Karel Lavrijsen

Identification of the cytochrome P450 enzymes involved in the metabolism of cisapride: in vitro studies of potential co‐medication interactions

Cisapride is a prokinetic drug that is widely used to facilitate gastrointestinal tract motility. Structurally, cisapride is a substituted piperidinyl benzamide that interacts with 5‐hydroxytryptamine‐4 receptors and which is largely without central depressant or antidopaminergic side‐effects. The aims of this study were to investigate the metabolism of cisapride in human liver microsomes and to determine which cytochrome P‐450 (CYP) isoenzyme(s) are involved in cisapride biotransformation. Additionally, the effects of various drugs on the metabolism of cisapride were investigated. The major in vitro metabolite of cisapride was formed by oxidative N‐dealkylation at the piperidine nitrogen, leading to the production of norcisapride. By using competitive inhibition data, correlation studies and heterologous expression systems, it was demonstrated that CYP3A4 was the major CYP involved. CYP2A6 also contributed to the metabolism of cisapride, albeit to a much lesser extent. The mean apparent Km against cisapride was 8.6±3.5 μM (n=3). The peak plasma levels of cisapride under normal clinical practice are approximately 0.17 μM; therefore it is unlikely that cisapride would inhibit the metabolism of co‐administered drugs. In this in vitro study the inhibitory effects of 44 drugs were tested for any effect on cisapride biotransformation. In conclusion, 34 of the drugs are unlikely to have a clinically relevant interaction; however, the antidepressant nefazodone, the macrolide antibiotic troleandomycin, the HIV‐1 protease inhibitors ritonavir and indinavir and the calcium channel blocker mibefradil inhibited the metabolism of cisapride and these interactions are likely to be of clinical relevance. Furthermore, the antimycotics ketoconazole, miconazole, hydroxy‐itraconazole, itraconazole and fluconazole, when administered orally or intravenously, would inhibit cisapride metabolism.

Interaction of miconazole ketoconazole and itraconazole with rat-liver microsomes

The interaction of the antimycotics miconazole, ketoconazole and itraconazole with liver microsomes from untreated rats or from rats pretreated with phenobarbital or 3-methylcholanthrene, gave rise to type II difference spectra. The interactions of the antimycotics with control, phenobarbital-induced or 3-methylcholanthrene-induced microsomes were biphasic, except for the monophasic binding of ketoconazole to phenobarbital-induced microsomes. The N-demethylation of N,N-dimethylaniline, the O-demethylation of p-nitroanisole and the hydroxylation of aniline in microsomes from untreated and inducer-treated rats were lowered by miconazole and ketoconazole, the former being the more potent inhibitor. Control microsomes were less sensitive than induced microsomes. Itraconazole was almost devoid of inhibitory properties. The three antimycotics were non-competitive (mixed) inhibitors of enzyme activities in phenobarbital-induced microsomes. The Ki values were of the same order of magnitude as the Ks values, except for itraconazole. For the latter drug, Ki values were much greater than could be expected from the spectral studies. It is concluded that the antimycotics affect microsomal enzyme activities via a direct interaction of an azole-nitrogen with the haem group of cytochrome P-450. The interaction with mammalian cytochrome P-450 decreases from miconazole greater than ketoconazole much greater than itraconazole and is much weaker than the interaction of the antimycotics with yeast cytochrome P-450.

Comparative metabolism of flunarizine in rats, dogs and man: an in vitro study with subcellular liver fractions and isolated hepatocytes

1. The biotransformation of 3H-flunarizine ((E)-1-[bis(4-fluorophenyl)methyl]-4-(3-phenyl-2-propenyl)piperazine dihydrochloride, FLUN) was studied in subcellular liver fractions (microsomes and 12,000 g fraction) and in suspensions or primary cell cultures of isolated hepatocytes of rats, dogs and man. The major in vitro metabolites were characterized by h.p.l.c. co-chromatography and/or by mass spectrometric analysis. 2. The kinetics of FLUN metabolism was studied in microsomes of dog and man. The metabolism followed linear Michaelis-Menten kinetics over the concentration range 0.1-20 microM FLUN. 3. A striking sex difference was observed for the in vitro metabolism of FLUN in rat. In male rats, oxidative N-dealkylation at one of the piperazine nitrogens, resulting in bis(4-fluorophenyl) methanol, was a major metabolic pathway, whereas aromatic hydroxylation at the phenyl of the cinnamyl moiety, resulting in hydroxy-FLUN, was a major metabolic pathway in female rats. In incubates with hepatocytes, these two metabolites were converted to the corresponding glucuronides. 4. In human subcellular fractions, aromatic hydroxylation to hydroxy-FLUN was the major metabolic pathway. In primary cell cultures of human hepatocytes, oxidative N-dealkylation at the 1- and 4-piperazine nitrogen and glucuronidation of bis(4-fluorophenyl)methanol were observed. The in vitro metabolism of FLUN in humans, resembled more than in female rats and in dogs than that in male rats. 5. The present in vitro results are compared with data of previous in vivo studies in rats and dogs. The use of subcellular fractions and/or isolated hepatocytes for the study of species differences in the biotransformation of xenobiotics is discussed.

Induction potential of fluconazole toward drug-metabolizing enzymes in rats.

The induction of drug-metabolizing enzymes in rat liver was studied after subchronic administration of the new triazole antifungal agent fluconazole. The administered doses were 10, 40, and 160 mg/kg per day for 7 days. Fluconazole behaved as a high-magnitude inducer and significantly increased cytochrome P-450 concentrations already at 10 mg/kg (+42%). Cytochrome P-450 induction by fluconazole was dose dependent and reached a value of 302% of the control value at the dose of 160 mg/kg. The induction effects on cytochrome P-450 were also reflected in the drug-metabolizing enzyme activities in hepatic microsomes of pretreated rats. Fluconazole (160 mg/kg per day) preferentially induced the demethylase activities of N,N-dimethylaniline and p-nitroanisole to 258 and 281% of the control values, respectively. The detoxification enzyme UDP-glucuronosyltransferase was significantly lowered by fluconazole at the highest dose. A possible link between the induction potential and the pharmacokinetic properties of triazole antifungal agents is discussed.

Is the Metabolism of Alfentanil Subject to Debrisoquine Polymorphism? A Study Using Human Liver Microsomes

The present study was designed to investigate whether the metabolism of the opiate analgesic alfentanil in humans is subject to the debrisoquine 4-hydroxylation polymorphism. The role of a specific cytochrome P-450 form, debrisoquine 4-hydroxylase, in the metabolism of alfentanil was investigated by competitive inhibition experiments over the concentration range 4–100 μM. Alfentanil was incubated with human liver microsomes in the presence of an NADPH-generating system. Alfentanil and its major metabolites were quantified in the incubates by reversed phase high-performance liquid chromatography (HPLC). Alfentanil was rapidly metabolized, yielding noralfentanil as the main metabolite. Kinetically, alfentänil metabolism occurred monophasically and the kinetic parameters were 22.8 μM for Km app and 3.86 nmol alfentanil metabolized min-1 ·mg protein-1 for Vm app. Debrisoquine was a weak, noncompetitive inhibitor of alfentanil metabolism and of the formation of its major metabolites, with K1 values between 2.00 and 3.21 mM. It can be concluded that alfentanil is not metabolized tit vitro by the human cytochrome P-450 form involved in debrisoquine 4-hydroxylation; therefore, the in vivo disposition of the drug is most likely not affected by deficiency of this enzyme.

Recombinant Escherichia coli cells immobilized in Ca-alginate beads for metabolite production

Milligram amounts of metabolites of drug candidates are required to identify toxic products. Human drug metabolites are currently produced selectively in a time- and cost-efficient manner in bioreactor systems containing recombinant Escherichia coli co-expressing a human cytochrome P450 isoenzyme/NADPH cytochrome P450 reductase (hCYP/HR) complex. For further optimization, immobilization of the catalytic system in Ca-alginate microbeads was considered. This new concept was designed for CYP3A4 with testosterone as substrate. Immobilized E. coli cells had a high maximal and homogeneously distributed biomass. Viability was stable over at least 1 week of culture and even longer during storage. Gene expression was ideally initiated 6 h after immobilization. Although immobilized E. coli cells expressed a highly functional enzyme system after 2 days, they did not metabolize testosterone, probably due to cell permeability problems resulting from immobilization. Therefore, immobilized cell membranes displaying testosterone bioconversion activity, even after long-term storage, will be used in bioreactors with high organic solvent content.

Metabolism of alfentanil by isolated hepatocytes of rat and dog

1. The biotransformation of 3H-alfentanil was studied using suspension cultures of isolated hepatocytes of male and female rats and of dogs. 2. In hepatocytes of the male rat, alfentanil was readily metabolized, following linear Michaelis-Menten kinetics over the concentration range 5-400 microM. The metabolism was strongly inhibited by the cytochrome P-450 inhibitors metyrapone, alpha-naphthoflavone and piperonyl butoxide. 3. The major metabolites of alfentanil, which were formed in suspension cultures of male rat hepatocytes, were identified by h.p.l.c. co-chromatography and by mass spectrometry and included N-[4-(hydroxymethyl)-4-piperidinyl]-N-phenylpropanamide, N-[4-(methoxymethyl)-4-piperidinyl]-N-phenylpropanamide or noralfentanil and N-[1-[2-(4-ethyl-4,5-dihydro-5-oxo-1-H-tetrazol-1-yl)ethyl]- 4-(hydroxymethyl)-4-piperidinyl]-N-phenylpropanamide or desmethylalfentanil. 4. The major in-vitro metabolic pathways of alfentanil in hepatocytes of the three sources were oxidative N-dealkylation at the piperidine nitrogen and oxidative O-demethylation at the methoxymethyl moiety.