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Anti-inflammatory activity of indomethacin in copper deficient rats
2 54
Pharmacological Research, Vol. 25, Supplement 2, 1992
ANTI-INFLAMMATORY ACTIVITY OF INDOMETHACIN IN COPPER DEFICIENT RATS R . Milanino, M . Marrella, U. Moretti, M . Pasqualicchio, R . Gasperini and G.P. Velo Istituto di Farmacologia, UniversitA di Verona, Policlinico di Borgo Roma, 37134, Verona, Italy Key Words: acute inflammation, copper-deficiency, indomethacin, rat INTRODUCTION
In 1976 Sorenson proposed in his classical paper (1) that the coordination compounds formed in vivo between the copper present in serum and the clinically used non-steroidal antiinflammatory agents could represent the active metabolites of these drugs . However in vitro and computer simulation studies carried out by Arena and co-workers have subsequently shown that, at least for salicylic and acetylsalicylic acids, the complexes with copper do not seem to exist under plasma conditions being the ligands incapable of effectively competing for the metal (2) . Nevertheless in a recent paper copper-aspirinate has been shown to be significantly more active than an appropriate mixture of copper and aspirin when given orally to rats with local inflammation, indicating that, despite of any theoretical prevision, some intact complex seemed to survive at the acid pH present in rat stomach (3) . These latter results suggest that both in vitro and computer simulation studies may not be always adequately predictive of the in vivo situations . Thus, in the attempt to provide some in vivo evidence capable of sustaining or further questioning the Sorenson's hypothesis we decided to study, in a preliminary experiment, the acute anti-inflammatory activity of indomethacin in rats deprived of copper . MATERIALS AND METHODS
Female Sprague-Dawley rats (CD-COBS from Charles River, Italy) weighing 150±10 g at the beginning of the experiment were used . The animals were housed in groups of 5 to 10, on a 12h light-dark cycle, at constant temperature (20°±1°C) and humidity (55±5%) . Rats were fed ad libitum either on a standard diet (12 mg/kg of copper) or on a deficient diet (0 .2 mg/kg of copper) (both purchased from Ditta Piccioni, Brescia, Italy), and bi-distilled copper-free water. After 1 month of normal or deficient feeding paw oedema was induced by injecting the rats into the plantar surface of the right hind paw with 0 .1 ml of 1% carrageenan (X-carrageenan, Marine Colloids Inc .) suspended into sterile saline . Foot volume was measured by means of a water displacement device (Ugo Basile, Milano, Italy) . One hour before the carrageenan injection the rats were orally treated either with 2 mg/kg indomethacin suspended into 1% acacia gum (10 ml/kg) or with the vehicle alone . Copper determinations were carried out by flame atomic absorption spectrophotometry (Perkin Elmer 3030) on non-haemolytic plasma deproteinized by addition of an equal amount of 15% trichloroacetic acid. RESULTS
Following 1 month of copper deficient diet the general status of copper deprived rats was satisfactory, and no statistically significant differences of body weight were observed between copper deficient and normally fed animals (207±18 vs 219±15 g respectively) . On the other hand within 2-3 weeks from the beginning of the 0 .2 mg/kg copper deprived diet plasma copper concentration of the copper depleted group decreased below 10% of the control rats (15 .9±8 .1 vs 177 .0±28 .6 µg/100 ml respectively) . As expected from previous experiments (4,5), the induction of a copper deficiency status clearly promoted a statistically significant enhancement of the acute inflammatory response at all the examined times (3h = + 31%; 5h = + 18% ; 22h = + 27%) (see table) . The treatment with indomethacin at the oral dose of 2 mg/kg was found to be effective in reducing the foot oedema development in both normally fed and copper deprived rats (see table). However the per cent inhibition observed was by no means comparable in the two groups of animals being indomethacin significantly less active in the copper depleted group at all the considered times (3h = - 54% ; 5h = - 65% ; 22h = - 33%) (see table) . 1043-6618/92/25110254-02/$03 .00/0
© 1992 The Italian Pharmacological Society
Pharmacological Research, Vol . 25, Supplement 2, 1992
25 5
Table - Effect of indomethacin treatment on the foot-oedema developed by normal and copper-deficient rats . Group
3h Oedema vol . % Inh. ml (SD) (SD)
Normal Untreated
0.78 (0.08)
Normal Treated
0 .38 * (0.10)
Cu def. Untreated
1 .02 * (0 .14)
Cu def. Treated
0 .78 0 (0.13)
5h Oedema vol . ml (SD)
% Inh . (SD)
1 .00 (0.10) 51 .1 (18.9)
0 .54 * (0.12)
0.62 (0.09) 46 .9 (20 .1)
1 .18 * (0.08) 23 .7 ' (10.2)
0 .99 0 (0.13)
22h Oedema vol . % Inh . ml (SD) (SD)
0 .41 * (0.12)
34.1 (13 .1)
0 .79 (0 .10) 16.2 ' (11 .2)
0 .61 ° (0 .09)
22 .9 0 (9 .9)
* P<0 .01 vs Normal Untreated, ° P<0.01 vs Cu-def . Untreated, ' P<0.01 vs Normal Treated ; Student's "t" test . n = 10 rats per group . DISCUSSION
Although preliminary, the results described in this paper seem to support the hypothesis of Sorenson (1) suggesting that the presence of a normal amount of copper in plasma is needed in order to obtain a full expression of indomethacin anti-inflammatory potential . We like to note that, as previously observed studying female Sprague-Dawley rats maintained for one month on a 0 .2 mg/kg copper deficient diet (5), the copper deficiency status does not induce any biologically significant variations at the histological, haematological and haematochemical levels . The only change observed is a dramatic decrease of copper in plasma whose apparent consequences are an exacerbation of the acute inflammatory reaction as well as a reduction of indomethacin efficacy . Further work is however needed to better verify Sorenson's hypothesis, and it would be especially relevant to see whether or not the loss of anti-inflammatory activity in copper deficiency conditions is also shared by other non- steroidal anti-inflammatory drugs .
REFERENCES
1) Sorenson JRJ. Copper chelates as possible active forms of the antiarthritic agents . J Med Chem 1976,19 :135-48 . 2) Arena G, Kavu G, Williams DR . Metal-ligand complexes involved in rheumatoid arthritis . V . Formation constants for calcium(II)-, magnesium(H)- and copper(II)salicylate and acetylsalicylate interactions . J inorg nucl Chem 1978, 40 :1221-26 . 3) Korolkiewicz Z, Hac E, Gagalo I, Gorczyca P, Lodzinska A . The pharmacological activity of complexes and mixtures with copper and salicylates or aminopyrine following oral dosing in rats. Agents and Actions 1989, 26 :355-59 . 4) Milanino R, Conforti A, Fracasso ME, Franco L, Leone R, Passarella E, Tarter G, Velo GP . Concerning the role of endogenous copper in the acute inflammatory process . Agents and Actions 1979, 9 :581-588 . 5) Milanino R, Velo GP. Multiple actions of copper in control of inflammation : studies in copper deficient rats . In : Rainsford KD,Brune K, Whitehouse MW, eds. Trace elements in the pathogenesis and treatment of inflammation (AAS Vol . 8) . Basel : Birkhauser Verlag, 1981 :209-230 .
Pharmacological Research, Vol. 25, Supplement 2, 1992
ANTI-INFLAMMATORY ACTIVITY OF INDOMETHACIN IN COPPER DEFICIENT RATS R . Milanino, M . Marrella, U. Moretti, M . Pasqualicchio, R . Gasperini and G.P. Velo Istituto di Farmacologia, UniversitA di Verona, Policlinico di Borgo Roma, 37134, Verona, Italy Key Words: acute inflammation, copper-deficiency, indomethacin, rat INTRODUCTION
In 1976 Sorenson proposed in his classical paper (1) that the coordination compounds formed in vivo between the copper present in serum and the clinically used non-steroidal antiinflammatory agents could represent the active metabolites of these drugs . However in vitro and computer simulation studies carried out by Arena and co-workers have subsequently shown that, at least for salicylic and acetylsalicylic acids, the complexes with copper do not seem to exist under plasma conditions being the ligands incapable of effectively competing for the metal (2) . Nevertheless in a recent paper copper-aspirinate has been shown to be significantly more active than an appropriate mixture of copper and aspirin when given orally to rats with local inflammation, indicating that, despite of any theoretical prevision, some intact complex seemed to survive at the acid pH present in rat stomach (3) . These latter results suggest that both in vitro and computer simulation studies may not be always adequately predictive of the in vivo situations . Thus, in the attempt to provide some in vivo evidence capable of sustaining or further questioning the Sorenson's hypothesis we decided to study, in a preliminary experiment, the acute anti-inflammatory activity of indomethacin in rats deprived of copper . MATERIALS AND METHODS
Female Sprague-Dawley rats (CD-COBS from Charles River, Italy) weighing 150±10 g at the beginning of the experiment were used . The animals were housed in groups of 5 to 10, on a 12h light-dark cycle, at constant temperature (20°±1°C) and humidity (55±5%) . Rats were fed ad libitum either on a standard diet (12 mg/kg of copper) or on a deficient diet (0 .2 mg/kg of copper) (both purchased from Ditta Piccioni, Brescia, Italy), and bi-distilled copper-free water. After 1 month of normal or deficient feeding paw oedema was induced by injecting the rats into the plantar surface of the right hind paw with 0 .1 ml of 1% carrageenan (X-carrageenan, Marine Colloids Inc .) suspended into sterile saline . Foot volume was measured by means of a water displacement device (Ugo Basile, Milano, Italy) . One hour before the carrageenan injection the rats were orally treated either with 2 mg/kg indomethacin suspended into 1% acacia gum (10 ml/kg) or with the vehicle alone . Copper determinations were carried out by flame atomic absorption spectrophotometry (Perkin Elmer 3030) on non-haemolytic plasma deproteinized by addition of an equal amount of 15% trichloroacetic acid. RESULTS
Following 1 month of copper deficient diet the general status of copper deprived rats was satisfactory, and no statistically significant differences of body weight were observed between copper deficient and normally fed animals (207±18 vs 219±15 g respectively) . On the other hand within 2-3 weeks from the beginning of the 0 .2 mg/kg copper deprived diet plasma copper concentration of the copper depleted group decreased below 10% of the control rats (15 .9±8 .1 vs 177 .0±28 .6 µg/100 ml respectively) . As expected from previous experiments (4,5), the induction of a copper deficiency status clearly promoted a statistically significant enhancement of the acute inflammatory response at all the examined times (3h = + 31%; 5h = + 18% ; 22h = + 27%) (see table) . The treatment with indomethacin at the oral dose of 2 mg/kg was found to be effective in reducing the foot oedema development in both normally fed and copper deprived rats (see table). However the per cent inhibition observed was by no means comparable in the two groups of animals being indomethacin significantly less active in the copper depleted group at all the considered times (3h = - 54% ; 5h = - 65% ; 22h = - 33%) (see table) . 1043-6618/92/25110254-02/$03 .00/0
© 1992 The Italian Pharmacological Society
Pharmacological Research, Vol . 25, Supplement 2, 1992
25 5
Table - Effect of indomethacin treatment on the foot-oedema developed by normal and copper-deficient rats . Group
3h Oedema vol . % Inh. ml (SD) (SD)
Normal Untreated
0.78 (0.08)
Normal Treated
0 .38 * (0.10)
Cu def. Untreated
1 .02 * (0 .14)
Cu def. Treated
0 .78 0 (0.13)
5h Oedema vol . ml (SD)
% Inh . (SD)
1 .00 (0.10) 51 .1 (18.9)
0 .54 * (0.12)
0.62 (0.09) 46 .9 (20 .1)
1 .18 * (0.08) 23 .7 ' (10.2)
0 .99 0 (0.13)
22h Oedema vol . % Inh . ml (SD) (SD)
0 .41 * (0.12)
34.1 (13 .1)
0 .79 (0 .10) 16.2 ' (11 .2)
0 .61 ° (0 .09)
22 .9 0 (9 .9)
* P<0 .01 vs Normal Untreated, ° P<0.01 vs Cu-def . Untreated, ' P<0.01 vs Normal Treated ; Student's "t" test . n = 10 rats per group . DISCUSSION
Although preliminary, the results described in this paper seem to support the hypothesis of Sorenson (1) suggesting that the presence of a normal amount of copper in plasma is needed in order to obtain a full expression of indomethacin anti-inflammatory potential . We like to note that, as previously observed studying female Sprague-Dawley rats maintained for one month on a 0 .2 mg/kg copper deficient diet (5), the copper deficiency status does not induce any biologically significant variations at the histological, haematological and haematochemical levels . The only change observed is a dramatic decrease of copper in plasma whose apparent consequences are an exacerbation of the acute inflammatory reaction as well as a reduction of indomethacin efficacy . Further work is however needed to better verify Sorenson's hypothesis, and it would be especially relevant to see whether or not the loss of anti-inflammatory activity in copper deficiency conditions is also shared by other non- steroidal anti-inflammatory drugs .
REFERENCES
1) Sorenson JRJ. Copper chelates as possible active forms of the antiarthritic agents . J Med Chem 1976,19 :135-48 . 2) Arena G, Kavu G, Williams DR . Metal-ligand complexes involved in rheumatoid arthritis . V . Formation constants for calcium(II)-, magnesium(H)- and copper(II)salicylate and acetylsalicylate interactions . J inorg nucl Chem 1978, 40 :1221-26 . 3) Korolkiewicz Z, Hac E, Gagalo I, Gorczyca P, Lodzinska A . The pharmacological activity of complexes and mixtures with copper and salicylates or aminopyrine following oral dosing in rats. Agents and Actions 1989, 26 :355-59 . 4) Milanino R, Conforti A, Fracasso ME, Franco L, Leone R, Passarella E, Tarter G, Velo GP . Concerning the role of endogenous copper in the acute inflammatory process . Agents and Actions 1979, 9 :581-588 . 5) Milanino R, Velo GP. Multiple actions of copper in control of inflammation : studies in copper deficient rats . In : Rainsford KD,Brune K, Whitehouse MW, eds. Trace elements in the pathogenesis and treatment of inflammation (AAS Vol . 8) . Basel : Birkhauser Verlag, 1981 :209-230 .