Showing papers by "Richard B. Kim published in 2004"

Polymorphisms in human MDR1 (P-glycoprotein): recent advances and clinical relevance.

Abstract: Drug transporters are increasingly recognized to be important to drug disposition and response. P-glycoprotein, the encoded product of the human MDR1 (ABCB1) gene, is of particular clinical relevance in that this transporter has broad substrate specificity, including a variety of structurally divergent drugs in clinical use today. Moreover, expression of this efflux transporter in certain tissue compartments such as the gastrointestinal tract and brain capillary endothelial cells limits oral absorption and central nervous system entry of many drugs. Recently, a number of single-nucleotide polymorphisms (SNPs) in MDR1 have been identified. An increasing number of studies have also implicated certain commonly occurring SNPs in MDR1 in problems including altered drug levels and host susceptibility to diseases such as Parkinson's disease, inflammatory bowel disease, refractory seizures, and CD4 cell recovery during human immunodeficiency virus therapy. However, in many such cases, the reported effects of MDR1 SNPs have been inconsistent and, in some cases, conflicting. In this review SNPs in MDR1 in relation to population frequencies, drug levels, and phenotypes are outlined. In addition, issues relating to MDR1 haplotypes, environmental factors, and study design, as potential confounding factors of the observed MDR1 polymorphism effect in vivo, are also discussed.

Pharmacogenetics of efavirenz and central nervous system side effects: an Adult AIDS Clinical Trials Group study.

Abstract: Objectives: Efavirenz is an effective antiretroviral agent, but central nervous system side effects occur commonly, and population (racial) differences in pharmacokinetics and response have been reported. Efavirenz is metabolized by cytochrome P4502B6 (CYP2B6). We investigated whether polymorphisms in CYP2B6, CYP3A4, CYP3A5, and MDR1 were associated with efavirenz central nervous system side effects and pharmacokinetics. Design: Twenty-four week cohort from a randomized study. Methods: Adult AIDS Clinical Trials Group study A5097s examined relationships between central nervous system side effects and efavirenz plasma concentration-time profiles in HIV-infected subjects. Efavirenz plasma pharmacokinetics were estimated by a population-based method. Central nervous system symptoms were assessed by questionnaires and neuropsychological testing. Results: Study subjects included 89 (57%) European-Americans, 50 (32%) African-Americans, and 15 (10%) Hispanics. The CYP2B6 T/T genotype at position 516 (Gln 172 His) was more common in African-Americans (20%) than in European-Americans (3%), and was associated with greater efavirenz plasma exposure (P < 0.0001). The median efavirenz [area-under-the-curve] (0-24 h) according to G/G, G/T, and T/T genotype was 44 (n=78), 60 (n=60), and 130 (n=14) μg.h/ml, respectively (P < 0.0001). The CYP2B6 G516T genotype was also associated with central nervous system symptoms at week 1 (P=0.036). Analysis of DNA from other subjects confirmed population differences in frequency of the G516T variant. No associations were apparent with the other polymorphisms studied. Conclusions: A CYP2B6 allelic variant that is more common in African-Americans than in Europeans-Americans was associated with significantly greater efavirenz plasma exposure during HIV therapy. Inter-individual differences in metabolism may, in part, explain susceptibility to efavirenz central nervous system side effects.

Genetic variability in CYP3A5 and its possible consequences.

Abstract: The cytochrome P450 3A (CYP3A) subfamily members are the most abundant and important drug-metabolizing enzymes in humans, and wide interindividual variability in CYP3A expression and function is present. CYP3A4 alone cannot fully explain the observed constitutive variability because its genetic variants are relatively uncommon and have limited functional significance, whereas CYP3A5 expression in humans is highly variable and may be contributory. However, it is difficult to delineate the relative contribution of CYP3A4 and CYP3A5, and to differentiate their effects on drug metabolism as their protein structure, function and substrates are so similar. By contrast, molecular biology methods provide the ability to identify CYP3A4 and CYP3A5 genotypes with certainty. This review collates currently available data on CYP3A5 polymorphisms, provides information on the population frequency of each genetic variant in major ethnic groups, and describes in vitro and in vivo studies that have attempted to identify genotype-phenotype associations.

Transporters and Renal Drug Elimination

Abstract: Carrier-mediated processes, often referred to as transporters, play key roles in the reabsorption and secretion of many endogenous and xenobiotic compounds by the kidney. The renal proximal tubule is the primary site of active transport for a wide variety of substrates, including organic anions/cations, peptides, and nucleosides. During the past decade, significant advances in molecular identification and characterization of transporter proteins have been made. Although it is generally noted that these transporters significantly contribute to renal drug handling and variability in drug disposition, the extent of our knowledge regarding the specific roles of such transporters in drug disposition and drug-drug interactions remains, for the most part, limited. In this review, we summarize recent progress in terms of molecular and functional characterization of renal transporters and their clinical relevance to drug therapy.

P-glycoprotein Expression, Localization, and Function in Sandwich-Cultured Primary Rat and Human Hepatocytes: Relevance to the Hepatobiliary Disposition of a Model Opioid Peptide

Abstract: Purpose. The isolation of hepatocytes from intact liver involves collagenase digestion of the tissue, resulting in loss of cell polarization and functional vectorial excretion. These studies examined re-polarization, localization of P-glycoprotein (P-gp) to the canalicular domain of the hepatocyte, and re-establishment of vectorial transport in sandwich-cultured (SC) rat and human primary hepatocytes.

Pharmacogenomics of the OATP and OAT families.

Abstract: Drug disposition is highly dependent on the interplay between drug metabolism and transport in organs such as the intestine, kidney, and liver. Genetically determined variation in drug transporter function or expression is now increasingly recognized to have a significant role as a determinant of intersubject variability in drug response. Similar to the discoveries of functional genetic variations in drug efflux transporters, such as multi-drug resistance proteins 1 and 2, there have been considerable advances in the identification of single nucleotide polymorphisms in transporters that facilitate cellular drug uptake. Among the uptake transporters, members of the organic anion-transporting polypeptides and organic anion transporters can mediate the cellular uptake of a large number of structurally divergent compounds. Accordingly, functionally relevant polymorphisms in these transporters may contribute to interindividual and interethnic variability in drug disposition and response. In this review, recent progress relating to pharmacogenomics of organic anion transporters will be outlined along with a compilation of currently known genetic polymorphisms.

Identification of Amino Acids in Rat Pregnane X Receptor that Determine Species-Specific Activation

Abstract: The pregnane X receptor (PXR) is a nuclear receptor significantly involved in the transcriptional regulation of drug-metabolizing enzymes and transporters. Interestingly, certain PXR ligands such as rifampin have been shown to readily induce human and rabbit but not rodent members of the cytochrome P450 3A. Because drugs of divergent chemical structures seem to be similarly affected, we hypothesized that specific amino acid residue(s) or domains in rat PXR affect receptor activation by certain human PXR ligands. To identify such a domain(s), an array of human-rat and rat-human chimeric PXR cDNAs in a tandem head-to-tail configuration were created using a random chimeragenesis method. Pharmacological characterization of these chimeras revealed a discreet segment within the ligand-binding domain of rat and human PXR to be essential for the rifampin effect. Within this region, the corresponding residues Leu308 and Phe305 of human and rat PXR, respectively, were found to be important for rifampin activation. Homology modeling derived from the recently determined crystal structure of human PXR indicates that these amino acids are located within or neighboring the flexible loop that forms part of the pore to the ligand-binding cavity. Rifampin, paclitaxel, and hyperforin sensitivity was conferred to rat PXR when Phe305 was converted to leucine, whereas attenuation of sensitivity was observed when Leu308 of human PXR was replaced with phenylalanine. Accordingly, our data provide compelling new insight into the importance of the amino acids comprising the pore to the ligand-binding cavity as a critical modulator of PXR response.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) and genetic variability (single nucleotide polymorphisms) in a hepatic drug uptake transporter: what's it all about?

Abstract: t o t c g S t o O a Historically, metabolism had been viewed as a major eterminant of drug disposition. In recent years, howver, clear evidence has emerged to suggest that memrane transporters are also critically important. Indeed, he role of efflux transporters such as the product of the DR1 gene, P-glycoprotein, in disposition and drug ffect has been clearly established and widely studied. oreover, single nucleotide polymorphisms (SNPs) in DR1 have also been a focus of numerous studies and ay play a role in the disposition of many drugs in linical use, including human immunodeficiency virus rotease inhibitors, digoxin, fexofenadine, and cycloporine (INN, ciclosporin). However, the directional ovement of drugs across organs such as the gastroinestinal tract, liver, and kidney requires the presence nd coordinate activity of drug uptake, as well as efflux ransporters. For example, uptake transporters exressed on the basolateral domain of hepatocytes faciltate the cellular accumulation of drug substrates, beore metabolism and efflux by transporter-mediated xcretion into bile (Fig 1). Among the uptake transporters, members of the oranic anion transporting polypeptides (OATPs) have

α1A-Adrenergic receptor polymorphism and vascular response

Abstract: Objective The α1A-adrenergic receptor is highly expressed in human vasculature including resistance arteries and veins, and its stimulation is primarily responsible for adrenergically mediated smooth muscle contraction. Variability in sensitivity to phenylephrine, an α1A adrenergic agonist, has a large genetic component. We examined the hypothesis that a common polymorphism of α1A-adrenergic receptor (Arg347Cys) affects in vivo response. Methods We measured vascular sensitivity to phenylephrine using the dorsal hand vein linear variable differential transformer technique and determined α1A-adrenergic receptor genotype in 74 healthy, nonsmoking adults (28 Arg/Arg, 30 Arg/Cys, and 16 Cys/Cys). Results Sensitivity to phenylephrine, expressed as the dose of phenylephrine resulting in 50% venoconstriction (Phe50), was not significantly different in subjects with the 3 α1A adrenergic receptor genotypes: Phe50 geometric mean (95% confidence interval) was 513 ng/min (287–9–8 ng/min) for Arg/Arg, 431 ng/min (274–680 ng/min) for Arg/Cys, and 471 ng/min (197–1124 ng/min) for Cys/Cys (P = .90). Conclusion We conclude that the Arg347Cys receptor polymorphism does not alter agonist-mediated venoconstriction in vivo. Clinical Pharmacology & Therapeutics (2004) 75, 539–545; doi: 10.1016/j.clpt.2004.02.006

Evaluation of herbal products as potential inhibitors of MDR1

Abstract: Herb-drug interactions are increasingly noted to be a potentially preventable cause of drug toxicity or loss of therapeutic efficacy. In addition to their effects on CYP enzymes, phytochemicals in St. John's wort and grapefruit juice are reported to interact with the drug efflux transporter, P-glycoprotein (P-gp)/MDR1. However, the potential effects of other popular, phytochemical rich herbs on MDR1 function have not been established. Ethanolic extracts of several herbal products were prepared. Bidirectional digoxin transport was determined across polarized Caco-2 cells containing P-gp. Herbal extracts were added to apical and basal compartments. Inhibitory effects of the herbal extracts were compared to verapamil 20 uM (positive control) and ethanol 2.5% (negative control). Viability of Caco-2 monolayer was confirmed by measuring inulin leak. Black Cohosh and St. John's wort were the most potent inhibitors of P-gp and completely abolished digoxin transport. The effect at 1.25 mg/ml was comparable to that of verapamil 20 uM. Echinacea, Feverfew, and Valerian inhibited digoxin transport by 70–80%. Ginseng and Perilla at similar concentrations did not affect digoxin transport. Kava and Garlic extracts appeared to compromise the integrity of the Caco-2 monolayer. Additional studies of Black Cohosh, Echinacea, Feverfew and Valerian are needed to determine the potential clinical relevance of these findings. Clinical Pharmacology & Therapeutics (2004) 75, P79–P79; doi: 10.1016/j.clpt.2003.11.301

P-glycoprotein Expression, Localization, and Function in Sandwich-Cultured Primary Rat and Human Hepatocytes: Relevance to the

Abstract: Purpose The isolation of hepatocytes from intact liver involves collagenase digestion of the tissue, resulting in loss of cell polarization and functional vectorial excretion These studies examined repolarization, localization of P-glycoprotein (P-gp) to the canalicular domain of the hepatocyte, and re-establishment of vectorial transport in sandwich-cultured (SC) rat and human primary hepatocytes Methods Protein localization and expression were determined in SC hepatocytes by confocal microscopy and Western blotting, respectively T ransporter f unction w as e valuated b y m easuring [D-penicillamine 2,5 ]enkephalin ( 3 H-DPDPE) and 5 (and 6)-carboxy2,7-dichlorofluorescein (CDF) biliary excretion in SC hepatocytes Results P-gp and the canalicular marker protein dipeptidyl peptidase IV (DPPIV) co-localized by Day 3 and Day 6 in SC rat hepatocytes and SC human hepatocytes, respectively, consistent with canalicular network formation visualized by light microscopy Co-localization of multidrug resistance associated protein 2 (MRP2) and P-gp in SC human hepatocytes was observed on Day 6 in culture Expression levels of P-gp increased slightly in both species over days in culture; similar expression was observed for MRP2 in SC human hepatocytes Oatp1a1 expression in SC rat hepatocytes was maintained over days in culture, whereas Oatp1a4 expression decreased OATP1B1 expression decreased slightly on Day 3 in SC human hepatocytes OATP1B3 expression was constant in SC human hepatocytes In vitro biliary excretion of the opioid peptide 3 H-DPDPE correlated with the proper localization of canalicular proteins in both species Excretion of CDF in SC human hepatocytes confirmed network formation and MRP2 function Conclusions These studies indicate that SC hepatocytes repolarize and traffic functional canalicular transport proteins to the appropriate cellular domain

Identification of nonsynonomous polymorphisms of human organic anion transporting polypeptide‐A (OATP‐A) associated with altered transport activity

Abstract: Organic anion transporting polypeptides (OATP) are a growing family of uptake transporters important to drug disposition. Studies from this laboratory have shown that OATP-A (SLC21A3) is selectively expressed in human intestine as well as kidney and brain. Accordingly, variability in the expression or function of this polyspecific drug uptake transporter may have important implications to bioavailability and tissue penetration of substrate drugs. In order to determine the extent of genetic heterogeneity in OATP-A, PCR-based genotyping analyses were carried out on ethnically defined genomic DNA samples (n=96 each for African-, Chinese-, European-, and Hispanic-Americans). We identified three novel nonsynonymous polymorphisms within the coding region of OATP-A; T38C (Ile38Thr), A516C (Glu172Asp) and G559A (Ala187Thr). Genotypic frequencies of these polymorphisms were dependent on race. In vitro functional assessment revealed that the A516C (Glu172Asp) variant had markedly reduced capacity for mediating the cellular uptake of OATP-A substrates, estrone sulfate and fexofenadine. Cell surface biotinylation experiments did not show altered plasma membrane expression of the transporter, suggesting the reduced transport activity associated with the A516C variant is likely due to altered intrinsic activity of the transporter. Our data suggest that OATP-A polymorphisms may contribute to inter-individual variability in intestinal absorption and CNS penetration of substrate drugs. Clinical Pharmacology & Therapeutics (2004) 75, P93–P93; doi: 10.1016/j.clpt.2003.11.355

Identification of single nucleotide polymorphisms in the multidrug resistance‐associated protein 4 (MRP4)

Abstract: The multidrug resistance-associated protein 4 (MRP4, ABCC4) is an ATP-binding cassette efflux transporter involved in the transport of nucleoside analogues. Genetic heterogeneity in MRP4 could influence the expression or intrinsic activity of this transporter and consequently alter the disposition profile or the efficacy of certain antiviral agents. In order to identify single nucleotide polymorphisms (SNPs), PCR was performed on genomic DNA samples from 96 Caucasians and analyzed by direct sequencing. In order to maximize the likelihood of identifying SNPs most likely to result in loss of transporter function, six exonic regions in MRP4 encompassing the key membrane spanning domain (MSD) or the nucleotide-binding domains (NBD) were assessed. (See Table) Three non-synonymous SNPs were identified. Two of these SNPs, G912T and G2269A, predict major amino acid changes within MSD1 and MSD2, respectively. No SNPs were identified in exons 12 and 19. The functional relevance of MRP4 variants is currently being evaluated using OAT-1/MRP4 double transfected HeLa cells. Clinical Pharmacology & Therapeutics (2004) 75, P93–P93; doi: 10.1016/j.clpt.2003.11.354 Table 1. MRP4 variant Amino acid change xon Domain Mutant allele frequency (%) * novel SNPs G912T K304N MSD1 9.4 G951A synonymous MSD1 37 G969A synonymous MSD1 35.9 G1035A* synonymous 8 MSD1 1 G1141A* synonymous 8 MSD1 0.5 A2230G* M7744V 3 MSD2 0.5 G2269A E757K 8 MSD2 1.6 G2844T synonymous NBD2 5.9 G3609A* synonymous 1.6

Modulation of adopted nuclear receptors, pregnane X receptor (PXR) and constitutive androstane receptor (CAR), by commonly prescribed chemotherapy agents

Abstract: The adopted nuclear receptors (NRs), CAR and PXR are key regulators of genes involved in drug disposition and may contribute to variable drug response and toxicity. Currently, little is known regarding the contribution of the NRs to chemotherapy-associated drug interactions or toxicities. We utilized a cell-based reporter assay to identify potential novel interactions between PXR or CAR and commonly prescribed chemotherapy drugs. In single agent studies, CAR-dependent activation of the CYP3A4 promoter was enhanced by vinblastine (EC50=28.6 nM) and CPT-11 (EC50=16.9 μM). Conversely, camptothecin (CPT) appeared to inhibit CAR activation (IC50=0.34 μM). CPT and cisplatin did not exert any effects as single agents; however both agents appeared to attenuate rifampin-mediated PXR activation in a dose-dependent manner, suggesting those agents may be antagonists of PXR. Interestingly, rifampin-mediated PXR activation was further enhanced by the co-incubation with etoposide or CPT-11. In primary human hepatocytes, rifampin-mediated CYP3A induction was similarly attenuated by the co-incubation with CPT and cisplatin, suggesting that NR-based reporter assays may be a useful tool for predicting conditions in vivo. Overall, our studies demonstrate that NRs interact with a number of commonly prescribed chemotherapy agents and that certain combination regimens may modulate NRs in an unexpected manner and contribute to the observed variability in efficacy and toxicity. Clinical Pharmacology & Therapeutics (2004) 75, P51–P51; doi: 10.1016/j.clpt.2003.11.194