INTRODUCTION Ziprasidone is second-generation antipsychotic drug and benzisoxazole derivative used in the treatment of schizophrenia, bipolar disorder (1, 2). Biotransformation is the process in which chemical transformation of drugs to desired products occurs by enzymes which are contributed either from microorganisms (bacteria and fungus) or animals (5,6). Biotransformation studies of drugs and the toxicity of metabolites are important in drug development (7). Transformation of drug by the application of whole cell microorganisms is more advantageous when compared with isolated enzymes. Recently microbial transformation is considered as economical and ecologically safe technology for the pharmaceutical and biotechnological specialists to provide new methods in production of pure useful metabolites, pharmaceutical and agrochemical compounds (8). Microbial transformation is extensively used method for production of new and useful metabolites of almost all classes of drugs which acts as substitute for chemical synthesis in preparation of pharmacologically active compounds (9). The cytochrome P450 (P450) enzyme systems are involved in the biotransformation of the majority of prescribed drugs and new drug entities. Among CYP450 superfamily, CYP3A4 is responsible for the biotransformation of approximately 55% of marketed drugs. Enzyme inhibition and induction studies are considered to be important in drug clearance, assessment and modeling of inhibition/induction of drug in drug discovery and development process (10). Present study was aimed at the confirmation of CYP3A4 like enzyme involvement for microbial biotransformation of ziprasidone by using CYP3A4 enzyme inhibitor and inducer by fermentation technique and HPLC analysis. EXRERIMENTAL WORK Microorganisms Aspergillus flavus (MTCC 1783), Aspergillus ochraceus (NCIM 1140), Aspergillus terreus (NCIM 657), Cunninghamella blakesleeana (MTCC 3729), Cunninghamella elegans (NCIM 689), Cunninghamella echinulata (NCIM 691), Gliocladium roseum (NCIM 1064), Rhizopus stolonifer (NCIM 880) were procured from National Chemical Laboratory (NCL) Pune, India and microbial type culture collection and Gene bank (MTCC), Chandigarh, India. Chemicals Ziprasidone was obtained as gift sample from Therdose Private Limited, Hyderabad, India. Fluoxetine and Carbamazepine were procured from Aurobindo pharma, Hyderabad, India. The solvents used for analytical study were HPLC grade. Other chemicals and culture media components were purchased from Qualigens & S.D. fine chemicals, Mumbai, India. Fermentation technique Biotransformation studies The fermentation was carried out in 250ml Erlenmeyer flask containing 50ml of broth media labelled as drug control, culture control and sample. For each organism two controls and one sample was used. Study consisted of one drug control which had drug being incubated without organism, culture control consisted of broth medium inoculated with a loopful of respective fungus. Sample flask consisted of both drug and culture. Two controls and sample flasks were incubated on orbital shaker under identical conditions to obtain the prominent growth of microorganisms for biotransformation study. Inhibition and induction studies The inhibition and induction studies were carried out for confirmation of enzyme CYP3A4 involvement in ziprasidone biotransformation by microorganisms. One sample and four controls were used. These include a substrate control, inhibitor/inducer control and inhibitor/inducer and substrate control to check the interference of substrate, inhibitor/inducer alone and in combination with the media. Culture control consisted of respective the microorganisms grown under identical conditions. The test was performed in two stages; in the first stage, drug control, culture control and inhibitor control were incubated in orbital shaker for 72 h. Inhibitor and substrate control and samples were incubated under the same conditions for 24 h by adding 20μg/ml of inhibitor/inducer. In second stage, i.e., after 24 h of incubation 0.5 ml of drug solution was added to the inhibitor/inducer and substrate control and to the sample incubated for 48hrs(11). The contents of the flask were extracted and analyzed by HPLC to determine the extent of inhibition/induction of metabolism of ziprasidone by microorganisms. The experiments were carried out in culture flasks (250 ml), each containing 50 ml of broth media as specified in experimental protocol for inhibition and induction studies given in Table 1 and incubated on a rotary shaker operated under identical conditions. The substrate (ziprasidone), CYP3A4 inhibitor (Fluoxetine) and inducer (Carbamazepine) stock solutions were prepared separately by dissolving 20 mg in 10 ml of methanol for the study. Table 1. Incubation protocol for inhibition and induction studies Contents of flask Medium broth Substrate Name of the flask Blank I Blank II Blank III Blank IV (drug (Culture (Substrate control) control) (inhibitor control) Blank V (inhibitor/ +Inhibitor) inducer +culture ) Sample (substrate +inhibitor/ inducer +culture) + + + + + + + - - + - + Culture Inhibitor/Inducer - + - - + + - - + + + + ‘+’ = added ; ‘-’ = Not added Extraction procedure The incubated flasks were taken out from shaker incubator and heated on water bath at 50°C for 30 min for inactivation of grown microbes. Then, these were transferred into centrifuge tubes and centrifuged at 3000 rpm for 10 min (R8C: Remi instruments, Mumbai, India). The supernatant obtained was collected in separate boiling tubes. The supernatant of drug and its metabolites were extracted by using dichloromethane (12). Then organic layer was collected and air dried. The dried extract was reconstituted with mobile phase for HPLC analysis. ANALYTICAL TECHNIQUES Ziprasidone and its metabolite in the extracted samples were estimated by High Performance Liquid Chromatography (HPLC) method. The HPLC system (Waters, USA) consisted of Waters 515 solvent delivery module and Waters 2489 UV-visible spectrophotometric detector. The mobile phase consisted of methanol: sodium acetate (75:25 v/v) with a flow rate of 1ml/m. The column used was C-18 (stainless steel column of 25 cm length and 4.6 mm internal diameter packed with porous silica spheres of 5 µ diameter, 100 Å pore diameter – II 5C-18 rs – 100a, 5 µm, 4.6 x 250 mm). The eluent was monitored at 314nm., sensitivity was set at 0.001 a.u.f.s(13). The elute of the metabolite peak was collected from HPLC and dried for further analysis by mass spectroscopy and PNMR for confirmation of its structure. Then the metabolite peak area was noted in inhibition and induction studies. The % of metabolite formed during inhibition and induction studies were calculated by comparing with peak area of metabolite obtained in biotransformation studies (without inducer and inhibitor). RESULTS In the present study inhibition and induction studies for microbial metabolism of ziprasidone was performed using CYP3A4 inhibitor (Fluoxetine) and inducer (Carbamazepine). The results of HPLC analysis of ziprasidone and its metabolite in culture extracts of Gliocladium roseum are represented in Figure 1. The peak at retention time of 2.4min. represented solvent peak and peak at 4.4min. represented culture contents. The peak at retention time of 11.8min. represented ziprasidone. Interestingly the sample of Gliocladium roseum has shown an extra metabolite peak at 7.1min. compared to its controls as shown in Figure 1. The metabolite structure was analysed and confirmed by Mass spectroscopy and PNMR spectroscopy. The metabolite collected from HPLC elute was dried. The mass spectrum of pure drug and its metabolite were compared. The mass spectrum of ziprasidone has shown a molecular ion peak at m/z 413 (M+1) and the mass spectrum of metabolite has shown a molecular ion peak at m/z 429 (M+1) as given in Figure 2 is equal to the molecular weight of ziprasidone sulphoxide. Inhibition and induction studies of microbial metabolism was performed as per the protocol given in Table 1 and the results are given in Table 3. Figure 1. HPLC chromatogram of Ziprasidone from culture extracts of Gliocladium roseum 700 D 600 drug control culture control sample 500 D - Drug M - Metabolite mV 400 300 M 200 100 0 0 2 4 6 min. 8 10 12 Figure 2. Mass spectrum of Ziprasidone Metabolite. 14 16 m/z-429(M+1) Table 3. Data of metabolite formation in induction and inhibition studies. Name of the drug Nature of drug Metabolite Area(mv) % Metabolite Ziprasidone Substrate for CYP3A4 2439695 23 Ziprasidone + Carbamazepine Ziprasidone + Fluoxetine Substrate + Inducer Substrate + Inhibitor 4280073 1113773 40.35 10.5 DISCUSSION The mass spectrum of pure ziprasidone exhibited a molecular ion peak at m/z 413(M+1), it was supported by prominent fragment ion peaks at m/z 194, 177. Ziprasidone metabolite indicated a protonated molecular ion peak at m/z 429(M+1) suggesting that a single atom of oxygen had been added to the molecule. Its product ion spectrum showed fragment ions at m/z 194 and 234, indicated that the oxindole moiety was unchanged as in Figure.2. Based on these data obtained the metabolite was identified as major metabolite of ziprasidone sulphoxide as in human beings (14). In the present study, a CYP3A4 inhibitor (Fluoxetine) and inducer (Carbamazepine) were used, during induction and inhibition studies to confirm the involvement of a specific CYP enzyme in metabolite formation by Gliocladium roseum. The % of metabolite formed was significantly decreased in presence of fluoxetine and the % of metabolite formed in presence of inducer was increased when compared with initial metabolism studies as shown in Table 3. It indicated that CYP3A4 inducer increased the metabolism of ziprasidone by Gliocladium roseum. The above induction and inhibition studies confirmed that the involvement of CYP3A4 in microbial metabolism of ziprasidone by Gliocladium roseum like in mammals. CONCLUSION Present study was aimed for the confirmation of the enzyme involved in metabolism of Ziprasidone a CYP3A4 substrate using the CYP3A4 inhibitor (Fluoxetine) and inducer (Carbamazepine). 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