Aims Rosuvastatin and pitavastatin have been proposed as probe substrates for the organic anion‐transporting polypeptide (OATP) 1B, but clinical data on their relative sensitivity and selectivity to OATP1B inhibitors are lacking. A clinical study was therefore conducted to determine their relative suitability as OATP1B probes using single oral (PO) and intravenous (IV) doses of the OATP1B inhibitor rifampicin, accompanied by a comprehensive in vitro assessment of rifampicin inhibitory potential on statin transporters. Methods The clinical study comprised of two separate panels of eight healthy subjects. In each panel, subjects were randomized to receive a single oral dose of rosuvastatin (5 mg) or pitavastatin (1 mg) administered alone, concomitantly with rifampicin (600 mg) PO or IV. The in vitro transporter studies were performed using hepatocytes and recombinant expression systems. Results Rifampicin markedly increased exposures of both statins, with greater differential increases after PO vs. IV rifampicin only for rosuvastatin. The magnitudes of the increases in area under the plasma concentration–time curve were 5.7‐ and 7.6‐fold for pitavastatin and 4.4‐ and 3.3‐fold for rosuvastatin, after PO and IV rifampicin, respectively. In vitro studies showed that rifampicin was an inhibitor of OATP1B1 and OATP1B3, breast cancer resistance protein and multidrug resistance protein 2, but not of organic anion transporter 3. Conclusions The results indicate that pitavastatin is a more sensitive and selective and thus preferred clinical OATP1B probe substrate than rosuvastatin, and that a single IV dose of rifampicin is a more selective OATP1B inhibitor than a PO dose.
Raloxifene is a selective estrogen receptor modulator which is effective in the treatment of osteoporosis in postmenopausal women. We report herein that cytochrome P450 (P450)3A4 is inhibited by raloxifene in human liver microsomal incubations. The nature of the inhibition was irreversible and was NADPH- and preincubation time-dependent, with K(I) and k(inact) values estimated at 9.9 microM and 0.16 min(-1), respectively. The observed loss of P450 3A4 activity was attenuated partially by glutathione (GSH), implying the involvement of a reactive metabolite(s) in the inactivation process. Subsequently, GSH adducts of raloxifene were identified in incubations with human liver microsomes; substitution with GSH occurred at the 5- or 7-position of the benzothiophene moiety or at the 3'-position of the phenol ring, with the 7-glutathionyl derivative being most abundant based on LC/MS and NMR analyses. These adducts are postulated to derive from addition of GSH to raloxifene arene oxides followed by dehydration and aromatization. Alternatively, raloxifene may be oxidized to an extended quinone intermediate, which then is trapped by GSH conjugation. The bioactivation of raloxifene most likely is catalyzed by P450 3A4, since the formation of GSH adducts was almost abolished when liver microsomes were pretreated with ketoconazole or with an inhibitory anti-P450 3A4 IgG. The GSH adducts also were detected in incubations of raloxifene with rat or human hepatocytes, while the corresponding N-acetylcysteine adducts were identified in the bile and urine from rats treated orally with the drug at 5 mg/kg. Taken together, these data indicate that P450 3A4-mediated bioactivation of raloxifene in vitro is accompanied by loss of enzyme activity. The significance of these findings with respect to the clinical use of raloxifene remains to be determined.
We studied the time course for the reversal of rifampin's effect on the pharmacokinetics of oral midazolam (a cytochrome P450 (CYP) 3A4 substrate) and digoxin (a P-glycoprotein (P-gp) substrate). Rifampin increased midazolam metabolism, greatly reducing the area under the concentration-time curve (AUC(0-∞)). The midazolam AUC(0-∞) returned to baseline with a half-life of ~8 days. Rifampin's effect on the AUC(0-3 h) of digoxin was biphasic: the AUC(0-3 h) increased with concomitant dosing of the two drugs but decreased when digoxin was administered after rifampin. Digoxin was found to be a weak substrate of organic anion-transporting polypeptide (OATP) 1B3 in transfected cells. Although the drug was transported into isolated hepatocytes, it is not likely that this transport was through OATP1B3 because the transport was not inhibited by rifampin. However, rifampin did inhibit the P-gp-mediated transport of digoxin with a half-maximal inhibitory concentration (IC(50)) below anticipated gut lumen concentrations, suggesting that rifampin inhibits digoxin efflux from the enterocyte to the intestinal lumen. Pharmacokinetic modeling suggested that the effects on digoxin are consistent with a combination of inhibitory and inductive effects on gut P-gp. These results suggest modifications to drug-drug interaction (DDI) trial designs.
A microdose cocktail containing midazolam, dabigatran etexilate, pitavastatin, rosuvastatin, and atorvastatin has been established to allow simultaneous assessment of a perpetrator impact on the most common drug metabolizing enzyme, cytochrome P450 (CYP)3A, and the major transporters organic anion-transporting polypeptides (OATP)1B, breast cancer resistance protein (BCRP), and MDR1 P-glycoprotein (P-gp). The clinical utility of these microdose cocktail probe substrates was qualified by conducting clinical drug interaction studies with three inhibitors with different in vitro inhibitory profiles (rifampin, itraconazole, and clarithromycin). Generally, the pharmacokinetic profiles of the probe substrates, in the absence and presence of the inhibitors, were comparable to their reported corresponding pharmacological doses, and/or in agreement with theoretical expectations. The exception was dabigatran, which resulted in an approximately twofold higher magnitude for microdose compared to conventional dosing, and, thus, can be used to flag a worst-case scenario for P-gp. Broader application of the microdose cocktail will facilitate a more comprehensive understanding of the roles of drug transporters in drug disposition and drug interactions.
The multidrug resistance protein Mrp2 is an ATP-binding cassette (ABC) transporter mainly expressed in liver, kidney, and intestine. One of the physiological roles of Mrp2 is to transport bilirubin glucuronides from the liver into the bile. Current in vivo models to study Mrp2 are the transporter-deficient and Eisai hyperbilirubinemic rat strains. Previous reports showed hyperbilirubinemia and induction of Mrp3 in the hepatocyte sinusoidal membrane in the mutant rats. In addition, differences in liver cytochrome P450 and UGT1a levels between wild-type and mutant rats were detected. To study whether these compensatory mechanisms were specific to rats, we characterized Mrp2 Ϫ/Ϫ mice. Functional absence of Mrp2 in the knockout mice was demonstrated by showing increased levels of bilirubin and bilirubin glucuronides in serum and urine, a reduction in biliary excretion of bilirubin glucuronides and total glutathione, and a reduction in the biliary excretion of the Mrp2 substrate dibromosulfophthalein. To identify possible compensatory mechanisms in Mrp2 Ϫ/Ϫ mice, the expression levels of 98 phase I, phase II, and transporter genes were compared in liver, kidney, and intestine of male and female Mrp2 Ϫ/Ϫ and control mice. Unlike in Mrp2 mutant rats, no induction of Mrp3 in Mrp2 Ϫ/Ϫ mice was detected. However, Mrp4 mRNA and protein in liver and kidney were increased ϳ6-and 2-fold, respectively. Phenotypic analysis of major cytochrome P450-mediated activities in liver microsomes did not show differences between wild-type and Mrp2 Ϫ/Ϫ mice. In conclusion, Mrp2Ϫ/Ϫ mice are a new valuable tool to study the role of Mrp2 in drug disposition.
Sitagliptin, a selective dipeptidyl peptidase 4 inhibitor recently approved for the treatment of type 2 diabetes, is excreted into the urine via active tubular secretion and glomerular filtration in humans. In this report, we demonstrate that sitagliptin is transported by human organic anion transporter hOAT3 (K m ϭ 162 M), organic anion transporting polypeptide OATP4C1, and multidrug resistance (MDR) P-glycoprotein (Pgp), but not by human organic cation transporter 2 hOCT2, hOAT1, oligopeptide transporter hPEPT1, OATP2B1, and the multidrug resistance proteins MRP2 and MRP4. Our studies suggested that hOAT3, OATP4C1, and MDR1 Pgp might play a role in transporting sitagliptin into and out of renal proximal tubule cells, respectively. Sitagliptin did not inhibit hOAT1-mediated cidofovir uptake, but it showed weak inhibition of hOAT3-mediated cimetidine uptake (IC 50 ϭ 160 M). hOAT3-mediated sitagliptin uptake was inhibited by probenecid, ibuprofen, furosemide, fenofibric acid, quinapril, indapamide, and cimetidine with IC 50 values of 5.6, 3.7, 1.7, 2.2, 6.2, 11, and 79 M, respectively. Sitagliptin did not inhibit Pgp-mediated transport of digoxin, verapamil, ritonavir, quinidine, and vinblastine. Cyclosporine A significantly inhibited Pgp-mediated transport of sitagliptin (IC 50 ϭ 1 M). Our data indicate that sitagliptin is unlikely to be a perpetrator of drug-drug interactions with Pgp, hOAT1, or hOAT3 substrates at clinically relevant concentrations. Renal secretion of sitagliptin could be inhibited if coadministered with OAT3 inhibitors such as probenecid. However, the magnitude of interactions should be low, and the effects may not be clinically meaningful, due to the high safety margin of sitagliptin.Sitagliptin [also known as MK-0431 ( Fig. 1)], is a selective, reversible inhibitor of dipeptidyl-peptidase 4 recently approved by the Food and Drug Administration for the treatment of type 2 diabetes (Deacon, 2005;Kim et al., 2005). Dipeptidyl-peptidase 4 inhibitors have a glucose-lowering effect by inhibiting the inactivation of incretin peptides, including glucagon-like peptide-1 and glucose-dependent insulinotropic peptide, which are released upon nutrient ingestion, stimulate meal-induced insulin secretion, and contribute to glucose homeostasis (Kieffer and Habener, 1999). The pharmacokinetics, metabolism, and excretion of sitagliptin have been investigated in humans and animals Herman et al., 2005Herman et al., , 2006Beconi et al., 2007). After a single oral dose of sitagliptin (1.5-600 mg) in healthy male volunteers, sitagliptin was well absorbed with an apparent terminal half-life ranging from 8 to 14 h. Sitagliptin was primarily renally eliminated as unchanged drug (Herman et al., 2005), with metabolism playing only a minor role (Vincent et al., 2007). Renal clearance of sitagliptin averaged approximately 388 ml/min in humans (Herman et al., 2005). Given that sitagliptin is approximately Article, publication date, and citation information can be found at
Inhibition of hepatic transporters such as organic anion transporting polypeptides (OATPs) 1B can cause drug-drug interactions (DDIs). Determining the impact of perpetrator drugs on the plasma exposure of endogenous substrates for OATP1B could be valuable to assess the risk for DDIs early in drug development. As OATP1B orthologs are well conserved between human and monkey, we assessed in cynomolgus monkeys the endogenous OATP1B substrates that are potentially suitable to assess DDI risk in humans. The effect of rifampin (RIF), a potent inhibitor for OATP1B, on plasma exposure of endogenous substrates of hepatic transporters was measured. From the 18 biomarkers tested, RIF (18 mg/kg, oral) caused significant elevation of plasma unconjugated and conjugated bilirubin, which may be attributed to inhibition of cOATP1B1 and cOATP1B3 based on in vitro to in vivo extrapolation analysis. To further evaluate whether cynomolgus monkeys are a suitable translational model to study OATP1B-mediated DDIs, we determined the inhibitory effect of RIF on in vitro transport and pharmacokinetics of rosuvastatin (RSV) and atorvastatin (ATV). RIF strongly inhibited the uptake of RSV and ATV by cOATP1B1 and cOATP1B3 in vitro. In agreement with clinical observations, RIF (18 mg/kg, oral) significantly decreased plasma clearance and increased the area under the plasma concentration curve (AUC) of intravenously administered RSV by 2.8-and 2.7-fold, and increased the AUC and maximum plasma concentration of orally administered RSV by 6-and 10.3-fold, respectively. In contrast to clinical findings, RIF did not significantly increase plasma exposure of either intravenous or orally administered ATV, indicating species differences in the rate-limiting elimination pathways.
The inhibitory effect of boceprevir (BOC), an inhibitor of hepatitis C virus nonstructural protein 3 protease was evaluated in vitro against a panel of drug-metabolizing enzymes and transporters. BOC, a known substrate for cytochrome P450 (P450) CYP3A and aldoketoreductases, was a reversible time-dependent inhibitor (k inact = 0.12 minute 21, K I = 6.1 mM) of CYP3A4/5 but not an inhibitor of other major P450s, nor of UDP-glucuronosyltransferases 1A1 and 2B7. BOC showed weak to no inhibition of breast cancer resistance protein (BCRP), P-glycoprotein (Pgp), or multidrug resistance protein 2. It was a moderate inhibitor of organic anion transporting polypeptide (OATP) 1B1 and 1B3, with an IC 50 of 18 and 4.9 mM, respectively. In human hepatocytes, BOC inhibited CYP3A-mediated metabolism of midazolam, OATP1B-mediated hepatic uptake of pitavastatin, and both the uptake and metabolism of atorvastatin. The inhibitory potency of BOC was lower than known inhibitors of CYP3A (ketoconazole), OATP1B (rifampin), or both (telaprevir). BOC was a substrate for Pgp and BCRP but not for OATP1B1, OATP1B3, OATP2B1, organic cation transporter, or sodium/taurocholate cotransporting peptide. Overall, our data suggest that BOC has the potential to cause pharmacokinetic interactions via inhibition of CYP3A and CYP3A/OATP1B interplay, with the interaction magnitude lower than those observed with known potent inhibitors. Conversely, pharmacokinetic interactions of BOC, either as a perpetrator or victim, via other major P450s and transporters tested are less likely to be of clinical significance. The results from clinical drug-drug interaction studies conducted thus far are generally supportive of these conclusions.
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