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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 cytosis, fever and rashes, occurs in a small proportion of the population (< 1: 2000) [ 14,15]. Stevens-Johnson syndrome has also been reported in patients on dapsone therapy [ 16]. The haemotoxicity of dapsone is mediated by the hydroxylamine metabolite, which is capable of being co-oxidized with haemoglobin (Hb) in the red blood cell to produce nitroso dapsone ( Figure 1) and methaemoglobin (Met-Hb) [9,10]. The nitroso compound can then be reduced back to the hydroxylamine by the action of glutathione, so that a futile oxidationreduction cycle exists in which the cell uses oxygen to deplete glutathione and NADPH [17]. Dapsoneinduced toxicity to white cells is poorly understood, but is also thought to be a consequence of hydroxylamine UDPGA Human liver samplesSamples were from histologically normal livers which had been removed and transferred to the laboratory within 30 min of death. The liver was sliced into 10-20 g portions, placed in vials and frozen in liquid nitrogen. These samples were stored at -80°C until the preparation of microsomes. Ethical approval was granted and consent was obtained from the donors' relatives before removal of the liver. Preparation ofmicrosomesThe frozen liver was thawed and minced in ice cold 0.067 M phosphate buffer (pH 7.5) containing 1.15% KCI (w/v). The livers were then homogenized using a motor driven Polytron homogenizer. The homogenates were centrifuged at 10 000 g for 20 min at 40 C to remove mitochondria, nuclei and cell debris. The supernatants were decanted off and centrifuged at 105 000 g for 60 min at 4°C to produce the microsomal pellet. lated using a Spectra-Physics Chromjet Integrator. An internal standard was not necessary due to a good correlation between radiometric and u.v. analysis of hydroxylamine formation (r=0.988). LC-MS analysis was performed using a j-Bondapak 10 C18 column (30 cm x 3.9 mm). Samples were eluted with a mobile phase consisting of ammonium formate (6 mm, pH 3.5) and acetonitrile (80:20 v/v) at a flow rate of 1 ml min-'. The mobile phase was delivered by two Jasco PU980 pumps (Jasco Corporation, Tokyo, Japan hydroxylamine (inhibition= 23.3 % at 5 giM ketoconazole, and 48.7% at 100 gIM sulphaphenazole), indicating a role for CYP3A and CYP2C9.In order to investigate inhibition further, the experiments with ketoconazole and sulphaphenazole wer...

1 The cytotoxicity of metabolites generated from phenytoin, sorbinil and mianserin by human and mouse liver microsomes was assessed by co-incubation with human mononuclear leucocytes as target cells. Cytotoxicity was determined by trypan blue dye exclusion. 2 Phenytoin and sorbinil were metabolised by NADPH-dependent murine microsomal enzymes to cytotoxic metabolites. Cytotoxicity produced by both drugs was significantly enhanced by the epoxide hydrolase inhibitor trichloropropane oxide (TCPO). No significant cytotoxicity was observed in the presence of human liver microsomes. 3 Mianserin was metabolised by both human and mouse liver microsomes to a cytotoxin. Cytotoxicity was greater in the presence of human liver microsomes (13.7 ± 2.2%; mean + s.d. for four livers, compared with 6.0 ± 2.4%, mean ± s.d., n = 4, with mouse liver microsomes), and was unaffected by pretreatment with TCPO. 4 Stable metabolites were quantified by radiometric high performance liquid chromatography. Phenytoin and sorbinil were metabolised to 5-(p-hydroxyphenyl)-5-phenylhydantoin (0.3-0.5% of incubated radioactivity) and 2-hydroxysorbinil (0.4-2.7% of incubated radioactivity), respectively, by both human and mouse liver microsomes. 5 Mianserin was metabolised to 8-hydroxymianserin and desmethylmianserin by both human and mouse liver microsomes. Desmethylmianserin was the major product in incubations with human liver microsomes (32.3 ± 12%, mean ± s.d. for four livers), whereas 8-hydroxymianserin was the predominant metabolite generated by mouse liver microsomes (25.9 ± 1.5%, mean ± s.d., n = 4). 6 Generation of electrophilic metabolites was assessed by determination of the amount of radiolabelled material which became irreversibly bound to protein. Only mouse liver microsomes activated phenytoin to a chemically reactive metabolite, whereas both mouse and human liver microsomes generated reactive metabolites from sorbinil and mianserin. 7 These studies show that drug cytotoxicity can be mediated by low concentrations (circa F.M) of metabolites generated by NADPH-dependent hepatic microsomal enzymes; however demonstration of cytotoxicity in vitro has not been established as a means of predicting in vivo toxicity.

1. We have attempted to reduce dapsone‐dependent methaemoglobinaemia formation in six dermatitis herpetiformis patients stabilised on dapsone by the co‐administration of cimetidine. 2. In comparison with control, i.e. dapsone alone, methaemoglobinaemia due to dapsone fell by 27.3 +/‐ 6.7% and 26.6 +/‐ 5.6% the first and second weeks after commencement of cimetidine administration. The normally cyanotic appearance of the patient on the highest dose of dapsone (350 mg day‐ 1), underwent marked improvement. 3. There was a significant increase in the trough plasma concentration of dapsone (2.8 +/‐ 0.8 x 10(‐5)% dose ml‐1) at day 21 in the presence of cimetidine compared with control (day 7, 1.9 +/‐ 0.6 x 10(‐5)% dose ml‐1, P less than 0.01). During the period of the study, dapsone‐mediated control of the dermatitis herpetiformis in all six patients was unchanged. 4. Trough plasma concentrations of monoacetyl dapsone were significantly increased (P less than 0.05) at day 21 (1.9 +/‐ 1.0 x 10(‐5)% dose ml‐ 1) compared with day 7 (1.6 +/‐ 0.9 x 10(‐5)% dose ml‐1:control). 5. Over a 12 h period, 20.6 +/‐ 8.9% (day 0) of a dose of dapsone was detectable in urine as dapsone hydroxylamine. Significantly less dapsone hydroxylamine was recovered from urine at day 14 (15.0 +/‐ 8.4) in the presence of cimetidine, compared with day 0 (control: P less than 0.05). 6. The co‐administration of cimetidine may be of value in increasing patient tolerance to dapsone, a widely used, effective, but comparatively toxic drug.