Identification of the cytochrome P450 isoenzymes involved in the metabolism of diazinon in the rat liver

The metabolism of diazinon, an organophosphorothionate pesticide, to diazoxon and pyrimidinol has been studied in incubations with hepatic microsomes from control Sprague–Dawley (SD) rats or SD rats treated with different P450‐specific inducers (phenobarbital, dexamethasone, β‐napthoflavone, and pyrazole). Results obtained indicate an involvement of CYP2C11, CYP3A2, and CYP2B1/2, whereas CYP2E1 and CYP1A1 do not contribute to the pesticide oxidative metabolism. Indeed, diazinon was metabolized by microsomes from control rats; among the inducers, phenobarbital and dexamethasone only increased the production of either metabolites, although to different extents. The production of the two metabolites is self‐limiting, due to P450 inactivation; therefore, the inhibition of CYP‐specific monooxygenase activities after diazinon preincubation has been used to selectively identify the competent CYPs in diazinon metabolism. Results indicate that, after diazinon preincubation, CYP3A2‐catalyzed reactions (2β‐ and 6β‐testosterone hydroxylation) are very efficiently inhibited; CYP2C11‐ and CYP2B1/2‐catalyzed reactions (2α‐ and 16β‐testosterone hydroxylation, respectively) are weakly inhibited, while CYP2E1‐, CYP2A1/2‐, and CYP1A1/2‐related activities were unaffected. Results obtained by using chemical inhibitors or antibodies selectively active against specific CYPs provide a direct evidence for the involvement of CYP2C11, CYP3A2, and CYP2B1/2, indicating that each of them contributed about 40–50% of the diazinon metabolism, in hepatic microsomes from untreated, phenobarbital‐, and dexamethasone‐treated rats, respectively. The higher diazoxon/pyrimidinol ratio observed after phenobarbital‐treatment together with the significantly more effective inhibition toward diazoxon production exerted by metyrapone in microsomes from phenobarbital‐treated rats supports the conclusion that CYP2B1/2 catalyze preferentially the production of diazoxon. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 53–61, 1999     

Inhibition of fipronil and nonane metabolism in human liver microsomes and human cytochrome P450 isoforms by chlorpyrifos

Previous studies have established that chlorpyrifos (CPS), fipronil, and nonane can all be metabolized by human liver microsomes (HLM) and a number of cytochrome P450 (CYP) isoforms. However, metabolic interactions between these three substrates have not been described. In this study the effect of either coincubation or preincubation of CPS with HLM or CYP isoforms with either fipronil or nonane as substrate was investigated. In both co- and preincubation experiments, CPS significantly inhibited the metabolism of fipronil or nonane by HLM although CPS inhibited the metabolism of fipronil more effectively than that of nonane. CPS significantly inhibited the metabolism of fipronil by CYP3A4 as well as the metabolism of nonane by CYP2B6. In both cases, preincubation with CPS caused greater inhibition than coincubation, suggesting that the inhibition is mechanism based.     

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.     

Interindividual variability of methadone response: impact of genetic polymorphism

Methadone, an opioid analgesic, is used clinically in pain therapy as well as for substitution therapy in opioid addiction. It has a large interindividual variability in response and a narrow therapeutic index. Genetic polymorphisms in genes coding for methadone-metabolizing enzymes, transporter proteins (p-glycoprotein; P-gp), and mu-opioid receptors may explain part of the observed interindividual variation in the pharmacokinetics and pharmacodynamics of methadone. Cytochrome P450 (CYP) 3A4 and 2B6 have been identified as the main CYP isoforms involved in methadone metabolism. Methadone is a P-gp substrate, and, although there are inconsistent reports, ABCB1 genetic polymorphisms also contribute slightly to the interindividual variability of methadone kinetics and influence dose requirements. Genetic polymorphism is the cause of high interindividual variability of methadone blood concentrations for a given dose; for example, in order to obtain methadone plasma concentrations of 250 ng/mL, doses of racemic methadone as low as 55 mg/day or as high as 921 mg/day can be required in a 70-kg patient without any co-medication. The clinician must be aware of the pharmacokinetic properties and pharmacological interactions of methadone in order to personalize methadone administration. In the future, pharmacogenetics, at a limited level, can also be expected to facilitate individualized methadone therapy.     

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.     

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.     

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.     

A comparison of aroclor 1254-induced and uninduced rat liver microsomes to human liver microsomes in phenytoin O-deethylation, coumarin 7-hydroxylation, tolbutamide 4-hydroxylation, S-mephenytoin 4'-hydroxylation, chloroxazone 6-hydroxylation and testosterone 6beta-hydroxylation

Aroclor 1254-induced rat liver homogenate supernatant (liver S-9) is routinely used as an exogenous metabolic activation system for the evaluation of mutagenicity of xenobiotics. The purpose of this study is to evaluate whether results obtained with Aroclor 1254-induced liver microsomes would be relevant to human. Aroclor 1254-induced and uninduced rat liver microsomes were compared to human liver microsomes in the metabolism of substrates which are known to be selectively metabolized by the major human cytochrome P450 (CYP) isoforms. The activities studied and the major CYP isoforms involved were as follows: phenacetin O-deethylation (CYP1A2); coumarin 7-hydroxylation, (CYP2A6); tolbutamide 4-hydroxylation (CYP2C9), S-mephenytoin 4'-hydroxylation (CYP2C19); dextromethorphan O-demethylation (CYP2D6); chloroxazone 6-hydroxylation (CYP2E1); and testosterone 6beta-hydroxylation (CYP3A4). We found that both induced and uninduced rat liver microsomes were active in all the pathways studied with the exception of coumarin 7-hydroxylation. Coumarin 7-hydroxylation was observed with human liver microsomes but not the rat liver microsomes. Aroclor-1254 was found to induce all activities measured, with the exception of coumarin 7-hydroxylation. Dextromethorphan O-deethylation activity was higher in the rat liver microsomes than the human liver microsomes. Testosterone 6beta-hydroxylation activity was found to be similar between the human liver microsomes and the induced rat liver microsomes. Our results suggest that experimental data obtained with Aroclor 1254-induced rat liver microsomes may not always be relevant to human.     

An evaluation of the P450 inhibition and induction potential of daptomycin in primary human hepatocytes

Two in vitro studies assessed the potential of daptomycin (Cubicin), a newly marketed antibiotic, to affect the cytochrome P450 (CYP450) isoforms in primary cultured human hepatocytes. Both induction and inhibition of isoforms 1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4 were evaluated. The highest concentrations of daptomycin used in both the induction and inhibition assays were approximately eight-fold higher than the peak total drug concentration (50-60 microg/mL), or the peak free drug concentration (estimated 5-6 microg/mL), in plasma at the clinical dose regimen of 4 mg/kg qd. Results in primary human hepatocytes indicate that daptomycin, at concentrations up to 400 microg total drug/mL, demonstrated no biologically significant induction of any of the CYP450 isoform activities in comparison with the negative control or known inducers. At daptomycin concentrations up to 40 microg free drug/mL, no biologically significant inhibition of the activities of these CYP450 isoforms was observed as compared with known inhibitors. The human hepatocyte results demonstrate that daptomycin has no effects on hepatic CYP450-mediated drug metabolism and, therefore, suggest that daptomycin is unlikely to show potential for pharmacokinetic interactions with concomitantly administered drugs that are metabolized by CYP450 isoforms.     

Contribution of cytochrome P450 and ABCB1 genetic variability on methadone pharmacokinetics, dose requirements, and response

Although the efficacy of methadone maintenance treatment (MMT) in opioid dependence disorder has been well established, the influence of methadone pharmacokinetics in dose requirement and clinical outcome remains controversial. The aim of this study is to analyze methadone dosage in responder and nonresponder patients considering pharmacogenetic and pharmacokinetic factors that may contribute to dosage adequacy. Opioid dependence patients (meeting Diagnostic and Statistical Manual of Mental Disorders, [4(th) Edition] criteria) from a MMT community program were recruited. Patients were clinically assessed and blood samples were obtained to determine plasma concentrations of (R,S)-, (R) and (S)-methadone and to study allelic variants of genes encoding CYP3A5, CYP2D6, CYP2B6, CYP2C9, CYP2C19, and P-glycoprotein. Responders and nonresponders were defined by illicit opioid consumption detected in random urinalysis. The final sample consisted in 105 opioid dependent patients of Caucasian origin. Responder patients received higher doses of methadone and have been included into treatment for a longer period. No differences were found in terms of genotype frequencies between groups. Only CYP2D6 metabolizing phenotype differences were found in outcome status, methadone dose requirements, and plasma concentrations, being higher in the ultrarapid metabolizers. No other differences were found between phenotype and responder status, methadone dose requirements, neither in methadone plasma concentrations. Pharmacokinetic factors could explain some but not all differences in MMT outcome and methadone dose requirements.     

Enantiomers of naringenin as pleiotropic,stereoselective inhibitors of cytochrome P450 isoforms

Interactions between naringenin and the cytochrome P450 (CYP) system have been of interest since the first demonstration that grapefruit juice reduced CYP3A activity. The effects of naringenin on other CYP isoforms have been less investigated. In addition, it is well known that interactions with enzymes are often stereospecific, but due to the lack of readily available pure naringenin enantiomers, the enantioselectivity of its effects has not been characterized. We isolated pure naringenin enantiomers by chiral high‐performance liquid chromatography and tested the ability of (R)‐,(S)‐ and rac‐naringenin to inhibit several important drug‐metabolizing CYP isoforms using recombinant enzymes and pooled human liver microsomes. Naringenin was able to inhibit CYP19, CYP2C9, and CYP2C19 with IC₅₀ values below 5 μM. No appreciable inhibition of CYP2B6 or CYP2D6 was observed at concentrations up to 10 μM. Whereas (S)‐naringenin was 2‐fold more potent as an inhibitor of CYP19 and CYP2C19 than (R)‐naringenin, (R)‐naringenin was 2‐fold more potent for CYP2C9 and CYP3A. Chiral flavanones like naringenin are difficult to separate into their enantiomeric forms, but enantioselective effects may be observed that ultimately impact clinical effects. Inhibition of specific drug metabolizing enzymes by naringenin observed in vitro may be exploited to understand pharmacokinetic changes seen in vivo. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Cytochrome P450 enzymes involved in the metabolism of tetrahydrocannabinols and cannabinol by human hepatic microsomes

In this study, tetrahydrocannabinols (THCs) were mainly oxidized at the 11-position and allylic sites at the 7alpha-position for Delta(8)-THC and the 8beta-position for Delta(9)-THC by human hepatic microsomes. Cannabinol (CBN) was also mainly metabolized to 11-hydroxy-CBN and 8-hydroxy-CBN by the microsomes. The 11-hydroxylation of three cannabinoids by the microsomes was markedly inhibited by sulfaphenazole, a selective inhibitor of CYP2C enzymes, while the hydroxylations at the 7alpha-(Delta(8)-THC), 8beta-(Delta(9)-THC) and 8-positions (CBN) of the corresponding cannabinoids were highly inhibited by ketoconazole, a selective inhibitor of CYP3A enzymes. Human CYP2C9-Arg expressed in the microsomes of human B lymphoblastoid cells efficiently catalyzed the 11-hydroxylation of Delta(8)-THC (7.60 nmol/min/nmol CYP), Delta(9)-THC (19.2 nmol/min/nmol CYP) and CBN (6.62 nmol/min/nmol CYP). Human CYP3A4 expressed in the cells catalyzed the 7alpha-(5.34 nmol/min/nmol CYP) and 7beta-hydroxylation (1.39 nmol/min/nmol CYP) of Delta(8)-THC, the 8beta-hydroxylation (6.10 nmol/min/nmol CYP) and 9alpha,10alpha-epoxidation (1.71 nmol/min/nmol CYP) of Delta(9)-THC, and the 8-hydroxylation of CBN (1.45 nmol/min/nmol CYP). These results indicate that CYP2C9 and CYP3A4 are major enzymes involved in the 11-hydroxylation and the 8-(or the 7-) hydroxylation, respectively, of the cannabinoids by human hepatic microsomes. In addition, CYP3A4 is a major enzyme responsible for the 7alpha- and 7beta-hydroxylation of Delta(8)-THC, and the 9alpha,10alpha-epoxidation of Delta(9)-THC.     

CYP3A4 and CYP2D6 inhibitory activities of Indonesian medicinal plants

Thirty samples of Indonesian medicinal plants were analyzed for their capacity to inhibit in vitro metabolism by human cytochrome P450 3A4 (CYP3A4) and CYP2D6 with a radiometric assay. The MeOH-soluble fractions of 25 samples, prepared from water extracts, demonstrated inhibitory activity more than 50% on the metabolism mediated by CYP3A4, and 21 samples on the metabolism mediated by CYP2D6. Among the MeOH-soluble fractions, Piper nigrum leaf showed the highest inhibitory activity against CYP3A4 (91.7%), and Punica granatum against CYP2D6 (98.1%). The water extracts of which MeOH-soluble fraction showed inhibitory activity more than 70% were fractionated with EtOAc. From the EtOAc-soluble fractions, Curcuma heyneana (67.0%), Pi. cubeba (75.0%), Pi. nigrum fruit (84.0%), Pi. nigrum leaf (85.8%), and Zingiber aromaticum (75.3%) demonstrated inhibitory activity more than 50% on the metabolism mediated by CYP3A4, but only Pi. nigrum fruit (72.8%) and Pi. nigrum leaf (69.1%) showed strong inhibitory activity against CYP2D6. For samples that showed more than 70% inhibition, their IC(50) values were determined. The most potent inhibitory activity against CYP3A4 (IC(50) value of 25 microg/ml) was found for the extract of Pi. nigrum leaf, while that of Catharanthus roseus showed the most potent inhibitory effect against CYP2D6 (IC(50) value of 11 microg/ml). These results should indicate once more the possibility of potential medicinal plant-drug interactions.     

In vitro metabolism of carbaryl by human cytochrome P450 and its inhibition by chlorpyrifos

Carbaryl is a widely used anticholinesterase carbamate insecticide. Although previous studies have demonstrated that carbaryl can be metabolized by cytochrome P450 (CYP), the identification and characterization of CYP isoforms involved in metabolism have not been described either in humans or in experimental animals. The in vitro metabolic activities of human liver microsomes (HLM) and human cytochrome P450 (CYP) isoforms toward carbaryl were investigated in this study. The three major metabolites, i.e. 5-hydroxycarbaryl, 4-hydroxycarbaryl and carbaryl methylol, were identified after incubation of carbaryl with HLM or individual CYP isoforms and analysis by HPLC. Most of the 16 human CYP isoforms studied showed some metabolic activity toward carbaryl. CYP1A1 and 1A2 had the greatest ability to form 5-hydroxycarbaryl, while CYP3A4 and CYP1A1 were the most active in generation of 4-hydroxycarbaryl. The production of carbaryl methylol was primarily the result of metabolism by CYP2B6. Differential activities toward carbaryl were observed among five selected individual HLM samples with the largest difference occurring in the production of carbaryl methylol. Co-incubations of carbaryl and chlorpyrifos in HLM greatly inhibited carbaryl metabolism. The ability of HLM to metabolize carbaryl was also reduced by pre-incubation of HLM with chlorpyrifos. Chlorpyrifos inhibited the generation of carbaryl methylol, catalyzed predominately by CYP2B6, more than other pathways, correlating with an earlier observation that chlorpyrifos is metabolized to its oxon primarily by CYP2B6. Therefore, carbaryl metabolism in humans and its interaction with other chemicals is reflected by the concentration of CYP isoforms in HLM and their activities in the metabolic pathways for carbaryl. (Supported by NCDA Environmental Trust Fund)     

Reduced dosing and liability in methadone maintenance treatment by targeting oestrogen signal for morphine addiction

Methadone maintenance treatment (MMT) is the major tapering therapy for morphine addictive patients. There have gender differences reported in response to MMT. This study discovered that the estrogen‐response element single nucleotide polymorphism (ERE‐SNP; rs16974799, C/T) of cytochrome 2B6 gene (cyp2b6; methadone catabolic enzyme) responded differently to MMT dosing. Oestradiol was associated with high MMT dosing, high enantiomer (R‐ or S‐) of 2‐ethylidene‐1,5‐dimethyl‐3,3‐dipheny‐pyrrolidine (EDDP; methadone metabolite) to methadone ratio and increased drug‐seeking behaviour, implicating oestradiol‐CYP‐EDDP/methadone axis decreasing MMT efficacy. In mouse model, oestrogen mitigates methadone antinociceptive response, facilitates methadone catabolism and up‐regulates methadone‐associated metabolizing enzymes. Oestrogen also ablates chronic methadone administration‐induced rewarding response. Mechanism dissection revealed the CC genotype of CYP2B6‐ERE‐SNP exerts higher ERE sequence alignment score, higher estrogenic response as compared to TT genotype. At last, preclinical study via targeting estrogen signal that tamoxifen (TMX; selective estrogen receptor modulator, SERM) could facilitate the tolerance phase rewarding response of methadone. Strikingly, TMX also reduces tapering/abstinence phases methadone liability in mice. In conclusion, this study demonstrates altering methadone metabolism through targeting estrogen signals might be able to free morphine addictive patients from the addiction of opioid replacement therapy. Therefore, the add‐on therapy clinical trial introducing SERM in MMT regimen is suggested.     

Specific human CYP 450 isoform metabolism of a pentachlorobiphenyl (PCB-IUPAC# 101)

Polychlorinated biphenyl IUPAC# 101-PCB 101 (chlorination pattern-2,2',4',5,5') is a common, persistent non-coplanar PCB congener found in the ambient environment but information related to its metabolism in humans is lacking. Previous studies indicate PCB 101 is rapidly metabolized in mammals through CYP 2B and 3A family enzymes. Recently, PCB metabolism through a 2A family isoform in hamsters was also reported. To specifically identify the human CYP 450 isoforms responsible for PCB 101 metabolism, we compared human microsome metabolism to metabolism using several specific recombinant human CYP isoforms. These data characterized selective and extensive metabolism by human CYP 2A6. The product formed was the 4-hydroxy-PCB 101 metabolite (4-hydroxy-2,2',4',5,5') and was the only major metabolite observed in the recombinant and human microsome investigation. This is important information for predicting human specific toxicokinetics of PCBs.     

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.     

Quantitative determination of the enantiomers of methadone and its metabolite (EDDP) in human saliva by enantioselective liquid chromatography with mass spectrometric detection

A sensitive enantioselective liquid chromatographic assay with mass spectrometric detection (LC-MS) has been developed and validated for the simultaneous determination of saliva concentrations of (R)- and (S)-methadone (Met) and (R)- and (S)-2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine (EDDP, a primary metabolite of Met). Saliva specimens were collected using Salivette devices (Sarsedt), and centrifuged; collected saliva was then spiked with deuterated internal standards, D3-Met and D3-EDDP, and directly injected into the LC-MS. Enantioselective separations were achieved on a liquid chromatographic chiral stationary phase (CSP) based upon immobilized alpha(1)-acid glycoprotein (AGP) using a mobile phase composed of acetonitrile: ammonium acetate buffer (10mM, pH 7.0) in a ratio of 18:82 (v/v), a flow rate of 0.9 ml/min and a temperature of 25 degrees C. Under these conditions, enantioselective separations were observed for methadone (alpha=1.30) and EDDP (alpha=1.17) within 15 min. Met, EDDP, D3-Met and D3-EDDP were detected using selected ion monitoring at m/z 310.20, 278.20, 313.20 and 281.20, respectively. Linear relationships between peak height ratio and drug-enantiomer concentrations were obtained for methadone in the range of 5.0-600.0 ng/ml, and for EDDP from 0.5 to 15.0 ng/ml per enantiomer with correlation coefficients better than 0.9994, where lower limit of quantification (LLOQ) for Met was 5 ng/ml and for EDDP 0.5 ng/ml. Acceptable intra- and inter-day precision of the method (CVs<4.0%) and accuracy (CVs<4.0%) were obtained. These findings demonstrate the accuracy and precision of the method used to successfully analyze saliva obtained from patients enrolled in a methadone-maintenance program.     

Cytochrome P450 2E1 is the primary enzyme responsible for low-dose carbon tetrachloride metabolism in human liver microsomes

We examined which human CYP450 forms contribute to carbon tetrachloride (CCl(4)) bioactivation using hepatic microsomes, heterologously expressed enzymes, inhibitory antibodies and selective chemical inhibitors. CCl(4) metabolism was determined by measuring chloroform formation under anaerobic conditions. Pooled human microsomes metabolized CCl(4) with a K(m) of 57 microM and a V(max) of 2.3 nmol CHCl(3)/min/mg protein. Expressed CYP2E1 metabolized CCl(4) with a K(m) of 1.9 microM and a V(max) of 8.9 nmol CHCl(3)/min/nmol CYP2E1. At 17 microM CCl(4), a monoclonal CYP2E1 antibody inhibited 64, 74 and 83% of the total CCl(4) metabolism in three separate human microsomal samples, indicating that at low CCl(4) concentrations, CYP2E1 was the primary enzyme responsible for CCl(4) metabolism. At 530 microM CCl(4), anti-CYP2E1 inhibited 36, 51 and 75% of the total CCl(4) metabolism, suggesting that other CYP450s may have a significant role in CCl(4) metabolism at this concentration. Tests with expressed CYP2B6 and inhibitory CYP2B6 antibodies suggested that this form did not contribute significantly to CCl(4) metabolism. Effects of the CYP450 inhibitors alpha-naphthoflavone (CYP1A), sulfaphenazole (CYP2C9) and clotrimazole (CYP3A) were examined in the liver microsome sample that was inhibited only 36% by anti-CYP2E1 at 530 microM CCl(4). Clotrimazole inhibited CCl(4) metabolism by 23% but the other chemical inhibitors were without significant effect. Overall, these data suggest that CYP2E1 is the major human enzyme responsible for CCl(4) bioactivation at lower, environmentally relevant levels. At higher CCl(4) levels, CYP3A and possibly other CYP450 forms may contribute to CCl(4) metabolism.     

Determination of methadone and its metabolites EDDP and EMDP in human hair by headspace solid-phase microextraction and gas chromatography–mass spectrometry

A simple method for analysis of methadone and its two main metabolites EDDP and EMDP in hair was developed using automatic headspace solid-phase microextraction (HS-SPME) at a multipurpose sampler and gas chromatography – mass spectrometry with electron impact ionization and selected ion monitoring (GC–MS-SIM). The washed hair pieces were digested in the closed headspace vial in 1 ml 1 M NaOH containing 0.5 g NaCl and each 10 ng of the internal standards D₉-methadone and D₃-EDDP at 110°C for 20 min. Then the HS-SPME was performed with a 65 μm polydimethylsiloxan/divinylbenzene fiber at the same temperature in the same vial for another 20 min followed by the desorption in the GC injection port. The calibration curves were linear between 0.1 and 3 ng/mg (methadone and EMDP) and 10 ng/mg (EDDP) respectively, at higher concentrations a negative deviation from linearity was found. The detection limits were 0.03 ng/mg (methadone) and 0.05 ng/mg (EDDP and EMDP), and the reproducibility was 9.2% for methadone and 11.2% for EDDP (n=12). The method was applied to hair samples of 26 drug fatalities. 19 cases were positive with 0.36–11.8 ng/mg methadone and 0.19 –10.8 ng/mg EDDP. EMDP was found only in two cases with 0.18 and 0.84 ng/mg. The methadone concentration range was in agreement with previous data, but the EDDP/methadone concentration ratios (0.19–0.67) were definitely higher than those determined by other methods.