INTRODUCTION:

Gout is most common painful clinical syndrome occurring due to hyperuricaemia (high serum uric acid level) and deposition of monosodium urate crystals in joints. Hyperuricemia is a condition in which uric acid concentration in serum is more than 7mg/dl that cause supersaturation of urate in plasma and deposition of uric acid crystal in salt form i.e. monosodium urate crystals (MSU). These crystals stimulate humoral and cellular pathway procedure that produces sever pain and inflammation in the joint^1-2.

Hyperuricemia can be caused by obesity, lipid abnormalities, hypertension and some other factor like high purine intake, increased alcohol consumption and drug therapy like diuretics hydrochlorothiazide and furosemide. The enzyme xanthine oxidase transformed purine bases are into uric acid by oxidation process. Uric acid is the last by product of purine nucleotides metabolism and it is being converted into alantion by uricase enzyme in liver of the animals only. Uricase enzymes absent in human body³. When the uric acid concentration more than 7mg/dl or supersaturation solubility limit of uric acid and it may turn to form crystals with deposition on various tissues and joints⁴.

Gout is categorized into primary gout and secondary gout. Primary gout is a genetic disorder characterized by an underlying biochemical defect resulting in hyperuricaemia⁵. Approximately 90% cases of gout are primary and more than 99% of those are idiopathic in nature. It may be occurs due to a combination of genetic, hormonal, and dietary causes. Secondary gout refers to those cases in which hyperuricaemia is due to other disorders such as an increased nucleic acid turnover or increased purine biosynthesis de novo¹.

The amount of serum uric acid depends on the rate of production and elimination of uric acid in the body. Due to specific enzyme hypoxanthine- guanine phosphoribosyl transferase and pp- ribose –P synthetase abnormalities cause over production of uric acid. Clinical manifestations of gout occur due to the formation and storage of uric acid crystals (monosodium urate crystals) in the body⁶. Uric acid crystals may deposit in various joints and other tissues like cartilage, tendons, bone and soft tissues. Chronic gout attack occurs, as evidenced by tophi that grow silently for years.⁴ Monosodium urate crystals increase the release of various inflammatory cytokines/ mediators but they rapidly phagocytized by neutrophilis during acute attack. Cytokines including interleukin 1, release during a gout attack responsible for fever and leukocytosis. The neutrophils flow is a vital for formulating acute crystal stimulated synovitis. Gouty inflammation of joints connected with cytokine directed synovial propagation, damage of cartilage and destruction of bone⁷. NSAIDs are most commonly utilized to cure the severe pain and swelling of gouty patient. Selection of drug of choice in gout depends upon the safety and efficiency⁸.

Figure 1. Pathway of MSU induce inflammation

Aceclofenac is a nonsteroidal anti-inflammatory drug having analgesic and anti-inflammatory activity. Chemically, aceclofenac is 2-[2, 6-Dichlorophenyl) amino] benzoic acetic acid carboxymethyl ester and utilized in various painful condition like rheumatoid arthritis, osteoarthritis and ankylosing spondylatis.^9-10 The mechanism of aceclofenac is to reduce the production of prostaglandin by inhibiting the enzyme cyclooxygenase, which is used in the prostaglandins synthesis. It also prevents the production of the inflammatory cytokines like interleukin (IL)–1β and tumor necrosis factor (TNF).¹¹ Aceclofenac is selectivity for COX2 receptor inhibitor. It is observed that diclofenac shows unwanted effect like gastric distress, indigestion and intestinal pain similar to naproxen and piroxicam in patients with osteoarthritis.¹²Now a day number of aceclofenac formulation are available in market with standard dose but because of short half life, its administration is repeated twice or thrice for compatible therapeutic effect. So, aim of this research to formulate slow release granules and study the effect on gouty arthritic model of rat.

MATERIALS AND METHODS:

Drug:

All the reagents used in this study were of analytical grade and the reagent solutions were prepared using double distilled water. Aceclofenac pure drug was obtained as a gift sample from Jackson Pharmaceutical Private Limited, Amritsar. Aceclofenac sustained release granules were prepared to study the effect on monosodium urate induce inflammation model in rat animal.

Formulation of Sustained Release granules of Aceclofenac:

All the ingredients were passed through sieve #40 and were subjected to drying to remove moisture content at 40⁰C to 45⁰C. Weighed amount of acelofenac and excipients were mixed properly by geometric addition method. Granulation had done with a binder solution of PVP K-30 with Isopropyl alcohol. The granules were passed through sieve #12 and kept for drying at 50^oC for half an hour. Dried granules were passed through sieve #20. Magnesium stearate and purified talc were passed through sieve #60, mixed and blended well with the initial formed mixture.

Animals:

Male Wister rats with average body weight of 150-180 gm were utilized in this study. The animals were conserved individually in metabolic stainless steel cages at light and optimum temperature maintained rooms with a 12 hour dark-light cycle and deliver the standard rat pellet and water under strict hygienic conditions.

Monosodium Urate Crystals Synthesis:

Monosodium urate crystals were developed by a combination of 1gm uric acid, 6ml of 1M sodium hydroxide and 194ml of purified water was heated. This mixture maintains pH 7.2 by 1N hydrochloric acid. The above mixture was beaten slowly and stored at 4 ℃ for 24 hours for formation of monosodium crystals and then washed. After washing, the crystals suspended in sterile saline solution of concentration 100 mg/ml^13-14.

Design of experimental study:

In this study, the different formulated granules of aceclofenac (3 mg/kg body weight) were received by rats (group IV to VI) in simple syrup (as vehicle) by intra-gastric administration once/twice daily for 14 successive days. Group-III also received simple syrup (as vehicle) for continue 14 days. On 14^th day, the gouty arthritis inflammatory model were induced 1 hour after aceclofenac granules, by single injection of 50 μl monosodium urate crystal suspension injected in the ankle joint of rat. The study was approve by IAEC.

Total thirty six (36) rats were randomly divide into six following groups (n=6).

Group I: Normal Group

Group II: Positive control Group

Group III: Vehicle control Group

Group IV: Conventional release granules of aceclofenac (once daily for 14 successive days)

Group V: Conventional release granules of aceclofenac (twice daily for 14 successive days)

Group VI: Sustained release granules of aceclofenac (once daily for 14 successive days)

Evaluation of the Joint Swelling:

The ankle joint volume was observed at 1st, 12th and 24th hrs after injection of monosodium urate crystal.

The percentage of joint swelling (Joint edema rate (%) = (joint swelling after modeling of rat / joint swelling before modeling of rat -1) ×100) could be measured.

Statistical Analysis - Results were expressed as mean ± S.D. and a statistical analysis was performed using ANOVA, to determine the significant differences between the groups, followed by Student’s Newman-Keul’s test. p< 0.05 implied significance.

RESULTS & DISCUSSION:

Formulation of Sustained Release granules of Aceclofenac - All the ingredients were used for formulation of sustain released granules of aceclofenac

Table 1. Formulation for aceclofenac sustain release granules by wet granulation

Ingredients	Quantity
Drug	200mg
Mannitol	8mg
Lactose	8mg
HPMC K 100	60mg
Binder solution PVP K-30 Isopropyl alcohol	9mg 100ml
Magnesium stearate	9mg
Purified talc	6mg
Total weight	300mg

Flow properties of Aceclofenac Granules:

The granules prepared for compression of aceclofenac sustain layer were evaluated for their flow properties. According to the USP limits Aceclofenac granules formulation had good flow whereas granules of all other formulations had excellent flow. Hausner ratio indicated that of Aceclofenac granules of formulations had good flow. Optimized formulation F8 with value of hausner’s ratio less that 1.18 had excellent flow. Compressibility index values of this formulation showed good flow (Table-2).

Anti-inflammatory effects of Aceclofenac granules in MSU crystal- induced gouty arthritis rats

As shown in table 3, the percentage of joint swelling rate of positive control group was increased significantly (p<0.05) when compared with normal group after 1 hr, 12hrs and 24 hrs with a single injection of MSU. The percentage of joint swelling rate was significantly (p<0.05) reduced with different formulated granules of aceclofenac when compared with positive control group after 12 hrs but not significantly reduced with vehicle treated groups. The results after 24 hrs showed that conventional release granules (BD) and sustained release granules (OD) significantly (p≤0.05) reduced percentage joint swelling rate as compared with positive control group (Figure .2). This shows therapeutic effect of sustained released granules (OD) is similar to conventional released granules (BD) and possesses an anti-inflammatory effect, which could provide relief for the gouty arthritis after administration of sustained release formulation of aceclofenac once in a day.

CONCLUSION

Current study evaluated the anti-inflammatory effect of slow release aceclofenac granules in mono sodium urate crystals produced gouty arthritis and also compared with conventional granules. This model responded with a local inflammatory reaction associated with edema and erythema of joints, together with severe pain after injection of exogenous manufactured MSU crystals. The results of present study concluded that the oral administration of formulated sustained release granules of aceclofenac could provide much more relief from joint swelling (Inflammation) in gouty arthritis when given once in a day (OD) instead of multiple administration (BD) of conventional release granules of aceclofenac. This will help in reducing the frequency (multiple dosing) of aceclofenac tablets formulated by conventional granules and also minimal the side effect of aceclofenac in gouty arthritis patient during long term use.

ACKNOWLEDGEMENT:

We are highly thankful to the Animal Ethical committee of MM College of Pharmacy, for the permission given.

CONFLICTS OF INTEREST:

The authors declare no conflict of interest.

REFERENCES:

1. Manjuladevi KR, Krishnan RS, Rahini P. A Review on gouty Arthritis. Research J. Pharm. and Tech. 2019; 12(11): 5583-5588

2. Jerine Peter S, et al. Evaluation of the Antioxidant and Potential binding affinity of Cyamopsis tetragonoloba seed against the receptor responsible for Gouty Arthritis. Research J. Pharm. and Tech 2020; 13(5): 2275-2281.

3. Gagliardi ACM, Miname MH, Santos RD. Uric acid: A marker of increased cardiovascular risk. Atherosclerosis. 2009; 202(1): 11-7.

4. Sherman MR, Saifer MGP, PerezRuiz F. PEG-uricase in the management of treatment-resistant gout and hyperuricemia. Advanced Drug Delivery Reviews 2008; 60(1): 59-68.

5. Wright JD, Pinto AB. Clinical manifestations and treatment of gout. Primary Care Update for OB/GYNS. 2003; 10(1): 19-23.

6. Kumaresan GD,Dhanraj M. Efficacy of Cox-2 inhibitors in the Clinical Management of TMJ Arthritis: A Review. Research J. Pharm. and Tech 2017; 10(12): 4439-4441.

7. Falasca GF. Metabolic diseases: gout. Clinics in dermatology. 2006; 24(6): 498-508.

8. Paul BJ. Crystal Arthritis - Past, present and future. Journal of Medical Education and Research. 2006; 8(4):232-4.

9. Shah R, et al. Validated spectroscopic method for estimation of aceclofenac from tablet formulation. Research J of Pharm and Tech. 2008; 1(4) 430-2.

10. Rajesh. M, and Narayanan. Comparative Study on Anti-inflammatory and Analgesic Activities of Pure Aceclofenac with the Aceclofenac Loaded Mucoadhesive Microparticles in Rats. Research J. Pharm. and Tech. 2015; 8 (2): 185-188

11. Dooley M, Spencer CM, Dunn CJ. Aceclofenac: a reappraisal of its use in the management of pain and rheumatic disease. Drug. 2001; 61(9): 1351-78

12. RasheedN., et al. Simultaneous Formulation, Evaluation and estimation of controlled release of NSAID Drug. Asian J. Pharm. Ana. 2017; 7(3): 176-184.

13. Ortiz-Bravo E, Sieck MS, Schumacher HR. Changes in the proteins coating monosodium urate crystals during active and subsiding inflammation. Arthritis and Rheumatology. 1993; 36(9): 1274-85.

14. Fam AG, et al. A comparison of five preparations of synthetic monosodium urate monohydrate crystals. Journal of Rheumatology. 1992; 19(5): 780-787.

Received on 22.04.2020 Modified on 19.10.2020

Research J. Pharm. and Tech 2021; 14(11):5729-5732.

DOI: 10.52711/0974-360X.2021.00996

Formulation Code	Angle of repose	Bulk volume	Tapped volume	Hauser ratio	Compressibility index (%)
F1	32.67	11.2	9.9	1.13	11.06

Groups	1hr	12hr	24hr
Normal group	5.67±3.23	5.42±2.66	5.45±2.90
Positive control group	34.41±4.01**	34.53±3.98**	33.83±3.80**
Vehicle treated group	33.39±3.34	33.30±4.26	31.17±3.96
Conventional release granules (OD)	33.44±3.40	24.61±3.26	23.95±3.14
Conventional release granules (BD)	32.43±3.75	24.81±3.97	14.56±3.70*
Sustained release granules (OD)	33.86±3.11	22.37±3.01*	13.32±2.37*