丹参-红花组分配伍对雌、雄性心肌缺血大鼠肝药酶亚型影响的比较研究

目的 比较丹参-红花组分配伍对不同性别大鼠肝药酶亚型CYP1A2、CYP2E1和CYP3A4的影响。方法 取SD大鼠按不同性别随机分为丹参-红花高、低剂量（生药4、2 g/kg）组及模型组和对照组,ip盐酸异丙肾上腺素制备心肌缺血模型,丹参-红花高、低剂量组给予相应提取物,模型、对照组给予等体积的蒸馏水,连续ig给药21 d后,取肝组织剪碎制备微粒体,加入非那西丁、氯唑沙宗、睾酮分别作为CYP1A2、CYP2E1和CYP3A4的特异性探针底物,体外温孵育,建立高效液相色谱（HPLC）法检测探针底物浓度,测定CYP1A2、CYP2E1和CYP3A4活性。结果 ①雄性大鼠实验结果显示,与对照组比较,模型组显著抑制CYP2E1和CYP3A4的活性（P<0.05、0.01）;与模型组比较,丹参-红花高剂量组显著抑制CYP2E1和CYP3A4的活性（P<0.05、0.01）;低剂量组显著抑制CYP2E1的活性（P<0.05）,诱导CYP3A4（P<0.01）。②雌性大鼠实验结果显示,与对照组比较,模型组显著抑制CYP2E1和CYP3A4的活性（P<0.05）,对CYP1A2的影响不显著;与模型组比较,丹参-红花高剂量组显著抑制CYP1A2、CYP2E1和CYP3A4的活性（P<0.05、0.01）;丹参-红花低剂量组抑制CYP2E1的活性（P<0.05）,对CYP1A2和CYP3A4的影响不显著。③实验结果对比显示,丹参-红花对雄性、雌性大鼠肝药酶亚型的影响相同点是均抑制了CYP2E1和CYP3A4活性,尤其对CYP2E1的抑制作用比较明显;不同点是丹参-红花低剂量诱导了雄性大鼠CYP3A4活性,但对雌性大鼠CYP3A4活性无明显影响。结论 丹参-红花组分配伍对不同性别大鼠部分肝药酶亚型活性的影响存在一定的差异。     

鸡尾酒法评价石蒜对大鼠细胞色素P450酶活性的影响

目的 运用鸡尾酒法评价石蒜对细胞色素P450酶活性的影响。方法 将大鼠随机分为对照组和石蒜低、高剂量组。对照组给予生理盐水，石蒜低、高剂量组大鼠灌胃给药0.5，1.0 g·kg^-1石蒜，连续给药15 d。然后第16天给予探针药物，UPLC-MS/MS检测探针药浓度。结果 与对照组对比，石蒜低剂量组和高剂量组的安非他酮AUC_（0-t）、C_max都显著升高（P<0.05），CL_z/F显著降低（P<0.05）。与对照组对比，石蒜高剂量组的美托洛尔、咪达唑仑和非那西丁AUC_（0-t）显著降低（P<0.05）、CL_z/F显著升高（P<0.05），而低剂量组与对照组比较差异无统计学意义。与对照组对比，石蒜组的甲苯磺丁脲AUC_（0-t）、CL_z/F差异无统计学意义，C_max显著降低（P<0.05）。结论 石蒜能抑制大鼠CYP2B1酶活性，诱导大鼠CYP2D1、CYP3A2和CYP1A2酶活性，稍有诱导大鼠CYP2C11酶活性的作用。     

雷公藤多苷片对大鼠药物代谢酶CYP2E1和CYP3A4活性的影响

目的:研究雷公藤多苷片对大鼠CYP450亚型酶CYP2E1和CYP3A4活性的影响方法:16只Wistar大鼠随机分成2组,每组8只,实验组给予雷公藤多苷片(1 mg·kg^-1·d^-1,ig,qd),对照组给予同体积的纤维素钠空白溶剂,连续给药10d后同时静脉给予探针药物氯唑沙宗(5 mg·kg^-1)和咪达唑仑(2 mg·kg^-1)。HPLC法测定两个探针药物的血药浓度,计算其药物动力学参数,评价CYP450亚型酶CYP2E1和CYP3A4的活性。结果:与对照组比较,实验组的氯唑沙宗主要药物动力学参数无明显变化（P>0.05）,而咪达唑仑的CL和AUC较对照组差异有统计学意义（P<0.05）。结论:雷公藤多苷片对大鼠的CYP2E1酶活性无明显影响,对CYP3A4酶活性有一定的抑制作用。     

中药止咳橘红颗粒对CYP3A4和CYP1A2抑制作用的研究

目的：在人体内研究止咳橘红对CYP3A4和CYP1A2的抑制作用，以预测止咳橘红与常用临床药物的相互作用。方法：咪哒唑仑和咖啡因分别作为CYP3A4和A2的探针药物，采取交叉设计，10名受试者在服用3d止咳橘红的前后均服用7.5mg咪哒唑仑和100mg咖啡因，服药后采血测定两者及代谢产物的代谢动力学参数，探讨针药物及代谢物的浓度用HPLC-MS法测定，Cmax,tmax从药时曲线中直接读出，AUC用梯形法计算，Ke用3P87程序进行拟合计算，分析服药前后CYP3A4和CYP1A2被抑制的情况，结果 服用止咳橘红后，咪哒唑仑的代谢受到了轻微的抑制，它的血药浓度，达峰时间和药时曲线下面积都有了升高趋势，但无显著差异。而咖啡因的代谢未受到影响。结论 止咳橘红对CYP3A4的活性有较弱的抑制作用，能够导致CYP3A4底物咪哒唑仑代谢的轻微抑制，而对CYP1A2的活性没有影响。止咳橘红长期使用或超过治疗剂量使用时是否会对CYP3A4产生显著性影响。尚需进一步的研究证明。     

速效救心丸对大鼠体内CYP450酶活性影响的研究

目的采用Cocktail探针药物法研究速效救心丸对大鼠体内药物代谢酶CYP2E1和CYP3A4的影响,为临床用药提供参考。方法 Wistar大鼠,随机分为,①实验组:灌胃给予速效救心丸(给药体积0.5mL/100g,给药剂量64.8mg·kg-1);②对照组:灌胃给予等体积的纤维素钠混悬液。采用高效液相色谱法(HPLC)测定探针底物氯唑沙宗和咪达唑仑的血药浓度,计算其药代动力学参数,考察大鼠体内CYP2E1及CYP3A4酶的活性。结果实验组氯唑沙宗的代谢无显著性差异,咪达唑仑的代谢明显减慢。结论速效救心丸对大鼠的CYP2E1酶活性无明显影响,对CYP3A4酶活性有抑制作用。     

酒精性肝损伤大鼠细胞色素P450 CYP2E1和细胞色素P450 CYP3A的代谢活性

目的观察酒精性肝损伤对大鼠细胞色素P450CYP3A(CYP3A)和细胞色素P450CYP2E1(CYP2E1)代谢活性的影响。方法采用ig给予白酒制备大鼠酒精性肝损伤模型,检测血清中谷丙转氨酶(GPT)和谷草转氨酶(GOT)活性,采用HE染色法光镜下观测酒精对肝脏损伤程度。大鼠ip给予CYP3A探针药物咪达唑仑10mg·kg-1或ig给予CYP2E1探针药物氯唑沙宗50mg·kg-1后,采用高效液相色谱法测定不同时间点大鼠血浆中咪达唑仑和氯唑沙宗的血药浓度,并应用3P87软件计算其药代动力学参数,以考察CYP2E1和CYP3A的代谢活性的变化。大鼠ig给予氯唑沙宗80mg·kg-1后,热板方法测定大鼠添足次数和添足反射潜伏期。结果酒精性肝损伤可致大鼠肝小叶结构不清,肝索排列紊乱,肝细胞体积增大,呈弥漫性中度水变性,肝窦受压,大部分肝细胞胞浆内见大小不等的脂肪空泡;与正常对照组相比,酒精性肝损伤组大鼠GPT和GOT活性分别增加了16.0%和20.0%(P<0.05,P<0.01)。酒精性肝损伤致大鼠CYP2E1对探针药物氯唑沙宗的代谢活性增强,AUC,t1/2和cmax分别降低了38.0%,30.5%和35.0%(P<0.05);酒精肝损伤组大鼠氯唑沙宗镇痛效果明显降低;酒精性肝损伤致大鼠CYP3A对探针药物咪达唑仑的代谢活性增强,AUC,t1/2和cmax分别降低了122.6%,54.9%和56.9%(P<0.01,P<0.05)。结论酒精性肝损伤可使大鼠CYP2E1和CYP3A代谢活性增强。     

糖尿病模型大鼠肝脏CYP2E1酶活性的变化

采用四氧嘧啶诱发糖尿病大鼠模型,测定肝苯胺羟化酶及其他药酶活性,同时用氯唑沙宗探针间接评价CYP2E1的活性。结果表明,糖尿病大鼠苯胺羟化酶活性增加80%,伴有其他药酶活性增加。大鼠单次po氯唑沙宗50mg·kg^-1,糖尿病组氯唑沙宗的C_max和AUC分别减少37%和34%,6 羟氯唑沙宗的Tpeak缩短,羟化指数(OH-CZX与CZX的AUC比或浓度比)升高表明糖尿病大鼠可诱导CYP2E1活性。提示糖尿病患者服用经CYP2E1酶代谢的药物应慎重。     

Cocktail探针药物法评价生、醋莪术对CYP450酶亚型的影响

目的用Cocktail探针药物法,研究生、醋莪术对大鼠CYP1A2、CYP3A4和CYP2E1活性的影响并进行比较,探讨其通过炮制增强或改变作用的趋向性。方法将SD大鼠随机分组,生、醋莪术组分别给予生、醋莪术提取液(9 g.kg-1),以生理盐水为空白对照,在一定时间点采集血样,用HPLC检测探针药物在大鼠体内的代谢情况,以评价生、醋莪术对CYP450酶的影响。结果生莪术的茶碱、氨苯酚和氯唑沙宗的代谢情况与空白组相比差异无显著性;但醋莪术与空白组相比,茶碱和氯唑沙宗的T12、Tmax和AUC明显增加,CL/F明显降低;氨苯酚的T12变化不明显,AUC明显降低,CL/F明显增加;氯唑沙宗的T12、Tmax、AUC明显增加,CL/F明显降低。结论单次给予大鼠生、醋莪术后,生莪术对CYP450酶的作用不明显,醋莪术对CYP1A2和CYP2E1具有抑制作用,对CYP3A4具有诱导作用。     

复方银杏叶胶囊对肝损伤大鼠CYP2E1、CYP3A4的影响

目的研究复方银杏叶胶囊(CGB)对酒精性肝损伤大鼠CYP2E1、CYP3A4活性的影响。方法正常组和酒精性肝损伤模型组均以CGB[(250 mg/(kg.d)]灌胃,分别在灌胃CGB前及灌胃1周后,灌胃探针药氯唑沙宗(50 mg/kg)及氨苯砜(20 mg/kg),于探针药灌后24 h内不同时间点采血,测定各探针药血药浓度。结果灌胃CGB前,模型组氯唑沙宗和氨苯砜的AUC0-24、Cmax均显著低于正常组(P<0.05或P<0.01)。灌胃CGB后,模型组氯唑沙宗和氨苯砜的AUC0-24、Cmax均较灌胃前显著升高(P<0.05或P<0.01);且氯唑沙宗的t1/2灌胃CGB后明显高于灌胃前(P<0.05)。结论 CGB能够明显抑制酒精性肝损伤大鼠CYP2E1、CYP3A4酶活性。     

木犀草素对大鼠CYP450酶活力和mRNA表达的影响

目的 研究木犀草素对大鼠细胞色素P450（CYP450）酶活力和mRNA表达的影响。方法 SD大鼠随机分为实验组和对照组,连续灌胃木犀草素或水14 d后,同时给予探针药非那西丁、安非他酮、氯唑沙宗和咪达唑仑,并用LC-MS测定给药后不同时间点的血药浓度,计算药动学参数并评估CYP1A2、CYP2B1、CYP2E1和CYP3A1酶活力变化。此外,实时荧光定量PCR （RT-PCR）测定CYP450的mRNA表达水平。结果 实验组大鼠非那西丁、安非他酮、咪达唑仑的药动学参数与对照组相比具有显著性差异,推测木犀草素能诱导大鼠CYP1A2、CYP2B1和CYP3A1活力,而氯唑沙宗的药动学参数2组间没有显著性差异。RT-PCR实验发现,木犀草素能显著上调大鼠CYP1A2、CYP2B1和CYP3A1的mRNA水平,对CYP2E1没有影响。结论 木犀草素会上调CYP1A2、CYP2B1和CYP3A1酶活力及mRNA表达水平。     

无梗五加果实提取物对大鼠CYP450活性的影响

目的研究无梗五加果实提取物对大鼠CYP450亚型CYP1A2、CYP2C19、CYP2E1、CYP3A4活性的影响。方法以咖啡因、奥美拉唑、氯唑沙宗和咪达唑仑作为CYP1A2、CYP2C19、CYP2E1和CYP3A4特异性探针药物,配制cocktail混合探针溶液,建立同时测定4种探针药物血药浓度的高效液相色谱法。将SD大鼠随机分为无梗五加果实70%乙醇提取物组、水提取物组和对照组,每天分别ig无梗五加果实70%乙醇提取物溶液、水提取物溶液和溶剂10 mL/kg,连续给药1周,于第8天ip混合探针药物10 mL/kg,分别在给药0、0.083、0.25、0.417、0.583、0.833、1、2、3、5、8、11、12 h于大鼠眼眶静脉丛采血,采用HPLC法对血浆样品进行分析,对给药组和对照组探针药物的药动学参数生物半衰期(t_(1/2))进行比较。结果与对照组比较,无梗五加果实水提取物组和70%乙醇提取物组咖啡因、咪达唑仑的t_(1/2)显著降低,奥美拉唑、氯唑沙宗的t_(1/2)显著增加,差异均具有统计学意义(P0.05、0.01)。结论无梗五加果实70%乙醇提取物和水提取物对大鼠的CYP2C19、CYP2E1有抑制作用,对大鼠的CYP1A2、CYP3A4有诱导作用。     

A clinical study to assess CYP1A2 and CYP3A4 induction by AZD7325, a selective GABA(A) receptor modulator - an in vitro and in vivo comparison

AIM(S)

To investigate the potential of AZD7325 to induce CYP1A2 and CYP3A4 enzyme activities.

METHODS

Induction of CYP1A2 and CYP3A4 by AZD7325 was first evaluated using cultured human hepatocytes. The effect of multiple doses of 10 mg AZD7325 on the pharmacokinetics of midazolam and caffeine was then examined in healthy subjects.

RESULTS

The highest CYP1A2 and CYP3A4 induction responses were observed in human hepatocytes treated with 1 or 10 µm of AZD7325, in the range of 17.9%–54.9% and 76.9%–85.7% of the positive control responses, respectively. The results triggered the further clinical evaluation of AZD7325 induction potential. AZD7325 reached a plasma C_max of 0.2 µm after 10 mg daily dosing to steady-state. AZD7325 decreased midazolam geometric mean AUC by 19% (0.81-fold, 90% CI 0.77, 0.87), but had no effect on midazolam C_max (90% CI 0.82, 0.97). The mean CL/F of midazolam increased from 62 l h⁻¹ (midazolam alone) to 76 l h⁻¹ when co-administered with AZD7325. The AUC and C_max of caffeine were not changed after co-administration of AZD7325, with geometric mean ratios (90% CI) of 1.17 (1.12, 1.23) and 0.99 (0.95, 1.03), respectively.

CONCLUSIONS

While AZD7325 appeared to be a potent CYP3A4 inducer and a moderate CYP1A2 inducer from in vitro studies, the expected efficacious dose of AZD7325 had no effect on CYP1A2 activity and only a weak inducing effect on CYP3A4 activity. This comparison of in vitro and in vivo results demonstrates the critical role that clinical exposure plays in evaluating the CYP induction risk of a drug candidate.     

海藻-昆布药对对大鼠肝微粒体代谢酶的影响及肝毒性研究

目的 发现不同剂量海藻-昆布药对提取物对大鼠肝微粒体代谢酶的诱导或抑制作用, 预测服用海藻-昆布药对时可能出现的药物-药物相互作用及肝脏毒性。方法 雌雄各半SD大鼠18只, 被随机分为海藻-昆布药对低、高剂量组和对照组, 低、高剂量组大鼠分别ig给予海藻-昆布药对提取物10.8、86.4 g/(kg·d), 连续经口给药15 d后麻醉处死, 取肝组织制备肝微粒体及HE染色石蜡切片。通过肝微粒体体外孵育方法测定3种肝脏CYP450同工酶特异性底物非那西丁(CYP1A2)、氯唑沙宗(CYP2E1)及咪达唑仑(CYP3A4)的降解和代谢产物生成量来评价肝药酶的诱导或抑制作用, 并以光镜下的组织病理切片检查来考察其肝毒性。结果 低剂量组大鼠无显著诱导或抑制3种CYP450代谢酶亚型1A2、2E1和3A4现象, 肝组织出现了肝窦扩张、轻度水肿等适应性改变, 高剂量组能显著诱导CYP3A4亚型, 但也不能显著的诱导或抑制肝微粒体代谢酶CYP1A2、CYP2E1亚型, 肝组织出现了脂肪变、点状坏死等可逆性损伤。结论 海藻-昆布药对具有诱导肝微粒体代谢酶CYP3A4的作用和轻微的肝细胞毒性, 高剂量经口给药能引起有临床意义的CYP450酶的诱导现象和肝脏损伤并可能导致不期望的药物-药物相互作用。     

Cocktail探针药物法评价灯盏花素对大鼠CYP450体内代谢活性的影响

目的用Cocktail探针药物法研究灯盏花素对大鼠CYP1A2、CYP2C9、CYP2E1和CYP2D6体内代谢活性的影响。方法将大鼠随机分为3组,空白对照组、低剂量组和高剂量组。低剂量组和高剂量组分别尾静脉给予灯盏花素粉针剂1.8,5.4 mg.kg-1.d-1,空白对照组给予生理盐水,共14 d。各组分别于第15 d注射Cocktail探针溶液,用HPLC检测各探针药物的血药浓度,用DAS2.0计算药代动力学参数,评价相应的CYP450亚型的体内代谢活性。结果高剂量组美托洛尔的AUC和t1/2显著高于空白对照组,CL显著低于空白对照组(P<0.05)。低剂量组美托洛尔虽然也有相同趋势,但差异无统计学意义(P>0.05)。各组咖啡因、氯唑沙宗和甲苯磺丁脲的主要药代动力学参数均无显著差异(P>0.05)。结论高剂量灯盏花素粉针剂可明显抑制大鼠CYP2D6的体内代谢活性,而对CYP1A2、CYP2E1和CYP2C9无显著性影响。     

The effect of organic solvents on enzyme kinetic parameters of human CYP3A4 and CYP1A2 in vitro

Abstract

Enzyme kinetic parameters provide essential quantitative information about characterization of individual steps in drug metabolism. Such enzymes are located in a (partially) aqueous environment. For in vitro measurements potential lipophilic substrates regularly require organic solvents to achieve concentrations sufficient for access of the drug to the binding site of the enzyme. However, solvents may interact with the enzymes. In this study, we investigated the effects of methanol, ethanol, acetonitrile and dimethyl sulfoxide (1% to 4%) on the assessment of k_m, V_max and Cl_int for the metabolism of midazolam via CYP3A4 to 1-hydroxymidazolam and the metabolism of caffeine to paraxanthine via CYP1A2 using expressed enzymes in vitro. The presence of acetonitrile proved the highest apparent V_max value for paraxanthine formation but the lowest values for 1-hydroxymidazolam formation. The k_m value for midazolam showed no systematic effects of organic solvents, while for caffeine k_m was up to 8-fold lower for solvent free samples compared to solvent containing samples. The present example suggests that effects of solvents may considerably influence enzyme kinetic parameters beyond a mere change in apparent activity. These effects illustrate a difference for individual enzyme--substrate pairs, solvents, and solvent concentrations. What remains is the determination to which extent these effects compromise in vitro–in vivo extrapolations, and which solvents are most appropriate.     

Pharmacogenomics of CYP2A6, CYP2B6, CYP2C19, CYP2D6, CYP3A4, CYP3A5 and MDR1 in Vietnam

Aim  The aim of this study was to obtain pharmacogenetic data in a Vietnamese population on genes coding for proteins involved in the elimination of drugs currently used for the treatment of malaria and human immunodeficiency virus/acquired immunodeficiency syndrome. Method  The main polymorphisms on the cytochrome P450 (CYP) genes, CYP2A6, CYP2B6, CYP2C19, CYP2D6, CYP3A4 and CYP3A5, and the multi-drug resistance 1 gene (MDR1) were genotyped in 78 healthy Vietnamese subjects. Pharmacokinetic metrics were available for CYP2A6 (coumarin), CYP2C19 (mephenytoin), CYP2D6 (metoprolol) and CYP3As (midazolam), allowing correlations with the determined genotype. Results  In the CYP2 family, we detected alleles CYP2A6*4 (12%) and *5 (15%); CYP2B6*4 (8%), *6 (27%); CYP2C19*2 (31%) and *3 (6%); CYP2D6*4, *5, *10 (1, 8 and 44%, respectively). In the CYP3A family, CYP3A4*1B was detected at a low frequency (2%), whereas CYP3A5 *3 was detected at a frequency of 67%. The MDR1 3435T allele was present with a prevalence of 40%. Allele proportions in our cohort were compared with those reported for other Asian populations. CYP2C19 genotypes were associated to the S-4′-OH-mephenytoin/S-mephenytoin ratio quantified in plasma 4 h after intake of 100 mg mephenytoin. While CYP2D6 genotypes were partially reflected by the α-OH-metroprolol/metoprolol ratio in plasma 4 h after dosing, no correlation existed between midazolam plasma concentrations 4 h post-dose and CYP3A genotypes. Conclusions  The Vietnamese subjects of our study cohort presented allele prevalences in drug-metabolising enzymes that were generally comparable with those reported in other Asian populations. Deviations were found for CYP2A6*4 compared to a Chinese population (12 vs. 5%, respectively; P = 0.023), CYP2A6*5 compared with a Korean population (15 vs. <1%, respectively; P < 0.0001), a Malaysian population (1%; P < 0.0001) and a Chinese population (1%; P < 0.0001); CYP2B6*6 compared with a Korean population (27 vs. 12%; P = 0.002) and a Japanese population (16%; P = 0.021). Pharmacokinetic metrics versus genotype analysis reinforces the view that the predictive value of certain globally common variants (e.g. CYP2D6 single nucleotide polymorphisms) should be evaluated in a population-specific manner.