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.

My life and times with enzymes and mediators.

The vascular endothelium plays a vital role in the control of the circulation. It metabolises various vasoactive substances, converts angiotensin I to angiotensin II and secretes the potent vasodilator prostacyclin, as well as nitric oxide and endothelin-1. The balance between these mediators determines the response of the cardiovascular system in diseases such as hypertension, atherosclerosis and myocardial infarction. In this article I describe some of my work over the past 50 years. In the process, I hope to show that it is not only Princes who travel the road to Serendip but also Scientists. The application of simple methods, luck, happy coincidences, serendipity and noticing the unusual have all played an important role in my scientific career. However, the choice of colleagues has also been vital.

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.

The fight against rheumatism: from willow bark to COX-1 sparing drugs.

Man has been fighting rheumatism for thousands of years. Early therapy began with the use around the world of decoctions or extracts of herbs or plants such as willow bark or leaves. Most or all of these turned out to contain salicylates. The first record was about 3,500 years ago in the Ebers papyrus. Hippocrates, Celsus, Pliny the Elder, Dioscorides and Galen all recommended decoctions containing salicylate for rheumatic pain. A country parson, the Reverend Edward Stone of Chipping Norton in Oxfordshire, made the first "clinical trial" of willow bark (1). He was surprised by its bitter taste, which reminded him of cinchona bark (containing quinine), then being used to treat malaria. He harvested a pound of willow bark, dried it, pulverized it and dispersed it in tea, small beer or water. He found in 50 patients that doses of 1 dram (1.8g) cured their fever. He concluded "I have no other motives for publishing this valuable specific, than that it may have a fair and full trial in all its variety of circumstances and situations, and that the world may reap the benefits accruing from it". Salicylic acid was chemically synthesised in 1860 by Kolbe in Germany and its ready supply led to even more extended usage as an external antiseptic, as an antipyretic and in the treatment of rheumatism.

Nonsteroid drug selectivities for cyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associated with human gastrointestinal toxicity: a full in vitro analysis.

The beneficial actions of nonsteroid anti-inflammatory drugs (NSAID) can be associated with inhibition of cyclo-oxygenase (COX)-2 whereas their harmful side effects are associated with inhibition of COX-1. Here we report data from two related assay systems, the human whole blood assay and a modified human whole blood assay (using human A549 cells as a source of COX-2). This assay we refer to as the William Harvey Modified Assay. Our aim was to make meaningful comparisons of both classical NSAIDs and newer COX-2-selective compounds. These comparisons of the actions of >40 NSAIDs and novel COX-2-selective agents, including celecoxib, rofecoxib and diisopropyl fluorophosphate, demonstrate a distribution of compound selectivities toward COX-1 that aligns with the risk of serious gastrointestinal complications. In conclusion, this full in vitro analysis of COX-1/2 selectivities in human tissues clearly supports the theory that inhibition of COX-1 underlies the gastrointestinal toxicity of NSAIDs in man.

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.

The history of inhibitors of angiotensin converting enzyme.

This review paper by Sir John Vane, The Nobel Prize Laureate for the first time reveals the insides of discovery of inhibitors of angiotensin converting enzyme (ACE-1), presently known as important drugs for the treatment of hypertension, congestive heart failure and coronary artery disease.

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.

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 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.

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.