An inhibition study of beauvericin on human and rat cytochrome P450 enzymes and its pharmacokinetics in rats

Beauvericin is a secondary metabolite natural product from microorganisms and has been shown to have a new potential antifungal activity. In this study, the metabolism and inhibition of beauvericin in human liver microsomes (HLM) and rat liver microsomes (RLM) were investigated. The apparent K_m and V_max of beauvericin in HLM were determined by substrate depletion approach and its inhibitory effects on cytochromes P450 (CYP) activities were evaluated using probe substrates, with IC₅₀ and the (K_i) values were 1.2 μM (0.5 μM) and 1.3 μM (1.9 μM), respectively for CYP3A4/5 (midazolam) and CYP2C19 (mephenytoin). Similarly, beauvericin was also a potent inhibitor for CYP3A1/2 (IC₅₀: 1.3 μM) in RLM. Furthermore, the pharmacokinetics of beauvericin in the rat were studied after p.o administration alone and co-administration with ketoconazole, which indicated a pharmacodynamic function may play a role in the synergistic effect on antifungal activity.     

In vitro metabolism of fipronil by human and rat cytochrome P450 and its interactions with testosterone and diazepam

Fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl)sulfinyl]-1H-pyrazole-3-carbonitrile) is a highly active, broad spectrum insecticide from the phenyl pyrazole family, which targets the gamma-amino butyric acid (GABA) receptor. Although fipronil is presently widely used as an insecticide and acaricide, little information is available with respect to its metabolic fate and disposition in mammals. This study was designed to investigate the in vitro human metabolism of fipronil and to examine possible metabolic interactions that fipronil may have with other substrates. Fipronil was incubated with human liver microsomes (HLM) and several recombinant cytochrome P450 (CYP) isoforms obtained from BD Biosciences. HPLC was used for metabolite identification and quantification. Fipronil sulfone was the predominant metabolite via CYP oxidation. The K(m) and V(max) values for human liver microsomes are 27.2 microM and 0.11 nmol/mg proteinmin, respectively; for rat liver microsomes (RLM) the K(m) and V(max) are 19.9 microM and 0.39 nmol/mg proteinmin, respectively. CYP3A4 is the major isoform responsible for fipronil oxidation in humans while CYP2C19 is considerably less active. Other human CYP isoforms have minimal or no activity toward fipronil. Co-expression of cytochrome b(5) (b(5)) is essential for CYP3A4 to manifest high activity toward fipronil. Ketoconazole, a specific inhibitor of CYP3A4, inhibits 78% of the HLM activity toward fipronil at a concentration of 2 microM. Oxidative activity toward fipronil in 19 single-donor HLMs correlated well with their ability to oxidize testosterone. The interactions of fipronil and other CYP3A4 substrates, such as testosterone and diazepam, were also investigated. Fipronil metabolism was activated by testosterone in HLM but not in CYP3A4 Supersomes. Testosterone 6beta-hydroxylation in HLM was inhibited by fipronil. Fipronil inhibited diazepam demethylation but had little effect on diazepam hydroxylation. The results suggest that fipronil has the potential to interact with a wide range of xenobiotics or endogenous chemicals that are CYP3A4 substrates and that fipronil may be a useful substrate for the characterization of CYP3A4 in HLM.     

In vitro metabolism of carbofuran by human, mouse, and rat cytochrome P450 and interactions with chlorpyrifos, testosterone, and estradiol

Carbofuran is a carbamate pesticide used in agricultural practice throughout the world. Its effect as a pesticide is due to its ability to inhibit acetylcholinesterase activity. Though carbofuran has a long history of use, there is little information available with respect to its metabolic fate and disposition in mammals. The present study was designed to investigate the comparative in vitro metabolism of carbofuran from human, rat, and mouse liver microsomes (HLM, RLM, MLM, respectively), and characterize the specific enzymes involved in such metabolism, with particular reference to human metabolism. Carbofuran is metabolized by cytochrome P450 (CYP) leading to the production of one major ring oxidation metabolite, 3-hydroxycarbofuran, and two minor metabolites. The affinity of carbofuran for CYP enzymes involved in the oxidation to 3-hydroxycarbofuran is significantly less in HLM (K_m = 1.950 mM) than in RLM (K_m = 0.210 mM), or MLM (K_m = 0.550 mM). Intrinsic clearance rate calculations indicate that HLM are 14-fold less efficient in the metabolism of carbofuran to 3-hydroxycarbofuran than RLM or MLM. A screen of 15 major human CYP isoforms for metabolic ability with respect to carbofuran metabolism demonstrated that CYP3A4 is the major isoform responsible for carbofuran oxidation in humans. CYP1A2 and 2C19 are much less active while other human CYP isoforms have minimal or no activity toward carbofuran. In contrast with the human isoforms, members of the CYP2C family in rats are likely to have a primary role in carbofuran metabolism. Normalization of HLM data with the average levels of each CYP in native HLM, indicates that carbofuran metabolism is primarily mediated by CYP3A4 (percent total normalized rate (% TNR) = 77.5), although CYP1A2 and 2C19 play ancillary roles (% TNR = 9.0 and 6.0, respectively). This is substantiated by the fact that ketoconazole, a specific inhibitor of CYP3A4, is an excellent inhibitor of 3-hydroxycarbofuran formation in HLM (IC₅₀: 0.31 μM). Chlorpyrifos, an irreversible non-competitive inhibitor of CYP3A4, inhibits the formation of 3-hydroxycarbofuran in HLM (IC₅₀: 39 μM). The use of phenotyped HLM demonstrated that individuals with high levels of CYP3A4 have the greatest potential to metabolize carbofuran to its major metabolite. The variation in carbofuran metabolism among 17 single-donor HLM samples is over 5-fold and the best correlation between CYP isoform activity and carbofuran metabolism was observed with CYP3A4 (r² = 0.96). The interaction of carbofuran and the endogenous CYP3A4 substrates, testosterone and estradiol, were also investigated. Testosterone metabolism was activated by carbofuran in HLM and CYP3A4, however, less activation was observed for carbofuran metabolism by testosterone in HLM and CYP3A4. No interactions between carbofuran and estradiol metabolism were observed.     

Effect of buprenorphine on CYP3A activity in rat and human liver microsomes

Buprenorphine is a partial opioid agonist available in France as an alternative to methadone in the treatment of opiate-dependent individuals. Twenty deaths have been reported in patients who have ingested buprenorphine in combination with benzodiazepines. Since buprenorphine and many benzodiazepines are CYP3A substrates, the effect of buprenorphine on CYP3A activity was examined in order to assess the likelihood of a pharmacokinetic interaction. The formation of 6beta-hydroxytestosterone was measured in dexamethasone-induced rat liver microsomes and in human liver microsomes under control conditions and in the presence of buprenorphine. Buprenorphine was found to be a weak inhibitor of CYP3A with a 50% decrease in enzyme activity occurring at a concentration of 118 microM (IC50) in human liver microsomes. IC50 was 0.3 microM for ketoconazole in the same system. Since the IC50 for buprenorphine is roughly 2000 times higher than typical plasma concentrations, this drug is unlikely to cause clinically significant inhibition of CYP3A in patients. Excessive CNS depression due to the combination of buprenorphine and benzodiazepines is most likely due to additive or synergistic pharmacologic effect unrelated to a pharmacokinetic interaction between the drugs.     

In vitro metabolism of BYZX in human liver microsomes and the structural elucidation of metabolite by liquid chromatography-mass spectrometry method

In vitro phase I metabolism of BYZX, a novel central-acting cholinesterase inhibitor for the treatment of the symptoms of Alzheimer's disease, was studied in human liver microsomes (HLM) and the metabolite formation pathways were investigated by chemical inhibition experiments and correlation analysis. The residual concentration of substrate and the metabolite formed in incubate were determined by HPLC method. The calibration curves of BYZX were linear over the concentration range from 5.07 microM to 200.74 microM. The relative standard deviations of within day and between day were less than 5% (n=5). The limit of detection (LOD) was 0.18 microg/mL (S/N=3) and the limit of quantification (LOQ) was 0.55 microg/mL (R.S.D.=5.2%, n=5). The determination recoveries of BYZX were in the range of 98.2-104.8%. The apparent K(m) of BYZX in HLM was 53.25+/-17.2 microM, the V(max) was 0.94+/-0.77 microM/min/mg protein, and the intrinsic clearance value (Cl(int)) was 0.018+/-0.02 mL/min/mg protein. Ketoconazole and cyclosporin A were the most potent inhibitors on BYZX metabolism in HLM with IC(50) being 0.89 microM and 18.17 microM, respectively. And the inhibition constant (K(i)) of ketoconazole was 0.42 microM. The metabolite of BYZX was N-des-ethyl-BYZX elucidated by LC-MS-MS. The results demonstrated that the developed HPLC method was reliability, simple technique, and was applicable to be used for the researches of in vitro metabolism of BYZX. CYP3A4 was the major isozyme responsible for BYZX metabolism; N-dealkylation was the major metabolic pathway of BYZX. The predominant metabolite of BYZX was N-des-ethyl-BYZX detected in vitro phase I metabolism in HLM.     

Stereoselective metabolism of tetrahydropalmatine enantiomers in rat liver microsomes

Tetrahydropalmatine (THP), with one chiral center, is an active alkaloid ingredient in Rhizoma Corydalis. The aim of the present paper is to study whether THP enantiomers are metabolized stereoselectively in rat, mouse, dog, and monkey liver microsomes, and then, to elucidate which Cytochrome P450 (CYP) isoforms are predominately responsible for the stereoselective metabolism of THP enantiomers in rat liver microsomes (RLM). The results demonstrated that (+)-THP was preferentially metabolized by liver microsomes from rats, mice, dogs, and monkeys, and the intrinsic clearance (Cl(int)) ratios of (+)-THP to (-)-THP were 2.66, 2.85, 4.24, and 1.67, respectively. Compared with the metabolism in untreated RLM, the metabolism of (-)-THP and (+)-THP was significantly increased in dexamethasone (Dex)-induced and β-naphthoflavone (β-NF)-induced RLM; meanwhile, the Cl(int) ratios of (+)-THP to (-)-THP in Dex-induced and β-NF-induced RLM were 5.74 and 0.81, respectively. Ketoconazole had stronger inhibitory effect on (+)-THP than (-)-THP, whereas fluvoxamine had stronger effect on (-)-THP in untreated and Dex-induced or β-NF-induced RLM. The results suggested that THP enantiomers were predominately metabolized by CYP3A1/2 and CYP1A2 in RLM, and CYP3A1/2 preferred to metabolize (+)-THP, whereas CYP1A2 preferred (-)-THP.     

Metabolism and transporter-mediated drug-drug interactions of the endothelin-A receptor antagonist CI-1034

CI-1034, an endothelin-A receptor antagonist was being developed for pulmonary hypertension. Drug-drug interaction studies using human hepatic microsomes were conducted to assess CYP1A2, CYP2C9, CYP2C19, CYP3A4 and CYP2D6 inhibition potential; CYP3A4 induction potential was evaluated using primary human hepatocytes. CI-1034 moderately inhibited CYP2C9 (IC(50) 39.6 microM) and CYP3A4 activity (IC(50) 21.6 microM); CYP3A4 inhibition was metabolism-dependent. In human hepatocytes, no increase in CYP3A4 activity was observed in vitro, while mRNA was induced 15-fold, similar to rifampin, indicating that CI-1034 is both an inhibitor and inducer of CYP3A4. A 2-week clinical study was conducted to assess pharmacokinetics, pharmacodynamics and safety. No significant changes were observed in [formula: see text] between days 1 and 14. However, reversible elevations of serum liver enzymes were observed with a 50mg BID dose and the program was terminated. To further understand the interactions of CI-1034 in the liver and possible mechanisms of the observed hepatotoxicity, we evaluated the effect of CI-1034 on bile acid transport and previously reported that CI-1034 inhibited biliary efflux of taurocholate by 60%, in vitro. This indicated that inhibition of major hepatic transporters could be involved in the observed hepatotoxicity. We next evaluated the in vitro inhibition potential of CI-1034 with the major hepatic transporters OATP1B1, OATP1B3, OATP2B1, MDR1, MRP2 and OCT. CI-1034 inhibited OATP1B1 (K(i) 2 microM), OATP1B3 (K(i) 1.8 microM) and OATP2B1 activity (K(i) 3.3 microM) but not OCT, MDR1 or MRP2 mediated transport. Our data indicates that CI-1034 is an inhibitor of major hepatic transporters and inhibition of bile efflux may have contributed to the observed clinical hepatotoxicity. We recommend that in vitro drug-drug interaction panels include inhibition and induction studies with transporters and drug metabolizing enzymes, to more completely assess potential in vivo interactions or toxicity.     

紫杉醇对食蟹猴和人CYP1A2、CYP3A4及CYP2A6酶代谢的影响

目的探讨紫杉醇对食蟹猴和人肝微粒体CYP1A2、CYP2A6和CYP3A4酶活性的影响。方法采用食蟹猴和人肝脏微粒体,分别以非那西汀、睾丸酮和香豆素分别作为CYP1A2、CYP2A6、CYP3A4的底物,建立CYP1A2、CYP2A6和CYP3A4体外代谢体系。采用不同浓度的紫杉醇分别与上述3种底物共同孵育于肝微粒体代谢体系中。用HPLC法分别测定各底物的代谢产物扑热息痛、6β-羟基睾丸酮、7-羟基香豆素的产生量,计算IC50值,以评估紫杉醇对CYP1A2、CYP2A6和CYP3A4代谢的影响。结果紫杉醇对食蟹猴肝微粒体3种酶的IC50值分别为570±5.9μmol/L、140±2.9μmol/L和无影响;紫杉醇对人肝微粒体3种酶的IC50值分别为193±6.6μmol/L、253±3.6μmol/L和24±1.6μmol/L。结论紫杉醇对食蟹猴肝微粒体CYP1A2和CYP3A4活性具有一定的抑制作用,但对CYP2A6酶的活性几乎没有影响。紫杉醇对人肝微粒体CYP1A2和CYP3A4活性的抑制作用较弱,但对CYP2A6酶的活性抑制作用较强,提示临床上紫杉醇与作为上述酶底物的药物联合用药时应慎重,以避免因中西药物相互作用所导致的不良反应发生。     

Hyperforin and its analogues inhibit CYP3A4 enzyme activity

Literature indicates that herb-drug interaction of St. John's wort is largely due to increased metabolism of the co-administered drugs that are the substrates of cytochrome P450 (CYP) 3A4 enzyme, alteration of the activity and/or expression of the enzyme. The major St. John's wort constituents, acylphloroglucinols, were evaluated for their effects on CYP3A4 enzyme activity to investigate their roles in herb-drug interaction. Hyperforin and four oxidized analogues were isolated from the plant and fully characterized by mass spectral and NMR analysis. These acylphloroglucinols inhibited activity of CYP3A4 enzyme potently in the fluorometric assay using the recombinant enzyme. Furoadhyperforin (IC(50) 0.072 microM) was found to be the most potent inhibitor of CYP3A4 enzyme activity, followed by furohyperforin isomer 1 (IC(50) 0.079 microM), furohyperforin isomer 2 (IC(50) 0.23 microM), hyperforin (IC(50) 0.63 microM) and furohyperforin (IC(50) 1.3 microM). As the acylphloroglucinols are potent inhibitors of the CYP3A4 enzyme, their modulation of the enzyme activity is unlikely to be involved in increased drug metabolism by St. John's wort.     

Description of a 96-well plate assay to measure cytochrome P4503A inhibition in human liver microsomes using a selective fluorescent probe

The standard method to evaluate CYP3A inhibition is to study the conversion of the specific CYP3A probe testosterone to its 6 beta-hydroxy metabolite in human liver microsomes, in the absence and presence of potential inhibitors. Quantification of the 6 beta-hydroxy metabolite is achieved by HPLC resulting in a tedious and time-consuming assay. In order to increase the P450 inhibition throughput, efforts were made to find a CYP3A probe that would produce a fluorescent metabolite. This paper reports the discovery of DFB as a potential CYP3A fluorescent probe. DFB was significantly metabolized in human microsomes (approximately 1-2 nmol/(min. mg protein)) to give the fluorescent compound DFH. The involvement of CYP3A in the metabolism of DFB was determined using multiple approaches. First, incubations conducted with microsomes made from cell lines expressing single CYPs (Gentest Supersomes) indicated that CYP3A played a major role in the metabolism of DFB. Secondly, immunoinhibition studies conducted with CYP3A antibody resulted in >95% inhibition of DFB metabolism in HLM. Thirdly, inhibition studies with specific CYP1A1, 1A2, 2C8/9, 2C19, 2D6, and 2E1 chemical inhibitors did not suppress DFB activity in HLM. However, ketoconazole, miconazole, nicardipine, and nifedipine, all known CYP3A inhibitors, completely abolished the formation of DFH in HLM. The potency of several inhibitors determined using DFB and testosterone as CYP3A probes was consistent (R = 0.98). Finally, a good agreement was obtained for the formation of DFH and production of 6 beta-hydroxytestosterone when DFB and testosterone were incubated separately with various human liver microsome preparations (R = 0.94, N = 11). In order to use DFH as a fluorescent CYP3A marker in a 96-well plate format, it was important to remove the excess of NADPH at the end of the incubation because the fluorescence of NADPH interferes with DFH detection. This was achieved by adding oxidized glutathione and glutathione reductase to convert NADPH to NADP(+) which is not fluorescent. The liquid-handling steps were fully automated in a 96-well plate format and a template was designed to generate IC(50) curves and to address potential fluorescent interferences from the test compounds. The assay was found to be reproducible (intraday variability <10% and interday variability indicated less than a 2-fold variation in the IC(50) values) and is now routinely used in our laboratory to evaluate CYP3A inhibition of NCEs.     

Effect of natural phenols on the catalytic activity of cytochrome P450 2E1

The effect of protocatechuic acid, tannic acid and trans-resveratrol on the activity of p-nitrophenol hydroxylase (PNPH), an enzymatic marker of CYP2E1, was examined in liver microsomes from acetone induced mice. trans-Resveratrol was found to be the most potent inhibitor (IC(50) = 18.5 +/- 0.4 microM) of PNPH, while protocatechuic acid had no effect on the enzyme activity. Tannic acid with IC(50) = 29.6 +/- 3.3 microM showed mixed- and trans-resveratrol competitive inhibition kinetics (K(i) = 1 microM and 2.1 microM, respectively). Moreover, trans-resveratrol produced a NADPH-dependent loss of PNPH activity, suggesting mechanism-based CYP2E1 inactivation. These results indicate that trans-resveratrol and tannic acid may modulate cytochrome P450 2E1 and influence the metabolic activation of xenobiotics mediated by this P450 isoform.     

Green chemistry appended synthesis,metabolic stability and pharmacokinetic assessment of medicinally important chromene dihydropyrimidinones

A green chemistry approach has been developed for the synthesis of chromene dihydropyrimidinone (CDHPM) using recyclable Fe/Al pillared clay catalyst. Pharmacokinetic parameters like aqueous solubility, lipophilicity, P-glycoprotein (P-gp) ATPase activity, permeability, plasma protein binding, red blood cell (RBC) partitioning, metabolic stability in liver microsomes and in silico computations have been studied for the most potent anticancer chromene dihydropyrimidinone hybrid 1. This compound exhibited low solubility, optimum lipophilicity, no P-gp inhibitory activity, intermediate permeability, high plasma protein binding, low RBC partitioning, acceptable metabolic stability in rat liver microsomes (RLM) as well as human liver microsomes (HLM) with transitional hepatic extraction ratio.     

Differential inhibition of human CYP3A4 and Candida albicans CYP51 with azole antifungal agents

The inhibition by azole antifungals of human cytochrome CYP3A4, the major form of drug metabolising enzyme within the liver, was compared with their inhibitory activity against their target enzyme, Candida albicans sterol 14alpha-demethylase (CYP51), following heterologous expression in Saccharomyces cerevisiae. IC(50) values for ketoconazole and itraconazole CYP3A4 inhibition were 0.25 and 0. 2 microM. These values compared with much lower doses required for the complete inhibition of C. albicans CYP51, where IC(50) values of 0.008 and 0.0076 microM were observed for ketoconazole and itraconazole, respectively. Additionally, stereoselective inhibition of CYP3A4 and CYP51 was observed with enantiomers of the azole antifungal compounds diclobutrazol and SCH39304. In both instances, the RR(+) configuration at their asymmetric carbon centres was most active. Interestingly, the SS(-) enantiomeric form of SCH39304 was inactive and failed to bind CYP3A4, as demonstrable by Type II binding spectra.     

Benzofuran- and furan-2-yl-(phenyl)-3-pyridylmethanols: synthesis and inhibition of P450 aromatase

A series of benzofuran-2-yl-(phenyl)-3-pyridylmethanol derivatives were prepared using an efficient 1-step procedure in good yields. In addition furan-2-yl-(phenyl)-3-pyridylmethanol derivatives were also prepared to determine the effect of the benzene ring in benzofuran with respect to inhibitory activity. The pyridylmethanol derivatives were all evaluated in vitro for inhibitory activity against aromatase (P450(AROM), CYP19), using human placental microsomes. The benzofuran-2-yl-(phenyl)-3-pyridylmethanol derivatives showed good to moderate activity (IC50 = 1.3-25.1 microM), which was either better than or comparable with aminoglutethimide (IC50 = 18.5 microM) but lower than arimidex (IC50 = 0.6 microM), with the 4-methoxyphenyl substituted derivative displaying optimum activity. Molecular modelling of the benzofuran-2-yl-(4-fluorophenyl)-3-pyridylmethanol derivative suggested activity to reside with the (S)-enantiomer. The furan-2-yl-(phenyl)-3-pyridylmethanol derivatives were devoid of activity indicating the essential role of the benzene ring of the benzofuran component for enzyme binding.     

Inhibition of cytochrome P450 activities by oleanolic acid and ursolic acid in human liver microsomes

Oleanolic acid (OA) and ursolic acid (UA), triterpene acids having numerous pharmacological activities including anti-inflammatory, anti-cancer, and hepato-protective effects, were tested for their ability to modulate the activities of several cytochrome P450 (CYP) enzymes using human liver microsomes. OA competitively inhibited CYP1A2-catalyzed phenacetin O-deethylation and CYP3A4-catalyzed midazolam 1-hydroxylation, the major human drug metabolizing CYPs, with IC50 (Ki) values of 143.5 (74.2) microM and 78.9 (41.0) microM, respectively. UA competitively inhibited CYP2C19-catalyzed S-mephenytoin 4'-hydroxylation with an IC50 (Ki) value of 119.7 (80.3) microM. However, other CYPs tested showed no or weak inhibition by both OA and UA. The present study demonstrates that OA and UA have inhibitory effects on CYP isoforms using human liver microsomes. It is thus likely that consumption of herbal medicines containing OA or UA, or administration of OA or UA, can cause drug interactions in humans when used concomitantly with drugs that are metabolized primarily by CYP isoforms. In addition, it appears that the inhibitory effect of OA on CYP1A2 is, in part, related to its anti-inflammatory and anticancer activities.     

In vitro inhibition of human liver drug metabolizing enzymes by second generation antihistamines

Cetirizine, terfenadine, loratadine, astemizole and mizolastine were compared for their ability to inhibit marker activities for CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 and for some glucuronidation isoenzymes in human liver microsomes. The most pronounced effects were observed with terfenadine, astemizole and loratadine which inhibited CYP3A4-mediated testosterone 6beta-hydroxylation (IC50 of 23, 21 and 32 microM, respectively) and CYP2D6-mediated dextromethorphan O-demethylation (IC50 of 18, 36 and 15 microM, respectively). In addition, loratadine markedly inhibited the CYP2C19 marker activity, (S)-mephenytoin 4-hydroxylation (Ki of 0.17 microM). Furthermore, loratadine activated the CYP2C9-catalyzed tolbutamide hydroxylation (ca. 3-fold increase at 30 microM) and inhibited some glucuronidation enzymes. Mizolastine appeared to be a relatively weak and unspecific inhibitor of CYP2E1, CYP2C9, CYP2D6 and CYP3A4 (IC50Ss in the 100 micromolar range). Cetirizine demonstrated no effect on the investigated activities. A comparison of the inhibitory potencies of cetirizine, terfenadine, loratidine, astemizole and mizolastine with their corresponding plasma concentrations in humans suggests that these antihistamines are not likely to interfere with the metabolic clearance of coadministered drugs, with the exception of loratidine, which appears to inhibit CYP2C19 with sufficient potency to warrant additional investigation.     

In vitro metabolism of zolmitriptan in rat cytochromes induced with beta-naphthoflavone and the interaction between six drugs and zolmitriptan

Zolmitriptan is a novel and highly selective 5-HT(1B/1D) receptor agonist used as an acute oral treatment for migraine. There are few reports regarding the in vitro metabolism of zolmitriptan. Previous studies indicated zolmitriptan was metabolized via CYP1A2 in human hepatic microsomes. In order to study the enzyme kinetics and drug interaction, the metabolism of zolmitriptan and possible drug-drug interactions were investigated in rat hepatic microsomes induced with different inducers. An active metabolite, N-demethylzolmitriptan, was detected and another minor, inactive metabolite that was reported in human hepatic microsomes was not detected in this study. The enzyme kinetics for the formation of N-demethylzolmitriptan from zolmitriptan in rat liver microsomes pretreated with BNF were 96+/-22 microM (K(m)), 11+/-3 pmol min(-1)mg protein(-1) (V(max)), and 0.12+/-0.02 microl min(-1)mg protein(-1) (CL(int)). Fluvoxamine and diphenytriazol inhibited zolmitriptan N-demethylase activity catalyzed by CYP1A2 (K(i)=3.8+/-0.3 and 3.2+/-0.1 microM, respectively). Diazepam and propranolol elicited a slight inhibitory effect on the metabolism of zolmitriptan (K(i)=70+/-11 and 90+/-18 microM, respectively). Cimetidine and moclobemide produced no significant effect on the metabolism of zolmitriptan. Fluvoxamine yielded a k(inactivation) value of 0.16 min(-1), and K(i) of 57 microM. The results suggest that rat hepatic microsomes are a reasonable model to study the metabolism of zolmitriptan, although there is a difference in the amount of minor, inactive metabolites between human hepatic microsomes and rat liver microsomes. The results of the inhibition experiments provided information for the interactions between zolmitriptan and drugs co-administrated in clinic, and it is helpful to explain the drug-drug interactions of clinical relevance on enzyme level. This study aso demonstrated that fluvoxamine may be a mechanism-based inactivator of CYP1A2.     

Purification and characterization of rat sterol 14-demethylase P450 (CYP51) expressed in Escherichia coli.

Sterol 14-demethylase P450 (CYP51) is an essential enzyme for sterol biosynthesis by eukaryotes. We have cloned rat and human CYP51 cDNAs [Aoyama, Y., Noshiro, M., Gotoh, O., Imaoka, S., Funae, Y., Kurosawa, N., Horiuchi, T., and Yoshida, Y. (1996) J. Biochem. 119, 926-933]. The cloned rat CYP51 cDNA was expressed in Escherichia coli with modification of the N-terminal amino acid sequence, and the expressed protein (CYP51m) was purified to gel-electrophoretic homogenity. The spectrophotometrically determined specific content of CYP51m was 16 nmol/mg protein and the apparent molecular weight was estimated to be 53,000 on SDS-PAGE. Soret peaks of the oxidized and reduced CO-complex of CYP51m were observed at 417 and 447 nm, respectively. The purified CYP51m catalyzed the 14-demethylation of lanosterol and 24,25-dihydrolanosterol upon reconstitution with NADPH-P450 reductase purified from rat liver microsomes. The apparent K(m) and V(max) values for lanosterol were 10.5 microM and 13.9 nmol/min/nmol P450, respectively, and those for 24, 25-dihydrolanosterol were 20.0 microM and 20.0 nmol/min/nmol P450, respectively. The lanosterol demethylase activity of the reconstituted system of CYP51m was inhibited by ketoconazole, itraconazole and fluconazole with apparent IC(50) values of 0.2, 0.7, and 160 microM, respectively.     

Demethylation of radiolabelled dextromethorphan in rat microsomes and intact hepatocytes.

Liver microsomal preparations are routinely used to predict drug interactions that can occur in vivo as a result of inhibition of cytochrome P450 (CYP)-mediated metabolism. However, the concentration of free drug (substrate and inhibitor) at its intrahepatic site of action, a variable that cannot be directly measured, may be significantly different from that in microsomal incubation systems. Intact cells more closely reflect the environment to which CYP substrates and inhibitors are exposed in the liver, and it may therefore be desirable to assess the potential of a drug to cause CYP inhibition in isolated hepatocytes. The objective of this study was to compare the inhibitory potencies of a series of CYP2D inhibitors in rat liver microsomes and hepatocytes. For this, we developed an assay suitable for rapid analysis of CYP-mediated drug interactions in both systems, using radiolabelled dextromethorphan, a well-characterized probe substrate for enzymes of the CYP2D family. Dextromethorphan demethylation exhibited saturable kinetics in rat microsomes and hepatocytes, with apparent Km and Vmax values of 2.1 vs. 2.8 microM and 0.74 nM x min(-1) per mg microsomal protein vs. 0.11 nM x min(-1) per mg cellular protein, respectively. Quinine, quinidine, pyrilamine, propafenone, verapamil, ketoconazole and terfenadine inhibited dextromethorphan O-demethylation in rat liver microsomes and hepatocytes with IC50 values in the low micromolar range. Some of these compounds exhibited biphasic inhibition kinetics, indicative of interaction with more than one CYP2D isoform. Even though no important differences in inhibitory potencies were observed between the two systems, most inhibitors, including quinine and quinidine, displayed 2-3-fold lower IC50 in hepatocytes than in microsomes. The cell-associated concentrations of quinine and quinidine were found to be significantly higher than those in the extracellular medium, suggesting that intracellular accumulation may potentiate the effect of these compounds. Studies of CYP inhibition in intact hepatocytes may be warranted for compounds that concentrate in the liver as the result of cellular transport.     

Polysaccharide peptides from Coriolus versicolor competitively inhibit model cytochrome P450 enzyme probe substrates metabolism in human liver microsomes

Polysaccharide peptide (PSP), isolated from COV-1 strain of Coriolus versicolor, is commonly used as an adjunct in cancer chemotherapy or health supplement in China. Previous studies have shown that PSP decreased antipyrine clearance and inhibited rat CYP2C11-mediated tolbutamide 4-hydroxylation and in human CYP2C9. In this study, the effects of the water extractable fraction of PSP on the metabolism of model CYP1A2, CYP2D6, CYP2E1 and CYP3A4 probe substrates were investigated in pooled human liver microsomes. PSP (1.25-20μM) dose-dependently decreased CYP1A2-mediated metabolism of phenacetin to paracetamol (IC(50) 19.7μM) and CYP3A4-mediated metabolism of testosterone to 6β-hydroxytestosterone (IC(20) 7.06μM). Enzyme kinetics studies showed the inhibition of CYP1A2 activity was competitive and concentration-dependent (K(i)=18.4μM). Inhibition of testosterone to 6β-hydroxytestosterone was also competitive and concentration-dependent (K(i)=31.8μM). Metabolism of dextromethorphan to dextrorphan (CYP2D6-mediated) and chlorzoxazone to 6-hydroxychlorzoxazone (CYP2E1-mediated) was only minimally inhibited by PSP, with IC(20) values at 15.6μM and 11.9μM, respectively. This study demonstrated that PSP competitively inhibited the CYP1A2- and CYP3A4-mediated metabolism of model probe substrates in human liver microsomes in vitro. The relatively high K(i) values for CYP1A2 and CYP3A4 would suggest a low potential for PSP to cause herb-drug interaction related to these CYP isoforms.