A comparative study of the formation of chemically reactive drug metabolites by human liver microsomes.

Abstract

1. The metabolism of amodiaquine (A), ethinyloestradiol (E), mianserin (M), phenytoin (Ph), sulphanilamide (S) and paracetamol (Pa) to both stable and chemically reactive, i.e. irreversibly protein bound, metabolites was investigated using microsomes prepared from histologically normal human liver obtained from eight kidney donors. 2. All drugs, except amodiaquine, were metabolized by NADPH‐dependent microsomal enzymes to chemically reactive metabolites. The degree of NADPH‐dependent binding varied between drugs (E, 11.5 +/‐ 5.8% incubated drug; M, 3.0 +/‐ 1.9%; Ph, 0.10 +/‐ 0.09%; S, 0.57 +/‐ 0.38%; Pa, 1.2 +/‐ 1.2%; mean of eight livers +/‐ s.d.). 3. Inclusion of glutathione (1 mM) or ascorbic acid (1 mM) in the incubation reduced the NADPH‐dependent binding for all substrates, indicating the involvement of electrophilic oxidation products. 4. Binding of M and Pa correlated with each other (Spearman's r = 0.86) and with total cytochrome P‐450 content (r = 0.76 and 0.78 respectively). E binding also correlated with the binding of M (r = 0.79) and Pa (r = 0.81) but not with cytochrome P‐450. Binding of Ph and S did not correlate with any of the other measured metabolic parameters.

An in vitro study of the microsomal metabolism and cellular toxicity of phenytoin, sorbinil and mianserin.

“…However, in these experiments, it was not possible to distinguish the irreversible binding of drug to microsomes from the binding to cells, which alone might have been related to the observed cytotoxicity. While the extent of a drug's irreversible binding to microsomes is a useful index of reactive metabolite formation in vitro, it is not a reliable indication of the drug's toxicity in vivo (Roberts & Jollow, 1979;Kitteringham et al, 1988).…”

“…The potential of a drug to form chemically reactive metabolites in man may be assessed by measuring the extent of its irreversible binding to protein in a human liver microsomal preparation (Kitteringham et al, 1988). Although this is a useful index of reactive metabolite formation in vitro, discrepancies are often observed between such indiscriminate irreversible binding and cytotoxicity (Devalia et al, 1982;Albano et al, 1985).…”

“…In the present study, we have extended this cytotoxicity assay by incorporating human liver microsomes, to create a system more relevant to adverse reactions in man, and by characterisation of the metabolites, both stable and reactive, formed under identical incubation conditions. Two of the drugs investigated, phenytoin and mianserin, have been shown to undergo metabolic activation by human liver microsomes to chemically reactive metabolites (Kitteringham et al, 1988) whilst the third, sorbinil, does not appear to produce a reactive species capable of covalent binding to proteins under identical conditions . Nevertheless, all three compounds (Figure 1) have been reported to cause adverse reactions in man.…”

Activation of Human Dendritic Cells byp-Phenylenediamine

“…14 C]pPD (50 M, 1 Ci) Ϯ glutathione (1 mM) was incubated with the cells (1 ϫ 10 6 ; total volume, 1 ml) in culture medium for 16 h, and irreversible binding was determined by exhaustive solvent extraction (method adapted from Kitteringham et al, 1988;Pirmohamed et al, 1995). Cellular and serum protein were separated by centrifugation and subsequently extracted using 100% acetonitrile (2 ml ϫ one wash and 1 ml ϫ three washes).…”

Metabolism of ciamexon by human liver microsomes: an investigation into the formation of stable, chemically reactive and cytotoxic metabolites.

“…Ciamexon (1(2-methoxy-6-methyl-(3)-pyridinyl)-methyl-2-aziridrine-carbonitrile), 6-hydroxy methyl ciamexon (ciamexon alcohol), 6-methanoyl ciamexon (ciamexon aldehyde), 6-carboxy ciamexon (ciamexon acid) and ["4C] Kitteringham et al (1988). Extracts were assayed for radioactivity to ensure that the removal of reversibly bound material was complete.…”