Metabolism of medroxyprogesterone acetate (MPA) via CYP enzymes in vitro and effect of MPA on bleeding time in female rats in dependence on CYP activity in vivo

Medroxyprogesterone acetate (MPA) is a drug commonly used in endocrine therapy for advanced breast cancer, although it is known to cause thrombosis as a serious side effect. Recently, we found that cytochrome P450 3A4 (CYP3A4) mainly catalyzed the metabolism of MPA via CYP in human liver microsomes. However, the metabolic products of MPA in humans and rats have not been elucidated. In addition, it is not clear whether thrombosis could be induced by MPA itself or by its metabolites. In this study, we determined the overall metabolism of MPA as the disappearance of the parent drug from an incubation mixture, and identified the enzymes catalyzing the metabolism of MPA via CYP in rats. Moreover, the effects of CYP-modulators on MPA-induced hypercoagulation in vivo were examined. Intrinsic clearance of MPA in rat liver microsomes was increased by treatment with CYP3A-inducers. The intrinsic clearance of MPA in liver microsomes of rats treated with various CYP-inducers showed a significant correlation with CYP3A activity, but not CYP1A activity, CYP2B activity or CYP2C contents. Among the eight recombinant rat CYPs studied, CYP3A1, CYP3A2 and CYP2A2 catalyzed the metabolism of MPA. However, since CYP3A2 and CYP2A2 are male-specific isoforms, CYP3A1 appears to be mainly involved in the metabolism of MPA in liver microsomes of female rats. In an in vivo study, pretreatment of female rats with SKF525A, an inhibitor of CYPs including CYP3A1, significantly (p < 0.05) enhanced MPA-induced hypercoagulation, whereas pretreatment with phenobarbital, an inducer of CYPs including CYP3A1, reduced it. These findings suggest that CYP-catalyzed metabolism of MPA is mainly catalyzed by CYP3A1 and that MPA-induced hypercoagulation is predominantly caused by MPA itself in female rats.     

Targeted label-free approach for quantification of epoxide hydrolase and glutathione transferases in microsomes

The aim of this study was to investigate the expression and organ distribution of cytochrome P450 (CYP450) enzymes, microsomal epoxide hydrolase (MEH), and microsomal glutathione-S-transferase (MGST 1, 2, 3) in human liver, lung, intestinal, and kidney microsomes by targeted peptide-based quantification using nano liquid chromatography–tandem multiple reaction monitoring (nano LC-MRM). Applying this method, we analyzed 16 human liver microsomes and pooled lung, kidney, and intestine microsomes. Nine of the CYP450s (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5) could be quantified in liver. Except for CYP3A4 and 3A5 existing in intestine, other CYP450s had little content (<0.1 pmol/mg protein) in extrahepatic tissues. MEH and MGSTs could be quantified both in hepatic and in extrahepatic tissues. The highest concentrations of MEH and MGST 1, 2 were found in liver; conversely MGST 3 was abundant in human kidney and intestine compared to liver. The targeted proteomics assay described here can be broadly and efficiently utilized as a tool for investigating the targeted proteins. The method also provides novel CYP450s, MEH, and MGSTs expression data in human hepatic and extrahepatic tissues that will benefit rational approaches to evaluate metabolism in drug development.     

Effect of ethanol on spectral binding, inhibition, and activity of CYP3A4 with an antiretroviral drug nelfinavir

Cytochrome P450 3A4 (CYP3A4) is the most abundant CYP enzyme in the liver and metabolizes approximately 50% of the drugs, including antiretrovirals. Although CYP3A4 induction by ethanol and impact of CYP3A4 on drug metabolism and toxicity is known, CYP3A4-ethanol physical interaction and its impact on drug binding, inhibition, or metabolism is not known. Therefore, we studied the effect of ethanol on binding and inhibition of CYP3A4 with a representative protease inhibitor, nelfinavir, followed by the effect of alcohol on nelfinavir metabolism. Our initial results showed that methanol, ethanol, isopropanol, isobutanol, and isoamyl alcohol bind in the active site of CYP3A4 and exhibit type I spectra. Among these alcohol compounds, ethanol showed the lowest K_D (5.9 ± 0.34 mM), suggesting its strong binding affinity with CYP3A4. Ethanol (20 mM) decreased the K_D of nelfinavir by >5-fold (0.041 ± 0.007 vs. 0.227 ± 0.038 μM). Similarly, 20 mM ethanol decreased the IC₅₀ of nelfinavir by >3-fold (2.6 ± 0.5 vs. 8.3 ± 3.1 μM). These results suggest that ethanol facilitates binding of nelfinavir with CYP3A4. Furthermore, we performed nelfinavir metabolism using LCMS. Although ethanol did not alter k_cat, it decreased the K_m of nelfinavir, suggesting a decrease in catalytic efficiency (k_cat/K_m). This is an important finding because alcoholism is prevalent in HIV-1-infected persons and alcohol is shown to decrease the response to antiretroviral therapy.     

Metabolism of carbamazepine by CYP3A6: a model for in vitro drug interactions studies

Carbamazepine (CBZ) is widely used in the treatment of epilepsy. The drug is principally metabolized by CYPs to 10, 11-epoxy carbamazepine (CBZ-E) but this metabolite more toxic than the parent drug, does possess anticonvulsant properties. In humans, CYP3A4, CYP2C8 and CYP1A2 have been shown to be implicated in CBZ biotransformation. Our purpose was to establish an experimental model to determine the interaction of CBZ with other antiepileptic drugs. We first identified the CYP isoforms that metabolized CBZ in rabbit. We used liver microsomes from rabbit treated with various compounds known to induce principally some CYPs subfamilies. Having tested all the compounds we demonstrated that only the animals treated with CYP3A inducers were able to metabolize CBZ strongly. The CBZ biotransformation was inhibited by anti CYP3A antibodies. All the CYP3A subfamily substrates specifically decrease CBZ-E formation. In our experiment we did not observe any inhibition with CYP2C substrate. These data provide evidence that in rabbit the CYP3A subfamily is primarily involved in CBZ metabolism. Using this model we investigated the interaction of CBZ with phenobarbital, phenytoin, ethosuccimide, primidone, progabide, vigabatrin and lamotrigine.     

Effect of Methamphetamine on Spectral Binding,Ligand Docking and Metabolism of Anti-HIV Drugs with CYP3A4

Cytochrome P450 3A4 (CYP3A4) is the major drug metabolic enzyme, and is involved in the metabolism of antiretroviral drugs, especially protease inhibitors (PIs). This study was undertaken to examine the effect of methamphetamine on the binding and metabolism of PIs with CYP3A4. We showed that methamphetamine exhibits a type I spectral change upon binding to CYP3A4 with δA_max and K_D of 0.016±0.001 and 204±18 μM, respectively. Methamphetamine-CYP3A4 docking showed that methamphetamine binds to the heme of CYP3A4 in two modes, both leading to N-demethylation. We then studied the effect of methamphetamine binding on PIs with CYP3A4. Our results showed that methamphetamine alters spectral binding of nelfinavir but not the other type I PIs (lopinavir, atazanavir, tipranavir). The change in spectral binding for nelfinavir was observed at both δA_max (0.004±0.0003 vs. 0.0068±0.0001) and K_D (1.42±0.36 vs.2.93±0.08 μM) levels. We further tested effect of methamphetamine on binding of 2 type II PIs; ritonavir and indinavir. Our results showed that methamphetamine alters the ritonavir binding to CYP3A4 by decreasing both the δA_max (0.0038±0.0003 vs. 0.0055±0.0003) and K_D (0.043±0.0001 vs. 0.065±0.001 nM), while indinavir showed only reduced K_D in presence of methamphetamine (0.086±0.01 vs. 0.174±0.03 nM). Furthermore, LC-MS/MS studies in high CYP3A4 human liver microsomes showed a decrease in the formation of hydroxy ritonavir in the presence of methamphetamine. Finally, CYP3A4 docking with lopinavir and ritonavir in the absence and presence of methamphetamine showed that methamphetamine alters the docking of ritonavir, which is consistent with the results obtained from spectral binding and metabolism studies. Overall, our results demonstrated differential effects of methamphetamine on the binding and metabolism of PIs with CYP3A4. These findings have clinical implication in terms of drug dose adjustment of antiretroviral medication, especially with ritonavir-boosted antiretroviral therapy, in HIV-1-infected individuals who abuse methamphetamine.     

Stereoselective metabolism of methadone N-demethylation by cytochrome P4502B6 and 2C19

Methadone is a clinically used opioid agonist that is oxidatively metabolized by cytochrome P450 (CYP) isoforms to a stable metabolite, EDDP. Methadone is a chiral drug administered as the racemic mixture of (R)-(-)- and (S)-(+)-methadone, but (R)-methadone is the active isomer. The cytochrome P450 (CYP) isoform involved in methadone's metabolism is thought to be CYP3A4, but human drug-drug interaction studies are not consistent with this. The ability of the common human drug-metabolizing CYPs (obtained from baculovirus-infected insect cell supersomes) to generate 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrilidine (EDDP) from racemic methadone was examined and then determined if the CYP isoforms metabolized methadone stereoselectively. Only CYP2B6, 2C19, and 3A4 generated measurable EDDP from 1 microg/ml of racemic methadone. The hierarchy of EDDP generation was CYP2B6 > CYP2C19 >/= CYP3A4. At 10 microg/ml of methadone, CYP2C9 and CYP2D6 also generated EDDP, but in at least 10-fold lower quantities than CYP2B6. Michaelis-Menten kinetic data demonstrated that CYP2B6 had the highest V(max) (44 ng/min/10pmol) and the lowest K(m) (12.6 microg/ml) for EDDP formation of all the CYP isoforms. In human liver microsomes with high and low CYP2B6 expression but equivalent CYP3A4 expression, high CYP2B6 expression microsomes generated twice the amount of EDDP from 10 microg/ml of methadone than low CYP2B6 expression microsomes. When stereoselective metabolism of racemic methadone by CYP2B6, 2C19, and 3A4 was examined using an enantiospecific methadone assay, CYP2B6 preferentially metabolized (S)-methadone, CYP2C19 preferentially metabolized (R)-methadone, and CYP3A4 showed no preference. These data suggest that multiple CYPs metabolized methadone but CYP2B6 had the highest V(max)/K(m). In addition, only CYP2B6 and 2C19 showed stereoselective metabolism. Our data could explain why the plasma concentration ratio of R/S methadone is variable and why drugs that induce CYP2B6 such as nevirapine and efavirenz also induce methadone metabolism, while the CYP3A4 inducer rifabutin has no effect on methadone pharmacokinetics.     

Determination of N-desethylamodiaquine by hydrophilic interaction liquid chromatography with tandem mass spectrometry: application to in vitro drug metabolism studies

The antimalarial drug amodiaquine is extensively metabolized to N-desethylamodiaquine (DEAQ) by cytochrome P450 2C8 (CYP2C8). DEAQ formation is an enzyme specific reaction that is used to quantify in vitro CYP2C8 activity. A rapid and sensitive method for the determination of DEAQ in human liver microsomes was developed using hydrophilic interaction liquid chromatography/tandem mass spectrometry (HILIC-MS/MS). Microsomal incubation samples were processed by protein precipitation with acetonitrile. The analytes were separated on a BETASIL Silica-100 (50mmx2.1mm, 5microm) column by isocratic elution at a flow rate of 220microl/min with a mobile phase consisting of 85% acetonitrile containing 5mM ammonium acetate and 0.1% formic acid. Detection was by positive electrospray ionization on a TSQ Quantum Discovery triple quadrupole mass spectrometer operated in the selective reaction monitoring mode. The precursor-product ion pair was m/z 328-->283 for DEAQ and m/z 331-->283 for DEAQ-d(3). The lower limit of quantification was 10nM for DEAQ and linearity was observed over the concentration range of 10-1500nM. Intra- and inter-day accuracy and precision were within 3.4 and 7.0%, respectively. The method was successfully applied to CYP2C8 drug metabolism studies in pooled human liver microsomes.     

Sorafenib Metabolism Is Significantly Altered in the Liver Tumor Tissue of Hepatocellular Carcinoma Patient

Background

Sorafenib, the drug used as first line treatment for hepatocellular carcinoma (HCC), is metabolized by cytochrome P450 (CYP) 3A4-mediated oxidation and uridine diphosphate glucuronosyl transferase (UGT) 1A9-mediated glucuronidation. Liver diseases are associated with reduced CYP and UGT activities, which can considerably affect drug metabolism, leading to drug toxicity. Thus, understanding the metabolism of therapeutic compounds in patients with liver diseases is necessary. However, the metabolism characteristic of sorafenib has not been systematically determined in HCC patients.

Methods

Sorafenib metabolism was tested in the pooled and individual tumor hepatic microsomes (THLMs) and adjacent normal hepatic microsomes (NHLMs) of HCC patients (n = 18). Commercial hepatic microsomes (CHLMs) were used as a control. In addition, CYP3A4 and UGT1A9 protein expression in different tissues were measured by Western blotting.

Results

The mean rates of oxidation and glucuronidation of sorafenib were significantly decreased in the pooled THLMs compared with those in NHLMs and CHLMs. The maximal velocity (V_max) of sorafenib oxidation and glucuronidation were approximately 25-fold and 2-fold decreased in the pooled THLMs, respectively, with unchanged K_m values. The oxidation of sorafenib in individual THLMs sample was significantly decreased (ranging from 7 to 67-fold) than that in corresponding NHLMs sample. The reduction of glucuronidation in THLMs was observed in 15 out of 18 patients’ samples. Additionally, the level of CYP3A4 and UGT1A9 expression were both notably decreased in the pooled THLMs.

Conclusions

Sorafenib metabolism was remarkably decreased in THLMs. This result was associated with the down regulation of the protein expression of CYP3A4 and UGT1A9.     

Metabolic transformations of antitumor imidazoacridinone, C-1311, with microsomal fractions of rat and human liver

The imidazoacridinone derivative C-1311 is an antitumor agent in Phase II clinical trials. The molecular mechanism of enzymatic oxidation of this compound in a peroxidase model system was reported earlier. The present studies were performed to elucidate the role of rat and human liver enzymes in metabolic transformations of this drug. C-1311 was incubated with different fractions of liver cells and the reaction mixtures were analyzed by RP-HPLC. We showed that the drug was more sensitive to metabolism with microsomes than with cytosol or S9 fraction of rat liver cells. Incubation of C-1311 with microsomes revealed the presence of four metabolites. Their structures were identified as dealkylation product, M0, as well as a dimer-like molecule, M1. Furthermore, we speculate that the hydroxyl group was most likely substituted in metabolite M3. It is of note that a higher rate of transformation was observed for rat than for human microsomes. However, the differences in metabolite amounts were specific for each metabolite. The reactivity of C-1311 with rat microsomes overexpressing P450 isoenzymes, of CYP3A and CYP4A families was higher than that with CYP1A and CYP2B. Moreover, the M1 metabolite was selectively formed with CYP3A, whereas M3 with CYP4A. In conclusion, this study revealed that C-1311 varied in susceptibility to metabolic transformation in rat and human cells and showed selectivity in the metabolism with P450 isoenzymes. The obtained results could be useful for preparing the schedule of individual directed therapy with C-1311 in future patients.     

Stereoselective Interaction Between Tetrahydropalmatine Enantiomers and CYP Enzymes in Human Liver Microsomes

Tetrahydropalmatine (THP), with one chiral center, is an alkaloid that possesses analgesic and many other pharmacological actives. The aim of the present study is to investigate stereoselective metabolism of THP enantiomers in human liver microsomes (HLM) and elucidate which cytochrome P450 (CYP) isoforms contribute to the stereoselective metabolism in HLM. Additionally, the inhibitions of THP enantiomers on activity of CYP enzymes are also investigated. The results demonstrated that (+)‐THP was preferentially metabolized by HLM. Ketoconazole (inhibitor of CYP3A4/5) inhibited metabolism of (?)‐THP or (+)‐THP at same degree, whereas the inhibition of fluvoxamine (inhibitor of CYP1A2) on metabolism of (+)‐THP was greater than that of (?)‐THP; moreover, the metabolic rate of (+)‐THP was 5.3‐fold of (?)‐THP in recombinant human CYP1A2. Meanwhile, THP enantiomers did not show obvious inhibitory effect on the activity of various CYP isoforms (CYP1A2, 2A6, 2C8, 2C9, 2C19, 2E1, and 3A4/5), whereas (?)‐THP, but not (+)‐THP, significantly inhibited the activity of CYP2D6 with the Ki value of 6.42 ± 0.38 μM. The results suggested that THP enantiomers were predominantly metabolized by CYP3A4/5 and CYP1A2 in HLM, and (+)‐THP was preferentially metabolized by CYP1A2, whereas CYP3A4/5 contributed equally to metabolism of (?)‐THP or (+)‐THP. Besides, the inhibition of CYP2D6 by (?)‐THP may cause drug–drug interaction, which should be considered. Chirality 25:43–47, 2013. © 2012 Wiley Periodicals, Inc.     

A gel-free MS-based quantitative proteomic approach accurately measures cytochrome P450 protein concentrations in human liver microsomes

The human cytochrome P450 (P450) superfamily consists of membrane-bound proteins that metabolize a myriad of xenobiotics and endogenous compounds. Quantification of P450 expression in various tissues under normal and induced conditions has an important role in drug safety and efficacy. Conventional immunoquantification methods have poor dynamic range, low throughput, and a limited number of specific antibodies. Recent advances in MS-based quantitative proteomics enable absolute protein quantification in a complex biological mixture. We have developed a gel-free MS-based protein quantification strategy to quantify CYP3A enzymes in human liver microsomes (HLM). Recombinant protein-derived proteotypic peptides and synthetic stable isotope-labeled proteotypic peptides were used as calibration standards and internal standards, respectively. The lower limit of quantification was approximately 20 fmol P450. In two separate panels of HLM examined (n = 11 and n = 22), CYP3A, CYP3A4 and CYP3A5 concentrations were determined reproducibly (CV or=0.87) and marker activities (r(2)>or=0.88), including testosterone 6beta-hydroxylation (CYP3A), midazolam 1'-hydroxylation (CYP3A), itraconazole 6-hydroxylation (CYP3A4) and CYP3A5-mediated vincristine M1 formation (CYP3A5). Taken together, our MS-based method provides a specific, sensitive and reliable means of P450 protein quantification and should facilitate P450 characterization during drug development, especially when specific substrates and/or antibodies are unavailable.     

Quantitative analysis of cytochrome P450 isoforms in human liver microsomes by the combination of proteomics and chemical probe‐based assay

Cytochrome P450 (CYP) is one of the most important drug‐metabolizing enzyme families, which participates in the biotransformation of many endogenous and exogenous compounds. Quantitative analysis of CYP expression levels is important when studying the efficacy of new drug molecules and assessing drug–drug interactions in drug development. At present, chemical probe‐based assay is the most widely used approach for the evaluation of CYP activity although there are cross‐reactions between the isoforms with high sequence homologies. Therefore, quantification of each isozyme is highly desired in regard to meeting the ever‐increasing requirements for carrying out pharmacokinetics and personalized medicine in the academic, pharmaceutical, and clinical setting. Herein, an absolute quantification method was employed for the analysis of the seven isoforms CYP1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1 using a proteome‐derived approach in combination with stable isotope dilution assay. The average absolute amount measured from twelve human liver microsomes samples were 39.3, 4.3, 54.0, 4.6, 10.3, 3.0, and 9.3 (pmol/mg protein) for 1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1, respectively. Importantly, the expression level of CYP3A4 showed high correlation (r = 0.943, p < 0.0001) with the functional activity, which was measured using bufalin—a highly selective chemical probe we have developed. The combination of MRM identification and analysis of the functional activity, as in the case of CYP3A4, provides a protocol which can be extended to other functional enzyme studies with wide application in pharmaceutical research.     

Metabolic activity and mRNA levels of human cardiac CYP450s involved in drug metabolism

Background

Tissue-specific expression of CYP450s can regulate the intracellular concentration of drugs and explain inter-subject variability in drug action. The overall objective of our study was to determine in a large cohort of samples, mRNA levels and CYP450 activity expressed in the human heart.

Methodology

CYP450 mRNA levels were determined by RTPCR in left ventricular samples (n = 68) of explanted hearts from patients with end-stage heart failure. Samples were obtained from ischemic and non-ischemic hearts. In some instances (n = 7), samples were available from both the left and right ventricles. A technique for the preparation of microsomes from human heart tissue was developed and CYP450-dependent activity was determined using verapamil enantiomers as probe-drug substrates.

Principal Findings

Our results show that CYP2J2 mRNA was the most abundant isoform in all human heart left ventricular samples tested. Other CYP450 mRNAs of importance were CYP4A11, CYP2E1, CYP1A1 and CYP2C8 mRNAs while CYP2B6 and CYP2C9 mRNAs were present at low levels in only some of the hearts analyzed. CYP450 mRNAs did not differ between ischemic and non-ischemic hearts and appeared to be present at similar levels in the left and right ventricles. Incubation of verapamil with heart microsomes led to the formation of nine CYP450-dependent metabolites: a major finding was the observation that stereoselectivity was reversed compared to human liver microsomes, in which the R-enantiomer is metabolized to a greater extent.

Conclusions

This study determined cardiac mRNA levels of various CYP450 isozymes involved in drug metabolism and demonstrated the prevalent expression of CYP2J2 mRNA. It revealed that cardiomyocytes can efficiently metabolize drugs and that cardiac CYP450s are highly relevant with regard to clearance of drugs in the heart. Our results support the claim that drug metabolism in the vicinity of a drug effector site can modulate drug effects.     

Novel pathways of bile acid metabolism involving CYP3A4

The hepatic predominating cytochrome P450, CYP3A4, plays an essential role in the detoxification of bile acids and is important in pathological conditions such as cholestasis where CYP3A4 is adaptively up-regulated. However, the mechanism that triggers the up-regulation of CYP3A4 is still not clear. In this study, using recombinant CYP3A4 and human liver microsomes, we demonstrate that CYP3A4 can metabolise lithocholic acid into 3-dehydrolithocholic acid, a potent activator of the nuclear receptors, pregnane X receptor and 1,25-dihydroxy vitamin D3 receptor, which are known to regulate the expression of CYP3A4. This process thus provides a feed-forward metabolism of toxic bile acid that may be of importance in maintaining bile acid homeostasis. We also provide evidence for a novel CYP3A4-mediated metabolic pathway of the secondary bile acid deoxycholic acid. Patients treated with the antiepileptic drug carbamazepine, a CYP3A4 inducer, had markedly elevated urinary excretion of 1beta-hydroxydeoxycholic acid compared to healthy controls. The importance of CYP3A4 in this process was verified by incubations with recombinant CYP3A4 and human liver microsomes, both of which efficiently converted deoxycholic acid into 1beta-hydroxydeoxycholic acid. Interestingly, CYP3A4 was also found to be active against the secondary bile acid ursodeoxycholic acid.     

UFLC-MS/MS研究胡椒碱在不同种属肝微粒体中的代谢稳定性和代谢表型

应用体外肝微粒体孵育体系,考察胡椒碱在人、SD大鼠、小鼠、恒河猴和比格犬5个种属肝微粒体中的代谢稳定性,比较代谢的种属差异,确定其在人肝微粒体中的代谢表型。通过UFLC-MS/MS检测方法,测定胡椒碱在各个种属肝微粒体中孵育后的剩余浓度,考察他们的代谢稳定性及体外代谢动力学参数。采用化学抑制法考察胡椒碱在人肝微粒体中的代谢表型。结果表明胡椒碱在人、SD大鼠、小鼠、恒河猴和比格犬的肝微粒体中,半衰期T1/2分别为31. 36、48. 46、138. 60、147. 45、165. 00 min;体外固有清除率CLint分别为0. 0442、0. 0286、0. 0100、0. 0094、0. 0084m L/(m L·mg);在人肝微粒体中,胡椒碱主要被CYP3A4和CYP2C9酶代谢。推测胡椒碱在各种肝微粒体中的代谢均相对较稳定,其中大鼠和人的肝微粒体代谢性质最相近,在后续的实验中可以考虑用大鼠的代谢结果预测人的代谢结果;人肝微粒体中参与胡椒碱代谢的酶主要有CYP3A4和CYP2C9。     

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.     

The stereoselective metabolism of halofantrine to desbutylhalofantrine in the rat: evidence of tissue-specific enantioselectivity in microsomal metabolism

The pharmacokinetics of the antimalarial drug (+/-)-halofantrine are stereoselective in humans and rats. To better understand the stereoselective metabolism of the drug to its primary metabolite, desbutylhalofantrine (DHF), a series of in vitro and in vivo experiments were undertaken in the rat. Formation of (-)-DHF exceeded that of (+)-DHF in liver microsomes [(-):(+) ratio of intrinsic formation clearances = 1.4]. In contrast, in intestinal microsomes no significant stereoselectivity was noted in the formation of the DHF enantiomers. Intestinal microsomes were also less efficient at producing the DHF enantiomers than were liver microsomes. Based on kinetic analysis of the DHF formation, there appeared to be more than one enzyme involved in the biotransformation. (+/-)-Ketoconazole (KTZ) effectively inhibited the formation of both DHF enantiomers by both liver and intestinal microsomes, although the reduction was more marked in liver microsomes. Through a combination of the use of CYP antibodies and recombinant CYP isoenzymes, the involvement of CYP 2B1/2, 3A1, 3A2, 1A1, 2C11, 2C6, 2D1, and 2D2 were implicated in the metabolism of halofantrine to DHF. Of these, CYP3A1/2 and CYP2C11 appeared to be the primary isoenzymes involved, although CYP2C11 showed greater (+)-DHF than (-)-DHF formation, whereas for CYP3A1 it was similar to the isolated rat liver microsomes. In vivo, oral (+/-)-KTZ caused significant increases in plasma halofantrine and decreases in DHF enantiomer plasma concentrations.     

Effects of Radix Astragali and Radix Rehmanniae, the components of an anti-diabetic foot ulcer herbal formula, on metabolism of model CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 probe substrates in pooled human liver microsomes and specific CYP isoforms

The present study investigated the effects of Radix Astragali (RA) and Radix Rehmanniae (RR), the major components of an anti-diabetic foot ulcer herbal formula (NF3), on the metabolism of model probe substrates of human CYP isoforms, CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4, which are important in the metabolism of a variety of xenobiotics. The effects of RA or RR on human CYP1A2 (phenacetin O-deethylase), CYP2C9 (tolbutamide 4-hydroxylase), CYP2D6 (dextromethorphan O-demethylase), CYP2E1 (chlorzoxazone 6-hydroxylase) and CYP3A4 (testosterone 6β-hydroxylase) activities were investigated using pooled human liver microsomes. NF3 competitively inhibited activities of CYP2C9 (IC(50)=0.98mg/ml) and CYP3A4 (IC(50)=0.76mg/ml), with K(i) of 0.67 and 1.0mg/ml, respectively. With specific human CYP2C9 and CYP3A4 isoforms, NF3 competitively inhibited activities of CYP2C9 (IC(50)=0.86mg/ml) and CYP3A4 (IC(50)=0.88mg/ml), with K(i) of 0.57 and 1.6mg/ml, respectively. Studies on RA or RR individually showed that RR was more important in the metabolic interaction with the model CYP probe substrates. RR dose-dependently inhibited the testosterone 6β-hydroxylation (K(i)=0.33mg/ml) while RA showed only minimal metabolic interaction potential with the model CYP probe substrates studied. This study showed that RR and the NF3 formula are metabolized mainly by CYP2C9 and/or CYP3A4, but weakly by CYP1A2, CYP2D6 and CYP2E1. The relatively high K(i) values of NF3 (for CYP2C9 and CYP3A4 metabolism) and RR (for CYP3A4 metabolism) would suggest a low potential for NF3 to cause herb-drug interaction involving these CYP isoforms.