JPM Vol. 31, No. 2 April 1994:95-98

Application of Papaya Latex-Induced Rat Paw Inflammation: Model for Evaluation of Slowly Acting Antiarthritic Drugs

0. P. Gupta, N. Sharma, and D. Chand

Department of Pharmacology, Regional Research Laboratory, Jammu-Tawi, India

Papaya latex-induced rat paw inflammation model for evaluating antiinflammatory activity has been developed and reported earlier. A number of drugs viz. aspirin, indomethacin, piroxicam, ibuprofen, prednisolone, levamisole, chloroquine, and boswellic acids showed antiinflammatory activity in this model. As the last three drugs showing the activity be- longed to the group of slowly acting antiarthritic drugs, this present study was undertaken to study in detail the sensitivity of this model for slowly acting, clinically effective, anti- arthritic drugs viz. chloroquine, levamisole, penicillamine, aurothioglucose, cyclophospha- mide, and boswellic acids. These drugs are known to show no appreciable activity in the known models of inflammation and arthritis. All these drugs tested in three graded doses showed dose-related significant antiinflammatory activity in this model, whereas those drugs in the carrageenan model tested in similar doses showed insignificant activity. Aspirin employed as a reference standard showed significant activity in both the models. Thus the slowly acting antiarthritic drugs will be identified as those displaying significant activity in the papaya latex model and insignificant activity in the carrageenan model and to be aspirin- like by their significant activity in both the above models of inflammation.

Keywords: Papaya latex; Rat Paw Inflammation; Antiarthritic drugs; Model for evaluation

Introduction

An ideal drug for treatment of rheumatoid arthritis still awaits discovery. Failure to discover such a drug may be attributed to a lack of both our exact knowledge about the etiology of human disease and an ideal exper- imental model of rheumatoid arthritis. The models de- veloped so far are good only for testing steroidal and aspirin-like antiinflammatory drugs. The slowly acting antiarthritic drugs effective clinically do not show any appreciable activity in either these models or the com- monly employed model of adjuvant arthritis in rats (Lewis et al., 1985).

In light of these facts, we decided to develop a new ideal model for antiarthritic evaluation. Given the pos- sibility for lysosomal enzymes to damage tissue and their role in adjuvant-induced arthritis (Anderson,

Address reprint requests to Dr. 0. P. Gupta, Department of Phar- macology, Regional Research Laboratory, Jammu-Tawi 180 001, India.

Received January 1, 1993; revised and accepted September 1, 1993.

Journal of Pharmacological and Toxicological Methods 31, 95-98 (1994) 0 1994 Elsevier Science Inc., 655 Avenue of the Americas, New York, NY 10010

1970; Ignarro and Slywka, 1972; Veto et al., 1972; Perse, 1974; Rainsford, 1987), we induced inflamma- tion/arthritis by employing a substance that mimicked lysosomal enzymes. Trials utilizing papaya latex which contain the hydrolytic enzymes papain and chymopo- pain proved to be rewarding. The inflammation pro- duced by papaya latex responded to steroidal, aspirin- like, and slowly acting anti-arthritic drugs as previ- ously reported (Gupta et al., 1992).

The present study was undertaken to investigate in detail all known slowly acting antiarthritic drugs by the papaya latex model as well as the conventionally employed carrageenan model and thereby understand the sensitivity of the new model for the slowly acting antiarthritic drugs.

Materials and Methods

Induction of In.ammation and Antiinflammatory Evaluation

Male albino Charles-Foster rats (150-200 g) were employed in this study. Test drugs were administered

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96 JPM Vol. 31, No. 2 April 1994:95-98

p.o. except for aurothioglucose which was adminis- tered intramuscularly. Paw volume was measured be- fore and 3 hr after the injection of inflammagen, using volume differential meter. model 7101 UC0 Basile.

Pretreatment Time

Because the test drugs employed are also called slowly acting ~ntir~eu~ati~ drugs, in addition to evalu- ating these drugs as per the conventional procedure that requires treating the animals 1 hr before injection of inflammagen, we also tested these drugs by treating the animals with test drugs 24 hr prior to drug adminis- tration.

Znflammagens

Papaya latex. This was obtained from unripe Pa- paya fruit as reported already (Gupta et al., 1992). We prepared 1% stock suspension of the latex in 0.5 M sodium acetate buffer, pH 4.5, containing 0.01% thy- mol (used as 1% solution in alcohol). The stock suspen- sion was diluted with buffer to make 0.25% suspension. Of this suspension, 0.1 mL was injected in the rat paw.

Carrageenan. We injected 0.1 mL of 1% aqueous suspension of this product in rat paw. (The product was obtained from Springfield, MA, U.S.A.)

Test Drugs

The following drugs were employed. Chloroquine phosphate (IPCA), levamisole hydrochloride (Olym- pus ~nte~rises), Cyclophosphamide (Khandelwal Laboratories), aurothioglucose (Sigma), D( -)penicillamine (Sigma), aspirin, and boswellic acids (RRL, Jammu). These test drugs were used as a fine homogenized suspension in gum acacia in the solution so that the dose for 100 g of body weight of rat was present in 1 mL of suspension. D( - )Penicillamine and aurothioglucose were used as aqueous solutions.

Results

Thus, from the above observations, it may be said that slowly acting antiarthritic drugs while showing sig- nificant activity in papaya latex model show insignifi- cant activity in the carrageenan model and if the test drug shows significant activity in both models of in- flammation, the the drug is likely to be steroidal or aspirin-like in nature. This generalization is likely to hold true for up-and-coming drugs as well since the drugs of diverse chemical nature belonging to either group have responded like the other members of that group. One of the main considerations in a screening program for any kind of drug is the ability to screen compounds of maximum number of chemical types. The papaya latex model seems to qualify this consider- ation.

All the slowly acting antiarthritic drugs viz. chlo- It is to be noted that it is the acute inflammation roquine , cyclophosphamide, levamisole, penicilla- induced by papaya latex which is being inhibited by mine, aurothioglucose, and boswellic acids employed slowly acting antiarthritic drugs, effective clinically in in three graded doses in effective range significantly arthritic conditions, associated with inflammation, and inhibited the rat paw inflammation induced by papaya chronic in nature. The inflammation induced by carra- latex as compared to those in control groups. Tested geenan is also acute in nature but is not being inhibited in the same doses, these drugs failed to show any signif- by slowly acting antiarthritic drugs. In the light of this, icant activity in ca~ageenan-induced rat paw inflam- it can be said that the mediators of in~ammation in- mation. Only chloroquine and levamisole showed sig- volved in the clinically encountered arthritic condi- nificant activity in the highest dose of the three doses tions and in papaya latex-induced inflammation are tested. Aspirin employed as a reference standard in common to a great extent, attributing to effectiveness this study showed significant activity in both papaya of slowly acting antia~hritic drugs in clinically encoun-

latex and carrageenan models of inflammation. These animals were treated with the test drugs 1 hr before the injection of inflammagen (Table I).

In the 24-hr pretreatment study also, the above drugs, except for penicillamine, showed significant ac- tivity. The percentage of inhibition of inflammation recorded as compared with control groups for these drugs are chloroquine 38.6 + 2.8, cyclophosphamide 30.5 + 3.3, levamisole 36.8 ? 3.0, penicillamine 16.2 f 1.5, aurothioglucose 28.4 ? 3.9, and boswellic acids 29.0 ? 2.4% in 25, 5, 20, 25, 10, and 100 mglkg p.o. doses, respectively (aurothioglucose injected intra- muscularly). These values are the mean * SEM ob- served in 15 animals for each drug. Aspirin too, tested in 200 mglkg p.o. dose showed 3 1.4% r 2.5% of inhibi- tion of intlammation as compared to that of control groups.

Discussion Slowly acting antiarthritic drugs employed in this

study showed significant antiinflammatory activity in papaya latex model and insigni~cant activity in the car- rageenan model as also reported in literature. On the other hand, steroidal and aspirin-like drugs exhibited significant antiinflammatory activity in both papaya latex and carrageenan models of inflammation as al- ready reported (Gupta et al., 1992).

0. P. GUPTA ET AL. RAT PAW INFLAMMATION

97

Table 1. Evaluation of Slowly Acting Antiarthritic Drugs by Carrageenan/Papaya Latex-Induced Rat Paw Inflammation Models

% Inhibition of Inflammation Compared with Control

Case Drug No. (mg/kg P.o.)

1. Chloroquine

2. Cyclophosphamide

3. Levamisole

4. Penicillamine

5. Aurothioglucose”

6. Boswellic acids

7. Aspirin

NOW: No. of animals in each group = 20. LI Administered intramuscularly. * p < 0.001. ** p < 0.02. *** p < 0.1. **** p < 0.5.

Dose

15 25 35 2.5 5.0 7.5

10 20 30 15 25 35 5

10 15 50

100 150 100 150 200

Group Mean ? SEM

Carrageenan Model Papaya Latex Model

4.0 * 1.6**** 28.9 2 3.F 7.0 f 2.7**** 34.9 2 3.5*

16.0 k 2.1** 40.0 ‘- 2.F 5.7 2 2.6**** 30.9 2 3.2*

10.5 2 2.7**** 31.2 f 3.3* 17.1 k 3.2** 38.3 z!z 3.4* 8.0 2 1.3**** 28.9 2 3.9*

11.9 f 3.8*** 36.3 2 3.2* 16.7 2 2.6** 37.7 * 4.0* 7.4 ? 3.5**** 18.3 t 2.8*

10.3 2 3.0*** 32.3 + 3.0* 12.3 2 2.5*** 36.5 k 2.2* 5.2 f 2.8**** 28.5 + 3.5* 6.4 k 2.9**** 35.2 + 3.8* 9.0 * 3.8*** 38.1 ” 3.7* 5.0 2 2.2**** 19.9 2 2.1*

10.7 k 2.7*** 26.7 ” 2.5* 11.5 t 3.3*** 29.1 ” 2.8* 10.5 k 2.1*** 29.4 + 3.5* 30.4 k 2.3* 34.0 2 2.8* 30.6 f 4.9* 36.8 2 3.0*

tered arthritic conditions and papaya latex-induced in- flammation.

That possibly the inflammation induced by papaya latex is being mediated through prostaglandins has been already pointed out in our earlier report (Gupta et al., 1992) as the drugs viz. aspirin, ibuprofen, indo- methacin, piroxicam, prednisolone, chloroquine, and boswellic acids were found to be effective against pa- paya latex-induced inflammation and are reported to be acting by inhibiting the prostaglandins. For aurothi- oglucose employed in the present study, its effective- ness in papaya latex-induced inflammation may be again attributed to the inhibition of prostaglandins as among the various mechanisms of action ascribed to gold salts. Studies maintain that these act by inhibiting the synthesis of prostaglandins (Flower et al., 1972; Store et al., 1975; Bray and Gorden, 1978). The effec- tiveness of penicillamine in papaya latex-induced in- flammation is likely to be due to its chemical reactivity as attributed to many of its effects including those on particular enzymes (Evans and Williamson, 1987).

Effectiveness of levamisole and cyclophosphamide in papaya latex inflammation may be again attributed to inhibition of prostaglandins. The two are reported

to be effective in rheumatoid arthritis due to immuno- stimulant and immunodepressant effects. However, pharmacologically it is not tenable that two drugs effec- tive in a situation could be acting by opposing pharma- cological actions. Most probably the two drugs are ef- fective in rheumatoid arthritis acting not through the immune system but through the same mechanism as is involved in inhibition of papaya latex-induced inflam- mation by the two drugs. This was possibly through the inhibition of prostaglandins as attributed to other drugs found effective in papaya latex-induced inflam- mation.

Aspirin employed as a prototype of antiinflamma- tory agents in this study exhibited almost the same ef- fect in 24 hr as did that in the I-hr pretreatment study in papaya latex-induced inflammation. This observa- tion is explainable from what has been reported in the literature that whole-body synthesis of prostaglandins in humans is depressed for at least 24 hr and sometimes as much as 48 hr after treatment with therapeutic doses of aspirin, indomethacin, and sodium salicylate (Hamberg, 1972; Horton et al., 1973; Koesis et al., 1973). Effectiveness of aspirin in papaya latex-induced inflammation even 24 hr after the treatment is explaina-

98 JPM Vol. 31, No. 2 April 1994:95-98

ble again on the basis of involvement of prostaglandins in the mediation of inflammation by papaya latex.

Thus, it may be concluded that the papaya latex model is sensitive for evaluating antiinflammatory drugs of various types including slowly acting anti- arthritic drugs which while showing significant activity in this model do show insigni~cant activity in carra- geenan model, and that inflammation induced by pa- paya latex seems to be mediated through prosta- glandins.

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