Relative roles of CYP2C19 and CYP3A4/5 in midazolam 1′-hydroxylation

1. During the characterization of recombinant CYP2C19, it was observed that this enzyme metabolized midazolam, which is generally regarded as CYP3A4/5 substrate, and we therefore decided to pursue this observation further.

2. CYP2C19 showed a Michaelis-Menten pattern for midazolam 1′-hydroxylation and was inhibited by (+)-N-3-benzylnirvanol and S-mephenytoin, which are a standard potent inhibitor and a substrate of CYP2C19, respectively.

3. The inhibitory potency by CYP3A4/5 inhibitor on the midazolam 1′-hydroxylation in human liver microsomes (HLM) was correlated with the CYP3A4/5 specific catalytic activity, but such correlation was not observed in CYP2C19 enzyme. The in vitro intrinsic clearance value for midazolam 1′-hydroxylation was not changed by the addition of (+)-N-3-benzylnirvanol in four individual HLM preparations.

4. These results indicated that although CYP2C19 is capable of catalyzing midazolam 1′-hydroxylation, CYP3A4/5 play a more important role.     

CYP2C19 genotype and S-mephenytoin 4′-hydroxylation phenotype in a Chinese Dai population

AIMS: To investigate the incidence of the CYP2C19 polymorphism in the Chinese Dai population. METHODS: One hundred and ninety-three healthy Chinese Dai volunteers were identified with respect to CYP2C19 by genotype and phenotype analyses. A polymerase chain reaction-restriction fragment length polymorphism method was performed for genotyping procedures. The 4'-hydroxymephenytoin (4'-OH-MP) and S/R-mephenytoin ( S/R-MP) excreted in the urine were determined by high-performance liquid chromatography and gas chromatography, respectively. RESULTS: Eighteen subjects were identified as poor metabolisers (PMs). The frequency of PMs in the Chinese Dai subjects was 9.3% (95% confidence interval 5.2, 13.4), which is lower than that in the Chinese Han population ( P<0.05). Chinese Dai subjects had a higher frequency of the mutant CYP2C19*2 allele (0.303) and a lower frequency of the mutant CYP2C19*3 allele (0.034). These two mutant alleles could explain all deficiencies of CYP2C19 activity in the Chinese Dai subjects. The frequency of the CYP2C19*3 allele is significantly lower than that in the Chinese Han population ( P<0.05). The mean S/R ratio was lower in the homozygous extensive metabolisers (EMs) compared with that in heterozygous EMs ( P<0.01), and the latter was lower than that in the PMs ( P<0.01). Furthermore, the mean S/R ratio in CYP2C19*3/ CYP2C19*2 heterozygous PMs was possibly lower than that in the CYP2C19*2/ CYP2C19*2 homozygous PMs ( P<0.05). CONCLUSION: The frequencies of PMs and CYP2C19*3 allele in the Chinese Dai population are significantly lower than those in the Han population. The CYP2C19 genotype analysis is largely consistent with the mephenytoin phenotype analysis. The variability of S/R ratios in EMs and PMs shows a gene-dosage effect.     

Activation of CYP3A-mediated testosterone 6β-hydroxylation by tanshinone IIA and midazolam 1-hydroxylation by cryptotanshinone in human liver microsomes

This study evaluated the in vitro activation of CYP3A-mediated midazolam 1-hydroxylation and testosterone 6β-hydroxylation by tanshinone I, tanshinone IIA, and cryptotanshinone. The abilities of tanshinones to activate CYP3A-mediated midazolam 1-hydroxylation and testosterone 6β-hydroxylation in human liver microsomes (HLMs) were tested. Substrate- and effector-dependent activation of CYP3A by tanshinones were both observed. Cryptotanshinone was shown to activate CYP3A-mediated midazolam 1-hydroxylation in a concentration-dependent manner. In contrast, tanshinone IIA and tanshinone I did not activate this hydroxylation reaction. In addition, tanshinone IIA activated CYP3A-mediated testosterone 6β-hydroxylation, whereas cryptotanshinone and tanshinone I did not. The results from our study enhance the understanding of CYP3A activation by tanshinone IIA and cryptotanshinone in HLMs. Additionally, these data allow for an accurate prediction of the magnitude and likelihood of Danshen-drug interactions.     

Activation of CYP3A-mediated testosterone 6β-hydroxylation by tanshinone IIA and midazolam 1-hydroxylation by cryptotanshinone in human liver microsomes

1. This study evaluated the in vitro activation of CYP3A-mediated midazolam 1-hydroxylation and testosterone 6β-hydroxylation by tanshinone I, tanshinone IIA, and cryptotanshinone.

2. The abilities of tanshinones to activate CYP3A-mediated midazolam 1-hydroxylation and testosterone 6β-hydroxylation in human liver microsomes (HLMs) were tested. Substrate- and effector-dependent activation of CYP3A by tanshinones were both observed.

3. Cryptotanshinone was shown to activate CYP3A-mediated midazolam 1-hydroxylation in a concentration-dependent manner. In contrast, tanshinone IIA and tanshinone I did not activate this hydroxylation reaction. In addition, tanshinone IIA activated CYP3A-mediated testosterone 6β-hydroxylation, whereas cryptotanshinone and tanshinone I did not.

4. The results from our study enhance the understanding of CYP3A activation by tanshinone IIA and cryptotanshinone in HLMs. Additionally, these data allow for an accurate prediction of the magnitude and likelihood of Danshen-drug interactions.

     

Evaluation of 4β-Hydroxycholesterol and 25-Hydroxycholesterol as Endogenous Biomarkers of CYP3A4: Study with CYP3A-Humanized Mice

Cytochrome P450 3A (CYP3A) enzymes metabolize approximately half of all drugs on the market. Since the endogenous compounds 4β-hydroxycholesterol (4β-HC) and 25-hydroxycholesterol (25-HC) are generated from cholesterol via CYP3A enzymes, we examined whether the plasma levels of 4β-HC and 25-HC reflect hepatic CYP3A4 activity by using a CYP3A-humanized mouse model, in which the function of endogenous Cyp3a was genetically replaced by human CYP3A. CYP3A-humanized mice have great advantages for evaluation of the relationship between hepatic CYP3A protein levels and plasma and hepatic levels of 4β-HC and 25-HC. Levels of CYP3A4 protein in the liver microsomes of CYP3A-humanized mice were increased by treatment with pregnenolone-16α-carbonitrile, a CYP3A inducer. Hepatic and plasma levels of 4β-HC and 25-HC normalized by cholesterol were significantly correlated with hepatic CYP3A4 protein levels. In addition, in vitro studies using human liver microsomes showed that the formation of 4β-HC was strongly inhibited by a CYP3A inhibitor, while the inhibitory effect of the CYP3A inhibition on the formation of 25-HC was weak. These results suggested that CYP3A mainly contributed to the formation of 4β-HC in human liver microsomes, whereas other factors may be involved in the formation of 25-HC. In conclusion, the in vivo studies using CYP3A-humanized mice suggest that plasma 4β-HC and 25-HC levels reflect hepatic CYP3A4 activity. Furthermore, taking the results of in vitro studies using human liver microsomes into consideration, 4β-HC is a more reliable biomarker of hepatic CYP3A activity.     

CYP3A4的配体结合机制

细胞色素P4503A4（CYP3A4）是药物研发过程中研究最多也最具挑战性的药物代谢酶。研究表明CYP3A4有一个活性极强的酶结合位点且具有多配体结合的可能性。     

Loss of C-terminal α-helix decreased SDF-1α-mediated signaling and chemotaxis without influencing CXCR4 internalization

AIM: To investigate the possibility that a novel s-helix-defective mutant of stromal cell-derived factor-1α(SDF-1α) (SDF-1/54R) acts as an antagonist of CXC chemokine receptor 4 (CXCR4). METHODS: According to the genetic sequence of natural SDF-1α, a recombinant α-helix-defective mutant of SDF-1α was designed and some biologic characteristics of this mutant were demonstrated. The migration of Jurkat cells was assessed with chemotactic assay. ERK phosphorylation was analyzed by Western blot with a specific anti-phospho-ERK1/2 antibody.Intracellular calcium influx was examined by flow cytometer with a calcium indicator dye Fluo-3AM. The CXCR4 on the cell surface was detected by flow cytometer with a PE conjoined anti-human CXCR4 antibody. RESULTS：Compared with native SDF-1α, SDF-1/54R displayed apparent decrease in chemotactic ability, ERK1/2 activation,and intracellular calcium influx in Jurkat cells. However, the binding to CXCR4 and inducing CXCR4 internalization of SDF-1/54R did not change outstandingly. Moreover, a competitive inhibitory effect of SDF-1/54R on the migration of Jurkat cells induced by native SDF-1α was confirmed. CONCLUSION: α-helix-defective mutant of SDF-1α, SDF-1/54R that remained both the N-terminus and the central β-sheet region, decreased SDF-1α-mediated signaling and chemotaxis but did not influence CXCR4 internalization, which suggested that SDF-1/54R might be developed as an anti-CHIV inhibitor with high biological potency and low side-effect.     

Effects of Intestinal CYP3A4 and P-Glycoprotein on Oral Drug Absorption—Theoretical Approach

Purpose. To evaluate the effects of gut metabolism and efflux on drug absorption by simulation studies using a pharmacokinetic model involving diffusion in epithelial cells. Methods. A pharmacokinetic model for drug absorption was constructed including metabolism by CYP3A4 inside the epithelial cells, P-gp-mediated efflux into the lumen, intracellular diffusion from the luminal side to the basal side, and subsequent permeation through the basal membrane. Partial differential equations were solved to yield an equation for the fraction absorbed from gut to the blood. Effects of inhibition of CYP3A4 and/or P-gp on the fraction absorbed were simulated for a hypothetical substrate for both CYP3A4 and P-gp. Results. The fraction absorbed after oral administration was shown to increase following inhibition of P-gp. This increase was more marked when the efflux clearance of the drug was greater than the sum of the metabolic and absorption clearances and when the intracellular diffusion constant was small. Furthermore, it was demonstrated that the fraction absorbed was synergistically elevated by simultaneous inhibition of both CYP3A4 and P-gp. Conclusions. The analysis using our present diffusion model is expected to allow the prediction of in vivo intestinal drug absorption and related drug interactions from in vitro studies using human intestinal microsomes, gut epithelial cells, CYP3A4-expressed Caco-2 cells, etc.     

Time-dependent inhibition (TDI) of CYP3A4 and CYP2C9 by noscapine potentially explains clinical noscapine–warfarin interaction

AIMS

To investigate the inhibition potential and kinetic information of noscapine to seven CYP isoforms and extrapolate in vivo noscapine-warfarin interaction magnitude from in vitro data.

METHODS

The activities of seven CYP isoforms (CYP3A4, CYP1A2, CYP2A6, CYP2E1, CYP2D6, CYP2C9, CYP2C8) in human liver microsomes were investigated following co- or preincubation with noscapine. A two-step incubation method was used to examine in vitro time-dependent inhibition (TDI) of noscapine. Reversible and TDI prediction equations were employed to extrapolate in vivo noscapine–warfarin interaction magnitude from in vitro data.

RESULTS

Among seven CYP isoforms tested, the activities of CYP3A4 and CYP2C9 were strongly inhibited with an IC₅₀ of 10.8 ± 2.5 µm and 13.3 ± 1.2 µm. Kinetic analysis showed that inhibition of CYP2C9 by noscapine was best fit to a noncompetitive type with K_i value of 8.8 µm, while inhibition of CYP3A4 by noscapine was best fit to a competitive manner with K_i value of 5.2 µm. Noscapine also exhibited TDI to CYP3A4 and CYP2C9. The inactivation parameters (K_I and k_inact) were calculated to be 9.3 µm and 0.06 min⁻¹ for CYP3A4 and 8.9 µm and 0.014 min⁻¹ for CYP2C9, respectively. The AUC of (S)-warfarin and (R)-warfarin was predicted to increase 1.5% and 1.1% using C_max or 0.5% and 0.4% using unbound C_max with reversible inhibition prediction equation, while the AUC of (S)-warfarin and (R)-warfarin was estimated to increase by 110.9% and 48.9% using C_max or 41.8% and 32.7% using unbound C_max with TDI prediction equation.

CONCLUSIONS

TDI of CYP3A4 and CYP2C9 by noscapine potentially explains clinical noscapine–warfarin interaction.     

α-Adrenoceptor activity of arylalkylimidazoles is improved byα-methylation and impaired byα-hydroxylation

Summary To investigate the effects of hydroxyl and methyl substitution of the alkyl bridge bond on the-adrenoceptor activity of arylalkylimidazole derivatives, the cardiovascular effects of the molecules were studied in anaesthetized and pithed rats. The compounds studied were 4(5)-substituted imidazole derivatives with a methano, ethano or etheno bridge between the imidazole and the 2-, 2,3- or 2,6-methyl substituted phenyl rings. The hypotensive and bradycardic activities of the molecules in the anaesthetized rat were always reduced by-hydroxylation and usually augmented by-methylation of the bridge between the imidazole and phenyl rings. Hydroxylation was associated with a consistent, marked decrease in vasopressor and sympatho-inhibitory activity in the cardiovascular system of the pithed rat, but a methyl moiety as a bulky substituent in the-position of the alkyl bridge did not decrease but even caused an increase in-adrenoceptor activity in this test system. The detrimental effect of-hydroxylation of the compounds at ₁- and ₂-adrenoceptors supports the notion that the interaction of the imidazoles at-adrenoceptor is different from that of the classical, noradrenaline-like phenethylamines. The results also suggest that the alkyl bridge between the phenyl and imidazole rings of the imidazoles may contribute directly to the binding process.     

CYP2C9、CYP2C19、CYP3A4基因多态性对磺脲类降糖药代谢的影响

磺脲类口服降糖药在人体内主要经过肝脏代谢。肝脏中的细胞色素氧化酶P450是一种重要的药物代谢酶系统,在人群中存在基因多态性,导致药物疗效和不良反应在个体间存在着较大的差异。本文将对CYP450中的几种重要的代谢酶亚型CYP2C9、CYP2C19、CYP3A4的基本结构、基因多态性、种族差异及其对磺脲类降糖药代谢的影响作一综述。     

丹参注射液对家兔体内CYP1A2、CYP2D6和CYP3A4活性的影响

目的：用Cocktail探针药物法研究丹参注射液对家兔细胞色素P450亚型酶活性的影响。方法：将家兔随机分成四组,耳缘静脉注射给予丹参注射液,分低、中、高剂量三个实验组,空白对照组给予生理盐水。用HPLC法测定各组Cock-tail探针药物（咖啡因、美托洛尔和氨苯砜）的血药浓度,比较药时曲线及药代动力学参数的变化,评价各组细胞色素P450亚型酶的活性。结果：丹参注射液低、中、高剂量组CYP2D6的活性与对照组相比降低,具有统计学意义（P〈0.05或P〈0.01）;CYP1A2和CYP3A4的活性与对照组相比差异均无统计学意义。结论：丹参注射液对家兔CYP2D6有抑制作用,对CYP1A2和CYP3A4的影响不显著。提示临床应用丹参注射液时,应关注其对CYP2D6的抑制作用,避免联合用药引发CYP2D6介导的药物相互作用,促进临床安全合理用药。     

Differential accumulation of HCBz and PeCBz in porcine ovarian follicles and their opposing actions on steroid secretion and CYP11, CYP17, 17β-HSD and CYP19 protein expression. A tissue culture approach

In the current study, we determined in vitro accumulation of hexachlorobenzene (HCBz) and pentachlorobenzene (PeCBz) in porcine ovarian follicles, the effect on steroidogenesis and the expression of enzymes responsible for steroid synthesis. Sixty percent of the HCBz and almost 100% of the PeCBz that was added to the culture medium accumulated in ovarian tissue, and only 1% of each was found in the medium. An inhibitory HCBz effect and stimulatory PeCBz effect on testosterone and estradiol secretion were noted. Immunoblot analyses showed an inhibitory effect of HCBz on CYP17, 17β-HSD and CYP19, a stimulatory effect of PeCBz on CYP17 and CYP19 and no effect on 17β-HSD protein expression. In conclusion, the greater exposure to an estrogenic action of PeCBz than anti-estrogenic HCBz would be a consequence of the preferential accumulation of PeCBz in the ovarian follicles. As one of the mechanisms of action, we propose modulation of steroidogenic enzymes expression.     

细胞色素P450(CYP450)4A11与CYP4F2基因多态性与心血管疾病的相关性

细胞色素P450(CYP)ω-羟化酶主要包括CYP4A11和CYP4F2,代谢花生四烯酸(AA)生成20-羟化二十碳四烯酸(20-HETE),20-HETE具有收缩血管和促进尿钠排泄等作用。CYP4A11和4F2基因具有高度多态性,这种多态性导致酶活性降低,并可能引起某些心血管疾病。     

中国汉族和维吾尔族药物代谢酶CYP3A4、CYP2C9、CYP2C19、CYP2D6基因多态性分析

目的对中国汉族、维吾尔族健康人群CYP3A4、CYP2C9、CYP2C19、CYP2D6进行基因多态性分析,并对汉族和维吾尔族人群等位基因频率和基因型频率进行比较。方法聚合酶链反应一限制性片段长度多态性(PCR-RFLP)法对CYP3A4、CYP2C9、CYP2C19、CYP2D6进行分型。结果汉族、维吾尔族健康人群CYP3A4*5等位基因频率为0,CYP3A4*18等位基因频率分别为0.183 8、0.140 2;CYP2C9*2等位基因频率分别为0.011 0、0.095 8,CYP2C9*13等位基因频率分别为0、0.002 3;CYP2C19*2等位基因频率分别为0.386 0、0.324 8,CYP2C19*3等位基因频率分别为0.051 5、0.021 0;CYP2D6*10等位基因频率分别为0.573 5、0.224 3。结论本研究在汉族、维吾尔族健康人群中未发现CYP3A4*5等位基因。汉族、维吾尔族健康人群CYP3A4*18、CYP2C9*13、CYP2C19*2、CYP2C19*3等位基因频率差异均无统计学意义。维吾尔族健康人群CYP2C9*2等位基因频率远高于汉族(P<0.01);CYP2D6*10等位基因频率远低于汉族(P<0.01),存在民族差异。     

川阿格雷对大鼠肝微粒体CYP1A2、CYP3A4和CYP2E1活性的影响

川阿格雷是基于传统中药机制,运用现代技术合成的新一代抗血小板药物,适用于预防和治疗因血小板高聚集态引起的心、脑及其他动脉的循环障碍疾病,具有很好的开发价值,而药物的代谢特性是药物研发的重要内容。有研究表明,CYP450酶可被药物等外源物诱导和抑制,导致其含量和活性的改变,从而直接影响药物在体内的血药浓度、动力     

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。