Design and application of synthetic 17B-HSD13 substrates reveals preserved catalytic activity of protective human variants.

Several hydroxysteroid dehydrogenase 17-beta 13 variants have previously been identified as protective against metabolic dysfunction-associated steatohepatitis (MASH) fibrosis, ballooning and inflammation, and as such this target holds significant therapeutic potential. However, over 5 years later, the function of 17B-HSD13 remains unknown. Structure-aided design enables the development of potent and selective sulfonamide-based 17B-HSD13 inhibitors.

Discovery of First Branched-Chain Ketoacid Dehydrogenase Kinase (BDK) Inhibitor Clinical Candidate PF-07328948.

Inhibition of branched-chain ketoacid dehydrogenase kinase (BDK or BCKDK), a negative regulator of branched-chain amino acid (BCAA) metabolism, is hypothesized to treat cardio-metabolic diseases. From a starting point with potential idiosyncratic toxicity risk, modification to a benzothiophene core and discovery of a cryptic pocket allowed for improved potency with 3-aryl substitution to arrive at PF-07328948, which was largely devoid of protein covalent binding liability. This BDK inhibitor was shown also to be a BDK degrader in cells and in vivo rodent studies.

High-throughput cytochrome P450 loss and metabolic intermediate complex assays to aid in designing out of CYP3A inactivation.

Time-dependent inactivation (TDI) of cytochrome P450 (CYP) enzymes may result in clinical drug-drug interactions (DDIs). Therefore, designing out of CYP TDI prior to advancing a compound to clinical development is highly desirable. As TDI of CYP3A is a common occurrence in small molecule drug discovery, high-throughput methods are sought to help identify the mechanism of inactivation and enable design strategies to mitigate CYP3A TDI.

Human Absorption, Distribution, Metabolism, and Excretion Studies: Origins, Innovations, and Importance.

Human absorption, distribution, metabolism, and excretion (hADME) studies represent one of the most important clinical studies in terms of obtaining a comprehensive and quantitative overview of the total disposition of a drug. This article will provide background on the origins of hADME studies as well as provide an overview of technological innovations that have impacted how hADME studies are carried out and analyzed. An overview of the current state of the art for hADME studies will be provided, the impacts of advances in technology and instrumentation on the timing of and approaches to hADME studies will be discussed, and a summary of the parameters and information obtained from these studies will be offered.

Static and Dynamic Projections of Drug-Drug Interactions Caused by Cytochrome P450 3A Time-Dependent Inhibitors Measured in Human Liver Microsomes and Hepatocytes.

Cytochrome P450 3A (CYP3A) is a frequent target for time-dependent inhibition (TDI) that can give rise to drug-drug interactions (DDI). Yet many drugs that exhibit in vitro TDI for CYP3A do not result in DDI. There were 23 drugs with published clinical DDI evaluated for CYP3A TDI in human liver microsomes (HLM) and hepatocytes (HHEP), and these data were used in static and dynamic models for projecting DDI caused by inactivation of CYP3A in both liver and intestine.

Cytochrome P450 3A Time-Dependent Inhibition Assays Are Too Sensitive for Identification of Drugs Causing Clinically Significant Drug-Drug Interactions: A Comparison of Human Liver Microsomes and Hepatocytes and Definition of Boundaries for Inactivation Rate Constants.

Time-dependent inhibition (TDI) of CYP3A is an important mechanism underlying numerous drug-drug interactions (DDIs), and assays to measure this are done to support early drug research efforts. However, measuring TDI of CYP3A in human liver microsomes (HLMs) frequently yields overestimations of clinical DDIs and thus can lead to the erroneous elimination of many viable drug candidates from further development. In this investigation, 50 drugs were evaluated for TDI in HLMs and suspended human hepatocytes (HHEPs) to define appropriate boundary lines for the TDI parameter rate constant for inhibition (k) at a concentration of 30 µM.

Identification of Glycochenodeoxycholate 3-O-Glucuronide and Glycodeoxycholate 3-O-Glucuronide as Highly Sensitive and Specific OATP1B1 Biomarkers.

The aim of this study was to investigate the sensitivity and specificity of endogenous glycochenodeoxycholate and glycodeoxycholate 3-O-glucuronides (GCDCA-3G and GDCA-3G) as substrates for organic anion transporting polypeptide 1B1 (OATP1B1) in humans. We measured fasting levels of plasma GCDCA-3G and GDCA-3G using liquid chromatography-tandem mass spectrometry in 356 healthy volunteers. The mean plasma levels of both compounds were ~ 50% lower in women than in men (P = 2.

Effective Application of Metabolite Profiling in Drug Design and Discovery.

At one time, biotransformation was a descriptive activity in pharmaceutical development, viewed simply as structural elucidation of drug metabolites, completed only once compounds entered clinical development. Herein, we present our strategic approach using structural elucidation to enable chemistry design/SAR development. The approach considers four questions that often present themselves to medicinal chemists optimizing their compounds for candidate selection: (1) What are the important clearance mechanisms that mediate the disposition of my molecule? (2) Can metabolic liabilities be modulated in a favorable way? (3) Does my compound undergo bioactivation to a reactive metabolite? (4) Do any of the metabolites possess activity, either on- or off-target? An additional question necessary to support compound development relates to metabolites in safety testing (MIST) and our approach also addresses this question.

Identification and Characterization of a Selective Human Carbonyl Reductase 1 Substrate.

During drug discovery efforts targeting inhibition of cytochrome P450 11B2 (CYP11B2)-mediated production of aldosterone as a therapeutic approach for the treatment of chronic kidney disease and hypertension, ()-6-(5-fluoro-4-(1-hydroxyethyl)pyridin-3-yl)-3,4-dihydro-1,8-naphthyridine-1()-carboxamide (1) was identified as a potent and selective inhibitor of CYP11B2. Preclinical studies characterized 1 as low clearance in both in vitro test systems and in vivo in preclinical species. Despite low metabolic conversion, an active ketone metabolite (2) was identified from in vitro metabolite-identification studies.

Dihydrobenzisoxazole-4-one compounds are novel selective inhibitors of aldosterone synthase (CYP11B2) with in vivo activity.

6,7-Dihydro-5H-2,1-benzisoxazol-4-one analogs are potent inhibitors of aldosterone synthase (CYP11B2) with selectivity over the highly homologous enzyme cortisol synthase (CYP11B1). These compounds are unique among inhibitors of CYP11B2 in their lack of a strong-heme binding group such as a pyridine or imidazole. Poor metabolic stability in hepatocyte incubations was found to proceed via a reduction of the isoxazole ring.

Leveraging of Rifampicin-Dosed Cynomolgus Monkeys to Identify Bile Acid 3-O-Sulfate Conjugates as Potential Novel Biomarkers for Organic Anion-Transporting Polypeptides.

In the search for novel bile acid (BA) biomarkers of liver organic anion-transporting polypeptides (OATPs), cynomolgus monkeys received oral rifampicin (RIF) at four dose levels (1, 3, 10, and 30 mg/kg) that generated plasma-free values (0.06, 0.66, 2.

Prevalence of Non-Cytochrome P450-Mediated Metabolism in Food and Drug Administration-Approved Oral and Intravenous Drugs: 2006-2015.

In recent years, claims of increased involvement of non-cytochrome P450 (non-P450) enzymes in the metabolism of drugs have appeared in the literature. However, no temporal summaries of the contribution of non-P450 enzymes to the metabolism of drugs have been published. Using data from human radiolabeled absorption, distribution, metabolism, and excretion studies available for a set of 125 orally or intravenously administered small-molecule drugs approved by the United States Food and Drug Administration from 2006 to 2015, the contributions of P450 and non-P450 enzymes to the formation of major metabolites (≥10% of dose) were assessed and tabulated.

Identification of Human Sulfotransferases Involved in Lorcaserin N-Sulfamate Formation.

Lorcaserin [(R)-8-chloro-1-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine] hydrochloride hemihydrate, a selective serotonin 5-hydroxytryptamine (5-HT) 5-HT(2C) receptor agonist, is approved by the U.S. Food and Drug Administration for chronic weight management.

Development of CYP11B1 and CYP11B2 assays utilizing homogenates of adrenal glands: Utility of monkey as a surrogate for human.

Elevated levels of aldosterone are associated with arterial hypertension, congestive heart failure, chronic kidney disease, and obesity. Aldosterone is produced predominantly in the zona glomerulosa of the cortex of the adrenal gland by the enzyme aldosterone synthase (CYP11B2). Treatment of the above indications by decreasing production of aldosterone is thought to be of therapeutic benefit by lessening the deleterious effects of aldosterone mediated through both the mineralocorticoid receptor and also through so called non-genomic pathways.

Cynomolgus monkey as a surrogate for human aldehyde oxidase metabolism of the EGFR inhibitor BIBX1382.

BIBX1382 was an epidermal growth factor receptor inhibitor under clinical investigation for treatment of cancer. This candidate possessed an attractive preclinical absorption, distribution, metabolism, and excretion profile, yet failed in clinical studies due in part to poor oral exposure, resulting from extensive metabolism by aldehyde oxidase (AO). In vitro metabolism studies were performed in liver cytosol and cryopreserved hepatocytes from multiple species.

G protein-coupled bile acid receptor 1 stimulation mediates arterial vasodilation through a K(Ca)1.1 (BK(Ca))-dependent mechanism.

Bile acids (BAs) and BA receptors, including G protein-coupled bile acid receptor 1 (GPBAR1), represent novel targets for the treatment of metabolic and inflammatory disorders. However, BAs elicit myriad effects on cardiovascular function, although this has not been specifically ascribed to GPBAR1. This study was designed to test whether stimulation of GPBAR1 elicits effects on cardiovascular function that are mechanism based that can be identified in acute ex vivo and in vivo cardiovascular models, to delineate whether effects were due to pathways known to be modulated by BAs, and to establish whether a therapeutic window between in vivo cardiovascular liabilities and on-target efficacy could be defined.

Progress towards clinically useful aldosterone synthase inhibitors.

Owing to the high degree of similarity between aldosterone synthase (CYP11B2) and cortisol synthase (CYP11B1), the design of selective inhibitors of one or the other of these two enzymes was, at one time, thought to be impossible. Through development of novel enzyme screening assays and significant medicinal chemistry efforts, highly potent inhibitors of CYP11B2 have been identified with selectivities approaching 1000-fold between the two enzymes. Many of these molecules also possess selectivity against other steroidogenic cytochromes P450 (e.

Identification of human UDP-glucuronosyltransferases involved in N-carbamoyl glucuronidation of lorcaserin.

Lorcaserin, a selective serotonin 5-HT(2C) receptor agonist, is a weight management agent in clinical development. Lorcaserin N-carbamoyl glucuronidation governs the predominant excretory pathway of lorcaserin in humans. Human UDP-glucuronosyltransferases (UGTs) responsible for lorcaserin N-carbamoyl glucuronidation are identified herein.

Synthetic inhibitors of cytochrome P-450 2A6: inhibitory activity, difference spectra, mechanism of inhibition, and protein cocrystallization.

A series of 3-heteroaromatic analogues of nicotine were synthesized to delineate structural and mechanistic requirements for selectively inhibiting human cytochrome P450 (CYP) 2A6. Thiophene, substituted thiophene, furan, substituted furan, acetylene, imidazole, substituted imidazole, thiazole, pyrazole, substituted pyrazole, and aliphatic and isoxazol moieties were used to replace the N-methylpyrrolidine ring of nicotine. A number of potent inhibitors were identified, and several exhibited high selectivity for CYP2A6 relative to CYP2E1, -3A4, -2B6, -2C9, -2C19, and -2D6.