Mechanism of Action of Nonsteroidal Anti-Inflammatory Drugs

MANAGEMENT OF PAIN

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MECHANISM OF ACTION OF NONSTEROIDAL ANTIINFLAMMATORY DRUGS Alexander Livingston, BSc, BVetMed, PhD, FRCVS

It is hard to imagine, but before 1971, no one knew how aspirin, the most commonly used nonprescription drug in the world, exerted its effects. The pharmacologic effects of aspirin and the other nonsteroidal anti-inflammatory drugs (NSAIDs) had been recognized since the active principle of willow bark had been characterized in 1876,14and their beneficial effects as anti-inflammatory, analgesic, and antipyretic agents as well as their unwanted effects in terms of gastric irritation, prolongation of bleeding time, and renal and hepatic damage were well known and documented. What was unknown, however, was the mechanisms by which these various effects were mediated. In 1971, Vane19 and Smith and Willis18 proposed separately in articles in the journal Nature that aspirin worked by inhibiting the production of prostaglandins. At that time, prostaglandins were a relatively esoteric group of compounds, best known for their presence in seminal fluid and their stimulant effects on smooth muscle. They were also known for their transitory nature because of their rapid breakdown in tissues and, consequently, the difficulty experienced by scientists trying to study them. Since that time, the significance of the prostaglandins and related compounds, known jointly as the eicosanoids, has been elaborated in a range of physiologic and pathologic processes. The term eicosanoids is derived from the precursor compound eicosatetraenoic acid, better known as arachdonic acid. An understanding of the processes that involve eicosanoids in the body has allowed the rational explanation of the many beneficial and harmful effects of the NSAIDs based on their ability to interfere with the synthesis of these compounds. There are many steps in the process of synthesis of eicosanoids from arachdonic acid, and there is more than one pathway by which the process may take From the Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

VETERINARY CLINICS OF NORTH AMERICA: SMALL ANIMAL PRACTICE VOLUME 30 NUMBER 4 JULY 2000

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place. Figure 1 shows the pathways available for the synthesis of eicosanoids from arachidonic acid in summary form. It should be noted that the two main alternatives concern the formation of either leukotrienes or thromboxane, prostacyclins, and prostaglandins depending on whether the arachdonic acid precursor is initially oxidized by the enzyme 5-lipoxygenase or by cyclo-oxygenase (COX). A series of further enzymatic steps results in the formation of a wide range of compounds as shown. It is important to note that Figure 1 is a summary of the various processes. Various cells and tissues contain different enzymes; thus, the processes shown represent a compilation of all the possible steps in all the possible tissues. In fact, particular cells in particular conditions only produce some of the active agents. Most of the effects attributable to NSAIDs seem to be confined to their ability to inhibit the enzyme COX early in the synthetic pathway. Nevertheless, other activities have been suggested to account for some of their actions such as inhibition of lipoxygenase activity and neutrophil activation.l*lo It has also been suggested that some NSAIDS can stimulate glycosaminoglycan synthesis in vitro.4 The mechanism of action of the NSAIDs on COX is considered to be that of a competitive inhibitor for most agents, although aspirin was believed to be covalently bound. About a decade ago, however, a second isoform of COX was recognized, resulting in the designation of COX-1 and COX-2J2 The most significant difference between the two isoforms is that in many of the situations examined, the COX-1 enzyme seems to be constitutive; that is, it is part of the normal enzyme complement of the cell and is apparently present at a fairly constant concentration, whereas the COX-2 enzyme seems to be inducible, that is, it appears and increases in concentration in response to some form of stimulus. These stimuli can be things such as cytokines, mitogens, or endotoxins, and an increase in concentration is commonly seen at sites of inflammation. The constitutive form of COX is normally associated with tissues, where prostaglandins serve a physiologic function; for instance, in the gastric mucosa, prostaglandins have a cytoprotective function and are synthesized via a COX-1 pathway. Similarly, the production of thromboxane in platelets is a COX-1mediated process. Because a significant proportion of the unwanted side effects of NSAIDs, namely, gastric ulceration and extended clotting time, were associated with these constitutive COX-1 effects, there was an immediate drive to focus on the inhibition of the COX-2-induced functions and the sparing of the COX-1 constitutive actions. This gave rise to the “good COX-bad C O X concept, where those NSAIDs that seemed to have some COX-2 inhibitory selectivity were presented as the solution to all problems associated with toxicity. As is always the case in biologic systems, the answer is probably not that simple, and specific COX-2 inhibitors are not going to answer all the problems. For instance the NSAID-induced enteropathy seen as small intestine damage may be more associated with the degree of enterohepatic recirculation of an NSAID than its COX specificity?’ The point is also raised that if nonspecific NSAID-induced gastric ulceration is present, the subsequent specific suppression of the inflammatory process by a COX-2-specific NSAID may in fact delay the healing process in the gastrointestinal tract. These points do not address the most worrying issue, however, and that is that the original classification may be an oversimplification. Not all COX-2 may be inducible; indeed, there is evidence7 for constitutive COX-2 in the central nervous system (CNS), and not all physiologic processes may be governed b j constitutive COX-1. For instance, we know that the renal perfusion in hypovo,

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transpeptidase

LEUKOTRIENE E4

Cysteinyl gbcinase

LEUKOTRIENE D4

1

Glutamyl transpeptidase

LEUKOTRIENE C4

Glutathione

LEUKOTRIENE A4

I I

I

4

LEUKOTRIENE B4 Dehydrase

, 5HPETE

5-Lipoxygenase

d

PGD,

Prostaglandm D-isomerase

PGF,

(TxA3

. PROSTACYCLIN WI2)

Prostacyclin synthetase

m

2

THROMBOXANE A2

Prostaglandin F-reductase

PGH2

Thromboxane synthetase

PROSTAGLANDIN HYDROPEROXIDE

\L Cyclo-ovgenase

Figure 1. Eicosanoid synthesis.

/

ARACHIDONIC ACID

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lemia is supported by prostaglandins, but in situ hybridization studies have shown that both COX-1 and COX-2 are present in the kidneys of some species. Consequently, the automatic assumption that COX-2-specific NSAIDs do not compromise renal perfusion responses in hypovolemia, because this is a constitutive process, may be in error. There also seems to be involvement of both isoenzymes in the prostaglandin-mediated physiologic processes associated with reproduction, although there may be species variation in this. The veterinary availability of NSAIDs is usually facilitated by the major human market for these drugs. In the United States alone, there are 14 million people who regularly use NSAIDs for arthritis and its associated pain; of those, up to 4% experience gastric toxicity effects each year. Consequently, any agent that can significantly reduce these side effects would have considerable market potential; thus, NSAIDs with high COX-2 specificity such as celecoxib are now available for human use and may well appear shortly on the veterinary market. Second-generation successors to celecoxib such as parecoxib and valdecoxib should soon be available for human use and, again, may have potential for veterinary use. Furthermore, those NSAIDs already in the veterinary market that have a significant degree of COX-2 versus COX-1 activity are being rigorously promoted on that basis. It is interesting to note that the assignment of COX-1 versus COX-2 activity in NSAIDs has led to some controversy, because the apparent activity for the respective isoenzyme may rest, in part, on the assay method applied. For instance, for the human studies, a variety of in vitro tests have been used. Perhaps the most reliable model has been the Chinese hamster ovary whole cell stably transfected with either hCOX-1 or hCOX-2, but most frequently, human whole-blood assays have been used. As the real test of any drug is in a pathologic situation, a variety of in vivo tests such as rat paw edema, rat paw hyperalgesia, rat adjuvant arthritis, lipopolysaccharide-inducedrat pyresis, and lipopolysaccharide-induced pyresis in monkeys have been used. Thus, it is not surprising that a variety of values have been obtained. Table 1 shows some such values derived from human whole-blood assays, and Table 2 shows data using assays based on canine tissues. Some considerable differences are apparent; however, it is interesting to note that the in vivo tests do not provide such a clear definition (Table 3) between the selective COX-2 inhibitors and the nonse-

Table 1. IN VlTRO HUMAN WHOLE-BLOOD CYCLO-OXYGENASE ASSAYS NSAlD

Ketoprofen Naproxen Ibuprofen Indomethacin (see Table 3 ) Diclofenac Meloxicam Etodolac Flosulide DFU (see Table 3 )

cox-1 (ICSO) 0.02 8.0

5.0

0.2 0.1

1.4 9.0 32.0 100.0

cox-2 (lCso) 1.0

74.0

>30.0 0.5 0.05 0.5 2.0

0.8 0.3

cox-1:cox-2 Ratio 0.02

0.1 0.2 0.4 2.0

3.0 5.0

40.0

300.0

IC, = concentration that produces 50% inhibition of enzyme activity (expressed in +M concentrations). From Wang Z Second generation COX-2 inhibitors. In Proceedings of the IBC Industry Symposium on COX-2 Inhibitors, San Diego, 1998; with permission.

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Table 2. IN VlTRO CANINE THROMBOXANE AND HISTIOCYTOMA COX-1 AND COX-2 ASSAYS

NSAID

cox-1 (IC5,)

Aspirin Ketoprofen Etodolac Flunixin Meloxicam Meclofenamic acid Tolfenamic acid Nimesulide Carprofen

34.0 0.03 1.3 0.008 0.9 0.7 0.2 2.2 13.2

cox-2 (lCSO) >100.0 0.12 2.6 0.01 0.3 0.05 0.01 0.06 0.1

cox-1:cox-2 Ratio

<0.3 0.25 0.5 0.7 3.0 15.0 15.0 38.0 129.0

IC, = concentration that produces 50% inhibition of enzyme activity (expressed in pM concentrations). From Ricketts AP, Lundy KM, Seibel SB:Evaluation of selective inhibition of canine cyclo-oxygenase 1 and 2 by carprofen and other non-steroidal anti-inflammatory drugs. Am J Vet Res 59:144-1446, 1998; with permission.

lective drugs, although these tests, of course, are comparing rat in vivo with human in vitro studies. The use of COX-l:COX-2 ratios can be confusing because of the use of an inhibitory concentration, that is, the concentration of the drug that inhibits the enzyme activity by 50%. Consequently, if the COX-l:COX-2 ratio is greater than 1, there is more COX-2 activity; if the ratio is less than 1, there is more COX-1 activity. Some authors have used a COX-2:COX-1 ratio, which turns everything upside down and confuses everyone. Because these ratios do not seem to be linearly related to clinical efficacy or toxicology, however, they should be used as guidelines only, and due notice should be taken of equally important factors like bioavailability, metabolism, and excretion. Table 4 lists the COX-1 and COX-2 activities of some common NSAIDs. The process whereby the release of arachidonic acid occurs in cells that are involved in the production of leukotrienes and prostaglandins and the mechanisms that control this process are quite complex. Initially, the cells are stimulated by a ligand-receptor interaction or some perturbation of the cell surface. This is followed by a G-protein-mediated process which, together with a rise in intracellular calcium, results in the release of arachidonic acid via the effects of phospholipase C or A, and the subsequent interaction of the arachdonic acid with activated 5-lipoxygenase or COX-1 or COX-2 to produce leukotrienes or Table 3. IN VlVO ASSAYS FOR CLINICAL EFFECTIVENESS Test (ED,, mglkg)

DFU (a highly specific COX-2 inhibitor)

lndomethacin

Rat paw edema Rat pyresis Rat paw hyperalgesia Adjuvant arthritis Monkey pyresis (approximtely 3 mp/ kg)

1.1 0.8 0.8 2.0 83%

2.0 1.0 1.4 0.2 Not detectable

ED,, = median effective dose. From Wang Z Second generation COX-2 inhibitors. In Proceedings of the IBC Industry Symposium on COX-2 Inhibiors, San Diego, 1998; with permission.

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prostaglandins. This process has been studied extensively6,l2 and is shown in a simplified schematic form in Figure 2. Although this process can explain the initiation of the inflammatory response in insulted cells, which, in turn, identifies the step at which NSAIDs exert their effects to idubit inflammation, the process whereby the actual induction of the COX-2 isoform of the enzyme is started is less well documented. Mechanisms have been proposed, however, and it is interesting to note that these are not confined to inflammatory cells but may well be seen in cancerous or dying cells as well. We know that platelet activating factor and interleukins, both of which are potent mediators of immune response and inflammatory changes, can be powerful inducers of transcription of the gene responsible for the production of COX-2. Experiments that can measure the amounts of COX-2 mRNA at different times under different conditions have allowed scientists to examine additional possible activities for this enzyme. Much of the process can be elucidated by using the inducers already described such as intracellular platelet activating factor to study their effects on the early genes such as c-fos and c-jun, where they can be shown to promote transcriptional activation by increasing the level of the appropriate mRNA.Z One of the outcomes of these studies13 has been the demonstration that corticosteroids can selectively inhibit the binding of transcription factors to DNA and hence inhibit COX-2 gene expression. The significance of these studies is threefold: first, they link the induction of the COX2 isoenzyme with inflammatory mediators; second, they propose a mechanism of action for some of the anti-inflammatory and analgesic effects of glucocorticoids; and third, they open up another possible therapeutic route for COX-2 inhibition if drugs that switch off the promoter genes can be developed. These studies have centered mainly on the mediation of the inflammatory processes, and although some of the analgesic effects of NSAIDs are associated with the peripheral inflammatory response and the sensitizing effects of some Table 4. NONSTEROIDAL ANTI-INFLAMMATORY DRUGS LICENSED FOR VETERINARY USE IN EUROPE AND NORTH AMERICA

Name Aspirin Benzydamine Carprofen Diclofenac Eltenac Flunixin Ibuprofen Ketoprofen Meclofenamic acid Meloxicam Metamizole (dipyrone) Niflumic acid Nimesulide Phenylbutazone Tolfenamic acid Vedaprofen

cox-1

++++ ++ + ++ ++ +++ +++ +++ + + ++ NA + ++++

++ NA

cox-2

Availability

-

Europe and North America Europe Europe and North America Europe Europe Europe and North America Europe and North America Europe and North America Europe and North America Europe and North America Europe and North America Europe Europe Europe and North America Europe and North America Europe

NA

+++

++ NA + + + +++ +++ NA NA

+++ -

++ NA

There are a great many studies using different assay systems that give a wide range of values for the activities listed above. The relative potencies are therefore a rough guide only. No. of plus signs indicates increasing activity. NA = not available. Datafrom Bishop Y The Veterinary Formulary, ed 4. London, Pharmaceutical Press, 1998.

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m

a

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of the inflammatory mediators on peripheral neurons and nerve endings, there is also clear evidence of a transmitter role for prostaglandins in the CNS.15,16 The presence of both COX-1 and COX-2 has been reported in the CNS, but it seems that although COX-1 is constitutive as in the periphery, the COX-2 isoenzyme may be either constitutive or induced.3r8It has been suggested that prostaglandins may play a significant role in the central sensitization process by which previously non-noxious stimuli are perceived as noxious after peripheral injury or insult via a feedback mechanism to potentiate depolarization-evoked transmitter release.” Intrathecal administration of nonspecific COX irhbitors such as ibuprofen has been shown to block hypersensitivity responses,lI but more interestingly, so has intrathecal administration of specific COX-2 inhibitorsz3;thus, it may be that this process is mediated by inducible COX-2. These findings would suggest that not only do NSAIDs have a central effect in reducing the perception of pain but that they might also act to reduce central hypersensitivity and that this effect might be susceptible to specific COX-2 inhibitory NSAIDs. There are continual developments in our understanding of the mechanisms of actions of NSAIDs that move forward with our understanding of the processes associated with the processing of painful stimuli. It is also interesting to note that some of the mechanisms by which NSAIDs reduce inflammatory changes and pain perception are also mechanisms by whch cells die or become cancerous, and active research is in progress to study the effects of these drugs on Alzheimer’s disease, stroke, and some forms of cancer, as the role of COX in these tissues is under active investigation.6,9, l3 The relevance of these studies to veterinary medicine, however, has not been demonstrated at this time. References 1. Altman RD: Neutrophil activation: An alternative to prostaglandin synthesis as the mechanism of actions for NSAIDs. Semin Arthritis Rheum 19(SuppI2):14,1990 2. Bazan NG, Fletcher BS, Herschman HR, et al: Platelet activating factor and retinoic acid synergistically activate the inducible prostaglandin synthase gene. Proc Natl Acad Sci USA 91:5252-5256, 1994 3. Beiche F, Scheuerer S, Brune K, et al: Upregulation of cyclooxygenase-2mRNA in the rat spinal cord following peripheral inflammation. FEBS Lett 390:165-169, 1996 4. Benton HP, Vasseur PB, Broderick-Villa GA, et al: Effect of carprofen on sulphated GAG metabolism, protein synthesis and prostaglandin release by cultured osteoarthritic canine chondrocytes. Am J Vet Res 58:286-292, 1997 5. Billah M: Regulation of phospholipase A. Annual Reports in Medicinal Chemistry 22223-233, 1987 6. Bonventre JV, Huang Z , Taheri MR, et al: Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A2. Nature 390:622-625, 1997 7. Breder CD, Dewitt D, Kraig RP: Characterization of inducible cyclooxygenase in rat brain. J Comp Neurol 355296315, 1995 8. Breder CD, Smith WL, Raz A, et al: Distribution and characterization of cyclooxygenase immunoreactivity in the ovine brain. J Comp Neurol322:409438, 1992 9. Chan TM, Morin PJ, Vogelstein B, et al: Mechanisms underlying nonsteroidal antiinflammatory drug-mediated apoptosis. Proc Natl Acad Sci USA 95:681-686, 1998 10. Dawson W, Boot JR, Havery J, et al: The pharmacology of benoxaprofen with particular reference to effects on lipoxygenase product formation. Eur J Rheumatol Inflamm 5:61-65, 1982 11. Ding DM, Yaksh TL: Spinal and systemic cyclooxygenase inhibitors suppress paw carrageenan-evoked thermal hyperalgesia in rats [abstract]. Anesthesiology 87A721, 1997

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12. Ford-Hutchinson AW: FLAP: A novel target for inhibiting the synthesis of leukotrienes. Trends in Pharmacological Sciences 1268-72, 1991 13. Lukiw WJ, Pelaez RP, Martinez J, et al: Budesonide epmer R or dexamethasone selectively inhibit platelet activating factor-induced or interleukin 1P-induced DNA binding activity of cis-acting transcription factors and cyclooxygenase 2 gene expression in human epidermal keratinocytes. Proc Natl Acad Sci USA 95:3914-3919, 1998 14. MacLagan T The treatment of acute rheumatism by salacin. Lancet i:342-383, 1876 15. Malmberg AB, Yaksh TL: Antinociceptive actions of spinal nonsteroidal anti-inflammatory agents on the formalin test in the rat. J Pharmacol Exp Ther 263:13&146, 1992 16. McCormack K: The spinal actions of non-steroidal anti-inflammatory drugs and the dissociation between their anti-inflammatory and analgesic effect. Drugs 47(Suppl 5):28-45, 1994 17. Ricketts AP, Lundy KM, Seibel SB: Evaluation of selective inhibition of canine cyclooxygenase 1 and 2 by carprofen and other non-steroidal anti-inflammatory drugs. Am J Vet Res 59:1441-1446, 1998 18. Smith JB, Willis AL: Aspirin selectively inhibits prostaglandin production in human platelets. Nature 231235-237, 1971 19. Vane JR: Inhibition of prostaglandin synthesis as a possible mechanism of action of aspirin-like drugs. Nature 231:232-235, 1971 20. Wallace JL, Tigley A W New insights into prostaglandins and mucosal defence. Aliment Pharmacol Ther 9:227-235, 1995 21. Wang 2: Second generation COX-2 inhibitors. In Proceedings of the IBC Industry Symposium on COX-2 Inhibitors, San Diego, 1998 22. Xie W, Chipman JG, Robertson DL, et al: Expression of a mitogen-responsive gene encoding prostaglandin synthase is regulated by mRNA splicing. Proc Natl Acad Sci USA 88:2692-2696, 1991 23. Yamamoto T, Nozaki-Taguchi N: Role of spinal cyclooxygenase (COX-2) on thermal hyperalgesia evoked by carageenan injection in the rat. Neuroreport 8:2179-2182, 1997

Address reprint requests to Alexander Livingston, BSc, BVetMed, PhD, FRCVS Dean's Office Western College of Veterinary Medicine University of Saskatchewan 52 Campus Drive Saskatoon, SK S7N 584 Canada