Monkey liver cytochrome P450 2C9 is involved in caffeine 7-N-demethylation to form theophylline

Abstract

1. Caffeine (1,3,7-trimethylxanthine) is a phenotyping substrate for human cytochrome P450 1A2. 3-N-Demethylation of caffeine is the main human metabolic pathway, whereas monkeys extensively mediate the 7-N-demethylation of caffeine to form pharmacological active theophylline.

2. Roles of monkey P450 enzymes in theophylline formation from caffeine were investigated using individual monkey liver microsomes and 14 recombinantly expressed monkey P450 enzymes, and the results were compared with those for human P450 enzymes.

3. Caffeine 7-N-demethylation activity in microsomes from 20 monkey livers was not strongly inhibited by α-naphthoflavone, quinidine or ketoconazole, and was roughly correlated with diclofenac 4′-hydroxylation activities. Monkey P450 2C9 had the highest activity for caffeine 7-N-demethylation. Kinetic analysis revealed that monkey P450 2C9 had a high V_max/K_m value for caffeine 7-N-demethylation, comparable to low K_m value for monkey liver microsomes. Caffeine could dock favorably with monkey P450 2C9 modeled for 7-N-demethylation and with human P450 1A2 for 3-N-demethylation.

4. The primary metabolite theophylline was oxidized to 8-hydroxytheophylline in similar ways by liver microsomes and by recombinant P450s in both humans and monkeys.

5. These results collectively suggest a high activity for monkey liver P450 2C9 toward caffeine 7-N-demethylation, whereas, in humans, P450 1A2-mediated caffeine 3-N-demethylation is dominant.     

Drug interactions of diclofenac and its oxidative metabolite with human liver microsomal cytochrome P450 1A2-dependent drug oxidation

Abstract

1.?The purpose of this study was to investigate the inhibitory effects of diclofenac on human cytochrome P450 1A2-, 2C19- and 3A4-mediated drug oxidations and to evaluate the drug interaction potential of diclofenac and 4′-hydroxydiclofenac.

2.?Diclofenac was converted to 4′-hydroxydiclofenac by recombinantly expressed human P450 1A2 with K_m and V_max values of 33?µM and 0.20?min^?1, respectively. Diclofenac and 4′-hydroxydiclofenac suppressed flurbiprofen 4′-hydroxylation by P450 2C9 strongly and moderately, respectively; however, they did not affect P450 2C19-dependent S-mephenytoin hydroxylation or P450 3A4-dependent midazolam hydroxylation.

3.?Although the caffeine 3-N-demethylation activity of liver microsomal P450 1A2 was inhibited by simultaneous incubation with diclofenac, the riluzole N-hydroxylation activities of recombinant P450 1A2 and human liver microsomes were inhibited after preincubation with diclofenac or 4′-hydroxydiclofenac for 20?min in the presence of NADPH. Using the inhibition constant (37?µM) of diclofenac on caffeine 3-N-demethylation and the reported 95th percentiles of maximum plasma concentration (10.5?µM) after an oral dose of diclofenac, the in vivo estimated increase in area under the plasma concentration–time curve was 29%.

4.?These results suggest that diclofenac could inhibit drug clearance to a clinically important degree that depends on P450 1A2. Clinically relevant drug interactions in vivo with diclofenac are likely to be invoked via human P450 1A2 function in addition to those caused by the effect of diclofenac on P450 2C9.     

Progesterone hydroxylation by cytochromes P450 2C and 3A enzymes in marmoset liver microsomes

1.?Common marmosets (Callithrix jacchus) are potentially useful nonhuman primate models for preclinical drug metabolism studies. However, the roles of marmoset cytochrome P450 (P450) isoforms in the oxidation of endobiotic progesterone have not been fully investigated. In this study, the roles of marmoset P450 isoforms in progesterone hydroxylation were extensively determined.

2.?The activities of liver microsomes from individual marmosets with respect to progesterone 21/17α- and 16α/6β-hydroxylation were significantly correlated with those for flurbiprofen 4-hydroxylation and midazolam 1′-hydroxylation, respectively, as similar correlations have been found in humans. Anti-P450 2?C and 3?A antibodies suppressed progesterone 21/17α- and 16α/6β-hydroxylation, respectively, in marmoset liver microsomes.

3.?Recombinant marmoset P450 2C58 and 2C19 catalyzed progesterone to form 21-hydroxyprogesterone and 16α-hydroxyprogesterone, respectively, as major products with high maximum velocity/K_m values of 0.53 and 0.089?mL/min/nmol, respectively. Recombinant marmoset P450 3A4/90 oxidized progesterone to form 6β-hydroxyprogesterone as a major product with homotropic cooperativity (>1 of Hill coefficients).

4.?These results indicate that the overall activities and roles of liver microsomal P450 enzymes in marmoset livers are similar to those in humans, especially for progesterone 21/17α- and 16α/6β-hydroxylation by marmoset P450 2?C and 3?A enzymes, respectively, suggesting important roles for these P450 enzymes in the metabolism of endobiotics in marmosets.     

Marmoset cytochrome P450 2J2 mainly expressed in small intestines and livers effectively metabolizes human P450 2J2 probe substrates,astemizole and terfenadine

1.?Common marmoset (Callithrix jacchus), a New World Monkey, has potential to be a useful animal model in preclinical studies. However, drug metabolizing properties have not been fully understood due to insufficient information on cytochrome P450 (P450), major drug metabolizing enzymes.

2.?Marmoset P450 2J2 cDNA was isolated from marmoset livers. The deduced amino acid sequence showed a high-sequence identity (91%) with cynomolgus monkey and human P450 2J2 enzymes. A phylogenetic tree revealed that marmoset P450 2J2 was evolutionarily closer to cynomolgus monkey and human P450 2J2 enzymes, than P450 2J forms in pigs, rabbits, rats or mice.

3.?Marmoset P450 2J2 mRNA was abundantly expressed in the small intestine and liver, and to a lesser extent in the brain, lung and kidney. Immunoblot analysis also showed expression of marmoset P450 2J2 protein in the small intestine and liver.

4.?Enzyme assays using marmoset P450 2J2 protein heterologously expressed in Escherichia coli indicated that marmoset P450 2J2 effectively catalyzed astemizole O-demethylation and terfenadine t-butyl hydroxylation, similar to human and cynomolgus monkey P450 2J2 enzymes.

5.?These results suggest the functional characteristics of P450 2J2 enzymes are similar among marmosets, cynomolgus monkeys and humans.     

Terfenadine t-butyl hydroxylation catalyzed by human and marmoset cytochrome P450 3A and 4F enzymes in livers and small intestines

1.?Roles of human cytochrome P450 (P450) 3A4 in oxidation of an antihistaminic drug terfenadine have been previously investigated in association with terfenadine–ketoconazole interaction. Several antihistamine drugs have been recently identified as substrates for multiple P450 enzymes. In this study, overall roles of P450 3A4, 2J2, and 4F12 enzymes in terfenadine t-butyl hydroxylation were investigated in small intestines and livers from humans, marmosets, and/or cynomolgus monkeys.

2.?Human liver microsomes and liver and small intestine microsomes from marmosets and cynomolgus monkeys effectively mediated terfenadine t-butyl hydroxylation. Ketoconazole and N-hydroxy-N′-(4-butyl-2-methylphenyl)-formamidine (a P450 4A/F inhibitor) almost completely and moderately inhibited these activities, respectively, in human liver microsomes; however, these chemicals did not show substantially suppression in marmoset liver. Anti-human P450 3A and 4F antibodies showed the roughly supportive inhibitory effects.

3.?Recombinant P450 3A4/90 and 4F12 showed high terfenadine t-butyl hydroxylation activities with substrate inhibition constants of 84–144?μM (under 26–76?μM of K_m values), in similar manners to liver and intestine microsomes.

4.?These results suggest that human and marmoset P450 3A4/90 and 4F12 in livers or small intestines played important roles in terfenadine t-butyl hydroxylation. Marmosets could be a model for humans during first pass extraction of terfenadine and related substrates.     

Model for the drug–drug interaction responsible for CYP3A enzyme inhibition. II: establishment and evaluation of dexamethasone-pretreated female rats

1.?Cytochrome P450 (CYP) 3A catalysis of testosterone 6β-hydroxylation in female rat liver microsomes was significantly induced, then reached a plateau level after pretreatment with 80?mg?kg^?1?day^?1 dexamethasone (DEX) for 3 days.

2.?Midazolam was mainly metabolized by CYP3A in DEX-treated female rat liver microsomes from an immuno-inhibition study, and the apparent K_m was 1.8?μM, similar to that in human microsomes.

3.?Ketoconazole and erythromycin, typical CYP3A inhibitors, demonstrated extensive inhibition of midazolam metabolism in DEX-treated female rat liver microsomes, and the apparent K_i values were 0.088 and 91.2?μM, respectively. The values were similar to those in humans, suggesting that DEX-treated female rat liver microsomes have properties similar to those of humans.

4.?After oral administration of midazolam, the plasma midazolam concentration in DEX-treated female rats significantly decreased compared with control female rats. The area under the plasma concentration curve (AUC) and elimination half-life were one-11th and one-20th of those of control female rats, respectively.

5.?Using DEX-treated female rats, the effect of CYP3A inhibitors on midazolam pharmacokinetics was evaluated. The AUC and maximum concentration in plasma (C_max) increased when ketoconazole was co-administered with midazolam.

6.?It was shown that the drug–drug interaction that occurs in vitro is also observed in vivo after oral administration of midazolam. In conclusion, the DEX-treated female rat could be a useful model for evaluating drug–drug interactions based on CYP3A enzyme inhibition.     

2,5-Dihydroxy-4-methoxybenzophenone: a novel major in vitro metabolite of benzophenone-3 formed by rat and human liver microsomes

1.?When benzophenone-3 (2-hydroxy-4-methoxybenzophenone; BP-3) was incubated with liver microsomes of untreated rats in the presence of NADPH, the 5-hydroxylated metabolite, 2,5-dihydroxy-4-methoxybenzophenone (5-OH-BP-3), was formed as a major novel metabolite of BP-3. The 4-desmethylated metabolite, 2,4-dihydroxybenzophenone (2,4-diOH-BP), previously reported as the major in vivo metabolite of BP-3, was also detected. However, the amount of 5-OH-BP-3 formed in vitro was about the same as that of 2,4-diOH-BP.

2.?The oxidase activity affording 5-OH-BP-3 was inhibited by SKF 525-A and ketoconazole, and partly by quinidine and sulfaphenazole. The oxidase activity affording 2,4-diOH-BP was inhibited by SKF 525-A, ketoconazole and α-naphthoflavone, and partly by sulfaphenazole.

3.?The oxidase activity affording 5-OH-BP-3 was enhanced in liver microsomes of dexamethasone-, phenobarbital- and 3-methylcholanthrene-treated rats. The activity affording 2,4-diOH-BP was enhanced in liver microsomes of 3-methylcholanthrene- and phenobarbital-treated rats.

4.?When examined recombinant rat cytochrome P450 isoforms catalyzing the metabolism of BP-3, 5-hydroxylation was catalyzed by P450 3A2, 1A1, 2B1, 2C6 and 2D1, while 4-desmethylation was catalyzed by P450 2C6 and 1A1.     

Marmoset cytochrome P450 2D8 in livers and small intestines metabolizes typical human P450 2D6 substrates,metoprolol, bufuralol and dextromethorphan

Abstract

• 1.?Although the New World non-human primate, the common marmoset (Callithrix jacchus), is a potentially useful animal model, comprehensive understanding of drug metabolizing enzymes is insufficient.

• 2.?A cDNA encoding a novel cytochrome P450 (P450) 2D8 was identified in marmosets. The amino acid sequence deduced from P450 2D8 cDNA showed a high sequence identity (83–86%) with other primate P450 2Ds. Phylogenetic analysis showed that marmoset P450 2D8 was closely clustered with human P450 2D6, unlike P450 2Ds of miniature pig, dog, rabbit, guinea pig, mouse or rat.

• 3.?Marmoset P450 2D8 mRNA was predominantly expressed in the liver and small intestine among the tissues types analyzed, whereas marmoset P450 2D6 mRNA was expressed predominantly in the liver where P450 2D protein was detected by immunoblotting.

• 4.?By metabolic assays using marmoset P450 2D8 protein heterologously expressed in Escherichia coli, although P450 2D8 exhibits lower catalytic efficiency compared to marmoset and human P450 2D6 enzymes, P450 2D8 mediated O-demethylations of metoprolol and dextromethorphan and bufuralol 1′-hydroxylation.

• 5.?These results suggest that marmoset P450 2D8 (also expressed in the extrahepatic tissues) has potential roles in drug metabolism in a similar manner to those of human and marmoset P450 2D6.

     

Identification of cytochrome P450 isoforms involved in the metabolism of loperamide in human liver microsomes

Objective: The purpose of the present study was to elucidate the cytochrome P450 (P450) isoform(s) involved in the metabolism of loperamide (LOP) to N-demethylated LOP (DLOP) in human liver microsomes. Methods: Three established approaches were used to identify the P450 isoforms responsible for LOP N-demethylation using human liver microsomes and cDNA-expressed P450 isoforms: (1) correlation of LOP N-demethylation activity with marker P450 activities in a panel of human liver microsomes, (2) inhibition of enzyme activity by P450-selective inhibitors, and (3) measurement of DLOP formation by cDNA-expressed P450 isoforms. The relative contribution of P450 isoforms involved in LOP N-demethylation in human liver microsomes were estimated by applying relative activity factor (RAF) values. Results: The formation rate of DLOP showed biphasic kinetics, suggesting the involvement of multiple P450 isoforms. Apparent K_m and V_max values were 21.1 M and 122.3 pmol/min per milligram of protein for the high-affinity component and 83.9 M and 412.0 pmol/min per milligram of protein for the low-affinity component, respectively. Of the cDNA-expressed P450 s tested, CYP2B6, CYP2C8, CYP2D6, and CYP3A4 catalyzed LOP N-demethylation. LOP N-demethylation was significantly inhibited when coincubated with quercetin (a CYP2C8 inhibitor) and ketoconazole (a CYP3A4 inhibitor) by 40 and 90%, respectively, but other chemical inhibitors tested showed weak or no significant inhibition. DLOP formation was highly correlated with CYP3A4-catalyzed midazolam 1-hydroxylation (r_s=0.829; P<0.01), CYP2B6-catalzyed 7-ethoxy-4-trifluoromethylcoumarin O-deethylation (r_s=0.691; P<0.05), and CYP2C8-catalyzed paclitaxel 6-hydroxylation (r_s=0.797; P<0.05). Conclusion: CYP2B6, CYP2C8, CYP2D6, and CYP3A4 catalyze LOP N-demethylation in human liver microsomes, and among them, CYP2C8 and CYP3A4 may play a crucial role in LOP metabolism at the therapeutic concentrations of LOP. Coadministration of these P450 inhibitors may cause drug interactions with LOP. However, the clinical significance of potential interaction of LOP metabolism by CYP2C8 and CYP3A4 inhibitors should be studied further.     

Fluvoxamine is a Potent Inhibitor of the Metabolism of Caffeine in vitro

Abstract: The selective serotonin re-uptake inhibitor, fluvoxamine, is a very potent inhibitor of CYP1A2, and accordingly causes pharmacokinetic interactions with drugs metabolised by CYP1A2, such as caffeine, theophylline, imipramine, tacrine and clozapine. Interaction between caffeine and fluvoxamine has been described in vivo, leading to lowering of total clearance of caffeine by 80% during fluvoxamine intake. The main purpose of the present study was to evaluate this interaction in vitro in human liver microsomes. A high-performance liquid chromatography method was developed in order to assay 1, 3-dimethylxanthine, 1, 7-dimethylxanthine, 3, 7-dimethylxanthine and 1, 3, 7-trimethyluric acid formed from caffeine by human liver microsomes. The limit of detection was 0.06 nmol · mg protein^?1 · hr^?1. As expected, fluvoxamine was a very potent inhibitor of the formation of the N-demethylated caffeine metabolites, displaying K_i values of 0.08–0.28 μM. The formation of 1, 7-dimethylxanthine was virtually abolished by 10 μM of fluvoxamine, indicating that the N3-demethylation of caffeine is almost exclusively catalysed by CYP1A2. The CYP3A4 inhibitors, ketoconazole and bromocriptine, inhibited 1, 3, 7-trimethyluric acid formation with K s of 0.75 μM and 5 μM, respectively, thus further supporting the involvement of CYP3A4 in the 8-hydroxylation of caffeine. The study shows that fluvoxamine, as expected, is a potent inhibitor of the metabolism of caffeine in vitro.     

Role of individual human cytochrome P450 enzymes in the in vitro metabolism of hydromorphone

1. The aim was to identify the individual human cytochrome P450 (CYP) enzymes responsible for the in vitro N-demethylation of hydromorphone and to determine the potential effect of the inhibition of this metabolic pathway on the formation of other hydromorphone metabolites.

2. Hydromorphone was metabolized to norhydromorphone (apparent K_m = 206?? 822?μM, V_max = 104 ? 834?pmol?min^?1?mg^?1 protein) and dihydroisomorphine (apparent K_m = 62 ? 557?μM, V_max = 17 ? 122?pmol?min^?1?mg^?1 protein) by human liver microsomes.

3. In pooled human liver microsomes, troleandomycin, ketoconazole and sulfaphenazole reduced norhydromorphone formation by an average of 45, 50 and 25%, respectively, whereas furafylline, quinidine and omeprazole had no effect. In an individual liver microsome sample with a high CYP3A protein content, troleandomycin and ketoconazole inhibited norhydromorphone formation by 80%.

4. The reduction in norhydromorphone formation by troleandomycin and ketoconazole was accompanied by a stimulation in dihydroisomorphine production.

5. Recombinant CYP3A4, CYP3A5, CYP2C9 and CYP2D6, but not CYP1A2, catalysed norhydromorphone formation, whereas none of these enzymes was active in dihydroisomorphine formation.

6. In summary, CYP3A and, to a lesser extent, CYP2C9 catalysed hydromorphone N-demethylation in human liver microsomes. The inhibition of norhydromorphone formation by troleandomycin and ketoconazole resulted in a stimulation of microsomal dihydroisomorphine formation.

     

Metabolism of (−)-fenchone by CYP2A6 and CYP2B6 in human liver microsomes

The in vitro metabolism of (?)-fenchone was examined in human liver microsomes and recombinant enzymes. The biotransformation of (?)-fenchone was investigated by gas chromatography-mass spectrometry. (?)-Fenchone was found to be oxidized to 6-exo-hydroxyfenchone, 6-endo-hydroxyfenchone and 10-hydroxyfenchone by human liver microsomal P450 enzymes. The formation of metabolites was determined by the relative abundance of mass fragments and retention times on gas chromatography (GC). CYP2A6 and CYP2B6 were major enzymes involved in the hydroxylation of (?)-fenchone by human liver microsomes, based on the following lines of evidence. First, of 11 recombinant human P450 enzymes tested, CYP2A6 and CYP2B6 catalysed the oxidation of (?)-fenchone. Second, oxidation of (?)-fenchone was inhibited by thioTEPA and (+)-menthofuran. Finally, there was a good correlation between CYP2A6, CYP2B6 contents and (?)-fenchone hydroxylation activities in liver microsomes of 11 human samples. CYP2A6 may be more important than CYP2B6 in human liver microsomes. Kinetic analysis showed that the V_max/K_m values for (?)-fenchone 6-endo-, 6-exo- and 10-hydroxylation catalysed by liver microsomes of human sample HG-03 were 24.3, 44.0 and 1.3?nM^?1?min^?1, respectively. Human recombinant CYP2A6 and CYP2B6 catalysed (?)-fenchone 6-exo-hydroxylation with V_max values of 2.7 and 12.9?nmol?min^?1?nmol^?1 P450 and apparent K_m values of 0.18 and 0.15?mM and (?)-fenchone 6-endo-hydroxylation with V_max values of 1.26 and 5.33?nmol?min^?1?nmol^?1 P450 with apparent K_m values of 0.29 and 0.26?mM. (?)-Fenchone 10-hydroxylation was catalysed by CYP2B6 with K_m and V_max values of 0.2?mM and 10.66?nmol?min^?1?nmol^?1 P450, respectively.     

Simultaneous pharmacokinetics evaluation of human cytochrome P450 probes,caffeine, warfarin,omeprazole, metoprolol and midazolam,in common marmosets (Callithrix jacchus)

1.?Pharmacokinetics of human cytochrome P450 probes (caffeine, racemic warfarin, omeprazole, metoprolol and midazolam) composite, after single intravenous and oral administrations at doses of 0.20 and 1.0?mg?kg^?1, respectively, to four male common marmosets were investigated.

2.?The plasma concentrations of caffeine and warfarin decreased slowly in a monophasic manner but those of omeprazole, metoprolol and midazolam decreased extensively after intravenous and oral administrations, in a manner that approximated those as reported for pharmacokinetics in humans.

3.?Bioavailabilities were ～100% for caffeine and warfarin, but <25% for omeprazole and metoprolol. Bioavailability of midazolam was 4% in marmosets, presumably because of contribution of marmoset P450 3A4 expressed in small intestine and liver, with a high catalytic efficiency for midazolam 1′-hydroxylation as evident in the recombinant system.

4.?These results suggest that common marmosets, despite their rapid clearance of some human P450 probe substrates, could be an experimental model for humans and that marmoset P450s have functional characteristics that differ from those of human and/or cynomolgus monkey P450s in some aspects, indicating their importance in modeling in P450-dependent drug metabolism studies in marmosets and of further studies.     

Studies on N-demethylation of methamphetamine by liver microsomes of guinea-pigs and rats: The role of flavin-containing mono-oxygenase and cytochrome P-450 systems

1. Relative participation of flavin-containing mono-oxygenase and cytochrome P-4S0 systems in N-hydroxylation of and formaldehyde release from methamphetamine were studied in vitro using liver microsomes of guinea-pigs and rats. In guinea pigs, only methimazole, an inhibitor of flavin-containing mono-oxygenase, significantly suppressed the above reactions.

2. Formaldehyde release from methamphetamine was significantly inhibited not only by methimazole but also by inhibitors of the cytochrome P-450 system in liver microsomes from rats, but not guinea-pigs.

3. Pretreatment of guinea-pigs with phenobarbital and 3-methylcholanthrene did not enhance the metabolism of methamphetamine.

4. Pretreatment of rats with phenobarbital but not 3-methylcholanthrene increased slightly the N-demethylation of methamphetamine by liver microsomes.

5. The results indicate that a marked species difference exists in the enzymes concerned with N-demethylation of methamphetamine. N-Oxidation predominates in guinea-pigs, whereas in rats, N-oxidation and C-oxidation of the methyl group participate equally as the initial reaction of the N-demethylation pathway.     

Marmoset cytochrome P450 2B6, a propofol hydroxylase expressed in liver

1. The common marmoset (Callithrix jacchus) is a useful experimental animal to evaluate the pharmacokinetics of drug candidates. Cytochrome P450 (P450) 2B enzyme in marmoset livers has been identified; however, only limited information on the enzymatic properties and distribution has been available.

2. Marmoset P450 2B6 amino acids showed high sequence identities (>86%) with those of primates including humans and cynomolgus monkeys. Phylogenetic analysis using amino acid sequences indicated that marmoset P450 2B6 was closer to human and cynomolgus monkey P450 2B6 than to P450 2B orthologs of other species, including pigs, dogs, rabbits and rodents.

3. Quantitative polymerase chain reaction analysis using specific primers showed P450 2B6 mRNA predominantly expressed in livers among the five marmoset tissues, similar to those of humans and cynomolgus monkeys.

4. Marmoset P450 2B6 heterologously expressed in Escherichia coli membranes oxidized 7-ethoxycoumarin, pentoxyresorufin, propofol and testosterone, at roughly similar rates to those of humans and/or cynomolgus monkeys. A high capacity of marmoset P450 2B6 with propofol 4-hydroxylation (at low ionic strength conditions) with a low K_m value was relatively comparable to that for marmoset livers.

5. These results collectively indicated a high propofol 4-hydroxylation activity of P450 2B6 expressed in marmoset livers.

     

Monkey liver cytochrome P450 2C19 is involved in R- and S-warfarin 7-hydroxylation

Cynomolgus monkeys are widely used as primate models in preclinical studies. However, some differences are occasionally seen between monkeys and humans in the activities of cytochrome P450 enzymes. R- and S-warfarin are model substrates for stereoselective oxidation in humans. In this current research, the activities of monkey liver microsomes and 14 recombinantly expressed monkey cytochrome P450 enzymes were analyzed with respect to R- and S-warfarin 6- and 7-hydroxylation. Monkey liver microsomes efficiently mediated both R- and S-warfarin 7-hydroxylation, in contrast to human liver microsomes, which preferentially catalyzed S-warfarin 7-hydroxylation. R-Warfarin 7-hydroxylation activities in monkey liver microsomes were not inhibited by α-naphthoflavone or ketoconazole, and were roughly correlated with P450 2C19 levels and flurbiprofen 4-hydroxylation activities in microsomes from 20 monkey livers. In contrast, S-warfarin 7-hydroxylation activities were not correlated with the four marker drug oxidation activities used. Among the 14 recombinantly expressed monkey P450 enzymes tested, P450 2C19 had the highest activities for R- and S-warfarin 7-hydroxylations. Monkey P450 3A4 and 3A5 slowly mediated R- and S-warfarin 6-hydroxylations. Kinetic analysis revealed that monkey P450 2C19 had high V_max and low K_m values for R-warfarin 7-hydroxylation, comparable to those for monkey liver microsomes. Monkey P450 2C19 also mediated S-warfarin 7-hydroxylation with V_max and V_max/K_m values comparable to those for recombinant human P450 2C9. R-warfarin could dock favorably into monkey P450 2C19 modeled. These results collectively suggest high activities for monkey liver P450 2C19 toward R- and S-warfarin 6- and 7-hydroxylation in contrast to the saturation kinetics of human P450 2C9-mediated S-warfarin 7-hydroxylation.     

Inhibition of antipyrine metabolite formation in rats in vivo

1. The effect of four inhibitors of cytochrome P-450-mediated drug oxidations (SKF 52SA, cimetidine, metyrapone and α-naphthoflavone) on the urinary metabolite pattern and ¹⁴CO₂ exhalation rate (CER)-time profile following [N-methyl-¹⁴C]antipyrine administration has been investigated.

2. The CER-time profiles indicated that inhibition of antipyrine metabolism was in the rank order SKF 525A >cimetidine > metyrapone >ANF.

3. The urinary metabolite patterns showed selectivity in action towards particular pathways, 3-hydroxylation being primarily decreased by SKF 525A and cimetidine, and N-demethylation by ANF. The results provide further evidence for involvement of multiple forms of cytochrome P-450 in antipyrine metabolism.     

Effects of inducers and/or inhibitors on metabolism of lysergic acid diethylamide in rat liver microsomes

1. When lysergic acid diethylamide (LSD) was incubated with liver microsomes obtained from untreated rats, SKF 525-A inhibited most potently the hydroxylation at the 13-position, moderately inhibited N-demethylation at the 6-position, and least affected the metabolism of the side-chain at the 8-position. Furthermore, an atmosphere of 80% CO and 20%O₂ (v/v) caused max. inhibition in N-demethylation, moderate inhibition in 13-hydroxylation, and the minimum in metabolism of the side-chain at the 8-position. These data suggested that the metabolism of LSD is catalysed by three separate enzyme systems.

2. The formation of 13-hydroxy-lysergic acid diethylamide (13-hydroxy-LSD) in liver microsomes obtained from 3-methylcholanthrene-treated rats was not inhibited by CO, although the hydroxylation required NADPH and oxygen.

3. The results of experiments using various inhibitors suggest that the 13-hydroxylation in liver microsomes from 3-methylcholanthrene-treated rats is catalysed by an enzyme system involving an unusual type of cytochrome P-448.     

Identification of the human cytochrome P450s responsible for the in vitro metabolism of a leukotriene B4 receptor antagonist,CP-195,543

1.?The major human cytochrome P450 (CYP) form(s) responsible for the metabolism of CP-195,543, a potent leukotriene B4 antagonist, were investigated.

2.?Incubation of CP-195,543 with human liver microsomes resulted in the formation of three major metabolites, M1–3. M1 and M2 were diastereoisomers and formed by oxidation on the benzylic position. M3 was formed by aromatic oxidation of the benzyl group attached to the 3-position of the benzopyran ring.

3.?The results from experiments with recombinant CYPs, correlation studies and inhibition studies with form-selective inhibitors and a CYP3A antibody strongly suggest that the CYP3A4 plays a major role in the metabolism of CP-195,543. Recombinant CYP3A5 did not metabolize CP-195,543.

4.?The apparent K_m and V_max for the formation of M1–3 in human liver microsomes were determined as 36?μM and 4.1?pmol?min^?1?pmol^?1 P450, 44?μM and 10?pmol?min^?1?pmol^?1 P450, and 34?μM and 2.0?pmol?min^?1?pmol^?1 P450, respectively. The average in vitro intrinsic clearance for M2 was the highest both in human liver microsomes and recombinant CYP3A4 compared with M1 and M3. Intrinsic clearance for M2 in human liver microsomes and recombinant CYP3A4 was 0.231 and 0.736 ml?min^?1?pmol^?1 P450, respectively. The intrinsic clearances for M1 and M3 in human liver microsomes and CYP3A4 were 0.114 and 0.060 and 0.197 and 0.088 ml?min^?1?pmol^?1 P450, respectively. This suggests that benzylic oxidation is the predominant phase I metabolic pathway of CP-195,543 in man.