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Home , Aspirin, Salicylic acid

Proc. Natl. Acad. Sci. USA Vol. 84, pp. 1417-1420, March 1987 Medical Sciences

Pharmacokinetics of and salicylate in relation to inhibition of arachidonate and antiinflammatory activity (//) GERALD A. HIGGS, JOHN A. SALMON, BRIAN HENDERSON, AND JOHN R. VANE* Department of , Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS, United Kingdom Contributed by John R. Vane, October 27, 1986

ABSTRACT Among the nonsteroid antiinflammatory We have now (i) measured the concentrations of aspirin there is generally a close correlation between the potency and salicylate in plasma and inflammatory exudates collected of their inhibition of arachidonate cyclooxygenase, and thus from rats 5 min to 6 hr after a single of production, and their antiinflammatory activity. either aspirin or salicylate; (ii) measured the effects on serum One anomaly in this generalization is that whereas aspirin and TXB2 concentrations and concentrations of PGE2 in the salicylate are equipotent as antiinflammatory agents, salicylate exudates; and (iii) compared the effects of aspirin and is less active than aspirin in inhibiting prostaglandin produc- salicylate, at a range of concentrations achieved in inflam- tion in vitro. Using rats, we have now measured the concen- matory exudates, on the production ofPGE2 by nonprolifera- trations of aspirin and salicylate in plasma and in inflammatory tive explants of acutely inflamed tissue. Some of these data exudates after their oral administration and determined their have been presented elsewhere (7). effects on production in clotting blood and prostaglandin (PG) E2 concentrations in the exudates. We have also investigated the effects of both drugs, at concentrations MATERIALS AND METHODS achieved in the exudates, on PGE2 production by nonprolifera- tive explants of acutely inflamed tissues. Aspirin is rapidly Groups of five male Wistar rats (190-210 g) were subcuta- metabolized, resulting in peak concentrations of salicylate in neously implanted with polyester sponges soaked in 1% the plasma and exudate that exceeded peak concentrations of carrageenan (Sigma), in sterile saline (8). Aspirin (Wellcome; aspirin by 30- to 50-fold. Furthermore, concentrations of 200 mg/kg) or salicylate (Wellcome; 200 mg/kg) was admin- aspirin rapidly declined, whereas high concentrations of sali- istered orally in aqueous solution 5 min to 6 hr before sponge cylate persisted in the plasma and in the exudate for up to 6 hr removal, whereas control animals received the vehicle alone. after a single administration of aspirin. Both drugs reduced After 6 hr, the rats were anesthetized, 5 ml of blood was PGE2 concentrations in inflammatory exudates by 50-70%, withdrawn from the abdominal aorta, and the sponges were but aspirin was considerably more potent than salicylate in removed. Blood (1 ml) was allowed to clot in glass test tubes inhibiting thromboxane B2 production in clotting blood. The at 37°C for 45 min, and the resulting serum was collected after concentration ofsalicylate found in inflammatory exudates 6 hr centrifugation. The remaining 4 ml of blood was added to after the administration of aspirin was sufficient to reduce tubes containing lithium (Sarstedt, UK) to prevent PGE2 production in explants by more than 50%. We conclude , plus aqueous potassium fluoride (10 ,ul/ml; 50% that the antiinflammatory action of both drugs depends on the wt/vol), to limit enzymic of aspirin. The sponge inhibition of PGE2 synthesis by salicylate. exudates were squeezed into plastic tubes containing 50 ,ul of the potassium fluoride solution and were centrifuged to yield The discovery that aspirin, indomethacin, and salicylate a cell-free supernatant. Aspirin and salicylate concentrations inhibit the synthesis ofprostaglandins (1-3) provided the first in plasma and exudates were measured using reversed-phase comprehensive explanation of the mechanism of action of HPLC (9). Briefly, internal standard (3,4-dimethylbenzoic these drugs. Subsequent studies have shown that the broad ; 3 ,g) was added to the sample that was then acidified group of nonsteroid antiinflammatory drugs inhibits arachi- with orthophosphoric acid and extracted with . donate cyclooxygenase, the that converts arachi- The chloroform was removed under nitrogen, and the residue donic acid to prostaglandin endoperoxides (for review see was reconstituted in HPLC mobile phase [: 0.072% ref. 4). However, anomalies exist in the potencies of these orthophosphoric acid, 55:45 (vol/vol)]. An aliquot was in- drugs with respect to their antiinflammatory activities and to jected onto a 5-Am Spherisorb ODS reversed-phase HPLC their ability to inhibit cyclooxygenase; of particular interest column. The compounds were eluted by pumping mobile has been the between the phase through the column at 1 ml/min and were detected at disparity potency of aspirin and 234 nm. salicylate in inhibiting cyclooxygenase. Aspirin and salicy- The concentrations of aspirin and in plasma late are equipotent as antiinflammatory agents, but aspirin is and inflammatory exudate were subjected to pharmacokinet- some 20 times more potent than salicylate in inhibiting an ic analysis using a model similar to that described by Rowland enzyme preparation from guinea pig lung in vitro (1). Fur- et al. (10). The data points were fitted by nonlinear least thermore, it has been reported that oral administration of squares to a two-compartment model for both aspirin and its aspirin (10 mg/kg) inhibited thromboxane (TX) production by metabolite salicylic acid using a computer package (11). The rat but 20 times this dose of salicylate had no effect concentration of the compounds in the plasma was assumed (5). These findings led to speculation that the antiinflamma- to represent the central compartment from which elimination tory activity of these drugs was not related to inhibition of occurred. The concentration of the compounds in the inflam- prostaglandin (PG) synthesis (5, 6). Abbreviations: PG, prostaglandin; TX, thromboxane. The publication costs of this article were defrayed in part by page charge *Present address: The William Harvey Research Institute, St. payment. This article must therefore be hereby marked "advertisement" Bartholomew's Hospital Medical College, Charterhouse Square, in accordance with 18 U.S.C. §1734 solely to indicate this fact. London EC1M 6BQ, United Kingdom.

Downloaded by guest on October 2, 2021 1417 1418 Medical Sciences: Higgs et al. Proc. Natl. Acad. Sci. USA 84 (1987) matory exudate was considered to reflect another compart- achieved about 1 min after administration ofthe (Fig. 1). ment linked to the central compartment. The rate constants After attaining a maximum concentration, the levels of for tissue distribution and elimination found to define the aspirin in plasma rapidly decreased to 10% ofthe peak values of salicylic acid after oral administration after 1 hr and to an undetectable level after 6 hr. were used in the evaluation of the pharmacokinetics of Salicylate was detected in all the plasma samples taken up aspirin and its major metabolite (salicylic acid). The rate to 6 hr after administration of aspirin. After 5 min, salicylate constants for absorption, tissue distribution, and elimination concentrations in the' plasma were 20 times higher than of aspirin and salicylic acid obtained by Rowland et al. (10) aspirin; the concentrations of salicylate reached a peak 30 provided a guide to these parameters for the present evalu- min to 2 hr after administration. At 6 hr, plasma salicylate ation. The only significant difference between the model concentrations had fallen but were still 20-30 times higher described by Rowland et al. (10) and that used in the present than peak aspirin concentrations (Fig. 1). study was that we had to assume that elimination of salicylic Aspirin was also detected in the inflammatory exudate, in acid from the central compartment occurred by a zero-order which the highest concentrations were reached after 30 min. process. 'All other rate constants were first order. Peak aspirin concentrations in plasma and exudates were TXB2 concentrations in the serum and in exudates and similar but aspirin remained in the exudate for longer than in PGE2 concentrations in the exudates were determined by the plasma (Fig. 2). Aspirin could not be detected in the specific radioimmunoassays (8). exudate 6 hr after administration. Salicylate concentrations In a separate series of experiments, sponges were implant- increased in the exudate after aspirin administration and ed in groups offive rats 24 hr before the animals were killed, reached concentrations at 2 hr 30-40 times higher than the and the inflamed tissue around the implant was dissected out. highest aspirin concentrations (Fig. 2). After 6 hr, salicylate The'tissue was cut into segments weighing 30-60 mg that was still present in the exudate at near maximal concentra- were then maintained individually in Trowell's nonprolifera- tions. tive adult organ maintenance culture under an atmosphere of Effects of Aspirin on Cyclooxygenase Activity. Serum from 95% 02/5% CO2 at 37TC for 24 hr (12). Aspirin (0.25-25 control animals contained TXB2 at 332.4 ± 65.7 ng/ml (mean ,ug/ml) or salicylate (2.5-100 Ag/ml) was added to the culture ± SEM, n = 5). Serum collected from rats 5 min to 6 hr after medium and at the end of the incubation period the concen- aspirin administration did not contain any detectable TXB2 tration of PGE2 was assayed by radioimmunoassay. (<0.5 ng/ml; Fig. 3). Inflammatory exudates collected from control animals 6 hr after sponge implantation contained TXB2 at 42.6 ± 4.1 ng/ml and PGE2 at 19.5 ± 2.4 ng/ml. The RESULTS administration ofaspirin 5-20 min before sponge removal had variable effects on TXB2 and PGE2 concentrations, but 30 Concentrations of Aspirin and Salicylate after Administra- min to 6 hr after aspirin administration PGE2 was reduced by tion ofAspirin. The highest concentration (2.28 ± 1.09 kkg/ml) 45-60% (Fig. 4) and TXB2 was reduced by 60-80% ofcontrol of aspirin in the plasma was observed at the first time point values (Fig. 3). measured, 5 min after aspirin administration. However, Concentrations of Salicylate after Administration of Salicy- evaluation of the data using a two-compartment pharmaco- late. Concentrations of salicylate in the plasma reached near kinetic model suggests that the highest concentrations of aspirin (-8 jig/ml) in the central compartment would be 0 160r . 00~~~~~~~ E A 120 T

._- i 80 _ e7z V)

V) 40 I- 0L -E 8. .c i 4 '._. cn a A <0l li 0 IL 120 240 -360 Time after administration of drug, min 120 240 FIG. 2. Concentrations of salicylate and aspirin in inflammatory Time after administration of drug, min exudates after oral doses of salicylate or aspirin at 200 mg/kg. (Upper) Concentrations of salicylate after salicylate (e) and after FIG. 1. Concentrations of salicylate and aspirin in plasma after aspirin (A); (lower) concentrations of aspirin after aspirin (m). All the oral doses of salicylate or aspirin at 200 mg/kg. (Upper) Concentra- exudates were collected 6 hr after the subcutaneous implantation of tions of salicylate after salicylate (e) and after aspirin (A). (Lower) sponges. Each point represents the mean ofvalues from five animals. Concentrations of aspirin after aspirin (n). Each point represents the The curves were fitted by least squares to a pharmacokinetic model mean of values from five animals. The' curves were fitted by least similar to that reported by Rowland et al. (10); the exudate was squares to a pharmacokinetic model similar to that reported by considered to be another compartment linked to the central plasma Rowland et al. (10), but with zero-order salicylate elimination. compartment. Downloaded by guest on October 2, 2021 Medical Sciences: Higgs et al. Proc. Natl. Acad. Sci. USA 84 (1987) 1419

120 A

100

80 601 401

'5 20 -5 0 c u 0 0 aL u ZHC)l B B m x

40

20

01 . . . 0 10 30 60 120 360 10 30 60 120 36s0 Time after administration of drug, min Time after administration of drug, min

FIG. 3. Effect of aspirin (A) or salicylate (B) at 200 mg/kg on FIG. 4. Effect of aspirin (A) or salicylate (B) at 200 mg/kg on TXB2 concentrations in serum (A) and inflammatory exudates (e). PGE2 concentrations in inflammatory exudates. Each point repre- Each point represents the mean of values from five animals, and the sents the mean of values from five animals, and the bars are 1 bars are ± 1 SEM. SEM.

peak values 5 min after salicylate administration and had not reduced by 40-90% but salicylate was significantly less active declined significantly in 6 hr (Fig. 1). These data could be than aspirin (Fig. 3). Salicylate had an effect similar to that of fitted to a two-compartment model quite well (Fig. 1); unlike aspirin on the concentrations of TXB2 and PGE2 in the the evaluation of the concentrations of aspirin in plasma, it inflammatory exudates, causing reductions of 40-80% by 30 was unnecessary to extrapolate back to peak levels of min to 6 hr after administration (Figs. 3 and 4). salicylate in the central compartment. Effect of Aspirin and Salicylate on PGE2 Production by No aspirin was detected after administration of salicylate. Tissue Explants. Explants of inflamed tissue produced PGE2 Salicylate accumulated more slowly in the exudate, reaching at 2.33 ± 0.74 ng/mg (wet weight) after 24 hr of culture. the highest concentrations 6 hr after administration (Fig. 2). Production of PGE2 was dose dependently inhibited by Effect of Salicylate on Cyclooxygenase Activity. The adding aspirin (0.25-25 ,ug/ml) or salicylate (2.5-100 Aug/ml) concentation of TXB2 in serum taken from animals treated to the culture medium (Fig. 5). Aspirin was approximately with salicylate 5 min to 6 hr before the collection ofblood was twice as potent as salicylate. 120

to 80

60

40

0.1 1.0 10 100 Drug in culture medium. /ig/ml

FIG. 5. Effect of aspirin (6) and salicylate (0) on PGE2 synthesis by explants of acutely inflamed tissue. Explants (30-60 mg) were incubated in nonproliferative culture for 24 hr, and then the PGE2 content ofthe medium was assayed by specific radioimmunoassay. Each point represents the mean of 4-12 values obtained in three separate experiments, and the bars are ± 1 SEM. Downloaded by guest on October 2, 2021 1420 Medical Sciences: Higgs et al. Proc. Natl. Acad Sci. USA 84 (1987) DISCUSSION the site of inflammation were sufficient at 30 min to inhibit prostaglandin formation, thereby contributing to the antiin- Aspirin irreversibly acetylates the active site of cyclooxy- flammatory effect, regeneration of cyclooxygenase would genase (13), thereby destroying its enzymic function. In overcome this inhibition, and the prolonged inhibition of platelets, which cannot make new protein, the effect of this TXB2 and PGE2 production in the exudate must be ascribed lasts for the life of the -up to 10 days. to the prolonged high concentrations of salicylate. There is now convincing evidence that platelet cyclooxy- We conclude, therefore, that aspirin has a direct effect on genase is inactivated by aspirin (given orally) in the platelet cyclooxygenase in the presystemic circulation but is presystemic circulation (14, 15) before the aspirin is substan- rapidly converted to salicylate, which, although a weaker tially deacetylated to salicylate on first pass through the cyclooxygenase inhibitor, attains sufficiently high concentra- portal circulation. tions in the periphery to have an effect. This explains why In cultures of smooth muscle cells (16) or endothelial cells aspirin is more potent in inhibiting platelet function after oral (17), the inhibitory effects of aspirin on cyclooxygenase are administration than it is as an antiinflammatory agent. Thus, the transient, and prostaglandin production recovers within 2-5 low levels ofaspirin and high levels ofsalicylate in inflammatory hr. This recovery is due to new enzyme synthesis (17). In exudates suggest that aspirin is a for salicylate (19) and humans, also, after administration of650 mg ofaspirin orally, that the antiinflammatory activity of both drugs is through the endothelial cells recover their capacity to generate prosta- inhibition of PGE2 production by salicylate. cyclin (18), an effect again attributed to new enzyme synthe- sis. We thank Dr. B. Weatherley for advice on the interpretation ofthe These observations led us to reexamine Dreser's proposal pharmacokinetic data and Mr. A. Padfield and Miss K. Jackson for that aspirin acts as a prodrug for salicylate (19) and that, skilled technical assistance. against inflammation, salicylate is the active moiety. All our 1. Vane, J. R. (1971) Nature (London) 231, 232-235. present results are consistent with this interpretation. 2. Smith, J. B. & Willis, A. L. (1971) Nature (London) 231, Plasma salicylate concentrations rapidly exceed plasma 235-237. aspirin concentrations after oral administration of aspirin, 3. Ferreira, S. H., Moncada, S. & Vane, J. R. (1971) Nature in the is (London) 231, 237-239. and the half-life of salicylate plasma considerably 4. Higgs, G. A., Moncada, S. & Vane, J. R. (1984) Ann. Clin. longer than that of aspirin (10, 15). We have also confirmed Res. 16, 287-299. that high concentrations of salicylate, lasting for several 5. Vargaftig, B. B. (1978) J. Pharm. Pharmacol. 30, 101-104. hours, are found in inflammatory exudates after oral admin- 6. Smith, M. J. H. (1975) Agents Actions 5, 315-317. istration of aspirin (20). 7. Henderson, B., Higgs, G. A., Salmon, J. A. & Vane, J. R. Despite its rapid hydrolysis to salicylate, aspirin has a (1986) Br. J. Pharmacol. 88, 400P (abstr.). differential effect on TXB2 production in the blood and at a 8. Higgs, G. A. & Salmon, J. A. (1979) Prostaglandins 17, peripheral site of inflammation. In clotting blood, TXB2 is 737-746. produced by aggregating platelets, and aspirin inhibited 9. Lo, L. Y. & Bye, A. (1980) J. Chromatogr. 181, 473-477. the 10. Rowland, M., Riegleman, S., Harris, P. A. & Sholkoff, S. D. platelet cyclooxygenase for the 6-hr duration of experi- (1972) J. Pharm. Sci. 61, 379-385. ment (Fig. 3). This confirms similar findings after oral 11. Chance, E. M., Curtis, A. R., Jones, I. P. & Kirby, C. R. administration of aspirin to humans (18). Salicylate is clearly (1977) FACSIMILE, A Computer Program for Flow and less potent than aspirin in inhibiting platelet TXB2 production Chemistry Simulation, and General Initial Value Problems in blood (Fig. 3). (H. M. Stationary Office, London, UK), C 13. In the sponge model ofinflammation, TXB2 is produced by 12. Poulter, L. W., Bitensky, L., Cashman, B. & Chayen, J. migrating leukocytes and platelets do not contribute to TXB2 (1970) Virchows Arch. (Cell Pathol.) 4, 303-309. concentrations in the exudates (21). The similar potency of 13. Roth, G. J. & Majerus, P. W. (1975) J. Clin. Invest. 56, aspirin and salicylate in reducing TXB2 and PGE2 concen- 624-632. trations in inflammatory exudates suggests that the effects of 14. Pedersen, A. K. & Fitzgerald, G. A. (1984) N. Engl. J. Med. both drugs are due to salicylate accumulating at the inflam- 311, 1206-1211. that the 15. Cerletti, C., Latini, R., Dejana, E., Tognoni, G., Garattini, S. matory site. This is supported by the observation & De Gaetano, G. (1985) Biochem. Pharmacol. 34, 1839-1841. maximum reduction of cyclooxygenase products in the exu- 16. Bailey, J. M., Muza, B., Hla, T. & Salata, K. (1985) J. Lipid dates coincides with the peak concentrations of salicylate in Res. 26, 54-61. the exudates after either aspirin or salicylate (Figs. 2, 3, and 17. Jaffe, E. A. & Weksler, B. B. (1979) J. Clin. Invest. 63, 4). The explant experiments indicate that salicylate inhibits 532-535. PGE2 productiontlby inflamed tissues at concentrations ob- 18. Heavey, D. J., Barrow, S. E., Hickling, N. E. & Ritter, J. M. tained in the exudtes after either aspirin or salicylate (1985) Nature (London) 318, 186-188. administration (Fig. 5). 19. Dreser, H. (1899) Pflugers Arch. 76, 306-318. The highest concentration of aspirin achieved in the 20. Smith, M. J. H., Ford-Hutchinson, A. W., Walker, J. R. & exudate was 1.5 ,ug/ml at 30 min (Fig. 2), and this concen- Slack, J. A. (1979) Agents Actions 9, 483-487. tration had minimal effects on prostaglandin production by 21. Higgs, G. A., Moncada, S., Salmon, J. A. & Seager, K. (1983) tissue explants (Fig. 5). Even if the aspirin concentration at Br. J. Pharmacol. 79, 863-868. Downloaded by guest on October 2, 2021