David J. Rance

Specificity of human UDP-glucuronosyltransferases and xenobiotic glucuronidation.

Several human liver UDP-Glucuronosyltransferases (UGTs) have been cloned and the cDNAs expressed in heterologous cell lines. This technological advance has allowed the assessment of the functional substrate specificity of these UGTs. The problems which may be encountered with the latency and assay of UGTs are briefly described. The data accumulated to date indicate that the Km, and possibly the Vmax/Km, for individual substrates are the best parameters to assess the specificity of the enzymes towards xenobiotic molecules. The substrate specificity of seven UGTs has been summarised from the currently available information. Of these, UGT1*02 and UGT2B8 appear to be key isoforms in the glucuronidation of a wide range of xenobiotic substrates. Additional UGTs have yet to be identified and characterised and their future inclusion may provide further insights. Finally, the functional role of each UGT in vivo has to be determined.

Species variation in hepatic aldehyde oxidase activity

SummaryThe activity of hepatic aldehyde oxidase from rabbit, guinea pig, rat, marmoset, dog, baboon and man was investigated in vitro with charged and uncharged N-heterocyclic substrates: Km and Vmax values were determined for phthalazine, 6,7-dimethoxy-l-[-4-(ethylcarbamoyloxy)piperidino]phthalazine (carbazeran), quinine and quinidine. The oxidation of N-phenylquinolinium chloride to N-phenyl-2-quinolone and N-phenyl-4-quinolone was followed spectrophotometrically. Rat or dog liver showed low and negligible enzyme activity respectively, whereas baboon liver contained a highly active aldehyde oxidase. Enzyme from marmoset and guinea pig liver had the closest spectrum of activity to human liver aldehyde oxidase. Unlike that from man, rabbit hepatic aldehyde oxidase was refractory towards carbazeran and converted N-phenylquinolinium chloride predominantly to the 2-quinolone. N-Phenyl-4-quinolone was the major oxidation product with enzyme from guinea pig, marmoset, baboon and man.

1-Substituted phthalazines as probes of the substrate-binding site of mammalian molybdenum hydroxylases

The interaction of a series of 1-substituted phthalazine derivatives with partially purified aldehyde oxidase from rabbit, guinea-pig and baboon liver, and with bovine milk xanthine oxidase, has been investigated. Of the 18 compounds examined, rabbit liver aldehyde oxidase metabolized 10, whereas guinea-pig and baboon liver enzyme oxidized 13 and 14, respectively. Where metabolites were characterized, oxidation was shown to occur at position four of the phthalazine ring. Km values ranged from 0.003 to 1.8 mM. In contrast, most compounds were competitive inhibitors of bovine milk xanthine oxidase with Ki values ranging from 0.015 to 1.3 mM; the cationic derivative 2-methylphthalazinium iodide was oxidized to 2-methyl-1-phthalazinone by both aldehyde oxidase and, with a much reduced affinity, by xanthine oxidase. In terms of structure-metabolism relationships, Vmax values were relatively insensitive to the electronic effects of substituents, but a trend for the more lipophilic derivatives to show increased affinities (Km and Vmax/Km) towards aldehyde oxidase could be seen. However, calculations of molecular size revealed a species-dependent cut-off threshold above which compounds were not metabolized. Results suggest that the relative size of the active site for hepatic aldehyde oxidase is in the order baboon greater than guinea-pig greater than rabbit, and that in spatial terms the active site of bovine milk xanthine oxidase is similar to that of baboon liver aldehyde oxidase. Thus, the binding site of rabbit liver aldehyde oxidase, a widely used source of the oxidase, is apparently more restricted than in some other species.

Pharmacokinetics, Pharmacodynamics, and Safety of the 5‐HT1B/1D Agonist Eletriptan following Intravenous and Oral Administration

Four separate studies were conducted to examine the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of eletriptan, a 5‐HT1B/1D receptor agonist being developed for the treatment of migraines, after oral and intravenous administration. Fifty‐five males received oral (1.5–30 mg or 30–120 mg) or intravenous (1.67‐50 μg/kg or 50–102 μg/kg) eletriptan in four double‐ and single‐blind, placebo‐controlled, ascending‐dose crossover studies. The maximum plasma concentration (Cmax) and area under the concentration curve (AUC) appeared linear over all dose ranges, with an apparent terminal half‐life of 4 to 5 hours. Clearance and volume of distribution remained constant with dose. The time to first occurrence of Cmax (tmax) for oral eletriptan was approximately 1 hour and was unaffected by dose. Comparison of AUC values suggested an absolute bioavailability of approximately 50%. A linear PK/PD model, fitted to the data, predicted small, transient elevations in diastolic blood pressure following eletriptan doses ± 60 mg. These effects were considered unlikely to be clinically significant. Eletriptan was well tolerated, and treatment‐related adverse events were mild to moderate and transient. These PK properties should result in eletriptan having a rapid onset and sustained duration of action in terms of migraine efficacy.

Tissue distribution of the molybdenum hydroxylases, aldehyde oxidase and xanthine oxidase, in male and female guinea pigs

SummaryThe activity of the molybdenum hydroxylase, aldehyde oxidase, was determined in crude homogenates and (NH4)2S04 fractions prepared from guinea pig liver, lung, kidney, intestine, spleen and heart. Xanthine oxidase was also measured in (NH4)2S04 fractions. In each case, xanthine oxidase levels were lower than those of aldehyde oxidase; activity of the latter enzyme was highest in the liver, whereas xanthine oxidase was predominant in the small intestine. There was no significant difference in the activity of either molybdenum hydroxylase between tissues taken from male and female guinea pigs.

Subcellular localisation of guinea pig hepatic molybdenum hydroxylases

Molybdenum hydroxylase activity in guinea pig liver has been compared with that of marker enzymes in mitochondria (succinate dehydrogenase), microsomes (glucose-6-phosphatase) and cytosol (lactate dehydrogenase). Aldehyde oxidase activity was highest in the cytosol, with about 10-fold activity of xanthine oxidase. Significant molybdenum hydroxylase activity was found in mitochondria with minimal levels in microsomes. Mitochondrial and cytosolic aldehyde oxidase varied in substrate specificity and electrophoretic mobility with two major bands in each fraction, one of which was common to cytosol and mitochondria.