I. Roots

Increase of cytochrome P450IA2 activity by omeprazole: Evidence by the 13C‐[N‐3‐methyl]‐caffeine breath test in poor and extensive metabolizers of S‐mephenytoin

Omeprazole has been shown to induce cytochrome P450IA1 and P450IA2 activity in vitro. To reflect cytochrome P450IA2 (CYP1A2) activity in vivo, the 13C‐[N‐3‐methyl]‐caffeine breath test was conducted in 18 volunteers: 12 extensive metabolizers, one intermediate metabolizer, and five poor metabolizers of S‐mephenytoin. Breath tests were performed before treatment with an oral dose of 40 mg omeprazole, on the seventh day of treatment, and after a 7‐day washout period. The mean percentage exhalation of the 13C test dose, as determined by 13CO2 in breath during 8 hours, was 23.0% ± 8.0% (n = 18) before treatment. The largest increases in exhalation rate of 13CO2 were observed in the poor metabolizers and the intermediate metabolizers (range, 12.8% to 62.9%; median, 38.9%); median area under the plasma concentration—time curves (AUC) of omeprazole was four times higher than in the extensive metabolizers. The change after omeprazole treatment in extensive metabolizers ranged from −9.8% to +47.7% (median, 12.3%; n = 12) of pretreatment values. In both groups exhalation rates of 13CO2 returned to near pretreatment values within the 7‐day washout period (24.2% ± 7.8%; n = 17). Changes in the 13C‐caffeine breath test correlated well with both the pretreatment value (R = −0.67, p = 0.003; n = 18) and the plasma AUC of omeprazole (R = 0.61, p = 0.007; n = 18). Therapeutic doses of omeprazole seem to induce CYP1A2 activity in poor metabolizers, whereas they exert minor inducing effects in extensive metabolizers of S‐mephenytoin.

Accelerated caffeine metabolism after omeprazole treatment is indicated by urinary metabolite ratios: Coincidence with plasma clearance and breath test

Omeprazole has previously been shown to induce hepatic cytochrome P4501A2 activity, as evidenced by an accelerated N‐3‐demethylation in the 13C‐[N‐3‐methyl]‐caffeine breath test. In this study we investigated whether the inducing potency of omeprazole can be quantified by the determination of urinary caffeine metabolite ratios, which are based on the urinary excretion of N‐3‐demethylated metabolites. These data were also compared with changes in plasma clearance.

Quantitative determination by HPLC of urinary 6β-hydroxycortisol, an indicator of enzyme induction by rifampicin and antiepileptic drugs

SummaryA method for determination of 6β-hydroxycortisol in urine by means of high performance liquid chromatography is described. After extraction of 10–30 ml aliquots of urine with ethylacetate, separation is accomplished on a silica gel column (30 cm, Lichrosorb Si 100) with a special two-phase four-component eluent of methylene chloride, n-hexane, ethanol and water. Complete separation of α- andβ-isomers requires 15 to 20 min. For routine determinations precolumn cleaning by backflush permits injections of samples at minimum time intervals. For quantitative determinations, each injection should contain at least 0.05–0.5 µg of 6β-hydroxycortisol, depending on the detector employed. The mean excretion rate in healthy male adults (26–40 years) was 273 µg/day (SD=74.5; n=12). In patients on long term mono-therapy with rifampicin, 6β-hydroxycortisol excretion had risen fourfold (1166 µg/d; SEM=248; n=7), paralleling the known enzyme-inducing effect of rifampicin. The relatively smaller increase to 498 µg/d observed in patients receiving triple therapy with rifampicin, isoniazid and ethambutol points to possible inhibition by isoniazid. The greatest stimulation of 6β-hydroxycortisol excretion (2352 µg/d) was found in patients receiving antiepileptic therapy (phenytoin and/or carbamazepine and other drugs). The HPLC technique for 6β-hydroxycortisol proved to be a tool routinely applicable to non-invasive evaluation of drug metabolizing enzyme activity in man.

Evaluation ofin vivo parameters of drug metabolizing enzyme activity in man after administration of clemastine, phenobarbital or placebo

SummaryThe 24 h urinary excretion of 6β-hydroxycortisol and D-glucaric acid, the plasma half-lives and total clearances of aminopyrine, and serum gamma-glutamyl-transpeptidase activity have been measured in nineteen healthy male volunteers. The study was done double blind and was conducted as a test of induction of microsomal drug metabolizing enzymes during and after daily doses of 6 mg clemastine, 300 mg phenobarbital or a placebo. The urinary excretion of 6β-hydroxycortisol and D-glucaric acid was significantly increased in the phenobarbital group, the standard for induction. No changes were observed after treatment with clemastine or placebo. Phenobarbital also reduced the half life of aminopyrine, but it was not affected by clemastine or placebo. Gamma-glutamyl-transpeptidase activity increased only in the phenobarbital group. The elimination constant k2 of aminopyrine and the excretion of glucaric acid in the pre-medication period were correlated (p<0.05). The results indicate that the tests were of diagnostic value in determination of microsomal enzyme induction by phenobarbital. Failure to observe similar changes after treatment with clemastine imply failure of induction of this activity under the experimental conditions.

Pharmacokinetic interaction between cyclosporin and diltiazem

SummaryPrevious reports have indicated that administration of the calcium antagonist diltiazem results in major changes in the pharmacokinetics of cyclosporin A (CyA). A new clinical trial was undertaken in 22 renal transplant patients receiving a constant dose of cyclosporin to further explore this interaction. Coadministration of diltiazem for one week produced an increase in the blood concentration of CyA and its metabolites 17 and 18 in almost all patients, but no increase in CyA metabolites 1 and 21. The mean whole blood CyA trough level determined by HPLC rose from 117 ng·ml−1 to 170 ng·ml−1 after one week on diltiazem, and the mean trough level of metabolite 17 rose similarly from 184 ng·ml−1 before to 336 ng·ml−1.Based on experiments with microsomes from human liver the effect of diltiazem was due to noncompetitve inhibition of CyA-metabolism by diltiazem, and the increased concentration of metabolite 17 might have been due to stronger inhibition of its secondary metabolism steps.

Antipyrine metabolite formation and excretion in patients with chronic renal failure.

SummaryIn the present study the influence of chronic renal insufficiency on antipyrine clearance, metabolite formation and excretion was investigated in 8 patients. After oral administration of antipyrine, the parent compound, its metabolites and their conjugates were assayed in plasma and urine. Besides the parent drug, 3-hydroxymethylantipyrine (HMA) was present in plasma in the free and conjugated forms, whereas 4-hydroxyantipyrine (OHA) and norantipyrine (NORA) were found only in the conjugated form. The same was true for urine. The plasma concentrations of these metabolites are too low to be measured in subjects with normal renal function.Plasma antipyrine clearance in the patients was in the same range as in healthy subjects. Investigation of metabolite kinetics, however, revealed that the rate of formation of NORA was preferentially decreased, whereas that of OHA and HMA were unaltered. Renal clearance of the metabolites of antipyrine was severely impaired in patients with renal insufficiency, and the resulting accumulation made it possible for the first-time to measure the antipyrine metabolites in plasma. Mean residence times of metabolites were longer than that of the parent compound. Renal clearances of the conjugates were correlated with the creatinine clearance, but were somewhat higher. Renal clearance of free HMA was lower and was also correlated with creatinine clearance.The mean clearance for glucuronidation of HMA was 93.1 ml/min. The results suggest that in healthy subjects Phase I metabolism is the rate-limiting step in the elimination of antipyrine, which is essential for its application as a model drug in metabolism studies.

Comparison of Methods to Study Enzyme Induction in Man

A combination of several in vivo parameters has been applied in male healthy volunteers to test the suitability of these parameters to indicate enzyme induction in man: Urinary excretion of D-glucaric acid and 6 beta-hydroxycortisol, activity of serum gamma-glut amyltranspeptidase, and pharmacokinetics of aminopyrine respond significantly to phenobarbital treatment. Glucaric acid excretion is enhanced about 7-fold. Its response to induction overcomes the large individual and inter-individual variations which exist in the untreated state for glucaric acid excretion and the other parameters applied, as well. Total body clearance of aminopyrine as obtained after an oral test dose increases more than twofold from 251 to 551 ml/min upon phenobarbital treatment. This arises from increases in both the elimination constant and the apparent volume of distribution, as well. Urinary excretion of aminoantipyrine during 24 hr is about doubled, whereas the elimination of acetyl-aminotipyrine is not much affected. 6 beta-hydroxycortisol excretion in urine and activity of serum gamma-glutamyltranspeptidase are significantly augmented to about 150% of control values. Half life times of phenobarbital measured after termination of treatment are in normal range, suggesting no self-induction of phenobarbital metabolism. Because of the complexity of drug metabolizing enzymes only a combination of different parameters reliably indicates alterations in this enzyme system by inducing agents.

Kinetics of hexobarbital and dipyrone in critical care patients receiving high-dose pentobarbital

SummaryThe effect of pentobarbital treatment in a mean dose of 30 mg/kg/day on the clearance of hexobarbital (Evipan) and dipyrone (Novalgin) has been evaluated in critical care patients receiving a large number of drugs as comedication. Eleven patients treated with pentobarbital showed a hexobarbital half-life of 2.79 h and a total plasma clearance of 9.80 ml·min−1·kg−1 as compared to 10 patients without pentobarbital administration in whom there was a significantly longer half life (6.92 h) and lower clearance (2.97 ml·min−1·kg−1). The kinetics of hexobarbital were correlated with the urinary excretion of D-glucaric acid, a non-invasive parameter of drug metabolising activity. In 10 patients on pentobarbital, the total plasma clearance of N-4-methylaminoantipyrine, the active form of dipyrone, did not differ from that in 8 patients not receiving pentobarbital. As drug kinetics show great variability in these patients, it is difficult to discriminate enzyme induction from other mechanisms, for example competitive inhibition or changes in volume of distribution. In the presence of pentobarbital, however, induction of drug metabolising enzymes should be considered as a possible reason for the higher clearance of hexobarbital.

Hybrid information provided by the 14C-aminopyrine breath test studies with 14C-monomethylaminoantipyrine in the guinea pig

SummaryN-Demethylation of 14C-aminopyrine (14C-AP), labelled at the methyl groups of the tertiary amino group, yields H14CHO and 14C-monomethylaminoantipyrine (14C-MMAAP) which also undergoes N-demethylation, however, at a slower rate as measured in hepatic microsomes. As after intraperitoneal application to male guinea pigs of 14C-AP (75 mg/kg; 50 μCi/kg), exhalation rate of 14CO2 declines in a biphasic manner, the hypothesis was tested whether the terminal part might reflect N-demethylation of MMAAP. The application of 14C-MMAAP (70 mg/kg; 10 μCi/kg), resulted in monophasic curves of 14CO2 exhalation rate. Their half lives were, however, longer than terminal half lives obtained after 14C-AP. Obviously, this terminal phase does not represent 14CO2 formation from the metabolite MMAAP only, but 14C-AP might still contribute to 14CO2 production. Confirmation was obtained by HPLC determination of AP and MMAAP in serum after injection of AP. Shortly after injection, high concentrations of AP and low ones of MMAAP were found in blood from the portal vein and systemic circulation. Thus, initial parts of 14CO2-exhalation rate curves reflect predominantly AP metabolism whereas later phases provide hybrid information.

IN VIVO PARAMETERS OF DRUG METABOLISM—DIFFERENCES IN SPECIFICITY TOWARDS INDUCING AGENTS

ABSTRACT The occurrence or absence of enzyme induction in man can be sufficiently described by simultaneous application of four different in vivo parameters of drug metabolism ( 1 , 2) . However, due to the existence of different types of inducers which provoke discernible states of induction, it can be presumed that the single parameters respond differently according to the type of inducer applied. Six in vivo and two in vitro parameters have been examined systematically in the male guinea pig, applying phenobarbital, pregnenolone-16α-carbonitrile, 3-methyl-cholanthrene, rifampicine and spironolactone as inducers. Thus, a typical pattern of response or lack of response of the parameters is obtained which characterizes the inducers in vivo . Since various agents induce various types of cytochrome P-450, and likewise various inducers affect different in vivo parameters of drug metabolism, these investigations might lead to the cytochrome P-450 specificity of various in vivo parameters of drug metabolism. The experiments further demonstrate that the lack of response of one parameter does not necessarily exclude the incidence of enzyme induction. In guinea pig, a combination of different parameters allows to establish an inducer-specific profile of in vivo parameters which perhaps can be transferred to situations existing in man.