Alfred G. Hildebrandt

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.

Hexobarbital-binding, hydroxylation and hexobarbital-dependent hydrogen peroxide production in hepatic microsomes of guinea pig, rat and rabbit

SummaryCytochrome P-450 dependent oxygenase (3′-hydroxy-hexobarbital) and oxidase activities (hydrogen peroxide) have been measured in hepatic microsomes from guinea pigs, rats and rabbits. A sensitive gas-chromatographic assay was developed to measure the hydroxylated product 3′-hydroxy-hexobarbital. The kinetics of its formation were determined and correlated to hexobarbital type I binding and compared with oxidase activity: in the rat, Vmax for 3′-hydroxy-hexobarbital formation and for hexobarbital-dependent H2O2 formation was 5.1 and 2.6 nmoles/mg/min, resp. This was increased by phenobarbital tratment to 15.2 and 13.4 nmoles/mg/min. In phenobarbital treated rabbits, Vmax was 15.0 nmoles/mg/min for hydroxylation and 40.8 for H2O2 formation. Spectral affinity constants (Ks) in control animals were 0.12 mM (rats) and 0.14 mM (rabbits). Phenobarbital treatment decreased these affinity constants, which were similar for each activity measured. In guinea pigs, however, hydroxylation of hexobarbital was low (3.1 nmoles/mg/min) and hexobarbital-dependent formation of H2O2 was higher than hydroxylation (Vmax:7.0 nmoles/mg/min). Phenobarbital treatment led here to two affinity constants for each activity measured, which however, were alike. The existence of low in addition to high affinity constants observed here might explain the difficulties seen hitherto in correlating hexobarbital binding and metabolism in this species. Total oxidase activity was higher than oxygenase activity in all species tested.It is suggested that oxygenase activity of cytochrome P-450 is not limited by binding but by a competition with oxidase activity for a common intermediary species. This might be peroxy-P-450 (substrate-Fe3+O22−), rendering either substrate-Fe3+O for hydroxylation reaction, or oxidized cytochrome P-450-substrate and hydrogen peroxide as product of oxidase function.

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.

Oxidative dealkylation and reductive denitrosation of nitrosomethylaniline in vitro.

N-Nitrosomethylaniline (NMA) was incubated with liver microsomes from female mice and rats. Both formaldehyde and nitrite formation were determined in the same incubation mixture under various experimental conditions. The animals were pretreated with phenobarbital (PB) or butylhydroxytoluene (BHT) in order to modify microsomal monooxygenase activities. Furthermore, various possibilities were tried to supply the microsomal system with reducing equivalents (NADPH-regenerating system, NADPH-regenerating system plus NADH or NADH alone). It can be deduced from these experiments that both enzymatic activities--oxidative demethylation and reductive denitrosation of NMA--do not proceed in a parallel manner. Thus both reactions are different from each other. They represent two separate pathways in nitrosamine metabolism.

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.

METABOLIC DENITROSATION OF N-NITROSO-N-METHYLANILINE : DETECTION OF AMINE-METABOLITES

The enzymatic denitrosation of N-nitroso-N-methylaniline (NMA) was investigated by measuring the resulting amine metabolites when NMA was incubated with liver microsomes of PB-pretreated mice. Aniline was found to be the main amine metabolite. Small amounts of the secondary amine, N-methylaniline (MA) and its metabolite, p-methylaminophenol (p-MAP), could also be detected. Incubation of MA resulted in the formation of aniline and p-MAP. The velocity of the metabolism of MA was somewhat faster than that of NMA. On the basis of the measured Vmax values the formation of aniline from MA or from NMA proceeded at nearly identical rates. The dissociation constants as a measure of binding affinity to cytochrome (cyt.) P-450 were determined by measuring the binding spectra. NMA has one Ks of 3.1 mM, whereas MA shows two apparent Ks values, 650 microM and 25 mM, respectively. The results are discussed in relation to the enzymatic mechanism of denitrosation of NMA.

Inhibition of bleomycin-induced DNA strand breaks in V 79 Chinese hamster cells by the antioxidant propylgallate

Bleomycin induced DNA single-strand breaks in Chinese hamster V 79 cells which were detected by the alkaline filter elution assay. In the presence of propylgallate, an antioxidant, the amount of DNA single-strand breaks was significantly reduced. The production of DNA strand breaks by methylnitronitrosoguanidine used as a positive control was not influenced by propylgallate. It is suggested that propylgallate inhibits the generation of DNA single-strand breaks by trapping reactive oxygen species produced by the bleomycin-iron(II) complex.

Urinary excretion ofD-glucaric acid, an indicator of drug metabolizing enzyme activity, in patients with impaired renal function

SummaryThe urinary excretion rate ofD-glucaric acid, an in vivo parameter of the activity of drug metabolizing enzymes, has been determined in patients with chronic renal insufficiency (glomerular filtration rate 4.5–80 ml/min/1.73 m2). The mean value of 22.3 µmoles/d (SD 7.2; n 28) was almost identical to that of healthy controls (22.1 µmoles/d, SD 7.3; n 22). Thus, no inhibitory or enhancing effect of renal insufficiency could be detected. The ability of this parameter to indicate alterations in the activity of hepatic drug metabolism, even in patients with renal insufficiency, was demonstrated by the increased excretion rate of glucaric acid (107 µmoles/d, SD 43.5; n 8; p<0.001) after treatment for 7 days with the enzyme inducer phenobarbital. No significant correlation was found between glucaric acid excretion and sex, age, body weight or body surface in 50 patients. Glucaric acid excretion, therefore, should not be related to the creatinine content of urine samples, since creatinine excretion decreases with severity of renal insufficiency and varies with sex, age, body weight and many other conditions. A single dose of dipyrone (Novalgin®), a further in vivo indicator of drug metabolism, increased glucaric acid excretion on the same day, but no interference was found after a single dose of cortisol.