Interplay of drug metabolizing enzymes with cellular transporters (original) (raw)
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Influence of Efflux Transporters on Drug Metabolism
Clinical Pharmacokinetics, 2011
Cytochrome P450 enzymes and efflux transporters, expressed in the intestine and or in the liver, play important roles in drug clearance and oral bioavailability. The relative contribution of transporters and enzymes in drug metabolism is still controversial. Some antiepileptic drugs, such as carbamazepine, phenytoin and phenobarbital (phenobarbitone), show time-dependent and dose-dependent pharmacokinetics due to their inductive effect on both efflux transporters and enzymes. However, steady-state plasma drug concentrations for each antiepileptic drug do not relate to oral daily dose in the same way, with decreased or increased apparent clearance according to the drug. A multicompartment pharmacokinetic model was developed in order to explain these different behaviours using a single mechanism of inductive action. The key for solving these apparent dissimilarities was to consider in the model the unique physiological connection that intestine, liver and bloodstream have. Efflux transporters not only enhance enzymatic competition in relation to first-order processes, but also change the predominance of some elimination routes. For instance, the carbamazepine-10,11-epoxide formation increases at the expense of other carbamazepine metabolites, enhancing both the systemic and presystemic elimination of parent drug. Conversely, the major hepatic metabolism of phenytoin diminishes in favour of its minor intestinal elimination, decreasing the total drug clearance.
Cytochrome P450 Inhibitory Properties of Common Efflux Transporter Inhibitors
Drug Metabolism and Disposition, 2014
Drug transporter inhibitors are important tools to elucidate the contribution of transporters to drug disposition both in vitro and in vivo. These inhibitors are often unselective and affect several transporters as well as drug metabolizing enzymes which can make experimental results difficult to interpret with confidence. We therefore tested fourteen commonly used P-glycoprotein (P-gp), Breast Cancer Resistance Protein (BCRP), and Multidrug-Resistance associated Protein (MRP) inhibitors as inhibitors of Cytochrome P450 (CYP) enzyme activities using recombinant enzymes. A subset of P-gp and/or CYP3A inhibitors were selected (cyclosporin A, elacridar, ketoconazole, quinidine, reserpine and tacrolimus) for a comparison of CYP-inhibition in human microsomes and hepatocytes. Most P-gp inhibitors showed CYP3A4 inhibition, with potencies often in a similar range as their P-gp inhibition, as well as less potent CYP2C19 inhibition. Other CYP enzymes were not strongly inhibited except a few cases of CYP2D6 inhibition. MRP and BCRP inhibitors showed limited CYP inhibition. Some inhibitors showed less CYP inhibition in human hepatocytes than human liver microsomes, for example elacridar, probably due to differences in binding, permeability limitations, or active, P-gp mediated efflux of the inhibitor from the hepatocytes. Quinidine was a potent CYP inhibitor in hepatocytes but only showed weak inhibition in microsomes. Quinidine shows an extensive cellular uptake, which may potentiate intracellular CYP inhibition. Elacridar is described as a potent and selective P-gp inhibitor and displayed modest CYP inhibition in this study, and is thus a useful model inhibitor to define the role of P-gp in drug disposition without interference with other processes.
Biotechnology Journal, 2008
21.1 INTRODUCTION Dietary effects on drug pharmacokinetics (PK) have been well documented. A number of mechanisms are responsible for food-drug interactions, including alterations in physiological conditions (e.g., gastric pH, gastric emptying, intestinal motility, and hepatic blood or bile flow rate), complexation of drugs with dietary components, and modulation of drug-metabolizing enzymes by dietary constituents [1, 2]. Over the past decade, numerous food-drug interaction studies have focused primarily on the effects of diet on drug-metabolizing enzymes. Alterations in activities of drug-metabolizing enzymes can subsequently change the PK of drugs that are substrates of these enzymes [2-4]. Foods that contain complex mixtures of phytochemicals, such as fruits, vegetables, herbs, and teas, can potentially modulate the activities of both phase I and phase II enzymes [2]. Many clinically significant metabolic food-drug interactions have been reported, as exemplified by the interactions between cytochrome P450 (CYP) enzymes and diet/dietary supplements, such as grapefruit juice (GFJ) and St. John's wort [2, 4, 5]. In recent years, great advances have been made in elucidating the roles of drug transporters in drug disposition [6-8]. It is now increas ingly recognized that drug transporters can also signi ficantly contribute to food-drug interactions. In this review, we focus on the interactions of diet/nutrients with drug trans porters arising from in vitro and in vivo studies, with specific emphasis on several major efflux transporters (e.g., P-glycoprotein (P-gp), Multidrug Resistance associated Protein (MRP) and Breast Cancer Resistance Protein (BCRP)) and uptake transporters (e.g., organic anion-transporting polypeptide (OATP) and Organic Anion Transporter (OAT)). 21.2 DIET/NUTRIENT INTERACTIONS WITH DRUG TRANSPORTERS 21.2.1 Interactions of Diet/Dietary Supplements with Drug Transporters Dietary supplements are products (other than tobacco) intended to supplement the diet. Many top-selling herbal products, such as St. John's wort, garlic, green tea, ginseng, and milk thistle, are classified as dietary supplements [9, 10]. Since their marketing does not require Food and Drug Administration (FDA) approval, the potential interactions of these herbal products with conventional drugs, in general, have not been carefully evaluated, raising a serious concern about the safety of using these products. Recent in vitro and in vivo studies have shown that many diet/dietary supplements can modulate the activities of both efflux and uptake transporters and alter the PK of various therapeutic agents, resulting in clinically important drug interactions (Table 21.1).
Unmasking the dynamic interplay between efflux transporters and metabolic enzymes
International journal of pharmaceutics, 2004
Drug efflux by intestinal P-glycoprotein (P-gp) is known to decrease the bioavailability of many CYP3A4 substrates. We have demonstrated that the interplay between P-gp and CYP3A4 at the apical intestinal membrane can increase the opportunity for drug metabolism by determining bidirectional extraction ratios across CYP3A4-transfected Caco-2 cells for two dual P-gp/CYP3A4 substrates, K77 (an experimental cysteine protease inhibitor) and sirolimus, as well as two negative control, CYP3A4 only substrates, midazolam and felodipine. Studies were carried out under control conditions, with a P-gp inhibitor (GG918) and with a dual inhibitor (cyclosporine). Measurement of intracellular concentration changes is an important component in calculating the extraction ratios. We hypothesize that the inverse orientation of P-gp and CYP3A4 in the liver will result in an opposite interactive effect in that organ. In vivo rat intestinal perfusion studies with K77 and rat liver perfusion studies with t...
Clinical Drug Investigation, 2001
Traditionally, drug-induced changes in cytochrome P450 isoenzyme activity, causing changes in drug metabolism and bioavailability, have been the main focus of drug interaction studies. Recent research, however, suggests that the drug transporters P-glycoprotein and organic anion transporting peptide (OATP), which can effect the efflux and influx of many classes of drugs, may contribute to drug interactions by mechanisms independent of oxidative metabolism. Experimental models designed to selectively probe the function of P-glycoprotein or OATP have demonstrated that changes in the activities of these transporters may have a significant effect on the bioavailability of clinically important drugs, leading to the potential for adverse drug interactions.
Transporters: Importance in Drug Absorption, Distribution, and Removal
Enzyme- and Transporter-Based Drug-Drug Interactions, 2009
There is an increasing appreciation of the role that transport proteins play in the absorption, distribution, and elimination of a wide variety of drugs in clinical use. These transporters can be divided into efflux transporters belonging to the ATP-binding cassette (ABC) family and solute carrier (SLC) family members that mediate the influx or bidirectional movement of drugs across the cell membrane. Their coordinated expression and activities at the basolateral and apical side of transporting epithelia are significant determinants of drug disposition, drug-drug interactions, and variability in drug response and toxicity. This chapter focuses on the major SLC and ABC drug transporters expressed in intestine, liver, and kidney, with special emphasis on their distribution, mode of action, and drug substrate specificity. 2.1 Introduction During the last 20 years, a large number of membrane transport proteins have been identified. These transporters are important determinants in the absorption, distribution, and elimination of drugs. The involvement of carrier-mediated processes in drug excretion was already appreciated long before the first transporters were cloned, and it has become increasingly apparent that transporters also play a critical role in drug absorption and tissue uptake. Drug transport proteins can be grouped into two major classes, the solute carriers (SLC) and ATP-binding cassette (ABC) transporters. Over 380 unique SLC sequences have been obtained from the human genome, which can be divided into 48 subfamilies (Fredriksson et al., 2008). The transport activities for xenobiotics for
Current Drug Metabolism, 2003
As discussed in earlier articles, predictions of in vivo drug-drug interactions from in vitro studies is a subject of high interest with obvious therapeutic as well as economic benefits. Up until now little attention has been given to the potential interplay between metabolic enzymes and transporters that could confound the in vivo-in vitro relationships. Drug efflux by intestinal Pglycoprotein (P-gp) is known to decrease the bioavailability of many CYP3A4 substrates. We have demonstrated that the interplay between P-gp and CYP3A4 at the apical intestinal membrane can increase the opportunity for drug metabolism by determining bidirectional extraction ratios across CYP3A4 transfected Caco-2 cells for two dual P-gp/CYP3A4 substrates, K77 (an experimental cysteine protease inhibitor) and sirolimus, as well as two negative control, CYP3A4 only substrates, midazolam and felodipine. Studies were carried out under control conditions, with a P-gp inhibitor (GG918) and with a dual inhibitor (cyclosporine). Measurement of intracellular concentration changes is an important component in calculating the extraction ratios. We hypothesize that the inverse orientation of P-gp and CYP3A4 in the liver will result in an opposite interactive effect in that organ. In vivo rat intestinal perfusion studies with K77 and rat liver perfusion studies with tacrolimus under control conditions and with inhibitors of CYP3A4 (troleandomycin), P-gp (GG918) and both CYP3A4/P-gp (cyclosporine) lend support to our hypotheses. These results serve as a template for predicting enzymetransporter (both absorptive and efflux) interactions in the intestine and the liver.
Molecular Pharmaceutics, 2012
The aim of this study was to characterize and utilize MDCK cell line expressing CYP3A4 and Pglycoprotein as an in vitro model for evaluating drug-herb and drug-drugs of abuse interactions. MDCK cell line simultaneously expressing P-gp and CYP3A4 (MMC) was developed and characterized by using expression and activity studies. Cellular transport study of 200 μM cortisol was performed to determine their combined activity. The study was carried across MDCK-WT, MDCK-MDR1 and MMC cell lines. Similar studies were also carried out in the presence of 50 μM naringin and 3 μM morphine. Samples were analyzed by HPLC for drug and its CYP3A4 metabolite. PCR, qPCR and western blot studies confirmed the enhanced expression of the proteins in the transfected cells. The vivid CYP3A4 assay and ketoconazole inhibition studies further confirmed the presence of active protein. Apical to basal transport of cortisol was found to be ten and three fold lower in MMC as compared to WT and MDCKMDR1 respectively. Higher amount of metabolite was formed in MMC than in MDCK-WT indicating enhanced expression of CYP3A4. Highest cortisol metabolite formation was observed in MMC cell line due to the combined metabolic activities of CYP3A4 and P-gp. Transport of cortisol increased fivefold in presence of naringin in MMC and doubled in MDCKMDR1. Cortisol transport in MMC was significantly lower than that in WT in presence of naringin. The permeability increased three fold in presence of morphine which is a weaker inhibitor of CYP3A4. Formation of 6β-hydroxy cortisol was found to decrease in presence of morphine and naringin. This new model cell line with its enhanced CYP3A4 and P-gp levels in addition to short culture time can serve as an invaluable model to study drug-drug interactions. This cell line can also be used to study the combined contribution of efflux transporter and metabolizing enzymes towards drug-drug interactions.
Pharmaceutical Excipients Influence the Function of Human Uptake Transporting Proteins
Molecular Pharmaceutics, 2012
Although pharmaceutical excipients are supposed to be pharmacologically inactive, solubilizing agents like Cremophor EL have been shown to interact with cytochrome P450 (CYP)-dependent drug metabolism as well as efflux transporters such as P-glycoprotein (ABCB1) and multidrug resistance associated protein 2 (ABCC2). However, knowledge about their influence on the function of uptake transporters important in drug disposition is very limited. In this study we investigated the in vitro influence of polyethylene glycol 400 (PEG), hydroxypropyl-β-cyclodextrin (HPCD), Solutol HS 15 (SOL), and Cremophor EL (CrEL) on the organic anion transporting polypeptides (OATP) 1A2, OATP2B1, OATP1B1, and OATP1B3 and the Na + / taurocholate cotransporting polypeptide (NTCP). In stably transfected human embryonic kidney cells we analyzed the competition of the excipients with the uptake of bromosulfophthalein in OATP1B1, OATP1B3, OATP2B1, and NTCP, estrone-3-sulfate (E 3 S) in OATP1A2, OATP1B1, and OATP2B1, estradiol-17β-glucuronide in OATP1B3, and taurocholate (TA) in OATP1A2 and NTCP cells. SOL and CrEL were the most potent inhibitors of all transporters with the strongest effect on OATP1A2, OATP1B3, and OATP2B1 (IC 50 < 0.01%). HPCD also strongly inhibited all transport proteins but only for substrates containing a sterane-backbone. Finally, PEG seems to be a selective and potent modulator of OATP1A2 with IC 50 values of 0.05% (TA) and 0.14% (E 3 S). In conclusion, frequently used solubilizing agents were shown to interact substantially with intestinal and hepatic uptake transporters which should be considered in drug development. However, the clinical relevance of these findings needs to be evaluated in further in vivo studies.
British Journal of Pharmacology, 2002
ABSTRACT P-glycoproteins (Pgps), encoded by the human MDR1 or rat Mdr1a/1b genes, are ATPdependent drug transporting proteins involved in the extrusion of toxic compounds from cells, whereas rat Mdr2-Pgp functions as a phosphatidylcholine translocase. The aim of this study was to evaluate the specificity of human MDR1-, and rat Mdr1b-and Mdr2-Pgps for relatively hydrophilic cationic drugs. ATP-dependent uptake of the monoquaternary diastereomers N-methylquinidine and N-methylquinine, and vincristine (as reference) was m e a s u r e d i n p l a s m a m e m b r a n e v e s i c l e s p r e p a r e d f r o m M D R 1 -, M d r 1 b -, o r M d r 2overexpressing insect cells. We observed ATP-dependent uptake of the compounds into MDR1-and Mdr1b-expressing vesicles. Mdr2 was not able to transport these substrates. MDR1-and Mdr1b-mediated transport was saturable, and could be inhibited by various compounds, including PSC-833. We noticed a remarkable difference between the transport of N-methylquinidine and N-methylquinine: for both MDR1 and Mdr1b the maximal initial uptake rates (V max ) of N-methylquinidine were greater than those determined for N-methylquinine, whereas the apparent K m constants differed only slightly. This stereoselective difference was also evident from differential inhibitory studies with the two isomers. Comparison of the normalized V max constants revealed that human MDR1 was more effective in transporting the tested substrates than rat Mdr1b. We conclude that: 1) MDR1 and Mdr1b, but not Mdr2, are able to transport permanentlycharged derivatives of quinidine and quinine, 2) both MDR1 and Mdr1b display stereospecificity in favor for the (R)-diastereomer N-methylquinidine as compared to the (S)-diastereomer N-methylquinine, and 3) human MDR1 is more efficient in transporting the cationic model compounds than its rat orthologue Mdr1b.