CYP3A4酶介导的人类药物代谢性别差异

药代动力学的性别差异是临床上药物疗效的重要影响因素。引起药代动力学性别差异的比较重要的分子因素包括药物代谢酶和药物转运体等。CYP3A4是人体含量最丰富的酶,具有广泛的代谢底物。大部分体内、外研究表明CYP3A4底物的代谢具有明显的性别差异,女性体内的代谢快于男性。该文系统综述人肝脏CYP3A4酶介导的药物代谢性别差异的研究进展。     

In vitro inhibitory effects of Friedelin on human liver cytochrome P450 enzymes

Context: Friedelin is a triterpenoid with several biological activities. However, the affects of Friedelin on the activity of human liver cytochrome P450 (CYP) enzymes remains unclear.

Objective: This study investigates the inhibitory effects of Friedelin on the major human liver CYP isoforms (CYP3A4, 1A2, 2A6, 2E1, 2D6, 2C9, 2C19 and 2C8).

Materials and methods: First, the inhibitory effects of Friedelin (100?μM) on the eight human liver CYP isoforms were investigated in vitro using human liver microsomes (HLMs), and then enzyme inhibition, kinetic studies, and time-dependent inhibition studies were conducted to investigate the IC₅₀, K_i and K_inact/K_I values of Friedelin.

Results: The results indicate that Friedelin inhibited the activity of CYP3A4 and 2E1, with the IC₅₀ values of 10.79 and 22.54?μM, respectively, but other CYP isoforms were not affected. Enzyme kinetic studies showed that Friedelin is not only a noncompetitive inhibitor of CYP3A4, but also a competitive inhibitor of CYP2E1, with K_i values of 6.16 and 18.02?μM, respectively. In addition, Friedelin is a time-dependent inhibitor of CYP3A4 with K_inact/K_i value of 4.84?nM/min.

Discussion and conclusion: The in vitro studies of Friedelin with CYP isoforms suggested that Friedelin has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP3A4 and 2E1. Further clinical studies are needed to evaluate the significance of this interaction.     

In vitro inhibitory effects of sophocarpine on human liver cytochrome P450 enzymes

Abstract

1.?Sophocarpine is a biologically active component isolated from the foxtail-like sophora herb and seed that is often orally administered for the treatment of cancer and chronic bronchial asthma. However, whether sophocarpine affects the activity of human liver cytochrome P450 (CYP) enzymes remains unclear.

2.?In this study, the inhibitory effects of sophocarpine on the eight human liver CYP isoforms (CYP1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19, and 2C8) were investigated in vitro using human liver microsomes (HLMs).

3.?The results indicate that sophocarpine could inhibit the activity of CYP3A4 and 2C9, with the IC₅₀ values of 12.22 and 15.96?μM, respectively, but that other CYP isoforms were not affected. Enzyme kinetic studies showed that sophocarpine is not only a noncompetitive inhibitor of CYP3A4 but also a competitive inhibitor of CYP2C9, with K_i values of 6.74 and 9.19?μM, respectively. Also, sophocarpine is a time-dependent inhibitor of CYP3A4 with K_inact/K_I value of 0.082/21.54?μM^?1?min^?1.

4.?The in vitro studies of sophocarpine with CYP isoforms suggested that sophocarpine has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP3A4 and 2C9. Further clinical studies are needed to evaluate the significance of this interaction.     

己烯雌酚对人肝微粒体内CYP3A4和CYP2C9酶的抑制作用

目的:体外实验考察己烯雌酚(DES)对细胞色素P450 3A4(CYP3A4)和细胞色素P450 2C9(CYP2C9)活性的抑制作用,以评佑DES通过抑制这两个重要的细胞色素P450(CYP)亚型而引发药物-药物相互作用的可能性.方法:混合人肝微粒体与不同浓度的DES(或阳性抑制剂),CYP3A4或CYP2C9的探针...     

Involvement of human cytochrome P450 3A4 in reduced haloperidol oxidation

Objective: The present study was conducted to identify in vitro the cytochrome P450(CYP) isoform involved in the metabolic conversion of reduced haloperidol to haloperidol using microsomes derived from human AHH-1 TK +/− cells expressing human cytochrome P450s. The inhibitory and/or stimulatory effects of reduced haloperidol or haloperidol on CYP2D6-catalyzed carteolol 8-hydroxylase activity were also investigated. Results: The CYP isoform involved in the oxidation of reduced haloperidol to haloperidol was CYP3A4. CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 2E1 were not involved in the oxidation. The k_M value for the CYP3A4 expressed in the cells was 69.7 μmol · l⁻¹, and the V_max was 4.87 pmol · min⁻¹ · pmol⁻¹ P450. Troleandomycin, a relatively selective probe for CYP3A enzymes, inhibited the CYP3A4-mediated oxidation of reduced haloperidol in a dose-dependent manner. Quinidine and sparteine competitively inhibited the oxidative reaction with a k_i value of 24.9 and 1390 μmol · l⁻¹, respectively. Carteolol 8-hydroxylase activity, which is a selective reaction probe for CYP2D6 activity, was inhibited by reduced haloperidol with a k_i value of 4.3 μmol · l⁻¹. Haloperidol stimulated the CYP2D6-mediated carteolol 8-hydroxylase activity with an optimum concentration of 1 μmol · l⁻¹, whereas higher concentrations of the compound (>10 μmol · l⁻¹) inhibited the hydroxylase activity. Conclusion: It was concluded that CYP3A4, not CYP2D6, is the principal isoform of cytochrome P450 involved in the metabolic conversion of reduced haloperidol to haloperidol. It was further found that reduced haloperidol is a substrate of CYP3A4 and an inhibitor of CYP2D6, and that haloperidol has both stimulatory and inhibitory effects on CYP2D6 activity. Received: 10 April 1997 / Accepted in revised form: 16 December 1997     

Terfenadine t-butyl hydroxylation catalyzed by human and marmoset cytochrome P450 3A and 4F enzymes in livers and small intestines

1.?Roles of human cytochrome P450 (P450) 3A4 in oxidation of an antihistaminic drug terfenadine have been previously investigated in association with terfenadine–ketoconazole interaction. Several antihistamine drugs have been recently identified as substrates for multiple P450 enzymes. In this study, overall roles of P450 3A4, 2J2, and 4F12 enzymes in terfenadine t-butyl hydroxylation were investigated in small intestines and livers from humans, marmosets, and/or cynomolgus monkeys.

2.?Human liver microsomes and liver and small intestine microsomes from marmosets and cynomolgus monkeys effectively mediated terfenadine t-butyl hydroxylation. Ketoconazole and N-hydroxy-N′-(4-butyl-2-methylphenyl)-formamidine (a P450 4A/F inhibitor) almost completely and moderately inhibited these activities, respectively, in human liver microsomes; however, these chemicals did not show substantially suppression in marmoset liver. Anti-human P450 3A and 4F antibodies showed the roughly supportive inhibitory effects.

3.?Recombinant P450 3A4/90 and 4F12 showed high terfenadine t-butyl hydroxylation activities with substrate inhibition constants of 84–144?μM (under 26–76?μM of K_m values), in similar manners to liver and intestine microsomes.

4.?These results suggest that human and marmoset P450 3A4/90 and 4F12 in livers or small intestines played important roles in terfenadine t-butyl hydroxylation. Marmosets could be a model for humans during first pass extraction of terfenadine and related substrates.     

In vitro inhibitory effects of dihydromyricetin on human liver cytochrome P450 enzymes

Context: Dihydromyricetin (DHM) is the most abundant and active flavonoid component isolated from Ampelopsis grossedentata (Hand-Mazz) W.T. Wang (Vitaceae) and it possesses numerous pharmacological activities. However, whether DHM affects the activity of human liver cytochrome P450 (CYP) enzymes remains unclear.

Materials and methods: The inhibitory effects of DHM on eight human liver CYP isoforms (i.e., 1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19 and 2C8) were investigated in vitro using human liver microsomes (HLMs).

Results: The results showed that DHM could inhibit the activity of CYP3A4, CYP2E1 and CYP2D6, with IC₅₀ values of 14.75, 25.74 and 22.69?μM, respectively, but that other CYP isoforms were not affected. Enzyme kinetic studies showed that DHM was not only a non-competitive inhibitor of CYP3A4 but also a competitive inhibitor of CYP2E1 and CYP2D6, with Ki values of 6.06, 9.24 and 10.52?μM, respectively. In addition, DHM is a time-dependent inhibitor for CYP3A4 with K_I/K_inact value of 12.17/0.057?min^?1?μM^?1.

Discussion and conclusion: The in vitro studies of DHM with CYP isoforms indicate that DHM has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP3A4, CYP2E1 and CYP2D6. Further clinical studies are needed to evaluate the significance of this interaction.     

Retinoids induce cytochrome P450 3A4 through RXR/VDR-mediated pathway

A panel of retinoids and carotenoids was screened as potential inducers of CYP3A4 through the RXR/VDR-mediated signaling pathway. Transient transfection assays revealed that 3 out of 12 retinoids screened transactivated RXR/VDR and induced CYP3A4 reporter activity. These three retinoids are the active metabolites of retinoids, 9-cis-retinal, 9-cis-retinoic acid (9-cis-RA), and all-trans-retinoic acid (all-trans-RA). 9-cis-RA and all-trans-RA preferentially transactivated the RXR/VDR heterodimers and RXR homodimers. Retinoids and VDR agonist 1, 25-dihydroxyvitamin D₃, but not PXR or CAR activator, could induce Cyp3a11 mRNA level in hepatocytes derived from PXR/CAR-double null mouse. Moreover, retinoids induced CYP3A4 enzyme activity in HepG2 human hepatoma and Caco-2 human colorectal adenocarcinoma cells. A direct role of retinoid-mediated CYP3A4 induction through RXR/VDR was proved by the results that 9-cis-retinal, 9-cis-RA, and all-trans-RA recruited RXR and VDR to CYP3A4 regulatory region pER6 (proximal everted repeat with a 6-nucleotide spacer) and dXREM (distal xenobiotic-responsive enhancer module). Thus, using various approaches, we have unequivocally demonstrated that retinoids transactivate RXR/VDR heterodimers and RXR homodimers and induce CYP3A expression at mRNA as well as enzyme activity levels in both liver and intestinal cells. It is possible that retinoids might alter endobiotic metabolism through CYP3A4 induction in vivo.     

Inhibitory effects of polyphenols on human cytochrome P450 3A4 and 2C9 activity

Polyphenols present in foods and supplements may contribute to human health by preventing cardiovascular diseases and cancer. Drug–food or drug–herb interactions have recently come into focus but, except for some phytochemicals, few components of food or herbs participate in such interactions. In this study, we systematically evaluated the inhibitory effects of 60 polyphenols and related compounds on human cytochrome P450 (CYP) 3A4 and CYP2C9 activity by in vitro assay to investigate whether some polyphenols induce drug interactions. In addition, the kinetics of potent CYP inhibitors was investigated by Lineweaver–Burk plot analysis. Three coumarins and 12 flavonoids significantly suppressed CYP3A4 or CYP2C9 activities. Lineweaver–Burk plot analysis indicated that apigenin and its dimer amentoflavone and imperatorin displayed a mixed type of inhibition on CYP3A4 or CYP2C9. Among the inhibitors, amentoflavone was the most potent inhibitor of CYP3A4 and CYP2C9 activities with IC₅₀ values of 0.07 and 0.03 μM, respectively. The K_i value of amentoflavone was significantly lower than that of the CYP2C9 inhibition positive control sulfaphenazole. These findings suggest that some dietary polyphenols may have the potential to inhibit the metabolism of clinical drugs.     

Contribution of cytochrome P450 3A4 and 3A5 to the metabolism of atorvastatin

Atorvastatin is a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor that is mainly metabolized by cytochrome P450 (CYP) 3A4. A recent study showed that the lipid-lowering effect of statins is affected by the CYP3A5 polymorphism. Therefore, it was investigated whether CYP3A5 contributes to the metabolism of atorvastatin. Two metabolites of atorvastatin, para- and ortho-hydroxyatorvastatin, were produced by human liver microsomes and human recombinant CYP3A enzymes, and the enzyme kinetic pattern exhibited substrate inhibition. The intrinsic clearance (CL_int) rates of para- and ortho-hydroxyatorvastatin by CYP3A4 were 2.4- and 5.0-fold of the respective CL_int rates of CYP3A5, indicating that CYP3A4 is the major P450 isoform responsible for atorvastatin metabolism. These results suggest that atorvastatin is preferentially metabolized by CYP3A4 rather than by CYP3A5, and thus the genetic CYP3A5 polymorphism might not be an important factor in the inter-individual variation of atorvastatin disposition and pharmacodynamics in human.     

Inhibitory effects of curculigoside on human liver cytochrome P450 enzymes

1.?Curculigoside possesses numerous pharmacological activities, and however, little data available for the effects of curculigoside on the activity of human liver cytochrome P450 (CYP) enzymes.

2.?This study investigates the inhibitory effects of curculigoside on the main human liver CYP isoforms. In this study, the inhibitory effects of curculigoside on the eight human liver CYP isoforms 1A2, 2A6, 2E1, 2D6, 2C9, 2C19, 2C8, and 3A4 were investigated in human liver microsomes.

3.?The results indicated that curculigoside could inhibit the activity of CYP1A2, CYP2C8, and CYP3A4, with IC₅₀ values of 15.26, 11.93, and 9.47?μM, respectively, but that other CYP isoforms were not affected. Enzyme kinetic studies showed that curculigoside was not only a noncompetitive inhibitor of CYP1A2, but also a competitive inhibitor of CYP2C8 and CYP3A4, with Ki values of 5.43, 3.54, and 3.35?μM, respectively. In addition, curculigoside is a time-dependent inhibitor for CYP1A2, with kinact/K_I values of 0.056/6.15?μM^?1?min^?1.

4.?The in vitro studies of curculigoside with CYP isoforms suggest that curculigoside has the potential to cause pharmacokinetic drug interactions with other coadministered drugs metabolized by CYP1A2, CYP2C8, and CYP3A4. Further in vivo studies are needed in order to evaluate the significance of this interaction.     

Effects of N-terminal modification of recombinant human cytochrome P450 1A2 on catalytic activity

• The high-level expression of mammalian cytochrome P450 in bacteria usually requires modification of the amino-terminal region of the enzyme. The effect of altering amino acids in the N-terminus of human recombinant CYP1A2 on its catalytic activity was investigated herein.

• Rates of 7-ethoxyresorufin O-deethylation by CYP1A2a (a form made by altering the amino acids LLL of CYP1A2 to RER at positions 3–5) in reconstituted systems were significantly low compared with those of other CYP1A2?N-terminal variants at a low ratio of cytochrome P450 to NADPH-cytochrome P450 reductase, but not at higher reductase concentrations.

• CYP1A2a-dependent ethoxyresorufin O-deethylase activity in a cumene hydroperoxide-supported system was approximately 2-fold higher than other CYP1A2?N-terminal variants.

• Our results suggest that modification of three N-terminal amino acids in CYP1A2 alters the interaction between CYP1A2 and the reductase in reconstituted phospholipid vesicles and in the bicistronic membranes.

     

Effects of cytochrome P450 3A4 and non-genetic factors on initial voriconazole serum trough concentrations in hematological patients with different cytochrome P450 2C19 genotypes

1.?Polymorphisms of cytochrome P450 2C19 (CYP2C19) is an important factor contributing to variability of voriconazole pharmacokinetics. Polymorphisms of CYP3A4, CYP3A5, CYP2C9 and non-genetic factors such as age, gender, body mass index (BMI), transaminase levels, concomitant medications might also affect voriconazole initial steady serum trough concentration (VIC_min) in haematological patients, but the effects were not clear.

2.?Eighteen single-nucleotide polymorphisms in CYP2C19, CYP3A4, CYP3A5, CYP2C9 were genotyped. Patients were stratified into two groups according to CYP2C19 genotype. Group 1 were patients with CYP2C19*2 or CYP2C19*3, and Group 2 were homozygous extensive metabolizers. The effects were studied in different groups. VIC_min was adjusted on daily dose (VIC_min/D) for overcoming effect of dose.

3.?A total of 106 blood samples from 86 patients were included. In final optimal scaling regression models, polymorphisms of rs4646437 (CYP3A4), age, BMI was identified to be factors of VIC_min/D in Group 1 (R^2?=^?.255, p?<?.001). Only age was confirmed as a factor of VIC_min/D in Group 2 (R^2?=^?0.144, p?=?.021).

4.?Besides polymorphisms of CYP2C19, in individualized medication of voriconazole in haematological patients, polymorphisms of CYP3A4, and non-genetic factors as BMI, age should also be taken into account, especially for individuals with CYP2C19*2 or CYP2C19*3.     

In vitro comparative inhibition profiles of major human drug metabolising cytochrome P450 isozymes (CYP2C9, CYP2D6 and CYP3A4) by HMG-CoA reductase inhibitors

Objective: The affinity of (+)-, (−)- and (±)-fluvastatin, a new synthetic HMG-CoA reductase inhibitor developed as a racemate, for specific human P450 monooxygenases in liver microsomes was compared with that of the pharmacologically active acidic forms of lovastatin, pravastatin and simvastatin. Methods: Affinity was determined as the inhibitory potency for prototype reactions for 3 major drug metabolising enzymes: diclofenac 4′-hydroxylation (CYP2C9), dextromethorphan O-demethylation (CYP2D6), and midazolam 1′-hydroxylation (CYP3A4). Results: Lovastatin acid, pravastatin and simvastatin acid displayed moderate affinity for all three P450 isozymes (estimated K_i > 50 μmol⋅l⁻¹). Racemic and (+)- and (−)-fluvastatin showed moderate affinity (estimated K_i > 50 μmol⋅l⁻¹) for CYP2D6 and CYP3A4, whereas their affinity for CYP2C9 was high (estimated K_i < 1 μmol⋅l⁻¹). Diclofenac 4′-hydroxylation was competitively and stereoselectively inhibited, with measured K_i’s of 0.06 and 0.28 μmol⋅l⁻¹ for (+)- and (−)-fluvastatin, respectively. Conclusion: Fluvastatin selectively inhibits a major drug metabolising enzyme (CYP2C9), the (+)-isomer (pharmacologically more active) showing 4–5 fold higher affinity. As already reported for lovastatin and simvastatin, in vivo drug interactions by inhibition of liver oxidation of CYP2C9 substrates (e.g. hypoglyceamic sulphonylureas and oral anticoagulants) may be expected. Received: 9 June 1995/Accepted in revised form: 7 November 1995     

肠道CYP3A和P-gp:口服药物的吸收屏障

细胞色素P4 5 0 3A (CYP3A)亚族是人类药物代谢最重要的I相酶。由MDR1基因编码的外向转运载体蛋白P糖蛋白 (P gp)为药物外排泵。这两种蛋白质在口服药物吸收的主要部位胃肠道均有高表达 ,同时二者的底物具有明显的重叠性。近来 ,大量研究表明 ,决定口服药物生物利用度的主要因素是肠道细胞CYP3A对已吸收药物的生物转化作用和肠道细胞中P gp对已吸收药物的主动外排作用。如果药物为CYP3A和 (或 )P gp的底物 ,当其与CYP3A和P gp的抑制剂同时服用后 ,药物的口服生物利用度将可能升高     

Identification of human cytochrome P450 enzymes involved in the formation of 4-hydroxyestazolam from estazolam

To predict drug interactions with estazolam, the biotransformation of estazolam to its major hydoxylated metabolite, 4-hydroxyestazolam was studied in vitro using pooled human liver microsomes and individual expressed human cytochrome P450 (CYP) enzymes. Estazolam was metabolized to 4-hydroxyestazolam according to the Hill kinetic model in pooled human liver microsomes. The K_m value for the 4-hydroxylation of estazolam was 24.1?µM, and the V_max value was 52.6?pmol?min^?1?mg^?1 protein. The formation of 4-hydroxyestazolam from estazolam in pooled human liver microsomes was significantly inhibited by itraconazole and erythromycin, specific CYP3A4 inhibitors, in a dose-dependent manner, with IC₅₀ values of 1.1 and 12.8?µM, respectively. When estazolam was incubated with expressed human CYP enzymes (CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4), it was metabolized only by CYP3A4. In conclusion, the biotransformation of estazolam to 4-hydroxyestazolam was catalyzed by CYP3A4.     

肠道CYP3A和P-gp:口服药物的吸收屏障

细胞色素P4 5 0 3A (CYP3A)亚族是人类药物代谢最重要的I相酶。由Mdr1基因编码的外向转运载体蛋白P糖蛋白 (P gp)为药物外排泵。这两种蛋白质在口服药物吸收的主要部位胃肠道均有高表达 ,同时二者的底物具有显著的重叠性。近来 ,大量研究表明 ,决定口服药物生物利用度的主要因素是肠道细胞CYP3A对已吸收药物的生物转化作用和肠道细胞中P gp对已吸收药物的主动外排作用。如果药物为CYP3A和 (或 )P gp的底物 ,当其与CYP3A和P gp的抑制剂同时服用后 ,药物的口服生物利用度将可能升高     

Potential impact of cytochrome P450 3A5 in human liver on drug interactions with triazoles

Cytochrome P450 3A is the main enzyme subfamily involved in the metabolism of a variety of marketed medicines. It is generally believed that the substrate specificity of polymorphic P450 3A5 is similar to that of the predominant P450 3A4 isoform, although some differences in catalytic properties have been found. It has been hypothesized that individuals with CYP3A5*1 (P450 3A5 expresser) might clear the HIV protease inhibitor saquinavir, administered by mouth, more rapidly than subjects lacking functional CYP3A5 alleles. Enhanced midazolam hydroxylation and cyclosporin metabolism occur in an in vitro P450 3A5 system and in liver microsomes expressing P450 3A5 in the presence of thalidomide. However, inhibition constants (K_i) of three triazole anti-fungal drugs (itraconazole, fluconazole, and voriconazole) for liver microsomal P450 3A5 are higher than for liver microsomal P450 3A4. To predict drug interactions in vivo, we estimated increases of areas under the curves (AUC) dependent on polymorphic P450 3A5 expression, using both 1 +[Inhibitor] / K_i (recommended in US FDA guidance), and 1 +[Inhibitor]_unbound / K_i (as recommended by Japanese MHLW Notice). Voriconazole would be expected to cause approximately a three-fold higher increase in AUC in subjects with CYP3A5*3/*3 than in those with CYP3A5*1/*3, especially when estimated using the FDA guidance. We conclude that drug interactions between marketed drugs may differ substantially between individuals with genetically distinct P450 3A5 catalytic functions.