Richard B. Kim

Membrane transporters in drug development

Membrane transporters can be major determinants of the pharmacokinetic, safety and efficacy profiles of drugs. This presents several key questions for drug development, including which transporters are clinically important in drug absorption and disposition, and which in vitro methods are suitable for studying drug interactions with these transporters. In addition, what criteria should trigger follow-up clinical studies, and which clinical studies should be conducted if needed. In this article, we provide the recommendations of the International Transporter Consortium on these issues, and present decision trees that are intended to help guide clinical studies on the currently recognized most important drug transporter interactions. The recommendations are generally intended to support clinical development and filing of a new drug application. Overall, it is advised that the timing of transporter investigations should be driven by efficacy, safety and clinical trial enrolment questions (for example, exclusion and inclusion criteria), as well as a need for further understanding of the absorption, distribution, metabolism and excretion properties of the drug molecule, and information required for drug labelling.

The drug transporter P-glycoprotein limits oral absorption and brain entry of HIV-1 protease inhibitors.

Currently available HIV-1 protease inhibitors are potent agents in the therapy of HIV-1 infection. However, limited oral absorption and variable tissue distribution, both of which are largely unexplained, complicate their use. We tested the hypothesis that P-glycoprotein is an important transporter for these agents. We studied the vectorial transport characteristics of indinavir, nelfinavir, and saquinavir in vitro using the model P-glycoprotein expressing cell lines L-MDR1 and Caco-2 cells, and in vivo after intravenous and oral administration of these agents to mice with a disrupted mdr1a gene. All three compounds were found to be transported by P-glycoprotein in vitro. After oral administration, plasma concentrations were elevated 2-5-fold in mdr1a (-/-) mice and with intravenous administration, brain concentrations were elevated 7-36-fold. These data demonstrate that P-glycoprotein limits the oral bioavailability and penetration of these agents into the brain. This raises the possibility that higher HIV-1 protease inhibitor concentrations may be obtained by targeted pharmacologic inhibition of P-glycoprotein transport activity.

Nuclear Receptors and the Regulation of Drug-Metabolizing Enzymes and Drug Transporters: Implications for Interindividual Variability in Response to Drugs

Erratic or unpredictable response to drugs remains a challenge of modern drug therapy. An important determinant of such interindividual differences in drug response is variability in the expression of drug‐metabolizing enzymes and/or transporters at sites of absorption and/or tissue distribution. Variable drug‐metabolizing enzyme and transporter expression can result in unpredictable exposure and tissue distribution of drugs and may manifest as adverse effects or therapeutic failure. In the past decade, important new insights have been made relating to the regulatory mechanisms governing the expression of drug‐metabolizing enzymes and transporters by ligand‐activated nuclear receptors. Specifically, there is compelling evidence to demonstrate that PXR, CAR, FXR, LXR, VDR, HNF4α, and AhR form a battery of nuclear receptors that regulate the expression of many important drug‐metabolizing enzyme and transporters. In this review, the authors focus on clinically important drug‐metabolizing enzymes such as CYP3A4, CYP2B6, CYP2C9, CYP2C19, UGT1A1, SULT2A1, and glutathione S‐transferases and their regulation by nuclear receptors. They also review the nuclear receptor–mediated regulation of drug transporters such as MDR1, MRP2, MRP4, BSEP, BCRP, NTCP, OATP1B3, and OATP1A2. Finally, they outline how the drug development process has been affected by the current understanding of the involvement of nuclear receptors in the regulation of drug disposition genes.

Drug Transporters in Drug Efficacy and Toxicity

Drug transporters are now widely acknowledged as important determinants governing drug absorption, excretion, and, in many cases, extent of drug entry into target organs. There is also a greater appreciation that altered drug transporter function, whether due to genetic polymorphisms, drug-drug interactions, or environmental factors such as dietary constituents, can result in unexpected toxicity. Such effects are in part due to the interplay between various uptake and efflux transporters with overlapping functional capabilities that can manifest as marked interindividual variability in drug disposition in vivo. Here we review transporters of the solute carrier (SLC) and ATP-binding cassette (ABC) superfamilies considered to be of major importance in drug therapy and outline how understanding the expression, function, and genetic variation in such drug transporters will result in better strategies for optimal drug design and tissue targeting as well as reduce the risk for drug-drug interactions and adverse drug responses.

Interaction of morphine, fentanyl, sufentanil, alfentanil, and loperamide with the efflux drug transporter P-glycoprotein.

Background The efflux transporter P-glycoprotein, a member of the adenosine triphosphate–binding cassette superfamily, is a major determinant of the pharmacokinetics and pharmacodynamics of the opioid loperamide, a well-recognized antidiarrheal agent. Animal studies indicate that P-glycoprotein limits morphine entry into the brain. In this study, the authors examined whether other opioids of importance to anesthesiologists such as fentanyl, sufentanil, and alfentanil, and also morphine-6-glucuronide and morphine-3-glucuronide, are P-glycoprotein substrates and whether, in turn, these opioids act also as P-glycoprotein inhibitors. Methods The transcellular movement of the various opioids, including loperamide and morphine, was assessed in L-MDR1 (expressing P-glycoprotein) and LLC-PK1 cell monolayers (P-glycoprotein expression absent). A preferential basal-to-apical versus apical-to-basal transport in the L-MDR cells but not the LLC-PK1 cells is seen for P-glycoprotein substrates. In addition, the effect of the various opioids on the transcellular movement of the prototypical P-glycoprotein substrate digoxin was examined in Caco-2 cell monolayers. IC50 values were calculated according to the Hill equation. Results Loperamide was a substrate showing high dependence on P-glycoprotein in that basal–apical transport was nearly 10-fold greater than in the apical–basal direction in L-MDRI cells. Morphine also showed a basal-to-apical gradient in the L-MDR1 cell monolayer, indicating that it too is a P-glycoprotein substrate, but with less dependence than loperamide in that only 1.5-fold greater basal–apical directional transport was observed. Fentanyl, sufentanil, and alfentanil did not behave as P-glycoprotein substrates, whereas the morphine glucuronides did not cross the cell monolayers at all, whether P-glycoprotein was present or not. Loperamide, sufentanil, fentanyl, and alfentanil inhibited P-glycoprotein–mediated digoxin transport in Caco-2 cells with IC50 values of 2.5, 4.5, 6.5, and 112 &mgr;m, respectively. Morphine and its glucuronides (20 &mgr;m) did not inhibit digoxin (5 &mgr;m) transport in Caco-2 cells, and therefore IC50 values were not determined. Conclusions Opioids have a wide spectrum of P-glycoprotein activity, acting as both substrates and inhibitors, which might contribute to their varying central nervous system–related effects.

Population differences in S‐warfarin metabolism between CYP2C9 genotype‐matched Caucasian and Japanese patients

Our objective was to investigate population differences in the metabolic activity of cytochrome P450 (CYP) 2C9 between genotypically matched Caucasian and Japanese patients by using the unbound oral clearance of S‐warfarin as an in vivo phenotypic trait measure.

Effect of drug transporter genotypes on pravastatin disposition in European- and African-American participants.

Objective Our aims were to evaluate the effects of polymorphisms in the hepatic drug uptake transporter organic anion transporting polypeptide 1B1 (OATP1B1, SLCO1B1) and efflux transporters multidrug resistance-associated protein 2 (MRP2, ABCC2), bile salt export pump (BSEP, ABCB11), and breast cancer-related protein (BCRP, ABCG2) on single-dose pravastatin pharmacokinetics in healthy European- and African-American participants. Methods The pharmacokinetics of a single oral 40 mg dose of pravastatin was determined in 107 participants (69 European-Americans and 38 African-Americans). Participants were genotyped for known OATP1B1, MRP2, BSEP, and BCRP polymorphisms. Baseline serum total and unconjugated plasma bilirubin concentrations were also determined. Results OATP1B1 genotypes were ethnicity-dependent with a 521C allele frequency of ∼15% in European-Americans and ∼1% in African-Americans. SLCO1B1 521TC genotype was associated with significantly higher pravastatin area under the curve [AUC(0–5)] (P=0.01) and Cmax values (P<0.05). When analyzed by diplotype, SLCO1B1*1a/*15 (N=8) participants exhibited 45 and 80% higher AUC values than SLCO1B1*1a/*1a (N=29) (P=0.013) and SLCO1B1*1b/*1b (N=34) (P=0.001) carriers, respectively. SLCO1B1*15/*15 (N=2) participants exhibited 92 and 149% higher AUC values than SLCO1B1*1a/*1a (P=0.017) and SLCO1B1*1b/*1b (P=0.011) carriers, respectively. European-Americans had significantly higher plasma pravastatin AUC(0–5) (P=0.01) and Cmax values (P=0.009) than African-Americans. Neither ABCC2, ABCB11, nor ABCG2 genotypes were associated with differences in pravastatin pharmacokinetics. We did not observe an effect of SLCO1B1 genotype on baseline total or unconjugated bilirubin levels. Conclusion SLCO1B1 genotype, in particular the 521C allele, had a significant effect on the pharmacokinetics of pravastatin. Even when adjusted for the presence of the SLCO1B1 521C or 388G variant allele, European-Americans demonstrated significantly higher pravastatin AUC and Cmax values than African-Americans.

Defining the Cellular Phenotype of “Ankyrin-B Syndrome” Variants: Human ANK2 Variants Associated With Clinical Phenotypes Display a Spectrum of Activities in Cardiomyocytes

Background— Mutations in the ankyrin-B gene (ANK2) cause type 4 long-QT syndrome and have been described in kindreds with other arrhythmias. The frequency of ANK2 variants in large populations and molecular mechanisms underlying the variability in the clinical phenotypes are not established. More importantly, there is no cellular explanation for the range of severity of cardiac phenotypes associated with specific ANK2 variants. Methods and Results— We performed a comprehensive screen of ANK2 in populations (control, congenital arrhythmia, drug-induced long-QT syndrome) of different ethnicities to discover unidentified ANK2 variants. We identified 7 novel nonsynonymous ANK2 variants; 4 displayed abnormal activity in cardiomyocytes. Including the 4 new variants, 9 human ANK2 loss-of-function variants have been identified. However, the clinical phenotypes associated with these variants vary strikingly, from no obvious phenotype to manifest long-QT syndrome and sudden death, suggesting that mutants confer a spectrum of cellular phenotypes. We then characterized the relative severity of loss-of-function properties of all 9 nonsynonymous ANK2 variants identified to date in primary cardiomyocytes and identified a range of in vitro phenotypes, including wild-type, simple loss-of-function, and severe loss-of-function activity, seen with the variants causing severe human phenotypes. Conclusions— We present the first description of differences in cellular phenotypes conferred by specific ANK2 variants. We propose that the various degrees of ankyrin-B loss of function contribute to the range of severity of cardiac dysfunction. These data identify ANK2 variants as modulators of human arrhythmias, provide the first insight into the clinical spectrum of “ankyrin-B syndrome,” and reinforce the role of ankyrin-B–dependent protein interactions in regulating cardiac electrogenesis.

Grapefruit juice ingestion significantly reduces talinolol bioavailability

Our objectives were to evaluate the effect of single and repeated grapefruit juice ingestion relative to water on the oral pharmacokinetics of the nonmetabolized and P‐glycoprotein‐transported drug talinolol in humans and to assess the potential impact of grapefruit juice ingestion on P‐glycoprotein and intestinal uptake transporters.

Blood−brain barrier transporters and response to CNS-active drugs

BackgroundThe capillary endothelial cells of the blood−brain barrier express an array of uptake and efflux drug transporters. Regulated expression and function of these transporters govern the central nervous system (CNS) penetration of essential nutrients, therapeutic drugs and, in some cases, toxins. Emerging evidence supports the notion of interplay between uptake and efflux drug transport as the determinants that define the extent of exposure of many drugs and their CNS action.ObjectiveIn this brief report, we review a number of key drug transporters known to be expressed at the blood−brain barrier and/or choroid plexus and focus on the implications of such transporters to CNS drug activity, side effects, and toxicity. Specifically, this report focuses on the uptake transporters OATP1A2 (organic anion-transporting polypeptide 1A2) and MCT1 (monocarboxylate transporter 1), which are known to have substrates that are either neuroactive or known to result in CNS side effects and/or toxicity. Furthermore, the efflux transporters P-gp (P-gp; MDR1/ABCB1), BCRP (breast-cancer-resistance protein), OAT3 (organic anion transporter 3), and MRP4 (multidrug-resistance-associated protein 4) are also reviewed.ConclusionsDrug transporters play a fundamental role in protecting the brain from exposure to drugs and other potential toxicants.