Aldehyde oxidase-catalysed oxidation of methotrexate in the liver of guinea-pig, rabbit and man.

Although 7-hydroxymethotrexate is a major metabolite of methotrexate during high-dose therapy, negligible methotrexate-oxidizing activity has been found in-vitro in the liver in man. The goals of this study were to determine the role of aldehyde oxidase in the metabolism of methotrexate to 7-hydroxymethotrexate in the liver and to study the effects of inhibitors and other substrates on the metabolism of methotrexate. Methotrexate, (+/-)-methotrexate and (-)-methotrexate were incubated with partially purified aldehyde oxidase from the liver of rabbit, guinea-pig and man and the products analysed by HPLC. Rabbit liver aldehyde oxidase was used for purposes of comparison. In-vitro aldehyde oxidase from the liver of man catalyses the oxidation of methotrexate to 7-hydroxymethotrexate, but the turnover is low. However, formation of 7-hydroxy-methotrexate from all forms of methotrexate by the liver in guinea-pig and man was significantly inhibited in the presence of 100 microM menadione and chlorpromazine, potent inhibitors of aldehyde oxidase. Allopurinol (100 microM) had a negligible inhibitory effect on liver aldehyde oxidase from guinea-pig and man. Allopurinol is a xanthine oxidase inhibitor. The production of 7-hydroxymethotrexate was enhanced in the presence of allopurinol. Although aldehyde oxidase is also responsible for some of this conversion, it is also possible that the closely related xanthine oxidase is responsible for the formation of 7-hydroxymethotrexate. By employing potent selective inhibitors of aldehyde oxidase, menadione and chlorpromazine, we have demonstrated for the first time that liver aldehyde oxidase from man is minimally involved in methotrexate oxidation.

Studies on the stability of trans-alpha-acetoxytamoxifen in Sprague-Dawley female rat liver slices, homogenate and subcellular fractions including microsomes and mitochondria.

This work examined the stability of trans alpha-acetoxytamoxifen in Krebs-Henseleit buffer (pH 7.4), in the presence of Sprague-Dawley female rat liver slices, rat liver homogenate and hepatic subcellular fractions, including microsomes and mitochondria, at pH 7.4 and at 37 degrees C over 300 min. The rate of hydrolysis was determined using high-performance liquid chromatography, and degradation profiles were obtained from which the rate and order of degradation were both evaluated. By applying zero-, first-, second- and third-order models of drug disappearance and the generation of by-products, first- and second-order appeared to produce the best fit. trans-alpha-Acetoxytamoxifen degraded rapidly in buffer and more slowly in the biological systems, probably due to the fact that the agent partitions into the hydrophobic component of the biological tissue and hence degrades at a much slower rate. The principal degradation products were trans-alpha-hydroxytamoxifen and, to a lesser extent, cis-alpha-hydroxytamoxifen. Another peak could not be identified. The production of trans-alpha-hydroxytamoxifen was enhanced in the presence of biological enzymes, whereas the concentration of the cis isomer remains relatively constant in buffer only (pH 7.4) and in the presence of biological enzymes. Therefore, the formation of identical adducts with DNA is consistent, because it has been shown that alpha-acetoxytamoxifen breaks down to form alpha-hydroxytamoxifen in vitro. The percentage of trans-alpha-acetoxytamoxifen remaining after 300 min was 40% in mitochondria and 32% in homogenate. The half-life (t1/2) was calculated for each condition by applying zero-, first- and second-order rate kinetics.

Effect of inhibitors on the biotransformation of tamoxifen by female rat and mouse liver slices and homogenates.

The metabolism of tamoxifen was studied in female Sprague-Dawley rat and mouse liver slices and homogenates, and the three principal tamoxifen metabolites, 4-hydroxytamoxifen, N-desmethyl-tamoxifen and tamoxifen N-oxide, were identified by HPLC using authentic standards. It was not possible to identify any of the minor metabolites such as the epoxides using this technique. The N-oxide metabolite only appeared when NADPH was added to the system; this is because the production of tamoxifen N-oxide is primarily mediated by microsomal flavin monooxygenase (FMO) which is NADPH dependent. However, this metabolite did appear in incubations with mouse liver slices only, because they are rich in flavin monooxygenases (FMOs). It did not appear in female rat or mouse liver homogenates, because the NADPH present is destroyed during homogenisation, therefore it was necessary to add NADPH to the system to produce the N-oxide metabolite. The purpose of this study was to investigate the effect of inhibitors on the biotransformation of tamoxifen by female rat and mouse liver slices and homogenates. Female rat liver slices and homogenates were incubated with the following inhibitors (1 mM): cimetidine, ascorbate, sodium azide and reduced glutathione. Cimetidine, a general P-450 inhibitor, inhibited the production of the N-desmethyl metabolite by about 80%; this is in agreement with the action of the other inhibitors. Reduced glutathione, ascorbate and sodium azide are mainly peroxidase inhibitors, so therefore from these novel and interesting results it was possible to suggest that peroxidases play a role in the metabolism of tamoxifen. This observation was also strengthened when the production of the N-desmethyl metabolite increased when horseradish peroxidase was added to the incubate. The production of 4-hydroxytamoxifen was reduced and the N-oxide metabolite was completely inhibited in the presence of peroxidase inhibitors. When rat liver homogenates was incubated with superoxide dismutase (SOD) and catalase, it was observed that the N-desmethyl metabolite disappeared completely at 60 min and the N-oxide and 4-hydroxy metabolites were completely inhibited. However, this phenomenon was only observed when SOD and catalase were preincubated for 30 min with the rat liver homogenate at 37 degrees C; without preincubation the production of these metabolites was unaffected. Finally, the effect of long incubation periods (300 min) on the production of metabolites was examined. It was found that there was a reduction in the concentration of metabolite produced after 60 min which was due to enzyme and co-factor degradation.

How an increase in the carbon chain length of the ester moiety affects the stability of a homologous series of oxprenolol esters in the presence of biological enzymes.

beta-Blockers including timolol and propranolol are administered in eye-drops for the treatment of glaucoma. Due to high incidence of cardiovascular and respiratory side-effects, their therapeutic value is limited. As a result of poor ocular bioavailability, many ocular drugs are applied in high concentrations, which give rise to both ocular and systemic side-effects. Therefore, some methods have been employed to increase ocular bioavailability such as (a) the development of drug delivery devices designed to release drugs at controlled rates, (b) the use of various vehicles that retard precorneal drug loss, and (c) the conversion of drugs to biologically reversible derivatives (prodrugs) with increased corneal penetration properties, from which the active drugs are released by enzymatic hydrolysis. A series of structurally related oxprenolol esters were synthesized and investigated as potential prodrugs for improved ocular use. The stability of each ester was studied in phosphate buffer (pH 7.4), also in the presence of (a) 30% human plasma, (b) aqueous humor, and (c) corneal extract at pH 7. 4 and at 37 degreesC. An account is given of how the stability of a homologous series of oxprenolol esters in the presence of biological enzymes is affected by an increase in the carbon chain length of the ester moiety.

A study of the relationship between the structure and physicochemical parameters of a homologous series of oxprenolol esters at various pH values and temperatures.

A number of beta-adrenergic blockers, including timolol and propranolol, are administered in eyedrops for the treatment of glaucoma, but their therapeutic value is limited by a relatively high incidence of cardiovascular and respiratory side effects. Because of poor ocular bioavailability, many ocular drugs are applied in high concentrations, which give rise to both ocular and systemic side effects. Methods to increase ocular bioavailability include (a) the development of drug delivery devices designed to release drugs at controlled rates, (b) the use of various vehicles that retard precorneal drug loss, and (c) the conversion of drugs to biologically reversible derivatives (prodrugs) with increased corneal penetration properties, from which the active drugs are released by enzymatic hydrolysis. A homologous series of aliphatic esters of oxprenolol were synthesized and investigated as potential prodrugs for ocular use. The stability of each O-acyl derivative was investigated in aqueous solutions over the pH range 2.2-9.0 at 37 degrees C. The observed rate constants (k[obs]), shelf-lives (t90), lipophilicities, and Arrhenius parameters were determined for each ester. A study of the relationship between the structure and physicochemical parameters of the homologous series of oxprenolol esters at various pH values and temperatures was made.