Characterization of metabolites of a NK1 receptor antagonist, CJ-11,972, in human liver microsomes and recombinant human CYP isoforms by liquid chromatography/tandem mass spectrometry

The in vitro metabolism of CJ-11,972, (2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-yl)-(5-tert-butyl-2-methoxybenzyl)amine, an NK1 receptor antagonist, was studied in human liver microsomes and recombinant human CYP isoforms. Liquid chromatography/mass spectrometry (LC/MS) and tandem mass spectrometry (LC/MS/MS) coupled to radioactive detection were used to detect and identify the metabolites. CJ-11,972 was extensively metabolized in human liver microsomes and recombinant human CYP 3A4/3A5 isoforms. A total of fourteen metabolites were identified by a combination of various MS techniques. The major metabolic pathways were due to oxidation of the tert-butyl moiety to form an alcohol (M6) and/or O-demethylation of the anisole moiety. The alcohol metabolite M6 was further oxidized to the corresponding aldehyde (M7) and carboxylic acid (M4). Two unusual metabolites (M13, M17), formed by C-demethylation of the tert-butyl group, were identified as 2-{3-[(2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-ylamino)methyl]-4-methoxyphenyl}propan-2-ol and (2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-yl)-(5-isopropenyl-2-methoxybenzyl)amine. A plausible mechanism for C-demethylation may involve oxidation of M6 to form an aldehyde metabolite (M7), followed by cytochrome P450-mediated deformylation leaving an unstable carbon-centered radical, which would quickly form either the alcohol metabolite M13 and the olefin metabolite M17.     

Studies on metabolites and metabolic pathways of bulleyaconitine A in rat liver microsomes using LC‐MSn combined with specific inhibitors

Bulleyaconitine A (BLA) from Aconitum bulleyanum plants is usually used as anti‐inflammatory drug in some Asian countries. It has a variety of bioactivities, and at the same time some toxicities. Since the bioactivities and toxicities of BLA are closely related to its metabolism, the metabolites and the metabolic pathways of BLA in rat liver microsomes were investigated by HPLC–MSⁿ. In this research, the 12 metabolites of BLA were identified according to the results of HPLC‐MSⁿ data and the relevant literature. The results showed that there are multiple metabolites of BLA in rat liver microsomes, including demethylation, deacetylation, dehydrogenation deacetylation and hydroxylation. The major metabolic pathways of BLA in rat liver microsomes were clarified by HPLC‐MS combined with specific inhibitors of CYP450 isoforms. As a result, CYP3A and 2C were found to be the principal CYP isoforms contributing to the metabolism of BLA. Moreover, CYP2D6 and 2E1 are also more important CYP isoforms for the metabolism of BLA. While CYP1A2 only affected the formation rate of M11, its effect on the metabolism of BLA is very small. Copyright © 2014 John Wiley & Sons, Ltd.     

UHPLC‐Q‐TOF‐MS/MS‐based screening and characterization of metabolites of cnidilin in human liver microsomes

Cnidilin is an active natural furocoumarin ingredient originating from well‐known traditional Chinese medicine Radix Angelicae Dahuricae . In the present study, an efficient approach was developed for the screening and identification of cnidilin metabolites using ultra‐high‐performance liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry. In this approach, an on‐line data acquisition method multiple mass defect filter combined with dynamic background subtraction was developed to trace all probable metabolites. Based on this analytical strategy, a total of 24 metabolites of cnidilin were detected in human liver microsomal incubation samples and the metabolic pathways were proposed. The results indicated that oxidation was the main biotransformation route for cnidilin in human liver microsomes. In addition, the specific cytochrome P450 (CYP) enzymes involved in the metabolism of cnidilin were identified using chemical inhibition and CYP recombinant enzymes. The results showed that CYP1A2 and CYP3A4 might be the major enzymes involved in the metabolism of cnidilin in human liver microsomes. The relationship between cnidilin and the CYP450 enzymes could provide us a theoretical basis of the pharmacological mechanism.     

In Vitro and In Vivo Primary Metabolic Characterization of F18, a Novel Histone Deacetylase-6 (HDAC6) Inhibitor,Using UHPLC–QqQ–MS/MS and Q-TOF–MS Methods

F18, N-hydroxy-4-(2-methoxy-5-(methyl (2-methylquinazolin-4-yl) amino) phenoxy) butanamide, is a novel selective HDAC6 inhibitor with good antitumor activity. In the early drug development, drug-metabolism studies are a crucial and indispensable part. In this study, we proposed to evaluate the in vitro primary metabolism of F18 in phase Ι in liver microsomes from human, rat, dog, monkey and mouse and investigate the metabolite profile both in vitro and in vivo using LC–MS/MS methods. F18 showed high metabolic stability in human, rat, dog, monkey and mouse liver microsomes over 120 min, with t _1/2 >8 h in human, rat, and dog, and t _1/2 <3.5 h in monkey, with almost no clearance in mouse. Human cytochrome P450 (P450) phenotyping showed that F18 was predominantly metabolized by CYP2C9, CYP2E1, CYP2D6 and CYP3A4. The investigation of the effect of F18 on CYP enzymes in HLM demonstrated that this compound did not significantly inhibit CYP 1A2 (IC₅₀ >100 μM), was a moderate inhibitor of CYP3A4 (IC₅₀ = 1.63 μM) and had negligible effects on CYP3A1/2 activity in rats. The results will be valuable in understanding drug–drug interactions (DDI) when F18 is co-administered with other drugs. The metabolites of F18 were investigated in rat plasma, urine, feces and different liver microsomes in NADPH samples, yielding at least 11 metabolites in these biological samples. The prominent metabolic pathways were de-methylation, de-amination, de-oxidation and O-glucuronidation. In summary, this work provides the first clues regarding F18 metabolism, providing important information for comprehensive understanding of F18 metabolites.

     

In vitro metabolism of a new H(+)/K(+) ATPase inhibitor DBM-819 in liver microsomes using HPLC and electrospray mass spectrometry.

The metabolism of 1-(2-methyl-4-methoxyphenyl)-4-[(3-hydroxypropyl)amino]-6-methyl-2,3-dihydropyrrolo[3,2c]quinoline (DBM-819), a new H(+)/K(+) ATPase inhibitor, has been studied by HPLC with spectrometric detection and on-line LC-electrospray mass spectrometry. In vitro incubation of DBM-819 with rat liver microsomes in the presence of NADPH resulted in the production of four metabolites (M1-4), whereas DBM-819 was oxidized to two metabolites, M2 and M4, by human liver microsomes. M2, M3 and M4 were identified as O-demethyl-DBM-819, 8-hydroxy-DBM-819 and N-dehydroxypropyl-DBM-819, respectively, based on LC/MS/MS analysis with authentic standards. M1 was tentatively identified as 1-(hydroxy-2-methyl-4-methoxyphenyl)-4-[(3-hydroxypropyl)amino]-6-methyl-2,3-dihydropyrrolo[3,2c]quinoline. Rat liver CYP1A1/2 catalyzed the oxidation of DBM-819 to 8-hydroxy-DBM-819 and N-dehydroxypropyl-DBM-819. Human CYP3A4 was a major isozyme for the formation of O-demethyl-DBM-819 as well as N-dehydroxypropyl-DBM-819.     

Characterization of in vitro metabolites of deoxypodophyllotoxin in human and rat liver microsomes using liquid chromatography/tandem mass spectrometry

The in vitro metabolism of deoxypodophyllotoxin (DPT), a medicinal herbal product isolated from Anthriscus sylvestris (Apiaceae), was investigated in rats and human microsomes and human recombinant cDNA-expressed CYPs. The incubation of DPT with pooled human microsomes in the presence of NADPH generated five metabolites while its incubation with dexamethasone (Dex)-induced rat liver resulted in seven metabolites (M1-M7) with major metabolic reactions including mono-hydroxylation, O-demethylation and demethylenation. Reasonable structures of the seven metabolites of DPT could be proposed, based on the electrospray tandem mass spectra. Chemical inhibition by ketoconazole and metabolism studies with human recombinant cDNA-expressed CYPs indicated that CYP 3A4 and 2C19 are the major CYP isozymes in the metabolism of DPT in human liver microsomes.     

Identification of cytochrome P450 isoforms involved in the metabolism of artocarpin and assessment of its drug–drug interaction

Artocarpin isolated from an agricultural plant Artocarpus communis has shows anti‐inflammation and anticancer activities. In this study, we utilized recombinant human UDP‐glucuronosyltransferasesupersomes (UGTs) and human liver microsomes to explore its inhibitory effect on UGTs and cytochrome p450 enzymes (CYPs). Chemical inhibition studies and screening assays with recombinant human CYPs were used to identify if CYP isoform is involved in artocarpin metabolism. Artocarpin showed strong inhibition against UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, CYP2C8 and CYP3A4. In particular, artocarpin exhibited competitive inhibition against CYP3A4 and noncompetitive inhibition against UGT1A3 and UGT1A7. The half inhibition concentration values for CYP3A4, UGT1A3 and UGT1A7 were 4.67, 3.82 and 4.82 μm , and the inhibition kinetic parameters for them were 0.78, 2.67 and 3.14 μm , respectively. After artocarpin was incubated in human liver microsomes and determined by HPLC, we observed its main metabolites (M1 and M2). In addition, we proved that CYP2D6 played the key role in the biotransformation of artocarpin in human liver microsomes. The result of molecular docking further confirmed that artocarpin interacted with CYP2D6, CYP2C8 and CYP3A4 through hydrogen bonds. This study provided preliminary results for further research on artocarpin or artocarpin‐containing herbs.     

Metabolism of a novel antiangiogenic agent KR-31831 in rats using liquid chromatography-electrospray mass spectrometry

KR-31831 ((2S,3R,4S)-4-(((1H-imidazol-2-yl)methyl)(4-chlorophenyl)amino)-6-amino-2-(dimethoxymethyl)-2-methyl-3,4-dihydro-2H-chromen-3-ol) is a novel antiangiogenic agent. In vitro and in vivo metabolism of KR-31831 in rats has been investigated using LC-MS and LC-MS/MS analysis. Incubation of rat liver microsomes and hepatocytes with KR-31831 produced three metabolites (M1-M3). M1, M2, and M3 were identified as N-((1H-imidazol-2-yl)methyl)-4-chlorobenzenamine, (2R,3R,4S)-4-(((1H-imidazol-2-yl)methyl)(4-chlorophenyl) amino)-6-amino-2-(hydroxymethyl)-2-methyl-3,4-dihydro-2H-chromen-3-ol, and N-((2S,3R,4S)-4- (((1H-imidazol-2-yl)methyl)(4-chlorophenyl)amino)-2-(dimethoxymethyl)-3-hydroxy-2-methyl-3,4-dihydro-2H-chromen-6yl)acetamide, respectively, by co-chromatography with the authentic standards and by comparison with product ion spectra of the authentic standards. Those in vitro metabolites were also detected in bile, plasma, or urine samples after an intravenous administration of KR-31831 to rats. The metabolic routes for KR-31381 included the metabolism of acetal group to hydroxymethyl group (M2), N-dealkylation to M1, and N-acetylation at the 6-amino group (M3).     

Galeon: A Biologically Active Molecule with In Silico Metabolite Prediction,In Vitro Metabolic Profiling in Rat Liver Microsomes,and In Silico Binding Mechanisms with CYP450 Isoforms

Galeon, a natural cyclic-diarylheptanoid (CDH), which was first isolated from Myrica gale L., is known to have potent cytotoxicity against A549 cell lines, anti-tubercular activity against Mycobacterium tuberculosis H37Rv, chemo-preventive potential, and moderate topoisomerase inhibitory activity. Here, in silico metabolism and toxicity prediction of galeon by CYP450, in vitro metabolic profiling study in rat liver microsomes (RLMs), and molecular interactions of galeon-CYP450 isoforms were performed. An in silico metabolic prediction study showed demethyl and mono-hydroxy galeon were the metabolites with the highest predictability. Among the predicted metabolites, mono-hydroxy galeon was found to have plausible toxicities such as skin sensitization, thyroid toxicity, chromosome damage, and carcinogenicity. An in vitro metabolism study of galeon, incubated in RLMs, revealed eighteen Phase-I metabolites, nine methoxylamine, and three glutathione conjugates. Identification of possible metabolites and confirmation of their structures were carried out using ion-trap tandem mass spectrometry. In silico docking analysis of galeon demonstrated significant interactions with active site residues of almost all CYP450 isoforms.     

液相色谱-质谱联用技术结合多探针底物法研究人参皂苷Rb1的体外代谢

以奥美拉唑、 苯妥英、 卡马西平和非那西丁为检测肝药酶细胞色素P450酶(CYP450)亚型的专属探针药物, 通过原型药物减少量测定法考察药物体外代谢的变化, 评价人参皂苷Rb1对CYP450不同亚型酶的作用. 结果表明, P2C9, P2C19和P3A4实验组与对照组差异不显著, P1A2实验组与对照组差异显著, 表明人参皂苷Rb1能诱导P1A2亚型酶的活性, 促进底物与酶反应, 加快底物的代谢, 而对P2C9, P2C19和P3A4三个亚型酶有弱的诱导或无诱导作用. 根据快速分离液相色谱-质谱联用(RRLC-MS/MS)检测结果推断, 人参皂苷Rb1在CYP450酶中的代谢产物可转化为人参皂苷Rb1氧化产物(Rb1+O)及人参皂苷Rd和F2.     

The use of liquid chromatography-atmospheric pressure chemical ionization mass spectrometry to explore the in vitro metabolism of cyanoalkyl piperidine derivatives

A LC/MS method using atmospheric pressure chemical ionization, positive ion mode and full scan to measure the in vitro metabolic stability of cyanoalkyl functionalized compounds with the human liver microsomes was employed. Percentage metabolism examined for the five cyanoalkyl piperidines revealed the optimal chain length and positioning of these functions to produce the most metabolically stable compound. The 4-cyanomethyl piperidine derivative was the most stable compound with 15% metabolism after 15 min incubation with human liver microsomes. In general, the major metabolites formed from the cyanoalkyl piperidine derivatives were due to oxidation of the cyanoalkyl chain or the piperidine fragment, resulting in a M+16 ion. However, the 2-cyanomethyl piperidine derivative exhibited an interesting biotransformation pathway with unusual metabolite peaks corresponding to M+5, M-11 and M+21 ions. Data-dependent MS/MS scanning was used to generate daughter ion spectra from the parent compound and its metabolite peaks. Based on the fragmentation analysis, a carboxylic acid, aldehyde and oxidative metabolite of the carboxylic acid structure have been proposed for M+5, M-11 and M+21 ions, respectively.     

Cytochrome P450 reaction phenotyping and inhibition and induction studies of pinostrobin in human liver microsomes and hepatocytes

Pinostrobin (PI, 5‐hydroxy‐7‐methoxyflavanone) is a natural flavonoid known for its rich pharmacological activities. The objective of this study was to identify the human liver cytochrome P450 (CYP450) isoenzymes involved in the metabolism of PI. A single hydoxylated metabolite was obtained from PI after an incubation with pooled human liver microsomes (HLMs). The relative contributions of different CYP450s were evaluated using CYP450‐selective inhibitors in HLMs and recombinant human CYP450 enzymes, and the results revealed the major involvement of CYP1A2, CYP2C9 and CYP2E1 in PI metabolism. We also evaluated the ability of PI to inhibit and induce human cytochrome P450 enzymes in vitro . High‐performance liquid chromatography and liquid chromatography–tandem mass spectrometry analytical techniques were used to estimate the enzymatic activities of seven drug‐metabolizing CYP450 isozymes in vitro . In HLMs, PI did not inhibit CYP 1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 or CYP3A4 (IC₅₀ > 100 μm ). In the induction studies, PI had minimal effects on CYP1A2, CYP2B6and CYP3A4 activity. Based on these results, PI would not be expected to cause clinically significant CYP450 inhibition or induction.     

In vitro metabolism of a novel phosphodiesterase-5 inhibitor DA-8159 in rat liver preparations using liquid chromatography/electrospray mass spectrometry

The in vitro metabolism of a new erectogenic, DA-8159, has been studied by LC with UV detection and on-line LC-electrospray mass spectrometry using rat hepatic microsomal incubation and rat liver perfusion. Both rat liver microsomal incubation of DA-8159 in the presence of NADPH and single-pass liver perfusion of DA-8159 resulted in the formation of three metabolites (M1-3). M1 was tentatively identified as hydroxy-DA-8159. M2 and M3 were identified as N-demethyl-DA-8159 and 5-(2-propyloxy-5-aminosulphonylphenyl)-1-methyl-3-propyl-1,6-dihydro-7H-pyrazolo(4,3-d)pyrimidin-7-one (DA-8164), respectively, on the basis of LC-MS/MS analysis with authentic standards. Rat CYP2D1 was a major isozyme for the formation of hydroxy-DA-8159 and N-demethyl-DA-8159. CYP2C12 and CYP1A1 catalysed the oxidation of DA-8159 to DA-8164.     

Enantioselective capillary electrophoresis for identification and characterization of human cytochrome P450 enzymes which metabolize ketamine and norketamine in vitro

Ketamine, a phencyclidine derivative, is used for induction of anesthesia, as an anesthetic drug for short term surgical interventions and in subanesthetic doses for postoperative pain relief. Ketamine undergoes extensive hepatic first-pass metabolism. Enantioselective capillary electrophoresis with multiple isomer sulfated β-cyclodextrin as chiral selector was used to identify cytochrome P450 enzymes involved in hepatic ketamine and norketamine biotransformation in vitro. The N-demethylation of ketamine to norketamine and subsequently the biotransformation of norketamine to other metabolites were studied via analysis of alkaline extracts of in vitro incubations of racemic ketamine and racemic norketamine with nine recombinantly expressed human cytochrome P450 enzymes and human liver microsomes. Norketamine was formed by CYP3A4, CYP2C19, CYP2B6, CYP2A6, CYP2D6 and CYP2C9, whereas CYP2B6 and CYP2A6 were identified to be the only enzymes which enable the hydroxylation of norketamine. The latter two enzymes produced metabolic patterns similar to those found in incubations with human liver microsomes. The kinetic data of ketamine N-demethylation with CYP3A4 and CYP2B6 were best described with the Michaelis–Menten model and the Hill equation, respectively. This is the first study elucidating the individual enzymes responsible for hydroxylation of norketamine. The obtained data suggest that in vitro biotransformation of ketamine and norketamine is stereoselective.     

Effect of ketoconazole on the transport and metabolism of drugs in the human liver cell model

The effect of ketoconazole on the biotransformation of amiodarone, rosiglitazone, and cyclophosphamide was studied using the human liver cell model based on differentiated HepaRG spheroids. The concentrations of major metabolites of amiodarone and cyclophosphamide were found to decrease in the presence of ketoconazole, a cytochrome P450 3A4 inhibitor. The concentration of the major metabolite of rosiglitazone, N-desmethyl rosiglitazone, decreased upon the addition of either sulfaphenazole, a cytochrome P450 2C9 inhibitor, or ketoconazole. The rosiglitazone metabolism involves CYP2C9 and CYP2C19. This result is attributable to the inhibitory effect of ketoconazole on p-glycoprotein, which decreases N-desmethyl rosiglitazone concentration in the culture medium. The utilization of the human liver cell model and selective inhibitors of transporters and cytochrome P450 isoforms can serve for standardization of the studies of drug-drug interactions involved in drug transport and metabolism.

     

In vitro metabolic profiling of synthetic cannabinoids by pooled human liver microsomes,cytochrome P450 isoenzymes,and Cunninghamella elegans and their detection in urine samples

As synthetic cannabinoids are extensively metabolized, there is an urgent need for data on which metabolites can be used for successful urine screening. This study examines the in vitro metabolism of EG-018 and its 5F-analogue EG-2201 by means of comparing three different in vitro models: pooled human liver microsomes, cytochrome P450 isoenzymes, and a fungal approach utilizing the filamentous fungus Cunninghamella elegans LENDNER, which is known for its ability to mimic human biotransformation of xenobiotics. In addition, this study includes the screening of two authentic urine samples from individuals with proven EG-018 consumption, for the evaluation of in vitro–in vivo extrapolations made in the study. Incubation with pooled human liver microsomes yielded 15 metabolites of EG-018 belonging to six different metabolite subgroups, and 21 metabolites of EG-2201 belonging to seven different metabolite subgroups, respectively. Incubation with cytochrome P450 isoenzymes incubation yielded a further three EG-018 and five EG-2201 metabolites. With reference to their summed metabolite peak abundancies, the isoenzymes CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5 were shown to contribute most to the microsomal metabolism of EG-018 and EG-2201. CYP2B6 was shown to make the lowest contribution, by far. As the phase I metabolism of both synthetic cannabinoids was shown to be distributed over a substantial number of different cytochrome P450 isoenzymes, it was concluded that it is likely to not be significantly affected by co-consumption of other drugs. Although fungal incubation with Cunninghamella elegans yielded an additional three EG-018 and four EG-2201 metabolites not observed after microsomal incubation, metabolites generated by Cunninghamella elegans were in good correlation with those generated by microsomal incubations. The fungal model demonstrated its ability to be an independent in vitro model in synthetic cannabinoid metabolism research. The three tested in vitro models enable sufficient predictive in vitro–in vivo extrapolations, comparable to those obtained from hepatocyte incubation published in the literature. In addition, with regard to the screening of authentic urine samples and comparison with the literature, one monohydroxylated EG-018 metabolite and two monohydroxylated EG-2201 metabolites can be recommended as urinary targets, on the basis of the tested in vitro models.

Graphical abstract

     

乌头类生物碱组分在CYP450中的代谢指纹图谱及对CYP450活性的影响

利用超高效液相色谱-串联质谱(UPLC-MS/MS)的多反应监测(MRM)技术, 结合多探针底物方法, 对单酯型及双酯型乌头类生物碱组分对细胞色素P450(CYP450)亚型的活性影响进行了研究; 同时利用超高效液相色谱-四极杆-飞行时间串联质谱(UPLC-Q-TOF-MS/MS)对单酯型和双酯型乌头类生物碱组分在CYP450中的代谢指纹图谱进行了研究. 活性影响研究结果表明, 单酯型生物碱组分对CYP2C及2D的抑制能力较强, 其IC₅₀值分别为7.44和6.74 μmol/L; 双酯型生物碱组分对CYP1A2, 3A, 2C和2D均有较弱的抑制作用, 其IC₅₀值分别为39.48, 70.44, 17.36和86.04 μmol/L. 代谢指纹图谱显示, 双酯型生物碱组分在大鼠肝微粒体中有6个特异性产物可以作为该反应的特征峰.     

Investigating the in vitro stereoselective metabolism of m‐nisoldipine enantiomers: characterization of metabolites and cytochrome P450 isoforms involved

m‐Nisoldipine, as a novel 1,4‐dihydropyridine calcium ion antagonist, was presented as a couple of enantiomers [(?), (+)‐m‐nisoldipine]. In this report, the in vitro metabolism of m‐nisoldipine enantiomers was investigated in rat liver microsomes (RLM) by the combination of two liquid chromatography mass spectrometric techniques for the first time. The metabolites were separated and assayed by ultra‐high performance liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry and further identified by comparison of their mass and chromatographic behaviors with reference substances. A total of 18 metabolites of (?)‐m‐nisoldipine and 16 metabolites of (+)‐m‐nisoldipine were detected, respectively, which demonstrated that (+)‐m‐nisoldipine is more metabolically stable than (?)‐m‐nisoldipine. In addition, the identified metabolic pathways of m‐nisoldipine enantiomers were involved in dehydrogenation, oxidation and ester hydrolysis. Afterwards, based on high‐performance liquid chromatography coupled to triple quadrupole linear ion trap mass spectrometry, various selective cytochrome P450 (CYP) enzyme inhibitors were employed to evaluate CYP isoforms. The results indicated that the inhibitors of CYP1A1/2, CYP2B1/2, 2D and 2C11 had no obvious inhibitory effects, yet the inhibitor of CYP 3A had a significant inhibitory effect on metabolism of m‐nisoldipine enantiomers. This showed that CYP 3A might primarily metabolize m‐nisoldipine in RLM. Copyright © 2015 John Wiley & Sons, Ltd.     

Interaction effects on cytochrome P450 both in vitro and in vivo studies by two major bioactive xanthones from Halenia elliptica D. Don

The major components, 1‐hydroxy‐2,3,5‐trimethoxy‐xanthone (HM‐1) and 1,5‐dihydroxy‐2,3‐dimethoxy‐xanthone (HM‐5) isolated from Halenia elliptica D. Don (Gentianaceae), could cause vasodilatation in rat coronary artery with different mechanisms. In this work, high‐performance liquid chromatography coupled to ion trap time‐of‐flight mass spectrometry (LCMS‐IT‐TOF) was used to clarify the metabolic pathways, and CYP450 isoform involvement of HM‐1 and HM‐5 were also studied in rat. At the same time, in vivo inhibition effects of HM‐1 and ethyl acetate extracts from origin herb were studied. Three metabolites of HM‐5 were found in rat liver microsomes (RLMs); demethylation and hydroxylation were the major phase I metabolic reactions for HM‐5. Multiple CYP450s were involved in metabolism of HM‐1 and HM‐5. The inhibition study showed that HM‐5 inhibited Cyp1a2, 2c6 and 2d2 in RLMs. HM‐1 inhibited activities of Cyp1a2, Cyp2c6 and Cyp3a2. In vivo experiment demonstrated that both HM‐1 and ethyl acetate extracts could inhibit Cyp3a2 in rats. In conclusion, the metabolism of xanthones from the origin herb involved multiple CYP450 isoforms; in vitro, metabolism of HM‐5 was similar to that of its parent drug HM‐1, but their inhibition effects upon CYP450s were different; in vivo, Cyp3a2 could be inhibited by HM‐1 and ethyl acetate extracts.     

Assessment of the stereoselective metabolism of methaqualone in man by capillary electrophoresis

Methaqualone (MQ) and its hydroxylated metabolites are quinazoline derivatives that exhibit atropisomerism. As a continuation of our previous work with these compounds (Electrophoresis 2001, 22, 3270-3280), chiral capillary zone electrophoresis with hydroxypropyl-beta-cyclodextrin as buffer additive and multiwavelength absorbance detection is shown to be an effective tool to provide insight into the stereoselectivity of the MQ metabolism. The five major monohydroxy MQ metabolites formed during biotransformation do not show enantiomerization at temperatures up to 85 degrees C. Enzymatic and acidic hydrolysis of urines that were collected after concomitant administration of 250 mg of MQ and 25 mg diphenhydramine (DH) chloride are both shown to provide stereoselective metabolic patterns with 4'-hydroxymethaqualone, the major urinary metabolite, being excreted almost exclusively as a single enantiomer. A stereoselectivity in the formation of 2'-hydroxymethaqualone and 2-hydroxymethaqualone was also observed in vitro using human liver microsomes and preparations containing the cytochrome P450 enzyme (CYP) CYP3A4 only. The presence of DH during incubation with human liver microsomes did not reveal a difference in the metabolic pattern obtained. Furthermore, CYP2D6 and CYP2C19 do not significantly contribute to the metabolism of MQ. This was independently observed in vitro and via analysis of urines of individuals that are either efficient metabolizer phenotypes or poor metabolizer phenotypes for the two polymorphic enzymes. Although interindividual differences in the monitored metabolic patterns were noted, no marked difference could be related to a CYP2D6 or CYP2C19 polymorphism.