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Purpose: A same-day PET imaging agent capable of measuring PD-L1 status in tumors is an important tool for optimizing PD-1 and PD-L1 treatments. Herein we describe the discovery and evaluation of a novel, fluorine-18 labeled macrocyclic peptide-based PET ligand for imaging PD-L1. Methods: [18F]BMS-986229 was synthesized via copper mediated click-chemistry to yield a PD-L1 PET ligand with picomolar affinity and was tested as an in-vivo tool for assessing PD-L1 expression. Results: Autoradiography showed an 8:1 binding ratio in L2987 (PD-L1 (+)) vs. HT-29 (PD-L1 (-)) tumor tissues, with >90% specific binding. Specific radioligand binding (>90%) was observed in human non-small-cell lung cancer (NSCLC) and cynomolgus monkey spleen tissues. Images of PD-L1 (+) tissues in primates were characterized by high signal-to-noise, with low background signal in non-expressing tissues. PET imaging enabled clear visualization of PD-L1 expression in a murine model in vivo, with 5-fold higher uptake in L2987 (PD-L1 (+)) than in control HT-29 (PD-L1 (-)) tumors. Moreover, this imaging agent was used to measure target engagement of PD-L1 inhibitors (peptide or mAb), in PD-L1 (+) tumors as high as 97%. Conclusion: A novel 18F-labeled macrocyclic peptide radioligand was developed for PET imaging of PD-L1 expressing tissues that demonstrated several advantages within a nonhuman primate model when compared directly to adnectin- or mAb-based ligands. Clinical studies are currently evaluating [18F]BMS-986229 to measure PD-L1 expression in tumors. [ABSTRACT FROM AUTHOR]

The incidence of systemic lupus erythematosus (SLE) is about nine times higher in women than in men, and the underlying mechanisms that contribute to this gender bias are not fully understood. Previously, using lupus‐prone (SWR × NZB)F1 (SNF1) mice, we have shown that the intestinal immune system could play a role in the initiation and progression of disease in SLE, and depletion of gut microbiota produces more pronounced disease protection in females than in males. Here, we show that the gut permeability features of lupus‐prone female SNF1 mice at juvenile ages directly correlate with the expression levels of pro‐inflammatory factors, faecal IgA abundance and nAg reactivity and the eventual systemic autoantibody levels and proteinuria onset. Furthermore, we observed that the disease protection achieved in female SNF1 mice upon depletion of gut microbiota correlates with the diminished gut inflammatory protein levels, intestinal permeability and circulating microbial DNA levels. However, faecal microbiota transplant from juvenile male and females did not result in modulation of gut inflammatory features or permeability. Overall, these observations suggest that the early onset of intestinal inflammation, systemic autoantibody production and clinical stage disease in lupus‐prone females is linked to higher gut permeability in them starting at as early as juvenile age. While the higher gut permeability in juvenile lupus‐prone females is dependent on the presence of gut microbes, it appears to be independent of the composition of gut microbiota. [ABSTRACT FROM AUTHOR]

BMS-932481 was designed to modulate ɣ-secretase activity to produce shorter and less amyloidogenic peptides, potentially averting liabilities associated with complete enzymatic inhibition. Although it demonstrated the intended pharmacology in the clinic, BMS-932481 unexpectedly caused drug-induced liver injury (DILI) in a multiple ascending dose study characterized by dose- and exposure-dependence, delayed onset manifestation, and a high incidence of hepatocellular damage. Retrospective studies investigating the disposition and probable mechanisms of toxicity of BMS-932481 are presented here. These included a mass balance study in bile-duct-cannulated rats and a metabolite profiling study in human hepatocytes, which together demonstrated oxidative metabolism followed by biliary elimination as the primary means of disposition. Additionally, minimal protein covalent binding in hepatocytes and lack of bioactivation products excluded reactive metabolite formation as a probable toxicological mechanism. However, BMS-932481 and 3 major oxidative metabolites were found to inhibit the bile salt export pump (BSEP) and multidrug resistance protein 4 (MRP4) in vitro. Considering human plasma concentrations, the IC50 values against these efflux transporters were clinically meaningful, particularly in the high dose cohort. Active uptake into human hepatocytes in vitro suggested the potential for hepatic levels of BMS-932481 to be elevated further above plasma concentrations, enhancing DILI risk. Conversely, measures of mitochondrial functional decline in hepatocytes treated with BMS-932481 were minimal or modest, suggesting limited contributions to DILI. Collectively, these findings suggested that repeat administration of BMS-932481 likely resulted in high hepatic concentrations of BMS-932481 and its metabolites, which disrupted bile acid transport via BSEP and MRP4, elevating serum biomarkers of liver injury. [ABSTRACT FROM AUTHOR]

It is estimated that more than 170 million people are infected with hepatitis C virus (HCV) worldwide (1,2). Clinical trials have demonstrated that, for the first time in human history, [...]

Progressive destruction of pancreatic islet β-cells by immune cells is a primary feature of type 1 diabetes (T1D) and therapies that can restore the functional β-cell mass are needed to alleviate disease progression. Here, we report the use of mesenchymal stromal/stem cells (MSCs) for the production and delivery of Gastrin, a peptide hormone that is produced by intestinal cells and foetal islets and can increase β-Cell mass, to promote protection from T1D. A single injection of syngeneic MSCs that were engineered to express Gastrin (Gastrin-MSCs) caused a significant delay in hyperglycaemia in non-obese diabetic (NOD) mice compared to engineered control-MSCs. Similar treatment of early-hyperglycaemic mice caused the restoration of euglycemia for a considerable duration, and these therapeutic effects were associated with the protection of, and/or higher frequencies of, insulin-producing islets and less severe insulitis. While the overall immune cell phenotype was not affected profoundly upon treatment using Gastrin-MSCs or upon in vitro culture, pancreatic lymph node cells from Gastrin-MSC treated mice, upon ex vivo challenge with self-antigen, showed a Th2 and Th17 bias, and diminished the diabetogenic property in NOD-Rag1 deficient mice suggesting a disease protective immune modulation under Gastrin-MSC treatment associated protection from hyperglycaemia. Overall, this study shows the potential of production and delivery of Gastrin in vivo, by MSCs, in protecting insulin-producing β-cells and ameliorating the disease progression in T1D. [ABSTRACT FROM AUTHOR]

This annual review is the sixth of its kind since 2016 (see references). Our objective is to explore and share articles which we deem influential and significant in the field of biotransformation and bioactivation. These fields are constantly evolving with new molecular structures and discoveries of corresponding pathways for metabolism that impact relevant drug development with respect to efficacy and safety. Based on the selected articles, we created three sections: (1) drug design, (2) metabolites and drug metabolizing enzymes, and (3) bioactivation and safety (Table 1). Unlike in years past, more biotransformation experts have joined and contributed to this effort while striving to maintain a balance of authors from academic and industry settings. Table 1. Articles covered in this review. Title First author Source Drug design 1 Metabolic and pharmaceutical aspects of fluorinated compounds BM Johnson J Med Chem 63: 6315–6386, 2020 2 Effective application of metabolite profiling in drug design and discovery MA Cerny J Med Chem 63: 6387–6406, 2020 3 Understanding the metabolism of proteolysis targeting chimeras (PROTACs): the next step toward pharmaceutical applications L Goracci J Med Chem 63: 11615–11638, 2020 4 Late-stage lead diversification coupled with quantitative nuclear magnetic resonance spectroscopy to identify new structure-activity relationship vectors at nanomole-scale synthesis: application to loratidine, a human histamine H1 receptor inverse agonist MS Lall J. Med. Chem 63: 7268–7292, 2020 5 GLORYx: prediction of the metabolites resulting from phase 1 and Phase 2 biotransformations of xenobiotics Christina de Bruyn Kops Chem Res Toxicol 34: 286–299, 2021 Metabolites & drug metabolizing enzymes 6 Inhibition of human UDP-glucuronosyltransferase (UGT) enzymes by kinase inhibitors: effects of dabrafenib, ibrutinib, nintedanib, trametinib and BIBF 1202. P. Korprasertthaworn Biochem Pharmacol 169: 113616, 2019 7 Highly selective inhibition of tyrosine kinase (TYK2) for the treatment of autoimmune diseases: discovery of the allosteric inhibitor BMS-986165 ST Wrobleski ST J Med Chem 62: 8973–8995, 2019 8 Discovery of a Novel deaminated metabolite of a single-stranded oligonucleotide in vivo by mass spectrometry Jing Li Bioanalysis 11: 1955–1966, 2019 9 In vitro metabolism of 2′‐ribose unmodified and modified phosphorothioate oligonucleotide therapeutics using liquid chromatography mass spectrometry Jaeah Kim Biomedical Chromatography 34: e4839, 2020 10 Excretion, mass balance, and metabolism of [14C]LY3202626 in humans: an interplay of microbial reduction, reabsorption, and aldehyde oxidase oxidation that leads to an extended excretion profile K Katyayan Drug Metab Dispos 48: 698–707, 2020 11 Novel homodimer metabolites of GDC-0994 via cytochrome P450-catalyed radical coupling RH Takahashi Drug Metab Dispos 48: 521–527, 2020 12 Metabolism and disposition of volanesorsen, a 2'- O-(2 methoxyethyl) antisense oligonucleotide, across species. N Post Drug Metab Dispos 47: 1164–1173, 2019 Bioactivation and safety 13 Novel o-toluidine metabolite in rat urine associated with urinary bladder carcinogenesis Y Tajima Chem Res Toxicol 33: 1907–1914, 2020 14 Identifying cysteine, N-Acetylcysteine, and glutathione conjugates as novel metabolites of aristolochic acid I: emergence of a new detoxifying pathway J Zhang Chem Res Toxicol. 33: 1374–1381, 2020 15 Significance of multiple bioactivation pathways for meclofenamate as revealed through modeling and reaction kinetics MA Schleiff Drug Metab Dispos 49: 133–141, 2021 16 Detoxication versus bioactivation pathways of lapatinib in vitro: UGT1A1 catalyzes the hepatic glucuronidation of debenzylated lapatinib. DT Nardone-White Drug Metab Dispos 49: 233–244, 2021 17 Discovery of JNJ-63576253: a clinical stage androgen receptor antagonist for F877L mutant and wild-type castration-resistant prostate cancer (mCRPC) Z Zhang J Med Chem 64: 909–924, 2021 18 Bioactivation of α,β-unsaturated carboxylic acids through acyl-glucuronidation T Mulder Drug Metab Dispos 48: 819–829, 2020 19 Strategies to mitigate the bioactivation of aryl amines C Zhang Chem Res Tox 33: 1950 − 1959, 2020 20 Investigation of clozapine and olanzapine reactive metabolite formation and protein binding by liquid chromatography-tandem mass spectrometry T Geib Chem Res Tox 33: 2420–2431, 2020 21 Application of a rat liver drug bioactivation transcriptional response assay early in drug development that informs chemically reactive metabolite formation and potential for drug-induced liver injury JJ Monroe Toxicol Sci 177: 281–299, 2020 [ABSTRACT FROM AUTHOR]

1. Terbinafine (TBF), a common antifungal agent, has been associated with rare incidences of hepatotoxicity. It is hypothesized that bioactivation of TBF to reactive intermediates and subsequent binding to critical cellular proteins may contribute to this toxicity. In the present study, we have characterized the bioactivation pathways of TBF extensively in human, mouse, monkey, dog and rat liver microsomes and hepatocytes. 2. A total of twenty glutathione conjugates of TBF were identified in hepatocytes; thirteen of these conjugates were also detected in liver microsomes. To the best of our knowledge, only two of these conjugates have been reported previously. The conjugates were categorized into three groups based on their mechanism of formation: (a) alkene/alkyne oxidation followed by glutathione conjugation, with or without N-demethylation, (b) arene oxidation followed by glutathione conjugation, with or without N-demethylation, and (c) N-dealkylation followed by glutathione conjugation of the allylic aldehyde, alcohol and acid intermediates. 3. Differences were observed across species in the contributions of these pathways toward overall metabolic turnover. We conclude that, in addition to the glutathione conjugates known to form by Michael addition to the allylic aldehyde, there are other pathways involving the formation of arene oxides and alkene/alkyne epoxides that may be relevant to the discussion of TBF-mediated idiosyncratic drug reactions. [ABSTRACT FROM AUTHOR]

Bacteroides fragilis (BF) is an integral component of the human colonic commensal microbiota. BF is also the most commonly isolated organism from clinical cases of intra-abdominal abscesses, suggesting its potential to induce proinflammatory responses upon accessing the systemic compartment. Hence, we examined the impact of mucosal and systemic exposures to BF on type 1 diabetes (T1D) incidence in NOD mice. The impact of intestinal exposure to BF under a chemically induced enhanced gut permeability condition, which permits microbial translocation, in T1D was also examined. While oral administration of heat-killed (HK) BF to prediabetic mice caused enhanced immune regulation and suppression of autoimmunity, resulting in delayed hyperglycemia, mice that received HK BF by intravenous injection showed rapid disease progression. Importantly, polysaccharide A-deficient BF failed to produce these opposing effects upon oral and systemic deliveries. Furthermore, BF-induced modulation of disease progression was observed in wild-type, but not TLR2-deficient, NOD mice. Interestingly, oral administration of BF under enhanced gut permeability conditions resulted in accelerated disease progression and rapid onset of hyperglycemia in NOD mice. Overall, these observations suggest that BF-like gut commensals can cause proinflammatory responses upon gaining access to the systemic compartment and contribute to T1D in at-risk subjects. [ABSTRACT FROM AUTHOR]

Summary: The dietary supplement and prebiotic values of β‐glucan‐rich products have been widely recognized and dietary approaches for modulating autoimmunity have been increasingly explored, we assess the impact of oral administration of high‐purity yeast β‐glucan (YBG) on gut immune function, microbiota and type 1 diabetes (T1D) using mouse models. Oral administration of this non‐digestible complex polysaccharide caused a dectin‐1‐dependent immune response involving increased expression of interleukin‐10 (IL‐10), retinaldehyde dehydrogenase (Raldh) and pro‐inflammatory cytokines in the gut mucosa. YBG‐exposed intestinal dendritic cells induced/expanded primarily Foxp3+, IL‐10+ and IL‐17+ T cells, ex vivo. Importantly, prolonged oral administration of low‐dose YBG at pre‐diabetic stage suppressed insulitis and significantly delayed the appearance of T1D in non‐obese diabetic (NOD) mice. Further, prolonged treatment with YBG showed increased Foxp3+ T‐cell frequencies, and a significant change in the gut microbiota, particularly an increase in the abundance of Bacteroidetes and a decrease in the Firmicute members. Oral administration of YBG, together with Raldh‐substrate and β‐cell antigen, resulted in better protection of NOD mice from T1D. These observations suggest that YBG not only has a prebiotic property, but also an oral tolerogenic‐adjuvant‐like effect, and these features could be exploited for modulating autoimmunity in T1D. [ABSTRACT FROM AUTHOR]

1. Due to its unique C-C and C-H bonding properties, conformational preferences and relative hydrophilicity, the cyclopropyl ring has been used as a synthetic building block in drug discovery to modulate potency and drug-like properties. During an effort to discover inhibitors of the hepatitis C virus non-structural protein 5B with improved potency and genotype-coverage profiles, the use of a pyrimidinylcyclopropylbenzamide moiety linked to a C6-substituted benzofuran or azabenzofuran core scaffold was explored in an effort to balance antiviral potency and metabolic stability. 2. In vitro metabolism studies of two compounds from this C6-substituted series revealed an NADPH-dependent bioactivation pathway leading to the formation of multiple glutathione (GSH) conjugates. Analysis of these conjugates by LC-MS and NMR demonstrated that the cyclopropyl group was the site of bioactivation. Based on the putative structures and molecular weights of the cyclopropyl-GSH conjugates, a multi-step mechanism was proposed to explain the formation of these metabolites by P450. This mechanism involves hydrogen atom abstraction to form a cyclopropyl radical, followed by a ring opening rearrangement and reaction with GSH. 3. These findings provided important information to the medicinal chemistry team which responded by replacing the cyclopropyl ring with a gem-dimethyl group. Subsequent compounds bearing this feature were shown to avert the bioactivation pathways in question. [ABSTRACT FROM AUTHOR]

A response from the authors of the study "pH of Drinking Water Influences the Composition of Gut Microbiome and Type 1 Diabetes Incidence" in the 2014 issue is presented.

β-Glucans are naturally occurring polysaccharides in cereal grains, mushrooms, algae, or microbes, including bacteria, fungi, and yeast. Immune cells recognize these β-glucans through a cell surface pathogen recognition receptor called Dectin-1. Studies using β-glucans and other Dectin-1 binding components have demonstrated the potential of these agents in activating the immune cells for cancer treatment and controlling infections. In this study, we show that the β-glucan from Saccharomyces cerevisiae induces the expression of immune regulatory cytokines (IL-10, TGF-β1, and IL-2) and a tolerogenic enzyme (IDO) in bone marrow-derived dendritic cells as well as spleen cells. These properties can be exploited to modulate autoimmunity in the NOD mouse model of type 1 diabetes (T1D). Treatment of prediabetic NOD mice with low-dose β-glucan resulted in a profound delay in hyperglycemia, and this protection was associated with increase in the frequencies of Foxp3+, LAP+, and GARP+ T cells. Upon Ag presentation, β-glucan-exposed dendritic cells induced a significant increase in Foxp3+ and LAP+ T cells in in vitro cultures. Furthermore, systemic coadministration of β-glucan plus pancreatic β cell Ag resulted in an enhanced protection of NOD mice from T1D as compared with treatment with β-glucan alone. These observations demonstrate that the innate immune response induced by low-dose β-glucan is regulatory in nature and can be exploited to modulate T cell response to β cell Ag for inducing an effective protection from T1D. [ABSTRACT FROM AUTHOR]

Recent mouse knockout studies identified adapter protein-2-associated kinase 1 (AAK1) as a viable target for treating neuropathic pain. BMS-986176/LX-9211 ( 4 ), as a highly selective, CNS-penetrable, and potent AAK1 inhibitor, has advanced into phase II human trials. On exploring the structure-activity relationship (SAR) around this biaryl alkyl ether chemotype, several additional compounds were found to be highly selective and potent AAK1 inhibitors with good druglike properties. Among these, compounds 43 and 58 showed very good efficacy in two neuropathic pain rat models and had excellent CNS penetration and spinal cord target engagement. Both compounds also exhibited favorable physicochemical and oral pharmacokinetic (PK) properties. Compound 58 , a central pyridine isomer of BMS-986176/LX-9211 ( 4 ), was 4-fold more potent than 4 in vitro and showed lower plasma exposure needed to achieve similar efficacy compared to 4 in the CCI rat model. However, both 43 and 58 showed an inferior preclinical toxicity profile compared to 4 .

Recent mouse knockout studies identified adapter protein-2 associated kinase 1 (AAK1) as a viable target for treating neuropathic pain. Potent small-molecule inhibitors of AAK1 have been identified and show efficacy in various rodent pain models. ( S )-1-((2',6-Bis(difluoromethyl)-[2,4'-bipyridin]-5-yl)oxy)-2,4-dimethylpentan-2-amine (BMS-986176/LX-9211) ( 34 ) was identified as a highly selective, CNS penetrant, potent AAK1 inhibitor from a novel class of bi(hetero)aryl ethers. BMS-986176/LX9211 ( 34 ) showed excellent efficacy in two rodent neuropathic pain models and excellent central nervous system (CNS) penetration and target engagement at the spinal cord with an average brain to plasma ratio of 20 in rat. The compound exhibited favorable physicochemical and pharmacokinetic properties, had an acceptable preclinical toxicity profile, and was chosen for clinical trials. BMS-986176/LX9211 ( 34 ) completed phase I trials with good human pharmacokinetics and minimum adverse events and is currently in phase II clinical trials for diabetic peripheral neuropathic pain (ClinicalTrials.gov identifier: NCT04455633) and postherpetic neuralgia (ClinicalTrials.gov identifier: NCT04662281).

While the discovery of immune checkpoint inhibitors has led to robust, durable responses in a range of cancers, many patients do not respond to currently available therapeutics. Therefore, an urgent need exists to identify alternative mechanisms to augment the immune-mediated clearance of tumors. Hematopoetic progenitor kinase 1 (HPK1) is a serine-threonine kinase that acts as a negative regulator of T-cell receptor (TCR) signaling, to dampen the immune response. Herein we describe the structure-based discovery of isofuranones as inhibitors of HPK1. Optimization of the chemotype led to improvements in potency, selectivity, plasma protein binding, and metabolic stability, culminating in the identification of compound 24 . Oral administration of 24 , in combination with an anti-PD1 antibody, demonstrated robust enhancement of anti-PD1 efficacy in a syngeneic tumor model of colorectal cancer., Competing Interests: The authors declare no competing financial interest., (© 2021 American Chemical Society.)

Background: Hematopoietic progenitor kinase 1 (HPK1 or MAP4K1) has been demonstrated as a negative intracellular immune checkpoint in mediating antitumor immunity in studies with HPK1 knockout and kinase dead mice. Pharmacological inhibition of HPK1 is desirable to investigate the role of HPK1 in human immune cells with therapeutic implications. However, a significant challenge remains to identify a small molecule inhibitor of HPK1 with sufficient potency, selectivity, and other drug-like properties suitable for proof-of-concept studies. In this report, we identified a novel, potent, and selective HPK1 small molecule kinase inhibitor, compound K (CompK). A series of studies were conducted to investigate the mechanism of action of CompK, aiming to understand its potential application in cancer immunotherapy., Methods: Human primary T cells and dendritic cells (DCs) were investigated with CompK treatment under conditions relevant to tumor microenvironment (TME). Syngeneic tumor models were used to assess the in vivo pharmacology of CompK followed by human tumor interrogation ex vivo., Results: CompK treatment demonstrated markedly enhanced human T-cell immune responses under immunosuppressive conditions relevant to the TME and an increased avidity of the T-cell receptor (TCR) to recognize viral and tumor-associated antigens (TAAs) in significant synergy with anti-PD1. Animal model studies, including 1956 sarcoma and MC38 syngeneic models, revealed improved immune responses and superb antitumor efficacy in combination of CompK with anti-PD-1. An elevated immune response induced by CompK was observed with fresh tumor samples from multiple patients with colorectal carcinoma, suggesting a mechanistic translation from mouse model to human disease., Conclusion: CompK treatment significantly improved human T-cell functions, with enhanced TCR avidity to recognize TAAs and tumor cytolytic activity by CD8+ T cells. Additional benefits include DC maturation and priming facilitation in tumor draining lymph node. CompK represents a novel pharmacological agent to address cancer treatment resistance., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

C-terminal Src kinase (CSK) functions as a negative regulator of T cell activation through inhibitory phosphorylation of LCK, so inhibitors of CSK are of interest as potential immuno-oncology agents. Screening of an internal kinase inhibitor collection identified pyridazinone lead 1 , and a series of modifications led to optimized compound 13 . Compound 13 showed potent activity in biochemical and cellular assays in vitro and demonstrated the ability to increase T cell proliferation induced by T cell receptor signaling. Compound 13 gave extended exposure in mice upon oral dosing and produced a functional response (decrease in LCK phosphorylation) in mouse spleens at 6 h post dose., Competing Interests: The authors declare the following competing financial interest(s): The authors of this manuscript are employees of Bristol-Myers Squibb., (Copyright © 2019 American Chemical Society.)

1. Due to its unique C-C and C-H bonding properties, conformational preferences and relative hydrophilicity, the cyclopropyl ring has been used as a synthetic building block in drug discovery to modulate potency and drug-like properties. During an effort to discover inhibitors of the hepatitis C virus non-structural protein 5B with improved potency and genotype-coverage profiles, the use of a pyrimidinylcyclopropylbenzamide moiety linked to a C6-substituted benzofuran or azabenzofuran core scaffold was explored in an effort to balance antiviral potency and metabolic stability. 2. In vitro metabolism studies of two compounds from this C6-substituted series revealed an NADPH-dependent bioactivation pathway leading to the formation of multiple glutathione (GSH) conjugates. Analysis of these conjugates by LC-MS and NMR demonstrated that the cyclopropyl group was the site of bioactivation. Based on the putative structures and molecular weights of the cyclopropyl-GSH conjugates, a multi-step mechanism was proposed to explain the formation of these metabolites by P450. This mechanism involves hydrogen atom abstraction to form a cyclopropyl radical, followed by a ring opening rearrangement and reaction with GSH. 3. These findings provided important information to the medicinal chemistry team which responded by replacing the cyclopropyl ring with a gem-dimethyl group. Subsequent compounds bearing this feature were shown to avert the bioactivation pathways in question.

Aim: High clearance is a commonly encountered issue in drug discovery. Here we present a centralized metabolic soft spot identification assay with adequate capacity and turnaround time to support the metabolic optimization needs of an entire discovery organization., Methodology: An integrated quan/qual approach utilizing both an orthogonal sample-pooling methodology and software-assisted structure elucidation was developed to enable the assay. Major metabolic soft spots in liver microsomes (rodent and human) were generated in a batch mode, along with kinetics of parent disappearance and metabolite formation, typically within 1 week of incubation., Results & Conclusion: A centralized metabolic soft spot identification assay has been developed and has successfully impacted discovery project teams in mitigating instability and establishing potential structure-metabolism relationships.

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., (The American Society for Pharmacology and Experimental Therapeutics.)

Daclatasvir is a first-in-class, potent, and selective inhibitor of the hepatitis C virus nonstructural protein 5A replication complex. In support of nonclinical studies during discovery and exploratory development, liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance were used in connection with synthetic and radiosynthetic approaches to investigate the biotransformation of daclatasvir in vitro and in cynomolgus monkeys, dogs, mice, and rats. The results of these studies indicated that disposition of daclatasvir was accomplished mainly by the release of unchanged daclatasvir into bile and feces and, secondarily, by oxidative metabolism. Cytochrome P450s were the main enzymes involved in the metabolism of daclatasvir. Oxidative pathways included δ-oxidation of the pyrrolidine moiety, resulting in ring opening to an aminoaldehyde intermediate followed by an intramolecular reaction between the aldehyde and the proximal imidazole nitrogen atom. Despite robust formation of the resulting metabolite in multiple systems, rates of covalent binding to protein associated with metabolism of daclatasvir were modest (55.2-67.8 pmol/mg/h) in nicotinamide adenine dinucleotide phosphate (reduced form)-supplemented liver microsomes (human, monkey, rat), suggesting that intramolecular rearrangement was favored over intermolecular binding in the formation of this metabolite. This biotransformation profile supported the continued development of daclatasvir, which is now marketed for the treatment of chronic hepatitis C virus infection., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

A recent medicinal chemistry campaign to identify positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGluR5) led to the discovery of potent compounds featuring an oxazolidinone structural core flanked by biaryl acetylene and haloaryl moieties. However, biotransformation studies of some of these mGluR5 PAMs demonstrated the formation of glutathione (GSH) conjugates. The conjugates in question were formed independently of NADPH as the main products in liver microsomes and liver cytosol (rat and human) and exhibited masses that were 307 u greater than their respective substrates, indicating the involvement of a reductive step in the formation of these metabolites. To further characterize the relevant metabolic sequences, GSH conjugates of (4R,5R)-5-(3-fluorophenyl)-4-(5-(pyrazin-2-ylethynyl)pyridin-3-yl)oxazolidin-2-one and (4R,5R)-5-(4-fluorophenyl)-4-(6-((3-fluoropyridin-2-yl)ethynyl)pyridin-2-yl)oxazolidin-2-one were biosynthesized and isolated. Subsequent analysis by NMR showed that GSH had reacted with the acetylene carbon atoms of these mGluR5 PAMs, suggesting a conjugate addition mechanism and implicating cytosolic and microsomal GSH S-transferases (GSTs) in catalysis. Interestingly, five closely related mGluR5 PAMs were not similarly prone to the formation of GSH conjugates in vitro. These compounds also featured acetylenes, but were flanked by either phenyl or cyclohexyl rings, which indicated that the formation of GSH conjugates was influenced by proximal functional groups that modulated the electron density of the triple bond and/or differences in enzyme-substrate specificity. These results informed an ongoing drug-discovery effort to identify mGluR5 PAMs with drug-like properties and a low risk of reactivity with endogenous thiols., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Certain aromatic nitriles are well-known inhibitors of cysteine proteases. The mode of action of these compounds involves the formation of a reversible or irreversible covalent bond between the nitrile and a thiol group in the active site of the enzyme. However, the reactivity of these aromatic nitrile-substituted heterocycles may lead inadvertently to nonspecific interactions with DNA, protein, glutathione, and other endogenous components, resulting in toxicity and complicating the use of these compounds as therapeutic agents. In the present study, the intrinsic reactivity and associated structure-property relationships of cathepsin K inhibitors featuring substituted pyridazines [6-phenylpyridazine-3-carbonitrile, 6-(4-fluorophenyl)pyridazine-3-carbonitrile, 6-(4-methoxyphenyl)pyridazine-3-carbonitrile, 6-p-tolylpyridazine-3-carbonitrile], pyrimidines [5-p-tolylpyrimidine-2-carbonitrile, 5-(4-fluorophenyl)pyrimidine-2-carbonitrile], and pyridines [5-p-tolylpicolinonitrile and 5-(4-fluorophenyl)picolinonitrile] were evaluated using a combination of computational and analytical approaches to establish correlations between electrophilicity and levels of metabolites that were formed in glutathione- and N-acetylcysteine-supplemented human liver microsomes. Metabolites that were characterized in this study featured substituted thiazolines that were formed following rearrangements of transient glutathione and N-acetylcysteine conjugates. Peptidases including γ-glutamyltranspeptidase were shown to catalyze the formation of these products, which were formed to lesser extents in the presence of the selective γ-glutamyltranspeptidase inhibitor acivicin and the nonspecific peptidase inhibitors phenylmethylsulfonyl fluoride and aprotinin. Of the chemical series mentioned above, the pyrimidine series was the most susceptible to metabolism to thiazoline-containing products, followed, in order, by the pyridazine and pyridine series. This trend was in keeping with the diminishing electrophilicity across these series, as demonstrated by in silico modeling. Hence, mechanistic insights gained from this study could be used to assist a medicinal chemistry campaign to design cysteine protease inhibitors that were less prone to the formation of covalent adducts.

The biphenyl derivatives 2 and 3 are prototypes of a novel class of NS5A replication complex inhibitors that demonstrate high inhibitory potency toward a panel of clinically relevant HCV strains encompassing genotypes 1-6. However, these compounds exhibit poor systemic exposure in rat pharmacokinetic studies after oral dosing. The structure-activity relationship investigations that improved the exposure properties of the parent bis-phenylimidazole chemotype, culminating in the identification of the highly potent NS5A replication complex inhibitor daclatasvir (33) are described. An element critical to success was the realization that the arylglycine cap of 2 could be replaced with an alkylglycine derivative and still maintain the high inhibitory potency of the series if accompanied with a stereoinversion, a finding that enabled a rapid optimization of exposure properties. Compound 33 had EC50 values of 50 and 9 pM toward genotype-1a and -1b replicons, respectively, and oral bioavailabilities of 38-108% in preclinical species. Compound 33 provided clinical proof-of-concept for the NS5A replication complex inhibitor class, and regulatory approval to market it with the NS3/4A protease inhibitor asunaprevir for the treatment of HCV genotype-1b infection has recently been sought in Japan.

Prostate cancer is the most commonly diagnosed cancer in men and accounts for significant morbidity and mortality in the western world. While traditional therapies are effective at clearing early stage cancer, they often fail to treat late stage metastatic disease. Thus, an effective therapy that targets prostate tumor growth and metastasis is desired for alleviating the disease and improving patient outcomes. Natural extracts have been the focus of recent investigation, particularly those with reduced cellular toxicity to healthy tissue. In this review, we discuss one potential candidate, ganoderic acid, an extract from the Ganoderma lucidum mushroom that has been tested in multiple cancer models. Interestingly, ganoderic acid DM (GA-DM) has shown toxicity to both androgen-dependent and independent prostate cancer cells with reduced osteoclastogenesis in late stage metastatic disease. This review will discuss the current knowledge on this GA-DM extract and the potential benefit in treating advanced prostate cancer. We will also provide an overview on the targeted delivery of GA-DM through nanoparticles that would reduce bystander toxicity and improve the drug's effectiveness. An improved understanding of this drug and its uses will advance the field of natural chemotherapeutics, particularly in treating advanced prostate cancer.

5-Isopropyl-6-(5-methyl-1,3,4-oxadiazol-2-yl)-N-(2-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)pyrrolo[2,1-f][1,2,4]triazin-4-amine (BMS-645737) is a potent and selective vascular endothelial growth factor receptor-2 antagonist. In this study, liquid chromatography/tandem mass spectrometry and NMR were used to investigate the biotransformation of BMS-645737 in vitro and in the cynomolgus monkey, dog, mouse, and rat. Metabolic pathways for BMS-645737 included multistep processes involving both oxidation and conjugation reactions. For example, the 2-methyl-1H-pyrrolo moiety underwent cytochrome P450-catalyzed hydroxylation followed by oxidation to a carboxylic acid and then conjugation with taurine. Alternatively, the 5-methyl-1,3,4-oxadiazol-2-yl moiety was metabolized by hydroxylation and then conjugation with sulfate. The pyridin-5-yl group underwent direct glucuronidation in hepatocytes (dog, monkey, human) and conjugation with N-acetylglucosamine in the monkey. Conjugation with glutathione and processing along the mercapturic acid pathway was a minor metabolic pathway in vivo, although BMS-645737 did not form conjugates in the presence of glutathione-supplemented liver microsomes. Other minor biotransformation pathways included oxidative dehydrogenation, dihydroxylation, and hydrolytic opening of the oxadiazole ring followed by either deacetylation or hydrolysis of the resulting diacyl hydrazide. Whereas previous studies have shown the formation of N-acetylglucosamine conjugates of alcohols, arylamines, and other small molecules, this report describes the biotransformation of a heterocyclic aromatic amine via direct conjugation with N-acetylglucosamine.

Botanical dietary supplements containing Cimicifuga racemosa (Actaea racemosa; black cohosh) are used commonly by women to assuage menopausal symptoms including hot flashes and sleep disorders. Despite the popularity of such supplements, little is known about the metabolism or possible toxicity of many compounds that could be concentrated therein. The aim of this study was to selectively identify phase I metabolites resulting from metabolic bioactivation of constituents of black cohosh in vitro and to determine whether evidence of such metabolites could be found in the urine of perimenopausal women taking black cohosh oral supplements. A variation of an ultrafiltration mass spectrometric assay devised previously was used to screen an extract of black cohosh for the formation of electrophilic phase I metabolites that had been trapped as GSH conjugates. Mercapturates (N-acetylcysteine conjugates) corresponding to the GSH conjugates identified during screening were synthesized and characterized using LC-MS/MS with product-ion scanning. During a phase I clinical trial of black cohosh in perimenopausal women, urine was collected from seven subjects, each of whom took a single oral dose of either 32, 64, or 128 mg of the black cohosh extract. These urine samples were analyzed for the presence of mercapturate conjugates using positive-ion electrospray LC-MS and LC-MS/MS. On the basis of their propensity to form GSH adducts following metabolic activation by hepatic microsomes and NADPH in vitro, a total of eight electrophilic metabolites of black cohosh were detected, including quinoid metabolites of fukinolic acid, fukiic acid, caffeic acid, and cimiracemate B. Additional quinoid metabolites were formed from hydroxytyrosol and dihydroxyphenyl lactic acid, neither of which had been isolated previously from black cohosh. However, mercapturate conjugates of these black cohosh constituents were not detected in urine samples from women who consumed single oral doses of up to 256 mg of a standardized black cohosh extract. Therefore, for moderate doses of a dietary supplement containing black cohosh, this study found no cause for safety concerns over the formation of quinoid metabolites in women.

Dietary supplements containing Piper methysticum Forst. (kava) have been implicated in multiple cases of liver injury in humans, including 10 recently reviewed cases in which patients required liver transplantation following the usage of kava-containing products (Centers for Disease Control and Prevention, reprinted. (2003) J. Am. Med. Assoc. 289, 36-37). To investigate a possible mechanism(s) of kava-induced hepatotoxicity, an extract of kava was incubated in vitro with hepatic microsomes, NADPH, and GSH. Electrophilic intermediates that were generated via metabolic activation were trapped as GSH conjugates and removed from the protein mixture using ultrafiltration. Positive ion electrospray LC-MS/MS with precursor ion scanning was used for the selective detection of GSH conjugates, and LC-MS(n) product ion scanning was used to elucidate their structures. Using this in vitro MS-based screening assay, two novel electrophilic metabolites of kava, 11,12-dihydroxy-7,8-dihydrokavain-o-quinone and 11,12-dihydroxykavain-o-quinone, were identified. Mercapturic acids of these quinoid species were not detected in the urine of a human volunteer following ingestion of a dietary supplement that contained kava; instead, the corresponding catechols were metabolized extensively to glucuronic acid and sulfate conjugates. These observations indicate that quinoid metabolites, under most circumstances, are probably not formed in substantial quantities following the ingestion of moderate doses of kava. However, the formation of electrophilic quinoid metabolites by hepatic microsomes in vitro suggests that such metabolites might contribute to hepatotoxicity in humans when metabolic pathways are altered (e.g., because of a drug interaction, genetic difference in enzyme expression, etc.) or if conjugation pathways become saturated.