Application of Covalent Binding Body Burden in the HµREL Human Hepatocyte Coculture Model for Reactivity Risk Assessment of Metabolically Low Turnover Drugs.

The human hepatocyte suspension model has been a valuable tool to study covalent binding (CVB) for compounds that form reactive metabolites. However, accurately measuring CVB values with the suspension model becomes challenging for metabolically low turnover compounds. In this study, we evaluated the HµREL human hepatocyte coculture model relative to existing literature using human hepatocyte suspension for drugs of known drug-induced liver injury category. Our results indicate that this coculture model provides ample metabolic turnover to reproducibly measure CVB. It is sufficiently robust to apply a predefined 1 mg/day CVB body burden threshold for risk assessment to guide our discovery programs, allowing for expanded coverage to include metabolically low turnover compounds.

Metabolism and disposition of JNJ-10450232 (NTM-006) in rats, dogs, monkeys and humans.

JNJ-10450232 (NTM-006), a novel non-opioid, non-nonsteroidal anti-inflammatory drug with structural similarities to acetaminophen, demonstrated anti-pyretic and/or analgesic activities in preclinical models and humans and reduced potential to cause hepatotoxicity in preclinical species. Metabolism and disposition of JNJ-10450232 (NTM-006) following oral administration to rats, dogs, monkeys and humans are reported. Urinary excretion was the major route of elimination based on recovery of 88.6% (rats) and 73.7% (dogs) of oral dose. The compound was extensively metabolized based on low recovery of unchanged drug in excreta from rats (11.3%) and dogs (18.4%). Clearance is driven by O-glucuronidation, amide hydrolysis, O-sulfation and methyl oxidation pathways. The combination of metabolic pathways driving clearance in human is covered in at least one preclinical species despite a few species-dependent pathways. O-Glucuronidation was the major primary metabolic pathway of JNJ-10450232 (NTM-006) in dogs, monkeys and humans, although amide hydrolysis was another major primary metabolic pathway in rats and dogs. A minor bioactivation pathway to quinone-imine is observed only in monkeys and humans. Unchanged drug was the major circulatory component in all species investigated. Except for metabolic pathways unique to the 5-methyl-1H-pyrazole-3-carboxamide moiety, metabolism and disposition of JNJ-10450232 (NTM-006) are similar to acetaminophen across species.

Bifunctionalized isotopologs as calibrants for standard-free quantitation of drug metabolites in plasma by liquid chromatography coupled with radiometry and mass spectrometry.

The reported method involves a novel workflow that eliminates the need for authentic reference standards for the quantitation of drug metabolites in biological samples using a single multi-isotopically labeled compound bearing both radio and stable isotopes. The resulting radio and stable bifunctionalized isotopolog (RADSTIL) of the parent drug is employed as a substrate for in vitro biotransformation to targeted RADSTILs of metabolites as calibrants. Inclusion of a radio label enables both radiometric and mass spectrometric detection. The addition of stable labels ensures the subsequent isotopic interference-free quantitation of unlabeled metabolites in preclinical and clinical samples. This affords a more accurate quantitation workflow compared with the current semi-quantitation method, which utilizes isotopic interfering radio isotopologs of metabolites alone as calibrants. The proof-of-concept is illustrated with (14 C,13 C2 )-acetaminophen where in vitro biotransformation produced (14 C,13 C2 )-sulfate and (14 C,13 C2 )-glucuronide calibrants. Absolute quantitation of the acetaminophen metabolites was then achieved by liquid chromatography coupled with radiometry and mass spectrometry. Quantitative data obtained by this method fell within 82-86% of the values from conventional LC-MS/MS method.

Spirocyclic Thiohydantoin Antagonists of F877L and Wild-Type Androgen Receptor for Castration-Resistant Prostate Cancer.

Androgen receptor (AR) transcriptional reactivation plays a key role in the development and progression of lethal castration-resistant prostate cancer (CRPC). Recurrent alterations in the AR enable persistent AR pathway signaling and drive resistance to the treatment of second-generation antiandrogens. AR F877L, a point mutation in the ligand binding domain of the AR, was identified in patients who acquired resistance to enzalutamide or apalutamide. In parallel to our previous structure-activity relationship (SAR) studies of compound 4 (JNJ-pan-AR) and clinical stage compound 5 (JNJ-63576253), we discovered additional AR antagonists that provide opportunities for future development. Here we report a highly potent series of spirocyclic thiohydantoins as AR antagonists for the treatment of the F877L mutant and wild-type CRPC.

Discovery of JNJ-63576253: A Clinical Stage Androgen Receptor Antagonist for F877L Mutant and Wild-Type Castration-Resistant Prostate Cancer (mCRPC).

Persistent androgen receptor (AR) activation drives therapeutic resistance to second-generation AR pathway inhibitors and contributes to the progression of advanced prostate cancer. One resistance mechanism is point mutations in the ligand binding domain of AR that can transform antagonists into agonists. The AR F877L mutation, identified in patients treated with enzalutamide or apalutamide, confers resistance to both enzalutamide and apalutamide. Compound 4 (JNJ-pan-AR) was identified as a pan-AR antagonist with potent activity against wild-type and clinically relevant AR mutations including F877L. Metabolite identification studies revealed a latent bioactivation pathway associated with 4. Subsequent lead optimization of 4 led to amelioration of this pathway and nomination of 5 (JNJ-63576253) as a clinical stage, next-generation AR antagonist for the treatment of castration-resistant prostate cancer (CRPC).

Reproducing human and cross-species drug toxicities using a Liver-Chip.

Nonclinical rodent and nonrodent toxicity models used to support clinical trials of candidate drugs may produce discordant results or fail to predict complications in humans, contributing to drug failures in the clinic. Here, we applied microengineered Organs-on-Chips technology to design a rat, dog, and human Liver-Chip containing species-specific primary hepatocytes interfaced with liver sinusoidal endothelial cells, with or without Kupffer cells and hepatic stellate cells, cultured under physiological fluid flow. The Liver-Chip detected diverse phenotypes of liver toxicity, including hepatocellular injury, steatosis, cholestasis, and fibrosis, and species-specific toxicities when treated with tool compounds. A multispecies Liver-Chip may provide a useful platform for prediction of liver toxicity and inform human relevance of liver toxicities detected in animal studies to better determine safety and human risk.

Discovery of a GPR40 Superagonist: The Impact of Aryl Propionic Acid α-Fluorination.

GPR40 is a G-protein-coupled receptor which mediates fatty acid-induced glucose-stimulated insulin secretion from pancreatic beta cells and incretion release from enteroendocrine cells of the small intestine. GPR40 full agonists exhibit superior glucose lowering compared to partial agonists in preclinical species due to increased insulin and GLP-1 secretion, with the added benefit of promoting weight loss. In our search for potent GPR40 full agonists, we discovered a superagonist which displayed excellent in vitro potency and superior efficacy in the Gαs-mediated signaling pathway. Most synthetic GPR40 agonists have a carboxylic acid headgroup, which may cause idiosyncratic toxicities, including drug-induced-liver-injury (DILI). With a methyl group and a fluorine atom substituted at the α-C of the carboxylic acid group, 19 is not only highly efficacious in lowering glucose and body weight in rodent models but also has a low DILI risk due to its stable acylglucuronide metabolite.

Reductive Desulfuration as an Important Tool in Detection of Small Molecule Modifications to Payload of Antibody Drug Conjugates.

A high resolution accurate mass LC-MS method was developed to facilitate the characterization of a subset of antibody drug conjugate (ADC) biotherapeutics, where the payload is linked to the antibody by a thioether bond. Desulfuration of the thioether linker was optimized for release of the payload to take advantage of the high resolution and high mass accuracy of the Orbitrap to characterize metabolism of the payload. Two model ADCs, trastuzumab emtansine (T-DM1) and SigmaMAb dansyl-cadavarine-SMCC (SigmaMAb ADC mimic) were selected for optimization of the desulfuration reaction as a function of reaction time, pH, organic solvent, and chaotropic reagents (urea, guanidine HCl) by monitoring the yield of released desulfurated DM1 from T-DM1 and desulfurated dansyl-cadaverine-SMCC from SigmaMAb ADC mimic, respectively. The optimized desulfuration technique was successfully applied to enable characterization of the ADC following its incubation in hepatocytes, liver microsomes, and buffers, as illustrated by the identification of a hydrolyzed thiosuccinimide ring of SigmaMAb ADC mimic following incubation in buffer for 48 h. The results from this study demonstrate that the chemical cleavage of thioether bond by desulfuration is simple, efficient, and specific. This technique is useful in characterization of metabolism on the payload of ADC to provide guidance for improvement of its biopharmaceutical profile. This is the first report on characterization of modification to payload of ADC following desulfuration.

Fasiglifam (TAK-875): Mechanistic Investigation and Retrospective Identification of Hazards for Drug Induced Liver Injury.

TAK-875, a GPR40 agonist, was withdrawn from Phase III clinical trials due to drug-induced liver injury (DILI). Mechanistic studies were conducted to identify potential DILI hazards (covalent binding burden (CVB), hepatic transporter inhibition, mitochondrial toxicity, and liver toxicity in rats) associated with TAK-875. Treatment of hepatocytes with radiolabeled TAK-875 resulted in a CVB of 2.0 mg/day, which is above the threshold of 1 mg/day considered to be a risk for DILI. Covalent binding to hepatocytes was due to formation of a reactive acyl glucuronide (AG) and, possibly, an acyl-CoA thioester intermediate. Formation of TAK-875AG in hepatocytes and/or in vivo was in the order of non-rodents > human (in vitro only) > rat. These data suggest that non-rodents, and presumably humans, form TAK-875AG more efficiently than rats, and that AG-mediated toxicities in rats may only occur at high doses. TAK-875 (1000 mg/kg/day) formed significant amounts of AG metabolite (≤32.7 µM) in rat liver that was associated with increases in ALT (×4), bilirubin (×9), and bile acids (×3.4), and microscopic findings of hepatocellular hypertrophy and single cell necrosis. TAK-875 and TAK-875AG had similar potencies (within 3-fold) for human multi-drug resistant associated protein 2/4 (MRP2/4) and bile salt export pump, but TAK-875AG was exceptionally potent against MRP3 (0.21 µM). Inhibition of MRPs may contribute to liver accumulation of TAK-875AG. TAK-875 also inhibited mitochondrial respiration in HepG2 cells, and mitochondrial Complex 1 and 2 activities in isolated rat mitochondria. In summary, formation of TAK-875AG, and possibly TAK-875CoA in hepatocytes, coupled with inhibition of hepatic transporters and mitochondrial respiration may be key contributors to TAK-875-mediated DILI.

Discovery of novel benzo[b]thiophene tetrazoles as non-carboxylate GPR40 agonists.

GPR40 partial agonism is a promising new mechanism for the treatment of type 2 diabetes mellitus with clinical proof of concept. Most of the GPR40 agonists in the literature have a carboxylic acid functional group, which may pose a risk for idiosyncratic drug toxicity. A novel series of GPR40 agonists containing a tetrazole as a carboxylic acid bioisostere was identified. This series of compounds features a benzo[b]thiophene as the center ring, which is prone to oxidation during phase 1 metabolism. Following SAR optimization targeting GPR40 agonist activity and intrinsic clearance in microsomes (human and rat), potent and metabolically stable compounds were selected for in vivo evaluation. The compounds are efficacious at lowering blood glucose in a SD rat oGTT model.

Standard-Free Bioanalytical Approach for Absolute Quantitation of Drug Metabolites Utilizing Biosynthesis of Reciprocal Radio and Stable Isotopologues and Its Application.

The following work describes a combined enzymatic and bioanalytical method that permits absolute quantitation of metabolites in biological samples without the requirement for reference metabolite standards. This technique was exemplified using a radio (14C) isotopologue and a stable (13C6) isotopologue of acetaminophen as substrates for in vitro biosynthesis of the corresponding radio and stable isotope labeled metabolites, namely, 14C- and 13C6-glucuronides and sulfates. By supplanting the use of authentic metabolite standards, traditionally used to calibrate 13C6-metabolites via liquid chromatography-tandem mass spectrometry (LC-MS/MS), 13C6-metabolites were radiocalibrated by their 14C-isotopologues via liquid chromatography coupled with radioactivity detection and mass spectrometry (LC-RAD/MS). The radiocalibrated 13C6-isotopologues were in turn used to quantitate acetaminophen and its corresponding metabolites in rat plasma samples by LC-MS/MS. Variation between this and a conventional LC-MS/MS method using authentic standards for calibration was within ±17%, permitting its use in preclinical and clinical applications. Since authentic metabolite standards are not required under the concept of radio and stable isotopologues using adapted LC-RAD/MS protocols, significantly fewer resources are required to support accurate metabolite quantitation which in turn enables efficient analysis of simple and complex metabolite profiles.

In vitro and physiologically-based pharmacokinetic based assessment of drug-drug interaction potential of canagliflozin.

AIMS: Canagliflozin is a recently approved drug for use in the treatment of type 2 diabetes. The potential for canagliflozin to cause clinical drug-drug interactions (DDIs) was assessed. METHODS: DDI potential of canagliflozin was investigated using in vitro test systems containing drug metabolizing enzymes or transporters. Basic predictive approaches were applied to determine potential interactions in vivo. A physiologically-based pharmacokinetic (PBPK) model was developed and clinical DDI simulations were performed to determine the likelihood of cytochrome P450 (CYP) inhibition by canagliflozin. RESULTS: Canagliflozin was primarily metabolized by uridine 5'-diphospho-glucuronosyltransferase 1A9 and 2B4 enzymes. Canagliflozin was a substrate of efflux transporters (P-glycoprotein, breast cancer resistance protein and multidrug resistance-associated protein-2) but was not a substrate of uptake transporters (organic anion transporter polypeptide isoforms OATP1B1, OATP1B3, organic anion transporters OAT1 and OAT3, and organic cationic transporters OCT1, and OCT2). In inhibition assays, canagliflozin was shown to be a weak in vitro inhibitor (IC50 ) of CYP3A4 (27 µmol l -1 , standard error [SE] 4.9), CYP2C9 (80 µmol l -1 , SE 8.1), CYP2B6 (16 µmol l-1 , SE 2.1), CYP2C8 (75 µmol l -1 , SE 6.4), P-glycoprotein (19.3 µmol l -1 , SE 7.2), and multidrug resistance-associated protein-2 (21.5 µmol l -1 , SE 3.1). Basic models recommended in DDI guidelines (US Food & Drug Administration and European Medicines Agency) predicted moderate to low likelihood of interaction for these CYPs and efflux transporters. PBPK DDI simulations of canagliflozin with CYP probe substrates (simvastatin, S-warfarin, bupropion, repaglinide) did not show relevant interaction in humans since mean areas under the concentration-time curve and maximum plasma concentration ratios for probe substrates with and without canagliflozin and its 95% CIs were within 0.80-1.25. CONCLUSIONS: In vitro DDI followed by a predictive or PBPK approach was applied to determine DDI potential of canagliflozin. Overall, canagliflozin is neither a perpetrator nor a victim of clinically important interactions.

Cytochrome P450 induction response in tethered spheroids as a three-dimensional human hepatocyte in vitro model.

Cytochrome P450 (CYP) induction is a key risk factor of clinical drug-drug interactions that has to be mitigated in the early phases of drug discovery. Three-dimensional (3D) cultures of hepatocytes in vitro have recently emerged as a potentially better platform to recapitulate the in vivo liver structure and to maintain long-term hepatic functions as compared with conventional two-dimensional (2D) monolayer cultures. However, the majority of published studies on 3D hepatocyte models use rat hepatocytes and the response to CYP inducers between rodents and humans is distinct. In the present study, we constructed tethered spheroids on RGD/galactose-conjugated membranes as an in vitro 3D model using cryopreserved human hepatocytes. CYP3A4 mRNA expression in the tethered spheroids was induced to a significantly greater extent than those in the collagen sandwich cultures, indicating the transcriptional regulation was more sensitive to the CYP inducers in the 3D model. Induction of CYP1A2, CYP2B6 and CYP3A4 activities in the tethered spheroids were comparable to, if not higher than that observed in the collagen sandwich cultures. The membrane-based model is readily integrated into multi-well plates for higher-throughput drug testing applications, which might be an alternative model to screen the CYP induction potential in vitro with more physiological relevance.

Contribution of metabolites to P450 inhibition-based drug-drug interactions: scholarship from the drug metabolism leadership group of the innovation and quality consortium metabolite group.

Recent European Medicines Agency (final) and US Food and Drug Administration (draft) drug interaction guidances proposed that human circulating metabolites should be investigated in vitro for their drug-drug interaction (DDI) potential if present at ≥ 25% of the parent area under the time-concentration curve (AUC) (US Food and Drug Administration) or ≥ 25% of the parent and ≥ 10% of the total drug-related AUC (European Medicines Agency). To examine the application of these regulatory recommendations, a group of scientists, representing 18 pharmaceutical companies of the Drug Metabolism Leadership Group of the Innovation and Quality Consortium, conducted a scholarship to assess the risk of contributions by metabolites to cytochrome P450 (P450) inhibition-based DDIs. The group assessed the risk of having a metabolite as the sole contributor to DDI based on literature data and analysis of the 137 most frequently prescribed drugs, defined structural alerts associated with P450 inhibition/inactivation by metabolites, and analyzed current approaches to trigger in vitro DDI studies for metabolites. The group concluded that the risk of P450 inhibition caused by a metabolite alone is low. Only metabolites from 5 of 137 drugs were likely the sole contributor to the in vivo P450 inhibition-based DDIs. Two recommendations were provided when assessing the need to conduct in vitro P450 inhibition studies for metabolites: 1) consider structural alerts that suggest P450 inhibition potential, and 2) use multiple approaches (e.g., a metabolite cut-off value of 100% of the parent AUC and the R(met) strategy) to predict P450 inhibition-based DDIs caused by metabolites in the clinic.

A nonradioactive approach to investigate the metabolism of therapeutic peptides by tagging with 127i and using inductively-coupled plasma mass spectrometry analysis.

The metabolic fate of adrenocorticotropic hormone (ACTH) fragment 4-10 (4-10) was evaluated following incorporation of a nonradioactive (127)I-tag and with selective detection of I(+) at m/z 127 by inductively coupled plasma mass spectrometry (ICP-MS). (127)I has all the advantages of radioactive (125)I as a metabolite tracer and, together with its detection in the femtogram range, has led to a successful metabolite profiling of (127)I-ACTH (4-10) in vitro. The observed metabolic stability of this peptide in tissue preparations from human was plasma > kidney S9 > liver microsomes > liver cytosol, liver S9. Metabolic turnover of (127)I-ACTH (4-10) was not NADPH-dependent and, together with inhibition by protease inhibitor cocktail and EDTA, is consistent with metabolism exclusively by proteases. Our preliminary studies using chemical inhibitors suggested the involvement of metalloprotease, serine peptidase, and aminopeptidase in (127)I-ACTH (4-10) metabolism. The liver is the primary site of metabolic clearance of (127)I-ACTH (4-10), with kidney S9 taking four times longer to produce a metabolite profile comparable to that produced by liver S9. A total of six metabolites retaining the (127)I-tag was detected by ICP-MS, and their structures were elucidated using a LTQ/Orbitrap. (127)I-ACTH (4-10) underwent both N- and C-terminal proteolysis to produce (127)I-Phe as the major metabolite. The (127)I-tag had minimal effect on the metabolic turnover and site of proteolysis of ACTH (4-10), which, together with ICP-MS providing essentially equimolar responses, suggests that the use of a (127)I-tag may have general utility as an alternative to radioiodination to investigate the metabolism of peptide therapeutics.

Metabolism and excretion of canagliflozin in mice, rats, dogs, and humans.

Canagliflozin is an oral antihyperglycemic agent used for the treatment of type 2 diabetes mellitus. It blocks the reabsorption of glucose in the proximal renal tubule by inhibiting the sodium-glucose cotransporter 2. This article describes the in vivo biotransformation and disposition of canagliflozin after a single oral dose of [(14)C]canagliflozin to intact and bile duct-cannulated (BDC) mice and rats and to intact dogs and humans. Fecal excretion was the primary route of elimination of drug-derived radioactivity in both animals and humans. In BDC mice and rats, most radioactivity was excreted in bile. The extent of radioactivity excreted in urine as a percentage of the administered [(14)C]canagliflozin dose was 1.2%-7.6% in animals and approximately 33% in humans. The primary pathways contributing to the metabolic clearance of canagliflozin were oxidation in animals and direct glucuronidation of canagliflozin in humans. Unchanged canagliflozin was the major component in systemic circulation in all species. In human plasma, two pharmacologically inactive O-glucuronide conjugates of canagliflozin, M5 and M7, represented 19% and 14% of total drug-related exposure and were considered major human metabolites. Plasma concentrations of M5 and M7 in mice and rats from repeated dose safety studies were lower than those in humans given canagliflozin at the maximum recommended dose of 300 mg. However, biliary metabolite profiling in rodents indicated that mouse and rat livers had significant exposure to M5 and M7. Pharmacologic inactivity and high water solubility of M5 and M7 support glucuronidation of canagliflozin as a safe detoxification pathway.

LC-ESI-MS/MS quantification of 4ß-hydroxycholesterol and cholesterol in plasma samples of limited volume.

A liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) assay was developed and qualified for analyzing 4ß-hydroxycholesterol and cholesterol in 5 µl of human and mouse plasma. Stable isotope-labeled d7-analogs of both analytes were used as internal standards and 4.2% (w/v) human serum albumin in phosphate-buffered saline was used as the surrogate matrix for preparation of calibration curves and QCs. The assay is capable of quantification of 4ß-hydroxycholesterol and cholesterol from 5 to 500 ng/ml and 50 to 2000 µg/ml, respectively, with acceptable accuracy and precision following evaluation of recovery of analytes, autosampler stability and potential contribution of chemical oxidation to the formation of 4ß-hydroxycholesterol. The final reconstituted solution was diluted for quantification of cholesterol typically present at 1000 fold higher concentration than 4ß-hydroxycholesterol in the same samples used for 4ß-hydroxycholesterol quantification. The successful quantification using a low plasma volume was achieved by quantification of total forms (free and conjugated) of both analytes after alkaline hydrolysis, followed by derivatization to form electrospray ionization-sensitive picolinyl esters, which upon collision-induced dissociation gave high mass precursor-product ion pair for selective detection by multiple reaction monitoring. In addition, chromatographic separation using a 16-min reversed phase gradient elution on a 1.9 µm particle size, C18 column, overcame interference from other isobaric plasma oxysterols during detection by multiple-reaction monitoring. This assay was compared to an orthogonal enzymatic assay for cholesterol and all samples, but one, provided values that were within 10% of each other. In addition, this assay passed the incurred sample tests for both analytes in human and mouse plasma samples according to reported acceptance criteria for incurred sample reanalysis. The quantification of both analytes permitted the determination of 4ß-hydroxycholesterol compared to its ratio to cholesterol as an endogenous biomarker for CYP3A4/5 activity. The LC-ESI-MS/MS assay was also successfully applied to quantification of 4ß-hydroxycholesterol and cholesterol in plasma samples from untreated human and mice including FRG™ KO C57Bl/6 chimeric mice with humanized livers. The preliminary data indicated that the plasma 4ß-hydroxycholesterol concentrations or their ratio to cholesterol from mice including chimeric mice were higher than those from human.

Bio-generation of stable isotope labeled internal standards for absolute and relative quantitation of drug metabolites in plasma samples by LC-MS/MS.

In order to achieve a better understanding of the toxicity of drug candidates, quantitative characterization of circulatory drug metabolites has been of increasing interest in current pharmaceutical research. Stable isotope labeled (STIL) internal standards (IS) are ideally used to simplify drug metabolite quantitation via liquid chromatography and tandem mass spectrometry (LC-MS/MS) analysis, primarily due to their capability to compensate matrix effects, thereby leading to faster method establishment by using generic assay conditions. However, chemical synthesis of STIL metabolites can often be resource intensive, requiring lengthy exploratory synthesis route development and/or extensive optimization to achieve the required stability for some metabolites. To overcome these challenges, we developed a general method that could generate STIL metabolites in a matter of hours from STIL parent drugs through the utilization of an appropriate in vitro metabolic incubation. This methodology can potentially save valuable synthesis resources, as well as provide timely availability of STIL IS. The following work demonstrates the proof-of-concept that multiple STIL metabolites can be generated simultaneously to provide satisfactory performance for both absolute quantitation of drug metabolites and for potential use in assessment of relative exposure coverage across species in safety tests of drug metabolites (MIST).

Free radical metabolism of raloxifene in human liver microsomes.

Raloxifene was metabolized predominantly by CYP3A4 in human liver microsomes to a pair of carbon-carbon (RD1­2) and ether (RD3­4) linked homodimers in an nicotinamide adenine dinucleotide phosphate-dependent manner. The major homodimer formed by human liver microsomes (RD3) was different from the major homodimer formed by peroxidases (RD1). RD1, 3 and 4 were identified by both mass spectrometry (MS) and nuclear magnetic resonance (NMR) as symmetrical carbon-carbon (both carbon 7 from benzo[b]thiopen-6-ol) linked homodimer, asymmetrical ether (oxygen from 4-hydroxyphenyl and carbon 7 from benzo[b]thiopen-6-ol) linked homodimer and asymmetrical ether (oxygen and carbon 7 from benzo[b]thiopen-6-ol) linked homodimer, respectively. The structures of the homodimers RD1, 3 and 4 provided evidence for free radical metabolism of raloxifene by predominantly CYP3A4 in human liver microsomes to oxygen-centered phenoxy radicals from 4-hydroxyphenyl and benzo[b]thiopen-6-ol moieties. Further delocalization to ortho carbon-centered radical was only observed for benzo[b]thiopen-6-ol derived phenoxy radical.

Design and characterization of optimized adenosine A2A/A1 receptor antagonists for the treatment of Parkinson's disease.

The design and characterization of two, dual adenosine A(2A)/A(1) receptor antagonists in several animal models of Parkinson's disease is described. Compound 1 was previously reported as a potential treatment for Parkinson's disease. Further characterization of 1 revealed that it was metabolized to reactive intermediates that caused the genotoxicity of 1 in the Ames and mouse lymphoma L51784 assays. The identification of the metabolites enabled the preparation of two optimized compounds 13 and 14 that were devoid of the metabolic liabilities associated with 1. Compounds 13 and 14 are potent dual A(2A)/A(1) receptor antagonists that have excellent activity, after oral administration, across a number of animal models of Parkinson's disease including mouse and rat models of haloperidol-induced catalepsy, mouse and rat models of reserpine-induced akinesia, and the rat 6-hydroxydopamine (6-OHDA) lesion model of drug-induced rotation.