Many xenobiotics including the pharmacoenhancer cobicistat increase serum creatinine by inhibiting its renal active tubular secretion without affecting the glomerular filtration rate. This study aimed to define the transporters involved in creatinine secretion, applying that knowledge to establish the mechanism for xenobiotic-induced effects. The basolateral uptake transporters organic anion transporter OAT2 and organic cation transporters OCT2 and OCT3 were found to transport creatinine. At physiologic creatinine concentrations, the specific activity of OAT2 transport was over twofold higher than OCT2 or OCT3, establishing OAT2 as a likely relevant creatinine transporter and further challenging the traditional view that creatinine is solely transported by a cationic pathway. The apical multidrug and toxin extrusion transporters MATE1 and MATE2-K demonstrated low-affinity and high-capacity transport. All drugs known to affect creatinine inhibited OCT2 and MATE1. Similar to cimetidine and ritonavir, cobicistat had the greatest effect on MATE1 with a 50% inhibition constant of 0.99 μM for creatinine transport. Trimethoprim potently inhibited MATE2-K, whereas dolutegravir preferentially inhibited OCT2. Cimetidine was unique, inhibiting all transporters that interact with creatinine. Thus, the clinical observation of elevated serum creatinine in patients taking cobicistat is likely a result of OCT2 transport, facilitating intracellular accumulation, and MATE1 inhibition.

Tenofovir alafenamide (TAF) is a prodrug of tenofovir (TFV) currently in clinical evaluation for treatment for HIV and hepatitis B virus (HBV) infections. Since the target tissue for HBV is the liver, the hepatic delivery and metabolism of TAF in primary human hepatocytes in vitro and in dogs in vivo were evaluated here. Incubation of primary human hepatocytes with TAF resulted in high levels of the pharmacologically active metabolite tenofovir diphosphate (TFV-DP), which persisted in the cell with a halflife of >24 h. In addition to passive permeability, studies of transfected cell lines suggest that the hepatic uptake of TAF is also facilitated by the organic anion-transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3, respectively). In order to inhibit HBV reverse transcriptase, TAF must be converted to the pharmacologically active form, TFV-DP. While cathepsin A is known to be the major enzyme hydrolyzing TAF in cells targeted by HIV, including lymphocytes and macrophages, TAF was primarily hydrolyzed by carboxylesterase 1 (CES1) in primary human hepatocytes, with cathepsin A making a small contribution. Following oral administration of TAF to dogs for 7 days, TAF was rapidly absorbed. The appearance of the major metabolite TFV in plasma was accompanied by a rapid decline in circulating TAF. Consistent with the in vitro data, high and persistent levels of TFV-DP were observed in dog livers. Notably, higher liver TFV-DP levels were observed after administration of TAF than those given TDF. These results support the clinical testing of once-daily low-dose TAF for the treatment of HBV infection. Chronic hepatitis B (CHB) is a major global health problem, and the World Health Organization (WHO) estimates that ϳ240 million people worldwide are chronically infected by hepatitis B virus (HBV) (1) (http://www.who.int/mediacentre /factsheets/fs204/en/). The small-molecule anti-HBV agents currently approved by U.S. Food and Drug Administration (FDA) are all nucleoside/nucleotide analogs targeting HBV reverse transcriptase. Tenofovir disoproxil fumarate (TDF), a prodrug of tenofovir (TFV), is a current first-line treatment for CHB (2) and has demonstrated efficacy in both hepatitis B e antigen (HBeAg)-positive and HBeAg-negative patients (3-5). TDF is also widely used as the backbone of current anti-HIV combination regimens (6, 7). For both HIV and HBV, the mechanism of action of TDF is intracellular metabolism to its pharmacologically active form, tenofovir diphosphate (TFV-DP), followed by competition with endogenous 2=-dATP for incorporation by the viral reverse transcriptase and subsequent chain termination of viral DNA replication (8, 9). TFV-DP is an effective inhibitor of HBV reverse transcriptase, with an inhibitor constant (K i ) of 0.18 M in vitro (9).A new prodrug of tenofovir, tenofovir alafenamide (TAF or GS-7340), was selected to more efficiently load HIV target cells while lowering circulating levels of TFV, resulting in reduced offtarget exposure (10-13). The administration of 25 mg of TAF in HIV-i...

The experimental pharmacoenhancer cobicistat (COBI), a potent mechanism-based inhibitor of cytochrome P450 3A enzymes, was found to inhibit the intestinal efflux transporters P-glycoprotein and breast cancer resistance protein. Consistent with its transporter inhibition, COBI significantly increased the absorptive flux of potential candidates for clinical coadministration, including the HIV protease inhibitors atazanavir and darunavir and the lymphoid cell-and tissue-targeted prodrug of the nucleotide analog tenofovir, GS-7340, through monolayers of Caco-2 cells in vitro.C obicistat (COBI) is being developed as a pharamacoenhancer (booster) for coformulation with drugs that are metabolized by cytochrome P450 3A (CYP3A) enzymes. Similar to ritonavir (RTV; currently used as a pharmacoenhancer of protease inhibitors [PIs] used to treat human immunodeficiency virus [HIV]), COBI is a potent mechanism-based CYP3A inhibitor, and its coadministration with CYP3A substrates can lead to desired boosting effects and unintended drug-drug interactions. COBI has been found to have a number of potentially differentiating attributes relative to RTV: (i) it has more selective CYP3A inhibition over other CYP enzymes, (ii) it has improved solubility and coformulatability, (iii) it has a reduced induction potential mediated by the pregnane X receptor (PXR; also known as the nuclear receptor subfamily 1, group 1, member 2 [NR1I2]), and (iv) it has decreased effects on adipocytes in vitro (21). COBI's lack of anti-HIV activity also eliminates the potential for selection of PI resistance mutations when boosting non-HIV PI drugs (21). Clinically, COBI increases systemic levels of the CYP3A substrates midazolam and elvitegravir (EVG) to a similar extent as RTV (7, 13), and coadministration with COBI allows EVG to be administered once daily (16). A fixed-dose, once-daily, single-tablet regimen that includes EVG/COBI and the nucleos(t)ide reverse transcriptase inhibitors tenofovir disoproxil fumarate and emtricitabine, colloquially known as "QUAD," has completed registrational trials, including meeting its primary endpoints in phase 3 studies (2,3,19,20).Inhibition of efflux transporters expressed in the intestine can serve as a secondary mechanism for a pharmacoenhancer to increase systemic exposure to coadministered drugs by increasing their absorption. P-glycoprotein (Pgp; also known as multidrug resistance protein 1 [MDR1] or ATP-binding cassette subfamily B member 1 [ABCB1]) and the breast cancer resistance protein (BCRP; also known as ATP-binding cassette subfamily G member 2 [ABCG2]), both expressed at the apical side of the small intestine, have been highlighted by regulatory agencies and in the literature as key transporters affecting xenobiotic pharmacokinetics (5, 6). In addition to the role of CYP3A enzymes in RTV boosting, HIV PIs are known to be substrates for transporters, including Pgp (11). Determination of the relative roles of transport and CYP3A inhibition in limiting HIV PI exposure is difficult due to their being sub...

The biguanide metformin is widely used as first-line therapy for the treatment of type 2 diabetes. Predominately a cation at physiological pH’s, metformin is transported by membrane transporters, which play major roles in its absorption and disposition. Recently, our laboratory demonstrated that organic cation transporter 1, OCT1, the major hepatic uptake transporter for metformin, was also the primary hepatic uptake transporter for thiamine, vitamin B1. In this study, we tested the reverse, i.e., that metformin is a substrate of thiamine transporters (THTR-1, SLC19A2, and THTR-2, SLC19A3). Our study demonstrated that human THTR-2 (hTHTR-2), SLC19A3, which is highly expressed in the small intestine, but not hTHTR-1, transports metformin (Km = 1.15 ± 0.2 mM) and other cationic compounds (MPP+ and famotidine). The uptake mechanism for hTHTR-2 was pH and electrochemical gradient sensitive. Furthermore, metformin as well as other drugs including phenformin, chloroquine, verapamil, famotidine, and amprolium inhibited hTHTR-2 mediated uptake of both thiamine and metformin. Species differences in the substrate specificity of THTR-2 between human and mouse orthologues were observed. Taken together, our data suggest that hTHTR-2 may play a role in the intestinal absorption and tissue distribution of metformin and other organic cations and that the transporter may be a target for drug–drug and drug–nutrient interactions.

BackgroundInhibition of the transporter-mediated hepatobiliary elimination of bile salts is a putative mechanism for liver toxicity observed with some endothelin receptor antagonists (ERAs).MethodsSandwich-cultured human hepatocytes were used to study the hepatobiliary distribution and accumulation of exogenous taurocholate, ERAs and endogenous bile acids. The molecular mechanisms for findings in hepatocytes or clinical observations were further explored using either vesicular assays (efflux transporters) or transfected cell-lines (uptake transporters). Inhibition constants (IC50) were measured for the human hepatobiliary transporters bile salt export pump (BSEP), sodium taurocholate cotransporting polypeptide (NTCP), multidrug resistance protein 2 (MRP2), P-glycoprotein (Pgp), breast cancer resistance protein (BCRP), organic anion-transporting polypeptide 1B1 (OATP1B1) and OATP1B3.ResultsThe ERAs showed dose-dependent reductions in exogenous taurocholate cellular accumulation in human hepatocytes, with macitentan having the greatest effect. Consistent with their effects on bile acids, the ERAs inhibited bile transporters. IC50 values for OATP1B1 and OATP1B3 ranged from 2 µM for macitentan to 47 µM for ambrisentan. Macitentan and bosentan also inhibited NTCP with IC50 values of 10 and 36 µM, respectively. Similar to previously reported findings with sitaxsentan, BSEP inhibition was observed for bosentan and macitentan with IC50 values of 42 and 12 µM, respectively. In contrast, ambrisentan showed little or no inhibition of these transporters. Other transporters tested were weakly inhibited by the ERAs. Accumulation in hepatocytes was also a factor in the effects on bile transport. Macitentan demonstrated the greatest accumulation in human hepatocytes (∼100x) followed by sitaxsentan (∼40x), bosentan (∼20x) and ambrisentan (∼2x).ConclusionsSignificant differences in the inhibition of hepatic transporters were observed between the evaluated ERAs in vitro. Macitentan had the highest level of cellular accumulation and caused the greatest effects on bile acid distribution in human hepatocytes followed by sitaxsentan and bosentan. Ambrisentan showed a low potential to affect bile acids.