Addressing time-dependent CYP 3A4 inhibition observed in a novel series of substituted amino propanamide renin inhibitors, a case study.

Time-dependent inhibitors of CYPs have the potential to perpetrate drug-drug interactions in the clinical setting. After finding that several leading compounds in a novel series of substituted amino propanamide renin inhibitors inactivated CYP3A4 in an NADPH-dependent and time-dependent manner, a search to identify the cause of this liability was initiated. Extensive SAR revealed that the amide bridge present in compound 1 as a possible culprit. Through the installation of a metabolic soft spot distal to this moiety, potent renin inhibitors with improved CYP profile were identified.

Design and optimization of a substituted amino propanamide series of renin inhibitors for the treatment of hypertension.

The discovery and SAR of a new series of substituted amino propanamide renin inhibitors are herein described. This work has led to the preparation of compounds with in vitro and in vivo profiles suitable for further development. Specifically, challenges pertaining to oral bioavailability, covalent binding and time-dependent CYP 3A4 inhibition were overcome thereby culminating in the identification of compound 50 as an optimized renin inhibitor with good efficacy in the hypertensive double-transgenic rat model.

The discovery of 4-{1-[({2,5-dimethyl-4-[4-(trifluoromethyl)benzyl]-3-thienyl}carbonyl)amino]cyclopropyl}benzoic acid (MK-2894), a potent and selective prostaglandin E2 subtype 4 receptor antagonist.

The discovery of highly potent and selective second generation EP(4) antagonist MK-2894 (34d) is discussed. This compound exhibits favorable pharmacokinetic profile in a number of preclinical species and potent anti-inflammatory activity in several animal models of pain/inflammation. It also shows favorable GI tolerability profile in rats when compared to traditional NSAID indomethacin.

High-throughput cytochrome P450 inhibition assays using laser diode thermal desorption-atmospheric pressure chemical ionization-tandem mass spectrometry.

This paper describes the development of a high-throughput method for the analysis of cytochrome P450 (CYP) inhibition assay incubation samples using laser diode thermal desorption interfaced with atmospheric pressure chemical ionization mass spectrometry (LDTD-APCI-MS). Data for the CYP isoforms 3A4, 2D6, 2C9, and 1A2 from competitive inhibition assays are shown. The potential for inhibition of the CYP isoforms was measured by monitoring the level of the metabolites 6beta-hydroxytestosterone (3A4), dextrorphan (2D6), 4'-hydroxydiclofenac (2C9), and acetaminophen (1A2) formed in the presence of drug candidates using an eight-point titration. The analytical method involves plating of the inhibition samples on specially designed 96-well plates with stainless steel bottoms, followed by direct analysis using the LDTD source. Validation of the LDTD-MS method was performed by testing for interferences, reproducibility, dynamic range, ion suppression, and the ability of the source to produce comparable results to previously validated LC-MS methods. IC50 values for each CYP isoform using 33 different test compounds showed excellent agreement between LDTD-APCI-MS and LC-MS methods and literature values where available. Assay analysis time using the LDTD-APCI source is reduced to less than 30 min for a single 96-well plate compared to greater than 10 h using the LC-MS method. The LDTD-APCI-MS and LC-MS methods and results are compared and limitations and future potential for LDTD-APCI-MS are discussed.

Discovery, biosynthesis, and structure elucidation of new metabolites of norandrostenedione using in vitro systems.

The aim of our study was to demonstrate the positive impact that in vitro systems could have on the synthesis and characterization of unknown metabolites of banned doping agents. Using norandrostenedione (estr-4-en-3,17-dione), we were able to identify and characterize by GC/MS and LC/UV/MS several new hydroxylated metabolites formed in human hepatocyte incubations. The site of hydroxylation of M1, M2, M3, and M5 was demonstrated to be at C-6beta position by incubating estr-4-en-6beta-ol-3,17-dione (M4), which is the direct 6beta-hydroxylated metabolite of norandrostenedione. The structure of M5 was confirmed to be estr-4-en-6beta,17beta-diol-3-one (6beta-hydroxynortestosterone) using a commercially available authentic standard. For the other metabolites, M1, M2, and M3, no standards were available. Due to limited access to fresh human liver tissues, in vitro incubation conditions in rat liver subcellular fractions and hepatocytes were optimized as an alternative to produce sufficient quantities of the unknown metabolites for MS and/or NMR characterization. The structure of M1 was assigned to 5alpha-estran-3alpha,6beta-diol-17-one (6beta-hydroxynorandrosterone) and M3 to 5alpha-estran-3beta,6beta-diol-17-one (6beta-hydroxynorepiandrosterone) based on NMR data. M2 is proposed to be 5beta-estran-3alpha,6beta-diol-17-one (6beta-hydroxynoretiocholanolone) based on GC/MS fragmentation of the TMS-enol bis-TMS-ether derivative. The in vitro approach reported here, in addition to urinary excretion studies in humans, could contribute significantly to the discovery, the synthesis, and structure elucidation of new markers of doping agents.

Evaluation of human hepatocyte incubation as a new tool for metabolism study of androstenedione and norandrostenedione in a doping control perspective.

Human hepatocyte incubations were used to study the metabolism of precursors of testosterone and nortestosterone and to evaluate qualitatively the correlation between in vitro and published in vivo urinary metabolic profiles. Both phase I and phase II biotransformations were observed in vitro: oxidoreduction at C-3 and C-17, reduction at C-4,5, hydroxylation at C-6 beta and C-16, glucuronidation and sulfation. All major metabolites detected in urine following oral administration of androstenedione and norandrostenedione were present in human hepatocyte incubations. The good correlation between in vitro and in vivo metabolic profiles indicates that hepatocyte incubations can be a useful tool to identify and characterize metabolites that could be potential urinary markers for detection of steroid abuse by athletes.