Synthesis of prodrugs of mefenamic acid and their in vivo evaluation

Abstract

Objective: The purpose of the study was to synthesize prodrugs of mefenamic acid, to be used as Anti inflammatory drug with fewer adverse effects. Methods: The drug was covalently bonded to PEG 1500 (polyethylene glycol) and PEG 6000 as such and with a linker glycine. The prodrugs were characterized by FT-I.R and N.M.R. For the drug release studies, all the prodrugs were subjected to pH 1.2 and pH 7.2. For the anti inflammatory activity, Carrageenan induced rat paw edema method was followed and for Ulcer protecting activity, Pylorus ligation method was used, the prodrugs were administered to male Sprague-Dawley rats. Results: The results suggested that the prodrugs of mefenamic acid, the drug release was higher at pH 7.2 than at pH 1.2. The result obtained for anti inflammatory activity was comparable to the standard drug of mefenamic acid. For ulcers, the prodrugs were found to possess Ulcer curing property higher than the standard drug. Conclusion: The prodrugs thus synthesized possess anti inflammatory activity as well as good ulcer protecting activity, can be used instead of standard drug. INTRODUCTION Mefenamic acid is an NSAID, derivative of N-aryl anthranilic acid belonging to class of fenamic acids. It is chemically N-(2,3-xylyl) anthranilic acid [1, 2, 3]. The drug is used for its analgesic, anti inflammatory activity [4], and primary dysmenorrhea. The dose of the drug used is 500 mg as initial dose followed by 250 mg every 6 hrs and not to continue more than a week. The mechanism of action [5, 6, 7] of mefenamic acid is inhibition of cyclooxygenase (COX) enzymes which are required for production of prostaglandins. The adverse effects of the drug include ulcer formation in the upper GI tract [2, 3].

Full text

SYNTHESIS OF PRODRUGS OF MEFENAMIC ACID AND THEIR IN VIVO EVALUATION
Original Article
DURGA PRASAD KEMISETTI*1, SARANGAPANI MANDA2, JITHAN AUKUNURU3, KRISHNA MOHAN CHINNALA4,
NAGA KISHORE RAPAKA5
1Department of Pharmaceutical Chemistry, Mother Teresa College of Pharmacy, N.F.C Nagar, Ghatkesar, R.R Dist, Andhra Pradesh India,
2Department of Pharmaceutical Chemistry, University College of Pharmaceutical Sciences, Kakatiya University, Warangal, Andhra
Pradesh, India, 3Department of Pharmaceutics, Mother Teresa College of Pharmacy, N.F.C Nagar, Ghatkesar, R.R Dist, Andhra Pradesh,
India, 4Department of Pharmacology, Nalla Narsimha Reddy School of Pharmacy, Chowdariguda, Ghatkesar, R.R Dist, Andhra Pradesh,
India,5
Received: 06 May 2014 Revised and Accepted: 15 June 2014
Department of Pharmacology, Geethanjali College of Pharmacy, Keesara, Ghatkesar, R.R Dist, Andhra Pradesh, India.
Email: kdp251999@gmail.com
ABSTRACT
Objective: The purpose of the study was to synthesize prodrugs of mefenamic acid, to be used as Anti inflammatory drug with fewer adverse
effects.
Methods: The drug was covalently bonded to PEG 1500 (polyethylene glycol) and PEG 6000 as such and with a linker glycine. The prodrugs were
characterized by FT-I.R and N.M.R. For the drug release studies, all the prodrugs were subjected to pH 1.2 and pH 7.2. For the anti inflammatory
activity, Carrageenan induced rat paw edema method was followed and for Ulcer protecting activity, Pylorus ligation method was used, the
prodrugs were administered to male Sprague-Dawley rats.
Results: The results suggested that the prodrugs of mefenamic acid, the drug release was higher at pH 7.2 than at pH 1.2. The result obtained for
anti inflammatory activity was comparable to the standard drug of mefenamic acid. For ulcers, the prodrugs were found to possess Ulcer curing
property higher than the standard drug.
Conclusion: The prodrugs thus synthesized possess anti inflammatory activity as well as good ulcer protecting activity, can be used instead of
standard drug.
Keywords: Polyethylene glycol, Mefenamic acid, Ulcer protecting, Anti inflammatory, Prodrugs.
INTRODUCTION
Mefenamic acid is an NSAID, derivative of N-aryl anthranilic acid
belonging to class of fenamic acids. It is chemically N-(2,3-xylyl)
anthranilic acid [1, 2, 3]. The drug is used for its analgesic, anti
inflammatory activity [4], and primary dysmenorrhea. The dose of the
drug used is 500 mg as initial dose followed by 250 mg every 6 hrs and
not to continue more than a week. The mechanism of action [5, 6, 7] of
mefenamic acid is inhibition of cyclooxygenase (COX) enzymes which
are required for production of prostaglandins. The adverse effects of the
drug include ulcer formation in the upper GI tract [2, 3].
Prodrugs
Polymeric prodrug approach is one of the method where in a drug is
covalently bonded to a polymer [8]. The requirements of prodrug
are it should be chemically or enzymatically cleavable, non toxic and
no pharmacological activity as such [9, 10]. Prodrugs are classified
as Carrier linked prodrugs, Tripartite prodrugs, Mutual prodrugs,
Polymeric prodrugs and Bioprecursor [10]. The use of polymers was
first introduced by Prof. H. Ringsdorf [11]. Polymers have been used
as carriers and backbone for many drugs which are biodegradable
[12]. The commonly used polymers are polyethylene glycol,
polyvinyl pyrrolidone (PVP). Polyethylene glycol is a non
biodegradable polymer but, excreted easily [13]. Many drugs are
conjugated to polyethylene glycol of varying molecular weights [14,
15]. Polyethylene glycol-acyclovir prodrugs [16] were synthesized
and prodrugs of aspirin were synthesized using HEMA (Hydroxy
ethyl methacrylate) as polymers [17]. PEG 6000 was used as
polymer for preparation of solid dispersions of aceclofenac and was
evaluated for particle size and dissolution rate [18]. Polymer-drug
conjugates are synthesized covalently by ester, anhydride, amide
linkages [19, 20]. Use of polyethylene glycol as polymeric backbone
to drugs for drug delivery is known as PEGylation [21]. Mefenamic
acid sustained release tablets have been formulated using sodium
alginate to form water insoluble gel [22]. Mefenamic acid solubility
was increased using HPMC (Hydroxy propyl methyl cellulose) by
spherical agglomeration technique [23]. Mefenamic acid mutual prodrug
was synthesized using glucosamine [24]. Based on the facts, the thought
for synthesizing prodrugs of mefenamic acid using polyethylene glycol
1500 and 6000 as polymeric backbone has turned up.
MATERIALS AND METHODS
Mefenamic acid drug was obtained from Hetero Drugs, Hyderabad.
PEG 1500, PEG 6000, DMF (di methyl formamide), DCC (dicyclohexyl
carbodiimide), DMAP (dimethyl amino pyridine), Glycine and other
solvents of reagent grade were purchased from SD Fine chemicals.
Animal studies were done in department of Pharmacology,
Geethanjali College of Pharmacy, Keesara, Ghatkesar and it was
approved by Institutional Animal Ethics Committee, Regd no:
1648/PO/a/12/CPCSEA-GCOP-IAEC-03/2013 for anti inflammatory
and ulcer protecting activity.
Synthesis of PEG 1500/6000-Mefenamic acid
PEG 1500/6000 1.5 gms and 1.6 ml of pyridine were taken in a
round bottomed flask, to it a solution of DCC 1gm and 0.6 gms of
DMAP in 10 ml DMF was added. The flask was kept in an ice bath
and temperature was maintained 0o
Synthesis of PEG 1500/6000 glycine
C, to this mefenamic acid was
added for 10 mints. The flask was then placed on a magnetic stirrer,
attached with a condenser and was allowed for coupling reaction for
7 days at room temperature. The residue obtained was dissolved in
DCM (dichloro methane) and reprecipitated by excess of cold diethyl
ether [25]. The product obtained was subjected to TLC (thin layer
chromatography) using mobile phase DCM: methanol 3:2. Finally
structure was confirmed (Scheme I) by FT-I.R and N.M.R.
PEG 1500/6000 1.5 gms and 1.6 ml of pyridine were taken in a two
necked round bottomed flask and 20 ml DMF was added and placed
on a magnetic stirrer. To this 0.18 gms of glycine was added in small
International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491 Vol 6, Issue 7, 2014
Innovare
Academic Sciences

Kemisetti et al.
Int J Pharm Pharm Sci, Vol 6, Issue 7, 437-442
438
portions for 3 hrs maintaining room temperature. Then the contents
were refluxed by attaching a condenser, maintaining a temperature
of 130o
HO OOOH
32
POLY ETHYLENE GLYCOL 1500/6000
OO
32
POLY ETHYLENE GLYCOL 1500/6000- MEFENAMIC ACID
H
N
COOH CH
3
CH
3
MEFENAMIC ACID
NH OC
CH
3
CH
3
HN
CO
H
3
C
H
3
C
Scheme I
C for 21 hrs. The residue obtained was dissolved in DCM and
reprecipitated by excess of cold diethyl ether. The formation of the
product was subjected to TLC, mobile phase DCM: methanol 3:2 and
structures (Scheme II) were confirmed by FT-I.R and N.M.R
HN
H
3
CCH
3
HO OOOH
32
POLY ETHYLENE GLYCOL 1500/6000
H
2
NOH
O
GLYCINE
H
2
NO
O
OO
32
NH
2
O
O
POLY ETHYLENE GLYCOL 1500/6000-GLYCINE-MEFENAMIC ACID
H
N
COOH CH
3
CH
3
MEFENAMIC ACID
N
H
H
3
C
H
3
C
C
O
OC
H
NO
O
OO
32 N
H
O
O
Scheme II
Synthesis of PEG 1500/6000-glycine-Mefenamic acid
A solution of DCC 1 gm in 10 ml of DMF and 0.6 gms of DMAP in 10
ml of DMF were taken in a beaker. The mixture was added drop by
drop to another beaker containing a solution of 0.4 gms of PEG
1500/6000-glycine in 20 ml of DMF. Mefenamic acid 1 gm was
added in portions to the above mixture at 0o
The coupling reaction was carried out for 7 days at room
temperature. The residue obtained was dissolved in DCM and
reprecipitated by excess of cold diethyl ether. The product was
subjected to TLC, mobile phase DCM: methanol 3:2 and structures
(Scheme II) were confirmed by FT-I.R and N.M.R.
C for 10 mints. The
contents were transferred to a round bottomed flask fitted with a
condenser and placed on magnetic stirrer.
In vitro Drug Release Studies
The synthesized prodrugs were checked for the drug release at pH
1.2 and 7.2, temperature was maintained at 37o C. The λmax was
determined for mefenamic acid and found to be 285 nm, aliquots of
5 ml were collected at intervals of 0, 5, 10, 15, 30, 45, 60 mints, sink
conditions were maintained. A standard graph was plotted and %
drug release was found. A graph of time v/s cumulative drug release
was plotted at pH 1.2 and 7.2.
Anti inflammatory activity
Male Sprague-Dawley rats weighing 100-150 gms were divided into
6 groups. The method for this activity followed was Carrageenan
induced rat paw edema. Ist group control, IInd group standard drug,
IIIrd, IVth, Vth, VIth groups received mefenamic acid prodrugs,
injected into the subplantar region in the left and right hind paws
and the swelled volume was measured by Dolphin, India
Plethysmometer. The measurements were taken at intervals of 1, 3,
6 hrs [26]. The % inhibition was calculated by
Ulcer protecting activity
For this activity, the method followed was Pylorus-ligation. The
animals weighing 100-150 gms were fastened overnight,
anaesthetized, incised 1 cm long in abdomen below the sternum. The
stomach was exposed and a thread was passed round pyloric
sphincter, a knot was tied. Abdomen was closed with sutures and
animals were kept in separate cage, allowed to recover.
Ist group received control, IInd group received standard drug
mefenamic acid 10 mg/kg, IIIrd, IVth, Vth, VIth groups were injected
mefenamic acid prodrugs and after performing pylorus ligation kept
in separate cages, after 4 hrs the animals were sacrificed and
abdomen was cut open, stomach was removed and washed under
running tap water, then placed on glass slide and observed on
microscope at 10 X magnification for ulcers [27].
Ulcer Index was calculated by
RESULTS
Table 1: Physical properties of Mefenamic acid prodrugs
S.
No.
Compound
Solubility
Colour
Melting
Pint
I.R Spectra
N.M.R Spectra
1
Mefenamic acid
Methanol
White
220-229o
OH of COOH-2569, 2
C
0
2.1δ-2.4 δ (t, 6H) 2.5δ-(d, 2H) 6.7-7.5 δ
(m, 7H) 7.9 δ (s, 1H), 9.5 δ (s, 1H).
NH-
3348, C=O-1647.
2
PEG 1500-Mefenamic
acid
Dichloro
methane
White
200-208o
2
C
0
1.1-1.5δ-(m, 12H), 1.6-4.5δ (m, 44H),
5.6-7.7δ (m, 14H), 7.9δ (d, 2H).
NH-3327, C-O-C-1157, CH
str-2850, C=O-1627.
3
PEG 1500-Glycine-
Mefenamic acid
Dichloro
methane
White
225-234o
2
C
0
1.1-1.5δ (m, 12H) 1.6-4.5δ (m, 44H)
5.6-7.5δ (m, 14H) 7.9δ(d, 2H).
NH-3327, 3124, C-O-C-
1157, CH-str-2850, C=O-1629.
4
PEG 6000-Mefenamic
acid
Dichloro
methane
White
224-233o
2
C
0
1.1-1.5δ-(m, 12H), 1.6-4.5δ (m, 44H),
5.6-7.7δ (m, 14H), 7.9δ (d, 2H).
NH-3327, C-O-C-1157, CH-
str-2850, C=O-1626.
5
PEG 6000-Glycine-
Mefenamic acid
Dichloro
methane
White
221-230o
2
C
0
1.1-1.5δ (m, 12H) 1.6-4.5δ (m, 44H)
5.6-7.5δ (m, 14H) 7.9δ(d, 2H).
NH-3321, 2937, C-O-C-
1161, CH-str-2852, C=O-1693.

Fig. 1: I.R Spectra of Mefenamic acid
Fig. 2: I.R Spectra of PEG 1500/6000-mefenamic acid
Fig. 3: I.R Spectra of PEG 1500/6000-Glycine-Mefenamic acid
Fig. 4: N.M.R Spectra of Mefenamic acid

Kemisetti et al.
Int J Pharm Pharm Sci, Vol 6, Issue 7, 437-442
440
Fig. 5: N.M.R Spectra of PEG 1500/6000-mefenamic acid
Fig. 6: N.M.R Spectra of PEG 1500/6000-Glycine-mefenamic acid
Fig. 7: In vitro drug release profile of PEG 1500 prodrugs at pH
1.2 and 7.2.
Fig. 8: In vitro drug release profile of PEG 6000 prodrugs at pH
1.2 and 7.2.
Table 2: Anti inflammatory Activity
Groups Change in Paw volume (ml) mean±SEM
& % Inhibition
1 hr
3 hr
6 hr
Control
0.52±0.02
0.61±0.02
0.65±0.03
Mefenamic acid
0.19±0.01
(63.46)
0.29±0.02
(52.46)
0.20±0.02
(69.23)
PEG1500-Mefenamic
acid
0.27±0.02
(48.08)
0.41±0.01
(32.79)
0.22±0.01
(66.15)
PEG 1500-Gly-
Mefenamic acid
0.21±0.03
(59.62)
0.42±0.01
(31.15)
0.23±0.01
(63.16)
PEG6000-Mefenamic
acid
0.20±0.01
(61.54)
0.32±0.02
(47.54)
0.21±0.01
(67.69)
PEG6000-Gly-
Mefenamic acid
0.24±0.02
(43.08)
0.36±0.02
(40.98)
0.24±0.02
(63.08)
Values are mean±SEM, n=6, one way ANOVA p<0.05 vs control
Fig. 9: Anti inflammatory activity of mefenamic acid prodrugs

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441
Table 3: Gross Ulcer Index of Mefenamic acid prodrugs
S. No.
Treatment
Ulcer Index
1
Control
10
2
Mefenamic acid
18
3
PEG1500-Mefenamic acid
16
4
PEG1500-Gly-Mefenamic acid
16
5
PEG6000-Mefenamic acid
19
6
PEG6000-Gly-Mefenamic acid
17
Fig. 10: Ulcer Index of mefenamic acid prodrugs
DISUSSION
Prodrug approach is currently one of the popular approaches in the
development of new drugs [28-35]. In this study, prodrugs of
mefenamic acid were developed and investigated. The prodrugs
were successfully synthesized. Upon synthesis, these were
characterized for various parameters. The result obtained from
spectral data (Sl.no 1 of table 1) indicated the standard drug had a
COOH group by I.R and N.M.R. The structures of PEG 1500/6000-
mefenamic acid (Sl.No 2 and 4) were confirmed by I.R and N.M.R,
that COOH group of mefenamic acid had formed an ester, the
structures were given in scheme I.The structures for PEG
1500/6000-glycine-mefenamic acid (Sl.No 3 and 5) by I.R and N.M.R,
the carboxylic acid group of mefenamic acid had formed an amide
linkage with the amino terminal of glycine, the structures were given
in scheme II. Fig.1 and Fig.4 I.R and N.M.R spectras of standard drug
mefenamic acid, Fig.2 and Fig.5 I.R and N.M.R spectras of PEG
1500/6000-mefenamic acid, Fig.3 and Fig.6 I.R and N.M.R spectras
of PEG 1500/6000-glycine-mefenamic acid.
The drug release studies for the synthesized prodrugs were given in
Fig.7 and Fig.8, the study revealed that drug release for PEG 1500-
mefenamic acid, PEG 1500-glycine-mefenamic acid, PEG 6000-
mefenamic acid and PEG 6000-glycine-mefenamic acid was more at
pH 7.2 than at pH 1.2. Also the comparision study indicated that
there is a role of spacer (glycine) in the drug release.
Table 2 and Fig.9 were the results obtained for anti inflammatory
activity of the prodrugs synthesized. The results were taken for 1 hr,
3 hrs and 6 hrs for change in paw volume, out of the 4 prodrugs, 1 of
the compound had better anti inflammatory activity than standard
drug.
Table 3 and Fig.10 indicated ulcer protecting activity for the
synthesized prodrugs. The ulcer index was measured and out of 4
prodrugs synthesized 3 of them were found to be more protecting
towards ulcers than standard drug. The prodrugs which had more
ulcer protecting activity are PEG 1500-mefenamic acid, PEG 1500-
glycine-mefenamic acid and PEG 6000-glycine-mefenamic acid.
CONCLUSION
The prodrugs of Mefenamic acid synthesized have retained their anti
inflammatory activity similar to standard drug Mefenamic acid. An
important finding of the result regarding ulcer protection that
prodrugs of mefenamic acid have shown good ulcer protection than
standard drug.
CONFLICT OF INTERESTS
Declared None
ACKNOWLEDGEMENTS
The authors thank Mother Teresa College of Pharmacy and Geethanjali
College of Pharmacy for providing the facilities for this work.
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