Donglu Zhang

Formation of mammalian metabolites of cyclobenzaprine by the fungus, Cunninghamella elegans.

The fungus, Cunninghamella elegans, was used as a microbial model of mammalian drug metabolism to biotransform a tricyclic antidepressant, cyclobenzaprine. Seventy-five percent of this drug at a concentration of 1 mM was metabolized within 72 h by C. elegans grown on Sabouraud dextrose broth. Milligram amounts of fungal metabolites were isolated by reversed-phase high performance liquid chromatography (HPLC) and their structures were characterized by 1H NMR spectroscopy, mass spectrometry, and UV spectroscopy analyses. The major fungal metabolites of cyclobenzaprine were 2-hydroxycyclobenzaprine (59%), N-desmethylcyclobenzaprine (21%), cyclobenzaprine trans-10,11-dihydrodiol (5%), N-desmethyl-2-hydroxy-cyclobenzaprine (3%), 3-hydroxycyclobenzaprine (3%), and cyclobenzaprine N-oxide (1%). These fungal metabolites were used as standards to investigate the metabolism of cyclobenzaprine by rat liver microsomes. Rat liver microsomes also biotransformed cyclobenzaprine to produce similar metabolites as the fungus. The isotope labeling of 2-hydroxycyclobenzaprine by 18O2 and the trans-configuration of the dihydrodiol suggested that these reactions were catalyzed by cytochrome P-450 monooxygenases in C. elegans. These results also demonstrated that the fungal biotransformation system could be used to predict and synthesize the mammalian drug metabolites.

Transformation of Amoxapine by Cunninghamella elegans

ABSTRACT We examined Cunninghamella elegans to determine its ability to transform amoxapine, a tricyclic antidepressant belonging to the dibenzoxazepine class of drugs. Approximately 57% of the exogenous amoxapine was metabolized to three metabolites that were isolated by high-performance liquid chromatography and were identified by nuclear magnetic resonance and mass spectrometry as 7-hydroxyamoxapine (48%),N-formyl-7-hydroxyamoxapine (31%), andN-formylamoxapine (21%). 7-Hydroxyamoxapine, a mammalian metabolite with biological activity, now can be produced in milligram quantities for toxicological evaluation.

Fungal transformations of antihistamines : metabolism of cyproheptadine hydrochloride by Cunninghamella elegans

1. Metabolites formed during incubation of the antihistamine cyproheptadine hydrochloride with the zygomycete fungus Cunninghamella elegans in liquid culture were determined. The metabolites were isolated by hple and identified by mass spectrometric and proton nmr spectroscopic analysis. Two C elegans strains, ATCC 9245 and ATCC 36112, were screened and both produced essentially identical metabolites. 2. Within 72 h cyproheptadine was extensively biotransformed to at least eight oxidative phase-I metabolites primarily via aromatic hydroxylation metabolic pathways. Cyproheptadine was biotransformed predominantly to 2-hydroxycyproheptadine. Other metabolites identified were 1- and 3-hydroxycyproheptadine, cyproheptadine 10,11-epoxide, N-desmethylcyproheptadine, N-desmethyl-2-hydroxycyproheptadine, cyproheptadine N-oxide, and 2-hydroxycyproheptadine N-oxide. Although a minor fungal metabolite, cyproheptadine 10,11-epoxide represents the first stable epoxide isolated from the microbial biotransformation of drugs. 3. The enzymatic mechanism for the formation of the major fungal metabolite, 2-hydroxycyproheptadine, was investigated. The oxygen atom was derived from molecular oxygen as determined from 18O-labelling experiments. The formation of 2-hydroxycyproheptadine was inhibited 35, 70 and 97% by cytochrome P450 inhibitors metyrapone, proadifen and 1-aminobenzotriazole respectively. Cytochrome P450 was detected in the microsomal fractions of C. elegans. In addition, 2-hydroxylase activity was found in cell-free extracts of C. elegans. This activity was inhibited by proadifen and CO, and was inducible by naphthalene. These results are consistent with the fungal epoxidation and hydroxylation reactions being catalysed by cytochrome P450 monooxygenases. 4. The effects of types of media on the biotransformation of cyproheptadine were investigated. It appears that the glucose level significantly affects the biotransformation rates of cyproheptadine; however it did not change the relative ratios between metabolites produced.

Biotransformation of protriptyline by filamentous fungi and yeasts

1. The potential of various fungi to metabolize protriptyline (an extensively used antidepressant) was studied to investigate similarities between mammalian and microbial metabolism. 2. Metabolites produced by each organism were isolated by high-pressure liquid chromatography and identified by nuclear magnetic resonance and mass spectrometry. The metabolites identified in one or more fungi were 2-hydroxyprotriptyline, N-desmethylprotriptyline, N-acetylprotriptyline, N-acetoxyprotriptyline, 14-oxo-N-desmethylprotriptyline, 2-hydroxy-acetoxyprotriptyline and 3-(5-hydrodibenzo[bf][7]annulen-5-yl)propanoic acid. 3. Among 27 filamentous fungi and yeast species screened, Fusarium oxysporum f. sp. pini 2380 metabolized 97% of the protriptyline added. Several other fungi screened gave significant metabolism of protriptyline, including Cunninghamella echinulata ATCC 42616 (67%), C. elegans ATCC 9245 (17%), C. elegans ATCC 36112 (22%), C. phaeospora ATCC 22110 (50%), F. moniliforme MRC-826 (33%) and F. solani 3179 (12%). 4. F. oxysporum f. sp. pini produced phase I and phase II metabolites and thus is a suitable microbial model for protriptyline metabolism.

Oxidation of phenothiazine and phenoxazine by Cunninghamella elegans

1. To determine the ability of fungi to metabolize sulphur- and oxygen-containing azaarenes, Cunninghamella elegans ATCC 9245 was grown in 125-ml flasks containing fluid Sabouraud medium. The cultures and controls were incubated at 28°C with shaking and dosed with 16.7 mM phenothiazine or phenoxazine. After incubation for 72h, the mycelia and filtrates were extracted with ethyl acetate and the combined residues analysed by high-performance liquid chromatography. Residual phenothiazine and phenoxazine were 21 and 22%, respectively, of the total UV absorbance at 254u2009nm. 2. The metabolites were identified by mass spectrometry and proton nuclear magnetic resonance spectroscopy. The fungus oxidized phenothiazine to phenothiazine sulphoxide, 3-hydroxyphenothiazine sulphoxide, phenothiazin-3-one, and 3-hydroxyphenothiazine and oxidized phenoxazine to phenoxazin-3-one. 3. Three of the four compounds produced by C. elegans from phenothiazine were identical to those produced by mammals, supporting the use of the fungus as a microbial model for drug metabolism.