Jean Paul Cano

Involvement of the macrolide antibiotic inducible cytochrome P-450 LM3c in the metabolism of midazolam by microsomal fractions prepared from rabbit liver

This report characterizes the cytochrome P-450 isozyme involved in midazolam metabolism. This study was undertaken into liver microsomal fractions prepared from untreated rabbits or animals treated with drugs known to specifically induce various cytochrome P-450 isozymes such as form LM2 by phenobarbital, LM4 and LM6 by 3-methylcholanthrene and beta-naphthoflavone, LM3a by ethyl alcohol and acetone, and LM3c by macrolide antibiotics (rifampicin, erythromycin and triacetyloleandomycin). Among this library of characterized microsomal preparations, only those obtained from macrolide antibiotic-treated rabbits exhibited a Type I binding spectrum upon addition of midazolam (Ks = 3.2-5.3 micrograms/ml; 10.6-17.5 microM) and significantly metabolized midazolam to its various hydroxylated metabolites (Km = 2.52 +/- 0.22 micrograms/ml; 8.32 +/- 0.73 microM and Vmax = 20 micrograms metabolites formed/min/mg proteins; 66 nmoles metabolites formed/min/mg proteins). The following observations further confirmed the specific involvement of the cytochrome P-450 LM3c isozyme: (i) only anti-cytochrome P-450 LM3c isozyme antibodies intensively inhibited midazolam metabolism, (ii) incubation of microsomes, prepared from TAO-treated rabbits, with midazolam in the presence of potassium ferricyanide which restored the functional cytochrome P-450 LM3c isozyme, increased midazolam metabolism to a similar extent, and (iii) in the presence of Cyclosporin A, a specific substrate of the rabbit cytochrome P-450 LM3c isozyme, midazolam metabolism was inhibited in a concentration-dependent manner. These data demonstrated that the rabbit cytochrome P-450 LM3c isozyme was predominantly involved in midazolam metabolism.

Human hepatocytes as a key in vitro model to improve preclinical drug development

SummaryOver past decades, numerous in vitro and/or ex vivo models have been developped to investigate drug metabolism. In the order of complexity we found the isolated perfused liver, hepatocytes in co-culture with epithelial cells, hepatocytes in suspension and in primary culture and subcellular hepatic microsomal fractions. Because they can be easily prepared from both animals (pharmacological and toxicological species) and humans (whole livers as well as biopsies obtained during surgery) hepatocytes in primary culture provide the most powerfull model to better elucidate drug behavior at an early stage of preclinical develompent such as: the characterization of main biotransformation reactions, the identification of phase I and phase II isozymes involved in such reactions, the evaluation of inter-species differences allowing the selection of a second toxicological animal species more closely related to man on the basis of metabolic profiles, the detection of the inducing and/or inhibitory effects of a drug on metabolic enzymes, the prediction of drug interactions, the estimation of inter-individual variability in biotransformation reactions. The use of hepatocytes, and in particular those obtained from humans, at an early stage of drug development allows the obtention of more predictive preclinical data and a better knowledge of drug behavior in humans before the first administration of the drug in healthy volunteers.

Plasma concentrations and pharmacokinetics of midazolam during anaesthesia.

Midazolam and 1‐hydroxymidazolam plasma concentrations have been monitored and pharmacokinetic parameters of midazolam estimated during anaesthesia induced and Maintained by its repeated injection according to two protocols (3 × 0.3 mg kg−1 at 45 min intervals or an induction dose of 0.3 mg kg−1 with maintenance doses of 0.15 mg kg−1 at 30 min intervals). Minimum plasma concentrations of midazolam measured just before each injection were 258.8 ± 108.4 ng ml−1 for the first protocol and 353.1 ± 55.2 ng ml−1 for the second protocol; maximum midazolam concentrations, measured 5 min after the last administration, were 1103.1 ± 237.9 ng ml−1 and 743.0 ± 103.2 ng ml−1, respectively, suggesting that a continuous infusion of midazolam after a loading dose should be better than repeated injections at keeping the concentration close to the sedative level of 400 ng ml−1. The estimated pharmacokinetic parameters were similar to those already published, except for the β elimination half‐life of midazolam (3.24 ± 0.90 h for protocol 1 and 3.34 ± 1.47 h for protocol 2) which was slightly longer than that reported for single dose studies. The comparison of plasma determinations, obtained either by gas‐liquid chromatography or by a radioreceptor assay technicjue, clearly showed that 1‐hydroxymidazolam, even after repeated midazolam administration, was not present at a concentration sufficient to affect the overall pharmacological activity of the parent drug.

Determination of Chloroquine and Monodesethylchloroquine in Hair

Using thin-layer and gas chromatography and mass spectrometry, chloroquine and its major metabolite (monodesethylchloroquine) were identified in hair samples of numerous patients who received this antimalarial drug for several months. In two patients the amounts of chloroquine were, respectively, 310 and 145 mg/kg hair and those of the monodesethylchloroquine 23 and 11 mg/kg. The respective proportions (93 and 7%) are the same in the two subjects. The chloroquine percentage was near those in the spleen or stomach wall after poisoning. Other metabolites in hair are being identified. Hair analysis may provide a good toxicologic and forensic science complement to the blood, urine, and tissues. It may be useful for the control of chloroquine therapy.

Methotrexate and 7-hydroxy-methotrexate pharmacokinetics following intravenous bolus administration and high-dose infusion of methotrexate

The pharmacokinetics of methotrexate and 7-hydroxy-methotrexate were studied in patients undergoing very high-dose methotrexate monotherapy. The patients received, first, two methotrexate intravenous bolus test doses (50 mg/m2) one with and one without concomitant administration of folinic acid (15 mg every 6 h) in a random sequence, and, second, an 8 h infusion, individualized to achieve a peak plasma concentration of 5 X 10(-4) M methotrexate (infusion rates greater than 1000 mg/h). Methotrexate and 7-hydroxy-methotrexate concentrations were measured by specific radioimmunoassays and the data were analysed simultaneously by an integrated pharmacokinetic model. Following test dose administration, methotrexate and 7-hydroxy-methotrexate plasma concentration kinetics were best described by assuming that methotrexate elimination (and 7-hydroxy-methotrexate formation) occurred from a peripheral compartment reaching rapid equilibrium with the plasma. Folinic acid administration did not influence the disposition of either compound. Following the infusion, a significant (P less than 0.01) decrease of methotrexate total plasmatic clearance occurred without modification of 7-hydroxy-methotrexate formation and elimination.

Pharmacokinetics of isosorbide dinitrate and its mononitrate metabolites after intravenous infusion

Plasma concentrations of isosorbide dinitrate (ISDN) and its two active metabolites 2-isosorbide mononitrate (2-ISMN) and 5-isosorbide mononitrate (5-ISMN) have been measured during and for 6 hr after intravenous infusion at a rate of 2.5mg/hr during 1.75 hr in six cardiac patients, by a capillary gas chromatographic method. Data were analyzed by simultaneous modeling of the observed kinetics of the three compounds. Two or three phases were detected on the postinfusion ISDN concentration-time curves. ISDN concentrations declined with a mean terminal half-life of 2.81 hr±0.7 SD. The mean systemic clearance of ISDN (2.9 L/min ±0.7 SD) and its mean total volume of distribution (259 L +- 48 SD) were relatively high. Plasma 5-ISMN concentrations were 5- to 6-fold greater than those of 2-ISMN during the whole observation period. Maximum levels of 2-ISMN (6.7 ng/ml ± 0.9 SD) and of 5-ISMN (27 ng/ml ± 6 SD) occurred within a few minutes after the end of infusion. The mean half-lives of 2-ISMN (1.59 hr± 0.19 SD) and of 5-ISMN (3.78 hr± 0.79 SD) estimated by the model were smaller than those calculated by a model-independent method (2.95 hr± 0.41 SD and 5.98 hr± 2.22, respectively), but were in good agreement with those reported in the literature following separate administration of both metabolites to man. This study shows how such modeling can distinguish between metabolite formation and elimination processes and allow the determination of metabolite half-lives after administration of the precursor drug.

DELTA 2-VALPROATE BIOTRANSFORMATION USING HUMAN LIVER MICROSOMAL FRACTIONS

The metabolism of 2-n-propyl-2-pentenoate (Δ2-VPA) was evaluated in human hepatic microsomal fractions. Two biotransformation pathways have been particularly investigated. In the presence of the cytochrome P-450 co-factor, NADPH, the main metabolites recovered were Δ3-VPA, Δ2,4-VPA and VPA. The glucuronidation of Δ2-VPA was also studied on various hepatic microsomal fractions using Brij® 35 as activator and UDP-glucuronic acid as co-factor. A large interindividual variability occurred in this metabolic pathway.Km andVmax were 0.85 mmol/l and 1.75 nmol·min−1·mg−1, respectively, for Δ2-VPA and 1.11 mmol/l and 5.71 nmol·min−1·mg−1 for VPA, respectively. The good correlationr=0.82; p<0.001) observed between the glucuronidation of VPA and Δ2-VPA as well as the mutual inhibition of each others glucuronidation strongly suggests that (a) common single UDP-glucuronosyltransferase isoenzyme(s) was (were) involved in this glucuronidation step. The glucuronidation of specific substrates for various UDP-glucuronosyltransferase isoenzymes showed a good relationship between the glucuronidations of Δ2-VPA and morphine, a substrate for UDP-glucuronosyltransferase-2B. Moreover, morphine competitively inhibits A -VPA glucuronidation. It seems the same isoenzyme or, at least, (a) very closely related isoenzyme(s) belonging to UDP-glucuronosyltransferase-2 isoenzyme, is involved in Δ2-VPA glucuronidation.