黄芪颗粒和黄芪注射液对CYP1A2、CYP2D、CYP2C亚酶活性影响的实验研究

目的通过体内和体外实验研究黄芪颗粒和黄芪注射液对CYP1A2、CYP2D、CYP2C亚酶的活性影响。方法建立体外"cocktail"反应体系,利用LC/MS/MS法测定酶代谢产物,计算黄芪颗粒和黄芪注射液对CYP1A2、CYP2D6、CYP2C9和CYP2C19亚酶活性的影响;大鼠随机分为对照组和实验组,不同浓度黄芪颗粒和黄芪注射液连续灌胃10 d,制备肝微粒体并进行"cocktail"反应,评价两种药物体内对大鼠CYP1A2、CYP2D1、CYP2C6和CYP2C11亚酶活性影响。结果在体外"cocktail"实验中,黄芪颗粒和黄芪注射液明显地抑制了CYP2D6、CYP2C19和CYP1A2亚酶活性,而二者对于CYP2C9亚酶活性无影响。在大鼠灌胃给药实验中,黄芪颗粒在剂量32、160和800 mg.kg-1.d-1提高CYP1A2亚酶活性2.13、3.23和2.20倍,黄芪注射液在剂量0.16、0.8、4 g.kg-1.d-1提高CYP1A2亚酶活性1.65、2.26和2.89倍,而二者均未诱导CYP2D1、CYP2C6和CYP2C11亚酶活性增加。结论黄芪颗粒和黄芪注射液对CYP2D6、CYP2C19活性有抑制作用,对大鼠CYP1A2活性有诱导作用。     

氟他胺在大鼠肝微粒体经细胞色素P450 1A2代谢的性别差异

目的体外研究大鼠肝微粒体细胞色素P450 1A2(CYP1A2)对氟他胺(flutamide Flu)代谢的性别差异影响。方法制备正常♀♂大鼠肝微粒体,用CYP1A2抗体与氟他胺(2 mg·L^-1)共同温孵,测定氟他胺主要代谢产物2-羟基氟他胺(2-hydroxyflutamide, HF)和原药的浓度比(HF/Flu),评价氟他胺在大鼠肝微粒体代谢的性别差异。结果在CYP1A2抗体浓度为1∶400,孵育时间为30 min条件下,氟他胺在♂大鼠肝微粒体中的HF/Flu为(1.5±0.6),而♀动物为(0.9±0.4)。不同性别大鼠肝微粒体对氟他胺的代谢存在性别差异(P<0.01)。结论Flu在♂大鼠肝微粒体中代谢快,而在♀大鼠肝微粒体中代谢较慢。♂大鼠体内的CYP1A2酶活性高于♀大鼠。     

丹参注射液对人肝微粒体酶CYP2C9,CYP2C19,CYP2D6体外抑制作用的研究

目的 评价丹参注射液在体外对人肝微粒体酶CYP2C9、CYP2C19、CYP2D6活性的影响。方法 将丹参注射液与CYP450酶3种亚型(CYP2C9、CYP2C19、CYP2D6)的特异性探针底物甲苯磺丁脲、奥美拉唑、右美沙芬与大鼠肝微粒体进行孵育,采用LC-MS/MS法测定对应3种代谢产物4-羟基甲苯磺丁脲、5-羟基奥美拉唑、右啡烷的浓度,求算出IC₅₀。结果 丹参注射液对CYP2C9、CYP2C19和CYP2D6的IC₅₀值均>50 μg/mL。结论 丹参注射液对人肝微粒体酶CYP2C9、CYP2C19、CYP2D6没有抑制作用。     

混合探针底物法测定五味子成分对大鼠肝脏细胞色素P450同工酶的影响

研究大鼠多次口服五味子成分和体外对肝脏细胞色素P450酶(CYP450)6种同工酶的影响。采用超速离心法制备正常及多次口服五味子醇/水提物的大鼠肝微粒体并与探针药进行体外温孵,应用液相色谱?串联质谱分析方法测定CYP450的6种同工酶特异性探针底物非那西丁(CYP1A2)、右美沙芬(CYP2D2)、双氯芬酸钠(CYP2C6)、美芬妥英(CYP2C11)、氯唑沙宗(CYP2E1)、咪达唑仑(CYP3A1/2)在大鼠肝微粒体的代谢产物生成(对乙酰氨基酚、右啡烷、4-羟基双氯芬酸钠、4-羟基美芬妥英、6-羟基氯唑沙宗、1-羟基咪达唑仑)以反映各CYP450同工酶活性。五味子醇提物(28～120μg.mL-1)体外对大鼠肝微粒体CYP450的6个同工酶均有不同程度的抑制作用。大鼠多次口服五味子醇提物(1.5 g.kg-1,qd×7d)对肝脏CYP3A1/2和CYP2E1有显著诱导作用,对CYP2D2和CYP2C11有明显抑制作用,而对CYP2C6和CYP1A2无明显影响。五味子水提物(100～500μg.mL-1)体外对大鼠肝脏CYP450同工酶亦有抑制作用;体内多次给药(1.5 g.kg-1,qd×7d)对肝脏CY...     

和厚朴酚、厚朴酚、栀子苷、绿原酸和黄芪甲苷对人和大鼠体外CYP1A2、CYP3A和CYP2D的抑制作用

目的 观察和厚朴酚、厚朴酚、栀子苷、绿原酸和黄芪甲苷5种中药成分体外对人和大鼠肝CYP1A2、CYP3A和CYP2D的抑制作用。方法 在人和大鼠肝微粒体孵育体系中,分别以非那西丁、咪达唑仑和右美沙芬为探针,应用HPLC检测受试物对探针代谢产物生成量的影响,评估5种中药成分对CYP1A2、CYP3A和CYP2D在该体系中的活性影响,并计算得到抑制率和IC₅₀。结果 和厚朴酚对人和大鼠CYP1A2、CYP2D的IC₅₀值分别为5.5、3.9、35.3和46.7 μmol·L^-1;厚朴酚对人CYP1A2、大鼠CYP1A2和CYP2D的IC50值分别为23.8,29.1和39.9 μmol·L^-1;栀子苷、绿原酸和黄芪甲苷对3种CYP酶亚型的IC₅₀均>100 μmol·L^-1;和厚朴酚对人和大鼠CYP3A的IC₅₀均>100 μmol·L^-1;厚朴酚对人CYP3A、CYP2D和大鼠CYP3A的IC₅₀均>100 μmol·L^-1。结论 和厚朴酚体外对人和大鼠CYP1A2和CYP2D有抑制作用,厚朴酚体外对人CYP1A2、大鼠CYP1A2和CYP2D有抑制作用,均呈浓度依赖性。     

注射用丹参总酚酸(冻干)对人CYP450酶和P-糖蛋白体外抑制作用及对大鼠CYP1A2和CYP3A体内诱导作用(英文)

目的探讨注射用丹参总酚酸(冻干)(SLI)对人CYP450酶和P-糖蛋白体外抑制作用以及对大鼠CYP1A2和CYP3A体内诱导作用。方法①应用P450-GloTMCYP450检测试剂盒,通过化学发光法测定SLI和经典抑制剂对细胞色素P4501A2(CYP1A2),CYP2D6,CYP3A4,CYP2C19和CYP2C9的IC50值,通过比较SLI和经典抑制剂对相应细胞色素P450亚型的IC50值来判断SLI对人CYP450酶的体外抑制作用。②Wistar大鼠分别iv给予SLI 3,10和30 mg·kg-1和诱导剂苯巴比妥钠20 mg·kg-1,采用探针底物法,通过比较代谢产物的生成速率来评价SLI对大鼠CYP1A2和CYP3A的诱导作用。③应用ATP酶检测试剂盒,通过化学发光法测定ATP酶活性来评价SLI是否为P-gp的底物或抑制剂。结果①CYP1A2,CYP2C9,CYP2C19,CYP2D6和CYP3A4抑制剂的IC50与SLI对其的IC50进行比较(CYP1A2:0.12μmol·L-1vs 840μmol·L-1;CYP2C9:3.362μmol·L-1vs 704μmol·L-1;CYP2C19:3.236μmol·L-1vs 306μmol·L-1;CYP2D6:0.117μmol·L-1vs 2660μmol·L-1;CYP3A4:0.078μmol·L-1vs 1780μmol·L-1)。②与空白对照组(86.4±6.3)nmol·g-1.min-1相比,SLI 3,10和30 mg·kg-1组CYP1A2活性分别为83.4±6.6,82.5±4.0和(83.4±6.6)nmol·g-1.min-1。与空白对照组(16.1±0.9)nmol·g-1.min-1比较,SLI 3,10和30 mg·kg-1组CYP3A活性分别为15.7±0.6,15.9±0.7和(15.9±1.0)nmol·g-1.min-1,无显著性差异。③以临床血药浓度为依据设计的一系列浓度的SLI 0.0002,0.0006,0.002,0.006,0.017,0.052,0.156和0.468 g.L-1的ATP酶活性分别与空白对照组进行比较(5.8,5.3,5.8,5.5,5.8,5.2,,5.8,5.3,vs 5.75μmol·g-1.min-1),无显著性差异。结论SLI临床给药剂量既不能体外抑制人CYP1A2,CYP2D6,CYP3A4,CYP2C19和CYP2C9酶活性,也不能诱导大鼠CYP1A2和CYP3A,同时也不是P-gp的体外抑制剂或底物。     

双环醇对肝脏部分切除大鼠CYP450酶活性和表达的影响(英文)

本研究考察双环醇对肝脏部分切除(PH)后大鼠肝脏微粒体细胞色素P450(CYP)活性、基因和蛋白表达的影响及相关机制。大鼠PH前灌胃给予双环醇(300 mg.kg-1)3次,PH后处死大鼠,取其血清和肝组织进行检测,依次测定血清谷丙转氨酶(ALT)、肝微粒体丙二醛(MDA)和肝脏总CYP含量、4种CYP同工酶活性、基因和蛋白表达。结果显示,双环醇可显著抑制PH大鼠血清ALT和肝微粒体MDA的升高,抑制肝脏总CYP含量的减少,抑制CYP2C6、2C11活性和mRNA表达的下降,明显抑制CYP3A1/2活性的下降,并上调CYP3A1和2E1的mRNA和蛋白表达。结果表明,双环醇对PH大鼠肝脏CYP450酶及部分同工酶活性和表达的改变有明显改善作用,其作用机制可能与其抗氧化作用和酶诱导作用密切相关。     

五味子醇甲在大鼠肝微粒体内的代谢动力学和性别差异

体外研究五味子醇甲(schizandrin，SZ)在大鼠肝微粒体内的代谢动力学和性别差异。制备正常雌、雄大鼠肝微粒体，与SZ共同温孵，以高效液相色谱法测定SZ及其代谢产物。SZ在雄鼠肝微粒体内代谢反应的最大速率V_max、米氏常数K_m和清除率Cl_int分别为(21.88±2.30) μmol·L^-1·min^-1·mg^-1(protein)，(389.00±46.26) μmol·L^-1和(0.056 3±0.000 7) min·mg^-1(protein)；在雌鼠肝微粒体内代谢反应的最大速率V_max、米氏常数K_m和清除率Clint_{分别为(0.61±0.07) μmol·L^-1·min^-1·mg^-1(protein)，(72.64±13.61) μmol·L^-1和(0.008 4±0.000 8) min·mg^-1(protein)，雌、雄鼠肝微粒体内SZ的主要代谢物不同，分别为7,8-顺二羟基五味子醇甲(M1)和7,8-顺二羟基-2-去甲基五味子醇甲(M2b)。酮康唑、奎尼丁和奥芬得林对SZ的在雌、雄大鼠肝微粒体内代谢均有不同程度的抑制作用，西咪替丁对其在雄鼠肝微粒体内的代谢也有一定的抑制作用。SZ在雌、雄大鼠肝微粒体中代谢动力学及代谢产物存在明显的性别差异，这种差异可能主要是由CYP3A和CYP2C11在大鼠肝微粒体内的性别差异引起的。}     

尼扎替丁对大鼠CYP1A2亚型的影响

目的通过尼扎替丁的大鼠体内、外实验,观察尼扎替丁对大鼠CYP1A2亚型的影响。方法通过HPLC法测定全血中咖啡因的代谢率,观测尼扎替丁对大鼠CYP1A2活性的影响;通过W estern b lot法测定尼扎替丁对大鼠肝微粒体CYP1A2蛋白表达的调控;通过HPLC法测定肝微粒体重组系统对乙酰氨基酚的含量,确定尼扎替丁对大鼠肝微粒体CYP1A2亚型的作用。结果实验组中给予大鼠不同浓度的尼扎替丁(14、27、54 mg.kg-1),其咖啡因代谢率为29.6%±12.5%、32.4%±13.4%、37.5%±15.0%,对照组为26.9%±11.9%,各剂量组及对照组间差异均无显著性(P>0.05);实验各剂量组与对照组的CYP1A2蛋白表达差异无显著性;肝微粒体体外重组系统中,实验组各浓度尼扎替丁对CYP1A2没有抑制作用,CYP1A2的活性>100%;对照组α-萘黄酮有明显的抑制作用,IC50=0.0306μmol.L-1。结论体内、外实验结果均表明尼扎替丁对大鼠CYP1A2没有抑制作用。     

异鼠李素对人肝微粒体CYPs活性及大鼠原代肝细胞毒性作用研究

目的 研究异鼠李素对肝脏6种CYPs的体外抑制作用,以及对大鼠原代肝细胞的毒性作用。方法 采用人肝微粒体（HLMs）体外温孵法研究异鼠李素对6种细胞色素P450酶（CYPs）——CYP2C19、CYP2D6、CYP3A4、CYP2E1、CYP1A2和CYP2C9的体外抑制作用;使用HPLC-MS/MS法检测异鼠李素和HLMs共同孵育后的代谢产物;利用体外培养的低CYPs活性的大鼠原代肝细胞,考察不同剂量异鼠李素对细胞培养液中乳酸脱氢酶（LDH）、丙氨酸氨基转移酶（ALT）、天门冬氨酸氨基转移酶（AST）的影响。结果 50 μmol/L的异鼠李素对CYP2E1和CYP1A2有一定的抑制作用,抑制率分别为59.48%和39.91%;异鼠李素和HLMs共同孵育后,产生去甲基化代谢产物3,3'',4'',5,7-五羟基黄酮,转化为极性和水溶性较高的代谢物;30、100、300 μmol/L的异鼠李素会使大鼠原代肝细胞培养液中的ALT和LDH显著上升（P＜0.01）,100、300 μmol/L异鼠李素使AST显著上升（P＜0.05、0.01）,呈浓度相关性。结论 异鼠李素在体外主要经HLMs代谢,同时对CYP2E1和CYP1A2有一定的抑制作用,可能会使CYP2E1和CYP1A2的底物药物在体内的浓度产生变化,导致一系列药物的相互作用;大量使用异鼠李素可能会造成一定程度的肝细胞损伤,且呈现浓度相关性。临床应用应合理设置剂量,并注意潜在的药物之间的相互作用。     

In vitro metabolism of chloroquine: identification of CYP2C8, CYP3A4, and CYP2D6 as the main isoforms catalyzing N-desethylchloroquine formation.

In humans, the antimalarial drug chloroquine (CQ) is metabolized into one major metabolite, N-desethylchloroquine (DCQ). Using human liver microsomes (HLM) and recombinant human cytochrome P450 (P450), we performed studies to identify the P450 isoform(s) involved in the N-desethylation of CQ. In HLM incubated with CQ, only DCQ could be detected. Apparent Km and Vmax values (mean +/- S.D.) for metabolite formation were 444 +/- 121 microM and 617 +/- 128 pmol/min/mg protein, respectively. In microsomes from a panel of 16 human livers phenotyped for 10 different P450 isoforms, DCQ formation was highly correlated with testosterone 6beta-hydroxylation (r = 0.80; p < 0.001), a CYP3A-mediated reaction, and CYP2C8-mediated paclitaxel alpha-hydroxylation (r = 0.82; p < 0.001). CQ N-desethylation was diminished when coincubated with quercetin (20-40% inhibition), ketoconazole, or troleandomycin (20-30% inhibition) and was strongly inhibited (80% inhibition) by a combination of ketoconazole and quercetin, which further corroborates the contribution of CYP2C8 and CYP3As. Of 10 cDNA-expressed human P450s examined, only CYP1A1, CYP2D6, CYP3A4, and CYP2C8 produced DCQ. CYP2C8 and CYP3A4 constituted low-affinity/high-capacity systems, whereas CYP2D6 was associated with higher affinity but a significantly lower capacity. This property may explain the ability of CQ to inhibit CYP2D6-mediated metabolism in vitro and in vivo. At therapeutically relevant concentrations ( approximately 100 microM CQ in the liver), CYP2C8, CYP3A4, and, to a much lesser extent, CYP2D6 are expected to account for most of the CQ N-desethylation.     

Trimethoprim and sulfamethoxazole are selective inhibitors of CYP2C8 and CYP2C9, respectively.

To evaluate the inhibitory effects of trimethoprim and sulfamethoxazole on cytochrome P450 (P450) isoforms, selective marker reactions for CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 were examined in human liver microsomes and recombinant CYP2C8 and CYP2C9. The in vivo drug interactions of trimethoprim and sulfamethoxazole were predicted in vitro using [I]/([I] + K(i)) values. With concentrations ranging from 5 to 100 microM, trimethoprim exhibited a selective inhibitory effect on CYP2C8-mediated paclitaxel 6alpha-hydroxylation in human liver microsomes and recombinant CYP2C8, with apparent IC(50) (K(i)) values of 54 microM (32 microM) and 75 microM, respectively. With concentrations ranging from 50 to 500 microM, sulfamethoxazole was a selective inhibitor of CYP2C9-mediated tolbutamide hydroxylation in human liver microsomes and recombinant CYP2C9, with apparent IC(50) (K(i)) values of 544 microM (271 microM) and 456 microM, respectively. With concentrations higher than 100 microM trimethoprim and 500 microM sulfamethoxazole, both drugs lost their selectivity for the P450 isoforms. Based on estimated total hepatic concentrations (or free plasma concentrations) of the drugs and the scaling model, one would expect in vivo in humans 80% (26%) and 13% (24%) inhibition of the metabolic clearance of CYP2C8 and CYP2C9 substrates by trimethoprim and sulfamethoxazole, respectively. In conclusion, trimethoprim and sulfamethoxazole can be used as selective inhibitors of CYP2C8 and CYP2C9 in in vitro studies. In humans, trimethoprim and sulfamethoxazole may inhibit the activities of CYP2C8 and CYP2C9, respectively.     

Chlorpropamide 2-hydroxylation is catalysed by CYP2C9 and CYP2C19 in vitro: chlorpropamide disposition is influenced by CYP2C9, but not by CYP2C19 genetic polymorphism

AIMS: We evaluated the involvement of cytochrome P450 (CYP) isoforms 2C9 and 2C19 in chlorpropamide 2-hydroxylation in vitro and in chlorpropamide disposition in vivo. METHODS: To identify CYP isoforms(s) that catalyse 2-hydroxylation of chlorpropamide, the incubation studies were conducted using human liver microsomes and recombinant CYP isoforms. To evaluate whether genetic polymorphisms of CYP2C9 and/or CYP2C19 influence the disposition of chlorpropamide, a single oral dose of 250 mg chlorpropamide was administered to 21 healthy subjects pregenotyped for CYP2C9 and CYP2C19. RESULTS: In human liver microsomal incubation studies, the formation of 2-hydroxychlorpropamide (2-OH-chlorpropamide), a major chlorpropamide metabolite in human, has been best described by a one-enzyme model with estimated K(m) and V(max) of 121.7 +/- 19.9 microm and 16.1 +/- 5.0 pmol min(-1) mg(-1) protein, respectively. In incubation studies using human recombinant CYP isoforms, however, 2-OH-chlorpropamide was formed by both CYP2C9 and CYP2C19 with similar intrinsic clearances (CYP2C9 vs. CYP2C19: 0.26 vs. 0.22 microl min(-1) nmol(-1) protein). Formation of 2-OH-chlorpropamide in human liver microsomes was significantly inhibited by sulfaphenazole, but not by S-mephenytoin, ketoconazole, quinidine, or furafylline. In in vivo clinical trials, eight subjects with the CYP2C9*1/*3 genotype exhibited significantly lower nonrenal clearance [*1/*3 vs.*1/*1: 1.8 +/- 0.2 vs. 2.4 +/- 0.1 ml h(-1) kg(-1), P < 0.05; 95% confidence interval (CI) on the difference 0.2, 1.0] and higher metabolic ratios (of chlorpropamide/2-OH-chlorpropamide in urine: *1/*3 vs.*1/*1: 1.01 +/- 0.19 vs. 0.56 +/- 0.08, P < 0.05; 95% CI on the difference - 0.9, - 0.1) than did 13 subjects with CYP2C9*1/*1 genotype. In contrast, no differences in chlorpropamide pharmacokinetics were observed for subjects with the CYP2C19 extensive metabolizer vs. poor metabolizer genotypes. CONCLUSIONS: These results suggest that chlorpropamide disposition is principally determined by CYP2C9 activity in vivo, although both CYP2C9 and CYP2C19 have a catalysing activity of chlorpropamide 2-hydroxylation pathway.     

Metabolic activation of o-phenylphenol to a major cytotoxic metabolite, phenylhydroquinone: role of human CYP1A2 and rat CYP2C11/CYP2E1

1. The in vitro metabolic activation of o-phenylphenol has been evaluated as yielding a toxic metabolite, 2,5-dihydroxybiphenyl (phenylhydroquinone), by p-hydroxylation in liver microsomes of rat and human. The involvement of rat CYP2C11, CYP2E1 and human CYP1A2 in the p-hydroxylation of o-phenylphenol is suggested. 2. 2,3- and phenylhydroquinone, which induced DNA single-strand scission in the presence of 1 microM CuCl2, were the most cytotoxic chemicals examined to cultured mammalian cell lines among o-phenylphenol, m-phenylphenol, p-phenylphenol, 2,2'-, 4,4'-, 2,3- and phenylhydroquinone. 3. Rat and human liver microsomes catalysed the formation of phenylhydroquinone, but not 2,3-dihydroxybiphenyl, using o-phenylphenol as a substrate. A higher rate of metabolic activation of o-phenylphenol was observed with livers of the male than the female rats by 5.6- and 2.6-fold respectively. 4. Inhibitory antibodies against the male-specific CYP2C11 inhibited hepatic o-phenylphenol p-hydroxylation in the male F344 and Sprague-Dawley rat by > 70%. Liver microsomes from the isoniazid-treated rats produced 1.8- and 3-fold induction of o-phenylphenol p-hydroxylation and chlorzoxazone 6-hydroxylation (a CYP2E1-dependent activity) respectively. 5. Human CYP1A2, expressed by baculovirus-mediated cDNA expression systems, exhibited a remarkably higher capacity for o-phenylphenol p-hydroxylation at concentrations of 5 (> 5-fold), 50 (> 2-fold) and 500 microM (> 2-fold) than CYP2A, CYP2B, CYP2Cs, CYP2D6, CYP2E1 and CYP3A4 on the basis of pmol P450. 6. Among various CYP inhibitors tested here, 7,8-benzoflavone and furafylline, typical human CYP1A2 inhibitors, inhibited the microsomal p-hydroxylation of o-phenylphenol in human livers most potently by 70 and 50% respectively. 7. The results thus indicate the involvement of rat CYP2C11/CYP2E1 and human CYP1A2 in the hepatic p-hydroxylation of o-phenylphenol.     

In vitro inhibition of cytochrome P450 enzymes in human liver microsomes by a potent CYP2A6 inhibitor, trans-2-phenylcyclopropylamine (tranylcypromine), and its nonamine analog, cyclopropylbenzene.

Currently, there are no selective, well characterized inhibitors for CYP2A6. Therefore, the effects of trans-(+/-)-2-phenylcyclopropylamine (tranylcypromine), a potent CYP2A6 inhibitor, on human liver microsomal cytochromes P450 (CYP) were studied to elucidate its selectivity. The IC50 value of tranylcypromine in coumarin 7-hydroxylation (CYP2A6 model activity) was 0.42 +/- 0.07 microM and in chlorzoxazone 6-hydroxylation (CYP2E1 model activity) 3.0 +/- 1.1 microM. The IC50 values for CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 activities were >10 microM. Potency and selectivity of tranylcypromine were strongly dependent on the amine group, because its nonamine analog cyclopropylbenzene was much less potent inhibitor of CYP1A, CYP2A6, CYP2C19, and CYP2E1 activities and did not inhibit at all CYP2C9, CYP2D6, or CYP3A4 activities. In human liver microsomes tranylcypromine induced type II and cyclopropylbenzene type I difference spectrum. According to the double reciprocal analysis of these spectral responses both tranylcypromine and cyclopropylbenzene may have at least two P450-related binding sites in liver microsomes. The K(a) values of tranylcypromine varied from 4.5 to 15.1 microM and -34.3 to 167 microM in microsomes derived from three different livers and of cyclopropylbenzene from -1.6 to 10.1 microM and -34.6 and 75.2 microM in the same liver microsomes. Based on these results, tranylcypromine seems an adequately selective CYP2A6 inhibitor for in vitro use.     

In vitro inhibition of the cytochrome P450 (CYP450) system by the antiplatelet drug ticlopidine: potent effect on CYP2C19 and CYP2D6

AIMS: To examine the potency of ticlopidine (TCL) as an inhibitor of cytochrome P450s (CYP450s) in vitro using human liver microsomes (HLMs) and recombinant human CYP450s. METHODS: Isoform-specific substrate probes of CYP1A2, 2C19, 2C9, 2D6, 2E1 and 3A4 were incubated in HLMs or recombinant CYPs with or without TCL. Preliminary data were generated to simulate an appropriate range of substrate and inhibitor concentrations to construct Dixon plots. In order to estimate accurately inhibition constants (Ki values) of TCL and determine the type of inhibition, data from experiments with three different HLMs for each isoform were fitted to relevant nonlinear regression enzyme inhibition models by WinNonlin. RESULTS: TCL was a potent, competitive inhibitor of CYP2C19 (Ki = 1.2 +/- 0.5 microM) and of CYP2D6 (Ki = 3.4 +/- 0.3 microM). These Ki values fell within the therapeutic steady-state plasma concentrations of TCL (1-3 microM). TCL was also a moderate inhibitor of CYP1A2 (Ki = 49 +/- 19 microM) and a weak inhibitor of CYP2C9 (Ki > 75 microM), but its effect on the activities of CYP2E1 (Ki = 584 +/- 48 microM) and CYP3A (> 1000 microM) was marginal. CONCLUSIONS: TCL appears to be a broad-spectrum inhibitor of the CYP isoforms, but clinically significant adverse drug interactions are most likely with drugs that are substrates of CYP2C19 or CYP2D6.     

In vitro metabolism and drug interaction potential of a new highly potent anti-cytomegalovirus molecule, CMV423 (2-chloro 3-pyridine 3-yl 5,6,7,8-tetrahydroindolizine 1-carboxamide)

AIMS: To identify the enzymes involved in the metabolism of CMV423, a new anticytomegalovirus molecule, to evaluate its in vitro clearance and to investigate its potential involvement in drug/drug interactions that might occur in the clinic. METHODS: The enzymes involved in and the kinetics of CMV423 biotransformation were determined using pools of human liver subcellular fractions and heterologously expressed human cytochromes P450 (CYP) and FMO. The effect of CMV423 on CYP probe activities as well as on indinavir and AZT metabolism was determined, and 26 drugs were tested for their potential to inhibit or activate CMV423 metabolism. RESULTS: CMV423 was oxidized by CYP and not by FMO or cytosolic enzymes. The Km values for 8-hydroxylation to rac-RPR 127025, an active metabolite, and subsequent ketone formation by human liver microsomes were 44 +/- 13 microM and 47 +/- 11 microM, respectively, with corresponding Vmax/Km ratios of 14 and 4 microl min(-1) nmol(-1) P450. Inhibition with selective CYP inhibitors indicated that CYP1A2 was the main isoform involved, with some participation from CYP3A. Expressed human CYP1A1, 1A2, 2C9, 3A4 and 2C8 catalysed rac-RPR 127025 formation with Km values of < 10 microM, 50 +/- 21 microM, 55 +/- 19 microM, circa 282 +/- 61 microM and circa 1450 microM, respectively. CYP1B1, 2A6, 2B6, 2C19, 2D6, 2E1 or 3A5 did not catalyse the reaction to any detectable extent. CYP1A1 and 3A4 also catalysed ketone formation from rac-RPR 127025. In human liver microsomes, CMV423 at 1 and 10 microM inhibited CYP1A2 activity up to 31% and 63%, respectively, CYP3A4 activity up to 40% (10 microM) and CYP2C9 activity by 35% (1 and 10 microM). No effect was observed on CYP2A6, 2D6 and 2E1 activities. CMV423 had no effect on indinavir and AZT metabolism. Amongst 26 drugs tested, none inhibited CMV423 metabolism in vitro at therapeutic concentrations. CONCLUSIONS: CMV423 is mainly metabolized by CYP1A2 and 3A4. Its metabolism should not be saturable at the targeted therapeutic concentrations range (Cmax < 1 microM). CMV423 will probably affect CYP1A2 and 1A1 activities in vivo to some extent, but no other drug-drug interactions are expected.     

Relative contributions of CYP2C9 and 2C19 to phenytoin 4-hydroxylation in vitro: inhibition by sulfaphenazole, omeprazole, and ticlopidine

OBJECTIVES: To determine the relative contribution of cytochromes P450 (CYP) 2C9 and 2C19 to the formation of 5-(-4-hydroxyphenyl)-5-phenylhydantion (HPPH) from phenytoin (PPH). DESIGN: Hydroxylation of PPH to form HPPH was studied in vitro using human liver microsomes and microsomes from cDNA-transfected human lymphoblastoid cells. RESULTS: Formation of HPPH from PPH in liver microsomes had a mean (+/- SEM) apparent Km [substrate concentration corresponding to 50% of maximal reaction velocity (Vmax)] of 23.6 +/- 1.8 mumol/l. Coincubation with the CYP2C9 inhibitor, sulfaphenazole (SPA), at 5 mumol/l reduced reaction velocity to less than 15% of control values. The mean inhibitor concentration at which 50% inhibition is achieved (IC50 value) for SPA versus PPH hydroxylation (0.49 microM) was similar to the SPA IC50 versus flurbiprofen hydroxylation (0.46 microM) and tolbutamide hydroxylation (0.7-1.5 microM). In contrast, the CYP2C19 inhibitor omeprazole (OME) at 10 mumol/l produced only a small degree of inhibition. Incubation of PPH with microsomes from cDNA-transfected human lymphoblastoid cells containing CYP1A2, 2A6, 2B6, 2C8, 2D6, 2E1, or 3A4 yielded no detectable formation of HPPH. Only CYP2C9 and 2C19 had PPH hydroxylation activity, with apparent Km values for the high-affinity component of 14.6 mumol/l and 24.1 mumol/l, respectively. Based on Vmax values in liver microsomes, the Vmax and Km values in expressed CYPs and the relative abundance of the two isoforms in human liver, CYP2C9, and 2C19 were estimated to have relative contributions of 90% and 10%, respectively, to net intrinsic clearance. CONCLUSIONS: Formation of HPPH from PPH is mediated exclusively by CYP2C9 and 2C19, with CYP2C9 playing the major role.     

Contribution of CYP2C9, CYP2A6, and CYP2B6 to valproic acid metabolism in hepatic microsomes from individuals with the CYP2C9*1/*1 genotype.

The present study investigated the role of specific human cytochrome P450 (CYP) enzymes in the in vitro metabolism of valproic acid (VPA) by a complementary approach that used individual cDNA-expressed CYP enzymes, chemical inhibitors of specific CYP enzymes, CYP-specific inhibitory monoclonal antibodies (MAbs), individual human hepatic microsomes, and correlational analysis. cDNA-expressed CYP2C9*1, CYP2A6, and CYP2B6 were the most active catalysts of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA formation. The extent of 4-OH-VPA and 5-OH-VPA formation by CYP1A1, CYP1A2, CYP1B1, CYP2C8, CYP2C19, CYP2D6, CYP2E1, CYP4A11, CYP4F2, CYP4F3A, and CYP4F3B was only 1-8% of the levels by CYP2C9*1. CYP2A6 was the most active in catalyzing VPA 3-hydroxylation, whereas CYP1A1, CYP2B6, CYP4F2, and CYP4F3B were less active. Correlational analyses of VPA metabolism with CYP enzyme-selective activities suggested a potential role for hepatic microsomal CYP2A6 and CYP2C9. Chemical inhibition experiments with coumarin (CYP2A6 inhibitor), triethylenethiophosphoramide (CYP2B6 inhibitor), and sulfaphenazole (CYP2C9 inhibitor) and immunoinhibition experiments (including combinatorial analysis) with MAb-2A6, MAb-2B6, and MAb-2C9 indicated that the CYP2C9 inhibitors reduced the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA by 75-80% in a panel of hepatic microsomes from donors with the CYP2C9*1/*1 genotype, whereas the CYP2A6 and CYP2B6 inhibitors had a small effect. Only the CYP2A6 inhibitors reduced VPA 3-hydroxylation (by approximately 50%). The extent of inhibition correlated with the catalytic capacity of these enzymes in each microsome sample. Overall, our novel findings indicate that in human hepatic microsomes, CYP2C9*1 is the predominant catalyst in the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA, whereas CYP2A6 contributes partially to 3-OH-VPA formation.