Ute I. Schwarz

Identification of functionally variant MDR1 alleles among European Americans and African Americans

MDR1 (P‐glycoprotein) is an important factor in the disposition of many drugs, and the involved processes often exhibit considerable interindividual variability that may be genetically determined. Single‐strand conformational polymorphism analysis and direct sequencing of exonic MDR1 deoxyribonucleic acid from 37 healthy European American and 23 healthy African American subjects identified 10 single nucleotide polymorphisms (SNPs), including 6 nonsynonymous variants, occurring in various allelic combinations. Population frequencies of the 15 identified alleles varied according to racial background. Two synonymous SNPs (C1236T in exon 12 and C3435T in exon 26) and a nonsynonymous SNP (G2677T, Ala893Ser) in exon 21 were found to be linked (MDR1ast;2) and occurred in 62% of European Americans and 13% of African Americans. In vitro expression of MDR1 encoding Ala893 (MDR1ast;1) or a site‐directed Ser893 mutation (MDR1ast;2) indicated enhanced efflux of digoxin by cells expressing the MDR1‐Ser893 variant. In vivo functional relevance of this SNP was assessed with the known P‐glycoprotein drug substrate fexofenadine as a probe of the transporters activity. In humans, MDR1ast;1 and MDR1ast;2 variants were associated with differences in fexofenadine levels, consistent with the in vitro data, with the area under the plasma level–time curve being almost 40% greater in the *1/*1 genotype compared with the *2/*2 and the *1/*2 heterozygotes having an intermediate value, suggesting enhanced in vivo P‐glycoprotein activity among subjects with the MDR1ast;2 allele. Thus allelic variation in MDR1 is more common than previously recognized and involves multiple SNPs whose allelic frequencies vary between populations, and some of these SNPs are associated with altered P‐glycoprotein function.

Fruit juices inhibit organic anion transporting polypeptide–mediated drug uptake to decrease the oral availability of fexofenadine

Our objective was to examine the effect of different fruits and their constituents on P‐glycoprotein and organic anion transporting polypeptide (OATP) activities in vitro and on drug disposition in humans.

A genome-wide scan for common genetic variants with a large influence on warfarin maintenance dose

Warfarin dosing is correlated with polymorphisms in vitamin K epoxide reductase complex 1 (VKORC1) and the cytochrome P450 2C9 (CYP2C9) genes. Recently, the FDA revised warfarin labeling to raise physician awareness about these genetic effects. Randomized clinical trials are underway to test genetically based dosing algorithms. It is thus important to determine whether common single nucleotide polymorphisms (SNPs) in other gene(s) have a large effect on warfarin dosing. A retrospective genome-wide association study was designed to identify polymorphisms that could explain a large fraction of the dose variance. White patients from an index warfarin population (n = 181) and 2 independent replication patient populations (n = 374) were studied. From the approximately 550 000 polymorphisms tested, the most significant independent effect was associated with VKORC1 polymorphisms (P = 6.2 x 10(-13)) in the index patients. CYP2C9 (rs1057910 CYP2C9*3) and rs4917639) was associated with dose at moderate significance levels (P approximately 10(-4)). Replication polymorphisms (355 SNPs) from the index study did not show any significant effects in the replication patient sets. We conclude that common SNPs with large effects on warfarin dose are unlikely to be discovered outside of the CYP2C9 and VKORC1 genes. Randomized clinical trials that account for these 2 genes should therefore produce results that are definitive and broadly applicable.

Intestinal Drug Transporter Expression and the Impact of Grapefruit Juice in Humans

The goals of this study were to assess the extent of human intestinal drug transporter expression, determine the subcellular localization of the drug uptake transporter OATP1A2, and then to assess the effect of grapefruit juice consumption on OATP1A2 expression relative to cytochrome P450 3A4 and MDR1. Expression of drug uptake and efflux transporters was assessed using human duodenal biopsy samples. Fexofenadine uptake by different transporters was measured in a transporter‐transfected cell line. We investigated the influence of grapefruit juice on pharmacokinetics of orally administered fexofenadine. The effect of grapefruit juice on the expression of intestinal transporters was determined using real‐time polymerase chain reaction and Western blot analysis. In the duodenum of healthy volunteers, an array of CYP enzymes as well as uptake and efflux transporters was expressed. Importantly, uptake transporters thought to be liver‐specific, such as OATP1B1 and 1B3, as well as OATP2B1 and 1A2 were expressed in the intestine. However, among OATP transporters, only OATP1A2 was capable of fexofenadine uptake when assessed in vitro. OATP1A2 colocalized with MDR1 to the brush border domain of enterocytes. Consumption of grapefruit juice concomitantly or 2 h before fexofenadine administration was associated with reduced oral fexofenadine plasma exposure, whereas intestinal expression of either OATP1A2 or MDR1 remained unaffected. In conclusion, an array of drug uptake and efflux transporters are expressed in the human intestine. OATP1A2 is likely the key intestinal uptake transporter for fexofenadine absorption whose inhibition results in the grapefruit juice effect. Although short‐term grapefruit juice ingestion was associated with reduced fexofenadine availability, OATP1A2 or MDR1 expression was unaffected.

Coordinate induction of both cytochrome P4503A and MDR1 by St John's wort in healthy subjects

Many drugs are cosubstrates of cytochrome P450 (CYP) 3A and MDR1; furthermore, their disposition is markedly affected by pretreatment with inducing agents, including St Johns wort. Such drug interactions reflect induction of both proteins through a common mechanism involving the steroid X receptor/pregnane X receptor. However, the relative contributions of enhanced metabolism and efflux transport to the overall induction process are unknown.

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.

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.

Human Skeletal Muscle Drug Transporters Determine Local Exposure and Toxicity of Statins

Rationale: The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, are important drugs used in the treatment and prevention of cardiovascular disease. Although statins are well tolerated, many patients develop myopathy manifesting as muscle aches and pain. Rhabdomyolysis is a rare but severe toxicity of statins. Interindividual differences in the activities of hepatic membrane drug transporters and metabolic enzymes are known to influence statin plasma pharmacokinetics and risk for myopathy. Interestingly, little is known regarding the molecular determinants of statin distribution into skeletal muscle and its relevance to toxicity. Objective: We sought to identify statin transporters in human skeletal muscle and determine their impact on statin toxicity in vitro. Methods and Results: We demonstrate that the uptake transporter OATP2B1 (human organic anion transporting polypeptide 2B1) and the efflux transporters, multidrug resistance–associated protein (MRP)1, MRP4, and MRP5 are expressed on the sarcolemmal membrane of human skeletal muscle fibers and that atorvastatin and rosuvastatin are substrates of these transporters when assessed using a heterologous expression system. In an in vitro model of differentiated, primary human skeletal muscle myoblast cells, we demonstrate basal membrane expression and drug efflux activity of MRP1, which contributes to reducing intracellular statin accumulation. Furthermore, we show that expression of human OATP2B1 in human skeletal muscle myoblast cells by adenoviral vectors increases intracellular accumulation and toxicity of statins and such effects were abrogated when cells overexpressed MRP1. Conclusions: These results identify key membrane transporters as modulators of skeletal muscle statin exposure and toxicity.

Breast cancer resistance protein (ABCG2) and drug disposition : intestinal expression, polymorphisms and sulfasalazine as an in vivo probe

Breast cancer resistance protein (BCRP) is an efflux transporter expressed in tissues that act as barriers to drug entry. Given that single nucleotide polymorphisms (SNPs) in the ABCG2 gene encoding BCRP are common, the possibility exists that these genetic variants may be a determinant of interindividual variability in drug response. The objective of this study is to confirm the human BCRP-mediated transport of sulfasalazine in vitro, evaluate interindividual variation in BCRP expression in human intestine and to determine the role of ABCG2 SNPs to drug disposition in healthy patients using sulfasalazine as a novel in vivo probe. To evaluate these objectives, pinch biopsies were obtained from 18 patients undergoing esophagogastro–duodenoscopy or colonoscopy for determination of BCRP expression in relation to genotype. Wild-type and variant BCRP were expressed in a heterologous expression system to evaluate the effect of SNPs on cell-surface trafficking. A total of 17 healthy individuals participated in a clinical investigation to determine the effect of BCRP SNPs on sulfasalazine pharmacokinetics. In vitro, the cell surface protein expression of the common BCRP 421 C>A variant was reduced in comparison with the wild-type control. Intestinal biopsy samples revealed that BCRP protein and mRNA expression did not significantly differ between patients with 34GG/421CC versus patients with 34GG/421CA genotypes. Remarkably, in subjects with 34GG/421CA genotype, sulfasalazine area under the concentration–time curve was 2.4-fold greater compared with 34GG/421CC subjects (P<0.05). This study links commonly occurring SNPs in BCRP with significantly increased oral sulfasalazine plasma exposure in humans. Accordingly, sulfasalazine may prove to have utility as in vivo probe for assessing the clinical impact of BCRP for the disposition and efficacy of drugs.

Induction of intestinal P-glycoprotein by St John's wort reduces the oral bioavailability of talinolol.

St Johns wort (SJW) is known to induce cytochrome P450 (CYP) 3A4 and P‐glycoprotein through pregnane X‐receptor activation. Our study evaluated the effects of long‐term SJW administration on oral and intravenous pharmacokinetics of the nonmetabolized in vivo probe of P‐glycoprotein, talinolol, in relation to intestinal P‐glycoprotein expression. In a controlled, randomized study (N=9), the pharmacokinetics of oral (50 mg) and intravenous talinolol (30 mg) was determined before and after 12 days SJW (900 mg daily, Jarsin 300®). Duodenal biopsies were taken and MDR1 genotypes assessed. SJW reduced the oral talinolol bioavailability by 25% (P=0.049) compared with water control. A 93% increase in oral clearance (P=0.177) and a 31% reduction in area under the serum concentration time curve (AUC; P=0.030) were observed. Renal and nonrenal clearance (CLNR), elimination half‐life, peak serum drug concentration (Cmax), and time to reach Cmax were not significantly altered. After intravenous talinolol, SJW affected only CLNR (35% increase compared with water, P=0.006). SJW increased MDR1 messenger ribonucleic acid (mRNA) as well as P‐glycoprotein levels in the duodenal mucosa. Subjects with the combined MDR1 genotype comprising 1236C>T, 2677G>T/A, and 3435C>T polymorphisms had lower intestinal MDR1 mRNA levels and displayed an attenuated inductive response to SJW as assessed by talinolol disposition. Long‐term SJW decreased talinolol AUC with a corresponding increase in intestinal MDR1 expression, suggesting that SJW has a major inductive effect on intestinal P‐glycoprotein. Interestingly, the magnitude of induction appeared to be affected by MDR1 genotype.