Furafylline is a potent and selective inhibitor of cytochrome P450IA2 in man.

1. Furafylline (1,8-dimethyl-3-(2'-furfuryl)methylxanthine) is a methylxanthine derivative that was introduced as a long-acting replacement for theophylline in the treatment of asthma. Administration of furafylline was associated with an elevation in plasma levels of caffeine, due to inhibition of caffeine oxidation, a reaction catalysed by one or more hydrocarbon-inducible isoenzymes of P450. We have now investigated the selectivity of inhibition of human monooxygenase activities by furafylline. 2. Furafylline was a potent, non-competitive inhibitor of high affinity phenacetin O-deethylase activity of microsomal fractions of human liver, a reaction catalysed by P450IA2, with an IC50 value of 0.07 microM. 3. Furafylline had either very little or no effect on human monooxygenase activities catalysed by other isoenzymes of P450, including P450IID1, P450IIC, P450IIA. Of particular interest, furafylline did not inhibit P450IA1, assessed from aryl hydrocarbon hydroxylase activity of placental samples from women who smoked cigarettes. 4. It is concluded that furafylline is a highly selective inhibitor of P450IA2 in man. 5. Furafylline was a potent inhibitor of the N3-demethylation of caffeine and of a component of the N1- and N7-demethylation. This confirms earlier suggestions that caffeine is a selective substrate of a hydrocarbon-inducible isoenzyme of P450 in man, and identifies this as P450IA2. Thus, caffeine N3-demethylation should provide a good measure of the activity of P450IA in vivo in man. 6. Although furafylline selectively inhibited P450IA2, relative to P450IA1, in the rat, this was at 1000-times the concentration required to inhibit the human isoenzyme, suggesting a major difference in the active site geometry between the human and the rat orthologues of P50IA2.     

Gemfibrozil is a potent inhibitor of human cytochrome P450 2C9.

The in vitro inhibitory effects of gemfibrozil on cytochrome P450 (CYP) 1A2 (phenacetin O-deethylation), CYP2A6 (coumarin 7-hydroxylation), CYP2C9 (tolbutamide hydroxylation), CYP2C19 (S-mephenytoin 4'-hydroxylation), CYP2D6 (dextromethorphan O-deethylation), CYP2E1 (chlorzoxazone 6-hydroxylation), and CYP3A4 (midazolam 1'-hydroxylation) activities were examined using pooled human liver microsomes. The in vivo drug interactions of gemfibrozil were predicted in vitro using the [I]/([I] + K(i)) values. Gemfibrozil strongly and competitively inhibited CYP2C9 activity, with a K(i) (IC(50)) value of 5.8 (9.6) microM. In addition, gemfibrozil exhibited somewhat smaller inhibitory effects on CYP2C19 and CYP1A2 activities, with K(i) (IC(50)) values of 24 (47) microM and 82 (136) microM, respectively. With concentrations up to 250 microM, gemfibrozil showed no appreciable effect on CYP2A6, CYP2D6, CYP2E1, and CYP3A4 activities. Based on [I]/([I] + K(i)) values calculated using peak total (or unbound) plasma concentration of gemfibrozil, 96% (56%), 86% (24%), and 64% (8%) inhibition of the clearance of CYP2C9, CYP2C19, and CYP1A2 substrates could be expected, respectively. In conclusion, gemfibrozil inhibits the activity of CYP2C9 at clinically relevant concentrations, and this is the likely mechanism by which gemfibrozil interacts with CYP2C9 substrate drugs, such as warfarin and glyburide. Gemfibrozil may also impair clearance of CYP2C19 and CYP1A2 substrates, but inhibition of other CYP isoforms is unlikely.     

Ligand-based design of a potent and selective inhibitor of cytochrome P450 2C19

A series of omeprazole-based analogues was synthesized and assessed for inhibitory activity against CYP2C19. The data was used to build a CYP2C19 inhibition pharmacophore model for the series. The model was employed to design additional analogues with inhibitory potency against CYP2C19. Upon identifying inhibitors of CYP2C19, ligand-based design shifted to attenuating the rapid clearance observed for many of the inhibitors. While most analogues underwent metabolism on their aliphatic side chain, metabolite identification indicated that for analogues such as compound 30 which contain a heterocycle adjacent to the sulfur moiety, metabolism primarily occurred on the benzimidazole moiety. Compound 30 exhibited improved metabolic stability (Cl(int) = 12.4 mL/min/nmol) and was selective in regard to inhibition of CYP2C19-catalyzed (S)-mephenytoin hydroxylation in human liver microsomes. Finally, representative compounds were docked into a homology model of CYP2C19 in an effort to understand the enzyme-ligand interactions that may lead to favorable inhibition or metabolism properties.     

Climbazole is a new potent inducer of rat hepatic cytochrome P450

We examined the effect of climbazole on the induction of rat hepatic microsomal cytochrome P450 (P450), and compared the induction potency with other N-substituted azole drugs such as clorimazole. We found that climbazole is found to be a potent inducer of rat hepatic microsomal P450 as clorimazole. Induced level of P450 by climbazole was almost similar in extent to clorimazole when compared with other imidazole drugs in a dose- and time-dependent manner. Parallel to the increase in P450, climbazole increased aminopyrine and erythromycin N-demethylase, ethoxycoumarin O-deethylase, and androstenedione 16 beta- and 15 alpha/6 beta hydroxylase activities; however, clorimazole did not induce aminopyrine N-demethylase activity irrespective of its marked increase in P450 content. Immunoblot analyses revealed that climbazole induced CYP2B1, 3A2 and 4A1. The present findings indicate that climbazole is a new potent inducer of hepatic microsomal P450 and drug-metabolizing enzymes like clorimazole, but it may have some differential mechanism(s) for these enzymes' induction in rat liver.     

Acetylshikonin is a novel non‐selective cytochrome P450 inhibitor

Acetylshikonin is a biologically active compound with anti‐cancer and anti‐inflammatory activity, which is isolated from the roots of Lithospermum erythrorhizoma . An inhibitory effect of acetylshikonin against CYP2J2 activity was discovered recently. Based on this result, this study was expanded to evaluate the inhibitory effects of acetylshikonin against nine different cytochrome P450 (P450) isoforms in human liver microsomes (HLMs) using substrate cocktails incubation assay. Acetylshikonin showed a strong inhibitory effect against all P450s tested with IC₅₀ values of 1.4–4.0 μ m . Pre‐incubation of acetylshikonin with HLMs and NADPH did not alter the inhibition potency, indicating that acetylshikonin is not a mechanism‐based inhibitor. SKF‐525A, a widely used non‐specific P450 inhibitor, had no inhibitory activity against CYP1A2, 2A6, 2E1 and 2J2, while it showed an inhibitory effect against CYP2B6, CYP2C19 and 2D6 with IC₅₀ values of 2.5, 3.6 and 0.5 μ m , respectively. Our findings indicate that acetylshikonin may be a novel general P450 inhibitor, which could replace SKF‐525A.     

Ketoconazole: a potent inhibitor of cytochrome P-450-dependent drug metabolism in rat liver

Ketoconazole, an orally active imidazole antimycotic agent, is shown to be a potent inhibitor of drug N-demethylase activities of liver microsomes from rats pretreated with phenobarbital or pregnenolone-16 alpha-carbonitrile, and an inhibitor of 7-ethoxyresorufin O-deethylase activity of liver microsomes from rats pretreated with 5,6-benzoflavone. Spectrophotometric studies reveal that the imidazole compound binds to the cytochrome P-450 component of the monooxygenase complex, and has little effect on NADPH-cytochrome c (P-450) reductase activity. These results are strongly suggestive that cytochrome P-450 is the site of action of this potent inhibitor of drug metabolism in liver microsomes.     

Cytochrome P450 3A-dependent metabolism of a potent and selective gamma-aminobutyric acid Aalpha2/3 receptor agonist in vitro: involvement of cytochrome P450 3A5 displaying biphasic kinetics.

In vitro metabolism studies were conducted to determine the human cytochrome P450 enzyme(s) involved in the biotransformation of 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3b]pyridazine (TPA023), a selective agonist of human gamma-aminobutyric acid(A) receptor alpha2 and alpha3 subunits. Incubation of TPA023 with NADPH-fortified human liver microsomes resulted in the formation of t-butyl hydroxy TPA023, N-desethyl TPA023, and three minor metabolites. Both t-butyl hydroxylation and N-deethylation reactions were greatly inhibited (>85%) in the presence of CYP3A-selective inhibitory antibodies and chemical inhibitors, indicating that members of the CYP3A subfamily play an important role in TPA023 metabolism. Eadie-Hofstee plots of t-butyl hydroxylation and N-deethylation in pooled CYP3A5-rich human liver microsomes revealed a low K(m) (3.4 and 4.5 microM, respectively) and a high K(m) (12.7 and 40.0 microM, respectively) component. For both metabolites, the high K(m) component was not observed with a pool of microsomal preparations containing minimal levels of CYP3A5. Preincubation of liver microsomes with mifepristone (selectivity for CYP3A4 > CYP3A5) greatly inhibited both t-butyl hydroxylation and N-deethylation (>75%); however, the residual activities were significantly higher in the pooled CYP3A5-rich liver microsomes (p < 0.0005). In addition, elevated levels of residual t-butyl hydroxylase and N-deethylase activities were observed in the presence of both CYP3A5-rich and CYP3A5-deficient preparations when the substrate concentration increased from 4 to 40 microM. In agreement, metabolite formation catalyzed by recombinant CYP3A5 was described by a biphasic model. It is concluded that CYP3A4 plays a major role in TPA023 metabolism, and CYP3A5 may also contribute at higher concentrations of the compound.     

Coumarin metabolism by rat esophageal microsomes and cytochrome P450 2A3

The rat esophagus is strikingly sensitive to tumor induction by nitrosamines, and it has been hypothesized that this tissue contains cytochrome P450 enzymes (P450s) which catalyze the metabolic activation of these carcinogens. The metabolic capacity of the esophagus is not well characterized. In the study described here, the products of 14C-coumarin metabolism by rat esophageal microsomes were identified and quantified. Metabolite characterization was by LC/MS/MS and GC/MS and comparison to standards, quantification was by radioflow HPLC. The coumarin metabolites formed by rat esophageal microsomes were compared to those formed by P450 2A3. The major metabolites formed by esophageal microsomes were 8-hydroxycoumarin, o-hydroxyphenylacetaldehyde (o-HPA), and o-hydroxyphenylacetic acid (o-HPAA). A smaller amount of 5-hydroxycoumarin, about one-third the 8-hydroxycoumarin, was also formed. o-HPA and o-HPAA are products of coumarin 3,4-epoxidation. The relative rates of coumarin 8-hydroxylation and 3,4-epoxidation were similar. Coumarin 8-hydroxylation has not previously been reported as a major pathway in any tissue, and no P450s have yet been reported to catalyze this reaction. P450 2A3 catalyzed both the 7-hydroxylation and 3,4-epoxidation of coumarin. P450 2A3 was previously characterized as a coumarin 7-hydroxylase, however, in this study, we report that it catalyzes the formation of o-HPA more efficiently. The Km and Vmax were 1.3 +/- 0.35 microM and 0.65 +/- 0.06 nmol/min/nmol P450 for coumarin 7-hydroxylation and 1.4 +/- 0.58 microM and 3.1 +/- 0.46 nmol/min/nmol P450 for o-HPA formation.     

The alkaloid rutaecarpine is a selective inhibitor of cytochrome P450 1A in mouse and human liver microsomes.

Rutaecarpine, evodiamine, and dehydroevodiamine are quinazolinocarboline alkaloids isolated from a traditional Chinese medicine, Evodia rutaecarpa. The in vitro effects of these alkaloids on cytochrome P450 (P450)-catalyzed oxidations were studied using mouse and human liver microsomes. Among these alkaloids, rutaecarpine showed the most potent and selective inhibitory effect on CYP1A-catalyzed 7-methoxyresorufin O-demethylation (MROD) and 7-ethoxyresorufin O-deethylation (EROD) activities in untreated mouse liver microsomes. The IC(50) ratio of EROD to MROD was 6. For MROD activity, rutaecarpine was a noncompetitive inhibitor with a K(i) value of 39 +/- 2 nM. In contrast, rutaecarpine had no effects on benzo[a]pyrene hydroxylation (AHH), aniline hydroxylation, and nifedipine oxidation (NFO) activities. In human liver microsomes, 1 microM rutaecarpine caused 98, 91, and 77% decreases of EROD, MROD, and phenacetin O-deethylation activities, respectively. In contrast, less than 15% inhibition of AHH, tolbutamide hydroxylation, chlorzoxazone hydroxylation, and NFO activities were observed in the presence of 1 microM rutaecarpine. To understand the selectivity of inhibition of CYP1A1 and CYP1A2, inhibitory effects of rutaecarpine were studied using liver microsomes of 3-methylcholanthrene (3-MC)-treated mice and Escherichia coli membrane expressing bicistronic human CYP1A1 and CYP1A2. Similar to the CYP1A2 inhibitor furafylline, rutaecarpine preferentially inhibited MROD more than EROD and had no effect on AHH in 3-MC-treated mouse liver microsomes. For bicistronic human P450s, the IC(50) value of rutaecarpine for EROD activity of CYP1A1 was 15 times higher than the value of CYP1A2. These results indicated that rutaecarpine was a potent inhibitor of CYP1A2 in both mouse and human liver microsomes.     

Ridogrel: a selective inhibitor of the cytochrome P450-dependent thromboxane synthesis.

Ridogrel [(E)-5-[[[(3-pyridinyl)[3-(trifluoromethyl)phenyl] methylene]amino]oxy] pentanoic acid] is a potent inhibitor of the P450-dependent human platelet thromboxane A2 (TxA2) synthase. Fifty percent inhibition is already achieved at 5.0 +/- 0.37 nM. This IC50 value is close to half the P450 concentration used, i.e. 10.7 nM. Ridogrel binds to human platelet microsomal P450 as proven by the type II spectral changes induced by the addition of increasing concentrations of ridogrel to solubilized microsomes. The calculated half-maximal spectral change (SC50 value) is 3.78 +/- 1.79 nM. These results indicate that ridogrel binds stoichiometrically and suggest that inhibition of thromboxane synthesis may originate from liganding of its basic nitrogen to the haem-iron of P450 and from the attachment of the hydrophobic carboxylic side chain to or near the substrate binding place. Ridogrel is a selective inhibitor of the TxA2 synthase. At a high concentration (10 microM), ridogrel has a slight, if any, effect on the P450-mediated cholesterol synthesis in human liver and hepatoma cells and androgen synthesis from 17 alpha-hydroxy-20-dihydroprogesterone or pregnenolone in subcellular fractions from rat testes. These results indicate that ridogrel is a poor inhibitor of the P450-dependent 14 alpha-demethylase, 17 alpha-hydroxylase and 17,20-lyase. It has, up to 10 microM, no effect on the adrenal mitochondrial 11 beta-hydroxylase and cholesterol side-chain cleavage enzyme and does not inhibit aromatase activity in human placental microsomes. Ridogrel has no significant effect on the regio- and stereoselective P450-dependent oxidations of testosterone in liver microsomes from unpretreated or from 5-pregnen-3 beta-ol-20-one-16 alpha-carbonitrile-, phenobarbital- or 3-methylcholanthrene-pretreated male and female Sprague-Dawley rats. It does not interfere with the reduction of testosterone into 5 alpha-dihydrotestosterone and 5 alpha androstane 3 beta, 17 beta-diol.     

Triethylenethiophosphoramide is a specific inhibitor of cytochrome P450 2B6: implications for cyclophosphamide metabolism.

Cytochrome P450 2B6 is a genetically polymorphic enzyme that is important in the metabolism of a number of clinically used drugs. This enzyme is not as well studied as other cytochrome P450 (P450) isoforms because of the lack of specific antibodies, probe drugs, and inhibitors. Although recent progress has been made toward specific antibodies and probe drugs, a specific enzyme inhibitor is still lacking. Studies suggest that CYP2B6 plays an important role in the 4-hydroxylation of cyclophosphamide and that this reaction can be inhibited by triethylenethiophosphoramide (thioTEPA). We therefore wished to test the hypothesis that thioTEPA is an inhibitor of CYP2B6. Using human liver microsomes (HLMs) and recombinant P450 enzymes, we demonstrated that thioTEPA is a potent and specific inhibitor of CYP2B6. Enzyme activity was reduced 78.1 +/- 0.2% by 50 microM thioTEPA when CYP2B6 activity was measured by following the metabolism of 200 microM S-mephenytoin to nirvanol. thioTEPA did not significantly inhibit (<20% at 100 microM) the other isoforms tested (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4). thioTEPA seems to be a potent noncompetitive inhibitor of CYP2B6, with K(i) values of 4.8 +/- 0.3 and 6.2 +/- 0.7 microM for HLMs and recombinant CYP2B6, respectively, values that are within the plasma concentration range of thioTEPA at therapeutic doses (1.1-18.6 microM). We conclude that thioTEPA is a potent and specific inhibitor of CYP2B6 and that this is the likely mechanism by which thioTEPA inhibits the activation of cyclophosphamide. Furthermore, thioTEPA may prove to be a valuable new tool for the study of this important drug-metabolizing enzyme.     

The metabolism of clopidogrel is catalyzed by human cytochrome P450 3A and is inhibited by atorvastatin.

The prodrug clopidogrel (Plavix) is activated by cytochrome p450 (p450) to a metabolite that inhibits ADP-induced platelet aggregation. Clopidogrel is frequently administered to patients in conjunction with the CYP3A4 substrate atorvastatin (Lipitor). Since clinical studies indicate that atorvastatin inhibits the antiplatelet activity of clopidogrel, we investigated whether CYP3A4 metabolized clopidogrel in vitro. Microsomes prepared from dexamethasone-pretreated rats metabolized clopidogrel at a rate of 3.8 nmol min(-1) nmol of p450(-1), which is 65 and 1270% faster than the rate of metabolism by microsomes from control and beta-napthoflavone-treated rats, respectively. To identify the human p450s responsible for clopidogrel oxidation, genetically engineered microsomes containing a single human p450 isozyme were tested for their ability to oxidize clopidogrel. CYP3A4 and 3A5 metabolized clopidogrel at a significantly higher rate than eight other p450 isozymes, suggesting that CYP3A4 and 3A5 are primarily responsible for in vivo clopidogrel metabolism. Clopidogrel interacts with human CYP3A4 with a spectral dissociation constant (K(s)), K(m), and V(max) of 12 microM, 14 +/- 1 microM and 6.7 +/- 1 nmol min(-1) nmol p450(-1), respectively. Atorvastatin lactone, the physiologically relevant substrate, inhibits clopidogrel with a K(i) of 6 microM. When clopidogrel and atorvastatin are present at equimolar concentrations, clopidogrel metabolism is inhibited by greater than 90%. Since CYP3A4 and 3A5 metabolize clopidogrel faster than other human p450 isozymes and are the most abundant p450s in human liver, they are predicted to be predominantly responsible for the activation of clopidogrel in vivo.     

Rofecoxib is a potent inhibitor of cytochrome P450 1A2: studies with tizanidine and caffeine in healthy subjects

AIMS: Case reports suggest an interaction between rofecoxib and the CYP1A2 substrate tizanidine. Our objectives were to explore the extent and mechanism of this possible interaction and to determine the CYP1A2 inhibitory potency of rofecoxib. METHODS: In a randomized, double-blind, two-phase cross-over study, nine healthy subjects took 25 mg rofecoxib or placebo daily for 4 days and, on day 4, each ingested 4 mg tizanidine. Plasma concentrations and the urinary excretion of tizanidine, its metabolites (M) and rofecoxib, and pharmacodynamic variables were measured up to 24 h. On day 3, a caffeine test was performed to estimate CYP1A2 activity. RESULTS: Rofecoxib increased the area under the plasma concentration-time curve (AUC(0-infinity)) of tizanidine by 13.6-fold [95% confidence interval (CI) 8.0, 15.6; P < 0.001), peak plasma concentration (C(max)) by 6.1-fold (4.8, 7.3; P < 0.001) and elimination half-life (t(1/2)) from 1.6 to 3.0 h (P < 0.001). Consequently, rofecoxib markedly increased the blood pressure-lowering and sedative effects of tizanidine (P < 0.05). Rofecoxib increased several fold the tizanidine/M-3 and tizanidine/M-4 ratios in plasma and urine and the tizanidine/M-5, tizanidine/M-9 and tizanidine/M-10 ratios in urine (P < 0.05). In addition, it increased the plasma caffeine/paraxanthine ratio by 2.4-fold (95% CI 1.4, 3.4; P = 0.008) and this ratio correlated with the tizanidine/metabolite ratios. Finally, the AUC(0-25) of rofecoxib correlated with the placebo phase caffeine/paraxanthine ratio (r = 0.80, P = 0.01). CONCLUSIONS: Rofecoxib is a potent inhibitor of CYP1A2 and it greatly increases the plasma concentrations and adverse effects of tizanidine. The findings suggest that rofecoxib itself is also metabolized by CYP1A2, raising concerns about interactions between rofecoxib and other CYP1A2 substrate and inhibitor drugs.     

Role of rat liver cytochrome P450 3A and 2D in metabolism of imrecoxib

AIM: To investigate the in vitro metabolism of imrecoxib in rat liver microsomes and to identify the cytochrome P450 (CYP) forms involved in its metabolism. METHODS: Liver microsomes of Wistar rats were prepared using an ultracentrifuge. The in vitro metabolism of imrecoxib was studied by incubation with rat liver microsomes. To characterize the CYP forms involved in the 4 '-methyl hydroxylation of imrecoxib, the effects of typical CYP inducers (such as dexamethasone, isoniazid and beta-naphthoflavone) and of CYP inhibitors (such as ketoconazole, quinine, alpha-naphthoflavone, methylpyrazole, and cimetidine) on the formation rate of 4 '-hydroxymethyl imrecoxib were investigated. RESULTS: Imrecoxib was metabolized to 3 metabolites by rat liver microsomes: 4'-hydroxymethyl imrecoxib (M4), 4'-hydroxymethyl-5-hydoxyl imrecoxib (M3), and 4 '-hydroxymethyl-5-carbonyl imrecoxib (M5). Over the imrecoxib concentration range studied (5-600 micromol/L), the rate of 4'-methyl hydroxylation conformed to monophasic Michaelis-Menten kinetics. Dexamethasone significantly induced the formation of M4. Ketoconazole markedly lowered the metabolic rate of imrecoxib in a concentration-dependent manner. Moreover, a significant inhibitory effect of quinine on the formation of M4 was observed in microsomes obtained from control rats, isoniazid-induced rats, and b-naphthoflavone-induced rats. In contrast, a-naphthoflavone, cimetidine, and methylpyrazole had no inhibitory effects on this metabolic pathway. CONCLUSION: Imrecoxib is metabolized via 4'-methyl hydroxylation in rat liver microsomes. The reaction is mainly catalyzed by CYP 3A. CYP 2D also played a role in control rats, in isoniazid-induced rats and in beta-naphthoflavone-induced rats.     

Porcine cytochrome P450 and metabolism

The pig and especially the minipig are becoming increasingly used as a test animal both in pharmacological and toxicological testing of new compounds. The minipig is used because of its size, it is easy to handle and less test substrate is required. When using an animal species for testing it is of importance to know if the test animal's posses the same abilities to metabolize drugs as humans. Some of the P450 enzymes have been characterized in the pig regarding substrate specificity, inhibition and regulation. The porcine enzymes CYP1A, CYP2A and CYP3A all metabolize the same test substrates as the human enzymes, whereas the enzymes CYP2B, CYP2D, and CYP2E in pig on the other hand seem to be different from the human enzymes concerning metabolism of the well know test substrates. Some of the porcine enzymes have been sequenced i.e. CYP1A, CYP2A, CYP2B, CYP2D, CYP2E and CYP3A and not surprisingly the porcine CYPs that metabolize the human test substrates are about 75% identical in cDNA sequences. What is needed is inhibitory antibodies against each of the porcine enzymes, in order to test whether a test compound is metabolized by one or the other enzyme. Until now chemical inhibitors have been used, but they are rarely 100% specific. Anti-human inhibitory antibodies have also been used, but they may not recognize the porcine enzyme and therefore will not inhibit the reaction. Antibodies for immunoblotting would also make it possible to estimate how much of the total P450 the individual enzymes comprise. From what is known about the porcine P450, it can be concluded that the pig seems to be a good test species if CYP1A, CYP2A or CYP3A are involved in the metabolism of the test compound, depending on the contribution of other enzymes in competing pathways.     

Inhibition and activation of the human liver microsomal and human cytochrome P450 3A4 metabolism of testosterone by deployment-related chemicals.

Cytochrome P450 (P450) enzymes are major catalysts involved in the metabolism of xenobiotics and endogenous substrates such as testosterone (TST). Major TST metabolites formed by human liver microsomes include 6beta-hydroxytestosterone (6beta-OHTST), 2beta-hydroxytestosterone (2beta-OHTST), and 15beta-hydroxytestosterone (15beta-OHTST). A screen of 16 cDNA-expressed human P450 isoforms demonstrated that 94% of all TST metabolites are produced by members of the CYP3A subfamily with 6beta-OHTST accounting for 86% of all TST metabolites. Similar K(m) values were observed for production of 6beta-, 2beta-, and 15beta-OHTST with human liver microsomes (HLM) and CYP3A4. However, V(max) and CL(int) were significantly higher for 6beta-OHTST than 2beta-OHTST (approximately 18-fold) and 15beta-OHTST (approximately 40-fold). Preincubation of HLM with a variety of ligands, including chemicals used in military deployments, resulted in varying levels of inhibition or activation of TST metabolism. The greatest inhibition of TST metabolism in HLM was following preincubation with organophosphorus compounds, including chlorpyrifos, phorate, and fonofos, with up to 80% inhibition noticed for several metabolites including 6beta-OHTST. Preincubation of CYP3A4 with chlorpyrifos, but not chlorpyrifos-oxon, resulted in 98% inhibition of TST metabolism. Phorate and fonofos also inhibited the production of most primary metabolites of CYP3A4. Kinetic analysis indicated that chlorpyrifos was one of the most potent inhibitors of major TST metabolites followed by fonofos and phorate. Chlorpyrifos, fonofos, and phorate inhibited major TST metabolites noncompetitively and irreversibly. Conversely, preincubation of CYP3A4 with pyridostigmine bromide increased metabolite levels of 6beta-OHTST and 2beta-OHTST. Preincubation of human aromatase (CYP19) with the test chemicals had no effect on the production of the endogenous estrogen, 17beta-estradiol.     

Hepatic metabolism of MK-0457, a potent aurora kinase inhibitor: interspecies comparison and role of human cytochrome P450 and flavin-containing monooxygenase.

MK-0457 (N-[4([4-(4-methylpiperazin-1-yl)-6-[(3-methyl-1H-pyrazol-5 -yl)amino]pyrimidin-2-yl]thio)phenyl]cyclopropanecarboxamide), an Aurora kinase inhibitor in development for the treatment of cancer, was evaluated for its in vitro metabolism in different species. This compound primarily underwent N-oxidation and N-demethylation in human, monkey, dog, and rat liver preparations. However, N-demethylation was less significant in dogs. The formation of minor metabolites varied with species, but all metabolites generated in human hepatocytes were observed in animals. Results of immunoinhibition, selective chemical inhibition, thermal inactivation, and metabolism by recombinant cytochromes P450 and flavin-containing monoogygenases (FMOs) strongly suggest that CYP3A4 and FMO3 comparably contributed to MK-0457 N-oxidation in human liver microsomes, where the reaction conformed to Michaelis-Menten kinetics. These studies indicate a major role of CYP2C8 in the N-demethylation reaction, whereas CYP3A4 only made a minor contribution. However, significant substrate inhibition was observed with MK-0457 N-demethylation at high substrate concentrations (>10 microM) in human liver microsomes relative to the anticipated therapeutic exposure. A multienzyme metabolic pathway such as this may mitigate the potential of drug interactions in clinical treatment with MK-0457.     

A novel rat hepatic clofibrate-inducible cytochrome P450 that is not a lauric acid hydroxylase.

2-Methoxy-6-[1-methylethyl]naphthalene (MMEN) was hydroxylated in an NADPH-dependent manner to the (omega-1)-alcohol and the (R)-omega- and (S)-omega-alcohols by rat hepatic microsomes. (S)-omega-Hydroxylation was selectively induced 7-fold by clofibrate treatment. Phenobarbital, 3-methyl-cholanthrene, dexamethasone, cholestyramine, and MMEN did not induce this activity to the same extent. Incubation of the racemic omega-alcohols with microsomes isolated from rats resulted in a greater rate of degradation of the (S)- than the (R)-omega-alcohol confirming (S)-omega-hydroxylation to be an initial catalytic event. MMEN and lauric acid were not competitive inhibitors of each other in microsomes from clofibrate-treated rats, indicating the (S)-omega-MMEN hydroxylase to be a different enzyme from the characterized clofibrate-inducible lauric acid hydroxylases, CYP4A1 and CYP4A3. This was confirmed by the observations that (1) lauric acid hydroxylation was inhibited by 0.02% Tween 20 or Tween 80 and 25 microM capric or myristic acids, whereas omega-MMEN hydroxylation was not, (2) omega-MMEN hydroxylation was inhibited by ketoconazole, cholesterol and acetone, whereas lauric acid hydroxylation was not, and (3) CYP4A1 and CYP4A3 expressed in Hep G2 cells did not catalyze MMEN hydroxylation. Microsomes from the lungs of rabbits treated with progesterone and kidney of untreated rats did not support selective (S)-omega-MMEN hydroxylation, indicating that this activity is not associated with CYP4A4 or CYP4A2, respectively. Leukotriene B4 (LTB4) hepatic microsomal hydroxylation was not inhibited by MMEN and microsomes from human neutrophils did not support the reaction. These data identify a hitherto uncharacterized cytochrome P450 which is selectively induced by clofibrate and does not catalyze the omega-hydroxylation of the fatty acids or prostaglandins investigated. It is proposed that the enzyme catalyzing the selective (S)-omega-hydroxylation of MMEN is a novel rat P450 and that it is either a new member of the CYP4 family or a clofibrate-inducible P450 from another gene family.     

Nefiracetam metabolism by human liver microsomes: role of cytochrome P450 3A4 and cytochrome P450 1A2 in 5-hydroxynefiracetam formation.

An in-vitro study was conducted to investigate the metabolism of nefiracetam in human liver microsomes and to identify the enzymes responsible for the metabolism. Nefiracetam was hydroxylated by human liver microsomes to 5-hydroxynefiracetam (5-OHN). Eadie-Hofstee plots for the formation of 5-OHN suggested substrate activation. The kinetic parameters, apparent Km, Vmax, and Hill coefficient, for the formation of 5-OHN by pooled human liver microsomes were 4012 microM, 2.66 nmol min(-1) (mg protein)(-1), and 1.65, respectively. The formation of 5-OHN was significantly correlated with cytochrome P450 (CYP)3A4-mediated testosterone 6beta-hydroxylase activity and dextromethorphan N-demethylase activity. The 5-OHN formation was inhibited (94%) by antibody to human CYP3A4/5. The 5-OHN formation was also inhibited by the CYP3A4 inhibitors ketoconazole and troleandomycin, but not significantly inhibited by several other P450 inhibitors. The microsomes containing cDNA-expressed CYP3A4 formed 5-OHN with sigmoidal kinetics. CYP3A5-containing microsomes did not form 5-OHN. These results indicated that CYP3A, most likely CYP3A4, was the major isozyme responsible for the formation of 5-OHN in human liver microsomes. CYP1A2 and CYP2C19 microsomes were also capable of forming 5-OHN. However, the contribution of CYP1A2 was considered to be relatively minor compared with that of CYP3A4, and the contribution of CYP2C19 was assumed to be negligible, based on the result of the immunoinhibition study and taking into account both the turnover rate by each isozyme and the relative abundance of each isozyme in human liver. We conclude that on average the formation of 5-OHN, the major metabolite of nefiracetam, is principally mediated by CYP3A4 with a relatively minor contribution by CYP1A2.     

Comparison of mouse and rat cytochrome P450-mediated metabolism in liver and intestine.

The liver is considered to be the major site of first-pass metabolism, but the small intestine is also able to contribute significantly. The improvement of existing in vitro techniques and the development of new ones, such as intestinal slices, allow a better understanding of the intestine as a metabolic organ. In this paper, the formation of metabolites of several human CYP3A substrates by liver and intestinal slices from rat and mouse was compared. The results show that liver slices exhibited a higher metabolic rate for the majority of the studied substrates, but some metabolites were produced at a higher rate by intestinal slices, compared with liver slices. Coincubation with ketoconazole inhibited the metabolic conversion in intestinal slices almost completely, but inhibition was variable in liver slices. To better understand the role of CYP3A in mice, we studied the relative mRNA expression of different CYP3A isoforms in intestine and liver from mice because, in this species, CYP3A expression has not been well described in these organs. It was found that in mice, CYP3A13 is more expressed in the intestine, whereas CYP3A11, CYP3A25, and CYP3A41 are more expressed in the liver, comparable to similar findings in the rat. Altogether, these data demonstrate that, in addition to liver, the intestine from mouse and rat may have an important role in the process of first-pass metabolism, depending on the substrate. Moreover, we show that intestinal slices are a useful in vitro technique to study gut metabolism.