Fungal biotransformation of ezetimibe

Structural transformation of ezetimibe was performed by fungi Beauvaria bassiana and Cunninghamella blakesleeana. The metabolites were identified by different spectroscopic techniques as (3R,4S)-1-(4-fluorophenyl)-3-((E)-3-(4-fluorophenyl) allyl)-4-(4-hydroxyphenyl) azetidin-2-one (2), (3R, 4S)-1-(4-fluorophenyl)-3-(3-(4fluorophenyl)-3-oxopropyl)-4-(4-hydroxyphenyl) azetidin-2-one (3), (3R,4S) 1-(4-fluorophenyl)-3-(3-(4-fluorophenyl) propyl)-4-(4-hydroxyphenyl) azetidin-2-one (4) and (2R,5S)-N, 5-bis (4-fluorophenyl)-5-hydroxy-2-(4-hydroxybenzyl) pentanamide (5). This study displays two important features of these fungi, viz., their ability to metabolize halogenated compounds, and their capacity to metabolize drugs that are targets of the UDP-Glucuronyl Transferase System, a phenomenon not commonly observed.


Introduction
Ezetimibe is a first prototype drug that represents the latest class of antihypercholesterolemic and lipid modifying agents exerting its effects via blocking cholesterol absorption. [1] It is believed to inhibit lipid absorption by preventing dietary and billiary cholesterol to move across the intestinal wall without influencing the absorption of fat soluble vitamins, triglycerides and bile acids. [2À4] Ezetimibe localizes in the brush border of the small intestinal enterocytes and decreases the uptake of cholesterol into the enterocytes. This has the net effect of inhibiting cholesterol absorption from the intestinal lumen facilitating its excretion. [5] Pre-clinical studies established that ezetimibe was glucuronidated to a single metabolite localized at the intestinal wall, where it prevented cholesterol absorption. [6] It was found that enterohepatic recirculation of ezetimibe and/or its glucuronide ascertained repeated delivery to the site of action and restrained peripheral exposure. [7] Ezetimibe has no effect on the action of major drug metabolizing enzymes (CYP450), which reduces the possible drugÀdrug interactions. [5] Ezetimibe and its active metabolite are highly bound to human plasma proteins (90%). Ezetimibe is primarily metabolized in the liver and in the small intestine via glucuronide conjugation with subsequent renal and biliary excretion. [6] Both the parent compound and its active metabolite are eliminated from plasma with a half-life of approximately 22 h allowing for once daily dosing. Ezetimibe is devoid of substantial inhibitory or inductive effects on cytochrome P-450 isoenzymes which explains its limited number of drug interactions. [7,8] The main objective of this study was to find a microbial metabolic system that mimics mammalian metabolism closely regarding halogenated drugs to aid the preclinical studies and exploring the enzymatic potential of microbes for drugs excreted via glucuronidation, thus, revealing the presence of a UDP-Glucuronyl transferase system, as well as its proposed activity in microbial biotransformation of pharmacologically active compounds. In addition, discovery of novel metabolites that are pharmacologically superior and less toxic in comparison to their predecessors [9] was also identified as an aim of the study.

Materials and methods Instrumentation
Electron impact mass spectrometry (EI-MS) was performed on Jeol JMS-600 H mass spectrometer. The 1 H-NMR, 13  Bruker Avance-500, 400, 300 and 100 MHz instruments. Chemical shift (d) in ppm and coupling constants (J) are in Hz, related to Tetramethylsilane (TMS) which was used as an internal standard. Ultraviolet (UV) experiments were recorded in spectroscopic grade methanol on a Shimadzu UV 240 (Shimadzu CorporationTokyo, Japan). Infrared (IR) spectra were measured in chloroform, as KBr disc on Shimadzu FT IR-8900 spectrophotometer or JASCO A-302 (JASCO International Co. Ltd., Japan).

Microbial cultures
The microbial cultures were purchased from American Type Culture Collection (ATCC), USA; Northern Regional Research Laboratories (NRRL) USA and Institute of Fermentation, Osaka, Japan (IFO). These microbial cultures were maintained on Sabouraud Dextrose Agar media (SDA) slants and incubated at 4 C before use.

General protocol for media preparation
Two-day-old spores of each fungus were aseptically transferred into broth medium flasks containing 100 mL of freshly prepared autoclaved medium. The seed flasks thus obtained were incubated on a shaker at 30 C for two days. These broth cultures were inoculated aseptically into 60 media flasks of 250 mL each containing 100 mL of medium, and fermentation was continued for further 24 h. In brief, 2000 mg ezetimibe designated as compound 1 was diluted with 60 mL methanol, the resulting solution was evenly distributed among 60 conical flasks having shake cultures and the fermentation was continued for 12 days for Beauvaria bassiana and nine days for Cunninghamella blakesleeana. Fifteen litres of ethyl acetate was used to wash, filter and extract the mycelia. The extracts were dried over anhydrous sodium sulphate and concentrated in vacuo to afford gums that were adsorbed on equal quantities of silica gel and eluted with various solvent gradients of petroleum ether, ethyl acetate and methanol.

Results and discussion
Ezetimibe (compound 1) (see Table 1) was fermented in broth cultures of B. bassiana and C. blakesleeana for 12 and nine days, respectively. Ethyl acetate was used to extract the parent compound and the metabolites from mycelia and the components were separated using silica gel columns. The fermentation resulted in the production of compounds 2 and 3 (see Tables 2 and 3) in 15.8% and 6.5% yields from B. bassiana, and compounds 4 and 5 (see Tables 4 and 5) in 5.2% yield from C. blakesleeana, respectively. Compound 2 was obtained from sub-column via further elution through addition of 42% ethyl acetate/       (Figures 1 and 2) were elucidated on the basis of their spectra of MS, 1 H and 13   This compound is UV active. The UV analysis showed absorbance at 236 nm for a ketonic group. The IR spectrum of the compound showed sharp absorption for a ketone at 1707 cm ¡1 with the disappearance of hydroxy absorption peak (3310 cm ¡1 ). The 1 H-NMR spectrum pointed out the disappearance of the ÀOH signals. The 13 C-NMR spectra (BB, DEPT-90 and DEPT-135) displayed the resonances for 4 methylene, 13 methine and 7 quarternary carbon atoms. The mass spectrum showed that the mass of the compound was 407.4081 g/mol which were 1 amu less than the substrate showing loss of one hydrogen atom. Henceforth, the metabolite was character- Compound (5) [(2R,5S)-N, 5-bis (4-fluorophenyl)-5-hydroxy-2-(4-hydroxybenzyl) pentanamide] had the molecular formula of C 24 H 23 F 2 NO 3 . EI-MS experiments indicated an M C peak at 411.1646 (calcd 412.1680). The molecular weight was estimated to be 411.4411. This compound is UV active. The mass spectrum showed that the mass of the compound of 411.4411 was approximately 1 amu higher than the substrate showing addition of one hydrogen atom. Henceforth, the metabolite was characterized as 1-(4-fluorophenyl)-3-(3-(4-fluorophenyl) propyl)-4-(4-hydroxyphenyl) azetidin-2-one (3). The 1 H NMR spectral data of metabolite 5 disclosed additional signals at 2.81 and 9.75 ppm. The signal at 9.75 ppm, which is not observed with ezetimibe, conforms to an exchangeable proton and is spatially close to protons at 6 and 6 0 positions, consequently it is assigned as ÀNH group. This could mean that the metabolite 5 has an extra ÀCH 2 group at 38.1 ppm as compared to that of ezetimibe, 1HÀ1H correlation revealed that the C4 is now ÀCH 2 and linked to two protons at 2.81 and 2.94 ppm and no more CH as it is in ezetimibe. The 13 C-NMR spectra (BB, DEPT-90 and DEPT-135) confirmed the presence of 14 methine, three methylene and seven quaternary carbon atoms. EI-MS m/z: 411.1646 (calcd 412.1680).IR (CHCl 3 ) n max cm ¡1 : 3466 (OH), 1707 (CDO).

Conclusion
Microbial biotransformation of ezetimibe resulted in four known compounds illustrating the enzymatic capacities of B. bassiana and C. blakesleeana. It also shows the ability of these strains to metabolize synthetic compounds in addition to their ability to metabolize natural compounds. [11] These fungi also display the ability to biotransform halogenated compounds, a difficult process as halogenated compounds often exhibit antimicrobial/antifungal properties. [12] These experiments shed light upon an important aspect that the fungi utilized have metabolising capacity for drugs metabolized via the UDP-Glucuronyl transferase system, as those drugs that are targets of CYP450 are more commonly known targets of fungal metabolism. [13] Thus, our experiments open the door for possibility of designing new analogues from synthetic drugs via biotransformation. These derivatives might show enhanced activity in terms of better pharmacokinetic profile. Alternatively these metabolites may exhibit off-target pharmacology.