The novel antifungal agent PLD-118 is neither metabolized by liver microsomes nor inhibits cytochrome P450 in vitro

PLD-118 is a novel, oral antifungal drug, under development for the treatment of Candida infections. Possible metabolism of PLD-118 by rat, dog and human S9 liver homogenates and inhibition of human cytochrome P450 (CYP) enzymes were investigated. PLD-118 (10 and 100 microM) incubated for 0-60 min with S9 fractions and NADPH was determined by HPLC, using the Waters AccQ.Tag method after derivatization of amino acids to stable, fluorescent derivatives. CYP assays were performed using pooled human liver microsomes with substrates, selective towards human CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A, incubated at concentrations around the Km. Incubation mixtures were preincubated with PLD-118 (0.1-100 microM) or control inhibitor for 5 min. No metabolism of PLD-118 was detected with rat and dog S9 fractions. A small (8%) decrease in PLD-118 at 100 microM (not detected at 10 microM) with human microsomes was considered to be biologically irrelevant. PLD-118 did not inhibit any of the tested CYPs. PLD-118, at concentrations up to 100 microM, is not metabolized by rat, dog or human liver S9 homogenates and does not inhibit human CYPs in vitro, suggesting little likelihood for interaction of PLD-118 with drugs metabolized by these enzymes.     

酮康唑对大鼠肝脏CYP450酶系的影响*

目的:观察酮康唑对大鼠肝细胞色素P450及其主要亚型的影响。方法:Sprague-Dawley大鼠用140,280,420μmol.kg-1.d-1酮康唑连续灌胃7 d,测定肝脏微粒体中总CYP450含量和CYP1A1,1A2,1B1,2B1/2,2E1和3A亚型活性。结果:不同剂量酮康唑给药后大鼠肝脏脏器系数、CYP1A1和1B1亚型活性明显增高(P<0.05,P<0.01);总CYP450含量和CYP3A活性显著降低(P<0.01);低剂量的酮康唑抑制CYP1A2和CYP2B1/2亚型的活性,高剂量却出现了诱导作用(P<0.05,P<0.01)。各剂量组对CYP2E1均无明显影响。结论:酮康唑对大鼠肝脏CYP450及主要药物代谢亚型CYP1A1,1A2,1B1,2B1/2和3A有影响,临床长期用药或与经肝脏CYP450代谢的药物联合应用时要注意监测血药浓度和肝脏功能,防止药物代谢减缓出现蓄积中毒或药物代谢加快而降低药效。     

细胞色素P450氧化还原酶的研究进展

细胞色素P450氧化还原酶(Cytochrom e P450 Reduc-tase,CPR)是细胞色素P450药物代谢酶系中为细胞色素P450提供电子的膜蛋白,本文从电子传递、底物作用及其基因调控三方面的特性对其进行了论述,并探讨了CPR领域一些待研究的问题。     

植物成分与细胞色素P450相互作用的研究进展

细胞色素P450（CYP450）是体内参与药物代谢的重要酶系,其活性在受到诱导或抑制后将干扰药物的作用。植物成分普遍存在于食物与药物中,与CYP450的相互作用将产生广泛影响。总结近年来植物成分与细胞色素P450的7个亚型CYP1A2、CYP2A6、CYP2C9、CYP2C19、CYP2D6、CYP2E1、CYP3A4相互作用的研究结果,为临床上合理用药提供参考。     

Involvement of microsomal NADPH‐cytochrome P450 reductase in metabolic reduction of drug ketones

Recently, it was found that the carbonyl group of 1‐[3‐(4‐phenoxyphenoxy)‐2‐oxopropyl]indole‐5‐carboxylic acid ( 5 ), an inhibitor of the pro‐inflammatory enzyme cytosolic phospholipase A₂α, is easily reduced by rat liver S9 fractions in vitro. Determination of the inhibitory potency of certain putative inhibitors of carbonyl reducing enzymes on the transformation of the ketone derivative 5 to its alcohol 6 by recombinant microsomal NADPH‐cytochrome P450 reductase and by recombinant cytosolic carbonyl reductase‐1 now reveals that these compounds show a lack of specificity for these two enzymes in part. Thus, an assignment of the roles of different carbonyl reductases in metabolic keto reduction by the use of inhibitors is problematic. In addition, the ability of NADPH‐cytochrome P450 reductase and carbonyl reductase‐1 to reduce the ketone groups of the drugs haloperidol and daunorubicin was examined. Under the conditions applied, a pronounced reductive metabolism was only observed for daunorubicin in the presence of microsomal NADPH‐cytochrome P450 reductase. Similarly, in rat liver S9 fractions a marked reduction of daunorubicin was seen, while haloperidol was only slightly metabolized to its alcohol. After separation of the S9 homogenate into a microsomal and a cytosolic fraction, it became evident that the ketone groups of daunorubicin, haloperidol and compound 5 were mainly reduced by cytosolic enzymes. However, since microsomes also catalysed these carbonyl reductions to some extent, it can be concluded that microsomal NADPH‐cytochrome P450 reductase can contribute to metabolic keto reductions in xenobiotics. Copyright © 2015 John Wiley & Sons, Ltd.     

Mechanism of cytochrome P450-3A inhibition by ketoconazole

Objectives Ketoconazole is extensively used as an index inhibitor of cytochrome P450‐3A (CYP3A) activity in vitro and in vivo, but the mechanism of ketoconazole inhibition of CYP3A still is not clearly established. Methods Inhibition of metabolite formation by ketoconazole (seven concentrations from 0.01 to 1.0 µm ) was studied in human liver microsomes (n = 4) at six to seven substrate concentrations for triazolam, midazolam, and testosterone, and at two substrate concentrations for nifedipine. Key findings Analysis of multiple data points per liver sample based on a mixed competitive–noncompetitive model yielded mean inhibition constant K_i values in the range of 0.011 to 0.045 µm . Ketoconazole IC50 increased at higher substrate concentrations, thereby excluding pure noncompetitive inhibition. For triazolam, testosterone, and midazolam α‐hydroxylation, mean values of α (indicating the ‘mix’ of competitive and noncompetitive inhibition) ranged from 2.1 to 6.3. However, inhibition of midazolam 4‐hydroxylation was consistent with a competitive process. Determination of K_i and α based on the relation between 50% inhibitory concentration values and substrate concentration yielded similar values. Pre‐incubation of ketoconazole with microsomes before addition of substrate did not enhance inhibition, whereas inhibition by troleandomycin was significantly enhanced by pre‐incubation. Conclusions Ketoconazole inhibition of triazolam α‐ and 4‐hydroxylation, midazolam α‐hydroxylation, testosterone 6β‐hydroxylation, and nifedipine oxidation appeared to be a mixed competitive–noncompetitive process, with the noncompetitive component being dominant but not exclusive. Quantitative estimates of K_i were in the low nanomolar range for all four substrates.     

The xenobiotic inhibitor profile of cytochrome P4502C8

AIMS: To investigate inhibition of recombinant CYP2C8 by: (i) prototypic CYP isoform selective inhibitors (ii) imidazole/triazole antifungal agents (known inhibitors of CYP), and (iii) certain CYP3A substrates (given the apparent overlapping substrate specificity of CYP2C8 and CYP3A). METHODS: CYP2C8 and NADPH-cytochrome P450 oxidoreductase were coexpressed in Spodoptera frugiperda (Sf21) cells using the baculovirus expression system. CYP isoform selective inhibitors, imidazole/triazole antifungal agents and CYP3A substrates were screened for their inhibitory effects on CYP2C8-catalysed torsemide tolylmethylhydroxylation and, where appropriate, the kinetics of inhibition were characterized. The conversion of torsemide to its tolylmethylhydroxy metabolite was measured using an h.p.l.c. procedure. RESULTS: At concentrations of the CYP inhibitor 'probes' employed for isoform selectivity, only diethyldithiocarbamate and ketoconazole inhibited CYP2C8 by > 10%. Ketoconazole, at an added concentration of 10 microM, inhibited CYP2C8 by 89%. Another imidazole, clotrimazole, also potently inhibited CYP2C8. Ketoconazole and clotrimazole were both noncompetitive inhibitors of CYP2C8 with apparent Ki values of 2.5 microM. The CYP3A substrates amitriptyline, quinine, terfenadine and triazolam caused near complete inhibition (82-91% of control activity) of CYP2C8 at concentrations five-fold higher than the known CYP3A Km. Kinetic studies with selected CYP3A substrates demonstrated that most inhibited CYP2C8 noncompetitively. Apparent Ki values for midazolam, quinine, terfenadine and triazolam ranged from 5 to 25 microM. CONCLUSIONS: Inhibition of CYP2C8 occurred at concentrations of ketoconazole and diethyldithiocarbamate normally employed for selective inhibition of CYP3A and CYP2E1, respectively. Some CYP3A substrates have the capacity to inhibit CYP2C8 activity and this may have implications for inhibitory drug interactions in vivo.     

Identification of human cytochrome P450 enzymes involved in the metabolism of IN-1130, a novel activin receptor-like kinase-5 (ALK5) inhibitor

1.?The in vitro metabolism of 3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide (IN-1130), a selective activin receptor-like kinase-5 (ALK5) inhibitor and a candidate drug for fibrotic disease, was studied.

2.?The cytochrome P450s (CYPs) responsible for metabolism of IN-1130 in liver microsomes of rat, mouse, dog, monkey and human, and in human CYP supersomes?, were identified using specific CYP inhibitors. The order of disappearance of IN-1130 in various liver microsomal systems studied was as follows: monkey, mouse, rat, human, and dog.

3.?Five distinct metabolites (M1–M5) were identified in all the above microsomes and their production was substantially inhibited by CYP inhibitors such as SKF-525A and ketoconazole. Among nine human CYP supersomes? examined, CYP3A4, CYP2C8, CYP2D6*1, and CYP2C19 were involved in the metabolism of IN-1130, and the production of metabolites were significantly inhibited by specific CYP inhibitors. IN-1130 disappeared fastest in CYP2C8 supersomes. CYP3A4 produced four metabolites of IN-1130 (M1–M4), whereas supersomes expressing human FMO cDNAs, such as FMO1, FMO3, and FMO5, produced no metabolites.

4.?Hence, it is concluded that metabolism of IN-1130 is mediated by CYP3A4, CYP2C8, CYP2D6*1, and CYP2C19.     

Influence of rifampicin on the expression and function of human intestinal cytochrome P450 enzymes

AIMS: To investigate the potential induction by rifampicin of intestinal CYP2C8, CYP2C9, CYP2D6 and CYP3A4 using preparations of human enterocytes. METHODS: Using a multilumen perfusion catheter shed human enterocytes were collected from 6 healthy subjects before and after 10 days of 600 mg day(-1) oral rifampicin administration. The protein expression of CYP2C8, CYP2C9, CYP2D6 and CYP3A4 as well as that of CYP3A4 mRNA was determined using Western blotting and RT-PCR, respectively. RESULTS: CYP3A4 mRNA expression in shed enterocytes increased from 74.6 +/- 44.2 to 143.2 +/- 68.4 a.u. (P < 0.05, 95% CI: 21.8-115.3). Expression of CYP2C8 and CYP2C9 increased from 5.1 +/- 0.9 to 10.4 +/- 2.3 pmol mg(-1) protein (P < 0.01, 95% CI: 2.8-7.7) and from 4.2 +/- 1.4 to 5.7 +/- 1.1 pmol mg(-1) protein (P < 0.01, 95% CI: 0.6-2.4), respectively. No significant difference in CYP2D6 expression before and during rifampicin intake was observed. Rifampicin administration also resulted in a significant induction of CYP3A4 protein (34.1 +/- 10.7 vs. 113.9 +/- 31.1 pmol mg(-1) protein (P < 0.001, 95% CI: 51.8-107.6)). Ex vivo incubation of enterocyte homogenates with verapamil resulted in a significantly increased production of the metabolites formed via CYP3A4 (D-617: 125.9 +/- 118.8 vs. 277.2 +/- 145.5 pmol min(-1) mg(-1) protein (P < 0.05, 95% CI: 30.1-272.5); norverapamil: 113.0 +/- 57.9 vs. 398.4 +/- 148.2 pmol min(-1) mg(-1) protein (P < 0.05, 95% CI: 47.2-523.6)). CONCLUSION: Our findings indicate that shed enterocytes are a useful tool to study the expression, regulation and function of drug metabolizing enzymes. Induction of intestinal CYP2C8 and CYP2C9 might contribute in part to rifampicin - mediated drug interactions, in addition to their hepatic counterparts and intestinal and hepatic CYP3A4.     

Interactions of phospholipase D and cytochrome P450 protein stability

Previous studies have suggested a relationship between cytochrome P450 (P450) 3A (CYP3A) conformation and the phospholipid composition of the associated membrane. In this study, we utilized a novel microsomal incubation system that mimics many of the characteristics of CYP3A degradation pathway that have been observed in vivo and in cultured cells to study the effects of phospholipid composition on protein stability. We found that addition of phosphatidylcholine-specific phospholipase D (PLD) stabilized CYP3A in this system, but that phosphatidylinositol-specific phospholipase C (PLC) was without effect. Addition of phosphatidic acid also stabilized CYP3A protein in the microsomes. The use of 1,10-phenanthroline (phenanthroline), an inhibitor of PLD activity, decreased CYP3A stability in incubated microsomes. Similarly, 6-h treatment of primary cultures of rat hepatocytes with phenanthroline resulted in nearly complete loss of CYP3A protein. Treatment of rats with nicardipine or dimethylsulfoxide (DMSO), which have been shown to affect CYP3A stability, altered the phospholipid composition of hepatic microsomes. It did not appear, though, that the changes in phospholipid composition that resulted from these in vivo treatments accounted for the change in CYP3A stability observed in hepatic microsomes from these animals.     

Effects of myricetin,an antioxidant,on the pharmacokinetics of losartan and its active metabolite,EXP‐3174, in rats: possible role of cytochrome P450 3A4, cytochrome P450 2C9 and P‐glycoprotein inhibition by myricetin

Objectives The effects of myricetin, a natural flavonoid, on the pharmacokinetics of losartan and its active metabolite, EXP‐3174, were investigated in rats. Losartan and myricetin interact with cytochrome P450 (CYP) enzymes and P‐glycoprotein, and the increase in the use of health supplements may result in myricetin being taken concomitantly with losartan as a combination therapy to treat or prevent cardiovascular diseases. Methods The pharmacokinetic parameters of losartan and EXP‐3174 were determined after oral administration of losartan (9 mg/kg) to rats in the presence or absence of myricetin (0.4, 2 and 8 mg/kg). The effects of myricetin on P‐glycoprotein as well as CYP3A4 and CYP2C9 activity were also evaluated. Key findings Myricetin inhibited CYP3A4 and CYP2C9 enzyme activity with a 50% inhibition concentration of 7.8 and 13.5 µm , respectively. In addition, myricetin significantly enhanced the cellular accumulation of rhodamine 123 in MCF‐7/ADR cells overexpressing P‐glycoprotein in a concentration‐dependent manner. The pharmacokinetic parameters of losartan were significantly altered by myricetin compared with the control. The presence of myricetin (2 or 8 mg/kg) increased the area under the plasma concentration–time curve of losartan by 31.4–61.1% and peak plasma concentration of losartan by 31.8–50.2%. Consequently, the absolute bioavailability of losartan in the presence of myricetin increased significantly (P < 0.05, 2 mg/kg; P < 0.01, 8 mg/kg) compared with the control. There was no significant change in the time to reach the peak plasma concentration, apparent volume of distribution at steady state or terminal half‐life of losartan in the presence of myricetin. Furthermore, concurrent use of myricetin (8 mg/kg) significantly decreased the metabolite–parent area under the plasma concentration–time curve ratio by 20%, implying that myricetin may inhibit the CYP‐mediated metabolism of losartan to its active metabolite, EXP‐3174. Conclusions The enhanced bioavailability of losartan may be mainly due to inhibition of the CYP3A4‐ and CYP2C9‐mediated metabolism of losartan in the small intestine or in the liver, and the P‐glycoprotein efflux pump in the small intestine by myricetin.     

细胞色素P450调节剂对DNA加合物形成的影响

人羊膜上皮细胞ＦＬ系分别接触ａ－萘黄酮（０．６ｍｍｏｌ·Ｌ￣（－１））β－萘黄酮（２０ｐｍｏｌ·Ｌ￣（－１））２４ｈ后，再用苯并（ａ）芘［Ｂ（ａ）Ｐ，１０ｕｍｏｌ·Ｌ￣（－１）］处理２４ｈ，用３２Ｐ后标记技术测定以Ｂ（ａ）－ＤＮＡ加合物。结果发现，阳性对照组，ａ－萘黄酮预处理组及β－萘黄酮预处理组加合物的量分别为（加合物个数／１０’个核苷酸）：４．７±０．２（１００％），１．８±０．９（３８．３％），１６．０±２．２（３４０．１％）．该实验结果直接显示了纳胞色素Ｐ４５０调节剂对肿瘤发生影响的作用水平。亦为药物对致癌物代谢影响的研究提供了一种方法．     

细胞色素氧化酶CYP3A4基因突变与表型研究进展

细胞色素P4503A4在许多内源性及外源性化合物的氧化及还原代谢中起关键作用，对CYP3A4基因表达的研究具有重要意义。本文对CYP3A4基因突变及其表型特征加以总结。     

植物成分与细胞色素P450相互作用的研究进展

细胞色素P450（CYP450）是体内参与药物代谢的重要酶系,其活性在受到诱导或抑制后将干扰药物的作用。植物成分普遍存在于食物与药物中,与CYP450的相互作用将产生广泛影响。总结近年来植物成分与细胞色素P450的7个亚型CYP1A2、CYP2A6、CYP2C9、CYP2C19、CYP2D6、CYP2E1、CYP3A4相互作用的研究结果,为临床上合理用药提供参考。     

The time-dependent effects of St John’s wort on cytochrome P450, uridine diphosphate-glucuronosyltransferase,glutathione S-transferase,and NAD(P)H-quinone oxidoreductase in mice

Hypericum perforatum [St. John’s wort (SJW)] is known to cause a drug interaction with the substrates of cytochrome P450 (P450, CYP) isoforms, mainly CYP3A. This study aims to determine the dose response and time course of the effects of SJW extract on P450s, UDP-glucuronosyltransferase (UGT), glutathione S-transferase (GST), and NAD(P)H-quinone oxidoreductase (NQO) in mice. The oral administration of SJW extract to male mice at 0.6 g/kg/d for 21 days increased hepatic oxidation activity toward a Cyp3a substrate nifedipine. By extending the SJW treatment to 28 days, hepatic nifedipine oxidation (NFO) and warfarin 7-hydroxylation (WOH) (Cyp2c) activities were increased by 95% and 34%, respectively. Immunoblot analysis of liver microsomal proteins revealed that the Cyp2c protein level was elevated by the 28-day treatment. However, the liver microsomal activities of the oxidation of the respective substrates of Cyp1a, Cyp2a, Cyp2b, Cyp2d, and Cyp2e1 remained unchanged. In the kidney, SJW increased the NFO, but not the WOH activity. The extended 28-day treatment did not alter mouse hepatic and renal UGT, GST, and NQO activities. These findings demonstrate that SJW stimulates hepatic and renal Cyp3a activity and hepatic Cyp2c activity and expression. The induction of hepatic Cyp2c requires repeated treatment for a period longer than the initial induction of Cyp3a.