Subsequent Glucuronidation Research Articles

Pharmacokinetics, mass balance, tissue distribution, metabolism, and excretion of [14C]aficamten following single oral dose administration to rats

The pharmacokinetics, metabolism, excretion, mass balance, and tissue distribution of [14C]aficamten were evaluated following oral administration of an 8 mg/kg dose in Sprague Dawley rats and in a quantitative whole-body autoradiography study in Long Evans rats. [14C]Aficamten accounted for ∼80% and a hydroxylated metabolite (M1) accounted for ∼12% of total radioactivity in plasma over 48-h (AUC0–48). Plasma t max was 4-h and the t 1/2 of total plasma radioactivity was 5.8-h. Tissues showing highest Cmax exposures were myocardium and semitendinosus muscle. Most [14C]aficamten-derived radioactivity was excreted within 48-h post-administration. Mean cumulative recovery in urine and faeces over 168-h was 8.3% and 90.7%, respectively. In urine and bile, unchanged aficamten was detected at <0.1 and <0.2% of dose, respectively; however, based on total radioactivity excreted in urine (8.0%) and bile (51.7%), approximately 60% of dose was absorbed. [14C]Aficamten was metabolised by hydroxylation with subsequent glucuronidation where the most abundant metabolite recovered in bile was M5 (35.2%), the oxygen-linked glucuronide of hydroxylated aficamten (M1a). The major metabolite detected in faeces was a 1,2,4-oxadiazole moiety ring-cleaved metabolite (M18, 35.3%), shown to be formed from the metabolism of M5 in incubations with rat intestinal contents solution.

In Vitro and In Vivo Biotransformation Profiling of 6PPD-Quinone toward Their Detection in Human Urine.

The antioxidant N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) and its oxidized quinone product 6PPD-quinone (6PPD-Q) in rubber have attracted attention due to the ecological risk that they pose. Both 6PPD and 6PPD-Q have been detected in various environments that humans cohabit. However, to date, a clear understanding of the biotransformation of 6PPD-Q and a potential biomarker for exposure in humans are lacking. To address this issue, this study presents a comprehensive analysis of the extensive biotransformation of 6PPD-Q across species, encompassing both in vitro and in vivo models. We have tentatively identified 17 biotransformation metabolites in vitro, 15 in mice in vivo, and confirmed the presence of two metabolites in human urine samples. Interestingly, different biotransformation patterns were observed across species. Through semiquantitative analysis based on peak areas, we found that almost all 6PPD-Q underwent biotransformation within 24 h of exposure in mice, primarily via hydroxylation and subsequent glucuronidation. This suggests a rapid metabolic processing of 6PPD-Q in mammals, underscoring the importance of identifying effective biomarkers for exposure. Notably, monohydroxy 6PPD-Q and 6PPD-Q-O-glucuronide were consistently the most predominant metabolites across our studies, highlighting monohydroxy 6PPD-Q as a potential key biomarker for epidemiological research. These findings represent the first comprehensive data set on 6PPD-Q biotransformation in mammalian systems, offering insights into the metabolic pathways involved and possible exposure biomarkers.

Identification and Quantification of Novel Major Metabolites of the Steroidal Aromatase Inhibitor, Exemestane.

Exemestane (EXE) is an aromatase inhibitor used for the prevention and treatment of estrogen receptor–positive breast cancer. Although the known major metabolic pathway for EXE is reduction to form the active 17β-dihydro-EXE (17β-DHE) and subsequent glucuronidation to 17β-hydroxy-EXE-17-O-β-D-glucuronide (17β-DHE-Gluc), previous studies have suggested that other major metabolites exist for exemestane. In the present study, a liquid chromatography–mass spectrometry (LC-MS) approach was used to acquire accurate mass data in MSE mode, in which precursor ion and fragment ion data were obtained simultaneously to screen novel phase II EXE metabolites in urine specimens from women taking EXE. Two major metabolites predicted to be cysteine conjugates of EXE and 17β-DHE by elemental composition were identified. The structures of the two metabolites were confirmed to be 6-methylcysteinylandrosta-1,4-diene-3,17-dione (6-EXE-cys) and 6-methylcysteinylandrosta-1,4-diene-17β-hydroxy-3-one (6-17β-DHE-cys) after comparison with their chemically synthesized counterparts. Both underwent biosynthesis in vitro in three stepwise enzymatic reactions, with the first involving glutathione conjugation. The cysteine conjugates of EXE and 17β-DHE were subsequently quantified by liquid chromatography–mass spectrometry in the urine and matched plasma samples of 132 subjects taking EXE. The combined 6-EXE-cys plus 6-17β-DHE-cys made up 77% of total EXE metabolites in urine (vs. 1.7%, 0.14%, and 21% for EXE, 17β-DHE, and 17β-DHE-Gluc, respectively) and 35% in plasma (vs. 17%, 12%, and 36% for EXE, 17β-DHE, and 17β-DHE-Gluc, respectively). Therefore, cysteine conjugates of EXE and 17β-DHE appear to be major metabolites of EXE in both urine and plasma.

Metabolism of primaquine in normal human volunteers: investigation of phase I and phase II metabolites from plasma and urine using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry

BackgroundPrimaquine (PQ), an 8-aminoquinoline, is the only drug approved by the United States Food and Drug Administration for radical cure and prevention of relapse in Plasmodium vivax infections. Knowledge of the metabolism of PQ is critical for understanding the therapeutic efficacy and hemolytic toxicity of this drug. Recent in vitro studies with primary human hepatocytes have been useful for developing the ultra high-performance liquid chromatography coupled with high-resolution mass spectrometric (UHPLC-QToF-MS) methods for simultaneous determination of PQ and its metabolites generated through phase I and phase II pathways for drug metabolism.MethodsThese methods were further optimized and applied for phenotyping PQ metabolites from plasma and urine from healthy human volunteers treated with single 45 mg dose of PQ. Identity of the metabolites was predicted by MetaboLynx using LC–MS/MS fragmentation patterns. Selected metabolites were confirmed with appropriate standards.ResultsBesides PQ and carboxy PQ (cPQ), the major plasma metabolite, thirty-four additional metabolites were identified in human plasma and urine. Based on these metabolites, PQ is viewed as metabolized in humans via three pathways. Pathway 1 involves direct glucuronide/glucose/carbamate/acetate conjugation of PQ. Pathway 2 involves hydroxylation (likely cytochrome P450-mediated) at different positions on the quinoline ring, with mono-, di-, or even tri-hydroxylations possible, and subsequent glucuronide conjugation of the hydroxylated metabolites. Pathway 3 involves the monoamine oxidase catalyzed oxidative deamination of PQ resulting in formation of PQ-aldehyde, PQ alcohol and cPQ, which are further metabolized through additional phase I hydroxylations and/or phase II glucuronide conjugations.ConclusionThis approach and these findings augment our understanding and provide comprehensive view of pathways for PQ metabolism in humans. These will advance the clinical studies of PQ metabolism in different populations for different therapeutic regimens and an understanding of the role these play in PQ efficacy and safety outcomes, and their possible relation to metabolizing enzyme polymorphisms.

Chemical synthesis, characterisation and in vitro and in vivo metabolism of the synthetic opioid MT-45 and its newly identified fluorinated analogue 2F-MT-45 with metabolite confirmation in urine samples from known drug users

PurposeThe detection of a novel psychoactive substance, 2F-MT-45, a fluorinated analogue of the synthetic opioid MT-45, was reported in a single seized tablet. MT-45, 2F-, 3F- and 4F-MT-45 were synthesised and reference analytical data were reported. The in vitro and in vivo metabolisms of MT-45 and 2F-MT-45 were investigated.MethodThe reference standards and seized sample were characterised using nuclear magnetic resonance spectroscopy, ultra-performance liquid chromatography–quadrupole time of flight mass spectrometry, gas chromatography–mass spectrometry, attenuated total reflectance-Fourier transform infrared spectroscopy and Raman spectroscopy. Presumptive tests were performed and physicochemical properties of the compounds determined. Metabolite identification studies using human liver microsomes, human hepatocytes, mouse hepatocytes and in vivo testing using mice were performed and identified MT-45 metabolites were confirmed in authentic human urine samples.ResultsMetabolic pathways identified for MT-45 and 2F-MT-45 were N-dealkylation, hydroxylation and subsequent glucuronidation. The major MT-45 metabolites identified in human in vitro studies and in authenticated human urine were phase I metabolites and should be incorporated as analytical targets to existing toxicological screening methods. Phase II glucuronidated metabolites were present in much lower proportions.Conclusions2F-MT-45 has been detected in a seized tablet for the first time. The metabolite identification data provide useful urinary metabolite targets for forensic and clinical testing for MT-45 and allows screening of urine for 2F-MT-45 and its major metabolites to determine its prevalence in case work.

Regioselectivity significantly impacts microsomal glucuronidation efficiency of R/S-6, 7-, and 8-hydroxywarfarin

Coumadin (R/S-warfarin) metabolism plays a critical role in patient response to anticoagulant therapy. Several cytochrome P450s oxidize warfarin into R/S-6-, 7-, 8-, 10, and 4′-hydroxywarfarin that can undergo subsequent glucuronidation by UDP-glucuronosyltransferases (UGTs); however, current studies on recombinant UGTs cannot be adequately extrapolated to microsomal glucuronidation capacities for the liver.Herein, we estimated the capacity of the average human liver to glucuronidate hydroxywarfarin and identified UGTs responsible for those metabolic reactions through inhibitor phenotyping. There was no observable activity toward R/S-warfarin, R/S-10-hydroxywarfarin or R/S-4′-hydroxywarfarin.The observed metabolic efficiencies (Vmax/Km) toward R/S-6-, 7-, and especially 8-hydroxywarfarin indicated a high glucuronidation capacity to metabolize these compounds.UGTs demonstrated strong regioselectivity toward the hydroxywarfarins. UGT1A6 and UGT1A1 played a major role in R/S-6- and 7-hydroxywarfarin glucuronidation, respectively, whereas UGT1A9 accounted for almost all of the generation of the R/S-8-hydroxywarfarin glucuronide.In summary, these studies expanded insights to glucuronidation of hydroxywarfarins by pooled human liver microsomes, novel roles for UGT1A6 and 1A9, and the overall degree of regioselectivity for the UGT reactions.

Comparative metabolites profiles of osthole in normal and osteoporosis rats using liquid chromatography quadrupole time-of-flight mass spectrometry

Osthole is a derivative of coumnarin, which has been used to treat several diseases, including osteoporosis. To investigate the metabolite profile of osthole in osteoporosis rats was utilized to understand its underlying mechanisms of its anti-osteoporosis effect. In this study, plasma samples were collected from normal and osteoporosis rats after oral administration of osthole and analyzed to identify the metabolites of osthole by high performance liquid chromatography quadrupole time-of-flight mass spectrometry. By comparing the molecular weight and MS fragmentation of the metabolites with those of parent drug and reference standards, a total of 36 metabolites in plasma were identified. Demethylation, hydroxylation, hydroxymethylene loss and reduction, and subsequent glucuronidation, methylation and sulfation were the major metabolic pathways of osthole in both normal and osteoporosis rats. A specific hydration metabolic pathway was found in osteoporosis rats. These results provided a meaningful basis for studying the underlying mechanism of the anti-osteoporosis effect of osthole.

Metabolic and pharmacokinetic characterization of a new synthetic cannabinoid APINAC in rats

Adamantan-1-yl 1-pentyl-1H-indazole-3-carboxylate (APINAC), a new synthetic cannabinoid, was recently isolated from a dietary supplement and identified in our laboratories. The metabolism and pharmacokinetics of APINAC were studied in vitro and in vivo with a rat model. APINAC (2.0 μM) was incubated with rat liver microsomes (RLMs) for up to 90 min to determine its phase I metabolic profile. APINAC was also administered to rats at a dose of 5 mg/kg intravenously (i.v.) or 10 mg/kg per os (p.o.). Blood samples were collected at specific time points, and urine samples were also collected for 1 day following APINAC administration. The analyses were conducted by both high- and low-resolution liquid chromatography–tandem mass spectrometry. Although APINAC was rapidly metabolized by RLMs (t 1/2, 15.2 ± 0.4 min), in vivo pharmacokinetic experiments in rats revealed moderate to long half-lives [11.3 h (i.v.) and 3.8 h (p.o.)]. A total of 22 APINAC metabolites could be detected in the RLMs and rat urine. APINAC was predominantly metabolized via ester hydrolysis to carboxylic acids M1 (with hydroxylation) and M18 (without hydroxylation), representative markers for APINAC intake. Hydroxylation of APINAC, with or without subsequent oxidation and glucuronidation, was observed in the case of the other metabolites. The diagnosis of illegal APINAC intake can be realized through the detection of several characteristic APINAC metabolites in human urine and/or APINAC itself in blood.

Reductive Metabolism Influences the Toxicity and Pharmacokinetics of the Hypoxia-Targeted Benzotriazine Di-Oxide Anticancer Agent SN30000 in Mice.

3-(3-Morpholinopropyl)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine 1,4-dioxide (SN30- 000), an analog of the well-studied bioreductive prodrug tirapazamine (TPZ), has improved activity against hypoxic cells in tumor xenografts. However, little is known about its biotransformation in normal tissues. Here, we evaluate implications of biotransformation of SN30000 for its toxicokinetics in NIH-III mice. The metabolite profile demonstrated reduction to the 1-N-oxide (M14), oxidation of the morpholine side-chain (predominantly to the alkanoic acid M18) and chromophore, and subsequent glucuronidation. Plasma pharmacokinetics of SN30000 and its reduced metabolites was unaffected by the presence of HT29 tumor xenografts, indicating extensive reduction in normal tissues. This bioreductive metabolism, as modeled by hepatic S9 preparations, was strongly inhibited by oxygen indicating that it proceeds via the one-electron (radical) intermediate previously implicated in induction of DNA double strand breaks and cytotoxicity by SN30000. Plasma pharmacokinetics of SN30000 and M14 (but not M18) corresponded closely to the timing of reversible acute clinical signs (reduced mobility) and marked hypothermia (rectal temperature drop of ∼8°C at nadir following the maximum tolerated dose). Similar acute toxicity was elicited by dosing with TPZ or M14, although M14 did not induce the kidney and lung histopathology caused by SN30000. M14 also lacked antiproliferative potency in hypoxic cell cultures. In addition M14 showed much slower redox cycling than SN30000 in oxic cultures. Thus a non-bioreductive mechanism, mediated through M14, appears to be responsible for the acute toxicity of SN30000 while late toxicities are consistent with DNA damage resulting from its one-electron reduction. A two-compartment pharmacokinetic model, in which clearance of SN30000 is determined by temperature-dependent bioreductive metabolism to M14, was shown to describe the non-linear PK of SN30000 in mice. This study demonstrates the importance of non-tumor bioreductive metabolism in the toxicology and pharmacokinetics of benzotriazine di-oxides designed to target tumor hypoxia.

In Vitro Metabolite Profiling of ADB-FUBINACA, A New Synthetic Cannabinoid.

Metabolite profiling of novel psychoactive substances (NPS) is critical for documenting drug consumption. N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide (ADB-FUBINACA) is an emerging synthetic cannabinoid whose toxicological and metabolic data are currently unavailable. We aimed to determine optimal markers for identifying ADB-FUBINACA intake. Metabolic stability was evaluated with human liver microsome incubations. Metabolites were identified after 1 and 3 h incubation with pooled human hepatocytes, liquid chromatography- high resolution mass spectrometry in positive-ion mode (5600+ TripleTOF®, Sciex) and several data mining approaches (MetabolitePilot™, Sciex). Metabolite separation was achieved on an Ultra Biphenyl column (Restek®); full-scan TOF-MS and information-dependent acquisition MS/MS data were acquired. ADB-FUBINACA microsomal half-life was 39.7 min, with a predicted hepatic clearance of 9.0 mL/min/kg and a 0.5 extraction ratio (intermediate-clearance drug). Twenty-three metabolites were identified. Major metabolic pathways were alkyl and indazole hydroxylation, terminal amide hydrolysis, subsequent glucuronide conjugations, and dehydrogenation. We recommend ADB-FUBINACA hydroxyalkyl, hydroxydehydroalkyl and hydroxylindazole metabolites as ADB-FUBINACA intake markers. N-dealkylated metabolites are not specific ADB-FUBINACA metabolites and should not be used as definitive markers of consumption. This is the first ADB-FUBINACA in vitro metabolism study; in vivo experiments enabling pharmacokinetic and pharmacodynamics studies or urine from authentic clinical/forensic cases are needed to confirm our results.

Role of the UGT2B17 deletion in exemestane pharmacogenetics

Exemestane (EXE) is an aromatase inhibitor used for the prevention and treatment of breast cancer. The major metabolic pathway for EXE is reduction to form the active 17β-dihydro-EXE (17β-DHE) and subsequent glucuronidation to 17β-hydroxy-EXE-17-O-β-D-glucuronide (17β-DHE-Gluc) by UGT2B17. The aim of the present study was to determine the effects of UGT2B17 copy number variation on the levels of urinary and plasma 17β-DHE-Gluc and 17β-DHE in patients taking EXE. Ninety-six post-menopausal Caucasian breast cancer patients with ER+ breast tumors taking 25 mg EXE daily were recruited into this study. UGT2B17 copy number was determined by a real-time PCR copy number variant assay and the levels of EXE, 17β-DHE and 17β-DHE-Gluc were quantified by UPLC/MS in patients’ urine and plasma. A 39-fold decrease (P<0.0001) in the levels of creatinine-adjusted urinary 17β-DHE-Gluc was observed among UGT2B17 (*2/*2) subjects vs subjects with the UGT2B17 (*1/*1) genotype. The plasma levels of 17β-DHE-Gluc was decreased 29-fold (P<0.0001) in subjects with the UGT2B17 (*2/*2) genotype vs subjects with UGT2B17 (*1/*1) genotype. The levels of plasma EXE-adjusted 17β-DHE was 28% higher (P=0.04) in subjects with the UGT2B17 (*2/*2) genotype vs subjects with the UGT2B17 (*1/*1) genotype. These data indicate that UGT2B17 is the major enzyme responsible for 17β-DHE-Gluc formation in vivo and that the UGT2B17 copy number variant may play a role in inter-individual variability in 17β-DHE levels in vivo.

Metabolites identification of berberine in rats using ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry

Berberine (BBR), the principle component for many medicinal plants such as Coptis chinensis Franch., Phellodendron chinense Schneid., and Mahonia bealei (Fort.) Carr., possesses diverse pharmacological activities, including anti-bacterial, anti-inflammatory, antitumor, hypolipidemic and antidiabetic activities. In this study, a rapid and reliable method using a five-step strategy based on the ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS), and metabolynx™ software with mass defect filter (MDF) technique was developed to investigate the metabolism of BBR. Plasma, bile, urine and feces samples were collected from rats after oral administration of BBR with a dose of 100mg/kg/day for three consecutive days and analyzed to characterize the metabolic profile of BBR. By comparing the molecular weights and MS fragmentations of the metabolites with those of the parent drug and reference standards, a total of 97 metabolites were identified, including 68 metabolites in urine, 45 metabolites in plasma, 44 metabolites in bile and 41 metabolites in feces. Demethylation, demethylenation, reduction, hydroxylation, and subsequent glucuronidation, sulfation and methylation were the major metabolic pathways of BBR in vivo.

Detection of metabolites of two synthetic cannabimimetics, MDMB-FUBINACA and ADB-FUBINACA, in authentic human urine specimens by accurate mass LC–MS: a comparison of intersecting metabolic patterns

The structures of two indazole-derived synthetic cannabimimetics, a methyl ester, MDMB-FUBINACA, and an amide, ADB-FUBINACA, differ only in the terminal groups on the side chains. Based upon liquid chromatography–quadrupole time-of-flight-mass spectrometry analysis of urine and blood samples collected from patients who were admitted to hospital with suspected drug intoxications and from postmortem forensic investigations, 38 metabolites were tentatively identified. Hydrolysis of the terminal groups (methyl ester and amide for MDMB-FUBINACA and ADB-FUBINACA, respectively) was found to be a common metabolic pathway for both compounds, leading to the formation of other common metabolites. Hydrolysed metabolites undergo subsequent monohydroxylation, dihydrodiol formation, fluorobenzyl loss and dehydrogenation. Most of the metabolites of MDMB-FUBINACA were products of ester hydrolysis. Metabolites formed by hydrolysis, additional monohydroxylation, dihydrodiol formation and fluorobenzyl loss were also detected in forms of glucuronides. Two unhydrolysed metabolites were identified as products of hydroxylation and fluorobenzyl loss with subsequent glucuronidation. In the case of ADB-FUBINACA, products of mono- and dihydroxylation, dihydrodiol formation, dihydrodiol formation combined with monohydroxylation, monohydroxylation combined with dehydrogenation and fluorobenzyl loss with monohydroxylation were identified. Monohydroxylated and dihydrodiol metabolites were also detected in the form of glucuronides. The most abundant metabolites were products of ester hydrolysis in free and glucuronidated forms (for MDMB-FUBINACA) and of dihydrodiol formation (for ADB-FUBINACA). These compounds are recommended for toxicological screening. To our knowledge, there are no reports dealing with the detection of metabolites of MBDB-FUBINACA and ADB-FUBINACA in authentic human urine specimens.

In vitro studies on flubromazolam metabolism and detection of its metabolites in authentic forensic samples.

Flubromazolam is a triazole benzodiazepine with high potency and long-lasting central nervous system depressant effects; however, limited data about its pharmacokinetics are available. Here, we report in vitro studies of the human flubromazolam metabolism analyzed by liquid chromatography high-resolution mass spectrometry (LC-HRMS). In vitro investigations were carried out in pooled human liver microsomes (pHLM) and recombinant cytochrome P450 (CYP)-enzymes. To confirm those metabolites detected in vitro, authentic samples obtained from two forensic cases were also analyzed by LC-HRMS. Additionally, determination of the unbound fraction of flubromazolam in pHLM and in plasma was performed by equilibrium dialysis with subsequent prediction of its hepatic clearance (CLH ) using well-stirred and parallel-tube models. Additional findings obtained by routine screening methods of these forensic cases are also reported. Studies using incubations with nicotinamide adenine dinucleotide phosphate-fortified pHLM with or without uridine 5'-diphosphoglucuronic acid and incubations with CYP-enzymes identified the main metabolic pathway of flubromazolam as hydroxylation on the α- and/or 4-position mediated by CYP3A4 and CYP3A5, with subsequent glucuronidation of the hydroxylated metabolites as well as of the parent drug. Further, α-hydroxy-flubromazolam and its corresponding glucuronide were detected in vivo together with the N-glucuronide of flubromazolam. The predicted CLH of flubromazolam using the well-stirred and parallel-tube models were 0.42 and 0.43 mL/min/kg, respectively. Based on the data presented here, flubromazolam is primarily metabolized by CYP3A4/5 with a high protein-binding and a predicted low clearance. Analysis of authentic samples suggested that analytical targets for flubromazolam should be the compound itself and α-hydroxy-flubromazolam. Copyright © 2016 John Wiley & Sons, Ltd.

In Vivo Metabolite Profiling of a Purified Ellagitannin Isolated from Polygonum capitatum in Rats.

Ellagitannin is a common compound in food and herbs, but there are few detailed studies on the metabolism of purified ellagitannins. FR429 is a purified ellagitannin with antitumor potential, which is from Polygonum capitatum Buch.-Ham.ex D. Don. The present study was designed to investigate the metabolic profiles of FR429 in rats in vivo. Using liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LC/MSn-IT-TOF), total eight metabolites were found in rat bile and urine after intravenous administration of FR429, but could not be detected in plasma. These metabolites were ellagic acid, mono-methylated FR429, ellagic acid methyl ether glucuronide, ellagic acid methyl ether diglucuronide, ellagic acid dimethyl ether glucuronide, and ellagic acid dimethyl ether diglucuronide. It was concluded that methylation and subsequent glucuronidation were the major metabolic pathways of FR429 in rats in vivo. This is the first report on the in vivo metabolism of the purified ellagitannin in rats.

In vitro and in vivo human metabolism of a new synthetic cannabinoid NM-2201 (CBL-2201).

In 2014, NM-2201 (CBL-2201), a novel synthetic cannabinoid (SC), was detected by Russian and United States laboratories. It was already added to the scheduled drugs list in Japan, Sweden and Germany. Unfortunately, no human metabolism data are currently available, making it challenging to confirm its intake because all previous investigated SCs were extensively metabolized. The present study aims to recommend appropriate marker metabolites by investigating NM-2201 metabolism in human hepatocytes and confirm the results in authentic human urine specimens. For the metabolic stability assay, 1 μM NM-2201 was incubated in human liver microsomes (HLMs) for up to 1 h; for metabolite profiling, 10 μM of NM-2201 was incubated in human hepatocytes for 3 h. Two authentic urine specimens from NM-2201 positive cases were analyzed after β-glucuronidase hydrolysis. Metabolite identification in hepatocyte samples and urine specimens was achieved with high-resolution mass spectrometry via information-dependent acquisition. NM-2201 was quickly metabolized in HLMs with an 8.0 min half-life. In human hepatocyte incubation samples, a total of thirteen NM-2201 metabolites were identified, generated mainly from ester hydrolysis and further hydroxylation, oxidative defluorination and subsequent glucuronidation. M13 (5-fluoro PB-22 3-carboxyindole) was the major metabolite. In the urine specimens, the parent drug NM-2201 was not detected; M13 was the predominant metabolite after β-glucuronidase hydrolysis. Therefore, based on our study, we recommend the M13 as a suitable urinary marker metabolite for confirming NM-2201 and/or 5F-PB-22 intake.

Metabolite profiling and enzyme reaction phenotyping of luseogliflozin, a sodium–glucose cotransporter 2 inhibitor, in humans

1. To understand the clearance mechanism of luseogliflozin, a sodium–glucose cotransporter 2 (SGLT2) inhibitor, we investigated its human metabolite profile and metabolic enzymes responsible for the primary metabolic pathways in human using reaction phenotyping.2. Sixteen metabolites of luseogliflozin were found in human plasma and/or urine and their structural information indicated that the drug was metabolized via multiple metabolic pathways. The primary metabolic pathways involve (1) O-deethylation to form M2 and subsequent glucuronidation to form M12, (2) ω-hydroxylation at ethoxy group to form M3 followed by oxidation to form the corresponding carboxylic acid metabolite (M17) and (3) direct glucuronidation to form M8.3. The reaction phenotyping studies indicated that the formation of M2 was mainly mediated by cytochrome P450 (CYP) 3A4/5, and subsequently M12 formation was catalyzed by UGT1A1, UGT1A8 and UGT1A9. The formation of M3 was mediated by CYP4A11, CYP4F2 and CYP4F3B, and the further oxidation of M3 to M17 was mediated by alcohol dehydrogenase and aldehyde dehydrogenase. The formation of M8 was catalyzed by UGT1A1.4. These results demonstrate that luseogliflozin is metabolized through multiple pathways, including CYP-mediated oxidation and glucuronidation, in human.

Metabolic disposition of AZD8931, an oral equipotent inhibitor of EGFR, HER2 and HER3 signalling, in rat, dog and man

1. This series of studies in rats, dogs and humans (Clinicaltrials.gov identifier: NCT01284595) investigated the pharmacokinetics, tissue distribution, metabolism and excretion of the EGFR, HER2 and HER3 signalling inhibitor AZD8931.2. Single oral or intravenous doses of 2-(4-[4-(3-chloro-2-fluoro[U-14C]-phenylamino)-7-methoxy-quinazolin-6-yloxy]-piperidin-1-yl)-N-methyl-acetamide difumarate ([14C]-AZD8931) were administered.3. AZD8931 absorption was rapid in all species. Following [14C]-AZD8931 administration to rats, radioactivity was widely and rapidly distributed, with the highest levels in organs of metabolism and excretion (gastrointestinal tract, liver). Following oral and intravenous [14C]-AZD8931 administration, excretion of radioactivity by all species occurred predominantly via the bile into faeces, with <5% of the dose being eliminated in urine. In all species, AZD8931 was principally cleared by metabolism. The major route of metabolism was hydroxylation and O-demethylation in rat, and aryl ring oxidation in dog. Metabolism of AZD8931 in humans was attributed to three pathways; oxidation and amine or ether cleavage around the piperidine ring with subsequent glucuronide or sulphate conjugation.4. AZD8931 is largely cleared by metabolism in the rat, dog and human. Excretory profiles indicate that there are no unique human metabolites.

Metabolite profiling and identification of L41, a novel cyclin-dependent kinase 1 inhibitor, in rat plasma and urine by liquid chromatography–tandem mass spectrometry

(E)-3-(2-Chlorophenyl)-1-(2-hydroxy-4,6-dimethoxy-3-(1,2,3,6-tetrahydropyridine-4-yl)phenyl)-2-propylene-1-one (L41) is a novel synthetic cyclin-dependent kinase 1 inhibitor. In this study, we proposed a strategy to systematically characterize associated phase I and phase II metabolites of L41 using rat plasma and urine samples. Biological samples were treated with an optimized sample preparation approach involving protein precipitation, liquid–liquid extraction and solid-phase extraction prior to LC–MS/MS analysis. Both an Agilent LC/MSD Trap VL and an AB SCIEX API 5000 triple quadrupole mass spectrometer were used for LC–MS/MS analysis. Based on the proposed strategy, 3 phase I and 4 phase II metabolites were detected by a highly sensitive MRM method. The major metabolites of L41 were found to be demethylation metabolites (M5, M6), C-4′,6′-di-demethylation metabolites followed by a C-2′ (OH) methylation metabolite (M7), and were shown to undergo subsequent glucuronidation processes. These results provide evidence for in vivo L41 metabolism, and the method may be applied to the further analysis of L41 and its metabolites in biological samples.

Absorption, metabolism and excretion of [14C]pomalidomide in humans following oral administration

PurposeTo investigate the pharmacokinetics and disposition of [14C]pomalidomide following a single oral dose to healthy male subjects.MethodsEight subjects were administered a single 2 mg oral suspension of [14C]pomalidomide. Blood (plasma), urine and feces were collected. Mass balance of radioactivity and the pharmacokinetics of radioactivity, pomalidomide and metabolites were determined. Metabolite profiling and characterization was performed. The enzymes involved in pomalidomide metabolism and the potential pharmacological activity of metabolites were evaluated in vitro.ResultsMean recovery was 88 %, with 73 and 15 % of the radioactive dose excreted in urine and feces, respectively, indicating good oral absorption. Mean C max, AUC0−∞ and t max values for pomalidomide in plasma were 13 ng/mL, 189 ng*h/mL and 3.0 h. Radioactivity and pomalidomide were rapidly cleared from circulation, with terminal half-lives of 8.9 and 11.2 h. Pomalidomide accounted for 70 % of the circulating radioactivity, and no circulating metabolite was present at >10 % of parent compound. Pomalidomide was extensively metabolized prior to excretion, with excreted metabolites being similar to those observed in circulation. Clearance pathways included cytochrome P450-mediated hydroxylation with subsequent glucuronidation (43 % of the dose), glutarimide ring hydrolysis (25 %) and excretion of unchanged drug (10 %). 5-Hydroxy pomalidomide, the notable oxidative metabolite, was formed primarily via CYP1A2 and CYP3A4. The hydroxy metabolites and hydrolysis products were at least 26-fold less pharmacologically active than pomalidomide in vitro.ConclusionsFollowing oral administration, pomalidomide was well absorbed, with parent compound being the predominant circulating component. Pomalidomide was extensively metabolized prior to excretion, and metabolites were eliminated primarily in urine.