Interconversions of haloperidol and reduced haloperidol in guinea pig and rat liver microsomes

An alcohol metabolite of haloperidol, reduced haloperidol, is present in the tissues of haloperidol-treated patients. We have studied whether rat and guinea pig liver microsomes have the capability to reduce haloperidol and thus serve as models for human haloperidol metabolism. Interestingly, the rat microsomes did not reduce haloperidol, but possessed an NADPH-dependent, carbon monoxide-inhibited mechanism to oxidize the reduced haloperidol back to haloperidol. Guinea pig microsomes efficiently reduced haloperidol molecules in a fashion not dependent on nicotinamide cofactors and not inhibited by carbon monoxide. Both of these activities were confined to the microsomal fraction. In guinea pigs, reduction of haloperidol was observed also in kidney slices, whereas brain slices proved inactive. Reduced haloperidol was also oxidized to haloperidol to a small extent in guinea pig microsomes. These in vitro experiments confirm our findings in vivo, which showed that in rats haloperidol is not reduced, while guinea pigs have a very active mechanism for reducing haloperidol. Thus, guinea pigs constitute a model for human haloperidol metabolism, and they should be used for further characterization of the reductive drug-metabolizing system.     

Metabolism of new-generation taxanes in human,pig, minipig and rat liver microsomes

The novel taxanes SB-T-1102, SB-T-1214 and SB-T-1216 are up to 1000-fold more cytotoxic for resistant tumour cells than clinically used paclitaxel and docetaxel, and the current study has examined the metabolism of these new taxanes in human, rat, pig and minipig liver microsomes. Metabolites were characterized by high-performance liquid chromatography (HPLC)/tandem mass spectrometry (MS/MS) analysis. Metabolic pathways derived from their structures were confirmed by investigating subsequent metabolism of purified metabolites. SB-T-1102, SB-T-1214 and SB-T-1216 were metabolized to 14, 10 and 11 products, respectively. In contrast to docetaxel, side-chain hydroxylation did not occur at their tert-butyl group, but on the isobutyl (SB-T-1102) or isobutenyl (SB-T-1214 and SB-T-1216) chains. Species differences in their metabolism were observed. For example, human and untreated rat microsomes hydroxylated SB-T-1216 preferentially at the side-chain, whereas pig and minipig microsomes preferentially metabolized more at the taxane core. The increased formation of secondary and tertiary metabolites in rat microsomes with high expression of CYP3A1/2 compared with uninduced rats confirmed the role of CYP3A in taxane metabolism. All major products were formed by human cDNA-expressed CYP3A4 and none by CYP1A2, 1B1, 2A6, 2C9 and 2E1, indicating the principal role of CYP3A orthologues in SB-T metabolism. The knowledge of metabolic pathways of the examined agents and of their rates of formation is important due to possible metabolic inactivation of these three novel drugs with a great potential for the therapy of taxane-resistant tumours. The relatively slow metabolism of SB-T-1102 could be favourable for its antitumour efficiency in vivo.     

Metabolism of new-generation taxanes in human, pig, minipig and rat liver microsomes

The novel taxanes SB-T-1102, SB-T-1214 and SB-T-1216 are up to 1000-fold more cytotoxic for resistant tumour cells than clinically used paclitaxel and docetaxel, and the current study has examined the metabolism of these new taxanes in human, rat, pig and minipig liver microsomes. Metabolites were characterized by high-performance liquid chromatography (HPLC)/tandem mass spectrometry (MS/MS) analysis. Metabolic pathways derived from their structures were confirmed by investigating subsequent metabolism of purified metabolites. SB-T-1102, SB-T-1214 and SB-T-1216 were metabolized to 14, 10 and 11 products, respectively. In contrast to docetaxel, side-chain hydroxylation did not occur at their tert-butyl group, but on the isobutyl (SB-T-1102) or isobutenyl (SB-T-1214 and SB-T-1216) chains. Species differences in their metabolism were observed. For example, human and untreated rat microsomes hydroxylated SB-T-1216 preferentially at the side-chain, whereas pig and minipig microsomes preferentially metabolized more at the taxane core. The increased formation of secondary and tertiary metabolites in rat microsomes with high expression of CYP3A1/2 compared with uninduced rats confirmed the role of CYP3A in taxane metabolism. All major products were formed by human cDNA-expressed CYP3A4 and none by CYP1A2, 1B1, 2A6, 2C9 and 2E1, indicating the principal role of CYP3A orthologues in SB-T metabolism. The knowledge of metabolic pathways of the examined agents and of their rates of formation is important due to possible metabolic inactivation of these three novel drugs with a great potential for the therapy of taxane-resistant tumours. The relatively slow metabolism of SB-T-1102 could be favourable for its antitumour efficiency in vivo.     

Metabolism of 4-chlorobiphenyl by guinea pig adrenocortical and hepatic microsomes

Studies were carried out to determine if 4-chlorobiphenyl (4-CB) was a substrate for adrenal monooxygenases and to compare its interactions with adrenal and hepatic microsomal enzymes. Addition of 4-CB to guinea pig adrenal microsomes produced a typical type I spectral change, indicative of binding to cytochrome(s) P-450 and similar to that seen in hepatic microsomal preparations. The activities of several adrenal and hepatic microsomal monooxygenases were decreased by 4-CB in vitro. High pressure liquid chromatographic analyses revealed that both adrenal and hepatic microsomes, in the presence of NADPH, converted 4-CB to a major metabolite which eluted with a retention time identical to that of 4-chloro-4'-biphenylol (4'-OH-4-CB). The identity of 4'-OH-4-CB was confirmed by mass spectrometry. The maximal rate of 4-CB metabolism was greater in adrenal, compared with liver microsomes, but 4-CB had a higher affinity for hepatic than for adrenal enzymes. The rate of adrenal 4-CB metabolism was four to five times greater in microsomes derived from the inner cortical zone (zona reticularis) than those from the outer zones (zona fasciculata and zona glomerulosa). Hepatic microsomes also converted 4-CB to a minor metabolite whose production was blocked by epoxide hydrolase inhibitors, suggesting it might be a diol. 4-CB metabolism was not demonstrable in adrenal mitochondrial preparations. The results indicate that chlorinated biphenyls can serve as substrates for adrenal microsomal monooxygenases, suggesting that local activation may contribute to their adrenocortical toxicity.     

N-Hydroxylation of 2-aminofluorene in the guinea pig and by guinea pig liver microsomes in vitro

Summary N-hydroxy-2-aminofluorene was found in the urine of guinea pigs intraperitoneally injected with 2-aminofluorene. The hydroxylamine was oxidized to the nitroso analogue and this was identified and determined in the carbon tetrachloride extract by its characteristic UV absorption, by thin-layer chromatography, and by the formation of a diazo compound in the reaction with nitrous acid. Only a small fraction of the 2-aminofluorene injected appeared in the urine as N-hydroxy derivative.Guinea pig liver microsomes were observed to N-hydroxylate 2-aminofluorene rather rapidly, the reaction proceeding at least as rapidly as the N-hydroxylation of aniline.The results of this paper were presented at meetings of the Deutsche Pharmakologische Gesellschaft in Mainz, April 26 to 28, 1965 (Kampffmeyer and Kiese) and Göttingen, September 27 to 30, 1965 (von Jagow, Kiese, Renner, and Wiedemann).     

Metabolism of doxophylline by rat liver microsomes

The metabolic transformation of the bronchospasmolytic agent doxophylline (2-(7'-theophyllinemethyl)-1,3-dioxolane) was studied in vitro with phenobarbital-induced rat liver microsomal fraction containing the NADPH-generating system. Doxophylline was poorly metabolized as 95% of the recovered material was parent compound. The major metabolite resulted: 2'-hydroxyethyl ester of theophylline acetic acid. This was an unusual metabolite which possibly arose from dioxolane C2 enzymatic oxydation and subsequent ring opening. Theophylline was a minor metabolite. The per cent ratio of the two metabolites was 4:1; as we did not detected the presence of any compounds formed from N-demethylation, we concluded that doxophylline underwent a regiospecific metabolism on the N7 side chain.     

罗红霉素在苯巴比妥诱导的大鼠肝微粒体中的代谢

目的:研究罗红霉素在大鼠肝微粒体中的代谢,并考察罗红霉素及其代谢物对细胞色素P-450的影响.方法:采用超离心法制备了苯巴比妥诱导的大鼠肝微粒体酶.罗红霉素的体外代谢采用微粒体孵化方法,代谢物经LC-MS方法分离和分析,并通过进一步与合成对照品比较其质谱和色谱行为确定其结构.结果:在微粒体孵化液中发现了N-去甲基,N-双去甲基及O-去烷基三种代谢物.罗红霉素及其代谢物与CYP450 Fe~(2 )形成复合物的能力较弱.结论:罗红霉素在苯巴比妥诱导的大鼠肝微粒体中主要经历N-去甲基化和肟醚侧链O-去烷基化途径,两种转化途径均为NADPH依赖性.罗红霉素及其代谢物对CYP450的抑制作用较弱,     

山冈橐吾碱在雌性大鼠肝微粒体内的代谢(英文)

研究了山冈橐吾碱 (clivorine)在雌性大鼠肝微粒体内的代谢 .山冈橐吾碱在雌性大鼠肝微粒体内的主要代谢物为两个非吡咯代谢物M1和M2 .与雄性大鼠不同 ,生成肝毒性的吡咯代谢物为其次要的代谢途径 .文献报道山冈橐吾碱在雄性大鼠肝微粒体内的主要代谢方式是形成相应的吡咯代谢物 .这提示山冈橐吾碱在雌雄大鼠肝微粒体内的主要代谢方式不同 .CYP4 5 0特异性抑制剂黄胺苯吡唑(CYP2C) ,毛果芸香碱 (CYP2A1) ,二乙基二硫代氨基甲酸钠 (CYP2E1)和酮康唑 (CYP3A)对M1和M2的形成无明显的影响 .黄素单氧化酶的特异性抑制剂甲巯咪唑可以显著地抑制M2 的形成 ,但对M1的形成无明显的抑制作用 ,且M1在肝微粒体中的形成为NADPH非依赖性 ,上述结果提示参与M1和M2代谢的酶分别为肝微粒体中的水解酶和黄素单氧化酶 .另一方面 ,毛果芸香碱 ,黄胺苯吡唑和二乙基二硫代氨基甲酸钠对山冈橐吾碱的吡咯代谢物的形成无明显的影响 ,而CYP3A的特异性抑制剂酮康唑可以显著地抑制吡咯代谢物的生成 ,且山冈橐吾碱在重组的大鼠肝CYP2C12 ,CYP2E1温孵液中无代谢 ,而在重组的大鼠肝CYP3A1和CYP3A2的温孵液中山冈橐吾碱被代谢成相应的吡咯代谢物 .这提示CYP3A作为主要的CYP4 5 0酶参与了山冈橐吾碱的肝毒性吡咯代谢物的形成 .山冈橐吾碱?     

Metabolism of alpha-naphthylthiourea by rat liver and rat lung microsomes

α-Naphthylthiourea (ANTU) is metabolized by rat liver and lung microsomes to α-naphthylurea (ANU) and atomic sulfur. A portion of the atomic sulfur formed in this reaction covalently binds to macromolecules of the liver and lung microsomes. Approximately half the atomic sulfur bound to the liver and lung microsomes appears to have reacted with cysteine side chains of the microsomal proteins to form a hydrodisulfide. The loss of cytochrome P-450 and monooxygenase activity seen on incubation of liver microsomes with ANTU is likely the result of the covalent binding of atomic sulfur to cytochrome P-450. The available evidence suggests that the pulmonary toxicity of ANTU results, at least in part, from the covalent binding of a cytochrome P-450 monooxygenase catalyzed metabolite of ANTU to pulmonary macromolecules. This metabolite is most likely atomic sulfur or alternatively, one containing the carbonyl carbon of ANTU. However, it is possible that the binding of both metabolites may be responsible for the lung toxicity.     

Metabolism of styrene oxide in the rat and guinea pig

The metabolism of styrene oxide has been studied in the rat and guinea pig, with emphasis upon bivalent sulfur metabolites. Methylthio analogs of phenylethylene glycol, with the methylthio group in both possible positions, were found as urinary metabolites in both species. These compounds were present in more than trace amounts. The excretion of 2-hydroxy-1-methylthio-1-phenylethane amounted to about 7% of the administered dose in the guinea pig, and about 2% in the rat, in o-24 hr urine samples. The positional isomer 1-hydroxy-2-methylthio-1-phenylethane was excreted in lesser amounts in both species. Acidic urinary metabolites derived from glutathione conjugates are species dependent. In this study, the only products observed in the rat were the mercapturic acids expected as a result of reaction of the oxide with glutathione. In the guinea pig, the major bivalent sulfur acids were the corresponding mercaptoacetic acids. Other related metabolites included a mercaptolactic and a mercaptopyruvic acid, together with one of the mercapturic acids. These metabolites result from partial acetylation or acetylation/deacetylation of cysteine or cysteinylglycine adducts. The hitherto unobserved dihydrodiol formed via an arene oxide was found as a minor metabolite for both styrene and styrene oxide.     

Metabolism of 1,1-dichloro-cis-diphenylcyclopropane by rat liver microsomes

The metabolic fate of the anti-estrogen 1,1-dichloro-cis-diphenylcyclopropane (Analog II), was studied in vitro with phenobarbital-induced rat liver microsomal fractions. The presence of five metabolites was directly or indirectly established. Biotransformation products were isolated by TLC and HPLC techniques and, when possible, the structures were confirmed through comparison with synthetic samples. The presence of an allyl chloride, highly reactive, metabolic intermediate was stated.     

地非三唑在鼠肝微粒体中的体外代谢

目的　为了解地非三唑 (Dip)在不同预处理的鼠肝微粒体中主要受何种酶代谢影响 ,为其临床合理应用和进一步开发利用提供科学依据。方法　将Dip与 6种不同诱导剂〔苯巴比妥 (PB)、地塞米松(Dex)、β 萘黄酮 (BNF)、Dip、吡啶和空白对照〕诱导的鼠肝微粒体进行体外共孵育 ,用氯仿终止反应 ,以地西泮为内标 ,采用反相高效液相色谱 (RP HPLC)法测定孵育后剩余的Dip的含量。结果　BNF诱导的鼠肝微粒体对Dip代谢具有强烈的催化活性 ,Dip诱导的微粒体的催化能力次之 ,PB诱导组也有一定的催化能力 ,其他几种诱导剂诱导的微粒体对Dip代谢能力与对照组无明显差别。测得Dip在BNF诱导的鼠肝微粒体中的Km 为 (6 0 .5± 1.3) μmol·L- 1,vm 为 (5 .6± 0 .4 )mmol·g- 1·min- 1。结论　由BNF诱导的鼠肝微粒体 (主要为细胞色素P4 5 0 1A)和PB诱导的鼠肝微粒体 (主要为细胞色素P4 5 0 2B)在Dip的体外代谢中可能起主导作用 ;Dip诱导的鼠肝微粒体对其自身的代谢也起了重要作用。     

Metabolism of simple quinones in guinea pig and rat cardiac tissue

• 1.1. The metabolism of the benzoquinone 2,5-dimethylbenzoquinone and of the naphthoquinone 2,3-dimethoxy-1,4-naphthoquinone was studied in subcellular fractions isolated from cardiac tissue of guinea pig and rat.
• 2.2. In both species the benzoquinone was mainly metabolized through the mitochondrial NADH-ubiquinoneoxidoreductase, whereas the naphthoquinone was metabolized to approximately equal extents by mitochondrial reductase and by soluble DT-diaphorase.
• 3.3. Guinea pig heart metabolized 3 times more naphthoquinone than rat heart.
• 4.4. As a consequence of quinone metabolism, marked amounts of O₂: were generated; naphthoquinoneinduced O₂₊: generation was about 4-fold higher in guinea pig than in rat heart.

     

Metabolism and metabolic inhibition of gambogic acid in rat liver microsomes

AIM: To study the metabolism of gambogic acid (GA) and the effects of selective cytochrome P-450 (CYP450) inhibitors on the metabolism of GA in rat liver microsomes in vitro. METHODS: Rat liver microsomes were used to perform metabolism studies. Various selective CYP450 inhibitors were used to investigate their effects on the metabolism of GA and the principal CYP450 isoform involved in the formation of major metabolite M(1) in rat liver microsomes. Types of inhibition in an enzyme kinetics model were used to model the interaction. RESULTS: GA was rapidly metabolized to two phase I metabolites, M(1) and M(2), in rat liver microsomes. M(1) and M(2) were tentatively presumed to be the hydration metabolite and epoxide metabolite of GA, respectively. alpha-Naphthoflavone uncompetitively inhibited the formation of M(1) while ketoconazole, sulfaphenazole, diethyl dithiocarbamate and quinidine had little or no inhibitory effects on the formation of M(1). CONCLUSION: GA is rapidly metabolized in rat liver microsomes and M(1) is crucial for the elimination of GA. Cytochrome P-450 1A2 is the major rat CYP involved in the metabolism of GA.     

Metabolism of lovastatin by rat and human liver microsomes in vitro

The metabolism of lovastatin (Mevacor) was examined using isolated microsomes derived from the livers of normal and phenobarbital-treated rats and from human liver samples. Incubation of lovastatin with rat liver microsomes resulted in the formation of several polar metabolites of lovastatin. The metabolites were isolated by HPLC and identified by NMR and mass spectrometry. One fraction consisted of a 2:1 mixture of 6-hydroxy-lovastatin and the rearrangement product delta 4,5-3-hydroxy lovastatin. Addition of a trace of acid to this mixture resulted in the formation of a single aromatized product, the desacyl-delta 4a,6,8-dehydro analog of lovastatin. Another microsomal metabolite was determined to be the delta 4,8a,1-3-hydroxy-lovastatin derivative. The chromatographic pattern of metabolites produced from lovastatin by human liver microsomes was similar to that obtained with rat liver microsomes. Metabolism of lovastatin by rat liver microsomes was both time and concentration dependent; optimal microsomal metabolism occurred with 0.1 mM lovastatin, whereas higher lovastatin concentrations inhibited the reaction. The open acid form of lovastatin was poorly metabolized by both the rat and the human liver microsomes.     

Metabolism of N,N-diethyl-meta-toluamide by rat liver microsomes

The NADPH- and oxygen-dependent metabolism of N,N-diethyl-m-toluamide (deet insect repellent) has been studied at 37 degrees C in suspensions of liver microsomes from phenobarbital-pretreated male Wistar rats. In pH 8 Tris-HCl buffer and at a substrate concentration of 200 microM, metabolic reactions corresponded to benzylic hydroxylation and N-deethylation, and to combinations of these reactions, leading to N,N-diethyl-m-hydroxymethylbenzamide, N-ethyl-m-toluamide, N-ethyl-m-hydroxymethylbenzamide, and m-toluamide. These compounds and N,N-diethyl-m-formylbenzamide were detected in methyl t-butyl ether extracts by capillary gas chromatography using three fused silica columns of different polarities. The structures of the in vitro metabolites were verified by comparisons with samples of authentic standards using combined gas chromatography-mass spectrometry. A selective derivatization procedure with acetic anhydride and pyridine facilitated the detection of the two alcohols. The major metabolites, N,N-diethyl-m-hydroxymethylbenzamide and N-ethyl-m-toluamide, were quantitatively determined in derivatized extracts with an internal standard of N,N-dipropyl-m-toluamide, a capillary column of DB-1, and a nitrogen-phosphorus detector. The mean enzymatic yield of these two products was 69% in three replicated experiments.