Aceclofenac is a nonsteroidal antiinflammatory drug that is used to relieve pain and inflammation in arthritic conditions like osteoarthritis, rheumatoid arthritis and ankylosing spodylitis. Aceclofenac is known to induce erosion and ulcers in the gastrointestinal tract but in contrast to other nonsteroidal anti-inflammatory agents, it has shown stimulatory effects on cartilage matrix synthesis. In order to lower the ulcerogenic potential and enhance effectiveness of aceclofenac as antiarthritic agent, a mutual prodrug was synthesized with neutraceutical carrier D-glucosamine that has shown to stimulate the biosynthesis of glucosaminoglycans and hyaluronic acid backbone needed for the formation of the proteoglycans found in the structural matrix of joints. The study showed that the prodrug not only lowered the ulcerogenic potential of aceclofenac but also enhanced its analgesic, antiinflammatory and antiarthritic activities thus proving the utility of D-glucosamine as an effective carrier in mutual prodrug synthesis.
Aceclofenac is a nonsteroidal antiinflammatory drug that is used to relieve pain and inflammation in arthritic conditions like osteoarthritis, rheumatoid arthritis and ankylosing spodylitis. Aceclofenac is known to induce erosion and ulcers in the gastrointestinal tract but in contrast to other nonsteroidal anti-inflammatory agents, it has shown stimulatory effects on cartilage matrix synthesis. In order to lower the ulcerogenic potential and enhance effectiveness of aceclofenac as antiarthritic agent, a mutual prodrug was synthesized with neutraceutical carrier D-glucosamine that has shown to stimulate the biosynthesis of glucosaminoglycans and hyaluronic acid backbone needed for the formation of the proteoglycans found in the structural matrix of joints. The study showed that the prodrug not only lowered the ulcerogenic potential of aceclofenac but also enhanced its analgesic, antiinflammatory and antiarthritic activities thus proving the utility of D-glucosamine as an effective carrier in mutual prodrug synthesis.
Aceclofenac is a nonsteroidal antiinflammatory drug that is used to relieve pain and inflammation in arthritic conditions like osteoarthritis, rheumatoid arthritis and ankylosing spodylitis. Aceclofenac is known to induce erosion and ulcers in the gastrointestinal tract but in contrast to other nonsteroidal anti-inflammatory agents, it has shown stimulatory effects on cartilage matrix synthesis. In order to lower the ulcerogenic potential and enhance effectiveness of aceclofenac as antiarthritic agent, a mutual prodrug was synthesized with neutraceutical carrier D-glucosamine that has shown to stimulate the biosynthesis of glucosaminoglycans and hyaluronic acid backbone needed for the formation of the proteoglycans found in the structural matrix of joints. The study showed that the prodrug not only lowered the ulcerogenic potential of aceclofenac but also enhanced its analgesic, antiinflammatory and antiarthritic activities thus proving the utility of D-glucosamine as an effective carrier in mutual prodrug synthesis.
Synthesis and Pharmacological Evaluation of Gastro-sparing Mutual Prodrug of Aceclofenac with Glucosamine
Suneela S. Dhaneshwar * 1 , Sanal Dev 1 , B.Rathi 2 and S. L. Bodhankar 2
1 Department of Pharmaceutical Chemistry, Bharati Vidyapeeth University, Poona College of Pharmacy, Erandwane, Pune, India. 2 Department of Pharmacology, Bharati Vidyapeeth University, Poona College of Pharmacy, Erandwane, Pune, India. * Author for correspondence: Dr. Suneela Dhaneshwar E-mail: suneeladhaneshwar@rediffmail.com
ABSTARCT Aceclofenac is a nonsteroidal antiinflammatory drug that is used to relieve pain and inflammation in arthritic conditions like osteoarthritis, rheumatoid arthritis and ankylosing spodylitis. Aceclofenac is known to induce erosion and ulcers in the gastrointestinal tract but in contrast to other nonsteroidal anti-inflammatory agents, it has shown stimulatory effects on cartilage matrix synthesis. In order to lower the ulcerogenic potential and enhance effectiveness of aceclofenac as antiarthritic agent, a mutual prodrug was synthesized with neutraceutical carrier D-glucosamine that has shown to stimulate the biosynthesis of glucosaminoglycans and hyaluronic acid backbone needed for the formation of the proteoglycans found in the structural matrix of joints. The study showed that the prodrug not only lowered the ulcerogenic potential of aceclofenac but also enhanced its analgesic, antiinflammatory and antiarthritic activities thus proving the utility of D-glucosamine as an effective carrier in mutual prodrug synthesis. Key words Aceclofenac, anti-arthritic, anti-inflammatory, glucosamine, gastrosparing, mutual prodrug.
INTRODUCTION
Inflammatory diseases are widely prevalent throughout the world. Nonsteroidal anti-inflammatory drugs (NSAIDs) are amongst the most extensively used drugs for the treatment of pain and inflammation associated with acute and chronic inflammatory disorders. The major drawback in the chronic administration of NSAIDs is the occurrence of serious side effects, mainly involving the gastrointestinal tract (GIT), that have serious impact on the public health due to the massive use of these drugs [1] . Prolonged use of these drugs produces gastroduodenal and mucosal erosions in 35-60% patients, ulcerations in 10-25% patients and severe hemorrhages or perforations in about 1% of patients [2] . Epidemiological studies have established that overall, NSAIDs enhance the risk of ulcer complications such as bleeding, perforations, hospitalization and death by approximately 3-10 folds [3] . NSAID-induced gastric damage has been attributed mainly to suppression of endogenous prostaglandins, which have cytoprotective effect on gastric mucosa [4] . Aryl acids have tendency to accumulate in the parietal cells of stomach and in the inflamed tissue. This pattern of preferential tissue distribution may contribute to the direct toxic effect on gastric epithelial cells such as apoptosis or necrosis [5] . These compounds inhibit the two isoforms of cyclooxygenases (COX-1 and COX-2) and thus suppress prostaglandin biosynthesis from arachidonic acid [6] . COX-1 has cytoprotective effect on GIT [7] while COX-2 mediates inflammation [8] . Aceclofenac is a nonsteroidal anti-inflammatory drug, which has gained a wide acceptance due to its ability to relieve pain and inflammation in arthritic conditions. It is indicated for Communique
18 | InPharm Communique Vol 2, No 3 management of osteoarthritis, rheumatoid arthritis and ankylosing spondylitis and is known to induce erosions and ulcers in the gastrointestinal tract [9] . In contrast to other NSAIDs, it has stimulatory effect on cartilage matrix synthesis [10] . Glucosamine, an amino sugar, is bodys starting material for the production of natural joint components like critical joint lubricants and shock absorbers [11] . Some scientists have theorized that when the body becomes unable to manufacture adequate supplies of glucosamine, susceptibility to developing osteoarthritis dramatically rises. Glucosamines role in halting or reversing joint degeneration appears to be directly due to its ability to act as an essential substrate for, and to stimulate the biosynthesis of the glycosaminoglycans and the hyaluronic acid backbone needed for the formation of the proteoglycans found in the structural matrix of joints [12] . Apparently, as we age, many of us lose the ability to produce the necessary levels of glucosamine that protect our joints. Consequently, the cartilage components of our skeletal frames lose their capacity to hold water, therefore, joint protection diminishes. The logical question here is, if the loss of glucosamine leads to arthritis, would restoring glucosamine slow the disease? In some clinical studies glucosamine was consistently shown to relieve pain in 3-4 week, restore joint flexibility, produce no known side effects and treat the cause and not just [11] . We have reported the utility of mutual prodrug concept in improving the safety profile of NSAIDs using glucosamine as a carrier. [13]
The present work reports the synthesis, physico-chemical characterization, in vitro hydrolysis and biological evaluation of mutual prodrug of aceclofenac with glucosamine. The objective of this work was to evaluate the utility of mutual prodrug concept in decreasing the ulcerogenic potential of aceclofenac by conjugating its carboxylic group with amino group of glucosamine. This mutual prodrug would have the additional advantage of producing a nutrient by-product, i.e. glucosamine on cleavage, which after its release is expected to be available to produce the above effects. MATERIALS AND METHODS Melting points were measured in open capillary tubes and are uncorrected. IR (KBr) spectrum was recorded on JASCO, FT- IR spectrophotometer, Model V5300 and 1 H- NMR spectrum on FT NMR, JEOL spectrophotometer (300 MHz) at University of Pune, using TMS as internal reference (chemical shift in , ppm). The mass spectrum was recorded on Jeol SX-102 (FAB) spectrometer. The max was determined on JASCO V530, UV-visible double beam spectrophotometer. Purity of the compounds was checked on silica gel 60F254 Merck plates using iodine vapours as visualizing agent. Aceclofenac was procured from IPCA Laboratories Mumbai, India as a gift sample. All chemicals used were of LR and AR grade and were obtained from Quali gens Fi ne Chemical s, Mumbai, India. Synthesis of glucosamine conjugate of aceclofenac (SA-004): Aceclofenac, (0.02 mol) was dissolved in 50 ml of chloroform and stirred for half an hour at 0 o C. N, N dicyclohexyl carbodimide (DCC) (0.0026 mol) was dissolved in 5 ml N, N dimethyl formamide in another beaker and was then added to a solution of aceclofenac at 0 o C and was stirred for 2 h. Glucosamine hydrochloride (0.026 mol) was suspended in 30 ml of chloroform. Triethylamine (0.052 mol) was then added drop wise, to the above mixture to liberate free glucosamine. The mixture was stirred for one hour at 0 o C.The solvent was distilled off to form dry residue. To this dry residue, mixture of aceclofenac and DCC was added drop wise at 0 o C.The reaction mixture was stirred for 12 h at 0 o C. It was filtered to remove the byproduct i.e. precipitated dicyclohexylurea. The filtrate was concentrated under vacuum. The crude product was recrystallized using alcohol and dried under vacuum. The route of synthesis is described in Scheme (Fig 1). C22H24Cl2N2O8, m.p130-132 o C, yield 83%, Rf 0.64, chloroform: methanol: acetic acid (4:1:0.1) IR (KBr) cm -1 ; 3377 (O-H); 1055, 1255 (C-O); 1718(C=O); 3319(N-H) cm -1 . 1 H-NMR (DMSO-d6): 2.26 (s, 4H, OH); 3.45-3.86 (d, 3H, CH, tetrahydropyran) ppm; 3.88 (s, 2H, CH2); 4.38 (s, 1H, ArNH); 4.48 (s, 1H, CH); 6.0 (d, 1H, CH); 6.82-6.84 (s, 1H, ArH); 6.92-6.98 (q, 4H, ArH); 7.28 (d, 1H, sec. amide). MS; m/z 514.09 (M+1). max: 271.0 (distilled water), 280.0 (CHCl3), 271.4 (phosphate buffer pH 7.4), 264 (HCl buffer, pH 1.2). Fig. 1. Scheme of synthesis
Research Article
InPharm Communique Vol 2, No 3 | 19 In vitro hydrolysis kinetics [14] : The synthesized compound was subjected to in vitro hydrolysis kinetic study. The kinetics of chemical hydrolysis of SA-004 was studied at 37 o C in aqueous buffer solutions of pH 1.2 and pH 7.4. The total buffer concentration was generally 0.05 M and a constant ionic strength () of 0.5 was maintained for each buffer by adding a calculated amount of potassium chloride. Free aceclofenac released after hydrolysis of conjugate was estimated on UV spectrophotometer for the amount of free aceclofenac released in HCl buffer and phosphate buffer at 264 nm and at 271.4 nm respectively. The assay was carried out in triplicate and was validated as per USP XXIV guidelines. The kinetics of hydrolysis was monitored by increase in free drug concentration with time and the order of reaction, half life (t 1 /2) and rate of hydrolysis were calculated. Hydrolysis in 0.05 M hydrochloric acid buffer (pH 1.2) [15] : SA-004 (10 mg) was introduced in 900 ml of HCl buffer (pH 1.2) taken in the basket and was kept in a constant temperature bath at 371 o C. The solution was occasionally stirred and 5 ml aliquot portions were withdrawn at various time intervals and were estimated on UV spectrophotometer at 264 nm for free aceclofenac released after hydrolysis of SA-004. Hydrolysis in 0.05 M phosphate buffer (pH 7.4): Same procedure as described above was followed for SA- 004, where instead of HCl buffer; phosphate buffer (pH 7.4) was used. Rate of hydrolysis was calculated using equation, K= (2.303/t) log (a/a-x), where K is hydrolysis constant, t is time in min, a represents initial concentration of prodrug, x is the amount of prodrug hydrolyzed and (a-x) is the amount of prodrug remaining. The K values from the plots were calculated separately and then average K and S.D. values were calculated. Biological screening: Materials: All the experimental procedures and protocols used in this study for pharmacological screening were reviewed and approved by the Institutional Animal Ethical Committee (IAEC) of Poona College of Pharmacy, Pune and were in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiment on Animals (CPCSEA), Government of India. Healthy male Wistar rats, weighing between 150-250 g were used. They were divided in various groups and housed under standard environmental conditions of temperature 231C and relative humidity of 505%. A 12 h light/dark cycle was followed. All animals had free access to water and standard pelleted laboratory animal diet. Statistical analysis was carried out by ANOVA followed by Dunnetts test to determine the significance of the difference between the control group and rats treated with the test compounds. Anti-inflammatory activity by carrageenan- induced rat paw edema method: This activity was preformed by carrageenan-induced rat paw edema method of [16] . Wistar rats of either sex having body weight 175-200 g were used in this study. The animals were divided randomly in four groups with 6 rats per group. A freshly prepared solution of carrageenan (1% w/v, 0.1 ml) solution was injected in to the planter region of right hind paw of each rat. One group was kept as a control and the animals of other group were pretreated with the test drugs suspended in 1.0% CMC given orally 1h before the carrageenan treatment. The paw volume was measured using UGO Basile Plethysmometer 7140 at 0 h, 1 h, 2 h, 3 h and 24 h after carrageenan treatment. Percent inhibition was calculated according to the formula given below: % Inhibition = {1- [(Vd Vp)/ (Vc Vp)] } 100 where, ( Vd Vp) is the difference in the paw volume after carrageenan injection (Vd) and before carrageenan injection (Vp) in drug treated group and (Vc Vp) is the difference in paw volume after carrageenan injection (Vc) and before carrageenan injection (Vp) in vehicle treated group. Data are expressed as mean SEM. Analgesic activity by Randall Selitto method [17]
Following the method of Randall Selitto, male Wistar rats weighing 100-150 g were previously selected for their response to an increasing paw pressure delivered by an analgesiometer (UGO Basile). Selected animals were distributed into groups of 6 animals and 0.1 ml of 1% w/v suspension of carrageenan in sterile saline was then injected into the subplantar tissue of the right hind paw while the left hind paw was taken as internal control. Test compounds were given orally 2 h later. Immediately before (0 value) and 1 h after administration, both inflamed and non-inflamed paws were subjected to an increasing pressure by the analgesiometer. The pressure at which each rat responded by withdrawing its hind paw or by struggling was recorded and considered as pain threshold. Mean pain threshold was calculated for each treatment group and percentage change versus the control group was determined. Analgesic activity by acetic acid- induced writhing method [18] : Swiss mice of either sex having body weight 24 to 26 g were used in the study. The animals were divided randomly in five groups with 6 mice per group. Mice were kept individually in the test cage, before acetic acid injection and habituated for 30 m. All compounds were dissolved in 1% CMC solution. The controlled group received p.o. administration of 1% CMC solution. After 1 h of drug administration 0.10 ml of 0.6% acetic acid was administered intraperitoneally. After five minutes they were observed for a period of ten minutes and the numbers of writhes were recorded for each animal. For scoring purposes, a writhe Communique
20 | InPharm Communique Vol 2, No 3 was indicated by stretching movements consisting of arching of the back, elongation of the body and extension of hind limbs. The formula for computing percent inhibition was: % Analgesic activity = (n - n / n) x 100 where n = mean number of writhes of control group, n = mean number of writhes of test group. Freunds complete adjuvant induced arthritis model: Antiarthritic activity was evaluated using prophylactic and therapeutic protocols of Freunds adjuvant induced arthritis method [19] . Male rats with an initial body weight of 130-200 g were used. On day 0, complete Freunds adjuvant (0.1 ml) was injected into the subplantar region of the left hind paw. Dosing with the test compounds or the standard was started depending on the protocol chosen. Paw volumes of both sides were recorded on the day of injection by using plethysmometer (7140-UGO Basile, Italy). On day 5, the volume of the injected paw was measured again. The severity of the non-injected paw (secondary lesions) was also recorded. The primary lesion was determined by measuring the paw volume on the 0, 5 th , 13 th , 18 th and 21 st day.
% Inhibition of edema of both the injected left paw and non-injected right paw over vehicle control group was calculated by using the same formula as mentioned for antiinflammatory activity by carrageenan-induced rat paw edema method. Assessment of gastric ulcerogenic effects in rats: The ulcerogenic activity was determined by the cold stress method of [20] . Groups of 6 male Wistar rats weighing 175 200 g were fasted for 24 h with free access to water. Test compounds were given orally to fasted animals. After oral administration, animals were stressed by exposure to cold (- 15 o C for 1 h). The animals were kept in separate polypropylene cages. After 2 h of drug administration, the animals were sacrificed using chloroform. The stomach was opened along the greater curvature and washed with saline and the glandular portions were examined macroscopically for the number and size of mucosal lesions. The severity of the gastric damage was determined for each stomach examined and scored accordingly to the scale: 0- no lesion; 1- redness; 2- limited number of petechiae/diffused, pronounced lesions, 3- localized, severe lesions, more than 5 small ulcers and/or 1-2 large ulcers; 4 - extended, severe lesions; 5- perforation. The ulcerogenic index was calculated by using formula, UI = Un + Us + Up 10 1 where, UI- ulcer index, Un- average of number of ulcers per animal, Us- average of severity score, Up- percentage of animals with ulcers. RESULTS AND DISCUSSION The free glucosamine was prepared by treating glucosamine hydrochloride with triethyl amine. Aceclofenac was treated with DCC to form a reactive intermediate o-acylisourea which was then coupled with the free glucosamine to form the glucosamine conjugate of aceclofenac (SA-004) (Scheme 1). The structure of the synthesized compound was assigned on the basis of elemental analysis, IR, 1 HNMR and mass spectral data. The synthesized compound did not undergo hydrolysis in 0.05M hydrochloric acid buffer (pH 1.2) whereas its hydrolysis in 0.05 M phosphate buffer (pH 7.4) followed first order kinetics with a half-life of 84.5 min. K value was found to be 0.009780.001. The anti-inflammatory activity of the synthesized compound was evaluated by carrageenan induced rat paw edema method of Winter et al. (Table 1). The compound was tested at equimolar dose to that of aceclofenac, which was used as a standard. SA-004 produced 49.25% inhibition of edema after 3 h, which was comparable to aceclofenac (46.27%). At 24 h, SA-004 produced 94.4% inhibition of edema, which was more as compared to aceclofenac (88.8%).
Table- I. Effect of prodrug on various parameters of inflammation and pain. Test Compound Carrageenan induced rat paw edema model * (% inhibition) Analgesic activity* (% inhibition) FCA induced arthritis* (% inhibition) 8 h 24 h Writhing test Randell- Selitto test Day 18 Day 21 Vehicle Aceclofenac (3mg/kg) Glucosamine (2.5mg/kg) SA-004 (4.47 mg/kg) - 46.27
50.75
49.25 - 88.89
83.33
94.44 - 44.57
48.58
51.44 - 57.1
64.3
67.9 - 45.59
10.98
54.76 - 71.37
60.03
76.89 Average of six readings; P < 0.05
Research Article
InPharm Communique Vol 2, No 3 | 21 Table - II. Gastric ulcerogenic effects of SA-004 and reference drugs after single oral administration to the rats. Compound Dose (mg/kg) Ulcer index S.D.# Control Aceclofenac Diclofenac SA-004 ---- 3 2 4.47* ---- 3.75 0.92 5.31 1.35 1.06 0.34 *Dose equimolar to aceclofenac, # Average of six readings.
The anti-arthritic activity of SA-004 was assessed by Freunds complete adjuvant induced arthritis model. Few hours after the induction of adjuvant arthritis by sub planter injection of 0.1 ml Mycobacterium butyricum to rats, the animals showed a local inflammatory reaction in the injected paw (primary response) with an increase in planter volume of about 60% over the baseline value. In addition, a disseminated arthritic reaction (secondary response) developed from day 1 after Freunds complete adjuvant (FCA) injection. Thus, swelling could be observed both in the injected paw and the non-injected contra-lateral paw. The effect of SA-004 on the development of adjuvant arthritis is shown in Table 1. The reduction of the planter volume was statistically significant for both hind legs and the reduction of edema was more pronounced in the injected paw than in the non-injected paw. For peripheral analgesic activity SA-004 was tested by acetic acid induced writhing in mice. The analgesic activity of SA- 004 in this test proved to be a little more (51.44%) than that of aceclofenac (44.57%). In the Randall-Selitto assay for analgesic activity, SA-004 significantly raised the pain threshold of the inflamed paw (67.9%) as compared to aceclofenac (57.1%). The compound was further screened for ulcerogenic activity. The extent of gastric damage caused by SA-004 and reference drugs after single oral administration to 24 h fasted male rats is shown in the Table 2. Ulcerogenic effect of SA-004 on the stomach was nonexistent at the dose at which it showed anti-inflammatory effect. Hence gastric tolerance to SA-004 was better than the reference drug CONCLUSION In conclusion, a mutual prodrug of aceclofenac was synthesized with an objective of developing a gastrosparing prodrug with better pharmacological profile. In order to lower the ulcerogenic potential and enhance effectiveness of aceclofenac as antiarthritic agent, a mutual prodrug was synthesized with neutraceutical carrier D-glucosamine that has shown to stimulate the biosynthesis of glucosaminoglycans and hyaluronic acid backbone needed for the formation of the proteoglycans found in the structural matrix of joints. It was interesting to note that conjugation of aceclofenac with glucosamine not only lowered its ulcerogenic potential but also enhanced its analgesic, antiinflammatory and antiarthritic activities noticeably thus proving the utility of D-glucosamine as an effective carrier and correctness of mutual prodrug hypothesis.
ACKNOWLEDGEMENTS The authors are thankful to IPCA Laboratories, Mumbai and Mr. V. Mohan, Consultant, Pune, for providing the gift samples of aceclofenac and glucosamine respectively. The authors are also thankful to the Department of Chemistry, University of Pune, for spectral analysis of the compound.
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