Inhibitors of cyclooxygenases: mechanisms, selectivity and uses.

The prostaglandins are lipid mediators, discovered in the 1930s by von Euler in Sweden and Goldblatt in the United Kingdom. They are made by the bifunctional enzyme, cyclooxygenase, which has both cyclooxygenase and peroxidase activities in the same molecule. Prostaglandins are involved in physiological functions such as protection of the stomach mucosa, aggregation of platelets and regulation of kidney function. They also have pathological functions such as their involvement in inflammation, fever and pain. Vane in 1971 elegantly showed that the pharmacological actions of aspirin and similar drugs were due to the inhibition of cyclooxygenase. Thus, aspirin-like drugs exert their anti-inflammatory, antipyretic and analgesic effects by inhibition of cyclooxygenase. In 1991, Simmons and his colleagues identified a second cyclooxygenase enzyme, designated cyclooxygenase-2, derived from a separate gene from cyclooxygenase-1. Cyclooxygenase-2 is upregulated by inflammatory mediators and forms prostaglandins which intensify the inflammatory response. Cyclooxygenase-1 is, therefore, a 'housekeeping' enzyme making prostaglandins, which are important for maintaining physiological functions and cyclooxygenase-2 makes prostaglandins which are important in inflammation. The discovery of cyclooxygenase-2 and the establishment of its structure led to the development of selective inhibitors of this enzyme, such as celecoxib and rofecoxib, with potent anti-inflammatory actions but with reduced gastrotoxic effects. A putative cyclooxygenase-3, has also been characterised and cloned. This enzyme is a product of the cyclooxygenase-1 gene, but retains intron 1 after transcription and translates into a cyclooxygenase enzyme with 34 additional amino acids. It is more sensitive to inhibition by paracetamol, aspirin and some other non-steroid anti-inflammatory drugs than cyclooxygenase-1 or cyclooxygenase-2. A cyclooxygenase enzyme induced in cultured cells by some non-steroid anti-inflammatory drugs is also more sensitive to inhibition by paracetamol than cyclooxygenase-2 induced by bacterial lipopolysaccharide.

The mechanism of action of aspirin.

The therapy of rheumatism began thousands of years ago with the use of decoctions or extracts of herbs or plants such as willow bark or leaves, most of which turned out to contain salicylates. Following the advent of synthetic salicylate, Felix Hoffman, working at the Bayer company in Germany, made the acetylated form of salicylic acid in 1897. This drug was named "Aspirin" and became the most widely used medicine of all time. In 1971, Vane discovered the mechanism by which aspirin exerts its anti-inflammatory, analgesic and antipyretic actions. He proved that aspirin and other non-steroid anti-inflammatory drugs (NSAIDs) inhibit the activity of the enzyme now called cyclooxygenase (COX) which leads to the formation of prostaglandins (PGs) that cause inflammation, swelling, pain and fever. However, by inhibiting this key enzyme in PG synthesis, the aspirin-like drugs also prevented the production of physiologically important PGs which protect the stomach mucosa from damage by hydrochloric acid, maintain kidney function and aggregate platelets when required. This conclusion provided a unifying explanation for the therapeutic actions and shared side effects of the aspirin-like drugs. Twenty years later, with the discovery of a second COX gene, it became clear that there are two isoforms of the COX enzyme. The constitutive isoform, COX-1, supports the beneficial homeostatic functions, whereas the inducible isoform, COX-2, becomes upregulated by inflammatory mediators and its products cause many of the symptoms of inflammatory diseases such as rheumatoid and osteoarthritis.

Mechanism of action of acetaminophen: is there a cyclooxygenase 3?

Acetaminophen, also known as paracetamol, is a nonsteroidal anti-inflammatory drug with potent antipyretic and analgesic actions but with very weak anti-inflammatory activity. When administered to humans, it reduces levels of prostaglandin metabolites in urine but does not reduce synthesis of prostaglandins by blood platelets or by the stomach mucosa. Because acetaminophen is a weak inhibitor in vitro of both cyclooxygenase (COX)-1 and COX-2, the possibility exists that it inhibits a so far unidentified form of COX, perhaps COX-3. In animal studies, COX enzymes in homogenates of different tissues vary in sensitivity to the inhibitory action of acetaminophen. This may be evidence that there are >2 isoforms of the enzyme. Recently, a variant of COX-2 induced with high concentrations of nonsteroidal anti-inflammatory drugs was shown to be highly sensitive to inhibition by acetaminophen. Therefore COX-3 may be a product of the same gene that encodes COX-2, but have different molecular characteristics.

Nociception in cyclooxygenase isozyme-deficient mice.

Prostaglandins formed by cyclooxygenase-1 (COX-1) or COX-2 produce hyperalgesia in sensory nerve endings. To assess the relative roles of the two enzymes in pain processing, we compared responses of COX-1- or COX-2-deficient homozygous and heterozygous mice with wild-type controls in the hot plate and stretching tests for analgesia. Preliminary observational studies determined that there were no differences in gross parameters of behavior between the different groups. Surprisingly, on the hot plate (55 degrees C), the COX-1-deficient heterozygous groups showed less nociception, because mean reaction time was longer than that for controls. All other groups showed similar reaction times. In the stretching test, there was less nociception in COX-1-null and COX-1-deficient heterozygotes and also, unexpectedly, in female COX-2-deficient heterozygotes, as shown by a decreased number of writhes. Measurements of mRNA levels by reverse transcription-PCR demonstrated a compensatory increase of COX-1 mRNA in spinal cords of COX-2-null mice but no increase in COX-2 mRNA in spinal cords of COX-1-null animals. Thus, compensation for the absence of COX-1 may not involve increased expression of COX-2, whereas up-regulation of COX-1 in the spinal cord may compensate for the absence of COX-2. The longer reaction times on the hot plate of COX-1-deficient heterozygotes are difficult to explain, because nonsteroid anti-inflammatory drugs have no analgesic action in this test. Reduction in the number of writhes of the COX-1-null and COX-1-deficient heterozygotes may be due to low levels of COX-1 at the site of stimulation with acetic acid. Thus, prostaglandins made by COX-1 mainly are involved in pain transmission in the stretching test in both male and female mice, whereas those made by COX-2 also may play a role in the stretching response in female mice.

Induction of an acetaminophen-sensitive cyclooxygenase with reduced sensitivity to nonsteroid antiinflammatory drugs.

The transformed monocyte/macrophage cell line J774.2 undergoes apoptosis when treated for 48 h with competitive inhibitors of cyclooxygenase (COX) isoenzymes 1 and 2. Many of these nonsteroid antiinflammatory drugs (NSAIDs), but in particular diclofenac, induce during this time period a COX activity that coincides with a robust induction of COX-2 protein. Induction of this activity requires high, apoptosis-inducing concentrations of diclofenac (>100 microM). Prolonged treatment of J774.2 cells with lower doses of diclofenac inhibits COX activity, indicating that diclofenac is a time-dependent, pseudoirreversible inhibitor of COX-2. It is difficult to wash out the inhibition. However, the activity evoked by high concentrations of diclofenac has a profoundly distinct COX active site that allows diclofenac, its inducer, to be washed readily from its active site. The diclofenac-induced activity also has the unusual property of being more sensitive to inhibition by acetaminophen (IC50 = 0.1-1.0 mM) than COX-2 induced with bacterial lipopolysaccharide. Moreover, relative to COX-1 or COX-2, diclofenac-induced enzyme activity shows significantly reduced sensitivity to inhibition by diclofenac or other competitively acting nonsteroid antiinflammatory drugs (NSAIDs) and the enzyme activity is insensitive to aspirin. If the robust induction of COX-2 observed is responsible for diclofenac-induced COX enzyme activity, it is clear that COX-2 can, therefore, exist in two catalytically active states. A luciferase reporter-construct that contains part of the COX-2 structure and binds into the membrane showed that chronic diclofenac treatment of fibroblasts results in marked mobilization of the fusion protein. Such a mobilization could result in enzymatically distinct COX-2 populations in response to chronic diclofenac treatment.

Anti-inflammatory drugs and their mechanism of action.

Nonsteroidal anti-inflammatory drugs (NSAIDs) produce their therapeutic activities through inhibition of cyclooxygenase (COX), the enzyme that makes prostaglandins (PGs). They share, to a greater or lesser degree, the same side effects, including gastric and renal toxicity. Recent research has shown that there are at least two COX isoenzymes. COX-1 is constitutive and makes PGs that protect the stomach and kidney from damage. COX-2 is induced by inflammatory stimuli, such as cytokines, and produces PGs that contribute to the pain and swelling of inflammation. Thus, selective COX-2 inhibitors should be anti-inflammatory without side effects on the kidney and stomach. Of course, selective COX-2 inhibitors may have other side effects and perhaps other therapeutic potential. For instance, COX-2 (and not COX-1) is thought to be involved in ovulation and in labor. In addition, the well-known protective action of aspirin on colon cancer may be through an action on COX-2, which is expressed in this disease. Moreover, NSAIDs delay the progress of Alzheimer's disease. Thus, selective COX-2 inhibitors may demonstrate new important therapeutic benefits as anticancer agents, as well as in preventing premature labor and perhaps even retarding the progression of Alzheimer's disease.

Mechanism of action of nonsteroidal anti-inflammatory drugs.

Salicylic acid and salicylates, obtained from natural sources, have long been used as medicaments. Salicylic acid was chemically synthesized in 1860 and was used as an antiseptic, an antipyretic, and an antirheumatic. Almost 40 years later, aspirin was developed as a more palatable form of salicylate. Soon after, other drugs having similar actions to aspirin were discovered, and the group was termed the "aspirin-like drugs" (also now termed the nonsteroidal anti-inflammatory drugs [NSAIDs]). Twenty-five years ago, it was proposed that the mechanism of action of NSAIDs was through their inhibition of prostaglandin biosynthesis. Since then, there has been general acceptance of the concept that these drugs work by inhibition of the enzyme cyclo-oxygenase (COX), which we now know to have at least two distinct isoforms: the constitutive isoform, COX-1, and the inducible isoform, COX-2. COX-1 has clear physiologic functions. Its activation leads, for instance, to the production of prostacyclin, which when released by the endothelium is antithrombogenic and when released by the gastric mucosa is cytoprotective. COX-2, discovered 6 years ago, is induced by inflammatory stimuli and cytokines in migratory and other cells. It is therefore attractive to suggest that the anti-inflammatory actions of NSAIDs are due to inhibition of COX-2, whereas the unwanted side-effects, such as irritation of the stomach lining, are due to inhibition of COX-1. Drugs that have the highest COX-2 activity and a more favorable COX-2: COX-1 activity ratio will have a potent anti-inflammatory activity with fewer side-effects than drugs with a less favorable COX-2: COX-1 activity ratio. The identification of selective inhibitors of COX-2 will therefore lead to advances in therapy.

Cyclooxygenases 1 and 2.

Cyclooxygenase (COX), first purified in 1976 and cloned in 1988, is the key enzyme in the synthesis of prostaglandins (PGs) from arachidonic acid. In 1991, several laboratories identified a product from a second gene with COX activity and called it COX-2. However, COX-2 was inducible, and the inducing stimuli included pro-inflammatory cytokines and growth factors, implying a role for COX-2 in both inflammation and control of cell growth. The two isoforms of COX are almost identical in structure but have important differences in substrate and inhibitor selectivity and in their intracellular locations. Protective PGs, which preserve the integrity of the stomach lining and maintain normal renal function in a compromised kidney, are synthesized by COX-1. In addition to the induction of COX-2 in inflammatory lesions, it is present constitutively in the brain and spinal cord, where it may be involved in nerve transmission, particularly that for pain and fever. PGs made by COX-2 are also important in ovulation and in the birth process. The discovery of COX-2 has made possible the design of drugs that reduce inflammation without removing the protective PGs in the stomach and kidney made by COX-1. These highly selective COX-2 inhibitors may not only be anti-inflammatory but may also be active in colon cancer and Alzheimer's disease.

Mechanism of action of antiinflammatory drugs.

In 1971, Vane showed that nonsteroid antiinflammatory drugs (NSAIDs) inhibited the biosynthesis of prostaglandins and proposed this as their mechanism of action. Much work around the world has followed. The aspirin-like drugs inhibit the binding of the prostaglandin substrate, arachidonic acid, to the active site of the enzyme. After characterization of the COX-1 enzyme in 1976, a second COX gene was discovered in 1991 encoding for the inducible COX-2. The constitutive isoform of COX, COX-1, has clear physiological functions. The inducible isoform, COX-2, is induced by pro-inflammatory stimuli in migratory cells and inflamed tissues. The range of activities of NSAIDs against COX-1 compared to COX-2 explains the variations in the side effects of NSAIDs at their antiinflammatory doses. Drugs which have the highest potency on COX-2 and less effect on COX-1 will have potent antiinflammatory activity with fewer side effects. All the results published so far support the hypothesis that the unwanted side effects of NSAIDs, such as damage to the gastric mucosa and kidneys, are due to their ability to inhibit COX-1, while their antiinflammatory (therapeutic effects) are due to inhibition of COX-2. Other roles for COX-2 inhibitors will surely be found in the next few years, for prostaglandin formation is under strong control in organs such as the kidney, lungs and uterus. COX-2 is also potently expressed in human colon cancer cells, and NSAIDs delay the progress of colon tumors possibly by causing apoptosis of the tumor cells. The risk of developing Alzheimer's disease, which may involve an inflammatory component, is lessened by chronic ingestion of NSAIDs. The new highly selective inhibitors of COX-2 will not only provide a means of delaying premature labor but will also lead to advances in cancer therapy and protection against Alzheimer's disease.

Mechanism of action of aspirin-like drugs.

Nonsteroid antiinflammatory drugs (NSAIDs) or aspirin-like drugs act by inhibiting the activity of the cyclooxygenase (COX) enzyme. Two isoforms of COX exist, COX-1, which is constitutively expressed, and COX-2, which is an inducible isoform. Prostaglandins synthesized by the constitutively expressed COX-1 are implicated in the maintenance of normal physiological function and have a 'cytoprotective' action in the stomach. COX-2 expression is normally low but is induced by inflammatory stimuli and cytokines. It is thought that the antiinflammatory actions of NSAIDs are caused by the inhibition of COX-2, whereas the unwanted side effects, such as gastrointestinal and renal toxicity, are caused by the inhibition of the constitutively expressed COX-1. Individual NSAIDs show different selectivities against the COX-1 and COX-2 isoforms. NSAIDs that are selective towards COX-2, such as meloxicam, may have an improved side-effect profile over current NSAIDs. In addition to their use as antiinflammatory agents in the treatment of rheumatoid arthritis and osteoarthritis, selective COX-2 inhibitors may also be beneficial in inhibiting colorectal tumor cell growth and in delaying premature labor.

Mechanism of action of anti-inflammatory drugs.

Cyclooxygenase (COX) is the pivotal enzyme in prostaglandin biosynthesis. It exists in two isoforms, constitutive COX-1 (responsible for physiological functions) and inducible COX-2 (involved in inflammation). Inhibition of COX explains both the therapeutic effects (inhibition of COX-2) and side effects (inhibition of COX-1) of non-steroidal anti-inflammatory drugs (NSAIDs). A NSAID which selectively inhibits COX-2 is likely to retain maximal anti-inflammatory efficacy combined with less toxicity. The activity of a number of NSAIDs has been investigated in several test systems, showing that most of those marketed have higher activities against COX-1 or are equipotent against both isoforms. Adverse event data of marketed NSAIDs show a relationship between a poor safety profile and more potent inhibition of COX-1 relative to COX-2. There are several new non-steroidal COX-2 inhibitors in development. The most clinically advanced is meloxicam, which consistently demonstrates higher activity against COX-2 than COX-1 in several test systems.

Co-induction of nitric oxide synthase and cyclo-oxygenase: interactions between nitric oxide and prostanoids.

1. Lipopolysaccharide (LPS) co-induces nitric oxide synthase (iNOS) and cyclo-oxygenase (COX-2) in J774.2 macrophages. Here we have used LPS-activated J774.2 macrophages to investigate the effects of exogenous or endogenous nitric oxide (NO) on COX-2 in both intact and broken cell preparations. NOS activity was assessed by measuring the accumulation of nitrite using the Griess reaction. COX-2 activity was assessed by measuring the formation of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) by radioimmunoassay. Western blot analysis was used to determine the expression of COX-2 protein. We have also investigated whether endogenous NO regulates the activity and/or expression of COX in vivo by measuring NOS and COX activity in the lung and kidney, as well as release of prostanoids from the perfused lung of normal and LPS-treated rats. 2. Incubation of cultured murine macrophages (J774.2 cells) with LPS (1 microgram ml-1) for 24 h caused a time-dependent accumulation of nitrite and 6-keto-PGF1 alpha in the cell culture medium which was first significant after 6 h. The formation of both 6-keto-PGF1 alpha and nitrite elicited by LPS was inhibited by cycloheximide (1 microM) or dexamethasone (1 microM). Western blot analysis showed that J774.2 macrophages contained COX-2 protein after LPS administration, whereas untreated cells contained no COX-2. 3. The accumulation of 6-keto-PGF1 alpha in the medium of LPS-activated J774.2 macrophages was concentration-dependently inhibited by chronic (24 h) exposure to sodium nitroprusside (SNP; 1-1000 microM). Sodium nitroprusside (1-1000 microM) also acutely (30 min) inhibited COX-2 activity in broken cell preparations of LPS-activated (12 h) J774.2 macrophages, in a similar concentration dependent manner. Addition of adrenaline (5 mM) and glutathione (0.1 mM) increased the activity of COX-2 in broken cell preparations. In the presence of these co-factors, SNP inhibited prostanoid production only at the highest concentration used (1 mM). When J774.2 cells were incubated in the presence of LPS (1 microg ml-1) and NG-monomethyl-L-arginine (L-NMMA: 1 mM) for 12 h, SNP at the highest concentration used (1 mM) acutely (30 min) inhibited the activity of COX-2 in cell homogenates with co-factors. However, when J774.2 macrophages were incubated for 24 or 12 h with LPS (1 microg ml-1)and L-NMMA (1 mM), the addition of SNP (0.001-1I000 microM) increased in a concentration-dependent manner the accumulation of 6-keto-PGF1a in intact cells (measured at 24 h) and COX-2 activity in cell homogenates in the presence of co-factors (determined at 12 h). SNP (1 mM; together with LPS for 12 h)decreased the amount of COX-2 protein induced by LPS in J774.2 macrophages.4. Indomethacin (30 1AM) abolished the formation of 6-keto-PGFa by LPS-activated macrophages, but had no effect on the release of nitrite. Conversely, L-NMMA, at the highest concentrations used (1 and 10 mM), increased the release of 6-keto-PGFIa an effect which was reversed by excess L-arginine (3 mM)but not by D-arginine. Similarly, the decrease in nitrite formation caused by L-NMMA was partially reversed by L-arginine (3 mM), but not by D-arginine. L-NMMA (10 mM; together with LPS for 12 h)increased the amount of COX-2 protein induced by LPS in J774.2 macrophages.5. In separate experiments, J774.2 macrophages were activated with LPS (1 microg ml-1), and L-NMMA(10 mM) was added for various times (0.5-24 h) before the collection of mediun at 24 h. L-NMMAenhanced the release of 6-keto-PGFI,, in a time-dependent manner, with the maximal enhancement seen when the NOS inhibitor was incubated with the cells for 24 h. 6. In experiments on male Wistar rats, we investigated the effect of L-NMMA on the release of prostanoids (6-keto-PGF1a prostaglandin E2, thromboxane B2) elicited by arachidonic acid (AA,30nmol) from ex vivo perfused kidneys and lungs. The release from the organs from normal and LPS-treated rats was unaffected by L-NMMA intraperitoneally (30 mg kg-1) for 6 h together with LPS(5 mg kg-1) or LPS vehicle. Similarly, acute (5 min) in vitro exposure to L-NMMA (1 mM) of the perfused organs from control and LPS-treated animals did not change the release of prostanoids elicited by AA (30 nmol).7. These results show that LPS causes the induction of iNOS and COX-2 in J774.2 macrophages. The co-release of NO and PGI2 induced by LPS is dependent on protein synthesis and occurs after a lag-time of 6-12 h. The formation of COX metabolites has no effect on NOS activity whereas NO inhibits both COX-2 activity and induction. These results demonstrate that NOS and COX can be co-induced in vitro and that under these conditions large amounts of NO inhibit the degree of COX expression and activity.In the absence of endogenous NO, lesser amounts of exogenous NO increase the activity of COX-2. In those situations in vivo when the level of NO induction is relatively low, NO does not regulate the increased activity of COX.

Pharmacodynamic profile of prostacyclin.

Since the 1930s and the discovery by von Euler of a vasoactive, lipid-soluble substance that he erroneously assumed was generated by the prostate gland and therefore should be called "prostaglandin," the family of prostaglandins has grown to some 90 substances. These lipid mediators are derived from arachidonic acid in the "arachidonic acid cascade." In 1976, while looking for the enzyme that generates the unstable prostanoid thromboxane A2 from arachidonic acid, Moncada and Vane discovered prostaglandin I2 and renamed it "prostacyclin." Prostacyclin is the main product of arachidonic acid in all vascular tissues tested to date and strongly vasodilates all vascular beds studied. It is also the most potent endogenous inhibitor of platelet aggregation yet discovered, both inhibiting aggregation and dispersing existing aggregates. It acts through activation of adenylate cyclase, leading to increased levels of cyclic adenosine monophosphate. It also appears to have a "cytoprotective" activity, as yet not completely understood. Its effects are short-lasting, disappearing within 30 minutes of cessation of infusion. A stable, freeze-dried preparation of prostacyclin (epoprostenol) is available for administration to humans, and several analogs with therapeutically desirable characteristics are currently being clinically tested and should become commercially available soon. Clinical application of prostacyclin is bedeviled by 2 characteristics: it is pharmacologically unstable, so care must be taken in its use, and the correct dosage regimens have not yet been established.

New insights into the mode of action of anti-inflammatory drugs.

The discovery of a second cyclooxygenase has provided fresh impetus to the search for new anti-inflammatory drugs. The second enzyme is effectively absent from healthy tissues but its levels rise dramatically during inflammation. It can be induced in migratory cells by bacterial lipopolysaccharide, cytokines and growth factors. The constitutive cyclooxygenase-1 (COX-1) can thus be considered a "housekeeping" enzyme, in contrast to cyclooxygenase-2 (COX-2) which is activated by tissue damage. Both enzymes have a molecular weight of around 70 kDa and similar Km and Vmax values for their reaction with arachidonic acid. Several non steroid anti-inflammatory drugs which have more than 1,000 fold selectivity for COX-2 over COX-1 are in the early stages of drug development.

Relationship between different isoforms of nitric oxide synthase and cyclooxygenase in various cell types.

Nitric oxide (NO) and prostacyclin (PGI2), formed by NO synthase (NOS) and cyclooxygenase (COX), respectively, are two potent anti-aggregatory vasodilators released from endothelial cells. Both NOS and COX exist as constitutive and inducible isoforms. We have shown that NOS and COX are co-induced in vitro and in vivo by bacterial endotoxin and that low amounts of NO increase whereas high amounts inhibit the activity and expression of inducible COX in vitro.

Regulatory mechanisms of the vascular endothelium: an update.

This review discusses recent experimental findings in prostacyclin, nitric oxide and endothelin research. Prostacyclin formation by endothelial cells in atherosclerosis and diabetes is reviewed and the synthesis of prostacyclin by cyclooxygenase 1 and 2 (COX-1 and COX-2) is discussed. Further work on nitric oxide describes its involvement in septic and haemorrhagic shock and its interactions with the cyclooxygenase pathway. Recent studies in endothelin research include the development of both selective and orally active receptor antagonists, characterization of endothelin converting enzymes and the involvement of endothelin-1 in inflammation and wound repair.