Donald J. Birkett

Tolbutamide hydroxylation by human liver microsomes: Kinetic characterisation and relationship to other cytochrome P-450 dependent xenobiotic oxidations

Tolbutamide hydroxylation has been investigated in human liver microsomes. Anti-human liver NADPH-cytochrome P-450 reductase IgG inhibited hydroxytolbutamide formation and this metabolite was not formed when NADPH-generating system was omitted from microsomal incubations. Tolbutamide hydroxylation followed Michaelis-Menten kinetics, consistent with the involvement of a single form of cytochrome P-450 in this reaction. Mean apparent Km and Vmax values for hydroxytolbutamide formation were 120 +/- 41 microM and 0.273 +/- 0.066 nmol min-1 mg-1, respectively. A range of clinically used drugs and xenobiotics used as probes for cytochrome P-450 activity in laboratory animals was screened for inhibitory effects on hydroxytolbutamide formation. Caffeine, paraxanthine, theophylline, theobromine, debrisoquine, erythromycin, phenacetin, propranolol, aminopyrine, benzo(a)pyrene and 7-ethoxycoumarin were all found not to inhibit tolbutamide hydroxylation. In contrast, sulphaphenazole, phenylbutazone, nifedipine, verapamil, cimetidine, aniline, dextropropoxyphene and mephenytoin were competitive inhibitors of tolbutamide hydroxylation. The respective apparent Ki values for these compounds were 0.12 microM, 11 microM, 15 microM, 118 microM, 140 microM, 182 microM, 225 microM and 375 microM. Sulphinpyrazone inhibited tolbutamide hydroxylation with atypical kinetics. The in vitro data is in good agreement with in vivo drug interactions with tolbutamide. The data also confirm that tolbutamide hydroxylation is not associated with the cytochromes P-450 responsible for methylxanthine metabolism or with the form responsible for the polymorphic oxidation of debrisoquine.

Tolbutamide and phenytoin hydroxylations by cDNA-expressed human liver cytochrome P4502C9☆

A human cytochrome P4502C9 cDNA clone has been isolated from a human liver bacteriophage Lambda gt11 library using oligonucleotide probes. Expression of the 1762 base pair cDNA in COS cells demonstrated that the encoded enzyme has a molecular mass of 55 kDa as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The expressed enzyme catalysed the methylhydroxylation of tolbutamide with an apparent Km of 131.7 microM, similar to that observed in human liver microsomes. P4502C9 also catalysed the 4-hydroylation of phenytoin, and inhibition experiments demonstrated that phenytoin was a competitive inhibitor of tolbutamide hydroxylation with an apparent Ki of 19.1 microM. Sulphaphenazole was a potent inhibitor of the expressed enzyme with respect to both tolbutamide and phenytoin hydroxylations. These data demonstrate that a single isozyme can catalyse the hydroxylations of both tolbutamide and phenytoin, and suggest that both reactions are mediated by the same isozyme(s) of cytochrome P450 in human liver.

Caffeine metabolism by human hepatic cytochromes p450: Contributions of 1A2, 2E1 and 3A isoforms

Caffeine (CA) N1-, N3- and N7-demethylase, CA 8-hydroxylase and phenacetin O-deethylase activities were measured in microsomes from 18 separate human livers which had been characterized previously for a range of cytochrome P450 (CYP) isoform-specific activities and immunoreactive CYP protein contents. Correlations between the high affinity components of the three separate CA N-demethylations were highly significant (r = 0.77-0.91, P < 0.001) and each of the three high affinity CA N-demethylations correlated significantly (r = 0.64-0.93, P < 0.05-0.001) with the high affinity phenacetin O-deethylase, 2-acetylaminofluorene N-hydroxylation and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) mutagenicity (all predominantly CYP1A2-mediated reactions). Consistent with these observations, cDNA-expressed human CYP1A2 catalyzed the N1-, N3- and N7-demethylation of CA and apparent Km values were similar (0.24-0.28 mM) for all three reactions and comparable to those observed previously with human liver microsomes. The low affinity components of CA N1- and N7-demethylation correlated significantly (r = 0.55-0.85, P < 0.05-0.001) with immunoreactive CYP2E1 content and the CYP2E1-specific activities 4-nitrophenol and chlorzoxazone hydroxylation. Diethyldithiocarbamate, a selective inhibitor of CYP2E1, inhibited the low affinity CA N1- and N7-demethylation, with IC50 values of 23 microM and 11 microM, respectively. The apparent Km values for CA N1- and N7-demethylation by cDNA-expressed CYP2E1 (namely 28 and 43 mM, respectively) were of a similar order to those calculated for the low affinity microsomal activities. Significant correlations (r = 0.87-0.97, P < 0.001) were observed between CA 8-hydroxylation and immunoreactive CYP3A content and the CYP3A-mediated reactions benzo(a)pyrene hydroxylation, omeprazole sulfoxidation and aflatoxin B1 mutagenesis. Effects of alpha-naphthoflavone, erythromycin, troleandomycin and nifedipine on microsomal CA 8-hydroxylation were generally consistent with CYP3A involvement. Taken together with previous data, the results indicate a major involvement of CYP1A2 in the high affinity component of all three human hepatic CA N-demethylations. In contrast, CYP2E1 appears to be the main enzyme involved in the low affinity components of CA N1- and N7-demethylation while CA 8-hydroxylation is catalysed predominantly by a CYP3A isoform(s).

Assessment of caffeine exposure: Caffeine content of beverages, caffeine intake, and plasma concentrations of methylxanthines

The caffeine content of all tea or coffee beverages consumed by 17 healthy adults over 24 hours was measured. Plasma caffeine, theophylline, theobromine, and paraxanthine concentrations were determined over the same 24 hours. The average caffeine content per drink was 60.4 ± 21.8 mg for instant coffee (14‐fold range), 80.1 ± 19.2 mg for brewed coffee (2.8‐fold range), and 28.8 ± 13.7 mg for tea (5.5‐fold range). The number of drinks of coffee and tea consumed was a poor index of actual caffeine intake (r2= 0.42). Caffeine intake correlated poorly with the 24‐hour average caffeine concentration (r2= 0.41), but there was a very good correlation between a single plasma caffeine concentration measured at 5 PM and the 24‐hour average concentration (r2= 0.94). The same was true for paraxanthine (r2= 0.86). Paraxanthine accounted for 67.3% of the total dimethylxanthines in plasma, while theobromine and theophylline accounted for 24.4% and 8.3%, respectively. Mean caffeine clearance was 1.2 ± 0.3 ml/min/ kg. Plasma caffeine concentration before the first drink in the morning correlated very poorly with caffeine clearance (r2= 0.07), even when adjusted for caffeine intake (r2= 0.21).

Validation of 4-nitrophenol as an in vitro substrate probe for human liver CYP2E1 using cDNA expression and microsomal kinetic techniques

The involvement of human cytochrome P450 (CYP) 2E1 in the hydroxylation of 4-nitrophenol (4NP) to 4-nitrocatechol (4NC) has been investigated using cDNA expression and liver microsomal kinetic and inhibitor techniques. 4NP hydroxylation by human liver microsomes and cDNA-expressed human CYP2E1 exhibited Michaelis-Menten kinetics; the respective apparent Km values were 30 +/- 7 and 21 microM. Mutual competitive inhibition was observed for 4NP and chlorzoxazone (CZ) (an alternative human CYP2E1 substrate) in liver microsomes, with close similarities between the calculated apparent Km and Ki values for each individual compound. 4NP and CZ hydroxylase activities in microsomes from 18 liver donors varied to a similar extent (3.3- and 3.0-fold, respectively) and 4NP hydroxylase activity correlated significantly (rs > or = 0.75, P < 0.005) with both CZ hydroxylation and immunoreactive CYP2E1 content. The prototypic CYP2E1 inhibitor, diethyldithiocarbamate, was a potent inhibitor of 4NC formation and decreased 4NP hydroxylation by cDNA-expressed CYP2E1 and human liver microsomes in parallel. Probes for other human CYP isoforms namely (alpha-naphthoflavone, coumarin, sulphaphenazole, quinidine, troleandomycin and mephenytoin) caused < 15% inhibition of liver microsomal 4NP hydroxylation. These data confirm that, as in animal species, 4NP hydroxylation is catalysed largely by CYP2E1 in human liver and 4NP may therefore be used as an in vitro substrate probe for the human enzyme.

Mechanism of action of paracetamol protective agents in mice in vivo

The mechanism of action of cysteine, methionine, N-acetylcysteine (NAC) and cysteamine in protecting against paracetamol (APAP) induced hepatotoxicity in male C3H mice in vivo has been investigated by, characterising the effect of the individual protective agents on the metabolism of an hepatotoxic dose of APAP, and determining the efficacy of the protective agents in animals treated with buthionine sulphoximine (BSO), a specific inhibitor of glutathione (GSH) synthesis. Co-administration of cysteine, methionine or NAC increased, while co-administration of cysteamine decreased, the proportion of GSH-derived conjugates of APAP excreted in the urine of mice administered APAP, 300 mg/kg. Pretreatment of animals with BSO abolished the protective effect of cysteine, methionine and NAC, whereas cysteamine still afforded protection against APAP after BSO treatment. In conjunction with other data, these results suggest the most likely mechanism for the protective effect of cysteine, methionine and NAC is by facilitating GSH synthesis, while the most likely mechanism for the protective effect of cysteamine is inhibition of cytochrome P-450 mediated formation of the reactive metabolite of APAP.

Characterisation of theophylline metabolism by human liver microsomes: Inhibition and immunochemical studies

Anti-human NADPH-cytochrome P-450 reductase inhibited all theophylline metabolic pathways confirming the involvement of cytochrome P-450 isozymes in the metabolism of theophylline. Tolbutamide, debrisoquine, mephenytoin, theobromine, phenylbutazone, sulphaphenazole and sulphinpyrazone did not inhibit theophylline metabolism by human liver microsomes. Verapamil and dextropropoxyphene were non-selective competitive inhibitors of theophylline metabolism. Cimetidine and caffeine selectively inhibited the two demethylations as Ki values for these two pathways were lower than for the 8-hydroxylation pathway. The effects of nifedipine, propranolol and alpha-naphthoflavone were atypical. The degree of inhibition by propranolol reached a plateau, which was greater for the two demethylations than for the 8-hydroxylation. Alpha-naphthoflavone (ANF) at low concentrations inhibited the demethylations to a greater extent than the 8-hydroxylation. At higher concentrations ANF activated all pathways, with this effect being most marked for the 8-hydroxylation. Nifedipine inhibited the theophylline demethylations but not the 8-hydroxylation. In some livers the 8-hydroxylation was markedly activated. The results confirm that there are at least two distinct cytochrome P-450 isozymes involved in theophylline metabolism, one isozyme being involved with the demethylations and a different isozyme involved in the 8-hydroxylation pathway. Preliminary correlation studies suggest that the human orthologue to the rabbit polycyclic hydrocarbon inducible P-450 Form 4 may be involved in the N-demethylations of theophylline.

Cigarette smoking and theophyiiine clearance and metabolism

Differences in plasma theophyiiine clearance (ClT) and metabolism between smoking and nonsmoking normal subjects were examined by analysis of plasma and urinary theophyiiine concentrations and of urinary metabolite concentrations under steady‐state oral dosing conditions. ClT in smokers (0.053 ± 0.006 l · hr−1 · kg−1) was greater than in nonsmokers (0.032 ± 0.002 l · hr−1 · kg−1, p < 0.005). Analyses of urinary metabolites revealed that clearance to l‐methyiuric acid (Cl1MU) and clearance to 3‐methylxanthine (Cl3MX) were increased in smokers 1.99‐fold and 2.10‐fold over nonsmoking controls (P < 0.005). Clearance to 1.3‐dimethyluric acid (ClDMU) was also enhanced in smokers 1.68‐fold compared to controls (P < 0.01). The positive relationship between Cl1MUand Cl3MX in both smokers and nonsmokers (r = 0.98, P < 0.001) supports the concept that the two 8‐demethylation pathways for theophyiiine metabolism are under common regulatory control and involve a form of cytochrome P450 distinct from that mediating 8‐hydroxylation of theophyiiine to DMU. These results suggest that cigarette smoking induces both of the cytochrome P450‐mediated pathways of theophyiiine metabolism but that N‐demethylation may be increased to a greater extent than 8‐hydroxylation.

Validation of the tolbutamide metabolic ratio for population screening with use of sulfaphenazole to produce model phenotypic poor metabolizers

The present study has validated kinetically a convenient method to measure tolbutamide hydroxylation capacity in human beings by use of urinary metabolic ratios. The known in vivo and in vitro inhibitory properties of sulfaphenazole were used to convert control phase subjects to phenotypically “poor” metabolizers of tolbutamide. Six healthy subjects were given a single 500 mg oral dose of tolbutamide with and without sulfaphenazole, 500 mg every 12 hours. Tolbutamide, hydroxytolbutamide, and carboxytolbutamide in urine were determined by newly developed HPLC procedures. Plasma tolbutamide clearance and half‐life were measured, as were the metabolic ratio (hydroxytolbutamide + carboxytolbutamide/tolbutamide) in successive 6‐hour urine collections. The mean tolbutamide plasma clearance decreased from 0.196 ± 0.026 ml/min/kg without sulfaphenazole to 0.039 ± 0.009 ml/min kg with sulfaphenazole, and the mean half‐life of tolbutamide increased from 7.28 ± 0.89 hours to 38.76 ± 13.30 hours. The metabolic ratio determined in the 6 to 12 hour urine collection period decreased from 794.0 ± 86.6 to 126.0 ± 79.3, and this collection period also gave the best separation of subjects between phases. There was a good correlation between tolbutamide plasma clearance and metabolic ratio (rs = 0.853, p < 0.01, n = 12) and between the percentage decrease in plasma tolbutamide clearance and the percentage decrease in metabolic ratio (r = 0.932, p < 0.01, n = 6). The tolbutamide urinary metabolic ratio therefore effectively distinguishes tolbutamide hydroxylase activity in “normal” subjects and in those converted to model phenotypically “poor” metabolizers by sulfaphenazole.

Caffeine as a probe for human cytochromes P450: validation using cDNA-expression, immunoinhibition and microsomal kinetic and inhibitor techniques.

The molecular basis for the use of caffeine (CA; 1,3,7-trimethylxanthine) as a probe for specific human cytochromes P450 has been investigated. The CA 1-, 3- and 7-demethylations (to form theobromine, paraxanthine and theophylline, respectively) all followed biphasic kinetics in human liver microsomes. Mean apparent Km values for the high- and low-affinity components of the demethylations ranged from 0.13-0.31 nM and 19.2-30.0 mM, respectively. cDNA-expressed CYP1A2 catalysed all three CA demethylations, and the apparent Km for CA 3-demethylation (the major metabolic pathway in humans) by the expressed enzyme was similar to the Km for the high-affinity liver microsomal CA 3-demethylase. IC50 values for inhibition of the CA demethylations by alpha-naphthoflavone were similar for both expressed CYP1A2 and the high-affinity microsomal demethylases. Moreover, CA was a competitive inhibitor of expressed CYP1A2 catalysed phenacetin O-deethylation, with the apparent Ki (0.080 mM) closely matching the apparent Km (0.082 mM) for CA 3-demethylation by the expressed enzyme. Expressed CYP1A1 was additionally shown to catalyse the 3-demethylation of CA, although activity was lower than that observed for CYP1A2. While these data indicate that CYP1A2 is responsible for the high-affinity component of human liver CA 3-demethylation, two limitations associated with the use of CA as an in vitro probe for CYP1A2 activity have been identified: (i) CA 3-demethylation reflects hepatic CYP1A2 activity only at appropriately low substrate concentrations; and (ii) CA is a non-specific CYP1A substrate and CYP1A1 may therefore contribute to CA 3-demethylase activity in tissues in which it is expressed. An anti-CYP3A antibody essentially abolished the 8-hydroxylation of CA to form trimethyluric acid, suggesting formation of this metabolite may potentially serve as a marker of CYP3A isozyme(s) activity.