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). According to the results obtained there was an increase in metabolite
production in presence of CYP3A4 enzyme inducer and decrease of metabolite formation in
presence of inhibitor during microbial biotransformation studies by Gliocladium roseum. Hence
it is concluded that Gliocladium roseum can be used as complementary in vitro tool for
prediction of CYP3A4 mediated mammalian biotransformation studies and drug-drug
interactions for newly marketed drugs.
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INTRODUCTION Ziprasidone is second