Pharmacokinetics and metabolism of zamifenacin in mouse, rat, dog and man.

1. Zamifenacin was rapidly metabolized in vitro by liver microsomes from rat, dog, and man. 2. Zamifenacin exhibited extensive plasma protein binding with human plasma showing 20 and 10-fold higher binding that that in rat and dog respectively. 3. Following oral administration to animals, metabolic clearance resulted in decreased bioavailability due to first-pass metabolism in rat and mouse. Oral clearance in man was low as a result of increased metabolic stability and increased plasma protein binding compared with animals. 4. Metabolism was the major route of clearance of zamifenacin with the primary metabolic step resulting in opening of the methylenedioxy ring to yield the catechol. In man, this metabolite was excreted as the glucuronide conjugate, whereas in the animal species it was further metabolized by mono-methylation of the catechol.

Evaluation of criteria for the acceptance of bioanalytical data.

Results from bioanalytical analyses for registration of a new drug entity are used to define its pharmacokinetics and bioavailability/bioequivalence. Whilst analytical data may be derived from the application of a validated method, it is essential to apply mathematical criteria to its acceptance, in order that the analyst can be assured that the assay is performing within defined limits and to its validated specification. Parameters evaluated for acceptability are the batch calibration curve, the minimum quantifiable concentration and the quality control (QC) sample acceptability. Specifically, six QC samples per analytical batch are used, two samples at each of three concentrations. The rationale for the definition of these criteria is evaluated together with a consideration of their applications and limitations. The relevance and use of Shewhart and Cusum plots to monitor assay performance is illustrated.

Altered acyl-CoA metabolism in riboflavin deficiency.

We have recently described the effects of riboflavin deficiency on the metabolism of dicarboxylic acids (Draye et al. (1988) Eur. J. Biochem. 178, 183-189). As both mitochondria and peroxisomes are thought to be involved, we have examined the activities of various enzymes in these organelles in the livers of riboflavin-deficient rats. Mitochondrial beta-oxidation of fatty acids was severely depressed due to loss of activity of the three fatty acyl-CoA dehydrogenases, whereas there was an enhancement of peroxisomal beta-oxidation due to an increased activity of the FAD-dependent fatty acyl-CoA oxidase, although the activities of other peroxisomal flavoproteins, D-amino acid oxidase and glycolate oxidase, were lowered. Hepatocyte morphometry revealed an increase in the numbers of peroxisomes, indicating a proliferation induced by the deficiency. The mitochondrial acyl-CoA dehydrogenases involved in branched-chain amino acid metabolism were also severely decreased leading to characteristic organic acidurias. There was some loss of activity of the flavin-dependent sections of the electron transport chain (complexes I and II), but these were probably not sufficient to affect normal function in vivo. The specificity of these effects allows the use of the riboflavin-deficient rat as a model for the study of dicarboxylate metabolism.

A study of the metabolism of [U-14C]3-methyl-2-oxopentanoate by rat liver mitochondria using h.p.l.c. with continuous on-line monitoring of radioactive intact acyl-coenzyme A intermediates.

A reverse-phase h.p.l.c. system for the resolution of the acyl-CoA intermediates of the degradation of 3-methyl-2-oxopentanoate is described. The validation of a method for the measurement of radioactive intermediates produced by the incubation of [U-14C]3-methyl-2-oxopentanoate with rat liver mitochondrial fractions is described. The absence of bicarbonate caused the accumulation of [14C]propionyl-CoA. The accumulation of [14C]2-methylbutyryl-CoA was observed in incubations with mitochondrial fractions derived from riboflavin-deficient animals. Studies of the accumulation of labelled intermediates with time suggest that there is regulation of the pathway of isoleucine degradation at methylmalonyl-CoA mutase, as suggested for valine [Corkey, Martin-Requero, Walajtys-Rode, Williams & Williamson (1982) J. Biol. Chem. 257, 9668-9676]. These studies demonstrate that h.p.l.c. with on-line continuous radiochemical detection is a powerful method for the investigation of the control of intermediary metabolism.