In vitro OATP1B1 and OATP1B3 inhibition is associated with observations of benign clinical unconjugated hyperbilirubinemia.

1. Transient benign unconjugated hyperbilirubinemia has been observed clinically with several drugs including indinavir, cyclosporine, and rifamycin SV. Genome-wide association studies have shown significant association of OATP1B1 and UGT1A1 with elevations of unconjugated bilirubin, and OATP1B1 inhibition data correlated with clinical unconjugated hyperbilirubinemia for several compounds. 2. In this study, inhibition of OATP1B3 and UGT1A1, in addition to OATP1B1, was explored to determine whether one measure offers value over the other as a potential prospective tool to predict unconjugated hyperbilirubinemia. OATP1B1 and OATP1B3-mediated transport of bilirubin was confirmed and inhibition was determined for atazanavir, rifampicin, indinavir, amprenavir, cyclosporine, rifamycin SV and saquinavir. To investigate the intrinsic inhibition by the drugs, both in vivo Fi (fraction of intrinsic inhibition) and R-value (estimated maximum in vivo inhibition) for OATP1B1, OATP1B3 and UGT1A1 were calculated. 3. The results indicated that in vivo Fi values >0.2 or R-values >1.5 for OATP1B1 or OATP1B3, but not UGT1A1, are associated with previously reported clinical cases of drug-induced unconjugated hyperbilirubinemia. 4. In conclusion, inhibition of OATP1B1 and/or OATP1B3 along with predicted human pharmacokinetic data could be used pre-clinically to predict potential drug-induced benign unconjugated hyperbilirubinemia in the clinic.

Prediction of clinical drug-drug interactions of veliparib (ABT-888) with human renal transporters (OAT1, OAT3, OCT2, MATE1, and MATE2K).

Veliparib (ABT-888) is largely eliminated as parent drug in human urine (70% of the dose). Renal unbound clearance exceeds glomerular filtration rate, suggesting the involvement of transporter-mediated active secretion. Clinically relevant pharmacokinetic interactions in the kidney have been associated with OAT1, OAT3, OCT2, MATE1, and MATE2K. In the present study, interactions of veliparib with these transporters were investigated. Veliparib inhibited OAT1, OAT3, OCT2, MATE1, and MATE2K with IC50 values of 1371, 505, 3913, 69.9, and 69.5 µM, respectively. The clinical unbound maximum plasma concentration of veliparib after single oral dose of 50 mg (0.45 µM) is manyfold lower than IC50 values for OAT1, OAT3, OCT2, MATE1, or MATE2K. These results indicate a low potential for drug-drug interaction (DDI) with OAT1/3, OCT2, or MATE1/2K. Additional studies demonstrated that veliparib is a substrate of OCT2. In Oct1/Oct2 double-knockout mice, the plasma exposure of veliparib was increased by 1.5-fold, and the renal clearance was decreased by 1.8-fold as compared with wild-type mice, demonstrating that organic cation transporters contribute to the renal elimination in vivo. In summary, the in vitro transporter data for veliparib predicts minimal potential for an OAT1/3-, OCT2-, and MATE1/2K-mediated DDI given the clinical exposure after single oral dose of 50 mg.

Development of in vitro methods to predict induction of CYP1A2 and CYP3A4 in humans.

The important role of cytochrome P450 (CYP) drug-metabolizing enzymes has been studied for many years, and the potential liabilities of inducing these enzymes are well understood. Though several mechanisms of induction have been studied, a growing consensus is developing that the aryl hydrocarbon receptor (AHR) and the pregnane X receptor (PXR) have evolved as the primary mechanisms responsible for clinically relevant drug-drug interactions caused by induction of drug-metabolizing factors. AHR and PXR have been identified as inducers of a variety of Phase I and Phase II drug-metabolizing enzymes, drug transporters, and other factors involved in drug metabolism. Though many genes are induced through these regulating factors, CYP1A2 and CYP3A4 have been the most reliable biomarkers to identify compounds with potential induction liabilities through AHR and PXR, respectively. Here are presented several in vitro methods to detect AHR- and PXR-mediated induction of CYP1A2 and CYP3A4 in fresh and cryopreserved primary human hepatocytes, stable transfectants, and transiently transfected immortalized cells.

Parabens inhibit human skin estrogen sulfotransferase activity: possible link to paraben estrogenic effects.

Parabens (p-hydroxybenzoate esters) are a group of widely used preservatives in topically applied cosmetic and pharmaceutical products. Parabens display weak associations with the estrogen receptors in vitro or in cell based models, but do exhibit estrogenic effects in animal models. It is our hypothesis that parabens exert their estrogenic effects, in part, by elevating levels of estrogens through inhibition of estrogen sulfotransferases (SULTs) in skin. We report here the results of a structure-activity-relationship of parabens as inhibitors of estrogen sulfation in human skin cytosolic fractions and normal human epidermal keratinocytes. Similar to reports of paraben estrogenicity and estrogen receptor affinity, the potency of SULT inhibition increased as the paraben ester chain length increased. Butylparaben was found to be the most potent of the parabens in skin cytosol, yielding an IC(50) value of 37+/-5 microM. Butylparaben blocked the skin cytosol sulfation of estradiol and estrone, but not the androgen dehydroepiandrosterone. The parabens were also tested as inhibitors of SULT activity in a cellular system, with normal human epidermal keratinocytes. The potency of butylparaben increased three-fold in these cells relative to the IC(50) value from skin cytosol. Overall, these results suggest chronic topical application of parabens may lead to prolonged estrogenic effects in skin as a result of inhibition of estrogen sulfotransferase activity. Accordingly, the skin anti-aging benefits of many topical cosmetics and pharmaceuticals could be derived, in part, from the estrogenicity of parabens.

Metabolic aromatization of N-alkyl-1,2,3,4-tetrahydroquinoline substructures to quinolinium by human liver microsomes and horseradish peroxidase.

Metabolic aromatization of xenobiotics is an unusual reaction with some documented examples. For instance, the oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine to the neurotoxic pyridinium ion metabolite 1-methyl-4-phenylpyridinium by monoamine oxidase (MAO) B in the brain has been of interest to a number of investigators. It has also been reported that although the aromatization of N-methyl-tetrahydroisoquinoline occurs with MAO B, the metabolism does not proceed for its isomer, N-methyl-tetrahydroquinoline, by the same enzyme. The aromatization of an N-alkyl-tetrahydroquinoline substructure was identified during in vitro metabolite profiling of compound A, which was designed as a potent renin inhibitor for the treatment of hypertension. The N-alkylquinolinium metabolite of compound A was identified by liquid chromatography-tandem mass spectrometry of human liver microsomal incubates and proton NMR of the isolated metabolite. Further in vitro metabolism studies with a commercially available chemical (compound B), containing the same substructure, also generated an N-alkylquinolinium metabolite. In vitro cytochrome P450 (P450) reaction phenotyping of compound A revealed that the metabolism was catalyzed exclusively by CYP3A4. Although compound B was a substrate for several P450 isoforms, its quinolinium metabolite was also generated predominantly by CYP3A4. Neither compound A nor compound B was a substrate of MAOs. The quinolinium metabolites were readily produced by horseradish peroxidase, suggesting that aromatization of the N-alkyltetrahydroquinoline could occur via a mechanism involving single electron transfer from nitrogen. Although dihydro intermediates from the tetrahydroquinoline substrates were not observed in the formation of quinolinium metabolites, cyanide trapping results indicated the occurrence of iminium intermediates.