Pharmacology & Pharmacy, 2011, 2, 370-374
doi:10.4236/pp.2011.24048 Published Online October 2011 (http://www.SciRP.org/journal/pp)
Copyright © 2011 SciRes. PP
In Vitro Competitive Metabolism Study of
Olmesartan Medoxomil in Rat Liver S9
Fractions Using LC/MS
Muruganathan Gandhimathi, Rahul Baghla, Sivalingam Subramanian, Thengungal Kochupapy Ravi
Department of Pharmaceutical Analysis, College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore, India.
Email: gands72@yahoo.co.in
Received January 12th, 2011; revised April 16th, 2011; accepted July 20th, 2011.
ABSTRACT
Olmesartan Medoxomil (OLM), Ramipril (RPL) & Fenofibric acid (FA) are used to treat hypertension and cardiovas-
cular disease. These drugs undergo hydrolytic metabolism by the enzyme liver esterase and converts into their respec-
tive active metabolites Olmesartan (OL), Ramiprilat (RPT) and Fenofibric acid (FA) for OLM, RPL and FEN respec-
tively. In this study the competitive metabolism of OLM, in presence of RPL and FEN was investigated in rat liver S9
fractions using a validated LC-MS method. Olmesartan Medoxomil was found to be highly reactive to the rat liver S9
fractions and formation of active metabolite Olmesartan is highest. The rate of formation of active metabolite Olme-
sartan reduced by 12.68% in the presence Ramipril and 6.56% in presence of Fenofibrate. A marked reduction of
18.96% was found in the formation of active metabolite Olmesartan from Olmesartan Medoxomil when all the three
drugs are in combination.
Keywords: In Vitro Metabolism, Omlesartan, Fenofibrate, Ramipril, Rat Liver, LC-MS
1. Introduction
Drug metabolism, basically an adaptive process, is a
rather useful property of the (liver) cell, as a whole. Drug
biotransformation usually leads to more polar compounds,
and thus to faster elimination, and to substances with
lower or no activity. Only rarely is an increase of activity
observed after biotransformation. A common challenge
during early development of a new drug candidate is to
determine an analytical approach that is capable of deliv-
ering a versatile, cost effective and efficient analytical
throughput in support of the preclinical program. The
major steps in the in vitro drug metabolism studies are the
identification and quantification of the drug and the me-
tabolites.
Carrying out in vitro study is inexpensive and serves
as an adequate screening mechanism that can rule out the
importance of a metabolic pathway and make in vivo
testing unnecessary. In vitro studies are able to help
clinical study design to impact directly on the labelling of
medicines, rationalise the contra-indications offered and
consolidate the patient group in which dosing is expected
to be safe and efficacious It may be concluded that the
metabolism of drugs understudy is affected by other,
hence combination/co-administration can be avoided.
It is common that a patient is taking several drugs for
the treatment of one or more health related issues at the
same time. A drug may affect or be affected by other
co-administered drugs. Metabolism-based drug-drug in-
teractions occur when a drug inhibits or induces the ac-
tivity of a drug metabolizing enzyme, which catalyzes
the metabolism of the concomitant drug. The metabo-
lism-based drug-drug interaction is one of the major fac-
tors that cause drug failures during drug development.
Early stage screening of compounds for potential drug-
drug interactions using in vitro techniques becomes nec-
essary in order to decrease late stage compound attrition.
This early in vitro screening of drug-drug interactions
facilitates the drug discovery and development process.
Olmesartan Medoxomil and Ramipril are antihyper-
tensive drugs belongs to ACE inhibitors and AT1 antago-
nist category respectively. Fenofibrate is a co-adminis-
tered drug with antihypertensives for its lipid lowering
nature. All the three drugs are prodrugs and in liver, they
undergo hydrolytic metabolism by the enzyme liver es-
In Vitro Competitive Metabolism Study of Olmesartan Medoxomil in Rat Liver S9 Fractions Using LC/MS371
terase to form their active metabolites Olmesartan, Rami-
prilat and Fenofibrate respectively, so there is a probabil-
ity of competitive metabolism interactions of RPL and
FA with OLM. A study was conducted to determine the
effect of two drugs on the metabolism of OLM when
available in combination or co administration. An exten-
sive literature survey revealed that, number of analytical
methods have been described for the estimation of
Olmesartan Medoxomil [1-5], Ramipril [6-9], Fenofi-
brate [10-12] from various matrices like formulation and
biological samples. No literatures are available for their
metabolic interaction study and hence this paper de-
scribes an invitro interaction study between them using
LC-MS method to predict the probability of interactions
in their metabolic pathway. As there is no such method
exists, this method would serve to analyze trace quantity
of them.
2. Materials and Methods
2.1. Incubation Conditions
Rat liver S9 fractions (0.5 mg/mL, Lot. No. 029K1126),
Sigma Aldrich Co. were used. Drug solutions were incu-
bated for 60 mins with RLF in 2.0 ml of incubation me-
dium consisting of a tris-HCL buffer (0.12 mM, pH 7.4 at
37˚C), MgCl2 (5 mM), Sodium pyrophosphate (6.25 mM),
D-glucose 6-phosphate (5 mM), D-glucose 6-phosphate
dehydrogenase (1 U/mL) and NADPH (β-Nicotinamide
adenine dinucleotide 2-phosphate reduced tetrasodium
salt, 0.5 mM). After 60min of incubation 1ml of ice
cooled methanol was added to stop the reaction by pre-
cipitation of proteins and the solution was centrifuged at
5000 rpm for 5 min to separate out the proteins and the
supernatant liquid was used for the study.
2.2. LC-MS Conditions
An end capped C18 reverse phase column (250 mm × 4.0
mm, 5 μm), E. Merck was used for the study. Mobile
phase consisted of acetonitrile, methanol and 0.35% v/v
formic acid (80:20:30 v/v). The study was carried out at
room temperature with a flow rate of 1.2 ml/min with
positive polarity mode by keeping MS scan range 200 -
850 AMU. Drying gas flow was adjusted at 10 L/Hr and
temperature was fixed at 250˚C.
2.3. LC-MS Analysis
All the measurements were performed by LC-MS with
ESI probe. The supernatants of the reaction mixtures
were injected into an endcapped C18 reverse phase col-
umn (250 mm × 4.0 mm, 5 μm) and eluted at a flow rate
of 1.2 ml/min in isocratic mode with a mobile phase con-
sist of acetonitrile, methanol and 0.35% v/v formic acid
(80:20:30 v/v). The mass peaks were observed respective
to their m/z values for the parent drugs and their active
metabolites.
2.4. Data Analysis
The incubated solutions of individual drugs Olmesartan
Medoxomil, Ramipril and Fenofibrate (30 µM of each
drug) at different incubation time intervals of 0 min, 30
min and 60 mins were injected in LC-MS system and the
M + 1 ion peaks for the drugs and the metabolites were
observed (OLM-559.35, OL-447.3, RPL-417.3, RPT-
389.2, FEN-361.2 and FA-319.1). Reduction in the drug
peak intensities and increase in the metabolite peak in-
tensities were observed carefully and relative intensities
were calculated for all the three drug peaks and their me-
tabolites. Relative percentage reduction of drugs peaks
and increase in metabolite peaks were calculated using
relative peak intensities for all the three drugs & the me-
tabolism pattern was studied using a plot with time on X
axis and relative percentage increase in active metabolite
peak on Y axis. Peak intensities of drugs and their ex-
pected metabolites were noted with single drug, as well
as in combinations. The effect of each drug on other
drug’s metabolism was calculated based on relative peak
intensity.
3. Results and Discussion
The biotransformation of Olmesartan Medoxomil to ac-
tive metabolite Olmesartan, ramipril to ramiprilat and
fenofibrate to fenofibric acid by the enzyme liver es-
terase present in S9 fraction are shown in Figures 1-3.
An accurate and sensitive LC-MS method was developed
and applied for carrying out in vitro metabolism interac-
tion study, which is a screening mechanism in drug dis-
covery. The drugs and metabolites were studied by MS
using their respective M + 1 value. Based on the peak
intensity of drug and expected metabolite, the metabo-
lism was studied. The mass spectrum of three drugs and
their metabolites are represented in Figures 4-6.
The percentage formation of active metabolite Olme-
sartan from Olmesartan Medoxomil in the presence of
Ramipril and Fenofibrate was compared with Olmesartan
Medoxomil alone (Table 1). Similarly for all drugs the
study was done and it was conducted for six times on
every occasion. The rate of formation of active metabo-
lite Olmesartan is highest among all. The rate of forma-
tion of active metabolite Olmesartan reduced by 12.68%
in the presence Ramipril and 6.56% in presence of Feno-
fibrate. A marked reduction of 18.96% was found in the
formation of active metabolite Olmesartan from Olme-
sartan Medoxomil when all the three drugs were in com-
bination. It may be concluded from the study that the
metabolism of OLM is affected by the presence of other
Copyright © 2011 SciRes. PP
In Vitro Competitive Metabolism Study of Olmesartan Medoxomil in Rat Liver S9 Fractions Using LC/MS
372
Figure 1. Metabolism of Olmesartan Medoxomil.
Figure 2. Metabolism of Ramipril.
Figure 3. Metabolism of Fenofibrate.
Copyright © 2011 SciRes. PP
In Vitro Competitive Metabolism Study of Olmesartan Medoxomil in Rat Liver S9 Fractions Using LC/MS373
Figure 4. Mass spectra of Olmesartan Medoximil (447) and Olmesartan (429).
Figure 5. Mass spectrum of Ramipril (417) and Ramiprilat (389).
Figure 6. Mass spectrum of Fenofibrate (361) and finofibric acid (319).
Copyright © 2011 SciRes. PP
In Vitro Competitive Metabolism Study of Olmesartan Medoxomil in Rat Liver S9 Fractions Using LC/MS
Copyright © 2011 SciRes. PP
374
Table 1. Relative percentage Olmesartan formation.
Relative Percentage of Olmesartan Formation
Drug
0 min % RSD 30 min % RSD 60 min %RSD
OLM 5.61 4.200 67.44 5.241 95.04 4.391
OLM + FEN 6.39 3.547 63.96 3.652 88.48 5.330
OLM + RPL 10.67 2.846 65.38 2.845 82.36 4.788
OLM + RPL + FEN 7.55 4.529 59.42 3.139 76.08 4.221
drugs; hence combination or co-administration of such
drugs could be avoided.
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