Application Of Microbial Transformation For Study On N-, O-Dealkylation Of Drugs

The N-, O-dealkylation metabolism of verapamil and naproxen via microbial transformation was studied in this paper, and the similarities and differences between fungi and humans were compared. Several metabolites were purified by semi-prepared HPLC. Fungal microsomes were prepared and their activity was estimated. By means of specific chemical substrates and the corresponding inhibitors of human cytochrome P450 isozymes, the drug metabolic enzymes in the fungi were investigated. Following are the details.1. Microbial transformation of verapamilThe potential of microorganism to metabolize verapamil and the similarities between humans and microbial metabolism were studied. Among 13 microorganism species screened, Cunninghamella blakesleeana AS 3 153 was selected to biotransform verapamil for further study. Based on the data of liquid chromatography-tandem mass spectrometry (LC-MSn), it can be inferred that the major products of verapamil transformed by C. blakesleeana AS 3.153 were mainly via cleavage of C-N or C-O bonds. Ten metabolites were identified, and five of them were purified and characterized by NMR. The influential factor experiments showed that in the condition of pH 6.5, 750 mg-L-1 of substrate concentration and 96 h of incubation, the total yield was 92.6% and the yield of the major metabolite, N-dealkylated verapamil, was >65%. The study showed that there were diverse pathways for C. blakesleeana AS 3.153 to metabolize verapamil, which were similar to humans, so it might be used as a useful model of human N-, O-dealkylated drug metabolism.2. Microbial transformation of naproxenMicrobial transformation of naproxen by C. blakesleeana AS 3.153 was investigated. When the initial pH of medium is 5-6, the concentration is 250 mg-L-1, naproxen was transformed into human phase I metabolite, (9-demethylnaproxen, and the yield was >95%. Furthermore, 50% of the (9-demethylated metabolite was transformed into human phase II metabolite, O-demethylnaproxen-6-0-sulfate. When the resting cell system was used, the incubation time could be decreased within 24 h, and the yield was still >95%. The conjugated metabolite was newly detected in microbial transformation samples. Other microorganisms in our study, such as C. elegans AS 3.156, transformed naproxen almost absolutely but the (9-demethylnaproxen was the only metabolite. It can be referred that there are not only phase I but phase II metabolites of drugs transformed by some fungi. It extended the application of fungus as models of mammalian drug metabolism and illustrated that the microorganism has diversity and specification on drug metabolism.3. Preparation of microsomes of AS 3.153 and estimation of their activity Microsomes of C. blakesleeana AS 3.153 were prepared byultra-centrifugation. The concentration of protein and cytochrom P450 were estimated respectively. Microbial microsomes were incubated, and the result indicated that fungal cytochrom P450 isozyme was involved in the biotransformation of verapamil. And it provided some useful data for further study of the mechanism of microbial transformation and the features of the enzymes in the filamentous fungus on sub cellular levels.4. Preliminary study of metabolism enzymes in C. blakesleeana AS 3.153Based on the results above, further investigations were made about the enzymes involved in N-, (9-dealkylation of drugs in C. blakesleeana AS 3.153 by means of some specific chemical substrates and inhibitors of human CYP450 isozymes. When the concentration is half of the verapamil, ketoconazole, the human specific inhibitor of CYP3A4 could inhibit theN-dealkylation reaction strongly and the rate of inhibition is 65%, but it had no effect on other metabolic pathways, such as O-dealkylation. It may be inferred that there were enzymes similar to human CYP3A4 involved in the W-dealkylation of verapamil in this fungus.Another drug, dextromethorphan, the specific substrate of human CYP2D6 isozyme, could be transformed into O-demethylated metab...