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.

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.

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.

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.

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.

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.

Overcoming the genotoxicity of a pyrrolidine substituted arylindenopyrimidine as a potent dual adenosine A(2A)/A(1) antagonist by minimizing bioactivation to an iminium ion reactive intermediate.

2-Amino-4-phenyl-8-pyrrolidin-1-ylmethyl-indeno[1,2-d]pyrimidin-5-one (1) is a novel and potent selective dual A(2A)/A(1) adenosine receptor antagonist from the arylindenopyrimidine series that was determined to be genotoxic in both the Ames and Mouse Lymphoma L5178Y assays only following metabolic activation. Compound 1 was identified as a frame-shift mutagen in Salmonella typhimurium tester strain TA1537 as indicated by a significant dose-dependent increase in revertant colonies as compared to the vehicle control. The metabolic activation-dependent irreversible covalent binding of radioactivity to DNA, recovery of 1 and its enamine metabolite from acid hydrolysis of covalently modified DNA, and protection of covalent binding to DNA by both cyanide ion and methoxylamine suggest that the frame-shift mutation in TA1537 strain involved covalent binding instead of simple intercalation to DNA. Compound 1 was bioactivated to endocyclic iminium ion, aldehyde, epoxide, and α,ß-unsaturated keto reactive intermediates from the detection of cyano, oxime, and glutathione conjugates by data-dependent high resolution accurate mass measurements. Collision-induced dissociation of these conjugates provided evidence for bioactivation of the pyrrolidine ring of 1. The epoxide and α,ß-unsaturated keto reactive intermediates were unlikely to cause the genotoxicity of 1 because the formation of their glutathione adducts did not ameliorate the binding of compound related material to DNA. Instead, the endocyclic iminium ions and amino aldehydes were likely candidates responsible for genotoxicity based on, first, the protection afforded by both cyanide ion and methoxylamine, which reduced the potential to form covalent adducts with DNA, and, second, analogues of 1 designed with low probability to form these reactive intermediates were not genotoxic. It was concluded that 1 also had the potential to be mutagenic in humans based on observing the endocyclic iminium ion following incubation with a human liver S9 preparation and the commensurate detection of DNA adducts. An understanding of this genotoxicity mechanism supported an evidence-based approach to selectively modify the structure of 1 which resulted in analogues being synthesized that were devoid of a genotoxic liability. In addition, potency and selectivity against both adenosine A(2A) and A(1) receptors were maintained.

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.

The conduct of in vitro studies to address time-dependent inhibition of drug-metabolizing enzymes: a perspective of the pharmaceutical research and manufacturers of America.

Time-dependent inhibition (TDI) of cytochrome P450 (P450) enzymes caused by new molecular entities (NMEs) is of concern because such compounds can be responsible for clinically relevant drug-drug interactions (DDI). Although the biochemistry underlying mechanism-based inactivation (MBI) of P450 enzymes has been generally understood for several years, significant advances have been made only in the past few years regarding how in vitro time-dependent inhibition data can be used to understand and predict clinical DDI. In this article, a team of scientists from 16 pharmaceutical research organizations that are member companies of the Pharmaceutical Research and Manufacturers of America offer a discussion of the phenomenon of TDI with emphasis on the laboratory methods used in its measurement. Results of an anonymous survey regarding pharmaceutical industry practices and strategies around TDI are reported. Specific topics that still possess a high degree of uncertainty are raised, such as parameter estimates needed to make predictions of DDI magnitude from in vitro inactivation parameters. A description of follow-up mechanistic experiments that can be done to characterize TDI are described. A consensus recommendation regarding common practices to address TDI is included, the salient points of which include the use of a tiered approach wherein abbreviated assays are first used to determine whether NMEs demonstrate TDI or not, followed by more thorough inactivation studies for those that do to define the parameters needed for prediction of DDI.

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.

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.

Pharmacokinetics of sirolimus (rapamycin) in subjects with mild to moderate hepatic impairment.

Eighteen adult subjects with mild to moderate hepatic impairment and 18 healthy control subjects were given a single 15-mg dose of sirolimus by oral solution. Mean whole-blood sirolimus weight-normalized oral-dose clearances (CL/F) were significantly decreased (P = .02) in subjects with mild to moderate hepatic impairment by -31.8% and -36.0%, respectively, compared with controls. There were no significant differences in mean sirolimus C(max) and t(max) values among groups. The observed decreases in CL/F may be relevant in renal transplant patients with mild to moderate hepatic impairment, based on the close similarity of sirolimus CL/F in controls and previously studied stable renal transplant patients receiving multiple-dose administration of sirolimus and cyclosporine. There was considerable overlap in the CL/F values of hepatic-impaired subjects and controls, suggesting that whole-blood sirolimus trough concentrations in renal transplant patients exhibiting mild to moderate hepatic impairment be initially monitored to assess the need for dose adjustments.

Production of GABA by cultured hippocampal glial cells.

Medium conditioned by cultured hippocampal glial contains an inhibitory factor that can hyperpolarize and suppress neuronal activity. Using biochemistry, electrophysiology, pharmacology, and mass spectrometry, we have identified the inhibitory factor as GABA (gamma-aminobutyric acid). Like GABA, the inhibitory factor increases chloride and potassium currents in neurons, which can be blocked by bicuculline. Mass spectrometry analysis of conditioned medium reveals peaks that are identical to that for GABA. Up to 500 micromolar GABA is found in conditioned medium from glial cultures. No GABA is found in conditioned medium from neuronal cultures. Hippocampal glia make much more GABA than cortical glia or glia from other brain regions. It is not clear how hippocampal glia synthesize GABA. Although they express GAD mRNA and adding glutamate to the culture medium increases the amount of GABA produced, other data suggest that glia do not use GAD to make GABA. Identifying the mechanism(s) by which GABA is produced by hippocampal glia would help clarify its role in modulating neuronal activity in the brain.

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).

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.

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.

A generic method to detect electrophilic intermediates using isotopic pattern triggered data-dependent high-resolution accurate mass spectrometry.

A need still exists for a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method that can detect broad classes of glutathione (GSH) conjugates and provide characterization of their structures. We now describe the development of a method that multiplexes high-resolution accurate mass analysis with isotope pattern triggered data-dependent product ion scans, for simultaneous detection and structural elucidation of GSH conjugates within a single analysis using a LTQ/Orbitrap. This method was initially developed to detect GSH conjugates generated from incubating 10 microM test compound with pooled human liver microsomes fortified with NADPH-regenerating system and a 2:1 ratio of 5 mM glutathione and [(13)C(2) (15)N-Gly]glutathione. The GSH conjugates were detected by isotope search of mass defect filtered and control subtracted full scan accurate MS data using MetWorks software. This was followed by elucidation of reactive intermediate structures using chemical formulae for both protonated molecules and their product ions from accurate masses in a single analysis. The mass accuracies measured for the precursor and product ions by the Orbitrap were <2 ppm in external mass calibration mode. Successful detection and characterization of GSH conjugates of acetaminophen, tienilic acid, clozapine, ticlopidine and mifepristone validated this method. In each case, the detected GSH conjugates were within the top five hits by isotope search. This method also has a broader detection capability since it is independent of the collision-induced dissociation behavior of the GSH conjugates. Furthermore, this method is amenable to a broad class of reactive intermediate trapping agents as exemplified by the simultaneous detection and structural elucidation of the cyano-N-methylene iminium ion conjugates of verapamil and its O-desmethyl metabolites, which we report for the first time. In addition to the chemically tagged reactive intermediates, this method also provides information on stable metabolites from the full scan accurate MS data.

Metabolite identification by data-dependent accurate mass spectrometric analysis at resolving power of 60,000 in external calibration mode using an LTQ/Orbitrap.

Performance evaluation of accurate mass measurement by the LTQ/Orbitrap, at a resolving power of 60,000 and in external calibration mode, indicated that the Orbitrap is capable of providing high mass accuracy of <2 ppm for over 24 h post-calibration. This, together with limited trade-off between sensitivity and resolving power plus a wide dynamic range for mass accuracy, suggested that the LTQ/Orbitrap is an ideal analytical tool for structural elucidation of metabolites. The application of the LTQ/Orbitrap to identification of human liver microsomal metabolites of carvedilol was evaluated, using parent mass list triggered data-dependent multiple-stage accurate mass analysis, at a resolving power of 60,000 in external calibration mode. A metabolite identification workflow was developed to utilize chemical formulas from high-resolution accurate mass measurements to confirm structures of product ions of a drug proposed by Mass Frontier, illustrated by identification of structures used to establish lineage of product ions of carvedilol, which later served as a template for identification of its metabolites. A total of 58 in vitro metabolites of carvedilol were detected using 5-ppm mass tolerance filters for theoretical m/z of protonated molecules of predicted metabolites in addition to product ions and neutral mass losses diagnostic of carvedilol. The chemical formulas with unsaturation numbers calculated from the accurate m/z of precursor and product ions can be used to assign, with a high degree of confidence, the structures of metabolites and the sites of metabolism. The mass accuracies obtained for all full scan MS and MSn spectra were <2 ppm. The majority of the metabolites identified agreed with those previously reported except for those that have not been reported before. For example, several glutathione conjugates of carvedilol were reported for the first time, which may explain the reported hepatotoxicity during clinical trials and recent clinical use.

Pharmacokinetics and metabolic disposition of sirolimus in healthy male volunteers after a single oral dose.

The pharmacokinetics and metabolic disposition of sirolimus (rapamycin, Rapamune), a macrocyclic immunosuppressive agent for the prevention of allograft rejection in organ transplantation, were investigated in 6 healthy male volunteers after a single nominal 40-mg oral dose of the C-radiolabeled drug, with the added aim of assessing the potential role of sirolimus metabolites in the clinical pharmacology of the parent drug. The absorption of parent drug and derived materials was rapid (tmax 1.3 +/- 0.5 hours, mean +/- SD), and the elimination of sirolimus was slow (t(1/2) 60 +/- 10 hours, mean +/- SD) in whole blood. The high whole blood to plasma (B/P) concentration ratio of sirolimus (142 +/- 39) was consistent with its extensive partitioning into formed blood elements. The markedly lower B/P value based on radioactivity (2.7 +/- 0.4) suggested that drug-derived products partitioned into formed blood elements to a much lesser extent. Based on AUC0-144h values, unchanged sirolimus represented an average 35% of total radioactivity in whole blood. Drug-derived products in whole blood were characterized by HPLC, LC/MS, and LC/MS/MS as 41-O-demethyl, 7-O-demethyl, and several hydroxy, dihydroxy, hydroxy-demethyl and didemethyl sirolimus metabolites. The percentage distribution of sirolimus metabolites in whole blood ranged from 3%-10% at 1 hour to 6%-17% at 24 hours after drug administration. Based on their low immunosuppressive activities and relative abundance in whole blood of humans after sirolimus administration, metabolites of sirolimus do not appear to play a major role in the clinical pharmacology of the parent drug. A majority of the administered radioactivity (91.0 +/- 8.0%) was recovered from feces, and only 2.2% +/- 0.9% was renally excreted.