Modulation of drug transport pathways by spices

MODULATION OF DRUG TRANSPORT PATHWAYS BY SPICES HAN YI (B.Sc. Pharm, Shenyang Pharmaceutical University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2007 ACKNOWLEDGEMENTS I would like to express my sincerest appreciation to my supervisor, Associate Professor Lim Lee Yong, for her help, stimulating suggestions and encouragement in all the time of research and writing of this thesis. Sincere gratitude is also expressed to Dr Tan May Chin, Theresa, A/P Go Mei Lin, for their precious guidance and help. I am also grateful to A/P Chan Sui Yung (Head of the Pharmacy Department), A/P Ho Chi Lui, Paul, A/P Li Shu Chuen, and Dr Koh Hwee Ling for their help and support. Special thanks are also extended to Ms Ng Sek Eng and Ms Wong Lai Peng for their technical assistance and my friends, especially Siok Lam, Mo Yun, Huang Min, Yupeng, Wenxia, Chunxia, Dahai and Weiqiang for their continuous support and encouragement. Especially, I would like to give my special thanks to my husband Xiaoqiang and my parents, whose patient love enabled me to complete my PhD thesis. I TABLE OF CONTENTS Content Pages ACKNOWLEDGEMENTS TABLE OF CONTENTS SUMMARY I II VII LIST OF FIGURES XI LIST OF TABLES XII LIST OF PUBLICATIONS XX Chapter Introduction 1.1 Oral drug bioavailability 1.2 Oral drug absorption 1.2.1 Passive diffusion 1.2.2 Carrier-mediated transport 1.3 Intestinal drug transporter systems 10 1.3.1 Introduction 10 1.3.2 The P-glycoprotein (P-gp) 14 1.3.2.1 Structure and mechanistic model 15 1.3.2.2 P-gp substrates 16 1.3.2.3 P-gp inhibitors 18 1.3.2.4 Role of P-gp in drug interactions 20 1.3.2.4.1 Drug absorption 20 1.3.2.4.2 Drug distribution 23 1.3.2.4.3 Drug disposition 24 1.4 Spice-drug interactions 25 1.4.1 Pepper 26 1.4.2 Capsicum 29 1.4.3 Garlic 31 II 1.4.4 Ginger 34 1.5 Statement of purpose 36 Chapter Screening of spices: cytotoxicity and effects on paracellular and P-gp-mediated transport pathways 39 2.1 Introduction 40 2.2 Materials 45 2.3 Methods 46 2.3.1 Spices 46 2.3.2 Dosing solutions 47 2.3.3 Cell culture 49 2.3.4 MTT assay 49 2.3.5 [14C]-mannitol transport across Caco-2 cell monolayers 50 2.3.6 [3H]-digoxin transport across LLC-PK1 and L-MDR1 cell monolayers 2.3.7 Statistical analyses 52 53 2.4 Results and discussion 53 2.4.1 MTT assay 53 2.4.2 [14C]-mannitol transport across Caco-2 cell monolayers 60 2.4.3 [3H]-digoxin transport across LLC-PK1 and L-MDR1 cell monolayers 65 2.5 Conclusions 67 Chapter Effect of piperine and capsaicin on cell proliferation 71 3.1 Introduction 72 3.2 Materials 75 3.3 Methods 75 3.3.1 MTT assay 75 3.3.2 Flow cytometric analysis of cell cycle 76 3.3.3 Statistical analysis 77 3.4 Results 77 III 3.4.1 Anti-proliferation activity 77 3.4.2 Cell cycle analysis 79 3.5 Discussion 82 3.6 Conclusions 84 Chapter Paracellular transport pathway of Caco-2 cell monolayers upon prolonged exposure to piperine and capsaicin 85 4.1 Introduction 86 4.2 Materials 90 4.3 Methods 91 4.3.1 Cell culture 91 4.3.2 Transepithelial electrical resistance and [14C]-mannitol transport 92 4.3.3 Preparation of detergent-soluble membrane and detergent–insoluble cytoskeleton fractions 92 4.3.4 Western blot 95 4.3.5 Immunofluorescence staining 93 4.3.6 Statistical analyses 94 4.4 Results 94 4.4.1 Transepithelial electrical resistance and [14C]-mannitol transport 94 4.4.2 Western blot 99 4.4.3 Immunofluorescence staining 105 4.5 Discussion 107 4.6 Conclusions 111 Chapter In vitro modulation of P-gp expression and function: a comparison of piperine and capsaicin 113 5.1 Introduction 114 5.2 Materials 119 5.3 Methods 119 5.3.1 [3H]-Digoxin transport studies 119 5.3.2 P-gp and MDR1 expression 121 IV 5.3.3 Western blot analysis of P-gp protein 121 5.3.4 Real time RT-PCR analysis of MDR1 mRNA 122 5.3.5 Reversibility study 123 5.3.6 In silico structure analysis of piperine and capsaicin 123 5.3.7 Statistical analyses 123 5.4 Results 124 5.4.1 [3H]-Digoxin transport studies 124 5.4.2 P-gp and MDR1 expression 125 5.4.3 Structural analysis of piperine and capsaicin 131 5.5 Discussion 132 5.6 Conclusions 136 Chapter In vivo modulation of P-gp expression: a comparison of piperine and capsaicin 138 6.1 Introduction 139 6.2 Materials 141 6.3 Methods 141 6.3.1 Animals 141 6.3.2 Spice administration and rat tissue collection 142 6.3.3 Histological evaluation 143 6.3.4 Western blot 143 6.3.5 RT-PCR 144 6.3.6 Statistical analyses 145 6.4 Results 145 6.4.1 Histological changes 145 6.4.2 Western blot analysis 147 6.4.3 RT-PCR analysis of rat mdr1a expression 154 6.5 Discussion 157 6.6 Conclusion 160 Chapter Final conclusions 161 V Chapter Proposed future studies 169 Chapter 10 Bibliography 172 VI SUMMARY Although much is known about food-drug interaction and its impact on the clinical outcome of a therapeutic drug, spice-drug interactions remain relatively unexplored despite the widespread use of spices on a daily basis by different people groups. The hypothesis for this project was that spices consumed as food supplements could affect oral drug bioavailability by interfering with intestinal drug transport. To prove our hypothesis, a series of spices commonly encountered in the South East Asian diet, i.e. piperine (active component of pepper), capsaicin (active component of chilli), the garlic derivatives, diallylsulfide (DAS) and diallyldisulfide (DADS), garlic aqueous extract and ginger ethanol extract, spices, were screened for their potential to modulate oral drug bioavailability. MTT assay results showed that the garlic aqueous extract at concentrations higher than mg/ml and the ginger ethanolic extract at ≥ 60 mg/ml were cytotoxic against the Caco-2, LLC-PK1 and L-MDR1 cells. Capsaicin at ≥ 500 µM was also cytotoxic towards the Caco-2 cells. In contrast, DAS (up to 300 µM), DADS (up to 300 µM) and piperine (up to 500 µM) did not affect the viability of the cell lines. Bi-directional [3H]-digoxin transport data across the polarized L-MDR1 cell monolayers indicated that the P-gp function was inhibited by sub-cytotoxic concentrations of piperine and capsaicin, with both spices capable of producing comparable inhibitory activity as the established P-gp inhibitor, verapamil. Both spices also modulated the paracellular transport of [14C]-mannitol across the Caco-2 cell monolayers. Although the ginger extract showed potential inhibitory action on the P-gp, its activity was significantly weaker. Garlic extract and its constituents, DAS and DADS, VII also modified digoxin transport, but not in a manner consistent with a P-gp inhibitor. On the basis of these results, piperine and capsaicin were selected for further experimentation. To delineate the action of the spices on the P-gp transporter, the cytotoxicity of piperine and capsaicin, as well as their effects on the paracellular transport pathways, were evaluated upon prolonged cellular exposure. MTT assay showed that the proliferation of Caco-2, LLC-PK1 and L-MDR1 cells was inhibited by 72h of co-incubation with piperine and capsaicin at 50 and 100 µM, concentrations which were non-cytotoxic upon short-term contact with the cells. The two spices exhibited greater anti-cell proliferation activity at higher concentrations and longer co-incubation times. Cell cycle analysis showed the accumulation of cells at the G1/G0 phase but, compared to established cell cycle modulators, piperine and capsaicin could only be regarded to have modest effects on the cell cycle progression of the epithelial cell lines evaluated. Concurrent exposure to piperine or capsaicin enhanced the paracellular transport of [14C]mannitol across the polarized Caco-2 cell monolayers. AB and BA mannitol Papp values were increased by 2.2 and 1.4 fold, respectively, by 100 µM of piperine, while capsaicin at the same concentration caused a 1.5-fold increase in both AB and BA mannitol Papp values. These effects were intensified by co-culturing the cell monolayers with either spice for 18 days prior to the transport experiments. Interestingly, the spices had to be present during the transport experiments, as spice removal from pre-exposed cell monolayers just prior to the transport experiments reduced the mannitol Papp values to levels comparable with those for unexposed cells. The mechanism underlying the spice VIII action had not been determined with certainty, but the tight junction proteins, ZO-1, occludin and F-actin, were unlikely to play major roles. Immunostaining of the Caco-2 cells revealed marginal changes in F-actin distribution by piperine and capsaicin, and in ZO-1 distribution by piperine. However, western blot analysis of Caco-2 cell monolayers cultured on TranswellTM inserts showed no change in ZO-1, occludin and actin expressions. Another study conducted on Caco-2 cell monolayers showed piperine and capsaicin to have very similar effects on P-gp expression and activity in vitro. This study was novel, because the biological activities of piperine and capsaicin have rarely been compared in parallel experiments despite their remarkably similar chemical structures. Exposure of the Caco-2 cells to piperine or capsaicin at 50 or 100 µM for h resulted in lower P-gp efflux activity, whereas a prolonged exposure of 48 or 72 h to either spice led to an upregulation of cellular P-gp expression accompanied by enhanced P-gp efflux activity. Western blot results indicated that capsaicin was a weaker P-gp protein inducer, although the resultant changes in bi-directional [3H]-digoxin transport profile produced by equivalent doses of capsaicin and piperine were comparable. Real-time PCR analysis demonstrated a concomitant up-regulation of the MDR1 mRNA level in the cells, suggesting a transcriptional mechanism by both spices. 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Mol Cell Biol 21:4188-4196. 225 [...]... efflux transport processes are saturable, and can be inhibited by structurally similar and diverse solutes 1.3 Intestinal drug transporter systems 1.3.1 Introduction A wide variety of transporters are located in the brush border and basolateral membranes of the intestine Some of these transporters facilitate drug absorption whereas others work as efflux pumps to hinder drug absorption Each transporter... transporters refer to the ATP-binding cassette family of transporter proteins often associated with tumor cells that exhibit multi -drug resistance (MDR) MDR is mediated by the transporters actively maintaining the intracellular drug level below the cell-killing threshold by expelling drug molecules out of the cells, the extrusion process being driven by the energy of ATP hydrolysis (Ambudkar et al., 1992) Notwithstanding... affect oral drug bioavailability will be elaborated The mechanisms of drug absorption and the membrane proteins involved in intestinal drug transport will be discussed In addition, brief descriptions of the spices of interest, as well as published data on their biological activity, are provided to rationalize our study 3 Chapter 1 Introduction 1.2 Oral drug absorption The human GI tract consists of the... that are transported by passive diffusion across the intestinal epithelia will traverse the cellular membranes The process of transcellular passive diffusion is believed to include the partitioning of the permeant between the contents of the GI lumen and the lipid bilayer of the enterocyte Rate of transport is defined by the Fick’s Law (Benet, 1973): V = PA·ΔC = (DK/h) A·ΔC where V is the transport. .. bioavailability of an orally administered drug is the fraction of the drug dose that reaches the general circulation unchanged Oral drug bioavailability is influenced by a variety of factors that lead to intra- and interindividual variability in drug response The influencing factors can be broken down into components that affect drug delivery to the intestine (gastric emptying rate, pH), drug absorption... for the transport of the small peptides PEPT-1 (Fei et al., 1994) is perhaps the best known of the intestinal peptide transporters PEPT-1 has been targeted as a gateway for enhancing oral drug delivery (Foltz et al., 2004) because of its capacity to also mediate the transport of peptide-like drugs, such as the β-lactam antibiotics (Okamura et al., 2003) 10 Chapter 1 Introduction Organic cationic transporters... however, demonstrated the involvement of transporters in the intestinal absorption of weak organic acids Such transporters include members of the organic anion transporter (OAT) family, organic anion transporting polypeptide (OATP) family and monocarboxylic acid 11 Chapter 1 Introduction transporter (MCT) family The MCT has been found to mediate the transport of various short-chain fatty acids (Halestrap... absorption pathway of drug) , drug metabolism (enzymes in intestinal lumen and enterocytes, first pass hepatic extraction, enterohepatic circulation), and active extrusion of drug from enterocytes into the lumen (drug efflux pumps) Among these factors, the effective passage of drug across the intestinal epithelial, and the intestinal phase І metabolism and active extrusion of absorbed drug have been recognized... as co-transporters, these simultaneously transport two substances by using the concentration gradient of one solute to transport the other solute against its concentration gradient) (Dai et al., 1996) or antiporters (also known as counter-transporters, these simultaneously transport two substances across the membrane in opposite directions) (Burdo et al., 2006) Active transport is characterized by high... passive, paracellular; (5) absorption limited by P-gp and/or other efflux transporters; (6) intestinal first-pass metabolism followed by absorption of parent and metabolite; and (7) receptor-mediated transport (Balimane et al., 2006) Apart from being transported into cells, a variety of substrates can be actively transported out of cells by membrane protein carriers (Funakoshi et al., 2003) This process, . MODULATION OF DRUG TRANSPORT PATHWAYS BY SPICES HAN YI (B.Sc. Pharm, Shenyang Pharmaceutical University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF. food -drug interaction and its impact on the clinical outcome of a therapeutic drug, spice -drug interactions remain relatively unexplored despite the widespread use of spices on a daily basis by. project was that spices consumed as food supplements could affect oral drug bioavailability by interfering with intestinal drug transport. To prove our hypothesis, a series of spices commonly