Metabolism of novel anti-HIV agent 3-cyanomethyl-4-methyl-DCK by human liver microsomes and recombinant CYP enzymes

Aim:

To investigate the metabolism of 3-cyanomethyl-4-methyl-DCK (CMDCK), a novel anti-HIV agent, by human liver microsomes (HLMs) and recombinant cytochrome P450 enzymes (CYPs).

Methods:

CMDCK was incubated with HLMs or a panel of recombinant cytochrome P450 enzymes including CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, and 3A5. LC-ion trap mass spectrometry was used to separate and identify CMDCK metabolites. In the experiments with recombinant cytochrome P450 enzymes, specific chemical inhibitors combined with CYP antibodies were used to identify the CYP isoforms involved in CMDCK metabolism.

Results:

CMDCK was rapidly and extensively metabolized by HLMs. Its intrinsic hepatic clearance estimated from the in vitro data was 19.4 mL·min⁻¹·kg⁻¹, which was comparable to the mean human hepatic blood flow rate (20.7 mL·min⁻¹·kg⁻¹). The major metabolic pathway of CMDCK was oxidation, and a total of 14 metabolites were detected. CYP3A4 and 3A5 were found to be the principal CYP enzymes responsible for CMDCK metabolism.

Conclusion:

CMDCK was metabolized rapidly and extensively in human hepatic microsomes to form a number of oxidative metabolites. CYP3A4 and 3A5 were the predominant enzymes responsible for the oxidation of CMDCK.     

Mechanism-based inhibition of cytochrome P450 (CYP)2A6 by chalepensin in recombinant systems, in human liver microsomes and in mice in vivo

BACKGROUND AND PURPOSE

Chalepensin is a pharmacologically active furanocoumarin compound found in rue, a medicinal herb. Here we have investigated the inhibitory effects of chalepensin on cytochrome P450 (CYP) 2A6 in vitro and in vivo.

EXPERIMENTAL APPROACH

Mechanism-based inhibition was studied in vitro using human liver microsomes and bacterial membranes expressing genetic variants of human CYP2A6. Effects in vivo were studied in C57BL/6J mice. CYP2A6 activity was assayed as coumarin 7-hydroxylation (CH) using HPLC and fluorescence measurements. Metabolism of chalepensin was assessed with liquid chromatography/mass spectrometry (LC/MS).

KEY RESULTS

CYP2A6.1, without pre-incubation with NADPH, was competitively inhibited by chalepensin. After pre-incubation with NADPH, inhibition by chalepensin was increased (IC₅₀ value decreased by 98%). This time-dependent inactivation (k_inact 0.044 min⁻¹; K_I 2.64 µM) caused the loss of spectrally detectable P450 content and was diminished by known inhibitors of CYP2A6, pilocarpine or tranylcypromine, and by glutathione conjugation. LC/MS analysis of chalepensin metabolites suggested an unstable epoxide intermediate was formed, identified as the corresponding dihydrodiol, which was then conjugated with glutathione. Compared with the wild-type CYP2A6.1, the isoforms CYP2A6.7 and CYP2A6.10 were less inhibited. In mouse liver microsomes, pre-incubation enhanced inhibition of CH activity. Oral administration of chalepensin to mice reduced hepatic CH activity ex vivo.

CONCLUSIONS AND IMPLICATIONS

Chalepensin was a substrate and a mechanism-based inhibitor of human CYP2A6. Formation of an epoxide could be a key step in this inactivation. ‘Poor metabolizers’ carrying CYP2A6*7 or *10 may be less susceptible to inhibition by chalepensin. Given in vivo, chalepensin decreased CYP2A activity in mice.     

IC50-based approaches as an alternative method for assessment of time-dependent inhibition of CYP3A4

1. The predictive utility of two in vitro methods (empirical IC₅₀-based and mechanistic k_inact/K_I) for the assessment of time-dependent cytochrome P450 3A4 (CYP3A4) inhibition has been compared.

2. IC₅₀ values were determined at multiple pre-incubation time points over 30?min for five CYP3A4 time-dependent inhibitors (verapamil, diltiazem, erythromycin, clarithromycin, and azithromycin). The ability of IC₅₀ data obtained following pre-incubation to predict k_inact/K_I parameters was investigated and its utility was assessed relative to the conventional k_inact/K_I model using 50 reported clinical drug–drug interactions (DDIs). Models with either hepatic or hepatic with intestinal components were explored.

3. For low/medium potency time-dependent inhibitors, 81% of the predicted k_inact/K_I(unbound) from IC₅₀ data were within an order of magnitude of the actual values, in contrast to 50% of potent inhibitors. An underprediction trend and >?50% of false-negatives were observed when IC₅₀ data were used in the DDI hepatic prediction model; incorporation of the intestine improved the prediction accuracy. On the contrary, 86% of the DDI studies were predicted within twofold using k_inact/K_I mechanistic approach and the combined hepatic and intestinal model.

4. Use of the empirical IC₅₀ approach as an alternative to the mechanistic k_inact/K_I model for in vivo DDI prediction is limited and is best restricted to preliminary investigations.

     

CYP2D6 and CYP3A4 involvement in the primary oxidative metabolism of hydrocodone by human liver microsomes

AIM: To determine the Michaelis-Menten kinetics of hydrocodone metabolism to its O- and N-demethylated products, hydromorphone and norhydrocodone, to determine the individual cytochrome p450 enzymes involved, and to predict the in vivo hepatic intrinsic clearance of hydrocodone via these pathways. METHODS: Liver microsomes from six CYP2D6 extensive metabolizers (EM) and one CYP2D6 poor metabolizer (PM) were used to determine the kinetics of hydromorphone and norhydrocodone formation. Chemical and antibody inhibitors were used to identify the cytochrome p450 isoforms catalyzing these pathways. Expressed recombinant cytochrome p450 enzymes were used to characterize further the metabolism of hydrocodone. RESULTS: Hydromorphone formation in liver microsomes from CYP2D6 EMs was dependent on a high affinity enzyme (Km = 26 microm) contributing 95%, and to a lesser degree a low affinity enzyme (Km = 3.4 mm). In contrast, only a low affinity enzyme (Km = 8.5 mm) formed this metabolite in the liver from the CYP2D6 PM, with significantly decreased hydromorphone formation compared with the livers from the EMs. Norhydrocodone was formed by a single low affinity enzyme (Km = 5.1 mm) in livers from both CYP2D6 EM and PM. Recombinant CYP2D6 and CYP3A4 formed only hydromorphone and only norhydrocodone, respectively. Hydromorphone formation was inhibited by quinidine (a selective inhibitor of CYP2D6 activity), and monoclonal antibodies specific to CYP2D6. Troleandomycin, ketoconazole (both CYP3A4 inhibitors) and monoclonal antibodies specific for CYP3A4 inhibited norhydrocodone formation. Extrapolation of in vitro to in vivo data resulted in a predicted total hepatic clearance of 227 ml x h-1 x kg-1 and 124 ml x h-1 x kg-1 for CYP2D6 EM and PM, respectively. CONCLUSIONS: The O-demethylation of hydrocodone is predominantly catalyzed by CYP2D6 and to a lesser extent by an unknown low affinity cytochrome p450 enzyme. Norhydrocodone formation was attributed to CYP3A4. Comparison of recalculated published clearance data for hydrocodone, with those predicted in the present work, indicate that about 40% of the clearance of hydrocodone is via non-CYP pathways. Our data also suggest that the genetic polymorphisms of CYP2D6 may influence hydrocodone metabolism and its therapeutic efficacy.     

Potent mechanism-based inhibition of CYP3A4 by imatinib explains its liability to interact with CYP3A4 substrates

BACKGROUND AND PURPOSE

Imatinib, a cytochrome P450 2C8 (CYP2C8) and CYP3A4 substrate, markedly increases plasma concentrations of the CYP3A4/5 substrate simvastatin and reduces hepatic CYP3A4/5 activity in humans. Because competitive inhibition of CYP3A4/5 does not explain these in vivo interactions, we investigated the reversible and time-dependent inhibitory effects of imatinib and its main metabolite N-desmethylimatinib on CYP2C8 and CYP3A4/5 in vitro.

EXPERIMENTAL APPROACH

Amodiaquine N-deethylation and midazolam 1′-hydroxylation were used as marker reactions for CYP2C8 and CYP3A4/5 activity. Direct, IC₅₀-shift, and time-dependent inhibition were assessed with human liver microsomes.

KEY RESULTS

Inhibition of CYP3A4 activity by imatinib was pre-incubation time-, concentration- and NADPH-dependent, and the time-dependent inactivation variables K_I and k_inact were 14.3 µM and 0.072 min⁻¹ respectively. In direct inhibition experiments, imatinib and N-desmethylimatinib inhibited amodiaquine N-deethylation with a K_i of 8.4 and 12.8 µM, respectively, and midazolam 1′-hydroxylation with a K_i of 23.3 and 18.1 µM respectively. The time-dependent inhibition effect of imatinib was predicted to cause up to 90% inhibition of hepatic CYP3A4 activity with clinically relevant imatinib concentrations, whereas the direct inhibition was predicted to be negligible in vivo.

CONCLUSIONS AND IMPLICATIONS

Imatinib is a potent mechanism-based inhibitor of CYP3A4 in vitro and this finding explains the imatinib–simvastatin interaction and suggests that imatinib could markedly increase plasma concentrations of other CYP3A4 substrates. Our results also suggest a possibility of autoinhibition of CYP3A4-mediated imatinib metabolism leading to a less significant role for CYP3A4 in imatinib biotransformation in vivo than previously proposed.     

Comparative analysis of substrate and inhibitor interactions with CYP3A4 and CYP3A5

To evaluate the role that cytochrome (CYP) 3A5 plays in hepatic drug metabolism, the substrate selectivity and inhibitory potential of over 60 compounds towards CYP3A4 and CYP3A5 were assessed using Escherichia coli recombinant cell lines. CYP3A4-mediated metabolism predominated for many of the compounds studied. However, a number of drugs gave similar CL_int estimates using CYP3A5 compared with CYP3A4 including midazolam (CL_int?=?3.4 versus 3.3?µl?min^–1?pmol^–1). Significant CYP3A5-mediated metabolism was also observed for several drugs including mifepristone (CL_int?=?10.3 versus 2.4?µl?min^–1?pmol^–1), and ritonavir (CL_int?=?0.76 versus 0.47?µl?min^–1?pmol^–1). The majority of compounds studied showed a greater inhibitory potential (IC₅₀) towards CYP3A4 compared with CYP3A5 (eightfold lower on average). A greater degree of time-dependent inhibition was also observed with CYP3A4 compared with CYP3A5. The range of compounds investigated in the present study extends significantly previous work and suggests that CYP3A5 may have a significant role in drug metabolism particularly in populations expressing high levels of CYP3A5 and/or on co-medications known to inhibit CYP3A4.     

Flavonoids diosmetin and luteolin inhibit midazolam metabolism by human liver microsomes and recombinant CYP 3A4 and CYP3A5 enzymes

We evaluated the effects of increasing concentrations of the flavonoids salvigenin, diosmetin and luteolin on the in vitro metabolism of midazolam (MDZ), a probe substrate for cytochrome P450 (CYP) 3A enzymes, which is converted into 1'-hydroxy-midazolam (1'-OH-MDZ) and 4-hydroxy-midazolam (4-OH-MDZ) by human liver microsomes. Salvigenin had only a modest effect on MDZ metabolism, whereas diosmetin and luteolin inhibited in a concentration-dependent manner the formation of both 1'-OH-MDZ and 4-OH-MDZ, with apparent K(i) values in the 30-50mumol range. Both diosmetin and luteolin decreased 1'-OH-MDZ formation by human recombinant CYP3A4, but not CYP3A5, whereas they decreased 4-OH-MDZ formation by both recombinant enzymes. To assess whether any relationship exists between the physico-chemical characteristics of flavones and their effects on MDZ metabolism, we tested the effects of three other flavones (flavone, tangeretin, chrysin) on MDZ metabolism by human liver microsomes. Whereas flavones possessing more than two hydroxyl groups (luteolin, diosmetin) inhibited MDZ biotransformation, flavones lacking hydroxyl groups in their A and B rings (flavone, tangeretin) stimulated MDZ metabolism. We also found close relationships between the maximum stimulatory or inhibitory effects of flavones on 1'-OH-MDZ and 4-OH-MDZ formation rates and their log of octanol/water partition coefficients (logP) or their total number of hydroxyl groups. The results of the study may be of clinical relevance since they suggest that luteolin and diosmetin may cause pharmacokinetic interactions with co-administered drugs metabolized via CYP3A.     

人细胞色素P450 3A4突变体CYP3A4.3,CYP3A4.4,CYP3A4.5和CYP3A4.18重组酶的异源表达与活性

目的重组表达人细胞色素P450(CYP)3A4突变体CYP3A4.3,CYP3A4.4,CYP3A4.5和CYP3A4.18蛋白,为CYP3A4代谢活性的体外研究提供单一酶源。方法应用杆状病毒表达系统构建含有上述各CYP3A4突变体基因序列的重组病毒,将其连同含人源还原型烟酰胺腺嘌呤二核苷磷酸-P450氧化还原酶(POR)和细胞色素b5基因的重组病毒共同感染昆虫草地夜蛾细胞Sf9得到CYP3A4突变体与POR和细胞色素b5共表达的重组蛋白,分别以高效液相色谱法和荧光分析法测定各重组酶对睾酮和7-甲氧基-4-三氟甲基香豆素的代谢活性。结果在mRNA分子水平上验证了CYP3A4突变体CYP3A4*3,CYP3A4*4,CYP3A4*5和CYP3A4*18基因在Sf9细胞中的转录。感染了各重组病毒的Sf9细胞裂解液对睾酮和7-苄氧基-4-三氟甲基香豆素有明显代谢。结论应用杆状病毒-昆虫细胞表达系统在体外成功表达了具有催化活性的人CYP3A4突变体CYP3A4.3,CYP3A4.4,CYP3A4.5和CYP3A4.18蛋白,其中CYP3A4.5活性显著低于野生型蛋白,CYP3A4.18活性显著高于野生型蛋白,而CYP3A4.3和CYP3A4.4与野生型蛋白活性近似。     

In vivo inhibition of CYP3A-mediated midazolam metabolism by anchusan in rats

Cytochrome P450 (CYP)-mediated drug interactions caused by Kampo medicine have not been investigated sufficiently. The current study was conducted to reveal the effect of anchusan, a commonly used Kampo formula for gastrointestinal disease, on CYP3A-mediated drug metabolism in rats. The pharmacokinetics of midazolam (MDZ) was investigated after the single or one-week administration of anchusan (500 mg/kg) to evaluate its inhibitory and inducible effect on CYP3A, respectively. MDZ was administrated 16 h after the last anchsan treatment in the multiple dose study, while their intervals were 2 or 16 h in the single dose study. Unexpectedly, the multiple-pretreatment of anchusan increased the AUC of MDZ by 2.4-fold rather than decreasing it, and the CYP3A contents and activities were unchanged in hepatic and intestinal microsomes of these rats. In contrast, no significant inhibitory effects on MDZ metabolism were observed by the single anchusan pretreatment. In vitro study showed that the preincubation of anchusan and some of its component extracts with rat liver microsomes reduced CYP3A activity in a time- and NADPH-dependent manner. These results suggested that anchusan increased the serum MDZ concentration in rats, at least in part, by the time-dependent inhibition of CYP3A.     

The human hepatic metabolism of simvastatin hydroxy acid is mediated primarily by CYP3A,and not CYP2D6

AIMS: To identify the cytochrome P450 (CYP) isoforms responsible for the metabolism of simvastatin hydroxy acid (SVA), the most potent metabolite of simvastatin (SV). METHODS: The metabolism of SVA was characterized in vitro using human liver microsomes and recombinant CYPs. The effects of selective chemical inhibitors and CYP antibodies on SVA metabolism were assessed in human liver microsomes. RESULTS: In human liver microsomes, SVA underwent oxidative metabolism to three major oxidative products, with values for Km and Vmax ranging from about 50 to 80 microM and 0.6 to 1.9 nmol x min(-1) x mg(-1) protein, respectively. Recombinant CYP3A4, CYP3A5 and CYP2C8 all catalysed the formation of the three SVA metabolites, but CYP3A4 was the most active. CYP2D6 as well as CYP2C19, CYP2C9, CYP2A6, CYP1A2 did not metabolize SVA. Whereas inhibitors that are selective for CYP2D6, CYP2C9 or CYP1A2 did not significantly inhibit the oxidative metabolism of SVA, the CYP3A4/5 inhibitor troleandomycin markedly (about 90%) inhibited SVA metabolism. Quercetin, a known inhibitor of CYP2C8, inhibited the microsomal formation of SVA metabolites by about 25-30%. Immunoinhibition studies revealed 80-95% inhibition by anti-CYP3A antibody, less than 20% inhibition by anti-CYP2C19 antibody, which cross-reacted with CYP2C8 and CYP2C9, and no inhibition by anti-CYP2D6 antibody. CONCLUSIONS: The metabolism of SVA in human liver microsomes is catalysed primarily (> or = 80%) by CYP3A4/5, with a minor contribution (< or = 20%) from CYP2C8. CYP2D6 and other major CYP isoforms are not involved in the hepatic metabolism of SVA.     

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     

Cytochrome P450 2C8 and CYP3A4/5 are involved in chloroquine metabolism in human liver microsomes

Chloroquine has been used for many decades in the prophylaxis and treatment of malaria. It is metabolized in humans through the N-dealkylation pathway, to desethylchloroquine (DCQ) and bisdesethylchloroquine (BDCQ), by cytochrome P450 (CYP). However, until recently, no data are available on the metabolic pathway of chloroquine. Therefore, the metabolic pathway of chloroquine was evaluated using human liver microsomes and cDNA-expressed CYPs. Chloroquine is mainly metabolized to DCQ, and its Eadie-Hofstee plots were biphasic, indicating the involvement of multiple enzymes, with apparent Km and Vmax values of 0.21 mM and 1.02 nmol/min/mg protein 3.43 mM and 10.47 nmol/min/mg protein for high and low affinity components, respectively. Of the cDNA-expressing CYPs examined, CYP1A2, 2C8, 2C19, 2D6 and 3A4/5 exhibited significant DCQ formation. A study using chemical inhibitors showed only quercetin (a CYP2C8 inhibitor) and ketoconazole (a CYP3A4/5 inhibitor) inhibited the DCQ formation. In addition, the DCQ formation significantly correlated with the CYP3A4/5-catalyzed midazolam 1-hydroxylation (r = 0.868) and CYP2C8-catalyzed paclitaxel 6alpha-hydroxylation (r = 0.900). In conclusion, the results of the present study demonstrated that CYP2C8 and CYP3A4/5 are the major enzymes responsible for the chloroquine N-deethylation to DCQ in human liver microsomes.     

Methadone N-demethylation in human liver microsomes: lack of stereoselectivity and involvement of CYP3A4

AIMS: To investigate the kinetics of CYP-mediated N-demethylation of methadone in human liver microsomes, and examine the role of stereoselectivity and CYP isoforms involved. METHODS: The kinetics of 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) formation via N-demethylation of rac-, (R)- and (S)-methadone in human liver microsomes prepared from six liver samples were determined by h.p.l.c., and inhibition of metabolic function was studied using isoform-specific chemical inhibitors and monoclonal antibodies. Microsomes containing expressed CYP3A4, CYP2D6 and CYP2C19 were also used to examine the formation of EDDP. RESULTS: The V max, Km, and CLint values for the formation of EDDP from rac-, (R)- and (S)-methadone were in the ranges of 20-77 nmol mg-1 protein h-1, 125-252 microm, and 91-494 ml h-1 g-1 protein. Km and CLint values for (R)- and (S)-methadone were not statistically significantly different (P >0.05), while V max values for (S)-methadone were 15% (P=0.045) lower than for (R)-methadone. Expressed CYP3A4 and CYP2C19 showed similar reaction rates for both (R)- and (S)-methadone, while CYP2D6 did not catalyse this reaction. Selective chemical inhibitors of CYP3A (troleandomycin, ketoconazole) and monoclonal human CYP3A4 antibodies significantly inhibited (P<0.05) the formation of EDDP in a concentration dependent manner by up to 80%. Sulphaphenazole (CYP2C9) also significantly inhibited (P<0.05) EDDP formation (range 14-25%). There were no statistically significant differences in the inhibition observed between the three substrates. Selective inhibitors of CYP1A2 (furafylline), CYP2A6 (coumarin), CYP2C19 ((S)-mephenytoin), CYP2D6 (quinidine) and CYP2E1 (diethyldithiocarbamic acid sodium salt and monoclonal human CYP2E1 antibodies) had no significant (P >0.05) effect. CONCLUSIONS: The N-demethylation of methadone in human liver microsomes is not markedly stereoselective, and is mediated mainly by CYP3A4 with the possible involvement of CYP2C9 and CYP2C19. Thus, the large interindividual variation reported for methadone pharmacokinetics may be due to variability in the expression of these CYP isoforms, and the reported stereoselectivity in the systemic clearance of methadone in vivo is not due to stereoselectivity in N-demethylation.     

The potent mechanism-based inactivation of CYP2D6 and CYP3A4 with fusidic acid in in vivo bioaccumulation

1.?The accumulation of fusidic acid (FA) after multiple doses of FA has been reported on in previous studies but the related mechanisms have not been clarified fully. In the present study, we explain the mechanisms related to the mechanism-based inactivation of CYP2D6 and CYP3A4.

2.?The irreversible inhibitory effects of FA on CYP2D6 and CYP3A4 were examined via a series of experiments, including: (a) time-, concentration- and NADPH-dependent inactivation, (b) substrate protection in enzyme inactivation and (c) partition ratio with recombinant human CYP enzymes. Metoprolol α-hydroxylation and midazolam 1′-hydroxylation were used as marker reactions for CYP2D6 and CYP3A4 activities, and HPLC-MS/MS measurement was also utilised.

3.?FA caused to the time- and concentration-dependent inactivation of CYP2D6 and CYP3A4. About 55.8% of the activity of CYP2D6 and 75.8% of the activity of CYP3A4 were suppressed after incubation with 10?μM FA for 15?min. K_I and k_inact were found to be 2.87?μM and 0.033?min^?1, respectively, for CYP2D6, while they were 1.95?μM and 0.029?min^?1, respectively, for CYP3A4. Inhibition of CYP2D6 and CYP3A4 activity was found to require the presence of NADPH. Substrates of CYP2D6 and CYP3A4 showed that the enzymes were protected against the inactivation induced by FA. The estimated partition ratio for the inactivation was 7 for CYP2D6 and 12 for CYP3A4.

4.?FA is a potent mechanism-based inhibitor of CYP2D6 and CYP3A4, which may explain the accumulation of FA in vivo.     

HPLC法测定利多卡因及其代谢产物估算人肝微粒体CYP3A的活性

目的　以利多卡因 (LDC)与其代谢产物的比值估算人肝微粒体中CYP3A的活性。方法　以 1 0g·L- 1微粒体蛋白浓度 3 7℃孵育利多卡因 60min ,以HPLC测定利多卡因及其代谢产物单乙基甘氨二甲基苯酰胺 (MEGX)和甘氨二甲基苯酰胺 (GX)的含量。结果　LDC、MEGX和GX的标准曲线方程分别为 ^Y =0 2 93 4X -0 0 0 5661(r =0 9997)、^Y =0 7913X -0 0 0 8916(r =0 9993 )和 ^Y =0 6799X -0 0 0 7770 (r =0 9985)。体外孵育的最佳条件为 2 0mg·L- 1的LDC在浓度为 1 0 g·L- 1的微粒体中 ,孵育 60min ,代谢产物与利多卡因的平均比值为 3 2 8。结论　(MEGX +GX) /LDC可用来估算人肝微粒体CYP3A的活性。     

Pharmacokinetics of midazolam in CYP3A4- and CYP3A5-genotyped subjects

Objective We investigated whether differences in pharmacokinetics of midazolam, a CYP3A probe, could be demonstrated between subjects with different CYP3A4 and CYP3A5 genotypes.Methods Plasma concentrations of midazolam, and of total (conjugated + unconjugated) 1OH-midazolam, and 4OH-midazolam were measured after the oral administration of 7.5 mg or of 75 µg of midazolam in 21 healthy subjects.Results CYP3A5*7, CYP3A4*1E, CYP3A4*2, CYP3A4*4, CYP3A4*5, CYP3A4*6, CYP3A4*8, CYP3A4*11, CYP3A4*12, CYP3A4*13, CYP3A4*17 and CYP3A4*18 alleles were not identified in the 21 subjects. CYP3A5*3, CYP3A5*6, CYP3A4*1B and CYP3A4*1F alleles were identified in 20, 1, 4 and 2 subjects, respectively. No statistically significant differences were observed for the AUC_inf values between the different genotypes after the 75-µg or the 7.5-mg dose.Conclusion Presently, CYP3A4 and CYP3A5 genotyping methods do not sufficiently reflect the inter-individual variability of CYP3A activity.     

Roles of CYP2A6 and CYP2B6 in nicotine C-oxidation by human liver microsomes

Nicotine C-oxidation by recombinant human cytochrome P450 (P450 or CYP) enzymes and by human liver microsomes was investigated using a convenient high-performance liquid chromatographic method. Experiments with recombinant human P450 enzymes in baculovirus systems, which co-express human nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH)-P450 reductase, revealed that CYP2A6 had the highest nicotine C-oxidation activities followed by CYP2B6 and CYP2D6; the K _m values by these three P450 enzymes were determined to be 11.0, 105, and 132 μM, respectively, and the V _max values to be 11.0, 8.2, and 8.6 nmol/min per nmol P450, respectively. CYP2E1, 2C19, 1A2, 2C8, 3A4, 2C9, and 1A1 catalysed nicotine C-oxidation only at high (500 μM) substrate concentration. CYP1B1, 2C18, 3A5, and 4A11 had no measurable activities even at 500 μM nicotine. In liver microsomes of 16 human samples, nicotine C-oxidation activities were correlated with CYP2A6 contents at 10 μM substrate concentration, whereas such correlation coefficients were decreased when the substrate concentration was increased to 500 μM. Contribution of CYP2B6 (as well as CYP2A6) was demonstrated by experiments with the effects of orphenadrine (and also coumarin and anti-CYP2A6) on the nicotine C-oxidation activities by human liver microsomes at 500 μM nicotine. CYP2D6 was found to have minor roles since quinidine did not inhibit microsomal nicotine C-oxidation at both 10 and 500 μM substrate concentrations. These results support the view that CYP2A6 has major roles for nicotine C-oxidation at lower substrate concentration and both CYP2A6 and 2B6 play roles at higher substrate concentrations in human liver microsomes. Received: 27 October 1998 / Accepted: 11 January 1999     

多药耐药蛋白和CYP3A4介导的中草药-西药相互作用

据近年的流行病学报告，国内外越来越多的人终身服用各种中草药，包括各类疾病患者（如：癌症等）和健康人群。随着中草药与处方西药联合应用治疗疾病的日益增多，越来越多的人注意到这两者的同时使用可能引起的中草药-西药相互作用会影响到治疗药物的活性。目前已知P-糖蛋白（P-gp）和CYP3A4一起能构成许多口服吸收药物的高效屏障，然而50％以上的临床用药都会被CYP3A4代谢或被P-gp转运。本文综述了多药耐药蛋白和CYP3A4介导的中草药-西药相互作用，同时也讨论了中草药-西药相互作用的关键因素。     

CYP3A5~*3和CYP3A4~*18B基因多态性对肾移植患者环孢素药代动力学的影响

目的回顾性研究肾脏移植后1mon,CYP3A5*3和CYP3A4*18B基因多态性对CsA药代动力学参数的影响。方法采用PCR-RFLP方法分析了63名肾脏移植患者CYP3A5*3和CYP3A4*18B基因型;荧光偏正免疫法用于检测肾移植患者静脉全血中的CsA浓度。结果在63名肾移植患者中,CYP3A5*3和CYP3A4*18B突变等位基因发生频率分别为0.770(95CI:0.767～0.773),0.235(95CI:0.235～0.241),而且这些等位基因表现出完全连锁不平衡。在移植术后1mon内,携带CYP3A4*1/*1野生型纯合子患者的C0以及剂量校正谷血浓度(C0/D)均明显高于携带CYP3A4*1/*18B杂合子或CYP3A4*18B/*18B突变型纯合子患者(P<0.05,Mann-WhitneyUtest);CYP3A5*1/*1基因型组的给药剂量明显高于CYP3A5*1/*3或CYP3A5*3/*3基因型组(P=0.004<0.01,Kruakal-Wallistest);CYP34*18B和CYP3A5*3联合考虑,对于CYP3A5表达组,同样发现C0、C0/D在CYP3A4*1/*1组C0以及C0/D均明显高于CYP3A4*1/*18B或CYP3A4*18B/*18B组(P<0.05,Mann-WhitneyUtest);而其他药动学参数在CYP3A5*3及CYP3A4*18B各组间相比差异则没有统计学意义。结论CYP3A5*3和(或)CYP3A4*18B基因多态性对肾移植后1monCsA药代动力学有一定影响,移植前CYP3A5*3基因型的分析仍需进一步研究。