Great strides have been made in identifying mediators of inflammation and pain. This has led to new targets for anti-inflammatory drug development. The purpose of this article is: 1) to provide a contemporary overview on mediators of inflammatory pain; 2) to provide information on the comparative efficacy of currently used NSAIDs (nonsteroidal anti-inflammatory drugs) in horses; 3) to introduce the pharmacology of new NSAIDs; and 4) to discuss targets for the development of future NSAIDs with a special discussion of the role of nitric oxide and pain.

The cardinal signs of inflammation are redness, swelling, heat, pain and loss of function. Pain is perhaps one of the most significant manifestations of inflammation which demands our attention and treatment. The expression of pain varies widely among animals. However, stimuli that cause pain are similar. Extremes of heat (e.g., burns) and cold (e.g., frostbite), high concentrations of hydrogen ions (e.g., lactate buildup in muscles), distension of a hollow viscus (e.g., intestinal obstruction and colic), traumatic injury (e.g., bone fractures) and ischemia (e.g., intestinal torsion) can cause pain. Pain elicits protective reflexes and often complex emotional responses. Persistent untreated pain can lead to hormonal, nervous, and psychological abnormalities in animals.

Horses with musculoskeletal disease, such as laminitis, often demonstrate signs of lameness. Administration of different anti-inflammatory drugs offers variable degrees and duration of pain relief, which are often assessed by gait analysis. Studies evaluating the efficacy and potency of these drugs in horses are ongoing at LSU.

Hyperalgesia refers to an increased sensitivity to pain and typically accompanies inflammation. It is produced by mediator substances in response to tissue injury and repair. Sources of these mediators include damaged tissue, blood vessels, neurons and immune cells. Bradykinin is released from damaged tissue and blood vessels. Substance P is liberated from excited or damaged free nerve endings. Neutrophils release leukotrienes (LTs) and activated macrophages release tumor necrosis factor alpha (TNFa) and interleukins (IL) 1 and 8. Fibroblasts, synoviocytes, and numerous other tissue cells release prostaglandins (PGs). These chemical mediators contribute collectively to hyperalgesia and pain.

Prostaglandins and LTs are produced from arachidonic acid when cell membranes are disrupted. Prostaglandins are formed via the action of cyclooxygenase (COX) which exists in two 'isoforms' (COX-1 and COX-2). Prostaglandins formed from'constitutive' COX-1 regulate the production of cytoprotective gastric mucus and control renal blood flow. Prostaglandins formed from 'inducible' COX-2 are generated by immune and tissue cells in the presence of inflammation. Leukotrienes are formed via the action of 5-lipoxygenase. Prostaglandin E2 and LTB4 products are thought to be important mediators of inflammatory pain or hyperalgesia.

Current NSAIDs are believed to act, at least in part, by inhibiting the COX enzyme. With few exceptions most NSAIDs inhibit both COX-1 and COX-2. Inhibition of COX-1 is thought to underlie the ulcer, hemorrhage, and nephropathy associated with long-term use of NSAIDs. Inhibition of COX-2 is thought to underlie the anti-inflammatory actions. In addition to their actions on COX, current NSAIDs may also inhibit neutrophil production of destructive superoxide radicals and proteases.

Currently available NSAIDs are indicated for the treatment of mild to moderately severe, acute and chronic pain. Nonsteroidal anti-inflammatory drugs differ widely in potency and therapeutic efficacy. In descending order, the therapeutic efficacy for NSAIDs in horses is flunixin, meclofenamic acid, ketoprofen, phenylbutazone, naproxen, and aspirin. Differences in efficacy can be explained by varying degrees of COX and cytokine inhibition, non-COX mechanisms, differing analgesia versus anti-inflammatory effects, and wide individual variation in response, within and between species.

Recent studies from our laboratory compared the therapeutic efficacy of phenylbutazone and ketoprofen (Ketofen® -- Rhome-Poulenc) in laminitic horses with chronic hoof pain. Four separate measures of analgesia were utilized including responses to an electronic hoof tester and subjective lameness assessment. The electronic hoof tester measured the amount of compressive force required to elicit a withdrawal reaction (i.e., hoof compression threshold or HCT) and the number of regions or 'loci' on the foot that were hyperalgesic. Lameness at a walk and trot were assessed using a modified Obel scale. Recommended therapeutic doses of phenylbutazone (4.4 mg/kg) and ketoprofen (2.2 mg/kg) were roughly equivalent in their ability to increase HCT and decrease lameness. However, a 3.63 mg/kg dose of ketoprofen (a phenylbutazone equimolar dose) produced a greater reduction in lameness and a greater increase in HCT which lasted for 24 hours. These results suggest that ketoprofen is more potent and, at appropriate doses, may be more efficacious than phenylbutazone for the production of analgesia. A similar study was recently performed comparing therapeutic doses of flunixin (Banamine®--Schering Plough Animal Health, Inc) (1.1 mg/kg) and phenylbutazone (4.4 mg/kg). Preliminary results indicate that flunixin (Banamine®): 1) was twice as efficacious as phenylbutazone in elevating HCT and reducing lameness; and 2) had roughly 8 times the analgesic potency (mg/kg) of phenylbutazone. Peak flunixin analgesia was observed 6 to 9 hours after dosing.

Although ketoprofen is the most recently approved NSAID for horses, new NSAIDs have been developed for other species. There is considerable effort on the part of the pharmaceutical industry to identify NSAIDs with superior anti-inflammatory efficacy, yet minimal gas-trointestinal toxicity. Their approach has been to develop NSAIDs that preferentially inhibit COX-2 with minimal effect on COX-1. A member of the oxicam chemical class of NSAIDs, such as meloxicam (Boehringer Ingelheim), has recently undergone extensive testing in Europe. Meloxicam, depending upon the bioassay utilized, has a 1 to 100 fold greater selectivity for COX-2 than COX-1. It is more selective for COX-2 than piroxicam, tenidap, diclofenac and indomethacin. Meloxicam also possesses greater anti-inflammatory potency (2-60 times) than piroxicam, indomethacin, diclofenac, tenidap, naproxen, aspirin and tenoxicam. This NSAID appears to be a potent suppressor of PG production from activated phagocytes. Meloxicam was rated as 'better than good' in human rheumatoid arthritis patients and 'analgesic' in osteoarthritic patients. Compared to piroxicam, diclofenac and naproxen, meloxicam (7.5-15mg/patient for 6 months) was associated with the least number of adverse GI events in arthritic patients. Meloxicam has also been studied in dogs. In a urate-induced canine synovitis model, meloxicam (0.1 and 0.5 mg/kg IV) increased limb loading from 4 to 12 hours. Significant reductions in standing lameness and pain score were also reported from 4-24 hours.

Meloxicam has been studied in horses. A dose of 0.6 mg/kg was administered to ponies, and responses to carrageenan-induced acute inflammation were recorded. Meloxicam reduced heat, protein, lactic dehydrogenase, leukocyte numbers, PGE2, and thromboxane B2 in exudates obtained from the site of inflammation. This effect was greatest at 4-8 hours. Meloxicam levels gradually increased over time at the inflamed site. Although the incidence of gastrointestinal side effects in the horse have not been elucidated, meloxicam may be an NSAID worthy of further study.

As greater knowledge of intracellular signaling pathways is gained, new targets for anti-inflammatory drugs are emerging. The intracellular signally molecule, nitric oxide (NO), has been implicated in the production of pain. Nitric oxide is produced from L-arginine via the action of two enzymes:'constitutive' NO synthase (cNOS) and 'inducible' NO synthase (iNOS). The constitutive enzyme is found in neurons and endothelial cells where it plays a role in neurotransmission and vasorelaxation, respectively. The inducible enzyme is found in immune cells where it gives rise to NO during inflammation. Nitric oxide has the beneficial role of being'microbiocidal' and a natural vasodilator, and the detrimental role of contributing to pain and inflammation.

A number of studies suggest a role for NO in hyperalgesia. For example, intracutaneous injection of NO produces dose-related pain in humans. Inhibitors of NOS, such as L-NAME, produce dose dependent analgesia in hyperalgesic rats. The actions of L-NAME are reversed by L-arginine, the natural substrate for NOS. Nitric oxide synthesis inhibitors such as L-NAME and L-NNA produce analgesia when: 1) administered systemically (e.g., intraperitoneally); 2) administered directly into the cerebral ventricles or spinal cord; or 3) administered directly at the site of inflammation.

L-arginine is the natural substrate for NOS and can increase NO levels in certain tissues. Although it antagonizes the action of the NOS inhibitors, some studies have shown that it produces a moderate degree of analgesia when administered alone. In fact, studies in horses suggest that substances that donate or increase NO may relieve pain. Other studies have shown that a 10% intravenous infusion of L-arginine caused vasodilation and improved the circulation in hooves of ponies with grass-induced acute laminitis. Topical application of nitroglycerine, a potent vasodilator and NO donor, was reported to reduce the bounding pulses, decrease lameness and lower blood pressure in the laminitic ponies. These data suggest that despite the fact that L-arginine and nitroglycerine produce NO (which contributes to pain), their ability to vasodilate, improve pedal circulation and ultimately relieve pain may be more important. Certainly, agents that affect the production of NO are worthy of further study in horses with laminitis and other inflammatory conditions.