Marmoset cytochrome P450 2B6, a propofol hydroxylase expressed in liver

1. The common marmoset (Callithrix jacchus) is a useful experimental animal to evaluate the pharmacokinetics of drug candidates. Cytochrome P450 (P450) 2B enzyme in marmoset livers has been identified; however, only limited information on the enzymatic properties and distribution has been available.

2. Marmoset P450 2B6 amino acids showed high sequence identities (>86%) with those of primates including humans and cynomolgus monkeys. Phylogenetic analysis using amino acid sequences indicated that marmoset P450 2B6 was closer to human and cynomolgus monkey P450 2B6 than to P450 2B orthologs of other species, including pigs, dogs, rabbits and rodents.

3. Quantitative polymerase chain reaction analysis using specific primers showed P450 2B6 mRNA predominantly expressed in livers among the five marmoset tissues, similar to those of humans and cynomolgus monkeys.

4. Marmoset P450 2B6 heterologously expressed in Escherichia coli membranes oxidized 7-ethoxycoumarin, pentoxyresorufin, propofol and testosterone, at roughly similar rates to those of humans and/or cynomolgus monkeys. A high capacity of marmoset P450 2B6 with propofol 4-hydroxylation (at low ionic strength conditions) with a low K_m value was relatively comparable to that for marmoset livers.

5. These results collectively indicated a high propofol 4-hydroxylation activity of P450 2B6 expressed in marmoset livers.

     

Metabolism of deltamethrin and cis- and trans-permethrin by human expressed cytochrome P450 and carboxylesterase enzymes

1. The metabolism of the pyrethroids deltamethrin (DLM), cis-permethrin (CPM) and trans-permethrin (TPM) was studied in human expressed cytochrome P450 (CYP) and carboxylesterase (CES) enzymes.

2. DLM, CPM and TPM were metabolised by human CYP2B6 and CYP2C19, with the highest apparent intrinsic clearance (CL_int) values for pyrethroid metabolism being observed with CYP2C19. Other CYP enzymes contributing to the metabolism of one or more of the three pyrethroids were CYP1A2, CYP2C8, CYP2C9*1, CYP2D6*1, CYP3A4 and CYP3A5. None of the pyrethroids were metabolised by CYP2A6, CYP2E1, CYP3A7 or CYP4A11.

3. DLM, CPM and TPM were metabolised by both human CES1 and CES2 enzymes.

4. Apparent CL_int values for pyrethroid metabolism by CYP and CES enzymes were scaled to per gram of adult human liver using abundance values for microsomal CYP enzymes and for CES enzymes in liver microsomes and cytosol. TPM had the highest and CPM the lowest apparent CL_int values for total metabolism (CYP and CES enzymes) per gram of adult human liver.

5. Due to their higher abundance, all three pyrethroids were extensively metabolised by CES enzymes in adult human liver, with CYP enzymes only accounting for 2%, 10% and 1% of total metabolism for DLM, CPM and TPM, respectively.

     

Topically injected adrenocorticotropic hormone induces mechanical hypersensitivity on a full‐thickness cutaneous wound model in rats

Cutaneous wound pain causes physical and psychological stress for patients with wounds. Previous studies reported that stress induces hyperalgesia and deteriorates wound healing. However, the effect of the stress response such as in hypothalamic‐pituitary‐adrenal (HPA) axis on local wound area is unclear. We aimed to investigate the effects of a stress response on the mechanical withdrawal threshold in the local wound area and describe the identification of a wound pain exacerbation. We topically injected adrenocorticotropic hormone (ACTH) into the granulation tissue of full‐thickness cutaneous wound model rats on the fifth day postwounding and measured the mechanical withdrawal thresholds, cytochrome P450 2Bs levels and concentration of 5,6‐epoxyeicosatrienoic acid in wound exudate. We found that ACTH induced mechanical hypersensitivity at 4 and 6 hours after injection (P = .004 and .021, respectively), and increased gene expression of cytochrome P450 2B12 expression (P = .046). Concentration of 5,6‐EET in the wound exudate was moderately correlated with the mechanical withdrawal threshold (r = ?.630). Finally, the mechanical withdrawal threshold in the 5,6‐EET group was significantly lower than that in the control group at 2 hours after the injection (P = .015). We propose that 5,6‐EET is one of the most promising contributors to the wound pain exacerbation. These findings could guide clinical wound and pain management.     

CYP2E1 and ALDH2 Genotypes and Alcohol Dependence in Japanese

The genotypes of the CYP2E1 and ALDH2 loci of alcoholic (alcohol dependence) and nonalcoholic (healthy) Japanese were investigated to examine the relationship between the polymorphism of CYP2E1 (C₁/C₂) and ALDH2 ( ALDH2*1/ALDH2*2 ), and the susceptibility to alcoholism. There was no significant difference in C₂ gene frequency between alcoholics (0.19) and nonalcoholics (controls) (0.20), whereas there was a significant difference in ALDH2 allele frequency, suggesting that, in Japanese, the C₂ genotype of CYP2E1 may have nothing to do with the risk of developing alcohol dependence. However, the ALDH2*1 allele may influence drinking behavior and the development of alcohol dependence. Furthermore, racial interethnic differences in the frequency of the mutated allele of the CYP2E1 gene (CJ were found, like the ALDH2 gene. Japanese healthy controls showed a significantly higher frequency of the C₂ allele than did Swedish healthy controls (0.05; reported by Persson et al., FEBS Lett. 319:207-211,1993).     

A drug interaction study between ticlopidine and cyclosporin in heart transplant recipients

Objectives: Previous uncontrolled studies have suggested an interaction between ticlopidine, a major antiplatelet agent, and cyclosporin in heart- and kidney-transplant recipients. The aims of this study were to examine in a randomised, double-blind fashion, the possible interaction between cyclosporin A and ticlopidine (250 mg per day) and the tolerability of this combination in heart-transplant recipients. Methods: Twenty heart-transplant recipients were randomised into either a treated or a placebo group. Blood samples were drawn for time-course evaluation of cyclosporin blood levels over a period of 12 h, following the morning intake of cyclosporin and, for platelet aggregation studies, before and after 14 days of ticlopidine administration. Twenty four-hour urine samples were collected for 6-β-hydroxycortisol measurements, before and after 14 days of ticlopidine. Results: Although given at half the recommended daily dosage, ticlopidine significantly reduced platelet aggregation. Pharmacokinetic parameters indicate that the bioavailability of cyclosporin A was not significantly modified by ticlopidine. However, one patient in the ticlopidine group was withdrawn because of a major fall in cyclosporin blood level within 3 days of treatment. Urinary excretion of 6-β-hydroxycortisol was augmented after treatment in the ticlopidine group compared with the placebo group, suggesting that induction of drug metabolism might have occurred. Data also show quite a large intra-individual variability in cyclosporin bioavailability in the placebo group, suggesting that poor absorption of the drug formulation and/or poor compliance might have contributed to the decreased cyclosporin blood levels in the patient withdrawn from this study and in previous uncontrolled studies. Conclusion: Cyclosporin bioavailability was not clearly modified by a half dosage of ticlopidine in this study. We, however, recommend closely monitoring cyclosporin blood levels when prescribing ticlopidine. Further studies will be needed with new formulations of cyclosporin or when using the full dosage of ticlopidine. Received: 20 July 1996 / Accepted in revised form: 12 February 1997     

Alteration of cytochrome P-450 by prolonged administration of imipramine and/or lithium to rats

Summary The aim of this study was to investigate imipramine-induced alterations of cytochrome P-450 and to determine whether prolonged concomitant administration of imipramine and lithium results in a pharmacokinetic interaction.Male Wistar rats received imipramine (10 mg/kg i. p.) at 12 h intervals or lithium chloride (100 mg/kg in drinking water) or they were treated with the combination of these drugs for 2 weeks. The long term treatment with imipramine produced a very complex alteration of cytochrome P-450: imipramine increased the level of the cytochrome, but it decreased the rate of its own aromatic hydroxylation in position 2. The rate of N-demethylation in the side chain was not changed. Consequently, in the case of both hydroxylation and demethylation, calculated molecular activities were decreased to 48% and 70% respectively. This differential change in activities corresponded well to the observed decrease of absorption in difference spectra (type I) produced in microsomes by imipramine. Carbamazepine-induced type I difference spectra were also decreased by imipramine pretreatment, but to a lesser extent. In contrast, hexobarbital type I binding was increased by imipramine treatment while type II difference spectra produced by metyrapone were not affected. The preliminary SDS-PAGE analysis of cytochrome P-450 isoenzymes of control and imipramine treated rats showed that the investigated antidepressant markedly intensified a protein band at 50.11 kD while bands at 51.28 kD, 56.20 kD and 56.88 kD were less intensive. These results indicate that the alteration of cytochrome P-450 by imipramine treatment is not only of quantitative but also of qualitative character. Lithium alone given to rats affected neither the concentration of cytochrome P-450 in microsomal protein nor the rate of imipramine metabolism in vitro. Lithium given jointly with imipramine reduced imipramine-induced elevation of cytochrome P-450. This, however, did not cause any change in the rate of imipramine metabolism in vitro and accordingly in imipramine pharmacokinetics in vivo. The concentration of lithium in the blood plasma tended to increase by concurrent administration of imipramine.Send offprint requests to K. J. Netter at the above address     

Characterization of the human cytochrome P450 enzymes involved in the metabolism of dihydrocodeine

Aims Using human liver microsomes from donors of the CYP2D6 poor and extensive metabolizer genotypes, the role of individual cytochromes P-450 in the oxidative metabolism of dihydrocodeine was investigated.
Methods The kinetics of formation of N- and O -demethylated metabolites, nordihydrocodeine and dihydromorphine, were determined using microsomes from six extensive and one poor metabolizer and the effects of chemical inhibitors selective for individual P-450 enzymes of the 1A, 2A, 2C, 2D, 2E and 3A families and of LKM1 (anti-CYP2D6) antibodies were studied.
Results Nordihydrocodeine was the major metabolite in both poor and extensive metabolizers. Kinetic constants for N -demethylation derived from the single enzyme Michaelis-Menten model did not differ between the two groups. Troleandomycin and erythromycin selectively inhibited N -demethylation in both extensive and poor metabolizers. The CYP3A inducer, α-naphthoflavone, increased N -demethylation rates. The kinetics of formation of dihydromorphine in both groups were best described by a single enzyme Michaelis-Menten model although inhibition studies in extensive metabolizers suggested involvement of two enzymes with similar K _m values. The kinetic constants for O -demethylation were significantly different in extensive and poor metabolizers. The extensive metabolizers had a mean intrinsic clearance to dihydromorphine more than ten times greater than the poor metabolizer. The CYP2D6 chemical inhibitors, quinidine and quinine, and LKM1 antibodies inhibited O -demethylation in extensive metabolizers; no effect was observed in microsomes from a poor metabolizer.
Conclusions CYP2D6 is the major enzyme mediating O -demethylation of dihydrocodeine to dihydromorphine. In contrast, nordihydrocodeine formation is predominantly catalysed by CYP3A.