Hepatitis B vaccine and neurotoxicity.
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Adult↗
Biomedical subjects
M Pirmohamed
Publications and source records attributed to M Pirmohamed.
Disposition of amodiaquine and related antimalarial agents in human neutrophils: implications for drug design.
The development and clinical use of 4-aminoquinoline antimalarial agents such as amodiaquine have been limited by toxicity to neutrophils. We have investigated the chemical basis of amodiaquine-induced toxicity and compared the findings with those for established antimalarial drugs proposed for human use. Amodiaquine, like chloroquine, mefloquine and halofantrine, was lysosomotropic and accumulated in human neutrophils. Amodiaquine did not lead to impairment of either cellular function or cell viability at therapeutic levels. In contrast to other antimalarial agents, amodiaquine (because it contains a 4-aminophenol function) depleted glutathione in activated neutrophils, by formation of an electrophilic quinoneimine metabolite. Bioactivation was accompanied by the expression of a drug-related antigen on the cell surface, which was recognized by drug-specific antibodies, suggesting that a type II hypersensitivity reaction is responsible for the observed toxicity. Similar bioactivation and accumulation were observed for the structurally related amopyroquine. The effects of chemical modifications at the 3'- and 5'-positions, which are known to enhance antimalarial activity, were also investigated. The introduction of a lipophilic 5'-chlorophenyl group and 3'-t-butyl group blocked bioactivation but enhanced cellular accumulation, with resultant impairment of function and neutrophil viability, whereas introduction of a second cationic dialkylamino group (bis-mannich compounds) blocked bioactivation and reduced cellular accumulation, without producing noticeable effects on cellular function and viability. These data provide a chemical rationale for the idiosyncratic agranulocytosis observed with amodiaquine, and they suggest that similar toxicity might be anticipated for amopyroquine but is less likely with bis-mannich antimalarial agents such as pyronaridine.
Amodiaquine↗
Characterization of the metabolites of carbamazepine in patient urine by liquid chromatography/mass spectrometry.
The urinary metabolites of carbamazepine (CBZ) in epileptic patients receiving long-term drug treatment have been characterized by LC/MS. CBZ-10,11-epoxide (9.6-15.0 micrograms/ml), trans-10, 11-dihydrodiol-CBZ (273.0-400.00 micrograms/ml), and CBZ (2.4-3.8 micrograms/ml) were measured by HPLC. The secondary N-glucuronide of CBZ, four phenolic O-glucuronides (including those of 2- and 3-OH-CBZ), two additional OH-CBZ O-glucuronides, and the N-glucuronide of CBZ-10,11-epoxide constituted the products of either direct conjugation or preliminary monoxygenation. Derivatives of these monoxygenated compounds, which were characterized as O-glucuronides, were represented by dihydroxylated (catechol) CBZ and its putative O-methyl metabolite and by 10,11-dihydrodiol-CBZ. 10,11-Dihydro-10-OH-CBZ O-glucuronide, a metabolite thought to be excreted only by uremic subjects, was not found. More complicated biotransformations of the 10,11-ene moiety were revealed by two carbinol products of azepine ring contraction: 9-OH-methyl-10-carbamoyl acridan and an hydroxylated derivative thereof, which were excreted as O-glucuronides. No polar sulfur-containing metabolites that might serve as indicators of reactive intermediate formation were found in human urine.
Adult↗
The relationship between the disposition and immunogenicity of sulfamethoxazole in the rat.
Idiosyncratic toxicity associated with sulfamethoxazole (SMX) is thought to be a consequence of bioactivation to the hydroxylamine metabolite (SMX-NOH) and further oxidation to the ultimate reactive metabolite, nitroso-sulfamethoxazole (SMX-NO). To establish the link between the formation of the ultimate reactive metabolite and SMX hypersensitivity, we have undertaken metabolism and immunogenicity studies in the rat by use of SMX and its metabolites. SMX was excreted in urine as N4-acetyl SMX and SMX-NOH, with approximately 10% remaining unchanged as parent amine. After administration of SMX-NOH (54 mg x kg(-1)) and SMX-NO (10 mg x kg(-1)), 38.3% and 46.1% of the doses, respectively, were excreted in urine as SMX and N4-acetyl SMX, which indicated extensive reduction of these metabolites in vivo. The immunogenic potential of SMX and its metabolites, SMX-NOH and SMX-NO, were assessed in rats by analyzing serum samples for the presence of anti-SMX IgG antibodies during a 4-week dosing period. No antibodies to SMX were detected in either control or SMX-treated rats. In contrast, a high titer of SMX-specific IgG antibody was present in sera from all the rats administered SMX-NO, reaching a maximum 14 to 21 days after the initial dose. Rats administered SMX-NOH only produced a weak IgG response after 3 weeks of dosing. These findings indicate that SMX-NO is highly immunogenic and may be responsible for the hypersensitivity reactions associated with SMX. Both SMX-NOH and SMX-NO undergo extensive reduction in vivo which may afford protection against SMX toxicity.
Animals↗
Comparison of the metabolism and toxicity of dapsone in rat, mouse and man.
The metabolism and toxicity of dapsone was compared in vitro and in vivo in rat, mouse and man. Metabolism was assessed by high-pressure liquid chromatography-mass spectrometry and methemoglobin formation has been used as a toxic endpoint. The greatest toxicity in vitro was seen in microsomes prepared from male Wistar rats (36.6 +/- 1.5% methemoglobin), although toxicity was also seen in microsomes from the female rat (8.2 +/- 1.3%), male CD1 (4.2 +/- 1.6%) and human (10. 9 +/- 1.1%). The rank order of toxicity agreed with the formation of the hydroxylamine metabolite in vitro. All microsomes were also capable of catalyzing the reverse reaction, i.e., reduction of the hydroxylamine to dapsone. However, in vivo administration of dapsone resulted in significant (P < 0.05) methemoglobinemia only in male rats and humans. This species difference in the susceptibility to dapsone toxicity could not be attributed solely to the sensitivity of the target erythrocytes, because the order of sensitivity to dapsone hydroxylamine was human > mouse > rat. Analysis of bile and urine revealed the formation of dapsone hydroxylamine and its glucuronide in male rats and humans, but not in female rats or mice. This species difference in the metabolism and toxicity of dapsone has important implications in the safety evaluation of related compounds for man.
Acetylation↗
Neutrophil cytotoxicity of the chemically reactive metabolite(s) of clozapine: possible role in agranulocytosis.
Clozapine is associated with a 0.8% incidence of agranulocytosis. Bioactivation to an unstable protein-reactive metabolite, identified as a nitrenium intermediate, has been implicated in the toxicity. In this study, we investigated whether the reactive metabolite is cytotoxic toward polymorphonuclear leukocytes and mononuclear leukocytes using horseradish peroxidase and H2O2 to generate the metabolite in situ. In the absence of a full metabolizing system (i. e., lack of horseradish peroxidase and/or H2O2), clozapine (0-100 microM) and its stable metabolites were not cytotoxic. With a full metabolizing system, both clozapine (30 microM) and demethylclozapine exhibited cytotoxicity toward polymorphonuclear leukocytes (50.7 +/- 7.7% and 17.6 +/- 1.2% cell death, respectively) and mononuclear leukocytes (36.6 +/- 2.1% and 24.6 +/- 4.1%, respectively), whereas clozapine N-oxide was not cytotoxic. Exogenous glutathione (GSH), N-acetylcysteine and ascorbic acid all protected the cells. Bioactivation of clozapine and demethylclozapine, but not the N-oxide, was accompanied by depletion of intracellular GSH. [14C]Clozapine was metabolized to the previously identified C6 and C9 glutathionyl conjugates; GSH conjugates were also detected when demethylclozapine and clozapine N-oxide were bioactivated by horseradish peroxidase and H2O2. In conclusion, using a novel in vitro assay, we have shown that clozapine and its stable metabolites are not cytotoxic per se but are bioactivated to cytotoxic metabolites. The cytotoxic metabolite of clozapine is identical to the protein-reactive metabolite that has been characterized previously. These cytotoxic metabolites may play an important role in the pathogenesis of clozapine agranulocytosis; the mechanism by which this occurs is currently being investigated.
Adult↗
Nurses' confidence in caring for patients with alcohol-related problems.
General nurses lack confidence and experience in caring for patients with alcohol-related problems. There is a need for better basic training and continuing education for nurses on alcohol issues. A specialist alcohol nurse could help support patients and staff.
Alcohol-Related Disorders↗
Characterisation of the toxic metabolite(s) of naphthalene.
The toxicity of naphthalene and its metabolites has been investigated in vitro. Both naphthalene and its metabolite 1-naphthol were bioactivated by human hepatic microsomes to metabolite(s) which were toxic to mononuclear leucocytes (MNL). However 1-naphthol was more cytotoxic than naphthalene (49.8 +/- 13.9% vs. 19.0 +/- 10.0% cell death; P < 0.01), indicating that the toxicity of naphthalene is dependent on the bioactivation of 1-naphthol. CYP2E1-induced rat liver microsomes increased metabolism of naphthalene by 13% compared to control microsomes with a concomitant increase in both 1-naphthol and dihydrodiol formation. The cytotoxicity of naphthalene but not of 1-naphthol was increased by CYP2E1 induction, indicating that separate enzymes are involved in the bioactivation of 1-naphthol. The metabolites of 1-naphthol, 1,2-naphthoquinone (51.4 +/- 6.6% cell death) and 1,4-naphthoquinone (49.1 +/- 3.4% cell death) were directly toxic to MNL and depleted glutathione to 1.0% of the control levels. Both quinones were also genotoxic to human lymphocytes. In contrast, the primary metabolite of naphthalene, the 1,2-epoxide (0-100 microM) was neither cytotoxic nor genotoxic, and did not deplete glutathione. In conclusion, our data suggests that the cytotoxicity and genotoxicity of naphthalene is associated with the formation of quinones from 1-naphthol rather than naphthalene-1,2-epoxide.
Adult↗
Lack association between schizophrenia and the CYP2D6 gene polymorphisms.
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Adult↗
Relevance of induction of human drug-metabolizing enzymes: pharmacological and toxicological implications.
1. Human drug-metabolizing systems can be induced, or activated, by a large number of exogenous agents including drugs, alcohol, components in the diet and cigarette smoke, as well as by endogenous factors. 2. Such perturbation of enzyme activity undoubtedly contributes to both intra-and inter-individual variation both with respect to the rate and route of metabolism for a particular drug. Induction may, in theory, either attenuate the pharmacological response or exacerbate the toxicity of a particular drug, or both. 3. The clinical impact of enzyme induction will depend upon the number of different enzyme isoforms affected and the magnitude of the inductive response within an individual, and also on the therapeutic indices of the affected substrates. 4. The toxicological implications will be determined either by any change in the route of metabolism, or by a disturbance of the balance between activation and detoxication processes, which may be isozyme selective.
Enzyme Induction↗
The effect of fluconazole and ketoconazole on the metabolism of sulphamethoxazole.
1. Cytochrome P450-mediated bioactivation of sulphamethoxazole to a hydroxylamine has been implicated in the hypersensitivity reactions associated with co-trimoxazole administration. Inhibiting the formation of the hydroxylamine may be one method of preventing the high frequency of toxicity which is observed in HIV-infected patients. Therefore, in this study, we have investigated the ability of fluconazole and ketoconazole, known cytochrome P450 inhibitors, to inhibit the formation of sulphamethoxazole hydroxylamine. 2. Ten healthy male volunteers were given co-trimoxazole (800 mg sulphamethoxazole and 160 mg trimethoprim) alone or 1 h after either fluconazole (150 mg) or ketoconazole (200 mg) in a randomized fashion with a washout period of at least 1 week between each phase. Urine was collected for 24 h, and sulphamethoxazole and its metabolites were quantified by electrospray LC-MS. 3. Ketoconazole had no effect on the urinary recovery of sulphamethoxazole or any of its metabolites. In contrast, fluconazole significantly (P < 0.001) inhibited the formation of sulphamethoxazole hydroxylamine by 50.0 +/- 15.1%. Fluconazole also inhibited the oxidation of sulphamethoxazole to the 5-methylhydroxy and 5-methylhydroxy acetate metabolites by 69.9 +/- 15.8% and 64.0 +/- 12.0%, respectively, but had no effect on the amount of sulphamethoxazole, N4-acetyl sulphamethoxazole, or sulphamethoxazole N1-glucuronide excreted in urine. 4. The potential clinical benefit of using fluconazole to prevent hypersensitivity to co-trimoxazole in patients with AIDS needs to be assessed in a prospective study using both metabolite formation and the clinical occurrence of adverse reactions as end-points.
Adult↗
Intracellular glutathione in the peripheral blood cells of HIV-infected patients: failure to show a deficiency.
OBJECTIVE: To determine whether HIV-infected patients have a deficiency of intracellular glutathione (GSH) in peripheral blood mononuclear cells (PBMC) and erythrocytes. DESIGN: Initial experiments determining the stability of intracellular GSH preceded the measurement of GSH levels in 33 HIV-positive patients and 40 control subjects within 1 h of isolation of their blood cells. In addition, the susceptibility of erythrocytes to dapsone hydroxylamine-induced methaemoglobinaemia was evaluated. METHODS: GSH levels were determined by an high-performance liquid chromatography method utilizing a fluorescent probe, monobromobimane. The bimane-GSH adduct formed in PBMC was also characterized by mass spectrometry. Methaemoglobin formation on exposure to dapsone hydroxylamine was determined spectrophotometrically. RESULTS: GSH levels remained stable for only 1 h after cell isolation, thereafter showing a decrease of 20 and 60% at 4 and 24H, respectively, There was no difference in the GSH levels in PBMC and erythrocytes of the HIV-positive patients compared with controls. The GSH levels were not related to the disease stage or to CD4+ cell counts. There was no difference in GSH levels in PBMC taken from trimethoprim-sulphamethoxazole-hypersensitive and non-hypersensitive patients. Methaemoglobinaemia on exposure of erythrocytes to dapsone hydroxylamine was concentration-dependent, but there was no significant difference between patients and controls. CONCLUSION: In contrast to previous studies, no deficiency of intracellular GSH in the PBMC and erythrocytes of HIV-infected patients was found. The discrepancy between studies may be methodological reflecting the instability of GSH, which requires prompt sample analysis.
Adult↗
Hydralazine-induced lupus: yet another autoantibody!
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Antibodies, Antinuclear↗
Idiosyncratic drug reactions. Metabolic bioactivation as a pathogenic mechanism.
The metabolism of drugs to chemically reactive metabolites may play a pivotal role in the pathogenesis of idiosyncratic drug toxicity. A large number of in vitro studies and a limited number of in vivo studies have demonstrated that many drugs are not toxic per se, but produce toxicity after undergoing enzyme-mediated bioactivation to chemically reactive species. Such reactive species may inflict a toxic insult on the cell either directly or indirectly by acting as a hapten and initiating an immune-mediated reaction. The enzymes responsible for bioactivation have been widely studied, both quantitatively and qualitatively, the most important being the enzymes of the cytochrome P450 (CYP) mixed function oxidase system. CYP enzymes are the most predominant drug metabolising enzymes in the liver and are also present in most other tissues of the body. The diversity of this enzyme system means that a wide range of xenobiotic substrates can be bioactivated by either a single CYP isoform or multiple isoforms of this enzyme superfamily. Other enzymes do, however, play an important role in drug bioactivation. In white blood cells, for example, myeloperoxidase has been shown to bioactivate a wide range of drugs. In other tissues low in CYP activity, prostaglandin H synthase may also be responsible for bioactivation; e.g. in the kidney paracetamol (acetaminophen) toxicity is though to result from activation via this enzyme. The phase II or conjugation enzymes may also be important in the ultimate bioactivation of drug molecules. Whilst activation by these enzymes is, to date, apparently confined to chemicals, most drugs are also substrates for these enzymes and bioactivation by them must remain a possibility.
Aryl Hydrocarbon Hydroxylases↗
Interindividual and interspecies variation in hepatic microsomal epoxide hydrolase activity: studies with cis-stilbene oxide, carbamazepine 10, 11-epoxide and naphthalene.
Microsomal epoxide hydrolase (HYL1) is a single-gene enzyme responsible for the hydrolysis of epoxides derived from the oxidative metabolism of xenobiotics. Variation in HYL1, therefore, may be an important determinant of drug toxicity. We have investigated HYL1 enzyme kinetics in six different species including man, for which a liver bank genotyped for polymorphisms in exons 3 and 4 of the HYL1 gene was used. Activity was measured by radiochromatography with high specific activity radiolabeled substrates, cis-stilbene oxide (CSO) and carbamazepine 10,11-epoxide (CBZ-E). In addition, naphthalene was used to investigate the hydrolysis of an epoxide (naphthalene 1,2-epoxide [N-E] generated in situ. There was marked species variation in enzyme activity that was substrate dependent. CSO was rapidly hydrolyzed by microsomes from all species, the rank order of specific activity being human > rabbit > dog > rat > hamster > mouse. In contrast, hydrolysis of CBZ-E was only observed with human liver microsomes. CBZ-E was only a weak (IC50 = 1 mM) inhibitor of CSO hydrolysis. The hydrolysis of N-E, determined as the diol-to-total metabolite ratio, was human > rabbit > dog > hamster > mouse > rat. Intraspecies variation in man was 4-fold, 7-fold and 2-fold for CSO, CBZ-E and N-E, respectively: none of this variation could be directly accounted for by the HYL1 polymorphisms in exons 3 and 4. These data emphasize the need for careful toxicokinetic evaluation of species used in the safety evaluation of compounds likely to form epoxide intermediates in vivo.
Adult↗
Kinetic parameters of lymphocyte microsomal epoxide hydrolase in carbamazepine hypersensitive patients. Assessment by radiometric HPLC.
Idiosyncratic hypersensitivity reactions with carbamazepine have been postulated to be due to a deficiency of microsomal epoxide hydrolase (HYL1), although this is based on indirect evidence. Using 3H-cis stilbene oxide (0.5 Ci/mmol) as a substrate, we have developed a radiometric HPLC assay sensitive enough to measure the kinetic parameters of HYL1 in lymphocytes. The intra-assay coefficient of variation was 8%. Enzyme activity has been measured in lymphocytes from six carbamazepine hypersensitive patients, six patients on carbamazepine without any adverse effects, and twelve drug-naive healthy volunteers. No significant difference was observed in three kinetic parameters of the enzyme among these three groups. The values for Km, Vmax, and intrinsic clearance ranged from 6.1-89.9 microM, 3.0-23.2 pmoles diol formed/min/mg protein, and 0.147-0.493 microliter/min/mg protein. There was no difference in enzyme activity between patients currently on carbamazepine and healthy volunteers, indicating a lack of induction of lymphocyte HYL1 by carbamazepine. Co-incubation of lymphocytes with 1,1,1-trichloropropene oxide, an inhibitor of hepatic HYL1, resulted in an 82% inhibition of activity, similar to that observed with the hepatic enzyme. The healthy volunteers were genotyped as being either GSTM1 positive (n = 6) or GSTM1 negative (n = 6). This did not affect the kinetic parameters of lymphocyte microsomal epoxide hydrolase. Our results suggest that there is normal HYL1 activity in lymphocytes of hypersensitive patients using cis-stilbene oxide as a substrate.
Adolescent↗
Genetic analysis of microsomal epoxide hydrolase in patients with carbamazepine hypersensitivity.
Carbamazepine therapy is occasionally complicated by hypersensitivity reactions, the mechanism of which is poorly understood. It has been suggested that affected individuals may have a genetically-determined defect of microsomal epoxide hydrolase. The aim of this study was to determine whether a single genetic mutation or pattern of mutations could be used to predict individual susceptibility to carbamazepine-hypersensitivity. DNA was isolated from 10 carbamazepine-hypersensitive patients and 10 healthy volunteers. The patients had developed various forms of toxicity with carbamazepine, including toxic epidermal necrolysis, Stevens-Johnson syndrome, hepatitis and pneumonitis. The technique of polymerase chain reaction single-strand conformation polymorphism analysis (PCR-SSCP) was used to screen for mutations in all nine exons of the microsomal epoxide hydrolase gene. Any new mutations detected by this method were characterised by direct sequencing of the DNA. In addition, in the most severely affected patient, we sequenced all nine exons of the gene. There was a higher frequency of mutations in the hypersensitive group when compared with the controls, but there was no consistent mutation (or pattern of mutations) in the microsomal epoxide hydrolase gene which was common to the hypersensitive group. DNA sequencing of all nine exons of the microsomal epoxide hydrolase gene from the most severely affected patient showed the sequence to be "wild-type," when compared to the previously published sequences. The results of this study suggest that a single mutation within the coding region of the microsomal epoxide hydrolase gene cannot be the sole determinant of the predisposition to carbamazepine hypersensitivity.
Adolescent↗
The role of cytochrome P450 enzymes in hepatic and extrahepatic human drug toxicity.
The human cytochrome P450 enzyme system metabolises a wide array of xenobiotics to pharmacologically inactive metabolites, and occasionally, to toxicologically active metabolites. Impairment of cytochrome P450 activity, which may be either genetic or environmental, may lead to toxicity caused by the parent compound itself. In practise, this usually only applies to drugs that have a narrow therapeutic index and when their clearance is critically dependent upon the fraction normally metabolised by that pathway. P450 enzymes may also convert the drug to a chemically reactive metabolite, which, if not detoxified, may lead to various forms of hepatic and extrahepatic toxicity, including cellular necrosis, hypersensitivity, teratogenicity, and carcinogenicity, depending on the site of formation and the relative stability of the metabolite, and the cellular macromolecule with which it reacts. Variation in the regulation and expression of the drug metabolising enzymes may play a key role in both interindividual variation in sensitivity to drug toxicity and tissue-specific damage. Avoidance of toxicity may be possible in rare instances by prediction of individual susceptibility or by designing new chemical entities that are metabolised by a range of enzymes (both cytochromes P450 and others) and do not undergo bioactivation.
Abnormalities, Drug-Induced↗