Hepatic microsomal metabolism of the potential memory-enhancing agent, CL 275,838, to its desbenzyl derivative.

1. The oxidation of the potential memory-enhancer and antidepressant agent CL 275,838 by rat liver microsomes was investigated. CL 275,838 was rapidly and extensively biotransformed in vitro to its desbenzyl derivative (II), the main metabolite observed in vivo. No other known metabolites could be detected in the incubation mixture except for trace amounts of a hydrolysis product (IV). 2. The formation of the desbenzylated derivative II required the presence of an NADPH-generating system and was significantly inhibited by carbon monoxide, SKF 525-A and cimetidine, indicating the participation of P450 in the oxidation of CL 275,838. The reaction was markedly enhanced by phenobarbital and by pregnenolone-16 alpha-carbonitrile [particularly in the female]. beta-Naphthoflavone did not significantly affect desbenzylation. 3. Kinetic studies indicate that there are sex-dependent differences in CL 275,838 metabolism in vitro, as observed in vivo in rat. Maximal velocity for the oxidation of CL 275,838 in microsomes isolated from the male rat was 17 times greater than in the female rat. The apparent Km for metabolism of CL 275,838 was similar in microsomes derived from the male and female rat. 4. CL 275,838 does not appreciably affect its own oxidation and does not cause significant hepatic microsomal enzyme induction in the male or female rat, except for slight enhancement of some components of the P450 system at doses (300 mg/kg once daily for 7 days) well above the effective pharmacological range.

Liquid chromatography of the potential memory-enhancing agent CL 275,838 and its main metabolites, using a post-column photochemical reactor and fluorimetric detection.

On irradiation with short-wavelength ultraviolet light, the potential memory-enhancing compound CL 275,838 (I) and its desbenzyl derivative CL 286,527 (metabolite II) are cleaved into the highly fluorescent derivative CL 228,346 (metabolite IV). This reaction was exploited for the sensitive and selective detection of these compounds in human and animal plasma, after reversed-phase high-performance liquid chromatography on a Supelco LC18 DB column (15 cm x 4.6 mm I.D.) at room temperature. The parent compound and its metabolites were isolated from plasma constituents using the Sep-Pak C18 Plus cartridge, with satisfactory recovery (76-90%) and selectivity. The detection limits were ca. 1.25, 5 and 0.3 ng/ml for I, II and IV, respectively, using 1 ml of plasma. The validation procedure, which includes analysis of multiple ascending calibration curves based on between-day values and replicate analysis of quality control samples analysed with each standard curve, indicated acceptable precision and accuracy of the method within the concentration ranges investigated, the overall coefficient of variation and relative error being less than 10%. The method was successfully applied to plasma samples from healthy volunteers and animals after single of multiple doses of compound I. Metabolites II and IV were detectable in plasma of all species, the former at higher concentrations than the parent compound and metabolite IV. Together with the fact that metabolite II retains much of the parent compound's biological activity in vivo and in vitro, this suggests that it may contribute to the pharmacological effects of compound I.