Role of cytochrome P450 3A in the metabolism of mefloquine in human and animal hepatocytes

We studied mefloquine metabolism in cells and microsomes isolated from human and animal (monkey, dog, rat) livers. In both hepatocytes and microsomes, mefloquine underwent conversion to two major metabolites, carboxymefloquine and hydroxymefloquine. In human cells and microsomes these metabolites only were formed, as already demonstrated in vivo, while in other species several unidentified metabolites were also detected. After a 48 hr incubation with human and rat hepatocytes, metabolites accounted for 55-65% of the initial drug concentration, whereas in monkey and dog hepatocytes, mefloquine was entirely metabolized after 15 and 39 hrs, respectively. The consumption of mefloquine was less extensive in microsomes, and unchanged drug represented 60% (monkey) to 85-100% (human, dog, rat) of the total radioactivity after 5 hr incubations. The involvement of the cytochrome P450 3A subfamily in mefloquine biotransformation was suggested by several lines of evidence. Firstly, mefloquine metabolism was strongly increased in hepatic microsomes from dexamethasone-pretreated rats, and also in human and rat hepatocytes after prior treatment with a cytochrome P450 3A inducer. Secondly, mefloquine biotransformation in rifampycin-induced human hepatocytes was inhibited in a concentration-dependent manner by the cytochrome P450 3A inhibitor ketoconazole and thirdly, a strong correlation was found between erythromycin-N-demethylase activity (mediated by cytochrome P450 3A) and mefloquine metabolism in human microsomes (r=0.81, P < 0.05, N=13). Collectively, these findings concerning the role of cytochrome P450 3A in mefloquine metabolism may have important in vivo consequences especially with regard to the choice of agents used in multidrug antimalarial regimens.     

25-Methoxylprotopanaxadiol derivatives and their anti-proliferative activities

(20R)-25-Methoxyl-dammarane-3β,12β,20-triol (25-OCH₃-PPD) is a dammarane-type sapogenin showing anti-proliferative potential. In our study, two series of analogs substituted at the C-3 or C-3 and C-12 positions with fatty acids were prepared conveniently. The cytotoxic activity of these compounds was evaluated using four different human tumor cell lines (A549, Hela, HT-29 and MCF-7) and a normal cell line (IOSE144). As compared with 25-OCH₃-PPD, compounds 1a, 1b, 2a and 2b showed better anti-proliferative activities against all tumor cell lines and all the derivatives, with low toxicities in the normal cell line. Compound 1a (C-3 monoformiate) exhibited the strongest activity with the IC₅₀ value of 5.2 μM towards HT29 cells. The results indicated that the difference in the substituents may affect the anti-proliferative activity of the compounds. The longer the side chain of 25-OCH₃-PPD is, the lower the anti-proliferative activity would be. This information may be useful for evaluating the structure–activity relationship of other dammarane-type sapogenins and for development of novel antineoplastic agents.     

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.     

Distribution, metabolism and excretion of a synthetic androgen 7alpha-methyl-19-nortestosterone, a potential male-contraceptive

A synthetic androgen 7α-Methyl-19-nortestosterone (MENT) has a potential for therapeutic use in ‘androgen replacement therapy’ for hypogonadal men or as a hormonal male-contraceptive in normal men. Its tissue distribution, excretion and metabolic enzyme(s) have not been reported. Therefore, the present study tested the distribution and excretion of MENT in Sprague-Dawley rats castrated 24 h prior to the injection of tritium-labeled MENT (³H-MENT). Rats were euthanized at different time intervals after dosing, and the amount of radioactivity in various tissues/organs was measured following combustion in a Packard oxidizer. The radioactivity (% injected dose) was highest in the duodenal contents in the first 30 min of injection. Specific uptake of the steroid was observed in target tissues such as ventral prostate and seminal vesicles at 6 h, while in other tissues radioactivity equilibrated with blood. Liver and duodenum maintained high radioactivity throughout, as these organs were actively involved in the metabolism and excretion of most drugs. The excretion of ³H-MENT was investigated after subcutaneous injection of ³H-MENT into male rats housed in metabolic cages. Urine and feces were collected at different time intervals (up to 72 h) following injection. Results showed that the radioactivity was excreted via feces and urine in equal amounts by 30 h.Aiming to identify enzyme(s) involved in the MENT metabolism, we performed in vitro metabolism of ³H-MENT using rat and human liver microsomes, cytosol and recombinant cytochrome P₄₅₀ (CYP) isozymes. The metabolites were separated by thin-layer chromatography (TLC). Three putative metabolites (in accordance with the report of Agarwal and Monder [Agarwal AK, Monder C. In vitro metabolism of 7α-methyl-19-nortestosterone by rat liver, prostate, and epididymis. Endocrinology 1988;123:2187-93]), [i] 3-hydroxylated MENT by both rat and human liver cytosol; [ii] 16α-hydroxylated MENT (a polar metabolite) by both rat and human hepatic microsomes; and [iii] 7α-methyl-19-norandrostenedione (a non-polar metabolite) by human hepatic microsomes, were obtained. By employing chemical inhibitors and specific anti-CYP antibodies, ³H-MENT was found to be metabolized specifically by rat CYP 2C11 and 3-hydroxysteroid dehydrogenase (3-HSD) enzymes whereas in humans it was accomplished by CYP 3A4, 17β-hydroxysteroid dehydrogenase (17β-HSD) and 3-HSD enzymes.     

In vitro metabolism of polychlorinated biphenyl congeners by beluga whale (Delphinapterus leucas) and pilot whale (Globicephala melas) and relationship to cytochrome P450 expression

We measured rates of oxidative metabolism of two tetrachlorobiphenyl (TCB) congeners by hepatic microsomes of two marine mammal species, beluga whale and pilot whale, as related to content of selected cytochrome P450 (CYP) forms. Beluga liver microsomes oxidized 3,3',4,4'-TCB at rates averaging 21 and 5 pmol/min per mg for males and females, respectively, while pilot whale samples oxidized this congener at 0.3 pmol/min per mg or less. However, rates of 3,3',4,4'-TCB metabolism correlated with immunodetected CYP1A1 protein content in liver microsomes of both species. The CYP1A inhibitor alpha-naphthoflavone inhibited 3,3',4,4'-TCB metabolism by 40% in beluga, supporting a role for a cetacean CYP1A as a catalyst of this activity. Major metabolites of 3,3',4,4'-TCB generated by beluga liver microsomes were 4-OH-3,3',4',5-TCB and 5-OH-3,3',4,4'-TCB (98% of total), similar to metabolites formed by other species CYP1A1, and suggesting a 4,5-epoxide-TCB intermediate. Liver microsomes of both species metabolized 2,2',5,5'-TCB at rates of 0.2-1.5 pmol/min per mg. Both species also expressed microsomal proteins cross-reactive with antibodies raised against some mammalian CYP2Bs (rabbit; dog), but not others (rat; scup). Whether CYP2B homologues occur and function in cetaceans is uncertain. This study demonstrates that PCBs are metabolized to aqueous-soluble products by cetacean liver enzymes, and that in beluga, rates of metabolism of 3,3',4,4'-TCB are substantially greater than those of 2,2',5,5'-TCB. These directly measured rates generally support the view that PCB metabolism plays a role in shaping the distribution patterns of PCB residues found in cetacean tissue.     

Preclinical metabolism of LB42908, a novel farnesyl transferase inhibitor, and its effects on the cytochrome P450 isozyme activities

Metabolism of LB42908, a novel farnesyl transferase inhibitor, was investigated for preclinical development. In vitro hepatic metabolism of LB42908 gave rise to at least 9 metabolites via phase I biotransformation pathways, which were characterized by HPLC-UV, LC-MS, and LC-MS/MS analyses. N-Dealkylation was shown to be a major phase I metabolic pathway. Species-specific in vitro metabolism of LB42908 was studied in liver fractions of rat, dog, monkey, and human. Order of metabolic stability is human≈dog>rat≈monkey in both S9 and microsomal fractions. Tissue-specific metabolism of LB42908 in various tissue homogenates of rats demonstrated that the liver was the major organ responsible for phase I metabolism of LB42908. The results from both qualitative and quantitative metabolism studies such as metabolic profiling and metabolic clearance indicated that dog would be the animal model of choice for preclinical toxicology studies. In addition, LB42908 was a potent CYP3A4 inhibitor in human liver microsomes and induced the activities of several CYP isozymes, implying that it has the potential for drug-drug interactions. Repeated dosing of LB42908 in rats did not significantly affect its own metabolism, indicating that long-term administration of LB42908 would not alter its pharmacokinetic profiles.     

Antitumor Activity of DMAKO-05, a Novel Shikonin Derivative,and Its Metabolism in Rat Liver Microsome

The antitumor activity of shikonin derivatives is largely dependent on the generation of superoxide radicals and the alkylation activity of their naphthoquinone moiety. However, our recent study showed that 1,4-dioxime-5,8-dimethoxynaphthalene (DMAKO-05), a novel shikonin derivative, displayed more potential antitumor activity and less toxicity compared to fluorouracil (5-FU) both in vitro and in vivo, even though the hydroxyl and carbonyl groups of its naphthoquinone structure were shielded. To understand the underlying mechanisms, we investigated the metabolism of DMAKO-05 in rat liver microsomes. The kinetic analysis indicated that DMAKO-05 underwent a biphasic metabolism in rat liver microsomes. The inhibition experiments showed that CYP1A and CYP3A were the major enzymes in the metabolism of DMAKO-05, along with partial contribution from CYP2A. In addition, the structures of eight DMAKO-05 metabolites, which were characterized by accurate mass and MS/MS fragmentograms, implied that DMAKO-05 was mainly metabolized through the oxygenation of its naphthoquinone nucleus and the hydrolysis of its side chain, instead of the oxidation of hydroxyimine to ketone. Therefore, DMAKO-05 should not be considered as a traditional naphthoquinone prodrug.KEY WORDS: antitumor, LC-TOF-MS/MS, metabolism, method validation, rat hepatic microsomes, shikonin oxime     

Species differences and interindividual variation in liver microsomal cytochrome P450 2A enzymes: effects on coumarin, dicumarol, and testosterone oxidation.

Antibody against purified CYP2A1 recognizes two rat liver microsomal P450 enzymes, CYP2A1 and CYP2A2, that catalyze the 7 alpha- and 15 alpha-hydroxylation of testosterone, respectively. In human liver microsomes, this antibody recognizes a single protein, namely CYP2A6, which catalyzes the 7-hydroxylation of coumarin. To examine species differences in CYP2A function, liver microsomes from nine mammalian species (rat, mouse, hamster, rabbit, guinea pig, cat, dog, cynomolgus monkey, and human) were tested for their ability to catalyze the 7 alpha- and 15 alpha-hydroxylation of testosterone and the 7-hydroxylation of coumarin. Antibody against rat CYP2A1 recognized one or more proteins in liver microsomes from all mammalian species examined. However, liver microsomes from cat, dog, cynomolgus monkey, and human catalyzed negligible rates of testosterone 7 alpha- and/or 15 alpha-hydroxylation, whereas rat and cat liver microsomes catalyzed negligible rates of coumarin 7-hydroxylation. Formation of 7-hydroxycoumarin accounted for a different proportion of the coumarin metabolites formed by liver microsomes from each of the various species examined. 7-Hydroxycoumarin was the major metabolite (greater than 70%) in human and monkey, but only a minor metabolite (less than 1%) in rat. The 7-hydroxylation of coumarin by human liver microsomes was catalyzed by a single, high-affinity enzyme (Km 0.2-0.6 microM), which was markedly inhibited (greater than 95%) by antibody against rat CYP2A1. The rate of coumarin 7-hydroxylation varied approximately 17-fold among liver microsomes from 22 human subjects. This variation was highly correlated (r2 = 0.956) with interindividual differences in the levels of CYP2A6, as determined by immunoblotting. These results indicate that CYP2A6 is largely or entirely responsible for catalyzing the 7-hydroxylation of coumarin in human liver microsomes. Treatment of monkeys with phenobarbital or dexamethasone increased coumarin 7-hydroxylase activity, whereas treatment with beta-naphthoflavone caused a slight decrease. These results suggest that environmental factors can increase or decrease CYP2A expression in cynomolgus monkeys, which implies that environmental factors may be responsible for the large variation in CYP2A6 levels in humans, although genetic factors may also be important. In contrast to rats and mice, the expression of CYP2A enzymes in cynomolgus monkeys and humans was not sexually differentiated. Despite their structural similarity to coumarin, the anticoagulants dicumarol and warfarin do not appear to be substrates for CYP2A6. The overall rate of dicumarol metabolism varied approximately 5-fold among the human liver microsomal samples, but this variation correlated poorly (r2 = 0.126) with the variation observed in CYP2A6 levels and coumarin 7-hydroxylase activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparative study of the oxidation of propranolol enantiomers in hepatic and small intestinal microsomes from cynomolgus and marmoset monkeys

Oxidative metabolism of propranolol (PL) enantiomers (R-PL and S-PL) to 4-hydroxypropranolol (4-OH-PL), 5-OH-PL and N-deisopropylpropranolol (NDP) was examined in hepatic microsomes from cynomolgus and marmoset monkeys and in small intestinal microsomes from monkeys and humans. In hepatic microsomes, levels of oxidation activities were similar between the two monkey species, and substrate enantioselectivity (R-PL < S-PL) was observed in the formation of 5-OH-PL and/or NDP. Kinetic experiments revealed that the formation of all metabolites was biphasic in cynomolgus monkeys, whereas only the formation of NDP was biphasic in marmosets. Inhibition experiments employing human CYP antibodies and chemical inhibitors suggested that mainly CYP2D enzymes and partially CYP1A and 2C enzymes are involved in the oxidation of PL in both monkey liver microsomes. In small intestinal microsomes, activity levels were much higher in cynomolgus monkeys than in marmosets and humans and reversed substrate enantioselectivity (R-PL > S-PL) was seen in the formation of NDP in cynomolgus monkeys and humans and in the formation of 5-OH-PL in marmosets. The formation of the three metabolites in cynomolgus monkeys and the formation of NDP in marmosets were biphasic, while the formation of 4-OH-PL in humans was monophasic. From the inhibition experiments using CYP antibodies, CYP2C9 and 2C19 were thought to be involved as N-deisopropylases and CYP2D6 and 3A4 as 4-hydroxylases in human small intestine. Furthermore, CYP1A, 2C and 3A enzymes could be involved in cynomolgus monkeys and CYP2C and 3A enzymes in marmosets. These results indicate that the oxidative profile of PL in hepatic and small intestinal microsomes differ considerably among cynomolgus monkeys, marmosets and humans.     

Metabolism of (−)-trans-(3R,4R)-dihydroxy-3,4-dihydrochrysene to diol epoxides by liver microsomes

Metabolism of biosynthetic (?)-trans-(3R,4R)-dihydroxy-3,4-dihydrochrysene by liver microsomes from control, phenobarbital-treated and 3-methylcholanthrene-treated rats was investigated. Although previous studies of the metabolism of related benzo[a]pyrene and benzo[e]pyrene dihydrodiols which also prefer the diaxial conformation had indicated that diol epoxides were minor metabolites, the diastereomeric chrysene 3,4-diol-1,2-epoxides-$\text{1}$ and $\text{?2}$ were major metabolites (66–90%). All three types of microsomes metabolized the chrysene 3,4-dihydrodiol at low but essentially similar rates (0.5–0.7 nmol substrate/nmol cytochrome P-450/min).     

Metabolism of 20-epimer of 1alpha,25-dihydroxyvitamin D3 by CYP24: species-based difference between humans and rats

The 20-epi form of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)-20-epi-D(3)) is expected as drugs for leukemia, other cancers or psoriasis, because it shows several-hundred fold enhanced ability to induce cell differentiation and growth inhibition than 1alpha,25-dihydroxyvitamin D(3) while its calcemic activity is only slightly elevated. In this study, we compared the human and rat CYP24-dependent metabolism of 1alpha,25(OH)(2)-20-epi-D(3) by using the Escherichia coli expression system. The HPLC and LC-MS analyses of the metabolites revealed that rat CYP24 converted 1alpha,25(OH)(2)-20-epi-D(3) to 25,26,27-trinor-1alpha(OH)-24(COOH)-20-epi-D(3) through 1alpha,24,25(OH)(3)-20-epi-D(3) and 1alpha,25(OH)(2)-24-oxo-20-epi-D(3). The binding affinity of trinor-1alpha(OH)-24(COOH)-20-epi-D(3) for vitamin D receptor (VDR) was less than 1/4000 of that of 1alpha,25(OH)(2)-20-epi-D(3). These results suggest that rat CYP24 can almost completely inactivate 1alpha,25(OH)(2)-20-epi-D(3). On the other hand, human CYP24 mainly converted 1alpha,25(OH)(2)-20-epi-D(3) to its putative demethylated compound with a hydroxyl group, via 1alpha,24,25(OH)(3)-20-epi-D(3), 1alpha,25(OH)(2)-24-oxo-20-epi-D(3), and 1alpha,23,25(OH)(3)-24-oxo-20-epi-D(3). All of these metabolites showed considerable affinity for vitamin D receptor. These results clearly demonstrate the species-based difference between human and rat on the CYP24-dependent metabolism of 1alpha,25(OH)(2)-20-epi-D(3).     

Use of electrospray ionization liquid chromatography-mass spectrometry to study the role of CYP2D^ in the in vitro metabolism of 5-hydroxytryptamine receptor antagonists

An electrospray ionization liquid chromatographic-mass spectrometric (ESI-LC-MS) method has been developed to study the involvement of the cytochrome P450 isoenzyme CYP2D6 in the in vitro metabolism of the indole containing 5-hydroxytryptamine (5-HT₃) receptor antagonists tropisetron, ondansetron and dolasetron in human liver microsomes. Compounds were eluted using linear gradients of acetonitrile-20 mM ammonium acetate, solvent A, (10:90, v/v) (ph 6.0) and solvent B, (60:40, v/v) (pH 6.0) and a Nucleosil C₄ column. Microsomal incubations were analysed using selected ion monitoring of the molecular ion of parent drug and the molecular ion of hydroxylated metabolites. The involvement of CYP2D6 in drug metabolism was assessed by inhibition studies using quinidine (5 μM), a specific inhibitor of human CYP2D6, as well as by incubating compounds with microsomes prepared from celss transfected with cDNA encoding human CYP2D6. Results showed that the oxidation of all three compounds involved CYP2D6, but only that of tropisetron was inhibited by over 90% in the presence of quinidine. The present method can be applied to pre-clinical compounds, at an early stage of drug discovery, to assess the involvement of CYP2D6 in their metabolism and to screen for those compounds where CYP2D6 is the only isoenzyme implicated in the formation of major metabolites.     

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.     

Metabolism of 1α,25-dihydroxyvitamin D2 by human CYP24A1

The metabolism of 1α,25-dihydroxyvitamin D₂ (1α,25(OH)₂D₂) by human CYP24A1 was examined using the recombinant enzyme expressed in Escherichia coli cells. HPLC analysis revealed that human CYP24A1 produces at least 10 metabolites, while rat CYP24A1 produces only three metabolites, indicating a remarkable species-based difference in the CYP24A1-dependent metabolism of 1α,25(OH)₂D₂ between humans and rats. LC-MS analysis and periodate treatment of the metabolites strongly suggest that human CYP24A1 converts 1α,25(OH)₂D₂ to 1α,24,25,26(OH)₄D₂, 1α,24,25,28(OH)₄D₂, and 24-oxo-25,26,27-trinor-1α(OH)D₂ via 1α,24,25(OH)₃D₂. These results indicate that human CYP24A1 catalyzes the C₂₄-C₂₅ bond cleavage of 1α,24,25(OH)₂D₂, which is quite effective in the inactivation of the active form of vitamin D₂. The combination of hydroxylation at multiple sites and C-C bond cleavage could form a large number of metabolites. Our findings appear to be useful to predict the metabolism of vitamin D₂ and its analogs in the human body.     

Oxidation of carbon tetrachloride,bromotrichloromethane, and carbon tetrabromide by rat liver microsomes to electrophilic halogens

In order to determine whether CCl₄, CBrCl₃, CBr₄ or CHCl₃ undergo oxidative metabolism to electrophilic halogens by liver microsomes, they were incubated with liver microsomes from phenobartital pretreated rats in the presence of NADPH and 2,6-dimethylphenol. The analysis of the reaction mixtures by capillary gas chromatography mass spectrometry revealed that 4-chloro-2,6-dimethylphenol was a metabolite of CCl₄ and CBrCl₃ whereas 4-bromo-2,6-dimethylphenol was a metabolite of CBr₄. The formation of the metabolites was significantly decreased when the reactions were conducted with heat denatured microsomes, in the absence of NADPH or under an atmosphere of N₂. These results indicate that the chlorines of CBrCl₃ and CCl₄ and the bromines of CBr₄ are oxidatively metabolized by rat liver microsomes to electrophilic and potentially toxic metabolites.     

Synthesis and structural assignment of two major metabolites of the LTA4H inhibitor DG-051

The same two major CYP mediated metabolites of DG-051 were produced in the presence of rat, dog, monkey and human liver microsomes. Their respective structures were hypothesized based on mass spectrometry data correlated with the parent structure and confirmed by comparison with authentic synthetic samples. The number of regioisomers synthesized as candidates for metabolite M1 was narrowed down using a metabolic study of a selectively deuterated DG-051 analogue.     

Formation of reactive 1-nitropyrene metabolites by lung microsomes and isolated lung cells

The metabolism and activation of 1-nitropyrene (1-NP) to reactive intermediates by lung microsomes and isolated lung cells was studied. Mutagenicity of 1-NP metabolites was assayed in Salmonella typhimurium TA98NR, a strain lacking a major component of nitroreductase activity. In the presence of NADPH, microsomes from rabbit, rat and hamster lung metabolized 1-NP to mutagenic products to a similar degree. Pretreatment with a mixture of polychlorinated biphenyls (PCB) decreased the formation of mutagenic metabolites by rabbit lung microsomes, but did not affect the production of mutagens by rat or hamster lung microsomes. ³H-1-NP was metabolized to covalently bound protein products at a rate of 82 and 10 pmol/mg by rabbit and hamster lung microsomes, respectively, whereas no binding was detected in rat lung microsomes. PCB-pretreatment increased covalent protein binding of ³ H-1-NP in lung microsomes from hamster and rat, but decreased the binding in rabbit lung microsomes. High performance liquid chromatography analysis indicated that ³H-1-NP was readily converted to ring-hydroxylated products by rabbit and hamster lung microsomes; the rate was much lower with rat lung microsomes. ³H-1-NP was activated to metabolites that covalently bound to protein in isolated rabbit lung cells, with the following rates being observed: Clara cells > lung digest > type II cells. In contrast, covalent protein binding in cells isolated from rat lung was very low. 1-NP was not activated to products mutagenic for S. typhimurium TA 98 N R when co-incubated with cells isolated either from rabbit or rat lung.Abbreviations 1-AP 1-aminopyrene - DMSO dimethyl sulfoxide - EGTA ethylene glycol-bis(ß-aminoethyl ether) - EM electron microscopy - HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid - HPBS HEPES-phosphate-buffered-saline - HPLC high performance liquid chromatography - NBT nitroblue tetrazolium - 1-NP 1-nitropyrene - 1-NP-4,5-diol trans-4,5-dihydro-4,5-dihydroxy-1-nitropyrene - 1-NP-9,10-diol trans-9,10-dihydro-9,10-dihydroxy-1-nitropyrene - 1-NP-4,5-oxide 1-nitropyrene-4,5-oxide - 1-NP-9,10-oxide 1-nitropyrene-9,10-oxide - 3-OH-1-NP 3-hydroxy-1-nitropyrene - 6-/8-OH-1-NP a mixture of 6- and 8-hydroxy-1-nitropyrene - PBS phosphate-buffered saline - PCB a mixture of polychlorinated biphenyls (Aroclor 1254) - TLC thin layer chromatography     

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.     

Metabolism of thalidomide in human microsomes, cloned human cytochrome P-450 isozymes, and Hansen's disease patients

Previous in vitro studies in rat microsomal preparations suggested that thalidomide is metabolized by the cytochrome P450 system (CYP). In this study, we examined the extent of thalidomide metabolism by preparations of pooled human microsomes, microsomes containing cloned human CYP isozymes (CYPIA2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4), and Hansen's disease patients. Results indicated that thalidomide was a poor substrate for CYP isozymes. Alteration of incubation buffer, pH, incubation time, and microsome and thalidomide concentrations did not increase the production of any metabolites. Thalidomide also did not inhibit metabolism of CYP-specific substrates and therefore any interactions with other drugs that are metabolized by the same enzyme system are unlikely. Hansen's patients were given a single oral dose of thalidomide (400 mg), and their blood and urine were collected at time points up to 72 hours, processed, and analyzed by tandem mass spectrometry. Although thalidomide was present in the plasma and urine, no metabolites were found in the plasma and very low amounts of the 5-OH thalidomide metabolite were present in the urine. These results suggest that thalidomide does not undergo significant metabolism by human CYP and that clinically important interactions between thalidomide and drugs that are also metabolized by this enzyme system are unlikely. The major route of thalidomide breakdown in humans and animals is through spontaneous hydrolysis with subsequent elimination in the urine.     

Inhibition of adrenocortical cytochrome P-450scc by (20R)-20-phenyl-5-pregnene-3 beta,20-diol

The effects of the cholesterol analogue, (20R)-20-phenyl-5-pregnene-3 beta,20-diol (20-PPD), on the catalytic and spectral properties of purified bovine adrenocortical cytochrome P-450scc were investigated. In contrast to results with cholesterol and (20R)-20-hydroxycholesterol, no conversion of 20-PPD to pregnenolone could be detected; instead, 20-PPD was found to be a potent inhibitor of cytochrome P-450scc. Kinetic analyses showed that the inhibition is reversible and competitive with respect to cholesterol with an apparent Ki = 30nM. Spectral binding studies with ferricytochrome P-450scc showed that 20-PPD formed a 1:1 complex with the enzyme, having an absorption spectrum similar to that produced by (20R)-20-hydroxycholesterol. These results indicate that 20-PPD binds with very high affinity to the substrate site on cytochrome P-450scc. The finding that the phenyl side chain is readily accommodated suggests the presence in this site of an open pocket which may be normally occupied by C-22 to C-27 of the cholesterol side chain.