Agents and Actions vol. 9, 5/6 (1979) Birkhiiuser Verlag, Basel
483
Aspirin, Salieylate and Prostaglandins by M.J.H. SMITH, A.W. FORD-HUTCHINSON, J.R. WALKER and J.A. SLACK Biochemical Pharmacology Research Unit, Department of Chemical Pathology, King's College Hospital Medical School, Denmark Hill, London, SE5 8RX
Abstract The effects of aspirin, salicylic acid and gentisie acid on the paw swellings in the araehidonic acid-potentiated and in the conventional carrageenan-induced oedema tests as well as on the content of prostaglandin-like activity and leucocyte migration in the exudate of inert implanted sponges in the rat have been studied. It is concluded that aspirin exerts two separate inhibitory effects on prostaglan din formation in vlvo, a rapid action of the intact molecule on easily accessible tissues and a later action due to its metabolic conversion to salicylic acid. Salicylic acid inhibits prostaglandin biosynthesis in vivo as the salieylate inn itself and there is no formation of a subsequent 'active' metabolite.
Introduction Acetylsalicylic acid (Aspirin) inhibits the activity of the prostaglandin synthetase complex in vitro and interferes with the conversion of arachidonie acid to its cascade of endoperoxides, thromboxanes and prostaglandins [1]. The mechanism of inhibition is irreversible involving a nonenzymatic acetylation either at or near the active site of the cyclo-oxygenase [2]. In contrast, salicylic acid, which is the immediate metabolite of aspirin in vivo, is a much weaker inhibitor of the enzyme in vitro and must do so by a quite different mechanism. Nevertheless the two acids, when administered orally to rats, are equipotent in reducing the content of prostaglandins in inflammatory exudates [3, 4]. There have been two explanations advanced to explain this apparent discrepancy. One is that salicylic acid requires a further metabolic transformation before it is fully active as a prostaglandin cyclo-oxygenase inhibitor in vivo. It has been suggested that the 'active' metabolite concerned is gentisic acid (2:5-dihydroxybenzoic acid) since this substance is not only over thirty times as potent against prostaglandin synthetase activity
in vitro as is salicylic acid [5] but also is one of its major metabolites in the rat [6]. An alternative view is that the salicylate ion, being only weakly bound to circulating proteins in the rat, is able to accumulate so rapidly in developing inflammatory exudates that it attains concentrations sufficient to inhibit prostaglandin biosynthesis in vivo [7]. Both hypotheses infer that the effects of aspirin on prostaglandin synthetase activity in vivo may be double, there being a rapid inhibition due to acetylation of the enzyme and a later action due to the metabolic formation either of salicylic acid or of gentisic acid. The present work describes the results of some experiments concerned with the effects of aspirin, salicylic and gentisic acids on prostaglandin formation and other aspects of inflammation in some animal models. Materials and methods Aspirin and salicylic acid were of British Pharmacopoeial grade, the gentisic acid being obtained from Sigma Chemical Co., USA, they were administered orally as a suspension in Tween 80 0.1% v/v. Female albino Wistar rats, 150-200 g, were obtained from Oxfordshire Laboratory Animal Colonies, Southern, Ltd. The sponge implantation, carrageenan-induced paw oedema, arachidonic acid-enhanced paw oedema and the measurement of prostaglandin-like activity and leucocyte counting were as described previously [8, 9] except that the carrageenan was dissolved in Tyrode's solution rather than in saline. Salicylic acid levels were assessed by the gas chromatography of the trimethylsilyl derivatives. Exudate (0.1 ml) was added to a test tube containing the internal standard (100 /tg of 3-ethyl-salicylic acid in 10 /B of methanol) and diluted with 0.8 ml distilled water. 0.1 ml of 1 NHC1 was added followed by 0.7 ml chloroform. The tubes were vortexed twice and the layers separated by centrifugation. The chloroform layer was placed in a reactivial, the chloroform removed under nitrogen, 20/tl of bis(trimethylsilyl)-trifluoroacetamide added and the vial heated at 45~
484
Aspirin, Salicylate and Prostaglandins
overnight. The derivatives were chromatographed on a column filled with 3% OV7 on Chromosorb W 100-120 A W - D M C S at 180 ~ (40 ml min -~ flow rate). A calibration curve was constructed by spiking control exudate with salicylic acid and following the same procedure for the test samples. The coefficient of variation for repeat extractions (n = 5) was 4% and for repeat injections (n = 5) of the same extract was 3%. The calibration curve covered the range 2 5 250 pg ml -~ of salicylic acid, the correlation coefficient being 0.998. Gentisic acid levels were determined as for salicylic acid using a calibration range of 1-100 pg ml-% the minim u m detection limit was 1/lg ml-L The presence of acetylsalicylic acid does not interfere with the determination of salicylic acid by this method.
Results The results in Table 1 show the effects of single doses of aspirin, salicylic acid and gentisic acid on arachidonic acid-potentiated and carrageenan-induced paw oedema tests in the rat. Aspirin (100 mg kg -~) significantly inhibited both
reactions but salicylic acid (100 mg kg -1) only reduced the paw swelling in the carrageenaninduced test producing an effect similar to that of the aspirin. This result confirms previous work [8] which used 50 and 200 mg kg -1 doses of the two drugs. If these results are extrapolated to compare equimolar doses of aspirin and sodium salicylate, again no significant difference can be observed between the two treatments. Gentisic acid (200 mg kg -1) did not significantly affect either paw oedema. In additional experiments with the arachidonic acid-potentiated system in which all three drugs were given orally in three divided doses, each of 100 mg kg -1 at 20, 3 and 0.5 h before the subplantar injection of the arachidonie acid-carrageenan mixture, both aspirin and salicylic acid produced significant inhibition of the paw swelling at 0.5 h whereas gentisic acid was inactive.
Table 1 Effects of single oral doses of aspirin, salicylic acid and gentisic acid on the increase in paw volume in the arachidonic acidpotentiated and in the carrageenan-induced oedema tests in the rat. Drug a
Dose (rag kg -1)
Increase in paw volume ~ 0.5h Arachidonic acid potentiated reaction % increase
Control Aspirin Salicylic acid Gentistic acid
44.5 26.7 40.3 39.8
100 100 200
3h Carrageenan4nduced reaction % inhibition of control
+_ 2.5 • 3.9 • 5.2 +_ 4.1
40 c 9 11
% increase
67.2 38.7 43.2 64.2
4-_9.2 _+ 5.4 _+ 2.7 • 9.7
% inhibition of control
42 a 36 ~ 5
a Drugs given orally 1 h before irritant injected into paw. b Results expressed as means _+ standard errors (5 rats per group). c p < 0.002. P < 0.05 from results of corresponding control group. Table 2 Effects of single oral doses (200 mg kg -1) of aspirin, salicylic acid and gentisic acid on prostaglandin-like activity, leucocyte emigration and concentrations of salicylate ion in 9-h sponge exudates in the rat. Drug
Aspirin Salicylic acid Gentisic acid
Mean % inhibition of control values~
Concentration of salicylate ion
Prostaglandinlike activity
Total leucocytes
pg m1-1
mM
83 b 83 ~ 0
47 b 43 b 0
228 + 21 192 + 14 --
1.6 1.4
a In the sponge exudates of the corresponding control group of rats the mean prostaglandin-like activity was equivalent to 10 ng m1-1 of P G E 2 and the total leucocyte count was 77 x l0 s/zl -x of exudate (10 rats per group). b p < 0.05 from results of control group.
485
Aspirin, Salicylate and Prostaglandins
Table 3
Effectsof local administration of aspirin and salicylicacid in 9-h spongeexudatesin the rat. Drug
Control Aspirin Salicylic acid
Dose (rag per sponge)
Prostaglandin-like activity a
Total leucocytes a
P G E 2 equivalents (ng m1-1 exudate)
% inhibition
(• 102/A-1 exudate)
% inhibition
0.5 0.5
11.2 + 1.2 1.8 + 0.3 9.4 + 0.7
84 b 16
66_+7 65 + 5 66 _+ 10
2 0
a Results given as means _+ standard errors (5 rats per group). b p < 0.05 from results of corresponding control group.
Table 2 shows the effects of the oral administration of a single dose (200 mg kg -1) of the three drugs on the accumulation of total leucocytes, the content of prostaglandin-like activity and the concentrations of salicylic acid in the 9-h exudates found in implanted sponges in the rat. Aspirin and salicylic acid produced similar effects whereas gentisic acid was inactive on both aspects of the inflammatory response. The salicylate concentrations found in the sponge exudates of the animals treated with either aspirin or salicylic acid were almost identical. Traces of gentisate (< 1 /lg m1-1) were found in exudates from three only of the animals treated with either aspirin or salicylic acid. The results in Table 3 show the effects of the direct administration of aspirin and salicylic acid into the sponge exudates. The local application of 0.5 mg quantities of both drugs produced no effect on the accumulation of leucocytes. Aspirin but not salicylate significantly reduced the prostaglandin-like activity in the exudate. Discussion
When given in single oral doses aspirin and salicylic acid differ in their effects on the arachidonic acid-potentiated carrageenan oedema reaction when the paw swellings are measured at 1.5 h after the drugs were administered (Table 1). This finding confirms earlier work [8] which used both smaller and larger doses of the drugs. It has been postulated that aspirin is active in this test because it acts as the intact molecule whereas salicylic acid, either administered as such or formed as a metabolic product of the aspirin, is inactive because the oedema forms so rapidly that there is not sufficient time for the formation of a subsequent metabolite, the so-called "active met abolite', which is a more potent inhibitor of prostaglandin
cyclo-oxygenase than is the parent molecule 110]. Pretreatment of the animals with several doses of salicylic acid caused a reduction of the paw swelling similar to that observed after the single dose of aspirin. While this result could be interpreted as evidence in favour of the formation of the 'active metabolite' of salicylic acid it is clear that this cannot be gentisic acid. Either a single oral dose, double that of the aspirin, or pretreatment with multiple doses of gentisic acid did not affect the development of the arachidonic acidpotentiated paw swelling in the rat. Furthermore aspirin and salicylic acid are equipotent in reducing the paw swelling occurring 4 h after the administration of the drugs in the conventional carrageenan-induced paw oedema test (Table 1). This time period should have been sufficient for the salicylic acid to undergo further metabolic transformation in the animals. Gentisic acid was again devoid of anti-inflammatory activity in this reaction. In the exudates formed after 9 h in subdermally implanted inert sponges in the rat, aspirin and salicylic acid, given as single oral doses of 200 mg kg -~, produced identical inhibitions both of prostaglandin-like activity and leucocyte accumulation (Table 2). At this time interval there is no dispute [10] that ample time should have elapsed for salicylic acid to form its 'active metabolite' but again the administration of gentisic acid proved to be totally ineffectual. In the rat salicylic acid is only weakly bound to the circulating proteins [11] and therefore accumulates rapidly in inflammatory exudates, It has been demonstrated by other workers [12] that after the oral administration of sodium salicylate the salicylate ion accumulates rapidly in sponge exudates reaching a maximum concentration in 1 h and there is little alteration over the next 12 h. In the present work it was observed that after the
486 single oral dose of salicylic acid the concentration of salicylate ion in the 9-h sponge exudates was 1.4 mM. This concentration is well in excess of IC50 values for salicylic acid as an inhibitor of prostaglandin synthetase activity in vitro, reported values being 0.312 m M [13], 0.66 m M [14], 0.75 m M [15] and 0.80 m M [10]. There is therefore no need to postulate the formation of an 'active metabolite', certainly not of gentisic acid, only traces of which were detected i n the exudates and which showed no activity in the test system. The salicylate ion itself is perfectly capable of reducing the prostaglandin-like activity in the sponge exudates after the oral administration of salicylic acid. The argument may be extended to include aspirin which is known to be hydrolysed very rapidly in vivo and the salicylate ion then accumulates in the sponge exudate at a rate equivalent to that observed after the oral administration of salicylic acid. The results observed following the administration of aspirin and salicylic acid directly into the sponge exudates (Table 3) provide evidence that aspirin is able to irreversibly inhibit prostaglandin biosynthesis in a limited environment by acting as the intact molecule. This effect is similar to its well-known action on the prostaglandin synthetase of blood platelets of many species in vivo and is explicable on the basis that the acetyl-salicylic acid is in direct contact with readily accessible tissues in vivo before it undergoes hydrolysis to salicylate. In contrast salicylic acid does not inhibit prostaglandin synthetase activity under similar conditions either in the sponge exudate (Table 3) or in circulating rat platelets [16]. It must also be the mechanism by which a single oral dose of aspirin inhibits the arachidonic acid-potentiated paw swelling in the rat (Table 1). Salicylic acid, however, although a much weaker inhibitor of the enzyme activity, is able to inhibit the arachidonic acid-potentiated paw swelling if a regime of pretreatment using several divided doses of the drug is instituted so that enough salicylate ion accumulates in advance in the injected paw. It is concluded that aspirin inhibits prostaglandin synthetase activity in the rat by two mechanisms. The first involves an irreversible acetylation of the enzyme protein by the intact molecule. This is how the drug acts in vitro and it can exert the same action in vivo providing first, that it is in direct contact with readily accessible tissues, e.g. blood platelets in the circulation,
Aspirin, Salieylate and Prostaglandins
vascular endothelium and adjacent tissues at injection sites, and secondly, that it is not removed too rapidly by hydrolysis. The second mechanism involves its metabolic conversion to salicylic acid and in the rat the salicylate ion accumulates so rapidly in developing inflammatory exudates that it achieves concentrations sufficient to inhibit the biosynthesis of prostaglandins. Furthermore it is the accumulation of the salicylate ion in vivo which causes the inhibition of leucocyte migration, an important aspect of acute and chronic inflammatory reactions which is independent of the prostaglandin system [17]. In this context salicylic acid is the 'active metabolite' of aspirin. Salicylic acid acts in vivo in the form of the salicylate ion both as an inhibitor of prostaglandin production and as an antiinflammatory agent. The results of the present work do not support the hypothesis that salicylic acid is further metabolized to a compound which is either a more potent inhibitor of prostaglandin formation [18] or a more powerful antiinflammatory agent. These two activities are not synonymous. Not only are there independent effects of the drug on prostaglandin biosynthesis and leucocyte migration but also the concentrations of salicylate ion which accumulate in inflammatory exudates in the rat are such that they interfere with many enzyme systems other than prostaglandin synthetase [19]. Some of these enzyme systems may play important roles in the development of inflammatory responses. Received 21 March 1979.
References [ 1] R.J. GRYGLEWSKt, Screening and Assessment of the Potency of Anti-inflammatory Drugs in vitro, in: AntiInflammatory Drugs (Eds. J.R. Vane and S.H. Ferreira; Springer-Verlag, Berlin, 1979), pp. 3-43. [21 T.Y. SHEN, Prostaglandin Synthetase Inhibitors, in: Prostaglandin and Thromboxanes (Eds. F. Berti, B. Samuelsson and G.P. Velo; Plenum Press, N.Y., 1977), pp. 111-136. [31 A.L. WILUS, R. DAVISON, P.W. RAMWELL, W.D. BROCKLEHU~SX and B. Sr~ITR, Release and Actions of
Prostaglandins in Inflammation and Fever: Inhibition by Anti-Inflammatory and Antipyretic Drugs, in: Prostaglandins in Cellular Biology (Eds. P.W. Ramwell and B.B. Pharris; Plenum Press, N.Y., 1972), pp. 227-259. [4] G.A. Hmas, E.A. HARVEY, S.H. FERREmA and J.R. VANE, The Effects of Anti-Inflammatory Drugs on the Production of Prostaglandins in vivo, in: Advances in Prostaglandin and Thromboxane Research, vol. 1 (Eds. B. Samuelsson and R. Paoletti; Raven Press, N.Y., 1976), pp. 105-110.
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Aspirin, Salicylate and Prostag!andins
[5] R.J. FLOWER and J.R. VANE, Some Pharmacologic and Biochemical Aspects of Prostaglandin Biosynthesis and its Inhibition, in: Prostaglandin Synthetase Inhibitors (Eds. H.J. Robinson and J.R. Vane; Raven Press, N.Y., 1974), pp. 9-18. [6] E. QUILLEY and M.J.H. SMITH, The Application of Paper Partition Chromatography to the Study of the Metabolism of Salicylate in the Rat, J. Pharm. Pharmac. 4, 624-630 (1952). [7] M.J.H. SMITH, Aspirin and Prostaglandins, Some Recent Developments, Agents and Actions 8, 427-429 (1978). [8] M.J.H. SMITH, A.W. FORD-HUTCHINSON and P.C. ELLIOTT, Prostaglandins and the Anti-Inflammatory Activities of Aspirin and Sodium Salicylate, J. Pharm. Pharmac. 27, 473478 (1975). [9] J.R. WALKER, M.J.H. SMITH and A.W. FORDHUTCHINSON, Anti-Inflammatory Drugs, Prostaglandins and Leucocyte Migration, Agents and Actions 6, 602-606 (1976). [ 10] S.H. FERREIRAand J.R. VANE,Mode of Action of AntiInflammatory Agents which are Prostaglandin Synthetase Inhibitors, in: Anti-Inflammatory Drugs (Eds. J.R. Vane and S.H. Ferreira; Springer-Verlag, Berlin, 1979), p. 373. [11] J.A. STURMAN and M.J.H. SMITH, The Binding of Salicylate to Plasma Proteins in Different Species, J. Pharm. Pharmac. 19, 621-623 (1967).
[12] N.S. DOHERTY, M. AATTILAand P.B. DEAN, Penetration of Naproxen and Salicylate into Inflammatory Exudates in the Rat, Ann. Rheum. Dis. 36, 244-248
(1977). [13] E.L. TOLMAN and R. PARTRIDGE, Multiple Sites of Interaction between Prostaglandins and Non-Steroidal Anti-Inflammatory Agents, Prostaglandins 9, 349-359 (1975). [14] B. FJALLAND, Inhibition by Non-Steroidal Anti-Inflammatory Agents of the Release of Rabbit Aorta Contracting Substance and Prostaglandins from Chopped Guinea Pig Lungs, J. Pharm. Pharmac. 26, 448-451 (1974). [15] J.R. VANE,Inhibition of Prostaglandin Synthesis as a Mechanism of Action for Aspirin-like Drugs, Nature (New Biol.) 231,232-235 (1971). [16] B.B. VARGAFa~O, Salicylic Acid Fails to lnhibit Generation of Thromboxane A z Activity after in vivo Administration to the Rat, J. Pharm. Pharmae. 30, 101-104 (1978). [17] A.W. FORD-HUTCHINSON, J.R. WALKER, N.S. CONNOR and M.J.H. SMITH, Prostaglandins and Leucocyte Migration in Inflammatory Reactions, Agents and Actions 7, 469-472 (1977). [18] J.R. VANE, The Mode of Action of Aspirin-like Drugs, Agents and Actions 8, 430-431 (1978). [19] M.J.H. SMITH and P.D. DAWKINS, Salicylate and Enzymes, J. Pharm. Pharmac. 23, 729-744 (1971).
483
Aspirin, Salieylate and Prostaglandins by M.J.H. SMITH, A.W. FORD-HUTCHINSON, J.R. WALKER and J.A. SLACK Biochemical Pharmacology Research Unit, Department of Chemical Pathology, King's College Hospital Medical School, Denmark Hill, London, SE5 8RX
Abstract The effects of aspirin, salicylic acid and gentisie acid on the paw swellings in the araehidonic acid-potentiated and in the conventional carrageenan-induced oedema tests as well as on the content of prostaglandin-like activity and leucocyte migration in the exudate of inert implanted sponges in the rat have been studied. It is concluded that aspirin exerts two separate inhibitory effects on prostaglan din formation in vlvo, a rapid action of the intact molecule on easily accessible tissues and a later action due to its metabolic conversion to salicylic acid. Salicylic acid inhibits prostaglandin biosynthesis in vivo as the salieylate inn itself and there is no formation of a subsequent 'active' metabolite.
Introduction Acetylsalicylic acid (Aspirin) inhibits the activity of the prostaglandin synthetase complex in vitro and interferes with the conversion of arachidonie acid to its cascade of endoperoxides, thromboxanes and prostaglandins [1]. The mechanism of inhibition is irreversible involving a nonenzymatic acetylation either at or near the active site of the cyclo-oxygenase [2]. In contrast, salicylic acid, which is the immediate metabolite of aspirin in vivo, is a much weaker inhibitor of the enzyme in vitro and must do so by a quite different mechanism. Nevertheless the two acids, when administered orally to rats, are equipotent in reducing the content of prostaglandins in inflammatory exudates [3, 4]. There have been two explanations advanced to explain this apparent discrepancy. One is that salicylic acid requires a further metabolic transformation before it is fully active as a prostaglandin cyclo-oxygenase inhibitor in vivo. It has been suggested that the 'active' metabolite concerned is gentisic acid (2:5-dihydroxybenzoic acid) since this substance is not only over thirty times as potent against prostaglandin synthetase activity
in vitro as is salicylic acid [5] but also is one of its major metabolites in the rat [6]. An alternative view is that the salicylate ion, being only weakly bound to circulating proteins in the rat, is able to accumulate so rapidly in developing inflammatory exudates that it attains concentrations sufficient to inhibit prostaglandin biosynthesis in vivo [7]. Both hypotheses infer that the effects of aspirin on prostaglandin synthetase activity in vivo may be double, there being a rapid inhibition due to acetylation of the enzyme and a later action due to the metabolic formation either of salicylic acid or of gentisic acid. The present work describes the results of some experiments concerned with the effects of aspirin, salicylic and gentisic acids on prostaglandin formation and other aspects of inflammation in some animal models. Materials and methods Aspirin and salicylic acid were of British Pharmacopoeial grade, the gentisic acid being obtained from Sigma Chemical Co., USA, they were administered orally as a suspension in Tween 80 0.1% v/v. Female albino Wistar rats, 150-200 g, were obtained from Oxfordshire Laboratory Animal Colonies, Southern, Ltd. The sponge implantation, carrageenan-induced paw oedema, arachidonic acid-enhanced paw oedema and the measurement of prostaglandin-like activity and leucocyte counting were as described previously [8, 9] except that the carrageenan was dissolved in Tyrode's solution rather than in saline. Salicylic acid levels were assessed by the gas chromatography of the trimethylsilyl derivatives. Exudate (0.1 ml) was added to a test tube containing the internal standard (100 /tg of 3-ethyl-salicylic acid in 10 /B of methanol) and diluted with 0.8 ml distilled water. 0.1 ml of 1 NHC1 was added followed by 0.7 ml chloroform. The tubes were vortexed twice and the layers separated by centrifugation. The chloroform layer was placed in a reactivial, the chloroform removed under nitrogen, 20/tl of bis(trimethylsilyl)-trifluoroacetamide added and the vial heated at 45~
484
Aspirin, Salicylate and Prostaglandins
overnight. The derivatives were chromatographed on a column filled with 3% OV7 on Chromosorb W 100-120 A W - D M C S at 180 ~ (40 ml min -~ flow rate). A calibration curve was constructed by spiking control exudate with salicylic acid and following the same procedure for the test samples. The coefficient of variation for repeat extractions (n = 5) was 4% and for repeat injections (n = 5) of the same extract was 3%. The calibration curve covered the range 2 5 250 pg ml -~ of salicylic acid, the correlation coefficient being 0.998. Gentisic acid levels were determined as for salicylic acid using a calibration range of 1-100 pg ml-% the minim u m detection limit was 1/lg ml-L The presence of acetylsalicylic acid does not interfere with the determination of salicylic acid by this method.
Results The results in Table 1 show the effects of single doses of aspirin, salicylic acid and gentisic acid on arachidonic acid-potentiated and carrageenan-induced paw oedema tests in the rat. Aspirin (100 mg kg -~) significantly inhibited both
reactions but salicylic acid (100 mg kg -1) only reduced the paw swelling in the carrageenaninduced test producing an effect similar to that of the aspirin. This result confirms previous work [8] which used 50 and 200 mg kg -1 doses of the two drugs. If these results are extrapolated to compare equimolar doses of aspirin and sodium salicylate, again no significant difference can be observed between the two treatments. Gentisic acid (200 mg kg -1) did not significantly affect either paw oedema. In additional experiments with the arachidonic acid-potentiated system in which all three drugs were given orally in three divided doses, each of 100 mg kg -1 at 20, 3 and 0.5 h before the subplantar injection of the arachidonie acid-carrageenan mixture, both aspirin and salicylic acid produced significant inhibition of the paw swelling at 0.5 h whereas gentisic acid was inactive.
Table 1 Effects of single oral doses of aspirin, salicylic acid and gentisic acid on the increase in paw volume in the arachidonic acidpotentiated and in the carrageenan-induced oedema tests in the rat. Drug a
Dose (rag kg -1)
Increase in paw volume ~ 0.5h Arachidonic acid potentiated reaction % increase
Control Aspirin Salicylic acid Gentistic acid
44.5 26.7 40.3 39.8
100 100 200
3h Carrageenan4nduced reaction % inhibition of control
+_ 2.5 • 3.9 • 5.2 +_ 4.1
40 c 9 11
% increase
67.2 38.7 43.2 64.2
4-_9.2 _+ 5.4 _+ 2.7 • 9.7
% inhibition of control
42 a 36 ~ 5
a Drugs given orally 1 h before irritant injected into paw. b Results expressed as means _+ standard errors (5 rats per group). c p < 0.002. P < 0.05 from results of corresponding control group. Table 2 Effects of single oral doses (200 mg kg -1) of aspirin, salicylic acid and gentisic acid on prostaglandin-like activity, leucocyte emigration and concentrations of salicylate ion in 9-h sponge exudates in the rat. Drug
Aspirin Salicylic acid Gentisic acid
Mean % inhibition of control values~
Concentration of salicylate ion
Prostaglandinlike activity
Total leucocytes
pg m1-1
mM
83 b 83 ~ 0
47 b 43 b 0
228 + 21 192 + 14 --
1.6 1.4
a In the sponge exudates of the corresponding control group of rats the mean prostaglandin-like activity was equivalent to 10 ng m1-1 of P G E 2 and the total leucocyte count was 77 x l0 s/zl -x of exudate (10 rats per group). b p < 0.05 from results of control group.
485
Aspirin, Salicylate and Prostaglandins
Table 3
Effectsof local administration of aspirin and salicylicacid in 9-h spongeexudatesin the rat. Drug
Control Aspirin Salicylic acid
Dose (rag per sponge)
Prostaglandin-like activity a
Total leucocytes a
P G E 2 equivalents (ng m1-1 exudate)
% inhibition
(• 102/A-1 exudate)
% inhibition
0.5 0.5
11.2 + 1.2 1.8 + 0.3 9.4 + 0.7
84 b 16
66_+7 65 + 5 66 _+ 10
2 0
a Results given as means _+ standard errors (5 rats per group). b p < 0.05 from results of corresponding control group.
Table 2 shows the effects of the oral administration of a single dose (200 mg kg -1) of the three drugs on the accumulation of total leucocytes, the content of prostaglandin-like activity and the concentrations of salicylic acid in the 9-h exudates found in implanted sponges in the rat. Aspirin and salicylic acid produced similar effects whereas gentisic acid was inactive on both aspects of the inflammatory response. The salicylate concentrations found in the sponge exudates of the animals treated with either aspirin or salicylic acid were almost identical. Traces of gentisate (< 1 /lg m1-1) were found in exudates from three only of the animals treated with either aspirin or salicylic acid. The results in Table 3 show the effects of the direct administration of aspirin and salicylic acid into the sponge exudates. The local application of 0.5 mg quantities of both drugs produced no effect on the accumulation of leucocytes. Aspirin but not salicylate significantly reduced the prostaglandin-like activity in the exudate. Discussion
When given in single oral doses aspirin and salicylic acid differ in their effects on the arachidonic acid-potentiated carrageenan oedema reaction when the paw swellings are measured at 1.5 h after the drugs were administered (Table 1). This finding confirms earlier work [8] which used both smaller and larger doses of the drugs. It has been postulated that aspirin is active in this test because it acts as the intact molecule whereas salicylic acid, either administered as such or formed as a metabolic product of the aspirin, is inactive because the oedema forms so rapidly that there is not sufficient time for the formation of a subsequent metabolite, the so-called "active met abolite', which is a more potent inhibitor of prostaglandin
cyclo-oxygenase than is the parent molecule 110]. Pretreatment of the animals with several doses of salicylic acid caused a reduction of the paw swelling similar to that observed after the single dose of aspirin. While this result could be interpreted as evidence in favour of the formation of the 'active metabolite' of salicylic acid it is clear that this cannot be gentisic acid. Either a single oral dose, double that of the aspirin, or pretreatment with multiple doses of gentisic acid did not affect the development of the arachidonic acidpotentiated paw swelling in the rat. Furthermore aspirin and salicylic acid are equipotent in reducing the paw swelling occurring 4 h after the administration of the drugs in the conventional carrageenan-induced paw oedema test (Table 1). This time period should have been sufficient for the salicylic acid to undergo further metabolic transformation in the animals. Gentisic acid was again devoid of anti-inflammatory activity in this reaction. In the exudates formed after 9 h in subdermally implanted inert sponges in the rat, aspirin and salicylic acid, given as single oral doses of 200 mg kg -~, produced identical inhibitions both of prostaglandin-like activity and leucocyte accumulation (Table 2). At this time interval there is no dispute [10] that ample time should have elapsed for salicylic acid to form its 'active metabolite' but again the administration of gentisic acid proved to be totally ineffectual. In the rat salicylic acid is only weakly bound to the circulating proteins [11] and therefore accumulates rapidly in inflammatory exudates, It has been demonstrated by other workers [12] that after the oral administration of sodium salicylate the salicylate ion accumulates rapidly in sponge exudates reaching a maximum concentration in 1 h and there is little alteration over the next 12 h. In the present work it was observed that after the
486 single oral dose of salicylic acid the concentration of salicylate ion in the 9-h sponge exudates was 1.4 mM. This concentration is well in excess of IC50 values for salicylic acid as an inhibitor of prostaglandin synthetase activity in vitro, reported values being 0.312 m M [13], 0.66 m M [14], 0.75 m M [15] and 0.80 m M [10]. There is therefore no need to postulate the formation of an 'active metabolite', certainly not of gentisic acid, only traces of which were detected i n the exudates and which showed no activity in the test system. The salicylate ion itself is perfectly capable of reducing the prostaglandin-like activity in the sponge exudates after the oral administration of salicylic acid. The argument may be extended to include aspirin which is known to be hydrolysed very rapidly in vivo and the salicylate ion then accumulates in the sponge exudate at a rate equivalent to that observed after the oral administration of salicylic acid. The results observed following the administration of aspirin and salicylic acid directly into the sponge exudates (Table 3) provide evidence that aspirin is able to irreversibly inhibit prostaglandin biosynthesis in a limited environment by acting as the intact molecule. This effect is similar to its well-known action on the prostaglandin synthetase of blood platelets of many species in vivo and is explicable on the basis that the acetyl-salicylic acid is in direct contact with readily accessible tissues in vivo before it undergoes hydrolysis to salicylate. In contrast salicylic acid does not inhibit prostaglandin synthetase activity under similar conditions either in the sponge exudate (Table 3) or in circulating rat platelets [16]. It must also be the mechanism by which a single oral dose of aspirin inhibits the arachidonic acid-potentiated paw swelling in the rat (Table 1). Salicylic acid, however, although a much weaker inhibitor of the enzyme activity, is able to inhibit the arachidonic acid-potentiated paw swelling if a regime of pretreatment using several divided doses of the drug is instituted so that enough salicylate ion accumulates in advance in the injected paw. It is concluded that aspirin inhibits prostaglandin synthetase activity in the rat by two mechanisms. The first involves an irreversible acetylation of the enzyme protein by the intact molecule. This is how the drug acts in vitro and it can exert the same action in vivo providing first, that it is in direct contact with readily accessible tissues, e.g. blood platelets in the circulation,
Aspirin, Salieylate and Prostaglandins
vascular endothelium and adjacent tissues at injection sites, and secondly, that it is not removed too rapidly by hydrolysis. The second mechanism involves its metabolic conversion to salicylic acid and in the rat the salicylate ion accumulates so rapidly in developing inflammatory exudates that it achieves concentrations sufficient to inhibit the biosynthesis of prostaglandins. Furthermore it is the accumulation of the salicylate ion in vivo which causes the inhibition of leucocyte migration, an important aspect of acute and chronic inflammatory reactions which is independent of the prostaglandin system [17]. In this context salicylic acid is the 'active metabolite' of aspirin. Salicylic acid acts in vivo in the form of the salicylate ion both as an inhibitor of prostaglandin production and as an antiinflammatory agent. The results of the present work do not support the hypothesis that salicylic acid is further metabolized to a compound which is either a more potent inhibitor of prostaglandin formation [18] or a more powerful antiinflammatory agent. These two activities are not synonymous. Not only are there independent effects of the drug on prostaglandin biosynthesis and leucocyte migration but also the concentrations of salicylate ion which accumulate in inflammatory exudates in the rat are such that they interfere with many enzyme systems other than prostaglandin synthetase [19]. Some of these enzyme systems may play important roles in the development of inflammatory responses. Received 21 March 1979.
References [ 1] R.J. GRYGLEWSKt, Screening and Assessment of the Potency of Anti-inflammatory Drugs in vitro, in: AntiInflammatory Drugs (Eds. J.R. Vane and S.H. Ferreira; Springer-Verlag, Berlin, 1979), pp. 3-43. [21 T.Y. SHEN, Prostaglandin Synthetase Inhibitors, in: Prostaglandin and Thromboxanes (Eds. F. Berti, B. Samuelsson and G.P. Velo; Plenum Press, N.Y., 1977), pp. 111-136. [31 A.L. WILUS, R. DAVISON, P.W. RAMWELL, W.D. BROCKLEHU~SX and B. Sr~ITR, Release and Actions of
Prostaglandins in Inflammation and Fever: Inhibition by Anti-Inflammatory and Antipyretic Drugs, in: Prostaglandins in Cellular Biology (Eds. P.W. Ramwell and B.B. Pharris; Plenum Press, N.Y., 1972), pp. 227-259. [4] G.A. Hmas, E.A. HARVEY, S.H. FERREmA and J.R. VANE, The Effects of Anti-Inflammatory Drugs on the Production of Prostaglandins in vivo, in: Advances in Prostaglandin and Thromboxane Research, vol. 1 (Eds. B. Samuelsson and R. Paoletti; Raven Press, N.Y., 1976), pp. 105-110.
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Aspirin, Salicylate and Prostag!andins
[5] R.J. FLOWER and J.R. VANE, Some Pharmacologic and Biochemical Aspects of Prostaglandin Biosynthesis and its Inhibition, in: Prostaglandin Synthetase Inhibitors (Eds. H.J. Robinson and J.R. Vane; Raven Press, N.Y., 1974), pp. 9-18. [6] E. QUILLEY and M.J.H. SMITH, The Application of Paper Partition Chromatography to the Study of the Metabolism of Salicylate in the Rat, J. Pharm. Pharmac. 4, 624-630 (1952). [7] M.J.H. SMITH, Aspirin and Prostaglandins, Some Recent Developments, Agents and Actions 8, 427-429 (1978). [8] M.J.H. SMITH, A.W. FORD-HUTCHINSON and P.C. ELLIOTT, Prostaglandins and the Anti-Inflammatory Activities of Aspirin and Sodium Salicylate, J. Pharm. Pharmac. 27, 473478 (1975). [9] J.R. WALKER, M.J.H. SMITH and A.W. FORDHUTCHINSON, Anti-Inflammatory Drugs, Prostaglandins and Leucocyte Migration, Agents and Actions 6, 602-606 (1976). [ 10] S.H. FERREIRAand J.R. VANE,Mode of Action of AntiInflammatory Agents which are Prostaglandin Synthetase Inhibitors, in: Anti-Inflammatory Drugs (Eds. J.R. Vane and S.H. Ferreira; Springer-Verlag, Berlin, 1979), p. 373. [11] J.A. STURMAN and M.J.H. SMITH, The Binding of Salicylate to Plasma Proteins in Different Species, J. Pharm. Pharmac. 19, 621-623 (1967).
[12] N.S. DOHERTY, M. AATTILAand P.B. DEAN, Penetration of Naproxen and Salicylate into Inflammatory Exudates in the Rat, Ann. Rheum. Dis. 36, 244-248
(1977). [13] E.L. TOLMAN and R. PARTRIDGE, Multiple Sites of Interaction between Prostaglandins and Non-Steroidal Anti-Inflammatory Agents, Prostaglandins 9, 349-359 (1975). [14] B. FJALLAND, Inhibition by Non-Steroidal Anti-Inflammatory Agents of the Release of Rabbit Aorta Contracting Substance and Prostaglandins from Chopped Guinea Pig Lungs, J. Pharm. Pharmac. 26, 448-451 (1974). [15] J.R. VANE,Inhibition of Prostaglandin Synthesis as a Mechanism of Action for Aspirin-like Drugs, Nature (New Biol.) 231,232-235 (1971). [16] B.B. VARGAFa~O, Salicylic Acid Fails to lnhibit Generation of Thromboxane A z Activity after in vivo Administration to the Rat, J. Pharm. Pharmae. 30, 101-104 (1978). [17] A.W. FORD-HUTCHINSON, J.R. WALKER, N.S. CONNOR and M.J.H. SMITH, Prostaglandins and Leucocyte Migration in Inflammatory Reactions, Agents and Actions 7, 469-472 (1977). [18] J.R. VANE, The Mode of Action of Aspirin-like Drugs, Agents and Actions 8, 430-431 (1978). [19] M.J.H. SMITH and P.D. DAWKINS, Salicylate and Enzymes, J. Pharm. Pharmac. 23, 729-744 (1971).
