The role of lymphatic transport on the systemic bioavailability of the Bcl-2 protein family inhibitors navitoclax (ABT-263) and ABT-199.

Navitoclax (ABT-263), a Bcl-2 family inhibitor and ABT-199, a Bcl-2 selective inhibitor, are high molecular weight, high logP molecules that show low solubility in aqueous media. While these properties are associated with low oral bioavailability (F), both navitoclax and ABT-199 showed moderate F in preclinical species. The objective of the described study was to determine if lymphatic transport contributes to the systemic availability of navitoclax and ABT-199 in dogs. The intravenous pharmacokinetics of navitoclax and ABT-199 were determined in intact (noncannulated) dogs. In oral studies, tablets (100 mg) of navitoclax and ABT-199 were administered to both intact and thoracic lymph duct-cannulated (TDC) dogs. The clearance of navitoclax and ABT-199 was low; 0.673 and 0.779 ml/min per kilogram, respectively. The volume of distribution of both compounds was low (0.5-0.7 l/kg). The half-lives of navitoclax and ABT-199 were 22.2 and 12.9 hours, respectively. The F of navitoclax and ABT-199 were 56.5 and 38.8%, respectively, in fed intact dogs. In fed TDC dogs, 13.5 and 4.67% of the total navitoclax and ABT-199 doses were observed in lymph with the % F of navitoclax and ABT-199 of 21.7 and 20.2%, respectively. The lower lymphatic transport of ABT-199 corresponds to the lower overall % F of ABT-199 versus navitoclax despite similar systemic availability via the portal vein (similar % F in TDC animals). This is consistent with the higher long chain triglyceride solubility of navitoclax (9.2 mg/ml) versus ABT-199 (2.2 mg/ml). In fasted TDC animals, lymph transport of navitoclax and ABT-199 decreased by 1.8-fold and 10-fold, respectively.

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.

Disposition and drug-drug interaction potential of veliparib (ABT-888), a novel and potent inhibitor of poly(ADP-ribose) polymerase.

The disposition of veliparib [(R)-2-(2-methylpyrrolidin-2-yl)-1H-benzo[d]imidazole-4-carboxamide, ABT-888], a novel and potent inhibitor of poly(ADP-ribose) polymerase for the treatment of cancers, was investigated in rats and dogs after intravenous and oral administration of [(3)H]veliparib and compared with that of humans. Veliparib absorption was high. Dosed radioactivity was widely distributed in rat tissues. The majority of drug-related material was excreted in urine as unchanged drug (approximately 54, 41, and 70% of the dose in rats, dogs, and humans, respectively). A lactam M8 and an amino acid M3 were two major excretory metabolites in animals. In the circulation of animals and humans, veliparib was the major drug-related component, and M8 was one of the major metabolites. Monooxygenated metabolite M2 was significant in the rat and dog, and M3 was also significant in the dog. Veliparib biotransformation occurred on the pyrrolidine moiety via formation of a lactam, an amino acid, and an N-carbamoyl glucuronide, in addition to oxidation on benzoimidazole carboxamide and sequential glucuronidation. In vitro experiments using recombinant human cytochrome P450 (P450) enzymes identified CYP2D6 as the major enzyme metabolizing veliparib with minor contributions from CYP1A2, 2C19, and 3A4. Veliparib did not inhibit or induce the activities of major human P450s. Veliparib was a weak P-glycoprotein (P-gp) substrate, showing no P-gp inhibition. Taken together, these studies indicate a low potential for veliparib to cause clinically significant P-gp or P450-mediated drug-drug interactions (DDIs). Overall, the favorable dispositional and DDI profiles of veliparib should be beneficial to its safety and efficacy.

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.

Hydrolysis of a series of parabens by skin microsomes and cytosol from human and minipigs and in whole skin in short-term culture.

Parabens are esters of 4-hydroxybenzoic acid and used as anti-microbial agents in a wide variety of toiletries, cosmetics and pharmaceuticals. It is of interest to understand the dermal absorption and hydrolysis of parabens, and to evaluate their disposition after dermal exposure and their potential to illicit localised toxicity. The use of minipig as a surrogate model for human dermal metabolism and toxicity studies, justifies the comparison of paraben metabolism in human and minipig skin. Parabens are hydrolysed by carboxylesterases to 4-hydroxybenzoic acid. The effects of the carboxylesterase inhibitors paraoxon and bis-nitrophenylphosphate provided evidence of the involvement of dermal carboxylesterases in paraben hydrolysis. Loperamide, a specific inhibitor of human carboxylesterase-2 inhibited butyl- and benzylparaben hydrolysis in human skin but not methylparaben or ethylparaben. These results show that butyl- and benzylparaben are more selective substrates for human carboxylesterase-2 in skin than the other parabens examined. Parabens applied to the surface of human or minipig skin were absorbed to a similar amount and metabolised to 4-hydroxybenzoic acid during dermal absorption. These results demonstrate that the minipig is a suitable model for man for assessing dermal absorption and hydrolysis of parabens, although the carboxylesterase profile in skin differs between human and minipig.

Specificity of procaine and ester hydrolysis by human, minipig, and rat skin and liver.

The capacity of human, minipig, and rat skin and liver subcellular fractions to hydrolyze the anesthetic ester procaine was compared with carboxylesterase substrates 4-methylumbelliferyl-acetate, phenylvalerate, and para-nitrophenylacetate and the arylesterase substrate phenylacetate. Rates of procaine hydrolysis by minipig and human skin microsomal and cytosolic fractions were similar, with rat displaying higher activity. Loperamide inhibited procaine hydrolysis by human skin, suggesting involvement of human carboxylesterase hCE2. The esterase activity and inhibition profiles in the skin were similar for minipig and human, whereas rat had a higher capacity to metabolize esters and a different inhibition profile. Minipig and human liver and skin esterase activity was inhibited principally by paraoxon and bis-nitrophenyl phosphate, classical carboxylesterase inhibitors. Rat skin and liver esterase activity was inhibited additionally by phenylmethylsulfonyl fluoride and the arylesterase inhibitor mercuric chloride, indicating a different esterase profile. These results have highlighted the potential of skin to hydrolyze procaine following topical application, which possibly limits its pharmacological effect. Skin from minipig used as an animal model for assessing transdermal drug preparations had similar capacity to hydrolyze esters to human skin.

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.

Comparison of paraben stability in human and rat skin.

Parabens are widely used preservatives in topical products, and are estrogenic in numerous experimental models. The typical cutaneous metabolism model, rat skin, hydrolyzes parabens much faster than human skin. Chronic application and absorption of parabens, combined with low metabolism rates, may lead to prolonged estrogenic effects in the skin.

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.

Comparison of skin esterase activities from different species.

PURPOSE: Many topically applied drugs contain esters that are hydrolyzed in the skin. Minipigs have emerged as potential models of human dermatology and, in some aspects, may be superior to commonly used rat skin. The aims of this study were to evaluate the suitability of minipig and rat skin as in vitro models of human epidermal esterase activity. METHODS: Naphthyl acetate and para-nitrophenyl acetate were tested as prototypical substrates of carboxylesterases from skin, plasma, and liver. Reaction products were monitored by high-performance liquid chromatography/ultraviolet analysis. RESULTS: Hydrolysis efficiency in skin was higher than plasma, but lower than liver. The esterase efficiency of rat skin microsomes (580-1100 min(-1) mg(-1)) was two to three orders of magnitude higher than human (1.3-4.2 min(-1) mg(-1)) and minipig microsomes (1.2-4.2 min(-1) mg(-1)). Rat skin cytosol (80-100 min(-1) mg(-1)) was 2- to 10-fold more efficient than human (2.4-67 min(-1) mg(-1)) or minipig cytosol (18-61 min(-1) mg(-1)). Most importantly, human skin fractions displayed kinetics of hydrolysis very similar to minipig skin. CONCLUSIONS: These studies show minipig skin as an appropriate, potentially valuable model for human epidermal ester metabolism and support the use of minipig skin in preclinical development of topically applied compounds.