Formation Of M3 Research Articles

Identification and characterization of the metabolites of sinomenine using liquid chromatography combined with benchtop Orbitrap mass spectrometry and nuclear magnetic resonance spectroscopy

RationaleSinomenine, a major bioactive compound isolated from Sinomenium acutum, has been used for the treatment of rheumatoid arthritis and other cardio‐cerebrovacular diseases. However, the metabolism of this drug has not been fully investigated. The current work was carried out to investigate the in vitro metabolism of sinomenine in liver microsomes.MethodsThe metabolites were generated by incubating sinomenine (3 μM) with the liver microsomes in the presence of NADPH at 37°C. The structure of the metabolites was characterized using liquid chromatography coupled to high‐resolution mass spectrometry (HRMS). Two major metabolites synthesized and their structures were further confirmed using nuclear magnetic resonance spectroscopy.ResultsUnder the current conditions, 12 metabolites were found and structurally identified using high resolution MS and MS2 spectra. Among these metabolites, M1, M2, M3, M4, M5, M6, M7, M9, M11, and M12 were first reported. The metabolites M8 and M10 were synthesized and unambiguously identified as N‐desmethyl‐sinomenine and sinomenine N‐oxide, respectively. The phenotyping study revealed that the formation of M8 was catalyzed by CYP2C8, 2C19, 2D6, and 3A4, whereas the formation of M3, M6, and M10 were exclusively catalyzed by CYP3A4. The metabolic pathways of sinomenine include N‐demethylation, O‐demethylation, dehydrogenation, oxygenation, and N‐oxygenation.ConclusionsN‐Demethylation and N‐oxygenation were the primary metabolic pathways of sinomenine. This study provides new insight into the in vitro metabolism of sinomenine, which would help prospects of sinomenine disposition and safety assessments.

In-vitro metabolism of LXY18, an orally available, potent blocker of AURKB relocation in mitosis

This study investigated the metabolism of LXY18, a quinolone-based compound that suppresses tumorigenesis by blocking AURKB localization. Metabolite profiling of LXY18 in liver microsomes from six species and human S9 fractions revealed that LXY18 undergoes various conserved metabolic reactions, such as N-hydroxylation, N-oxygenation, O-dealkylation, and hydrolysis, resulting in ten metabolites. These metabolites were produced through a combination of CYP450 enzymes, and non-CYP450 enzymes including CES1, and AO. Two metabolites, M1 and M2 were authenticated by chemically synthesized standards. M1 was the hydrolyzed product catalyzed by CES1 whereas M2 was a mono-N-oxidative derivative catalyzed by a CYP450 enzyme. AO was identified as the enzyme responsible for the formation of M3 with the help of AO-specific inhibitors and LXY18 analogs, 5b and 5c. M1 was the intermediate of LXY18 to produce M7, M8, M9, and M10. LXY18 potently inhibited 2C19 with an IC50 of 290 nM but had a negligible impact on the other CYP450s, indicating a low risk of drug-drug interaction. Altogether, the study provides valuable insights into the metabolic process of LXY18 and its suitability as a drug candidate. The data generated serves as a significant reference point for conducting further safety assessments and optimizing drug development.

Gas-Phase Thermochemistry of MX3 and M2X6 (M = Sc, Y; X = F, Cl, Br, I) from a Composite Reaction-Based Approach: Homolytic versus Heterolytic Cleavage.

A domain-based local-pair natural-orbital coupled-cluster approach with single, double, and improved linear-scaling perturbative triple correction via an iterative algorithm, DLPNO-CCSD(T1), was applied within the framework of the Feller-Peterson-Dixon approach to derive gas-phase heats of formation of scandium and yttrium trihalides and their dimers via a set of homolytic and heterolytic dissociation reactions. All predicted heats of formation moderately depend on the reaction type with the most and least negative values obtained for homolytic and heterolytic dissociation, respectively. The basis set size dependence, as well as the influence of static correlation effects not covered by the standard (DLPNO-)CCSD(T) approach, suggests that exploitation of the heterolytic dissociation reactions with the formation of M3+ and X- ions leads to the most robust heats of formation. The gas-phase formation enthalpies ΔHf°(0 K)/ΔHf°(298.15 K) and absolute entropies S°(298.15 K) were obtained for the first time for the Sc2F6, Sc2Br6, and Sc2I6 species. For ScBr3, ScI3, Sc2Cl6, and Y2Cl6, we suggest a reexamination of the experimental heats of formation available in the literature. For other compounds, the predicted values were found to be in good agreement with the experimental estimates. Extracted MX3 (M = Sc, Y; X = F, Cl, Br, and I) 0 K atomization enthalpies indicate weaker bonding when moving from fluorine to iodine and from yttrium to scandium. Likewise, the stability of yttrium trihalide dimers degrades when going from fluorine to iodine. Respective scandium trihalide dimers are less stable, with 0 K dimer dissociation energy decreasing in the row fluorine - chlorine - bromine ≈ iodine. Correlation of the (n - 1)s2p6 electrons on bromine and iodine, inclusion of zero-point energy, relativistic effects, and the effective-core-potential correction as well as amelioration of the DLPNO localization inaccuracy are shown to be of similar magnitude, which is critical if accurate heats of formation are a goal.

Non-Linear Pharmacokinetics of Oral Roscovitine (Seliciclib) in Cystic Fibrosis Patients Chronically Infected with Pseudomonas aeruginosa: A Study on Population Pharmacokinetics with Monte Carlo Simulations.

Roscovitine (Seliciclib), a new protein kinase inhibitor, was administered orally to adult patients with cystic fibrosis for the first time in the ROSCO-CF trial, a dose-escalation, phase IIa, randomized, controlled trial. Extensive pharmacokinetic sampling was performed up to 12 h after the first oral dose. Roscovitine and its main metabolite M3 were quantified by liquid chromatography coupled with tandem mass spectrometry. The pharmacokinetics analyses were performed by non-linear mixed effects modelling. Monte Carlo simulations were performed to assess the impact of dose on the pharmacokinetics of oral roscovitine. Twenty-three patients received oral doses ranging from 200 to 800 mg of roscovitine and 138 data points were available for both roscovitine and M3 concentrations. The pharmacokinetics was best described by a two-compartment parent-metabolite model, with a complex saturable absorption process modelled as the sum of Gaussian inverse density functions. The Monte Carlo simulations showed a dose-dependent and saturable first-pass effect leading to pre-systemic formation of M3. The treatment with proton-pump inhibitors reduced the rate of absorption of oral roscovitine. The pharmacokinetics of oral roscovitine in adult patients with cystic fibrosis was non-linear and showed significant inter-individual variability. A repeat-dose study will be required to assess the inter-occasional variability of its pharmacokinetics.

Impact of Inter‐individual Variability in CYP1A2 and CYP3A4 Activity on Sunitinib Metabolism

Sunitinib is an orally administered multi‐targeted tyrosine kinase inhibitor used in the treatment of renal cell carcinoma, gastrointestinal stromal tumors, and other types of cancer. The product label of sunitinib carries a boxed warning for idiosyncratic hepatotoxicity; however, the mechanisms of this toxicity remain unclear. Recent studies suggest that cytochrome P450‐mediated bioactivation may play a role in sunitinib‐induced liver injury. We have previously shown that CYP1A2 and CYP3A4 catalyze the metabolic activation of sunitinib via oxidative defluorination leading to formation of a chemically reactive, potentially toxic quinoneimine metabolite, which was trapped as a glutathione (GSH) conjugate. We hypothesize that inter‐individual variation in CYP1A2 and CYP3A activity affects hepatic exposure to sunitinib reactive metabolites and may influence the risk of developing sunitinib‐induced hepatotoxicity. The goals of this study were to 1) estimate the relative contributions of CYP1A2 and CYP3A to sunitinib metabolism and bioactivation, and 2) determine the impact of CYP1A2 and CYP3A enzyme activity on sunitinib metabolite formation in individual human liver microsomes. Sunitinib (5 ∝M) was incubated over time (0–10 min) with human liver microsomes from a single donor with high CYP1A2 activity (HH581) in the presence and absence of CYP1A2 inhibitor furafylline (25 ∝M) and CYP3A inhibitor ketoconazole (1 ∝M). Relative levels of sunitinib metabolites N‐desethylsunitinib (M1), defluorosunitinib (M3), and quinoneimine‐GSH conjugates (M5) were measured by LC‐MS/MS analysis. Sunitinib (10 ∝M) was also incubated with human liver microsomes from 12 individual donors, and metabolite formation was compared to individual CYP1A2 and CYP3A activities using probe substrates, phenacetin and midazolam, respectively. In human liver microsomes from a single donor with high CYP1A2 activity, co‐incubation with furafylline reduced M3 and M5 formation by 73% and 80%, respectively, compared to control; however, furafylline had a minimal effect on M1 formation (20% reduction). Ketoconazole reduced M1, M3, and M5 formation by 95%, 46% and 40%, respectively, compared to control. In human liver microsomes from 12 individual donors, formation of M3 and M5 was significantly correlated with CYP1A2 activity as estimated by phenacetin O‐deethylation (M3 vs. phenacetin O‐deethylation: r2 = 0.83) and (M5 vs. phenacetin O‐deethylation: r2 = 0.88). M1 formation was highly correlated with CYP3A activity as measured by midazolam 1′‐hydroxylation (r2 = 0.91). Collectively, these findings indicate that CYP1A2 plays a predominate role in sunitinib bioactivation leading to the quinoneimine‐GSH conjugates (M5), while CYP3A is the primary contributor to sunitinib N‐deethylation to form the major active metabolite M1. Future studies will be conducted in human hepatocytes to evaluate the effect of modulating P450 activity on sunitinib‐induced hepatocellular injury.Support or Funding InformationThis research is funded by the National Cancer Institute of the National Institutes of Health (K01CA190711). Research reported here is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Assessment of the pulmonary CYP1A1 metabolism of mavoglurant (AFQ056) in rat

1. AFQ056 phenotyping results indicate that CYP1A1 is responsible for the formation of the oxidative metabolite, M3. In line with the predominant assumption that CYP1A1 is mainly expressed in extrahepatic tissues, only traces of M3 were detected in hepatic systems. The aim of this study was to investigate the pulmonary CYP1A1 mediated metabolism of AFQ056 in rat.2. Western blot analysis confirmed that CYP1A1 is expressed in rat lung albeit at low levels. M3 formation was clearly observed in recombinant rat CYP1A1, lung microsomes and lung tissue slices and was strongly inhibited by ketoconazole in the incubations. As CYP3A4 and CYP2C9 metabolites were only observed at trace levels, we concluded that the reduced M3 formation was due to CYP1A1 inhibition.3. AFQ056 lung clearance (CLlung) as estimated from in vitro data was predicted to be negligible (<1% pulmonary blood flow). This was confirmed by in vivo experiments where intravenous and intra-arterial dosing to rats failed to show significant pulmonary extraction.4. While rat lung may make a contribution to the formation of M3, it is unlikely to be the only organ involved in this process and further experiments are required to investigate the potential metabolic elimination routes for AFQ056.

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.

Formation mechanism of layered double hydroxides in Mg2 +-, Al3 +-, and Fe3 +-rich aqueous media: Implications for neutralization in acid leach ore milling

Hydrotalcite-like layered double hydroxides (HT-LDHs) are important due to their extensive use in catalysis, polymer science, medicine, refractory materials, and the remediation of contaminated waters. The inclusion of several cations and anions in their structure can result in complex formation mechanisms depending on the chemical nature of the respective species. A systematic evaluation of the formation mechanism of HT-LDH from Mg2+Al3+Fe3+-rich aqueous media at ambient conditions was conducted to determine formation pathways in natural settings and in conditions observed in ore milling processes. Neutralization from pH1.7 to 12.5 (using NaOH) of individual M2+ and M3+ ions demonstrated that M3+-hydroxides precipitate first, followed by M2+-hydroxides in the order of Fe(OH)3, Al(OH)3, then Mg(OH)2. The rate of formation accelerates as pH increases. The addition of M2+ ions to M3+-hydroxides produces HT-LDH while the addition of M3+ ions to M2+-hydroxides does not. HT-LDH precipitates immediately under alkaline conditions (pH=13.2) with available M2++M3+ ions, while formation progresses slowly as initially acidic conditions (pH=1.7) are neutralized (to pH=12.5) via the precipitation of M3+-hydroxides and subsequently HT-LDH. This latter formation pathway is consistent with observations of the formation of MgFeAl HT-LDH during the neutralization process in an acid-leach process uranium mill: HT-LDH is first observed at pH~6.4 and is dominant at pH~8.0 or greater. At pH≥6.4, HT-LDH is co-associated with Fe3+-hydroxides. The early formation of M3+-hydroxides and substitution by M2+ ions into the structure appear to govern the overall HT-LDH formation mechanism, with the resulting excess positive charge counterbalanced by CO32− ions through atmospheric CO2 dissolution.

Metabolite profiling of anhuienoside C by rat intestinal bacteria using the LC–MS metabolomic approach

1. Anhuienoside C (A-C) is the main active component of the saponin exact of “Di Wu”, an oral drug for rheumatism treatment in China. In this study, we aimed to elucidate the metabolic pathways of A-C by intestinal bacteria using the metabolomic approach.2. Four deglycosylated metabolites (M1, M2, M3 and M4) were identified after A-C (50 µM) was incubated with rat fecal lysate. Chemical structures of these metabolites were determined by high-resolution masses and nuclear magnetic resonance (NMR).3. A one-compartment pharmacokinetic model was used to describe the formation of bacterial metabolites at a dose of 10 µM A-C. The results revealed that formation of M1 and M2 was rapid, whereas formation of M3 was rather slow. Further, it was found that the metabolites were generated by successive cleavage of the glycosyl residues.4. This is the first report that A-C is subjected to efficient bacterial metabolism in the gut with M1 and M2 as main metabolites. Our study should be helpful for a better understanding of in vivo disposition of oral A-C.

P450-mediated O-demethylated metabolite is responsible for rat hepatobiliary toxicity of pyridyltriazine-containing PI3K inhibitors.

The dysregulation of phosphatidylinositol 3-kinase (PI3K)-dependent pathways is implicated in several human cancers making it an attractive target for small molecule PI3K inhibitors. A series of potent pyridyltriazine-containing inhibitors of class Ia PI3Ks were synthesized and a subset of compounds was evaluated in exploratory repeat-dose rat toxicology studies. Daily oral dosing of compound 1: in Sprague Dawley rats for four consecutive days was associated with hepatobiliary toxicity that included biliary epithelial hyperplasia and hypertrophy, periductular edema, biliary stasis, and acute peribiliary inflammatory infiltrates. These histological changes were associated with clinical pathology changes that included increased serum liver enzymes, total bile acids, and bilirubin. The predominant clearance pathway of 1: was shown in vitro and in a bile-duct cannulated rat (14)C-ADME study to be P450-mediated oxidative metabolism. An O-demethylated pyridine metabolite, M3: , was identified as a candidate proximal metabolite that caused the hepatotoxicity. Co-administration of the pan-P450 inhibitor 1-aminobenzotriazole with 1: to rats significantly reduced the formation of M3: and prevented liver toxicity, whereas direct administration of M3: reproduced the toxicity. Structural changes were introduced to 1: to make the methoxypyridine ring less susceptible to P450 oxidation (compound 2: ), and addition of a methyl group to the benzylic carbon (compound 3: ) improved the pharmacokinetic profile. These changes culminated in the successful design of a clinical candidate 3: (AMG 511) that was devoid of liver toxicity in a 14-day rat toxicity study. Herein, we describe how a metabolism-based structure-activity relationship analysis allowed for the successful identification of a PI3K inhibitor devoid of off-target toxicity.

Oxidative metabolic pathway of lenvatinib mediated by aldehyde oxidase.

Lenvatinib is a multityrosine kinase inhibitor that inhibits vascular endothelial growth factor receptors, and is being developed as an anticancer drug. P450s are involved in one of the elimination pathways of lenvatinib, and mono-oxidized metabolites, such as N-oxide (M3) and desmethylated metabolite (M2), form in rats, dogs, monkeys, and humans. Meanwhile, two other oxidative metabolites are produced only in monkey and human liver S9 fractions, and their structures have been identified using high-resolution mass spectrometry as a quinolinone form of lenvatinib (M3') and a quinolinone form of desmethylated lenvatinib (M2'). The formation of M3' from lenvatinib occurred independently of NADPH and was effectively inhibited by typical inhibitors of aldehyde oxidase, indicating the involvement of aldehyde oxidase, but not P450s, in this pathway. M2' was a dioxidized metabolite arising from a combination of mono-oxidation and desmethylation and could only be produced from M2 in a NADPH-independent manner; M2' could not be generated from M3 or M3'. These results suggested that M2' is formed from lenvatinib by a unique two-step pathway through M2. Although both lenvatinib and M2 were substrates for aldehyde oxidase, an enzyme kinetic study indicated that M2 was a much more favorable substrate than lenvatinib. No inhibitory activities of lenvatinib, M2', or M3' and no significant inhibitory activities of M2 or M3 on aldehyde oxidase were observed, suggesting a low possibility of drug-drug interactions in combination therapy with substrates of aldehyde oxidase.

Variations of the tooth morphology of the Woolly Mammoth Mammuthus primigenius (Blumenbach, 1799) (Mammalia: Elephanthidae)

Cases of variations in the morphology of isolated upper teeth M3 of Mammuthus primigenius (Blumenbach, 1799) from the south-east of West Siberia and Yakutiya are described. The anomaly of M. primigenius tooth from Yakutiya is expressed in changing of the tooth crown shape and position of dental plates in M3. Development of the anomaly was probably associated with a delay of loosing the tooth of the previous generation (M2) and disorder of the formation of M3 at the stage of its mineralization. Two M3 teeth from Novosibirsk Province are characterized by changing of the wearing surface and root shape. One of them shows two wearing surfaces in parallel to initial masticatory surface, shifted lingually. The upper part of the root is bent buccally, that is connected with the falling out of the tooth from the alveolus. The appearance of such a change, presumably, is connected with a mechanical injury of the cheek region of skull. The reconstruction of the formation of this tooth anomaly in M. primigenius was carried out. The second of these teeth also has a wrong position of the masticatory surface, which has a gradual curvature from nearly perpendicular (to enamel plates) in the anterior part of the tooth to about 45° in the posterior plates.

In vitro identification of cytochrome P450 isoforms responsible for the metabolism of 1-hydroxyl-2,3,5-trimethoxy-xanthone purified from Halenia elliptica D. Don

1-Hydroxyl-2,3,5-trimethoxyxanthone (HM-1) is one of the main constituents extracted from Halenia elliptica D. Don, which is a traditionally used Tibetan medicinal plant. The aim of this study was to illustrate the proposed metabolic pathways of HM-1 and identify which cytochrome P450 (CYP450) isoforms involved in its metabolism by using pooled human liver microsomes (HLMs) and recombinant CYP450 isoforms with selective chemical inhibitors. Metabolites were identified by high performance liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LCMSn-ESI-IT-TOF) and nuclear magnetic resonance spectroscopy (hydrogen-1 NMR and carbon-13 NMR). Three metabolites (M1–M3) were identified, which demonstrated that demethylation and hydroxylation were the major Phase I metabolic reactions for HM-1 in HLMs. The structure of another metabolite (M4) was still unclear. The enzymatic kinetics of M1 (Km=23.19±14.20μM) and M2 (Km=32.06±17.09μM) exhibited substrate inhibition; whereas, the formation of M3 (Km=5.73±0.70μM) and M4 (Km=16.43±5.12μM) displayed Michaelis–Menten kinetics. The intrinsic clearance (Vmax/Km) of M3 was highest among these metabolites, suggesting that M3 was the major metabolite of HM-1. Moreover, CYP3A4 and CYP2C8 were the primary CYP450 isoform responsible for the metabolism of HM-1. CYP1A2, CYP2A6, CYP2B6, CYP2C9 and CYP2C19 were also involved in HM-1 metabolism, especially in the formation of M3. This study finally provides evidence of substrate inhibition and metabolism-based drug–drug interaction for the medicinal preparations containing HM-1 used in clinic.

In vitro metabolism of the 5-hydroxytryptamine1B receptor antagonist elzasonan

The metabolism of elzasonan has been examined in vitro using hepatic microsomes from human and recombinant heterologously expressed P450 enzymes (rCYP). Metabolism occurs primarily via oxidative N-demethylation to form M4 and oxidation reactions to form elzasonan N-oxide (M5) and 5-hydroxyelzasonan metabolite (M3). Additionally, elzasonan was shown to be metabolized to the novel cyclized indole metabolite (M6) which undergoes subsequent oxidation to form the iminium ion metabolite (M3a). The rCYP data was normalized relative to the levels of each CYP form in native human liver microsomes to better assess the contribution of each rCYP in the metabolism of elzasonan. Results demonstrated the involvement of CYP3A4 in the pathways leading to M3a, M3, M5 and M6 and CYP2C8 in the formation of M4. Kinetic constants for the formation of M3 were determined and correlation and inhibition studies suggested that CYP3A4 is primarily responsible for the formation of M3 and CYP2C19 plays a very minor role in its formation. Cytochrome b5 has shown to be an essential component in P450 3A4 catalyzed 5-hydroxyelzasonan formation and provides insights on the disconnect between human liver microsomes data and that of rCYP. Furthermore, rCYP3A4 containing b5 are useful models for predicting the rates for liver microsomes P450-dependent drug oxidations and should be utilized routinely.

Cytochrome P450 11A1 Bioactivation of a Kinase Inhibitor in Rats: Use of Radioprofiling, Modulation of Metabolism, and Adrenocortical Cell Lines to Evaluate Adrenal Toxicity

A drug candidate, BMS-A ((N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-1-(4-fluorophenyl) 2-oxo-1,2-dihydropyridine- 3-carboxamide)), was associated with dose- and time-dependent vacuolar degeneration and necrosis of the adrenal cortex following oral administration to rats. Pretreatment with 1-aminobenzotriazole (ABT), a nonspecific P450 inhibitor, ameliorated the toxicity. In vivo and in vitro systems, including adrenal cortex-derived cell lines, were used to study the mechanism responsible for the observed toxicity. Following an oral dose of the C-14 labeled compound, two hydroxylated metabolites of the parent (M2 and M3) were identified as prominent species found only in adrenal glands and testes, two steroidogenic organs. In addition, a high level of radioactivity was covalently bound to adrenal tissue proteins, 40% of which was localized in the mitochondrial fraction. ABT pretreatment reduced localization of radioactivity in the adrenal gland. Low levels of radioactivity bound to proteins were also observed in testes. Both M3 and covalent binding to proteins were found in incubations with mitochondrial fraction isolated from adrenal tissue in the presence of NADPH. In vitro formation of M3 and covalent binding to proteins were not affected by addition of GSH or a CYP11B1/2 inhibitor, metyrapone (MTY), but were inhibited by ketoconazole (KTZ) and a CYP11A1 inhibitor, R-(+)-aminoglutethimide (R-AGT). BMS-A induced apoptosis in a mouse adrenocortical cell line (Y-1) but not in a human cell line (H295R). Metabolite M3 and covalent binding to proteins were also produced in Y-1 and to a lesser extent in H295R cells. The cell toxicity, formation of M3, and covalent binding to proteins were all diminished by R-AGT but not by MTY. These results are consistent with a CYP11A1-mediated bioactivation to generate a reactive species, covalent binding to proteins, and subsequently rat adrenal toxicity. The thorough understanding of the metabolism-dependent adrenal toxicity was useful to evaluate cross-species adrenal toxicity potential of this compound and related analogues.

Elucidation of a Novel Bioactivation Pathway of a 3,4-Unsubstituted Isoxazole in Human Liver Microsomes: Formation of a Glutathione Adduct of a Cyanoacrolein Derivative after Isoxazole Ring Opening

Studies on the biotransformation of isoxazole rings have shown that molecules containing a C3-substituted isoxazole or a 1,2-benzisoxazole can undergo a two-electron reductive ring cleavage to form an imine. In the absence of a C3 substituent, the isoxazole ring opens via deprotonation of the C3 proton followed by N-O bond cleavage to yield an α-cyanoenol analog. We report the identification of a novel bioactivation pathway of a 3,4-unsubstituted isoxazole in human liver microsomes. After the enzyme-catalyzed cleavage of the 3,4-unsubstituted isoxazole ring of N-((2-isopropyl-7-methyl-1-oxoisoindolin-5-yl)methyl)isoxazole-5-carboxamide (P) in human liver microsomes, the formed α-cyanoenol (M1) condenses with formaldehyde to generate an α,β-unsaturated Michael acceptor intermediate (a cyanoacrolein derivative, VII), which further reacts with the cysteinyl thiol of glutathione to yield a GSH adduct of a cyanoacrolein derivative (M3). The same adduct also is formed when M1, generated in 0.1 N NaOH aqueous solution, reacts with formaldehyde and GSH. (13)C-labeled methanol was used to confirm that methanol from the drug stock solution was oxidized by liver microsomal enzymes to formaldehyde and the carbon atom from methanol was finally incorporated in the corresponding GSH adduct. The formation of isoxazole ring-opened products (M1 and M2) in human liver microsomes is NADPH-dependent. M1 and M2 were found in human liver microsomes preincubated with 1-aminobenzotriazole (1 mM) and NADPH (5 mM) at ∼ 10% of the levels found in the samples in the absence of 1-aminobenzotriazole, suggesting that this biotransformation pathway is primarily catalyzed by cytochrome P450. The formation of M3 also was inhibited by 1-aminobenzotriazole at a similar level.

Disposition and Metabolism of Semagacestat, a γ-Secretase Inhibitor, in Humans

Semagacestat is a functional gamma-secretase inhibitor that has been shown to reduce the rate of formation of amyloid-beta in vitro and in vivo. This study was conducted to characterize the disposition of semagacestat in humans. After a single 140-mg dose of [(14)C]semagacestat administered as an oral solution to six healthy male subjects, semagacestat was rapidly absorbed (T(max) approximately 0.5 h) and eliminated from the systemic circulation (terminal t(1/2) approximately 2.4 h). The major circulating metabolites of semagacestat, M2 (hydrolysis of the amide bond proximal to the benzazepine ring) and M3 (benzylic hydroxylation of the benzazepine ring), accounted for approximately 27 and 10% of total radioactivity exposure, respectively, as calculated from relative area under the plasma concentration versus time curve from 0 to 24 h derived from the plasma radiochromatograms. The radioactive dose was almost completely recovered after 7 days postdose, with 87% of the dose in urine and 8% in feces. Unchanged [(14)C]semagacestat in urine accounted for approximately 44% of the dose, which indicates that renal excretion played an important role in elimination. Metabolites M2 and M3, with their related secondary metabolites, each accounted for approximately 20% of the dose in excreta. In vitro data indicate the formation of M3 is primarily mediated by CYP3A, with cDNA-expressed CYP3A5 approximately 2 times more efficient than CYP3A4 in forming M3. Thus, the relative content of CYP3A4 and CYP3A5 in humans will likely determine the formation clearance of M3 after exposure to semagacestat.

Glucuronidation of piceatannol by human liver microsomes: major role of UGT1A1, UGT1A8 and UGT1A10

Piceatannol, a dietary polyphenol present in grapes and wine, is known for its promising anticancer and anti-inflammatory activity. The aim of this study was to analyse the concentration-dependent glucuronidation of piceatannol in vitro. To determine the glucuronidation of piceatannol, experiments were conducted with human liver microsomes as well as using a panel of 12 recombinant UDP-glucuronosyltransferase isoforms. Furthermore, the chemical structures of novel glucuronides were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Along with piceatannol it was possible to identify three metabolites whose structures were identified by LC-MS/MS as piceatannol monoglucuronides (M1-M3). Formation of M1 and M3 exhibited a pattern of substrate inhibition, with apparent K(i) and V(max)/K(m) values of 103 +/- 26.6 microm and 3.8 +/- 1.3 microl/mg protein per min, respectively, for M1 and 233 +/- 61.4 microm and 19.8 +/- 9.5 microl/mg protein per min, respectively, for M3. In contrast, formation of metabolite M2 followed classical Michaelis-Menten kinetics, with a K(m) of 18.9 +/- 8.1 microm and a V(max) of 0.21 +/- 0.02 nmol/mg protein per min. Incubation in the presence of human recombinant UDP-glucuronosyltransferases (UGTs) demonstrated that M1 was formed nearly equally by UGT1A1 and UGT1A8. M2 was preferentially catalysed by UGT1A10 and to a lesser extent by UGT1A1 and UGT1A8. The formation of M3, however, was mainly catalysed by UGT1A1 and UGT1A8. Our results elucidate the importance of piceatannol glucuronidation in the human liver, which must be taken into account in humans after dietary intake of piceatannol.

In-vitro sulfation of piceatannol by human liver cytosol and recombinant sulfotransferases

The aim of this study was to investigate the concentration-dependent sulfation of piceatannol, a dietary polyphenol present in grapes and wine and known for its promising anticancer and anti-inflammatory activity. Sulfation of piceatannol was investigated in human liver cytosol as well as using a panel of recombinant sulfotransferase isoforms. Furthermore, the chemical structures of novel sulfates were identified by liquid chromatography/mass spectrometry (LC/MS). In the presence of 3'-phosphoadenosine-5'-phosphosulfate, three metabolites could be detected whose structures were identified by LC/MS/MS as piceatannol disulfate (M1) and two monosulfates (M2, M3). The kinetics of M1 formation exhibited a pattern of substrate inhibition with a Ki of 21.8 +/- 11.3 microm and a Vmax/Km of 7.63 +/- 1.80 microl/mg protein per min. Formation of M2 and M3 showed sigmoidal kinetics with apparent Km and Vmax values of 27.1 +/- 2.90 microm and 118.4 +/- 4.38 pmol/mg protein per min, respectively, for M2; and 35.7 +/- 2.70 microm and 81.8 +/- 2.77 pmol/mg protein per min, respectively, for M3. Incubation in the presence of human recombinant sulfotransferases (SULTs) demonstrated that M1 was formed equally by SULT1A1*1 and SULT1B1 and to a lesser extent by SULT1A1*2. M2 was preferentially catalysed by SULT1A1*2, 1A3 and 1E1. The formation of M3, however, was mainly catalysed by SULT1A2*1 and SULT1A3. Our results elucidate the importance of piceatannol sulfation in human liver, which must be taken into account in humans after dietary intake of piceatannol.

In-vitro sulfation of piceatannol by human liver cytosol and recombinant sulfotransferases

Abstract Objectives The aim of this study was to investigate the concentration-dependent sulfation of piceatannol, a dietary polyphenol present in grapes and wine and known for its promising anticancer and anti-inflammatory activity. Methods Sulfation of piceatannol was investigated in human liver cytosol as well as using a panel of recombinant sulfotransferase isoforms. Furthermore, the chemical structures of novel sulfates were identified by liquid chromatography/mass spectrometry (LC/MS). Key findings In the presence of 3′-phosphoadenosine-5′-phosphosulfate, three metabolites could be detected whose structures were identified by LC/MS/MS as piceatannol disulfate (M1) and two monosulfates (M2, M3). The kinetics of M1 formation exhibited a pattern of substrate inhibition with a Ki of 21.8 ± 11.3 μM and a Vmax/Km of 7.63 ± 1.80 μl/mg protein per min. Formation of M2 and M3 showed sigmoidal kinetics with apparent Km and Vmax values of 27.1 ± 2.90 μM and 118.4 ± 4.38 pmol/mg protein per min, respectively, for M2; and 35.7 ± 2.70 μM and 81.8 ± 2.77 pmol/mg protein per min, respectively, for M3. Incubation in the presence of human recombinant sulfotransferases (SULTs) demonstrated that M1 was formed equally by SULT1A1*1 and SULT1B1 and to a lesser extent by SULT1A1*2. M2 was preferentially catalysed by SULT1A1*2, 1A3 and 1E1. The formation of M3, however, was mainly catalysed by SULT1A2*1 and SULT1A3. Conclusions Our results elucidate the importance of piceatannol sulfation in human liver, which must be taken into account in humans after dietary intake of piceatannol.