A critical evaluation of the experimental design of studies of mechanism based enzyme inhibition, with implications for in vitro-in vivo extrapolation.

The published literature on mechanism based inhibition (MBI) of CYPs was evaluated with respect to experimental design, methodology and data analysis. Significant variation was apparent in the dilution factor, ratio of preincubation to incubation times and probe substrate concentrations used, and there were some anomalies in the estimation of associated kinetic parameters (k(inact), K(I), r). The impact of the application of inaccurate values of k(inact) and K(I) when extrapolating to the extent of inhibition in vivo is likely to be greatest for those compounds of intermediate inhibitory potency, but this also depends on the fraction of the net clearance of substrate subject to MBI and the pre-systemic and systemic exposure to the inhibitor. For potent inhibitors, the experimental procedure is unlikely to have a material influence on the maximum inhibition. Nevertheless, the bias in the values of the kinetic parameters may influence the time for recovery of enzyme activity following re-synthesis of the enzyme. Careful attention to the design of in vitro experiments to obtain accurate kinetic parameters is necessary for a reliable prediction of different aspects of the in vivo consequences of MBI. The review calls for experimental studies to quantify the impact of study design in studies of MBI, with a view to better harmonisation of protocols.

In vitro identification of the P450 enzymes responsible for the metabolism of ropinirole.

The in vitro metabolism of ropinirole was investigated with the aim of identifying the cytochrome P450 enzymes responsible for its biotransformation. The pathways of metabolism after incubation of ropinirole with human liver microsomes were N-despropylation and hydroxylation. Enzyme kinetics demonstrated the involvement of at least two enzymes contributing to each pathway. A high affinity component with a K(M) of 5-87 microM and a low affinity component with a K(M) of approximately two orders of magnitude greater were evident. The high affinity component could be abolished by pre-incubation of the microsomes with furafylline. Additionally, incubation of ropinirole with microsomes derived from CYP1A2 transfected cells readily produced the N-despropyl and hydroxy metabolites. Some inhibition of ropinirole metabolism was also observed with ketoconazole, indicating a minor contribution by CYP3A. Multivariate correlation data were consistent with the involvement of the cytochrome P450 enzymes 1A2 and 3A in the metabolism of ropinirole. Thus, it could be concluded that the major P450 enzyme responsible for ropinirole metabolism at lower (clinically relevant) concentrations is CYP1A2 with a contribution from CYP3A, particularly at higher concentrations.

Determination of P4501A2 activity in human liver microsomes using [3-14C-methyl]caffeine.

1. Caffeine N3-demethylation, the major pathway of caffeine metabolism in man, is mediated by P4501A2. The carbon of the methyl group lost during N3-demethylation is eliminated as carbon dioxide in vivo, or as formaldehyde and formic acid in vitro. 2. A simple and sensitive assay was developed to quantify the [14C]formaldehyde/[14C]formic acid produced following incubation of human microsomes with [3-14C-methyl]caffeine. This assay, using solid-phase extraction, enables quantitation of [14C]formaldehyde/[14C]formic acid with acceptable precision (within 5%) and accuracy (within 10%). 3. Typical Km and Vmax for the N3-demethylation of caffeine were determined by this assay to be 500 (range 220-1200) microM, and 250 (range 115-450) pmol.mg protein-1.min-1 respectively. 4. The N3-demethylation activity determined in microsomes from a range of human livers correlated significantly with other P4501A2 activities (p < 0.001) and was inhibited (> 95%) by furafylline. In addition, caffeine N3-demethylation was catalysed by microsomes from cell lines transfected with human P4501A2 cDNA. 5. This assay, for quantitation of [14C]formaldehyde/[14C]formic acid in human liver microsomes, is suitable for use in in vitro drug interaction studies as a probe for P4501A2 activity.

Effects of routes of administration of TCNU on its plasma, tissue and tumour concentrations.

Tissue and plasma samples were taken from normal and tumour bearing mice at various time intervals following a dose of 30 mg kg-1 TCNU given by various routes. TCNU levels were measured by HPLC. The results show that the route of administration influences plasma concentrations, bioavailability and tissue and tumour concentrations. Intravenous administration gave 100% plasma bioavailability but only 15% of this level was seen following oral administration with consequently lower levels in tissues and tumours. The effect of gastric contents played a major role in this reduced bioavailability and plasma levels increased to 65% following overnight fasting. The differences in concentrations of TCNU observed here in plasma, tumour and normal tissues after oral and intravenous administration may have important clinical implications and may influence both anti-tumour activity and toxicity.

CYP3A4 drug interactions: correlation of 10 in vitro probe substrates.

AIMS: Many substrates of cytochrome P450 (CYP) 3A4 are used for in vitro investigations of drug metabolism and potential drug-drug interactions. The aim of the present study was to determine the relationship between 10 commonly used CYP3A4 probes using modifiers with a range of inhibitory potency. METHODS: The effects of 34 compounds on CYP3A4-mediated metabolism were investigated in a recombinant CYP3A4 expression system. Inhibition of erythromycin, dextromethorphan and diazepam N-demethylation, testosterone 6beta-hydroxylation, midazolam 1-hydroxylation, triazolam 4-hydroxylation, nifedipine oxidation, cyclosporin oxidation, terfenadine C-hydroxylation and N-dealkylation and benzyloxyresorufin O-dealkylation was evaluated at the apparent Km or S50 (for substrates showing sigmoidicity) value for each substrate and at an inhibitor concentration of 30 microM. RESULTS: While all CYP3A4 probe substrates demonstrate some degree of similarity, examination of the coefficients of determination, together with difference and cluster analysis highlighted that seven substrates can be categorized into two distinct substrate groups. Erythromycin, cyclosporin and testosterone form the most closely related group and dextromethorphan, diazepam, midazolam and triazolam form a second group. Terfenadine can be equally well placed in either group, while nifedipine shows a distinctly different relationship. Benzyloxyresorufin shows the weakest correlation with all the other CYP3A4 probes. Modifiers that caused negligible inhibition or potent inhibition are generally comparable in all assays, however, the greatest variability is apparent with compounds causing, on average, intermediate inhibition. Modifiers of this type may cause substantial inhibition, no effect or even activation depending on the substrate employed. CONCLUSIONS: It is recommended that multiple CYP3A4 probes, representing each substrate group, are used for the in vitro assessment of CYP3A4-mediated drug interactions.

Characterisation of the cytochrome P450 enzymes involved in the in vitro metabolism of granisetron.

1. The metabolism of granisetron was investigated in human liver microsomes to identify the specific forms of cytochrome P450 responsible. 2. 7-hydroxy and 9'-desmethyl granisetron were identified as the major products of metabolism following incubation of granisetron with human liver microsomes. At low, clinically relevant, concentrations of granisetron the 7-hydroxy metabolite predominated. Rates of granisetron 7-hydroxylation varied over 100-fold in the human livers investigated. 3. Enzyme kinetics demonstrated the involvement of at least two enzymes contributing to the 7-hydroxylation of granisetron, one of which was a high affinity component with a Km of 4 microM. A single, low affinity, enzyme was responsible for the 9'-desmethylation of granisetron. 4. Granisetron caused no inhibition of any of the cytochrome P450 activities investigated (CYP1A2, CYP2A6, CYP2B6, CYP2C9/8, CYP2C19, CYP2D6, CYP2E1 and CYP3A), at concentrations up to 250 microM. 5. Studies using chemical inhibitors selective for individual P450 enzymes indicated the involvement of cytochrome P450 3A (CYP3A), both pathways of granisetron metabolism being very sensitive to ketoconazole inhibition. Correlation data were consistent with the role of CYP3A3/4 in granisetron 9'-desmethylation but indicated that a different enzyme was involved in the 7-hydroxylation.

Lauric acid as a model substrate for the simultaneous determination of cytochrome P450 2E1 and 4A in hepatic microsomes.

In vitro techniques have been utilized to investigate the microsomal enzymes involved in the metabolism of lauric acid and to establish conditions in which it can be used as a model substrate for both cytochrome P450 4A and cytochrome P450 2E1 in human liver microsomes. Studies of enzyme kinetics of lauric acid omega-hydroxylation in human liver microsomes indicated the involvement of more than one enzyme in this pathway, a relatively low Km enzyme with a Km of 22 microM +/- 12 (n = 8) and a high Km enzyme with a Km an order of magnitude higher (550 microM +/- 310, n = 7). The apparent Vmax for this component correlated with the rate of cyclosporin metabolism and was highly sensitive to ketoconazole inhibition. These results indicated that this enzyme was a member of the 3A subfamily. The activity associated with the low Km enzyme (P450 4A) did not correlate with P450 1A2, 2A6, 2C9/8, 2C19, 2D6, 2E1, or 3A activities in a bank of human liver microsomes and was not appreciably inhibited by ketoconazole, furafylline, quinidine, sulfaphenazole, or diethyldithiocarbamate (DDC). Lauric acid omega-1 hydroxylation demonstrated simple Michaelis-Menten kinetics in each of the human liver microsomal samples examined, with a Km of 130 microM +/- 42 (n = 8). This activity was highly correlated with chlorzoxazone 6-hydroxylation in human liver microsomes (r = 0.98, n = 14, p < 0.001) and was inhibited by both DDC and chlorzoxazone. Additionally, rats treated with the P450 2E1 inducer isoniazid demonstrated a 3-fold increase in lauric acid omega-1 hydroxylation relative to the control group. Thus, the lauric acid hydroxylation assay, at a substrate concentration of 20 microM, appears to be an effective and specific P450 model substrate capable of determining simultaneously P450 4A and P450 2E1 related activities in hepatic microsomal samples.

The role of cytochrome P4502D6 in the metabolism of paroxetine by human liver microsomes.

Paroxetine is a selective serotonin reuptake inhibitor possessing anti-depressant activity. Demethylenation of the methylenedioxy phenyl group is the initial step in its metabolism, the liberated carbon appearing in vitro as formate. A radioassay involving [14C-methylenedioxy] paroxetine was developed and used to examine the role of cytochrome P4502D6 in paroxetine metabolism by human liver microsomes. The rate of formate production was much higher in microsomes from an extensive metaboliser of debrisoquine than from a poor metaboliser. Also, demethylenation of paroxetine was inhibited by the quinidine and quinine isomer pair in microsomes from the extensive metaboliser only. These observations strongly suggested that the process was catalysed by the enzyme cytochrome P4502D6. Metabolism could not be completely inhibited by quinidine, the residual activity representing the contribution of at least one other enzyme. The ability of microsomes from a poor metaboliser of debrisoquine to demethylenate paroxetine provided further evidence for the involvement of an enzyme distinct from P4502D6. This was confirmed by kinetic analysis of the process in microsomes from both poor and extensive metabolisers. It is concluded that, in man, the initial step of paroxetine metabolism is performed by at least two enzymes, one of which is cytochrome P4502D6.

Ketoconazole and sulphaphenazole as the respective selective inhibitors of P4503A and 2C9.

1. The potential of ketoconazole and sulphaphenazole to inhibit specific P450 enzyme activities (1A2, 2A6, 2B6, 2C9/8, 2C19, 2D6, 2E1, 3A and 4A) was investigated using human liver microsomes. 2. Ketoconazole demonstrated an inhibitory effect on cyclosporine oxidase and testosterone 6 beta-hydroxylase activities, with mean IC50's of 0.19 and 0.22 microM respectively. Ketoconazole inhibition of the other P450 activities investigated was significantly less, as illustrated by IC50's of at least a magnitude higher. 3. Sulphaphenazole was shown to have an inhibitory effect on tolbutamide hydroxylase activity, with a mean IC50 of 0.8 microM in incubations containing 100 microM tolbutamide. Sulphaphenazole (at concentrations of up to 100 microM) did not exhibit any significant inhibition of the other enzyme activities investigated. 4. Ketoconazole and sulphaphenazole are the respective selective inhibitors of P4503A and 2C9. Ketoconazole at 1 microM and sulphaphenazole at 10 microM can be used to establish the involvement of P4503A and 2C9 respectively in oxidative reactions in human liver microsomes.

Metabolism and disposition of 14C-granisetron in rat, dog and man after intravenous and oral dosing.

1. The disposition and metabolic fate of 14C-granisetron, a novel 5-HT3 antagonist, was studied in rat, dog, and male human volunteers after intravenous and oral administration. 2. Complete absorption occurred from the gastrointestinal tract following oral dosing, but bioavailability was reduced by first-pass metabolism in all three species. 3. There were no sex-specific differences observed in radiometabolite patterns in rat or dog and there was no appreciable change in disposition with dose between 0.25 and 5 mg/kg in rat and 0.25 and 10 mg/kg in dog. Additionally, there were no large differences in disposition associated with route of administration in rat, dog and man. 4. In rat and dog, 35-41% of the dose was excreted in urine and 52-62% in faeces, via the bile. Metabolites were largely present as glucuronide and sulphate conjugates, together with numerous minor polar metabolites. In man, about 60% of dosed radioactivity was excreted in urine and 36% in faeces after both intravenous and oral dosing. Unchanged granisetron was only excreted in urine (5-25% of dose). 5. The major metabolites were isolated and identified by MS spectroscopy and nmr. In rat, the dominant routes of biotransformation after both intravenous and oral dosing were 5-hydroxylation and N1-demethylation, followed by the formation of conjugates which were the major metabolites in urine, bile and plasma. In dog and man the major metabolite was 7-hydroxy-granisetron, with lesser quantities of the 6,7-dihydrodiol and/or their conjugates.

1-(Arylpiperazinylamidoalkyl)-pyrimidones: orally active inhibitors of lipoprotein-associated phospholipase A(2).

The lipophilic 1-substituent in a series of 1-((amidolinked)-alkyl)-pyrimidones, inhibitors of recombinant lipoprotein-associated phospholipase A(2), has been modified to give inhibitors of high potency in human plasma and enhanced physicochemical properties. Phenylpiperazineacetamide derivative 23 shows very promising oral activity.