Effect of methamphetamine on the pharmacokinetics of dextromethorphan and midazolam in rats.

Methamphetamine is the fourth most frequently reported compound associated with drug abuse on admission of patients to treatment centres after cocaine, heroin and marijuana. It is metabolized in the organism with a reaction that is catalyzed by cytochrome P450, mainly by the CYP2D and CYP3A subfamily, 4-hydroxyamphetamine and amphetamine being dominant metabolites. The present pharmacokinetic study was undertaken to investigate the possible influence of methamphetamine (10 mg/kg, i.p., once daily for six days) on the pharmacokinetics of dextromethorphane as a model substrate for rat cytochrome P-4502D2 and midazolam as a model substrate for CYP3A1/2. Animals received a single injection of dextromethorphane (10 mg/kg) or midazolam (5 mg/kg) in the tail vein 24 h after the last dose of methamphetamine or administration of placebo. The results of pharmacokinetic analysis showed a significantly increased rate of dextrorphane and 3-hydroxymorphinan formation, and a marked stimulatory effect of methamphetamine on CYP2D2 metabolic activity. Similarly, the kinetics of midazolam's metabolic conversion to hydroxy derivates of midazolam indicated a significant increase in CYP3A1/2 activity. The results showed that the administration of methamphetamine significantly stimulated the metabolic activity of CYP2D2 as well as that of CYP3A1/2. With regard to the high level of homology between human and rat CYP isoforms studied, the results may have a clinical impact on future pharmacotherapy for methamphetamine abuse.

Effect of St John's wort (Hypericum perforatum) on cytochrome P-450 activity in perfused rat liver.

St. John's wort (Hypericum perforatum) is a popular over-the-counter dietary supplement and a herbal antidepressant that has been implicated in drug interactions with substrates of several cytochrome P-450 (CYP) isozymes. The effects of the St. John's wort extract (100 mg/kg, i.p., once daily for 10 days) on metabolic activity of CYP450 were assessed in the system of isolated perfused rat liver. The substrates used in this study were tolbutamide (CYP2C6), dextromethorphan (CYP2D2) and midazolam (CYP3A2). Validated HPLC method was used to quantify all compounds of interest. St. John's wort administration affected CYP activity, causing a significant decline in AUC of dextromethorphan [F(4,31)=1511, p<0.001; PLSD, p<0.001] and AUC of midazolam [F(3,25)=221, p<0.001; PLSD, p=0.035] and a significant increase in AUC of tolbutamide [F(3,26)=200, p<0.001; PLSD, p<0.001]. St. John's wort administration resulted in a significant induction of CYP2D2 and CYP3A2, and in a significant inhibition of CYP2C6 metabolic activities.

Paroxetine-induced conversion of cytochrome P450 2D6 phenotype and occurence of adverse effects.

The paper is focused on a comparison of the distribution of side effects of treatment with paroxetine within a group of 30 patients genotyped and phenotyped for their CYP 2D6 metabolic status. Genotyping procedure showed that the patient group did not include any individual with poor metabolizer (PM) genotype; on the other hand, most patients (24) were classified as PMs by virtue of their phenotype, which suggests that a conversion to the poor metabolic phenotype ("phenocopy") occurred, probably as a consequence of a long-term administration of the strong CYP 2D6 inhibitor paroxetine. As to the occurence of common adverse effects, no marked difference between subjects converted into the PM group and those who had no history of such conversion was found. A significantly higher incidence of sexual dysfunction (p < 0.05) was, nevertheless, recorded in patients with the PM phenotype. The results of the study may provide evidence that it is the metabolic phenotype status, rather than the genetically given enzyme capacity (CYP 2D6 genotype), that is relevant for the actual toleration of treatment with CYP 2D6 inhibitors.

Effects of psychopharmacotherapy on phenotypic expression of cytochrome P450 2D6 in patients genotyped for CYP2D6 mutations.

No Abstract

Influence of pretreatment with immunosuppressants on O-demethylation of dextromethorphan in isolated perfused rat liver.

Changes in the immune system induced by exogenous or endogenous factors may be accompanied with modifications of the activity of the drug metabolising enzymes in the liver. Some immunostimulatory agents are known to suppress the oxidative metabolism mediated by cytochromes P450. Possible effects of substances which suppress the immune responses of the organism have not been fully understood yet. The present study was undertaken to investigate the influence of immunosuppressants cyclophosphamide and dexamethasone on the CYP2D 1-dependent metabolism of dextromethorphan (DEM) in the isolated perfused liver from male rat donors (Wistar albino, 250-310 g). Recirculatory perfusion system was used with Williams' medium E (Sigma Chemicals Co.) as a perfusion medium (120 mL). DEM was administered as a 1 mg bolus into the perfusion solution at the start of each experiment after 20 min preperfusion. Samples of perfusate for HPLC determination of DEM and its O-demethylated metabolite dextrorphan (DOR) were taken at 15 min intervals for 120 min. The results have shown a rapid conversion of DEM to DOR in the isolated rat liver preparations. Pharmacokinetic parameters in the livers from intact rats were as follows: t1/2 DEM = 19.1+/-4.10 min, k(m) = 0.035+/-0.008 min(-1), Cl(m) = 4.21+/-0.97 mL x min(-1), AUC(DOR) = 2160+/-201 microg x min x L(-1). Pretreatment of rats with dexamethasone (4 mg/kg/day, iv, x 3 days) led to a significant increase in the concentration of dextrorphan in the recirculating solution, but it did not substantially change the kinetic constants of DOR formation (km = 0.036+/-0.004 min(-1), Cl(m) = 4.27+/-0.43 mL x min(-1)). Cyclophosphamide (100 mg/kg, ip, 1 dose on day 5 before perfusion) induced nearly twofold increase in the DOR concentrations in perfusate and thus highly significant (p < 0.01) changes of the kinetic parameters characterizing the increased rate of conversion of DEM to DOR (t1/2 DEM = 12.1+/-0.90 min, km = 0.055+/-0.004 min(-1), Cl(m) = 7.09+/-1.37 mL x min(-1), AUC(DOR) = 3602+/-154 microg x min x L(-1)). Considering that both cyclophosphamide and dexamethasone belong to the most widely used immunosuppressive drugs, their potential to promote the CYP2D-mediated metabolism might have a clinical impact in combined therapy of autoimmune or other diseases.

Capillary zone electrophoresis determination of perphenazine in pharmaceutical preparations and in human serum.

Perphenazine belongs to the group of phenothiazine-based neuroleptic drugs frequently used in the long-term treatment of psychotic disorders. In this work, a new capillary zone electrophoresis (CZE) method for the rapid determination of perphenazine (PPZ) in pharmaceutical preparations and human blood serum was developed. Using solid-phase extraction (SPE) as a preclean/purification and preconcentration step before CZE analysis, a detection limit of 3 ng/mL for the monitoring of PPZ in blood serum (for a signal-to-noise ratio of 3) was reached.

Interferon- and streptolysin O-induced activation of protein kinases and inhibition of cytochrome P450-dependent monooxygenases in rats.

Immunostimulants known to initiate cytokine production were found to decrease the activity of hepatic microsomal drug oxidative enzymes but to activate protein kinase C (PKC). The present study investigated the effects of immunostimulating doses of rat interferon-gamma (IFN, 670,000 units i.p.) and streptolysin O (SLO, 100 HU/kg i.v. for 5 days) on hepatic soluble, membrane-bound and nuclear PKC, 7-ethylresorufin-O-deethylase (EROD) and 7-pentylresorufin-O-deethylase (PROD) in male Wistar rats. The SLO- and IFN-mediated decrease of EROD and PROD activity was associated with a characteristic activation of the hepatic and spleenic PKC. In SLO- and IFN-treated animals activities of the cytosolic, membrane-bound and nuclear PKC were significantly higher than in respective controls. Our results suggest that a decrease in hepatic cytochrome P450 content as well as the decrease in the EROD and PROD activities are inversely related to the function of PKC.

Direct determination of procainamide and N-acetylprocainamide by capillary zone electrophoresis in pharmaceutical formulations and urine.

In this work a new sensitive capillary zone electrophoresis method for the direct determination of procainamide (PA) and N-acetylprocainamide (NAPA) in pharmaceutical formulations and urine samples without any extraction and/or preconcentration steps has been developed. The determination was carried out in a fused-silica capillary of 43.5 cm (35.9 cm length to the detector) x 0.75 micron J.D. Phosphate 0.05 M buffer was used as the background electrolyte and 10 kV separation voltage was applied. The separation of PA and NAPA is possible in a wide range of pH from 1.7 to 9.7. However, in order to avoid the effect of the urine matrix, it is optimal to work at pH 7.7. The determination of PA and NAPA takes less than 5 min while high resolution is achieved. The detection limits obtained, 1.235 micrograms/ml and 0.359 microgram/ml for PA and NAPA respectively, are lower than those for GC method normally reported.

Influence of H2-receptor- and proton pump inhibitors on some functions of the oxydative and conjugative drug metabolism.

There are numerous investigations describing the influence of histamine H2-receptor antagonists and proton pump inhibitors on cytochrome P450-mediated hepatic oxydative and conjugative drug metabolizing enzymes. The aim of this study was to investigate the influence of the H2-receptor blockers cimetidine, ranitidine, famotidine, nizatidine and of the proton pump inhibitors omeprazole and lansoprazole on the acetylation capacity and on different microsomal monooxygenases of the rat liver. The experiments were performed in two randomized studies with male Wistar rats after a 7-day pretreatment of the animals with antisecretory, equipotent doses of the investigational products. The activities of the arylamine N-acetyltransferase (NAT) and the microsomal enzymes were determined in vitro. Cimetidine and ranitidine decreased the activity of NAT significantly, no effect on this enzyme was observed after nizatidine. Small doses of famotidine tended to lower, high doses of famotidine tended to enhance the NAT activity. The proton pump inhibitor omeprazole significantly increased the NAT activity, lansoprazole evoked a small increase of the enzyme activity. Ethyl-resorufin O-deethylase (EROD) and penthlresorufin O-depentylase (PROD) were sensitive to cimetidine, ranitidine and famotidine. Only omeprazole and lansoprazole treatment inhibited the detromethorphan O-demethylase (DXDM) activity.

Effects of streptolysin O, polyriboinosinic-polyribocytidylic acid and combinations with cyclophosphamide on the hepatic arylamine N-acetyltransferase and cytochrome P450-dependent monooxygenases in rats.

There is evidence that immunostimulation depresses the function of various cytochrome P450 (CYP)-dependent monooxygenases but activates the arylamine N-acetyltransferase (NAT). Therefore, the effects of the synthetic immunostimulator polyriboinosinic-polyribocytidylic acid (plC, 8 mg/kg i.p.), of sublytic doses of the streptococcal toxin streptolysin O (SLO, 50 HU/kg i.v. for 5 d) and of the immunosuppressor cyclophosphamide (CP, 100 mg/kg i.p.) on NAT and some monooxygenases were studied in rat liver. It was also evaluated whether CP might antagonize the effects of plC and SLO on drug metabolism. SLO, plC and CP reduced CYP content and the activities of some monooxygenases. NAT was significantly inhibited by CP (given 5 d before sacrifice) but not by plC, SLO or CP when given 2 d before sacrifice. CP lacked any effect on NAT if it was administered prior to SLO. However, it deteriorated synergistically the inhibition of the monooxygenases caused by SLO and plC.

Debrisoquine 4-hydroxylation and sulphamethazine N-acetylation in patients with schizophrenia and major depression.

Debrisoquine 4-hydroxylation and sulphamethazine N-acetylation phenotypes were determined in 115 Czech drug-free in-patients with schizophrenia (n = 64) or major depressive disorder (n = 51). These data were compared with a control group of 321 healthy volunteers from the North-East German area of Greifswald. The distribution of debrisoquine hydroxylator phenotypes was almost identical in patients and healthy controls. Thus, there were 8.7% (95% CI 5.4-12.0%) of poor metabolizers (PM) among patients while 8.7% (95% CI 23.6-13.8%) PM among the control group. The prevalences of PM amongst patients with chronic schizophrenia and major depression were 10.9% (95% CI 4.5-21.3%) and 5.9% (95% CI 1.24-16.3%), respectively (chi 2 schizophrenics vs control = 0.315, NS; chi 2 depressive patients vs control = 0.450, NS). However, within the group of EM patients there was a significant (P < 0.01) shift towards higher debrisoquine metabolic ratios, reflecting a lower hydroxylation capacity in EM patients compared with EM healthy controls. The proportion of slow acetylators (SA) was 60.0% (95% CI 51.0-68.9%) in the entire group of psychiatric patients and 57.5% (95% CI 52.1-62.9%) in the control group (chi 2 all patients vs control = 0.195, NS). Furthermore, there were no significant differences in the prevalence of the SA phenotype between controls and schizophrenics or patients with major depression. Although the results of this modest study were negative, the presence of subtle differences in the metabolic capacity between psychiatric patients and a healthy population cannot be ruled out.

Effects of streptolysin O, picibanil (OK 432) and interferon alpha 2A on cytochrome P-450-dependent monooxygenases and arylamine N-acetyltransferase in rat liver.

Streptolysin O, a thiol-activated exotoxin from group A beta-haemolytic streptococci, caused a dose-dependent depression of aniline hydroxylase, aminopyrine N-demethylase and ethylmorphine N-demethylase activities when added into the hepatic microsomal mixtures from male rats at concentrations 0.02-0.4 HU/mL in vitro. The activities of 7-ethoxycoumarin O-deethylase, 7-ethylresorufin O-deethylase and 7-pentylresorufin O-depentylase were not altered with the used concentrations of the toxin. Specific antibody against haemolytic action of streptolysin O added to incubation mixtures in vitro was not able to protect streptolysin-sensitive monooxygenases from the inhibition. The addition of streptolysin O (0.01-0.8 HU/mL) into the cytosol-containing medium did not significantly influence the activity of procainamide N-acetyltransferase. Immunomodulators picibanil (OK 432) and human recombinant interferon alpha 2A which are known to suppress oxidative metabolism in vivo in humans and animals, were without effect either on the cytochrome P-450-dependent monooxygenases or on the N-acetyltransferase activity when administered in vitro at the doses real in their clinical application (0.001-0.1 KE/mL of picibanil and 10-500 U/mL of alpha-interferon).

Relationship between acetylator phenotype and intensity of the hypoglycaemic effect of a suplhonamide preparation in children with insulin-dependent diabetes.

The distribution of acetylator phenotypes was determined in 95 healthy children (4-15 y) and in 48 children with Type I diabetes (5-15) using the sulphadimidine test for the determination of acetylation capacity. The frequency of slow acetylators within the diabetic group (60.4%) closely resembled that in the healthy population (59.0%). There was no significant sex difference within either group. Subgrouping according to age showed a more marked preponderance of slow acetylators (65.0%) among children under six years of age on the control group but not in the diabetics (60.0%). Despite a relatively low dose of sulphadimidine used for acetylator phenotyping (20 mg/kg orally), a marked hypoglycaemic effect was observed in diabetics with slow but not with fast acetylator phenotype. The result appears to be coherent with the marked difference in the plasma concentrations of non-metabolized sulphadimidine which 6 h after the dose was 3.5-fold higher in slow acetylators than in fast ones. (Tab. 2, Fig. 1, Ref. 53.)

Drug oxidation and N-acetylation in rats pretreated with subtoxic doses of streptolysin O.

SLO in sublytic doses (100 HU/kg body wt) depressed the hepatic microsomal cytochrome P450 and aminopyrine-N-demethylase but increased significantly the cytosolic N-acetyltransferase after acute and subacute (5 days) pretreatment of male Wistar rats. Aniline hydroxylase was reduced after acute and unchanged after subacute pretreatment. The effects of SLO on the oxidative enzymes and drug N-acetylation might have been caused by the membranal and immunological properties of the toxin.

N-acetylation in healthy and diseased children.

Acetylation capacity was examined in three groups of Czech children by measuring the plasma and urine concentrations of sulphamethazine and its acetylated metabolite 6 h after an oral test dose of 20 mg/kg sulphamethazine. Amongst 82 healthy children aged 4-15 y there were 32 (39%) fast acetylators; there was no significant difference between the number of boys and girls, or between children over or less than 6 years of age. In 41 patients aged 3-15 y with phenylketonuria, the acetylation indices showed a significantly higher proportion of fast acetylators - 24 (58.5%) using plasma measurements and 29 (70.7%) using urine data. In them the ratio between slow and fast acetylators was inverted compared to normal children. The preponderance of fast acetylators was greater in boys than in girls and in children over 6 years of age. An increased acetylation capacity in patients with phenylketonuria was apparent even in individuals classified as slow acetylators, because in them the plasma concentration of the acetylated metabolite was higher than in control acetylators. Amongst 48 young patients (5-15 y) with insulin-dependent diabetes there were 19 (39.6%) fast and 29 (60.4%) slow acetylators, which corresponded well to the phenotype distribution in control children. This did not support the suggested association between the fast acetylator phenotype and Type I diabetes.