Rheumatoid arthritis is a chronic inflammatory disease which chiefly affects synovial articulations and leads to damage of articular gristle and anchylosis of articulations. The consequence of assorted drug intercessions like the amethopterin, dipyridamole and ATP entirely and in combination on RA were studied. RA was introduced with the aid of Freund ‘s adjuvant ( 0.5ml/200gms wistar rat ) in 6 Separate Wistar rat groups. Freund ‘s complete adjuvant ( FCA ) is the combination of mineral oil, an emulsifier mannide monooleate with 1mg/ml heat killed dry mycobacteria TB H37Ra. The mycobacteria stimulate the immune system and heighten the immune response. On 11th twenty-four hours after FCA the assorted animate beings groups were treated with drug intercessions and after 10 yearss of intervention the paw hydrops of the several rats were assessed by a quicksilver supplanting plethysmograph ( INCO Corp. , India ) . During these surveies the alterations in paw hydrops by drug intervention of animate beings were analyzed. The consequences of this survey are discussed in this article.
Cardinal words: Freund ‘s adjuvant, dipyridamole, amethopterin, arthritis, ATP
Rheumatoid arthritis ( RA ) is a chronic, autoimmune, inflammatory upset of unknown aetiology that is characterized by symmetric synovitis and the leaning to do joint devastation, disablement and premature decease. Disease-modifying anti-rheumatic drugs ( DMARDs ) decelerate the natural class of the disease, cut down joint harm and hurting, and retard loss of map and disablement. Treatment plays a cardinal function in commanding the redness of arthritic arthritis and minimising joint harm. Treatment normally entails a combination of drug therapy and other non-drug therapies. The degrees of proinflammatory cytokines such as tumour mortification factor-alpha ( TNF-? ) , interleukin-1-i?? ( IL1-i?? ) and interleukin-6 ( IL-6 ) are increased in creaky articulations ( 1 ) , and these cytokines have been implicated in the joint devastation and tissue disfunction seen in arthritic arthritis ( 2 ) .
The advocation of low dose amethopterin ( MTX ) is the most widely used anti-rheumatic drug and it is the ‘gold criterion ‘ against which other systemic medicines are compared ( 3 ) . . It has been suggested that at least portion of the anti-inflammatory effects of the disease modifying anti-rheumatoid drugs amethopterin and sulphasalazine may be attributable to the release of the endogenous nucleoside adenosine by these drugs ( 4 ) . It has as its mark the enzyme dihydrofolate reductase, which is required for decrease of dihydrofolate to tetrahydrofolate ( 5 ) . Adenosine, an extra active metabolite of MTX has been found to be powerful anti-inflammatory effects, and earlier surveies strongly back up the impression that the powerful anti-inflammatory consequence via suppression of inflammatory cytokines such as tumour mortification factor ( TNF ) -alpha, interleukin-6, or macrophage repressive protein-1 alpha. It was further found that the anti-inflammatory consequence of adenosine is mediated via A2A and the A3 adenosine receptors.
The extremely selective A3 adenosine receptor ( A3AR ) agonist 1B-MECA had an anti-inflammatory consequence in collagen-induced arthritis in DBA1 mice and adjuvant-induced arthritis ( AIA ) in rats. Adenosine is progressively regarded as an of import endogenous regulator of inflammatory procedures. All the adenosine receptors presently recognised-A1, A2A, A2B and A3 can modify the release of inflammatory go-betweens or reactive O species from immune-competent cells ( 6, 7 ) . Most attending has been devoted to the A2 receptors, with the A2A subtype being shown to diminish the secernment of TNF-alpha ( 8, 9 ) and IL-12 ( 10,11 ) every bit good as stamp downing the mitochondrial respiratory explosion ( 12, 13 ) . There is besides grounds that similar activity may be shown by A2B receptors ( 14, 15 ) and A3 receptors ( 16, 17 ) , while A1 receptors can advance the release of some cytokines ( 18 ) but suppress IL-6 secernment. The construct of a cardinal function for adenosine receptors in inflammatory procedures is supported by the up-regulation, particularly of A2A and A2B receptors, that occurs in activated monocytes and macrophages. Dipyridamole is a widely established drug for the intervention of angina pectoris and cerebrovascular disease. Its chief mechanism of action is to suppress the cellular consumption and metamorphosis of adenosine, with ensuing vasodilatory and anti-aggregatory effects. On the other manus, ATP is besides found to be converted to adenosine and may hold outstanding functions endogenously ( 19 ) . We have now investigated the hypothesis that an addition in adenosine degrees in patients, induced by chronic intervention with dipyridamole, may in bend lead to a decrease in proinflammatory cytokine release, and could relieve the symptoms of arthritic arthritis. This survey was so far conducted to determine the anti-arthritic consequence of dipyridamole and ATP and interactive consequence with amethopterin in adjuvant induced arthritis in albino Wistar rats.
Materials and methods:
Dipyridamole and ATP were obtained from Sigma chemical company ( USA ) . Dipyridamole was dissolved in 0.1 N HCl and ATP was dissolved in unfertile H2O before utilizing it for injection. Methotrexate and H2O for injection was obtained from the CMC pharmaceutics, Vellore.
Trial animate beings:
Wistar Lewis rats of either sex weighing about 175 to 225 grams were used in the surveies. Animals were housed in coops under standard lab conditions ( 12:12 hour light dark rhythm at 25 ± 5 degree C ) .The rats were fed with commercial rat diet and H2O ad libitum and were divided in to 6 groups, incorporating 6 animate beings in each group. The ethical guidelines for the engagement of the animate beings used in experiments were followed in all the trials.
Initiation of arthritis:
Arthritis was induced by a modified method described earlier ( 20 ) . Arthritis was induced by shooting 0.5 milliliter Freund ‘s complete adjuvant ( FCA ) incorporating 1mg/ml of dry heat killed Mycobacterium butyricum of unfertile paraffin oil in to plantar surface of right hind pes of all the animate beings.
Animals were divided in to 6 groups of 6 rats in each group. On the 11nth twenty-four hours after shooting FCA, the animate beings in the assorted groups received the several drugs as follows: – Group 1 received saline 0.5 milliliter of 0.9 % NaCl and served as the control. Group 2 received MTX at a dosage of 0.2 mg/kg on alternate yearss for a entire period of 10 yearss. Group 3 received Dipyridamole at a dosage of 10mg/kg for 10 yearss. Group 4 received ATP at a dosage of 0.75 mg/kg for a period of 10 yearss. Group 5 received MTX 0.2mg/kg on alternate yearss along with Dipyridamole 10 mg/kg daily for 10 yearss. Group 6 received MTX 0.2 mg/kg on alternate yearss, along with ATP 0.75 mg/kg daily for a entire period of 10 yearss. After 10 yearss of intervention the paw hydrops of the several rats were assessed by a quicksilver supplanting plethysmograph ( INCO Corp. , India ) . The per centum suppression of paw hydrops was calculated utilizing the expression 1-Vt/Vc X100. Where by paw volume before arthritis ( Vt ) -paw volume after intercession ( Vc ) / paw volume before arthritis ( Vt ) X 100
Statistical analysis: The statistical analysis was conducted for control and trial group of animate beings and comparings were done between the control and trial groups incorporating n=6 figure of animate beings. The statistical trials applied were the Wilcoxon signed ranks trial and Man Whitney trial and it was used for the analyses of the information. P & A ; lt ; 0.05 were considered to be statistically important.
Consequences: The consequences of this survey are shown in Figure 1 and Table 1. Treatment of different animate being groups ( n=6 ) with dipyridamole ( 10mg/kg ) and amethopterin ( 0.2mg/kg ) caused significant and important betterment in paw hydrops caused by FCA injection. Similarly ATP ( 0.75mg/kg ) was injected to the rats and it besides caused a 36.11 % decrease in paw hydrops. The betterment in paw hydrops was evaluated by a quicksilver supplanting plethysmograph. The combination of amethopterin and dipyridamole produced a potentiated 46.39 % decrease in paw hydrops after intervention with these drugs for a continuance for 10 yearss. However the combination of amethopterin and ATP did non bring forth appreciable betterment as per our consequences and caused merely a 9.72 % decrease in accessory arthritis.
In our survey, MTX produced a good consequence on FCA induced arthritis and this can be attributed to assorted factors. Amongst the first line of drugs available for arthritic arthritis, the foremost of import is the drug MTX which is given at a low dosage. It is now widely accepted that the mechanism of action of MTX in RA is due to the suppression of the enzyme AICAR transformylase by the MTX polyglutamates, which are formed after MTX is taken up by the inflammatory cells ( 21,22 ) . Since AICARiboside straight inhibits ADA ( adenosine deaminase which deaminates adenosine in to inosine ) and AICAR inhibits AMP deaminase, the intracellular accretion of AICAR could take to let go of of adenosine and AMP ( converted by 5 ‘ nucleotidase to adenosine in the extracellular infinite ) in to the extracellular infinite. It is this addition in adenosine concentration in the extracellular infinite which is responsible for the anti inflammatory consequence seen in patients treated with MTX is proved in experiments conducted by Cronstein et Al ( 23,24 ) . To farther turn out that this adenosine released was responsible for the anti-inflammatory consequence, was apparent in other experiments where agents like adenosine kinase inhibitors ( 25-27 ) , sulfasalazine were used and were shown to hold anti inflammatory consequence as good by an adenosine dependant mechanism, in both in vitro and in vivo experiments. More support that MTX enhances adenosine release in worlds, was provided by recent surveies which showed that, kids treated with MTX for childhood leukemia, had increased adenosine concentration in the cerebrospinal fluid.
Similarly a decrease in paw hydrops was seen after intervention with dipyridamole and ATP therefore farther the impression that adenosine is responsible for relieving the symptoms of arthritic arthritis. ATP has been shown to be converted to adenosine endogenously and alleviate the symptoms of arthritis ( 19 ) , nevertheless it was less powerful so dipyridamole and MTX. The anti-inflammatory action caused by the drugs dipyridamole could be because of it ‘s reuptake suppression of adenosine at the redness site. The survey by German et Al ( 28 ) screening that dipyridamole enhances plasma adenosine degrees in human existences supports our claim that dipyridamole can do addition in adenosine degrees. And the concentrations of adenosine required to suppress inflammatory cell map are similar to those observed in vivo ( 29 ) and therefore an addition in adenosine caused by dipyridamole can really good hold an anti-inflammatory action. The adenosine therefore released extracellularly, interacts with adenosine receptors on the cell surface and produces decrease in redness by suppression of inflammatory cytokines such as tumour mortification factor ( TNF ) -alpha, interleukin-6 ( 30 ) . Of peculiar involvement is the interaction of adenosine on A2a receptor on stimulated neutrophils, that leads to suppression of release of toxic O metabolites, and adhesion to a assortment of different cell types, and suppresses neutrophil mediated hurt both in vitro and in vivo ( 31,32 ) . With the above grounds, we besides observed whether the adenosine reuptake-inhibitory action of dipyridamole would be sufficient to do an anti inflammatory action in the above experiment, and likewise whether the adenosine released from the metamorphosis of ATP would hold any anti inflammatory action every bit good. Our consequences portray that dipyridamole and methotrexate intervention per se significantly attenuated the paw hydrops. These consequences are fresh and interesting. We besides tried to measure, whether uniting the drugs dipyridamole with amethopterin had any interactive consequence, on the footing that the adenosine released from MTX and adenosine re-uptake suppression by dipyridamole, increased the extracellular concentration of adenosine and this could take to an enhanced anti inflammatory consequence in what can be described a possible drug synergy. Our consequences therefore confirm a outstanding function for dipyridamole and amethopterin combination in relieving the paw hydrops induced by FCA.
Consecutive No. Treatment % alteration from control ‘P’Value
1 ) Saline -9.72 P & A ; lt ; 0.05
2 ) Dipyridamole 40.83 P & A ; lt ; 0.05
( 10mg )
3 ) Methotrexate 37.5 P & A ; lt ; 0.05
( 0.2 milligram )
4 ) ATP 36.11 P & A ; lt ; 0.05
( 0.75mg )
5 ) Methotrexate + Dipyridamole 46.39 P & A ; lt ; 0.05
6 ) Methotrexate + ATP 9.72 P & A ; gt ; 0.05
Legend for Figures
Fig 1: Figure 1 illustrates the saloon graphs picturing the effects before and after injections of dipyridamole ( 10mg/kg ) , amethopterin ( 0.2mg/kg ) and ATP ( 0.75mg/kg ) on Freund ‘s complete adjuvant induced paw hydrops in albino rats. The values depicted are from 6 experiments each.
Table 1: Table 1 shows the consequence of dipyridamole ( 10mg/kg ) , amethopterin ( 0.2mg/kg ) and ATP ( 0.75mg/kg ) on paw hydrops induced by Freund ‘s complete accessory injection. The per centum alteration from control group ( saline treated ) is compared with treated group and statistical significance is given. The values depicted are from 6 experiments each.