selectivity, and substrate and inhibitor specificity, in comparison with other MRPs

Initially, MRP4/ABCC4 mRNA is restricted to only a few tissues

However, subsequent works demonstrated that MRP4/ABCC4 mRNA is wildly expressed in most tissues, including prostate [84], brain [85], kidney [86],

pancreas [87], the porcine coronary [88], the pulmonary arteries [88],

gastrointestinal tract [89], liver (barely detectable) [84] and a number of cell lines [90, 91] MRP4/ABCC4 protein is detected in the kidney [86], prostate [69], brain [85], pancreas [87], platelets [92], and erythrocytes [92] in humans

MRP4/ABCC4 can be present apically as well as basolaterally in polarized cells depending on the cell type The basolateral location of MRP4/ABCC4 protein was observed in the tubuloacinar cells of prostate [69], hepatocytes [78], the choroid plexus epithelium, and the ductular epithelial cells of pancreas [87] In contrast, the apical location of MRP4/ABCC4 protein was found in the proximal tubule of human kidney [86], and the endothelial cells of the brain capillaries [85] However, there is an almost equal distribution of MRP4/ABCC4 on the apical and

basolateral plasma membrane of the cultured bovine brain microvessel endothelial cells [93] In addition, MRP4/ABCC4 was found within the membrane of human platelet dense granules by immunoblotting and immunofluorescence microscopy [94] The expression of MRP4/ABCC4 in the membrane is cell and tissue specific

MRP4/ABCC4 is expressed on the apical membrane of the proximal tubule of human kidney This is ideal for transporting its substrates into urine

MRP4/ABCC4 is an organic anion pump capable of transporting

nucleoside and nucleotide analogs as well as cyclic nucleotide Urate, the end product of human purine metabolism, can be transported by MRP4/ABCC4 which

is a unidirectional efflux pump for urate with multiple allosteric substrate binding sites simultaneously with cAMP or cGMP [72] Additionally, MRP4/ABCC4 mediates efflux of GSH across the basolateral membrane of hepatocytes into blood [78] Depletion of intracellular GSH by DL-buthionine-(S,R)-sulphoximine (BSO), which is a potent and specific inhibitor of GSH, adversely affects the export of cAMP by MRP4/ABCC4 [73] In addition, in the presence of

physiological concentrations of GSH, MRP4/ABCC4 has a high affinity for the taurine and glycine conjugates of the common natural bile acids as well as the unconjugated bile acid cholate [95] Furthermore, several conjugated steroids such

as E217βG, and DHEAS which is the major circulating steroid made in the adrenal gland in humans could be transported by MRP4/ABCC4 [74] MRP4/ABCC4 also functions as a transporter of prostaglandin (PG) E1 and E2, while MRP1/ABCC1, MRP2/ABCC2, MRP3/ABCC3, and MRP5/ABCC5 do not transport PGE1 or PGE2 [75] Recently, Rius M et al found that MRP4/ABCC4 co-expressed with

PG synthesizing enzymes in the epithelial cells of human seminal vesicles in the male urogenital tract and the function of MRP4/ABCC4 as an export pump for PGE2, PGF2α and thromboxane B2 (TXB2) [96] Moreover, MRP4/ABCC4 is a novel p-aminohippurate (PAH) transporter that has high expression in kidney, and therefore may be important in renal PAH excretion [76]

Besides endobiotics, MRP4/ABCC4 is capable of transporting several xenobiotics Schuetz et al found that MRP4/ABCC4 participated in the efflux of PMEA and azidothymidine (AZT) [67] A statistically significant resistance for PMEA was detected in MRP4/ABCC4 cDNA transfected NIH3T3 cells, which can further confirm the active role of MRP4/ABCC4 in the transport of PMEA [69] In addition, substantial resistance to its precursor bis(pivaloxymethyl)-9-(2-phosphonylmethoxyethyl)adenine (bis-POM-PMEA) and 9-(2-

phosphonomethoxyethyl)-2,6-diaminopurine (PMEDAP) and its derivative

cyclopropyl-PMEDAP (cPr-PMEDAP) was found in MRP4/ABCC4

overexpressed HEK293 cells These MRP4/ABCC4 cells also showed a lower level of resistance to 9-(2-phosphonomethoxyethyl)guanine (PMEG) and the purine-based carbocyclic nucleoside analog abacavir [68] Only PMEA and bis-POM-PMEA were the nucleoside phosphonates can be affected by both

MRP4/ABCC4 and MRP5/ABCC5 [54, 68] Adachi et al reported that cells overexpressing MRP4/ABCC4 had significantly increased resistance to the

cytotoxicity of ganciclovir which is a purine-based antiretroviral agent [97] Futhermore, MRP4/ABCC4 is involved in tubular secretion of diuretics

(hydrochlorothiazide and furosemide) and antiviral drugs (adefovir which is previously called bis-POM PMEA, tenofovir and cidofovir whose urinary

excretion only a limited relationship with MRP4/ABCC4) [98, 99] Recently, four cephalosporins including ceftizoxime, cefazolin, cefmetazole, and cefotaxime had been identified as substrates of MRP4/ABCC4 In addition, MRP4/ABCC4 is involved in the luminal efflux of of ceftizoxime and cefazolin in the kidney by in vivo pharmacokinetic studies using MRP4 knock out mice [77]

MRP4/ABCC4 overexpressed NIH3T3 cells showed significantly

increased resistance to MTX in short-term drug exposure assays and exhibited decreased intracellular accumulation of MTX at 4 hours [69] Chen et al used the membrane vesicles prepared from insect cells infected with MRP4/ABCC4

baculovirus to define the role of MRP4/ABCC4 in the cellular pharmacology of MTX and related processes The results demonstrated that MTX is really

susceptible to MRP4/ABCC4-mediated transport In addition, the results also

showed that MRP4/ABCC4 also can transport folic acid and N5

-formyltetrahydrofolic acid (leucovorin) Furthermore, MRP4/ABCC4 only can mediate the transport of free drug, unable to mediate the transport of mono- or polyglutamated metabolites of MTX [100] 6-MP and 6-TG also exhibited higher levels of resistance in MRP4/ABCC4 transfected NIH3T3 cells compared with parental cells [53] 6-MP and 6-TG are prodrugs; therefore their cytotoxicity depends on their intracellular conversion to their metabolites (thionucleoside monophosphates) These thionucleoside monophosphates had been evaluated their substrate specificity of MRP4/ABCC4 and MRP5/ABCC5 in MRP4/ABCC4 or MRP5/ABCC5 transfected HE293 cell as well as the parental cells The results showed that thioinositol monophosphate (tIMP), thioguanosine monophosphate (tGMP) and 6-methyl-tIMP (MetIMP) were transported by both MRP4/ABCC4 and MRP5/ABCC5 MRP4/ABCC4 showed higher transport rate for tGMP and MetIMP, whereas only MRP5/ABCC5 can transport thioxanthosine

monophosphate (tXMP) [81] Many other anticancer agents have been identified

as substrates of MRP4/ABCC4, such as CPT-11 and its active metabolites SN-38 [71], and topotecan [70]

The commonly used inhibitors of OAT such as probenecid, and

sulfinpyrazone can inhibit the transport activities of MRP4/ABCC4 [76] Like MRP1/ABCC1 and MRP2/ABCC2, MRP4/ABCC4 is also inhibited by the

leukotriene D4 receptor antagonist MK571 [78] The flavonoid, genistein was demonstrated a strong inhibitor of MRP4/ABCC4 mediated transport of PMEA in the MRP4/ABCC4 -overexpressing microglia cells [79] In addition, the

phosphodiesterase 5 modulators including sildenafil and trequinsin are not only the high-affinity inhibitors of MRP5/ABCC5, but also potent inhibitors of

MRP4/ABCC4 [68] Furthermore, the inhibitors of nucleoside transport including dipyridamole, dilazep, and nitrobenzyl mercaptopurine riboside (NBMPR) also can inhibite MRP4/ABCC4 [68] Moreover, thecellular efflux of cGMP by both MRP4/ABCC4 and MRP5/ABCC5 is inhibitedby PGA1 and PGE1, the steroid progesterone and the anticancer drug estramustine (a combination of estrogen and mechlorethamine) [101] PGF1α, PGF2α, PGA1, and TXB2 are high-affinity inhibitors of MRP4/ABCC4 -mediated transport of PGE1 and PGE2 [75] Various NSAIDs including indomethacin, indoprofen, ketoprofen, and flurbiprofen

inhibited MRP4/ABCC4 mediated E217βG transport at physiologically relevant concentrations, whereas diclofenac, rofecoxib, and celecoxib were equally poor inhibitors [75] Recently, El-Sheikh et al reported that the inhibitory potency of the NSAIDs including salicylate, piroxicam, ibuprofen, naproxen, sulindac,

tolmetin, etodolac, diclofenac, indomethacin, ketoprofen, phenylbutazone and celecoxib was generally higher against MRP4/ABCC4- than MRP2/ABCC2-mediated MTX transport with therapeutically relevant concentrations [102] In addition, Ci et al found that most of the injectable cephalosporins are inhibitor of MRP4/ABCC4, except for cefepime, cefsulodin, and cephaloridine, while

aminocephalosporins have a weak inhibitory effect on MRP4/ABCC4 [77]

Although a variety of inhibitors of MRP4/ABCC4 has been identified, there are

no specific inhibitors of MRP4/ABCC4

MRP4/ABCC4 may be regulated at transcriptional, translational and posttranslational level Its expression is substantially increased in livers of mice with disruption of the farnesol X-activated receptor (FXR) which also known as bile acid receptor (BAR), which have increased levels of serum and hepatocellular bile acids, and MRP4/ABCC4 can be further upregulated by cholic acid feeding[103] In addition, the magnitude of MRP4/ABCC4 induction correlates with the amout of bile acid levels and is independent of FXR [104] The activation of the constitutive androstane receptor (CAR) is required to coordinately upregulate hepatic expression of MRP4/ABCC4 and Sult2a1, a cytosolic enzyme known to sulfate cholestatic bile acids and steroids Sult2a1 was down-regulated in

MRP4/ABCC4-null mice, further indicating an inter-relation between

MRP4/ABCC4 and Sult2a1 gene expression [105] In addition, MRP4/ABCC4 protein is up-regulated in liver but down-regulated in kidney in the rats with obstructive cholestasis However, no major alterations on MRP4/ABCC4 mRNA lever in liver or kidney in above rats, which indicating that the posttranscriptional mechanisms as predominant regulators of MRP4/ABCC4 expression in these rats [106] Futhermore, renal MRP4/ABCC4 expression is up-regulated in transport-deficient rats, which lack MRP2/ABCC2, suggesting that they may function together in the urinary excretion of some organic anions [107] Induction of

MRP4/ABCC4 and production of tumor necrosis factor alpha (TNF-α) were found correlated 24 h after human immunodeficiency virus infection in macrophages But no correlation with interleukin-6 (IL-6) production was observed [108]

However, Dreuw et al reported that IL-6 can up-regulate the expression of

MRP4/ABCC4 in normal human epidermal keratinocytes and dermal fibroblasts [109] In addition, AZT, one of the MRP4/ABCC4 substrates, increased the

expression of MRP4/ABCC4 in human immunodeficiency virus infected human monocyte-derived macrophages and lymphocytes, which decreased its

pharmacological activity [110] Moreover, several hepatotoxicants including acetaminophen, carbon tetrachloride and clofibrate (CFB) enhanced the

expression of MRP4/ABCC4 in mice [111, 112]

1.2 CYCLOPHOSPHAMIDE AND IFOSFAMIDE

1.2.1 Clinical activity and mechanism of action of cyclophosphamide and

CP is a widely used alkylating agent in the treatment for haematological malignancies and a variety of solid tumors, including leukemia [115], breast cancer [116], lung cancer [117], lymphomas [118], prostate cancer [119], ovarian cancer [120], and multiple myeloma [121] Although its role in the treatment for ovarian cancer and small-cell lung cancer is declining, CP continues to be used in the treatment of advanced breast cancer as a critical component of the CMF (CP,

methotrexate, and 5-fluorouracil), CEF (CP, epirubicin, and 5-fluorouracil), MVC (mitoxantrone, vinblastine, and CP) and TAC (docetaxel, doxorubicin and CP) regimen [122-125] Higher doses of CP are used in the treatment prior to bone marrow transplantation for aplastic anemia, leukemia and other malignancies for the mobilization of hematopoietic progenitor cellsfrom the bone marrow into peripheral blood [126] Moreover, CP has been used as an immunosuppressive drug to treat several autoimmune diseases including systemic lupus erythematosus [127] and rheumatoid arthritis [128]

In comparison with CP, IF is more effective in a wide range of malignant diseases IF has shown antitumor activity against a variety of tumors, including small-cell and non-small-cell lung carcinoma [129, 130], breast cancer [131], ovarian cancer [132], hepatoma [133], bladder cancer [134], cervical cancer [135], osteosarcoma [136], neuroblastoma [137], leukaemia [138], multiple myeloma [139], Ewing sarcoma [140], uterine sarcoma [141], and lymphomas [142] IF is used in combination with many other cytotoxic agents in clinical practice, such as etoposide, doxorubicin and mitomycin more often For example, VIM [IF,

etoposide (VP-16) and methotrexate] and MINE [mitoxantrone, mesna/IF and etoposide), IEV (IF, epirubicin and etoposide) regimens have been often used in the treatment of relapsed non-Hodgkin’s lymphoma and Hodgkin’s lymphoma, respectively [143-145] ICE (IF, carboplatin, and etoposide) is commonly used for the treatment of refractory small cell lung cancer [146], while MICE (mitomycin-

C, IF, cisplatin, and vindesine) is effective in non-small cell lund cancer[147] CP and IF are frequently used in combination with other anticancer agents in the management of cancer to obtain synergistic or additive anticancer effect resulting from complementary mechanisms of action

There is an increased understanding on the mechanisms of action of

oxazaphosphorines with regard to their anticancer and immuno-modulating effects The anticancer effects of oxazaphosphorines is generally considered to result from DNA crosslink formation through covalent bonding of highly reactive alkyl

groups of the alkylating nitrogen mustards of oxazaphosphorines with specific nucleophilic groups of DNA molecules CP and IF are prodrugs that are activated through 4-hydroxylation by hepatic cytochromeP450s (CYPs) such as CYP2B6, and CYP3A4 [148] The resultant ultimate bifunctional alkylating nitrogen

mustards are converted to chemically reactive carbonium ions at neutral pH and react with the 7-nitrogen atom of purine bases in DNA, especially when they are flanked by adjacent guanines The second arm in phosphoramide mustard can react with a second guanine moiety in an opposite DNA strand or in the same strand to form crosslinks [149] Additionally, the O6 atom of guanine is a target for reactive oxazaphosphorine mustards [150] The different intramolecular

distance between the chloroethyl groups in CP or IF results in a different range of cross-linked DNA Cross-links of DNA molecules were detected in cancer

patients within 3 hr following IF infusion, whereas DNA single strand break was not observed [151] In patients receiving continuous infusion of IF, a plateau of DNA cross-linking was achieved by 24 hr, while a marked decrease in the peak level of crosslinking was observed in the patients receiving IF infusion over 3 hr [151] In another study in 19 paediatric patients mostly with rhabdomyosarcoma

or Ewings sarcoma, the degree of DNA damage increased to a peak at 72 hr

following IF infusion, but returned to pretreatment values prior to the next dose of chemotherapy [152] There was a good correlation between the area of the plasma concentration-time curve (AUC) of IF and the cumulative percentage of cells with

DNA crosslinks, but only in those patients receiving fractionated dosing [152] The latter patients had more DNA damage than those patients in whom IF was administered by continuous infusion Following DNA crosslink formation, tumor cells will undergo apoptosis initiated by DNA damage and inhibition of DNA replication, modulation of cell cycle and other anti-proliferative effects

CP has modulating effects on both humoral and cell-mediated immunity and thus beneficial effects are obtained when used as an immunosuppressive drug

CP mediates the killing of circulating endothelial progenitors In addition, CP augmented the efficacy of antitumor immune responses in animals and humans by depleting CD4+/CD25+ regulatory T cells and increasing T lymphocyte

proliferation and T memory cells The immunostimulatory effect of CP is

associated with the marked inhibition of inducible nitric oxide synthase [153] These findings may provide a solid rationale for the use of CP as an

immunosuppressive agent in the treatment of autoimmune diseases or an integral component in combination with other immunotherapy in cancer treatment

1.2.2 Pharmacokinetics and pharmacodynamics of cyclophosphamide and

ifosfamide

When given orally, CP and IF are well absorbed with high oral

bioavailability (85−100%) A small fraction of the drug is metabolized in the liver and gut due to first-pass effect The peak concentration appears 1-2 hours

following oral drug administration CP is orally administered at low doses

(75−200 mg/day) when used as an immunosuppressive agent and in the treatment

of some malignancies IF is less commonly used as an oral agent, as oral

administration results in unacceptable neurotoxicity, probably due to increased formation of neurotoxic chloroacetaldehyde compared to intravenous (i.v.)

administration After oral or i.v administration, CP is rapidly distributed

throughout the body with ∼20% plasma protein binding, whereas the ability of protein binding is higher for its metabolite (<67%) [154] The distribution of IF is more extensive with lower plasma protein binding compared with CP Some studies in obese patients showed that a longer elimination half-life for IF due to the increased volume of distribution in obese patients [155] The majority of CP and IF elimination is by metabolic transformation in the liver; only 10-20%

remain unchanged in the urine and only 4% in the bile [156]

The initial activation of CP is 4-hydroxylation at C4 of oxazaphosphorine ring to form 4-hydroxy-CP (4-OH-CP) (shown in Figure 1-1) Multiple CYP enzymes including CYP2B6 and CYP3A4 in the liver are responsible for CP 4-hydroylation [156] 4-OH-CP is a major circulating metabolite of CP that enters tumor cells and decomposes through its tautomer aldophosphamide by

spontaneous β-elimination to form ultimate cytotoxic phosphoramide mustard and

an equimolaramount of the byproduct acrolein Alternatively, 4-OH-CP is

detoxified to 4-ketocyclophosphamide and carboxyphosphoramide by aldehyde dehydrogenases (ALDHs) An alternative metabolic pathway involves CP N-dechloroethylation to yield chloroacetaldehyde, which is also catalysed by

CYP3A4 [157]

N H P O N ClH2CH2C

ClH2CH2C

H P O N ClH2CH2C ClH2CH2C

O

OH

N H P O N ClH2CH2C ClH2CH2C

O

O

NH2P O N ClH2CH2C ClH2CH2C

O

O H

NH2P O N ClH2CH2C ClH2CH2C

O

O O H

NH2P OH N ClH2CH2C ClH2CH2C

O

O H

CYPs

N H P O N

degradation