Showing papers by "B.K. Park published in 1995"

Metabolism and bioactivation of clozapine by human liver in vitro.

Abstract: The metabolism of clozapine by human liver has been investigated in vitro. Irreversible protein-binding and conjunction with model nucleophiles have been used as markers for bioactivation of clozapine, while stable metabolite formation has been assessed using radiometric HPLC. In all nine liver microsomal preparations investigated, clozapine was extensively metabolized to the stable products desmethylclozapine (range 19%-27.2%), N-oxide (1.5-20.5%) and three polar metabolites (0-20.8%), and was bioactivated to a protein-reactive metabolite (0.6-2.1%). The CYP2D6 genotype did not influence the capacity of the livers to form these metabolites. All metabolic pathways were inhibited by ketoconazole, indicating the involvement of the cytochrome P450 enzymes. Isozyme-selective inhibitor studies demonstrated that whereas demethylation was performed by CYP1A2, N-oxidation and chemically reactive metabolite formation were dependent upon multiple forms of P450. The N-oxide was readily reduced back to clozapine in the presence of NADPH, this conversion being inhibited by ascorbic acid. Glutathione (1 mM) decreased covalent binding by 70%. The amount of putative adduct formed in the presence of glutathione (13.4 +/- 0.9%) was much greater than the covalent binding (mean 1.1 +/- 0.2%). The bioactivation of clozapine was, like the N-oxidation of clozapine, a reversible process. In summary, our results indicate clozapine undergoes extensive metabolism by human liver to both stable and chemically reactive metabolites, the formation of which is catalyzed by the cytochrome P450 enzymes. The role of the reactive metabolite, which may be a free radical, in the pathogenesis of clozapine agranulocytosis and hepatotoxicity requires further study.

The metabolic formation of reactive intermediates from clozapine, a drug associated with agranulocytosis in man.

Abstract: Clozapine, a dibenzodiazepine antipsychotic, is associated with a 0.8% incidence of agranulocytosis. This clinically restrictive toxicity has been attributed to its chemically reactive metabolites. The generation of such metabolites--assessed via covalent binding and formation of thioether adducts--was investigated using human, rat and mouse liver microsomes and human neutrophils and bone marrow cells. In every instance, one major glutathione adduct of clozapine--C-6 glutathionyl clozapine--was formed in the presence of added glutathione. Adduct formation by the neutrophils and myeloid cells was dependent on cell activation by phorbol myristate acetate. Small fractions of drug underwent covalent binding to microsomes (1-6.8%) and to protein coincubated with neutrophils (0.47%) and myeloid cells (0.21%). Clozapine did not deplete intracellular glutathione in activated neutrophils. Clozapine was also metabolized in vivo to glutathione conjugates in rats and mice, the conjugates eliminated in bile over a 3-hr period representing 38% and 33% of the dose, respectively. In addition to the principal clozapine adduct found in vitro, the C-8 glutathionyl derivative of deschloroclozapine was excreted by both species. It is concluded that clozapine undergoes bioactivation in several tissues and considerable bioactivation in vivo. The reactive metabolites generated by neutrophils and myeloid cells may play an important role in the metabolic causation of clozapine-induced agranuiocytosis.

N-Hydroxylation of dapsone by multiple enzymes of cytochrome P450 : implications for inhibition of haemotoxicity

Abstract: 1. The adverse reactions associated with the administration of dapsone are believed to be caused by metabolism to its hydroxylamine. Previous reports suggest that CYP3A4 is responsible for this biotransformation [1]. 2. Data presented in this paper illustrate the involvement of more than one cytochrome P450 enzyme in dapsone hydroxylamine formation using human liver microsomes. Eadie-Hofstee plots demonstrated bi-phasic kinetics in several livers. No correlation could be established between hydroxylamine formation and CYP3A concentrations in six human livers (r = -0.47; P = 0.34). 3. Studies with low molecular weight inhibitors illustrate the importance of CYP2C9 and CYP3A in dapsone N-hydroxylation. 4. Differential sensitivity of dapsone N-hydroxylation to selective CYP inhibitors indicated that the contribution of individual CYP enzymes varies between livers. Selective inhibition ranged from 6.8 to 44.1% by 5 microM ketoconazole, and from 24.0 to 68.4% by 100 microM sulphaphenazole. The extent of inhibition, by either ketoconazole or sulphaphenazole was dependent on the CYP3A content of the liver. 5. The levels of expression of these cytochrome P450 enzymes may be an important determinant of individual susceptibility to the toxic effects of dapsone, and may influence the ability of an enzyme inhibitor to block dapsone toxicity in vivo. Because of the inability to produce complete inhibition, selective CYP inhibitors are unlikely to offer any clinical advantage over cimetidine in decreasing dapsone hydroxylamine formation in vivo.

Role of hepatic metabolism in the bioactivation and detoxication of amodiaquine

Abstract: 1. The hepatic metabolism of the antimalarial drug amodiaquine was investigated in order to gain further insight into the postulated metabolic causation of the hepatotoxicity, which restricts the use of the drug. After intraportal (i.p.) administration (54 μmol/kg) to the anaesthetized rat, the drug was excreted in bile (23 ± 3% dose over 5 h; mean ± SD, n = 6) primarily as thioether conjugates.2. After i.p. administration, 20% of the dose was excreted into urine over 24 h as parent compound and products of N-dealkylation and oxidative deamination. Desethylamodiaquine accumulated in liver, but was not a substrate for bioactivation as measured by biliary elimination of a glutathione adduct.3. Prior administration of ketoconazole, an inhibitor of P450, reduced biliary excretion by 50% and effected a corresponding decrease in the amount of drug irreversibly bound to liver proteins. This indicated a role for P450 in the bioactivation of amodiaquine to a reactive metabolite that conjugates with glutathione and...

An investigation of the interaction between halofantrine, CYP2D6 and CYP3A4: studies with human liver microsomes and heterologous enzyme expression systems.

Abstract: 1. We have assessed the interaction of the antimalarial halofantrine with cytochrome P450 (CYP) enzymes in vitro, with the use of microsomes from human liver and recombinant cell lines. 2. Rac-halofantrine was a potent inhibitor (IC50 = 1.06 microM, Ki = 4.3 microM) of the 1-hydroxylation of bufuralol, a marker for CYP2D6 activity. Of a group of structurally related antimalarials tested, only quinidine (IC50 = 0.04 microM) was more potent. 3. Microsomes prepared from recombinant CYP2D6 and CYP3A4 cell lines were shown to catalyse halofantrine N-debutylation. 4. The metabolism of halofantrine to its N-desbutyl metabolite by human liver microsomes showed no correlation with CYP2D6 genotypic or phenotypic status and there was no consistent inhibition by quinidine. 5. The rate of halofantrine metabolism showed a significant correlation with both CYP3A4 protein levels (r = 0.88, P = 0.01) and the rate of felodipine metabolism (r = 0.86, P = 0.013), a marker substrate for CYP3A4 activity. Inhibition studies showed that ketoconazole is a potent inhibitor of halofantrine metabolism (IC50 = 1.57 microM). 6. In conclusion, we have demonstrated that halofantrine is a potent inhibitor of CYP2D6 in vitro and can also be metabolised by the enzyme. However, in human liver microsomes it appears to be metabolised largely by CYP3A4.

Determination of human hepatic cytochrome P4501A2 activity in vitro use of tacrine as an isoenzyme-specific probe.

Abstract: Oxidative metabolism of the cognition activator tacrine (1,2,3,4-tetrahydro-9-aminoacridine) is thought to be catalyzed by cytochrome P4501A2 (CYP1A2). In this study, the use of tacrine as a specific substrate to measure CYP1A2 activity in vitro was investigated. Tacrine metabolism was assessed in 16 human liver microsomal samples. Initially, the percentage conversion of tacrine to stable metabolites (i.e. 1-, 2-, 4-, and 7-hydroxytacrine) at a single time point was correlated with levels of CYP1A2 apoprotein. Apoprotein was detected by immunoquantification using a monospecific CYP1A2 antipeptide antibody. Significant correlations were seen between CYP1A2 content and the degree of 1-hydroxylation (r = 0.81, p < 0.001), 7-hydroxylation (r = 0.70, p < 0.001), and metabolism to all stable products (r = 0.82, p < 0.001). The major metabolite formed in all livers was 1-hydroxytacrine. The conversion of tacrine to this metabolite was examined in more detail. The rate of formation varied from 19.2 pmol min-1 mg-1 to 101.0 pmol min-1 mg-1. There was a significant correlation (r = 0.84, p < 0.001) between the rate of formation and CYP1A2 levels. Tacrine metabolism was also compared with the rate of formation of 3-methylxanthine, from theophylline, a reaction previously shown to be catalyzed by CYP1A2. Significant correlations were found between 3-methylxanthine formation and all quantified tacrine metabolites. The rate of 3-methylxanthine generation also correlated with CYP1A2 apoprotein levels. It is concluded, therefore, that tacrine is a valuable probe for the determination of human hepatic CYP1A2 activity in vitro.

Glutathione S-transferase mu genotype (GSTM1*0) in Alzheimer's patients with tacrine transaminitis.

Abstract: 1. Tacrine (1,2,3,4-tetrahydro-9-aminoacridine) which is used in Alzheimer's disease, causes elevation of liver transaminases ('tacrine transaminitis') in 40-50% of patients. This may be related to the formation of a chemically reactive metabolite from tacrine, which can be detoxified in vitro by glutathione. 2. Glutathione-S-transferase mu (GSTM1), a detoxication enzyme, is polymorphically expressed being absent in about 50% of patients. Its role in the detoxication of the reactive metabolite of tacrine is not known. 3. The frequency of the enzyme deficiency (GSTM1*0) has been investigated in patients with tacrine transaminitis using polymerase chain reaction (PCR) to determine whether the GSTM1 status can be used as an absolute predictive factor for susceptibility to tacrine transaminitis. 4. The frequency of the GSTM1*0 genotype in patients with tacrine transaminitis (n = 33; 45.5%) was not significantly different from that in patients treated with tacrine without liver dysfunction (n = 37; 43%), and when compared with all the controls used in the study (n = 167; 56%). 5. The frequency of the GSTM1*0 genotype in patients with Alzheimer's disease (n = 79; 46%) was not significantly different from that in healthy volunteers (n = 121; 59.5%). 6. Our results indicate that the GSTM1 status cannot be used clinically to predict individual susceptibility to tacrine transaminitis, and that patients with the GSTM1*0 genotype are unlikely to have an increased risk of tacrine-induced liver damage. Furthermore, the GSTM1 status was not associated with Alzheimer's disease.

Species variation in the bioactivation of tacrine by hepatic microsomes

Abstract: 1. The metabolite profile of tacrine (1,2,3,4-tetrahydro-9-amino acridine) was similar in hepatic microsomes from man, rat, dog, rabbit, mouse and hamster. Major metabolites were 1-, 2-, 4- and 7-OH tacrine. Only quantitative differences in metabolite profile were evident between species. 2. Bioactivation to protein-reactive metabolite(s) was seen in microsomes from all species. 3. 7-Methyl tacrine was found to undergo significantly less bioactivation than either 7-OH tacrine or tacrine itself. 4. In the presence of hepatic microsomes and thiol-containing agents protein-reactive metabolite formation was significantly reduced. With mercaptoethanol present a stable thioether adduct was generated from both tacrine and 7-OH tacrine. 5. Analysis of the thioether adduct by mass spectrometry yielded a molecular ion of m/z 290 consistent with the presence of a covalent adduct of 7-OH tacrine complexed in a 1:1 molar ratio with mercaptoethanol. 6. We have therefore provided further evidence for a two-step mechanism in the bioactivation of tacrine involving an initial 7-hydroxylation followed by a postulated 2-electron oxidation to yield a reactive quinone methide. This mechanism of bioactivation appears to be identical in human and animal hepatic microsomes.

Detection of autoantibodies directed against human hepatic endoplasmic reticulum in sera from patients with halothane‐associated hepatitis.

Abstract: 1. Previous studies have demonstrated the presence of antibodies to trifluoroacetylated hepatic proteins (TFA-proteins) in sera from patients with the severe form of halothane-associated hepatitis (halothane hepatitis). The TFA-proteins are produced via cytochrome P450-mediated metabolism of halothane to the reactive species TFA-chloride. 2. To investigate the presence of autoantibodies (which recognize various non-TFA-modified human hepatic polypeptides) in patients with halothane hepatitis immunoblotting experiments were performed using microsomal fractions prepared freshly from livers of five different (halothane-free) tissue donors. Blots were developed using 15 well-characterised sera from patients with halothane hepatitis. 3. Autoantibodies to human hepatic polypeptides were detected in most, but not all, of the patients' sera. The pattern of antibody reactivity varied markedly between sera. Although no common pattern of antibody recognition was observed, polypeptides of molecular mass between 60 and 80 kDa were the predominant targets. A similar protein recognition pattern was seen when each positive serum was tested against the five individual human liver samples. 4. Such autoantibodies were not detected in sera from 16 normal human blood donors, but were detected in three of six sera from patients exposed to halothane without developing hepatitis. 5. The autoantibodies are thought to arise in patients exposed to halothane as a consequence of a halothane-induced immune response to chemically-modified proteins. Such antibodies could contribute to the complex pathological processes involved in halothane hepatitis.

The effect of chemical substitution on the metabolic activation, metabolic detoxication, and pharmacological activity of amodiaquine in the mouse.

Abstract: The adverse reactions associated with the antimalarial amodiaquine (AQ), agranulocytosis and hepatotoxicity, have been attributed to the bioactivation of the drug to a quinone imine metabolite. Therefore the effect of chemical modification on the metabolism of AQ was studied, with particular reference to the prevention of bioactivation and the introduction of glucuronidation. Glutathione conjugates of AQ and desethylAQ were eliminated in bile after intraportal administration of [3H]AQ (54 mumol/kg, 20 microCi/kg) to anesthetized male CD1 mice. Thioether conjugates excreted into bile over 3 h accounted for 28% of the administered dose. Fluorine substitution at the C-4 position of AQ blocked bioactivation, as measured by formation of thioether conjugates, and resulted in a 5-fold decrease in biliary excretion of radiolabeled dose: ca 6% versus ca 29%. Additional substitution of a primary alcohol function into one of the ethyl moieties introduced glucuronidation as a pathway of elimination, with 10% of the dose being excreted in bile as an O-glucuronide of the parent compound over a 3-h period; excretion of total radioactivity in bile increased 2.5-fold. These substitutions resulted in a 2-fold greater excretion of radiolabel into urine: 41% and 39% for DFAQ and HDFAQ, respectively, versus 23% for AQ. Novel carboxylic acid and N-oxide metabolites of the fluorinated analogues were identified. AQ and the two fluorinated analogues had similar activity against Plasmodium berghei in mice. These results demonstrate that the metabolism of AQ can be diverted from extensive bioactivation to direct detoxication by simple chemical substitutions that do not impair pharmacological activity.