Brenda F. Leake

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.

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.

Polymorphisms in OATP-C IDENTIFICATION OF MULTIPLE ALLELIC VARIANTS ASSOCIATED WITH ALTERED TRANSPORT ACTIVITY AMONG EUROPEAN- AND AFRICAN-AMERICANS

The human organic anion transporting polypeptide-C (OATP-C) (gene SLC21A6) is a liver-specific transporter importantly involved in the hepatocellular uptake of a variety of endogenous and foreign chemicals. In this study, we demonstrate the presence of multiple functionally relevant single-nucleotide polymorphisms (SNPs) in OATP-C in a population of African- and European-Americans. Moreover, examination of 14 nonsynonymous polymorphisms indicated that genotypic frequencies were dependent on race. Functional assessment of 16OATP-C alleles in vitro revealed that several variants exhibited markedly reduced uptake of the OATP-C substrates estrone sulfate and estradiol 17β-d-glucuronide. Specifically, alterations in transport were associated with SNPs that introduce amino acid changes within the transmembrane-spanning domains (T217C (Phe-73 → Leu), T245C (Val-82 → Ala), T521C (Val-174 → Ala), and T1058C (Ile-353 → Thr)) and also with those that modify extracellular loop 5 (A1294G (Asn-432 → Asp), A1385G (Asp-462 → Gly), and A1463C (Gly-488 → Ala)). Cell surface biotinylation experiments indicated that the altered transport activity of some OATP-C variants was due, in part, to decreased plasma membrane expression. Given the relatively high genotypic frequency of the T521C (14%) transition in European-Americans and the G1463C (9%) transversion in African-Americans, SNPs in OATP-C may represent a heretofore unrecognized factor influencing drug disposition.

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.

Interrelationship between substrates and inhibitors of human CYP3A and P-glycoprotein.

AbstractPurpose. CYP3A and P-gp both function to reduce the intracellular concentration of drug substrates, one by metabolism and the other by transmembrane efflux. Moreover, it has been serendipitously noted that the two proteins have many common substrates and inhibitors. In order to test this notion more fully, systematic studies were undertaken to determine the P-gp-mediated transport and inhibitory characteristics of prototypical CYP substrates. Methods. L-MDR1, LLC-PK1, and Caco-2 cells were used to evaluate established CYP substrates as potential P-gp substrates and inhibitors in vitro, and mdrla deficient mice were used to assess the in vivo relevance of P-gp-mediated transport. Results. Some (terfenadine, erythromycin and lovastatin) but not all (nifedipine and midazolam) CYP3A substrates were found to be P-gp substrates. Except for debrisoquine, none of the prototypical substrates of other common human CYP isoforms were transported by P-gp. Studies in mdrla disrupted mice confirmed that erythromycin was a P-gp substrate but the CYP3A inhibitor ketoconazole was not. In addition, CYP3A substrates and inhibitors varied widely in their ability to inhibit the P-gp-mediated transport of digoxin. Conclusions. These results indicate that the overlap in substrate specificities of CYP3A and P-gp appears to be fortuitous rather than indicative of a more fundamental relationship.

The orphan nuclear receptor HNF4α determines PXR- and CAR-mediated xenobiotic induction of CYP3A4

The drug metabolizing enzyme cytochrome P450 3A4 (CYP3A4) is thought to be involved in the metabolism of nearly 50% of all the drugs currently prescribed. Alteration in the activity or expression of this enzyme seems to be a key predictor of drug responsiveness and toxicity. Currently available studies indicate that the ligand-activated nuclear receptors pregnane X receptor (PXR; NR1I2) and constitutive androstane receptor (CAR; NR1I3) regulate CYP3A4 expression. However, in cell-based reporter assays, CYP3A4 promoter activity was most pronounced in liver-derived cells and minimal or modest in non-hepatic cells, indicating that a liver-specific factor is required for physiological transcriptional response. Here we show that the orphan nuclear receptor hepatocyte nuclear factor-4α (HNF4α; HNF4A) is critically involved in the PXR- and CAR-mediated transcriptional activation of CYP3A4. We identified a specific cis-acting element in the CYP3A4 gene enhancer that confers HNF4α binding and thereby permits PXR- and CAR-mediated gene activation. Fetal mice with conditional deletion of Hnf4α had reduced or absent expression of CYP3A. Furthermore, adult mice with conditional hepatic deletion of Hnf4α had reduced basal and inducible expression of CYP3A. These data identify HNF4α as an important regulator of coordinate nuclear-receptor–mediated response to xenobiotics.

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.

Naringin is a Major and Selective Clinical Inhibitor of Organic Anion‐Transporting Polypeptide 1A2 (OATP1A2) in Grapefruit Juice

We showed previously that grapefruit and orange juices inhibited human enteric organic anion‐transporting polypeptide (OATP)1A2 in vitro and lowered oral fexofenadine bioavailability clinically. Inhibition of OATP1A2 transport by flavonoids in grapefruit (naringin) and orange (hesperidin) was conducted in vitro. Two randomized, crossover, pharmacokinetic studies were performed clinically. In one study, 120 mg of fexofenadine was ingested with 300 ml grapefruit juice, an aqueous solution of naringin at the same juice concentration (1,200 μM), or water. In the other study, fexofenadine was administered with grapefruit juice, with or 2 h before aqueous suspension of the particulate fraction of juice containing known clinical inhibitors of enteric CYP3A4, but relatively low naringin concentration (34 μM), or with water. Naringin and hesperidins half‐maximal inhibitions were 3.6 and 2.7 μM, respectively. Fexofenadine area under the plasma drug concentration–time curves (AUCs) with grapefruit juice and naringin solution were 55% (P<0.001) and 75% (P<0.05) of that with water, respectively. Fexofenadine AUCs with grapefruit juice and particulate fractions were 57% (P<0.001), 96% (not significant (NS)), and 97% (NS) of that with water, respectively. Individuals tested in both studies (n=9 of 12) had highly reproducible fexofenadine AUC with water (r2=0.85, P<0.001) and extent of reduction of it with grapefruit juice (r2=0.72, P<0.01). Naringin most probably directly inhibited enteric OATP1A2 to decrease oral fexofenadine bioavailability. Inactivation of enteric CYP3A4 was probably not involved. Naringin appears to have sufficient safety, specificity, and sensitivity to be a clinical OATP1A2 inhibitor probe. Inherent OATP1A2 activity may be influenced by genetic factors. This appears to be the first report of a single dietary constituent clinically modulating drug transport.

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.