Interaction of aromatic hydrocarbons and drugs with adrenal microsomal cytochrome P-450 in the guinea pig.

Addition of simple aromatic hydrocarbons (benzene, ethylbenzene, naphthalene) to guinea pig adrenal microsomes produced typical Type I difference spectra (ΔOD_385-420). Spectral dissociation constants (K_s) for each indicated a far higher affinity for adrenal than hepatic cytochrome P-450. Hydrocarbon affinities for adrenal cytochrome P-450 were similar to that for progesterone, an endogenous steroid substrate. Ethylmorphine and aniline produced Type I and Type II spectral changes respectively in adrenal microsomes. The K_s, and magnitude of spectrum for each in adrenals was similar to that in livers. Nonetheless, demethylation of ethylmorphine proceeded far more rapidly in adrenal than hepatic tissue. The Michaelis constants (K_m) for ethylmorphine metabolism in both tissues were similar. Although the aniline-induced difference spectra in adrenal and hepatic microsomes did not differ substantially, aniline hydroxylase activity was far greater in liver. Pretreatment of guinea pigs with phenobarbital or 3-methylcholanthrene increased hepatic but not adrenal ethylmorphine metabolism. Spironolactone pretreatment, in contrast, did not affect hepatic metabolism, but significantly lowered adrenal demethylase activity. The results indicate a relative non-specificity of guinea pig adrenal microsomal cytochrome P-450 and suggest that the adrenal cortex may represent a significant site for the extra-hepatic metabolism of foreign compounds in the guinea pig.     

Interaction of nitromethane with reduced hepatic microsomal cytochrome P-450.

Nitromethane interacts with sodium dithionite reduced rabbit liver microsomes to generate a difference spectrum characterized by maxima at 388, 423, 454 and 520 nm and minima at 405, 436, 480 and 550 nm. Spectral binding constants (K_s) of 0.306 ± 0.082 mM (A_max = 0.032 ± 0.004), 0.178 ± 0.029 mM (A_max = 0.040 ± 0.002), and 1.168 ± 0.250 mM (A_max = 0.035 ± 0.006) were calculated for the 388, 423 and 454 nm peaks respectively. These difference spectra are qualitatively different from those previously reported for aromatic nitro compounds [L. A. Sternson and R. E. Gammans, Drug Metab. Dispos.3, 266 (1975)]. Interaction of nitromethane with microsomes from rabbits pretreated with phenobarbital produced absorbance maxima and minima within 2 nm of the controls. Interaction of nitromethane with reduced microsomes from 3-methyl-cholanthrene (3-MC)-pretreated animals produced ferrohemochromes in which maximal absorbance changes were shifted to maxima at 384, 422, 451 and 514 nm and minima at 407, 433, 473 and 552nm. Peak-height ratios derived from difference spectra generated by the addition of 1 mM nitromethane to the sample cuvette were considerably different depending on whether the microsomes were obtained from control, phenobarbital- or 3-MC-pretreated rabbits and may indicate that phenobarbital, like 3-MC, induces qualitative changes in cytochrome P-450. Nitromethane apparently competes with carbon monoxide for a common binding site. Addition of nitromethane to CO-saturated microsomes reduced the magnitude of the 450 nm peak with a concomitant increase in the 423 nm peak of nitromethane. Similarly, addition of CO to reduced microsomes containing nitromethane caused reduction of the 423 nm peak of nitromethane with a corresponding increase of the 450 nm peak of CO. Nitromethane does not generate difference spectra with oxidized microsomes nor does it alter the K_s or A_max of aminopyrine, hexobarbital, aniline or zoxazolamine binding spectra. Nitromethane does inhibit the binding of the type II compound, nicotinamide. Addition of nitromethane to incubation flasks enhanced the metabolism of aniline while tending to inhibit the oxidative demethylation of ethylmorphine.     

Interaction of lapatinib with cytochrome P450 3A5

Lapatinib, an oral tyrosine kinase inhibitor used for breast cancer, has been reported to cause idiosyncratic hepatotoxicity. Recently, it has been found that lapatinib forms a metabolite-inhibitor complex (MIC) with CYP3A4 via the formation of an alkylnitroso intermediate. Because CYP3A5 is highly polymorphic compared with CYP3A4 and also oxidizes lapatinib, we investigated the interactions of lapatinib with CYP3A5. Lapatinib inactivated CYP3A5 in a time-, concentration-, and NADPH-dependent manner using testosterone as a probe substrate with K(I) and k(inact) values of 0.0376 mM and 0.0226 min(-1), respectively. However, similar results were not obtained when midazolam was used as the probe substrate, suggesting that inactivation of CYP3A5 by lapatinib is site-specific. Poor recovery of CYP3A5 activity postdialysis and the lack of a Soret peak confirmed that lapatinib does not form a MIC with CYP3A5. The reduced CO difference spectrum further suggested that a large fraction of the reactive metabolite of lapatinib is covalently adducted to the apoprotein of CYP3A5. GSH trapping of a reactive metabolite of lapatinib formed by CYP3A5 confirmed the formation of a quinoneimine-GSH adduct derived from the O-dealkylated metabolite of lapatinib. In silico docking studies supported the preferential formation of an O-dealkylated metabolite of lapatinib by CYP3A5 compared with an N-hydroxylation reaction that is predominantly catalyzed by CYP3A4. In conclusion, lapatinib appears to be a mechanism-based inactivator of CYP3A5 via adduction of a quinoneimine metabolite.     

Interaction of cotinine with rat hepatic microsomal P-450. Comparison with metyrapone and immunomodulation of cotinine and metyrapone binding by monoclonal anti-cotinine antibodies

The ability of the major nicotine metabolite, cotinine, to interact with rat liver microsomal cytochrome P-450 and the immunomodulatory effects of anti-cotinine antibodies were studied. Cotinine induced type II spectral changes with both microsomes from phenobarbital (PB)-induced rats and purified P-450 with apparent Ks values of 97 and 750 microM, respectively. In contrast, the Ks value was 0.3 microM for metyrapone and 5 microM for nicotine with both the microsomes and purified enzyme. The apparent Ki value for cotinine inhibition of 7-pentoxyresorufin O-dealkylase activity with the microsomes (87 microM) was approximately 87- and 870-fold higher than for nicotine and metyrapone, respectively. Monoclonal antibodies produced against cotinine cross-reacted equally well with metyrapone. They specifically blocked enzyme binding of both drugs based on dose-dependent inhibition of spectral changes, and reversed the metyrapone-induced inhibition of microsomal O-dealkylase activity. In contrast, antibodies to nicotine did not cross-react with cotinine or metyrapone and had no effect on their activity, although they did block the action of nicotine. These results demonstrate that cotinine binding to P-450 from PB-induced rats and inhibition of functional activity in vitro are qualitatively like the effects of metyrapone and nicotine, and that monoclonal anti-cotinine antibodies are useful molecular probes of the interactions between cotinine and metyrapone with the enzyme.     

The mechanism of halothane binding to microsomal cytochrome P450

Summary The unusual difference spectrum obtained with halothane and reduced rat liver microsomal cytochrome P450 can be simulated by addition of trifluoro diazoethane to dithionite reduced microsomes. Chemical evidence and model reactions suggest that in both cases a trifluoromethyl carbene complex is formed with the reduced hemoprotein. The spectral dissociation constants of the two species are similar as are their competitive reactions with carbon monoxide. The partial destruction of the carbenoid-cytochrome P450 complex characterizes the ligand as a highly reactive species. It is assumed that under anaerobic conditions this complex is formed by a two electron reduction of halothane and that covalent binding to microsomal proteins proceeds by this carbenoid species. A possible relationship to the hepatotoxicity of polyhalogenated compounds and anesthetics is discussed.     

Effect of p-amino-diphenyl ethers on hepatic microsomal cytochrome P450

The present paper aims to investigate whether p-amino-2',4'-dichlorodiphenyl ether and p-amino-4'-methyldiphenyl ether are inhibitors as well as inducers of P450. Mice were given daily intraperitoneal (ip) injections of p-amino-2',4'-dichlorodiphenyl ether (0.25 mmol/kg) or p-amino-4'-methyldiphenyl ether (0.25 mmol/kg) for 4 days and tested at 24 h and 48 h after the last dose injection. The results showed the mice pentobarbital sleeping time was shorter and the P450 content of hepatic microsome increased significantly in the group pretreated with p-amino-4'-methyldiphenyl ether when compared with the control group, while in mice pretreated with p-amino-2',4'-dichlorodiphenyl ether the hepatic microsome P450 content increased but the pentobarbital sleeping time was extended in clear contrast to the control group. The sleeping time of the phenobarbital group (80 mg/kg daily ip injection for 4 days) was shortened at 24 h after the last injection with increased P450 content of hepatic microsome, but it showed no difference at 48 h. The zoxazolamine-paralysis times of mice treated with p-amino-2',4'-dichlorodiphenyl ether were longer than those of the control mice, while the same dose of zoxazolamine did not lead to paralysis in mice pretreated with BNF. p-Amino-2',4'-dichlorodiphenyl ether and p-amino-4'-methyldiphenyl ether inhibited the activity of 7-ethoxyresorufin O-deethylase from rat hepatic microsome induced by BNF in vitro by 70.0% and 50.1% respectively. These results suggest that p-amino-2',4'-dichlorodiphenyl ether and p-amino-4'-methyldiphenyl ether are inhibitors as well as inducers of P450.     

Molecular mechanisms regulating the mitochondrial targeting of microsomal cytochrome P450 enzymes

Cytochrome P450 enzymes (CYPs) are a superfamily of monooxygenases found in almost all living organisms. CYPs are predominantly localized in the endoplasmic reticulum membranes as integral membrane proteins, where they metabolize a variety of endogenous and xenobiotic compounds. CYPs also reside in other subcellular compartments, including the plasma membranes and mitochondria. CYP localization in mitochondria is regulated in one of two ways: (1) direct targeting of inherent CYPs with canonical mitochondrial signals in their protein sequence after synthesis in the cytosol or (2) mitochondrial localization of microsomal CYPs after processing of the NH(2)-terminal region. Microsomal CYPs targeted to mitochondria demonstrate conventional or altered catalytic activities using electrons provided by the mitochondrial electron transport system. Mechanisms of microsomal CYP targeting to mitochondria, regulation of localization, and the implications of these in drug metabolism are described in the present review.     

Metabolism of Zaleplon by human hepatic microsomal cytochrome P450 isoforms

1. The metabolism of Zaleplon (CL-284,846; ZAL) has been studied in human liver microsomal preparations and in cDNA-expressed human cytochrome P450 (CYP) isoforms. 2. Human liver microsomes catalysed the NADPH-dependent N -deethylation of ZAL to DZAL (CL-284,859), but not to two other known in vivo metabolites, namely M1 (CL345,644) and M2 (CL-345,905). Sigmoidal kinetic plots were observed for ZAL deethylation indicating positive cooperativity. 3. The metabolism of ZAL to DZAL was determined in a characterized bank of 24 human liver microsomalpreparations.Good correlations (r² = 0.734-0.937) were observed with caffeine 8-hydroxylase, diazepam 3-hydroxylase, dextromethorphan N-demethylase and testosterone 2β-, 6β- and 15β-hydroxylase activities, which are allcatalysed by CYP3A isoforms. In contrast, poor correlations (r² 0.152-0.428) were observed for enzymatic markers for CYP1A2, CYP2A6, CYP2C9 10, CYP2D6, CYP2E1 and CYP4A9 11. 4. The metabolism of ZAL to DZAL in human liver microsomes was inhibited to 6-15% of control by 5-50 μM of the mechanism-based CYP3A inhibitor troleandomycin. Whereas some inhibition of DZAL formation was observed in the presence of 200 μM diethyldithiocarbamate, 5-50 μM furafylline, 2-20 μM sulphaphenazole, 50-500 μM S-mephenytoin and 1-10 μM quinidine had little effect. 5. Using human B-lymphoblastoid cell microsomes containing cDNA-expressed CYP isoforms, ZAL was metabolized to DZAL by CYP3A4, but not to any great extent by CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. 6. In contrast with ZAL, the NADPH-dependent N-deethylation of M2 to M1 proceeded at only a very low rate with both human liver microsomes and cDNA-expressed CYP3A4. 7. In summary, by correlation analysis, chemical inhibition studies and the use of cDNA-expressed CYPs, ZAL N -deethylation to DZAL in human liver appears to be catalysed by CYP3A isoforms.     

淫羊藿苷对不同月龄大鼠肝微粒体细胞色素P450的影响

目的 揭示淫羊藿苷(Ica)对大鼠肝微粒体细胞色素P450的含量及部分亚型的影响,并比较月龄的差异.方法 ig给予6月龄和18月龄的♂SD大鼠Ica( 60 mg· kg -1),4周后取肝脏,用钙沉淀法提取肝微粒体,BCA法测定微粒体蛋白浓度;用一氧化碳还原差示光谱法测定CYP450的含量;用ELISA法测定CYP1 A1、CYPb5的含量;用比色法测定苯胺羟化酶(反映CYP2E1活性)和红霉素-N-脱甲基酶(反映CYP3A活性)的活性;用real - time RT - PCR检测CYP1 A1、CYP2A3、CYP2E1、CYP3A1、CYP3A2和CYP4B1 mRNA的表达.结果 60 mg· kg-1 Ica明显增加了CYP450的总酶和CYP1 A1的含量、CYP3A的活性及CYP1 A1、CYP3A1、CYP3A2 mRNA的表达,降低了CYP2E1的活性及其mRNA的表达;但Ica对上述各指标的诱导或抑制作用在大鼠月龄方面差异不明显;Ica对CYPb5的含量及CYP2A3、CYP4B1 mRNA的表达未见明显影响.结论 Ica对大鼠肝微粒体CYP450总酶、CYPI A1和CYP3A具有诱导作用,对CYP2E1具有抑制作用,该作用未见明显月龄差异.     

Effects of propofol on human hepatic microsomal cytochrome P450 activities

1. The potential of propofol to inhibit the activity of major human cytochrome P450 enzymes has been examined in vitro using human liver microsomes. Propofol produced inhibition of CYP1A2 (phenacetin O -deethylation), CYP2C9 (tolbutamide 4-hydroxylation), CYP2D6 (dextromethorphan O -demethylation) and CYP3A4 (testosterone 6 beta hydroxylation) activities with IC = 40, 49, 213 and 32 mu M respectively. K for propofol against all of these enzymes with the exception of CYP2D6, where propofolishowed little inhibitory activity, was 30, 30 and 19 mu M respectively for CYPs 1A2, 2C9 and 3A4. 2. Furafylline, sulphaphenazole, quinidine and ketoconazole, known selective inhibitors of CYPs 1A2, 2C9, 2D6 and 3A4 respectively, were much more potent than propofol having IC = 0.8, 0.5, 0.2 and 0.1 mu M; furafylline and sulphaphenazole yielded K = 0.6 and 0.7 mu M respectively. i 3. The therapeutic blood concentration of propofol (20 mu M; 3-4 mu?g ml) together with the in vitro K estimates for each of the major human P450 enzymes have been used to i estimate the extent of cytochrome P450 inhibition, which may be produced in vivo by propofol. This in vitro - in vivo extrapolation indicates that the degree of inhibition of CYP1A2, 2C9 and 3A4 activity which could theoretically be produced in vivo by propofol is relatively low (40-51%); this is considered unlikely to have any pronounced clinical significance. 4. Although propofol has now been used in 190 million people since its launch in 1986,thereare onlysinglereportsofpossible druginteractions between propofoland either alfentanil or warfarin. Consequently, it is difficult to conclude from either the published literature or the ZENECA safety database whether there is any evidence to indicate that propofol produces clinically significant drug interactions through inhibition of cytochrome P450-related drug metabolism.     

Differential induction of cytochromes P450 and cytochrome P450-dependent arachidonic acid metabolism by 3,4,5,3',4'-pentachlorobiphenyl in the rat and the guinea pig

Differential induction of hepatic cytochromes P450 by 3,4,5,3',4'-pentachlorobiphenyl (PENCB) has been observed in the rat and the guinea pig: (1) in rat and guinea pig, treatment with the chosen dose levels resulted in significant induction of total, carbon monoxide-discernible cytochrome P450 content; the absorption maximum of the CO-adduct of the dithionite-reduced microsomes from PENCB-induced rat liver was shifted from 450 to 448 nm, whereas its counterpart in the guinea pig did not; (2) PENCB treatment significantly increased EROD activity in rat liver microsomes (up to 60-fold), but the increase in the guinea pig was less than fivefold; (3) PENCB-induced rat liver microsomes significantly induced the omega-1 hydroxylation of arachidonic acid (AA); however, omega-1 hydroxylation of AA was hardly affected by PENCB treatment in the guinea pig. Instead, omega-hydroxylation was significantly increased in this latter species. In addition to omega-1 hydroxylation in the rat or omega-hydroxylation in the guinea pig, an additional AA metabolite (designated peak III) was significantly induced by PENCB in both rat and guinea pig; (4) Western blot and ELISA analyses with polyclonal anti-P450 IA1/IA2 and IVA1 antibodies demonstrated that P450 IA1 was significantly induced in the rat but only slightly induced in the guinea pig, whereas P450 IVA1 was significantly suppressed in the rat but significantly induced in the guinea pig by PENCB treatment. The induction of the third arachidonic acid metabolite peak, Peak III, in both rat and guinea pig, particularly in the guinea pig, is obviously neither mediated by P450 IA1 nor by P450 IV A1. At present, it is still unclear which form(s) of cytochrome P450 isoenzymes is responsible for this latter hydroxylation of arachidonic acid.     

葛根素对小鼠和大鼠肝微粒体细胞色素P450的影响

葛根素(puerarin)是豆科植物葛根的主要有效成分,具有扩血管、抗血小板、降血压、降血糖、改善微循环等作用,广泛用于心脑血管疾病的防治[1].因联合用药日益普遍,使药物间的相互作用倍受重视.肝微粒体细胞色素P450(cytochrome P450, CYP)是参与药物代谢的重要酶系,药物对CYP活性的影响是药物间相互作用的重要机制之一.本研究探讨了葛根素腹腔注射对小鼠和大鼠肝微粒体CYP活性的影响.     

灯盏细辛注射液对小鼠肝微粒体细胞色素P450含量的影响

目的:研究灯盏细辛注射液对小鼠肝微粒体细胞色素P450含量的影响。方法:小鼠分为空白对照组、阳性对照组(苯巴比妥钠80mg/kg,i.p.,共7d)、灯盏细辛注射液正常剂量组、高剂量组(10、30mg/kg,i.p.,共14d)。用紫外分光光度法测定细胞色素P450及其同工酶的活性。结果:灯盏细辛注射液两剂量组小鼠肝微粒体的蛋白含量和细胞色素P450含量较空白对照组增高,对肝重指数基本无影响;可抑制红霉素-N-脱甲基酶(ERD)的活性(P<0.01),而对氨基比林-N-脱甲基酶(AMD)基本无影响(P>0.05);苯巴比妥钠组肝重指数、蛋白含量和细胞色素P450含量与其他各组比较均升高(P<0.05),对氨基比林-N-脱甲基酶、红霉素-N-脱甲基酶活性均有诱导作用(P<0.01)。结论:灯盏细辛注射液对小鼠肝微粒体蛋白、细胞色素P450含量有一定影响,对CYP3A可能有抑制作用,对氨基比林-N-脱甲基酶活性基本无影响。