Coproporphyrin-I: A Fluorescent, Endogenous Optimal Probe Substrate for ABCC2 (MRP2) Suitable for Vesicle-Based MRP2 Inhibition Assay.

Inside-out-oriented membrane vesicles are useful tools to investigate whether a compound can be an inhibitor of efflux transporters such as multidrug resistance-associated protein 2 (MRP2). However, because of technical limitations of substrate diffusion and low dynamic uptake windows for interacting drugs used in the clinic, estradiol-17ß-glucuronide (E17ßG) remains the probe substrate that is frequently used in MRP2 inhibition assays. Here we recapitulated the sigmoidal kinetics of MRP2-mediated transport of E17ßG, with apparent Michaelis-Menten constant (Km) and Vmax values of 170 ±17 µM and 1447 ± 137 pmol/mg protein/min, respectively. The Hill coefficient (2.05 ± 0.1) suggests multiple substrate binding sites for E17ßG transport with cooperative interactions. Using E17ßG as a probe substrate, 51 of 97 compounds tested (53%) showed up to 6-fold stimulatory effects. Here, we demonstrate for the first time that coproporphyrin-I (CP-I) is a MRP2 substrate in membrane vesicles. The uptake of CP-I followed a hyperbolic relationship, adequately described by the standard Michaelis-Menten equation (apparent Km and Vmax values were 7.7 ± 0.7 µM and 48 ± 11 pmol/mg protein/min, respectively), suggesting the involvement of a single binding site. Of the 47 compounds tested, 30 compounds were inhibitors of human MRP2 and 8 compounds (17%) stimulated MRP2-mediated CP-I transport. The stimulators were found to share the basic backbone structure of the physiologic steroids, which suggests a potential in vivo relevance of in vitro stimulation of MRP2 transport. We concluded that CP-I could be an alternative in vitro probe substrate replacing E17ßG for appreciating MRP2 interactions while minimizing potential false-negative results for MRP2 inhibition due to stimulatory effects.

Discovery of non-zwitterionic aryl sulfonamides as Nav1.7 inhibitors with efficacy in preclinical behavioral models and translational measures of nociceptive neuron activation.

Since zwitterionic benzenesulfonamide Nav1.7 inhibitors suffer from poor membrane permeability, we sought to eliminate this characteristic by replacing the basic moiety with non-basic bicyclic acetals and monocyclic ethers. These efforts led to the discovery of the non-zwitterionic aryl sulfonamide 49 as a selective Nav1.7 inhibitor with improved membrane permeability. Despite its moderate cellular activity, 49 exhibited robust efficacy in mouse models of neuropathic and inflammatory pain and modulated translational electromyogram measures associated with activation of nociceptive neurons.

Phosphocholine conjugation: an unexpected in vivo conjugation pathway associated with hepatitis c ns5b inhibitors featuring a bicyclo[1.1.1]pentane.

During a medicinal chemistry campaign to identify inhibitors of the hepatitis C virus nonstructural protein 5B (RNA-dependent RNA polymerase), a bicyclo[1.1.1]pentane was introduced into the chemical scaffold to improve metabolic stability. The inhibitors bearing this feature, 5-(3-(bicyclo[1.1.1]pentan-1-ylcarbamoyl)-4-fluorophenyl)-2-(4-fluorophenyl)-N-methyl-6-(3,3,3-trifluoropropyl)furo[2,3-b]pyridine-3-carboxamide (1) and 5-(3-(bicyclo[1.1.1]pentan-1-ylcarbamoyl)phenyl)-2-(4-fluorophenyl)-N-methyl-6-(3,3,3-trifluoropropyl)furo[2,3-b]pyridine-3-carboxamide (2), exhibited low turnover in incubations with liver S9 or hepatocytes (rat, human), with hydroxylation of the bicyclic moiety being the only metabolic pathway observed. In subsequent disposition studies using bile-duct-cannulated rats, the metabolite profiles of bile samples revealed, in addition to multiple products of bicyclopentane-oxidation, unexpected metabolites characterized by molecular masses that were 181 Da greater than those of 1 or 2. Further LC/MSn and NMR analysis of the isolated metabolite of 1 demonstrated the presence of a phosphocholine (POPC) moiety bound to the methine carbon of the bicyclic moiety through an ester bond. The POPC conjugate of the NS5B inhibitors was assumed to result from two sequential reactions: hydroxylation of the bicyclic methine to a tertiary alcohol and addition of POPC by CDP-choline: 1,2-diacylglycerol cholinephosphotransferase, an enzyme responsible for the final step in the biosynthesis of phosphatidylcholine. However, this pathway could not be recapitulated using CDP-choline-supplemented liver S9 or hepatocytes due to inadequate formation of the hydroxylation product in vitro. The observation of this unexpected pathway prompted concerns about the possibility that 1 and 2 might interfere with routine phospholipid synthesis. These results demonstrate the participation in xenobiotic metabolism of a process whose function is ordinarily limited to the synthesis of endogenous compounds.

Preclinical characterization of BMS-791325, an allosteric inhibitor of hepatitis C Virus NS5B polymerase.

BMS-791325 is an allosteric inhibitor that binds to thumb site 1 of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. BMS-791325 inhibits recombinant NS5B proteins from HCV genotypes 1, 3, 4, and 5 at 50% inhibitory concentrations (IC50) below 28 nM. In cell culture, BMS-791325 inhibited replication of HCV subgenomic replicons representing genotypes 1a and 1b at 50% effective concentrations (EC50s) of 3 nM and 6 nM, respectively, with similar (3 to 18 nM) values for genotypes 3a, 4a, and 5a. Potency against genotype 6a showed more variability (9 to 125 nM), and activity was weaker against genotype 2 (EC50, 87 to 925 nM). Specificity was demonstrated by the absence of activity (EC50s of >4 µM) against a panel of mammalian viruses, and cytotoxic concentrations (50%) were >3,000-fold above the HCV EC50. Resistance substitutions selected by BMS-791325 in genotype 1 replicons mostly mapped to a single site, NS5B amino acid 495 (P495A/S/L/T). Additive or synergistic activity was observed in combination studies using BMS-791325 with alfa interferon plus ribavirin, inhibitors of NS3 protease or NS5A, and other classes of NS5B inhibitor (palm site 2-binding or nucleoside analogs). Plasma and liver exposures in vivo in several animal species indicated that BMS-791325 has a hepatotropic disposition (liver-to-plasma ratios ranging from 1.6- to 60-fold across species). Twenty-four hours postdose, liver exposures across all species tested were ≥ 10-fold above the inhibitor EC50s observed with HCV genotype 1 replicons. These findings support the evaluation of BMS-791325 in combination regimens for the treatment of HCV. Phase 3 studies are ongoing.

Application of an in vitro OAT assay in drug design and optimization of renal clearance.

1. Optimization of renal clearance is a complex balance between passive and active processes mediated by renal transporters. This work aimed to characterize the interaction of a series of compounds with rat and human organic anion transporters (OATs) and develop quantitative structure-activity relationships (QSARs) to optimize renal clearance. 2. In vitro inhibition assays were established for human OAT1 and rat Oat3 and rat in vivo renal clearance was obtained. Statistically significant quantitative relationships were explored between the compounds' physical properties, their affinity for OAT1 and oat3 and the inter-relationship with unbound renal clearance (URC) in rat. 3. Many of the compounds were actively secreted and in vitro analysis demonstrated that these were ligands for rat and human OAT transporters (IC50 values ranging from <1 to >100 µM). Application of resultant QSAR models reduced renal clearance in the rat from 24 to <0.1 ml/min/kg. Data analysis indicated that the properties associated with increasing affinity at OATs are the same as those associated with reducing URC but orthogonal in nature. 4. This study has demonstrated that OAT inhibition data and QSAR models can be successfully used to optimize rat renal clearance in vivo and provide confidence of translation to humans.

Genome-wide analysis of FoxO1 binding in hepatic chromatin: potential involvement of FoxO1 in linking retinoid signaling to hepatic gluconeogenesis.

The forkhead transcription factor FoxO1 is a critical regulator of hepatic glucose and lipid metabolism, and dysregulation of FoxO1 function has been implicated in diabetes and insulin resistance. We globally identified FoxO1 occupancy in mouse hepatic chromatin on a genome-wide level by chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq). To establish the specific functional significance of FoxO1 against other FoxO proteins, ChIP-seq was performed with chromatin from liver-specific FoxO1 knockout and wild-type mice. Here we identified 401 genome-wide FoxO1-binding locations. Motif search reveals a sequence element, 5' GTAAACA 3', consistent with a previously known FoxO1-binding site. Gene set enrichment analysis shows that the data from FoxO1 ChIP-seq are highly correlated with the global expression profiling of genes regulated by FoxO1, demonstrating the functional relevance of our FoxO1 ChIP-seq study. Interestingly, gene ontology analysis reveals the functional significance of FoxO1 in retinoid metabolic processes. We show here that FoxO1 directly binds to the genomic sites for the genes in retinoid metabolism. Notably, deletion of FoxO1 caused a significantly reduced induction of Pck1 and Pdk4 in response to retinoids. As Pck1 and Pdk4 are downstream targets of retinoid signaling, these results suggest that FoxO1 plays a potential role in linking retinoid metabolism to hepatic gluconeogenesis.

Evaluation of cynomolgus monkey pregnane X receptor, primary hepatocyte, and in vivo pharmacokinetic changes in predicting human CYP3A4 induction.

Monkeys have been proposed as an animal model to predict the magnitude of human clinical drug-drug interactions caused by CYP3A4 enzyme induction. To evaluate whether the cynomolgus monkey can be an effective in vivo model, human CYP3A4 inducers were evaluated both in vitro and in vivo. First, a full-length pregnane X receptor (PXR) was cloned from the cynomolgus monkey, and the sequence was compared with those of rhesus monkey and human PXR. Cynomolgus and rhesus monkey PXR differed by only one amino acid (A68V), and both were highly homologous to human PXR (approximately 96%). When the transactivation profiles of 30 compounds, including known inducers of CYP3A4, were compared between cynomolgus and human PXR, a high degree of correlation with EC(50) values was observed. These results suggest that cynomolgus and human PXR respond in a similar fashion to these ligands. Second, two known human CYP3A4 inducers, rifampicin and hyperforin, were tested in monkey and human primary hepatocytes for induction of CYP3A enzymes. Both monkey and human hepatocytes responded similarly to the inducers and resulted in increased RNA and enzyme activity changes of CYP3A8 and CYP3A4, respectively. Lastly, in vivo induction of CYP3A8 by rifampicin and hyperforin was shown by significant reductions of midazolam exposure that were comparable with those in humans. These results show that the cynomolgus monkey can be a predictive in vivo animal model of PXR-mediated induction of human CYP3A4 and can provide a useful assessment of the resulting pharmacokinetic changes of affected drugs.

Development of New Benzenesulfonamides As Potent and Selective Nav1.7 Inhibitors for the Treatment of Pain.

By taking advantage of certain features in piperidine 4, we developed a novel series of cyclohexylamine- and piperidine-based benzenesulfonamides as potent and selective Nav1.7 inhibitors. However, compound 24, one of the early analogs, failed to reduce phase 2 flinching in the mouse formalin test even at a dose of 100 mpk PO due to insufficient dorsal root ganglion (DRG) exposure attributed to poor membrane permeability. Two analogs with improved membrane permeability showed much increased DRG concentrations at doses of 30 mpk PO, but, confoundingly, only one of these was effective in the formalin test. More data are needed to understand the disconnect between efficacy and exposure relationships.

Preclinical Pharmacokinetics and In Vitro Metabolism of Asunaprevir (BMS-650032), a Potent Hepatitis C Virus NS3 Protease Inhibitor.

Asunaprevir (ASV; BMS-650032), a low nanomolar inhibitor of the hepatitis C virus (HCV) NS3 protease, is currently under development, in combination with other direct-acting antiviral (DAA) agents for the treatment of chronic HCV infection. Extensive nonclinical and pharmacokinetic studies have been conducted to characterize the ADME properties of ASV. ASV has a moderate to high clearance in preclinical species. In vitro reaction phenotyping studies demonstrated that the oxidative metabolism of ASV is primarily mediated via CYP3A4; however, studies in bile-duct cannulated rats and dogs suggest that biliary elimination may contribute to overall ASV clearance. ASV is shown to have hepatotropic disposition in all preclinical species tested (liver to plasma ratios >40). The translation of in vitro replicon potency to clinical viral load decline for a previous lead BMS-605339 was leveraged to predict a human dose of 2 mg BID for ASV. Clinical drug-drug interaction (DDI) studies have shown that at therapeutically relevant concentrations of ASV the potential for a DDI is minimal. The need for an interferon free treatment combined with ASV's initial clinical trial data support development of ASV as part of a fixed dose combination for the treatment of patients chronically infected with HCV genotype 1.

Nonclinical pharmacokinetics of BMS-292655, a water-soluble prodrug of the antifungal ravuconazole.

The phosphate ester, BMS-292655, was developed as a water-soluble prodrug of the antifungal agent, ravuconazole (BMS-207147). BMS-292655 was comparatively stable in rat, beagle dog, cynomolgus monkey and human plasma, but was hydrolysed upon incubation with liver S9 preparations from all species. The major product in rat, monkey and human S9 was BMS-207147, while in dog S9, the intermediate ester, BMS-300043, predominated. BMS-300043 itself was more stable in dog S9 than in S9 preparations from the other species. Intravenous administration of BMS-292655 to rats, beagle dogs and cynomolgus monkeys indicated species differences in the extent of formation of BMS-207147 (monkeys>rats>dogs). The lower overall generation of BMS-207147 in dogs was consistent with the presence of circulating plasma levels of BMS-300043. BMS-300043 was present in monkey plasma but not detectable in rat plasma. The conversion of BMS-292655 to BMS-207147 in the presence of human S9 indicated the potential for BMS-292655 to function as a BMS-207147 prodrug in humans. The similarity in the hydrolysis of BMS-292655 when incubated with human and monkey S9 in vitro, coupled with the effective release of BMS-207147 from BMS-292655 upon i.v. administration to monkeys, is consistent with this conclusion.

Biaryl isoxazolinone antibacterial agents.

In an era of increasing resistance to classical antibacterial agents, the synthetic oxazolidinone series of antibiotics has attracted much interest. Zyvoxtrade mark was the first oxazolidinone to be approved for clinical use against infections caused by multi-drug resistant Gram-positive bacteria. In the course of studies directed toward the discovery of novel antibacterial agents, a new series of synthetic phenyl-isoxazolinone agents that displayed potent activity against Gram-positive bacterial strains was recently discovered at Bristol-Myers Squibb. Extensive investigation of various substitutions on the phenyl ring was then undertaken. We report here, the synthesis and antibacterial activity of a series of biaryl isoxazolinone compounds.

Cathepsin B-labile dipeptide linkers for lysosomal release of doxorubicin from internalizing immunoconjugates: model studies of enzymatic drug release and antigen-specific in vitro anticancer activity.

The anticancer drug doxorubicin (DOX) has been linked to chimeric BR96, an internalizing monoclonal antibody that binds to a Lewis(y)-related, tumor-associated antigen, through two lysosomally cleavable dipeptides, Phe-Lys and Val-Cit, giving immunoconjugates 72 and 73. A self-immolative p-aminobenzyloxycarbonyl (PABC) spacer between the dipeptides and the DOX was required for rapid and quantitative generation of free drug. DOX release from model substrate Z-Phe-Lys-PABC-DOX 49 was 30-fold faster than from Z-Val-Cit-PABC-DOX 42 with the cysteine protease cathepsin B alone, but rates were identical in a rat liver lysosomal preparation suggesting the participation of more than one enzyme. Conjugates 72 and 73 showed rapid and near quantitative drug release with cathepsin B and in a lysosomal preparation, while demonstrating excellent stability in human plasma. Against tumor cell lines with varying levels of BR96 expression, both conjugates showed potent, antigen-specific cytotoxic activity, suggesting that they will be effective in delivering DOX selectively to antigen-expressing carcinomas.