The formation and stability of 1-beta-glucuronide conjugate of the main metabolite of ipriflavone [7-(1-carboxy-ethoxy)-isoflavone] (CI)--were studied by using liver microsomes, hepatocytes, and isolated perfused liver of untreated and 3-methylcholanthrene (MC) treated dog and rat, and human liver microsomes. MC treatment enhanced the rate of conjugation twice as much as that of the control in the microsomes of both dogs and rats. Conjugation of CI by microsomes results in two metabolites, both sensitive to pH and temperature. Other two glucuronide forms appeared in experiments with hepatocytes and perfused liver. Mass spectrometry supported. The conclusion, assumption that both metabolites produced by microsomes are glucuronide conjugate isoforms of CI, and that they could be distinguished according to the intensity of peaks on FAB-MIKE spectra. The beta-glucuronidase enzyme hydrolysed only the 1-beta-glucuronide isomer, the other, migrated form remained unchanged. D-saccharic-acid-1,4-lactone, a specific inhibitor of beta-glucuronidase enzyme, decreased the rate of enzymatic cleavage. Standard curves of CI were prepared by HPLC, and 1-beta-CI-glucuronide was quantified according to the amount of CI formed by hydrolysis. The stability of conjugates greatly depends on pH and temperature, and the rate of degradation and isomerization is sensitive to the value of both. Lowering the pH from 7.4 to 5.0 and the temperature from 37 degrees C to 18 degrees C increased the stability of glucuronides. Increasing the pH to 12.0 results in very rapid acyl migration and hydrolysis.

1 Reduction of theophylline metabolism and elimination were observed in a theophylline-treated patient during ipri¯avone administration. After withdrawal of ipri¯avone, the serum theophylline level decreased to an extent similar to that found before administration of ipri¯avone. The e ects of ipri¯avone and its major metabolites 7-hydroxy-iso¯avone and 7-(1-carboxy-ethoxy)-iso¯avone on cytochrome P450 activities were studied in vitro in human liver microsomes from three donors. 2 Ipri¯avone and 7-hydroxy-iso¯avone competitively inhibited phenacetin O-deethylase and tolbutamide hydroxylase activity. The parent compound and its dealkylated metabolite were strong inhibitors exhibiting K i values around 10 ± 20 mM, while 7-(1-carboxy-ethoxy)-iso¯avone had no e ect on the cytochrome P450 activities investigated. 7-Hydroxy-iso¯avone is the only one that in¯uenced nifedipine oxidase activity. It competitively inhibited this activity with a K i value of 129.5 mM. 3 The steady state concentrations of ipri¯avone and 7-hydroxy-iso¯avone in plasma of patients receiving 36200 mg daily doses of ipri¯avone for 48 weeks were found to be 0.33+0.32 mM and 1.44+0.77 mM, respectively. 4 The results indicate that the decrease in theophylline metabolism observed in a patient treated with ipri¯avone may be due to a competitive interaction of ipri¯avone or its metabolite, 7-hydroxy-iso¯avone with CYP1A2. On the other hand, our in vitro ®ndings predict some more interaction with CYP2C9.

It is well established in the litrature, that selegiline is metabolised to its N-dealkylated metabolites, N-desmethylselegiline, methamphetamine and amphetamine. However, most studies on selegiline metabolism did not characterize the species differences in the formation of the metabolites. Therefore, in this study, we investigated the in vitro metabolism of selegiline in liver microsomes of different species. In addition, to the previously well-characterized metabolites, selegiline-N-oxide (selegiline-NO) was found to be formed as a metabolite of selegiline in rat liver microsomal preparation. The results of experiments with liver microsomes from other species indicated species differences in the rate and extent of formation of selegiline-NO. The dog and hamster liver microsomal preparations were the most active in terms of selegiline-NO production, whereas little selegiline was metabolized to its N-oxide in human liver microsomes. When selegiline-NO was incubated with rat liver microsomes, no metabolism occurred. When a short incubation time was applied in selegiline expriments no increase in the amount of selegiline-NO was detected. Accordingly, it was clear that selegiline was not metabolized to the N-dealkylated or N,N-bis-dealkylated compounds via selegiline-NO. Studies with different isoenzyme inhibitors indicated that the formation of selegiline-NO might be catalyzed at least partly by cytochrome P450 (CYP) 2D6 and CYP3A4. With the exception of hamster microsomes in the microsomal preparations in vitro, the formation of the R,S-stereoisomer of selegiline-NO was preferred.

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