Valdecoxib: assessment of cyclooxygenase-2 potency and selectivity.

The discovery of a second isoform of cyclooxygenase (COX) led to the search for compounds that could selectively inhibit COX-2 in humans while sparing prostaglandin formation from COX-1. Celecoxib and rofecoxib were among the molecules developed from these efforts. We report here the pharmacological properties of a third selective COX-2 inhibitor, valdecoxib, which is the most potent and in vitro selective of the marketed COX-2 inhibitors that we have studied. Recombinant human COX-1 and COX-2 were used to screen for new highly potent and in vitro selective COX-2 inhibitors and compare kinetic mechanisms of binding and enzyme inhibition with other COX inhibitors. Valdecoxib potently inhibits recombinant COX-2, with an IC(50) of 0.005 microM; this compares with IC values of 0.05 microM for celecoxib, 0.5 microM for rofecoxib, and 5 microM for etoricoxib. Unique binding interactions of valdecoxib with COX-2 translate into a fast rate of inactivation of COX-2 (110,000 M/s compared with 7000 M/s for rofecoxib and 80 M/s for etoricoxib). The overall saturation binding affinity for COX-2 of valdecoxib is 2.6 nM (compared with 1.6 nM for celecoxib, 51 nM for rofecoxib, and 260 nM for etoricoxib), with a slow off-rate (t(1/2) approximately 98 min). Valdecoxib inhibits COX-1 in a competitive fashion only at very high concentrations (IC(50) = 150 microM). Collectively, these data provide a mechanistic basis for the potency and in vitro selectivity of valdecoxib for COX-2. Valdecoxib showed similar activity in the human whole-blood COX assay (COX-2 IC(50) = 0.24 microM; COX-1 IC(50) = 21.9 microM). We also determined whether this in vitro potency and selectivity translated to significant potency in vivo. In rats, valdecoxib demonstrated marked potency in acute and chronic models of inflammation (air pouch ED(50) = 0.06 mg/kg; paw edema ED(50) = 5.9 mg/kg; adjuvant arthritis ED(50) = 0.03 mg/kg). In these same animals, COX-1 was spared at doses greater than 200 mg/kg. These data provide a basis for the observed potent anti-inflammatory activity of valdecoxib in humans.

Biochemical and clinical evidence that aspirin-intolerant asthmatic subjects tolerate the cyclooxygenase 2-selective analgetic drug celecoxib.

BACKGROUND: Subjects with aspirin-intolerant asthma (AIA) respond with bronchoconstriction and extrapulmonary adverse reactions to conventional nonsteroidal anti-inflammatory drugs (NSAIDs) that inhibit the cyclooxygenase (COX) step in the biosynthesis of prostaglandins. Recently, 2 isotypes of COX have been identified, and COX-2-selective NSAIDs have been developed for treatment of inflammatory disorders. OBJECTIVE: We investigated whether 33 subjects with a typical history of AIA tolerated the new COX-2-selective NSAID celecoxib. METHODS: All subjects displayed current aspirin sensitivity in oral or inhalation challenge tests. The subjects first underwent a double-blind, randomized, cross-over, increasing-dose challenge with placebo or celecoxib (10, 30, or 100 mg in suspension) on 2 occasions 7 days apart. Thereafter, all subjects were exposed to 400 mg of celecoxib administered during an open challenge session as two 200-mg doses 2 hours apart. Lung function, clinical symptoms, and urinary excretion of leukotriene E(4) (LTE(4)) were monitored, with the latter being a sensitive biochemical marker of aspirin intolerance. RESULTS: There were no changes in lung function or extrapulmonary symptoms during the double-blind sessions or in urinary excretion of LTE(4). Also, the highest recommended daily dose of celecoxib was well tolerated, with no symptoms, lung function changes, or alterations in urinary LTE(4) levels. CONCLUSIONS: A group of subjects with clinically well-documented AIA tolerated acute challenge with the selective COX-2 inhibitor celecoxib. The findings indicate that the intolerance reaction in AIA is due to inhibition of COX-1. Large long-term studies of COX-2 inhibitors in AIA should be undertaken.

Neuronal overexpression of COX-2 results in dominant production of PGE2 and altered fever response.

Cyclooxygenases catalyze the first committed step in the formation of prostaglandins and thromboxanes from arachidonic acid. Cyclooxygenase-2 (COX-2), the inducible isoform of cyclooxygenase, is expressed in brain selectively in neurons of hippocampus, cerebral cortex, amygdala, and hypothalamus. Prostaglandins function in many processes in the CNS, including fever induction, nociception, and learning and memory, and are upregulated in paradigms of excitotoxic brain injury such as stroke and epilepsy. To address the varied functions of COX-2 and its prostaglandin products in brain, we have developed a transgenic mouse model in which COX-2 is selectively overexpressed in neurons of the CNS. COX-2 transgenic mice demonstrate elevated levels of all prostaglandins and thromboxane, albeit with a predominant induction of PGE(2) over other prostaglandins, followed by more modest inductions of PGI(2), and relatively smaller increases in PGF(2alpha),PGD(2), and TxB(2). We also examined whether increased neuronal production of prostaglandins would affect fever induction in response to the bacterial endotoxin lipopolysaccharide. COX-2 induction in brain endothelium has been previously determined to play an important role in fever induction, and we tested whether neuronal expression of COX-2 in hypothalamus also contributed to the febrile response. We found that in mice expressing transgenic COX-2 in anterior hypothalamus, the febrile response was significantly potentiated in transgenic as compared to non-transgenic mice, with an accelerated onset of fever by 1 2 hours after LPS administration, suggesting a role for neuronally derived COX-2 in the fever response.

Characterization of celecoxib and valdecoxib binding to cyclooxygenase.

Two compounds (celecoxib and valdecoxib) from the diarylheterocycle class of cyclooxygenase inhibitors were radiolabeled and used to characterize their binding to cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), several single-point variants of COX-2 (Val523Ile, Tyr355Ala, Arg120Ala, Arg120Gln, Arg120Asn) and one triple-point variant of COX-2 [Val523Ile, Arg513His, Val434Ile (IHI)]. We demonstrate highly specific and saturable binding of these inhibitors to COX-2. Under the same assay conditions, little or no specific binding to COX-1 could be detected. The affinity of [(3)H]celecoxib for COX-2 (K(D) = 2.3 nM) was similar to the affinity of [(3)H]valdecoxib (K(D) = 3.2 nM). The binding to COX-2 seems to be both rapid and slowly reversible with association rates of 5.8 x 10(6)/M/min and 4.5 x 10(6)/M/min and dissociation rates of 14 x 10(-3)/min (t(1/2) = 50 min) and 7.0 x 10(-3)/min (t(1/2) = 98 min) for [(3)H]celecoxib and [(3)H]valdecoxib, respectively. These association rates increased (4- to 11-fold) when the charged arginine residue located at the entrance to the main hydrophobic channel was mutated to smaller uncharged amino acids (Arg120Ala, Arg120Gln, and Arg120Asn). Mutation of residues located within the active site of COX-2 that define a 'side pocket' (Tyr355Ala, Val523Ile, IHI) of the main channel had a greater effect on the dissociation rate than the association rate. These mutations, which modified the shape of and access to the 'side pocket', affected the binding affinity of [(3)H]valdecoxib more than that of [(3)H]celecoxib. These binding studies provide direct insight into the properties and binding constants of celecoxib and valdecoxib to COX-2.

Pharmacology of celecoxib in rat brain after kainate administration.

Prostaglandin E(2) (PGE(2)) is the major prostaglandin produced both centrally and in the periphery in models of acute and chronic inflammation, and its formation in both locations is blocked by cyclooxygenase-2 (COX-2) inhibitors such as celecoxib. In animal models of inflammation, PGE(2) inhibition in the brain may occur secondarily to a peripheral action by inhibiting local PG formation that elicits increased firing of pain fibers and consequent activation of PG synthesis in the central nervous system (CNS). Celecoxib was studied in the kainate-induced seizure model in the rat, a model of direct central prostaglandin induction, to determine whether it can act directly in the CNS. In the kainate-treated rat brain there was increased PGE(2), PGF(2alpha), and PGD(2) production, with COX activity and PGE(2) formation increased about 7-fold over normal. We quantitated mRNA levels for enzymes involved in the prostaglandin biosynthetic pathways and found that both COX-2 and PGE synthase (PGEs) mRNA levels were increased in the brain; no changes were found for expression of COX-1 or PGD synthase mRNA. By Western blot analysis, COX-2 and PGEs were induced in total brain, hippocampus, and cortex, but not in the spinal cord. Immunohistological studies showed that COX-2 protein expression was enhanced in neurons. Dexamethasone treatment reduced the expression of both COX-2 and PGEs in kainate-treated animals. Celecoxib reduced the elevated PGE(2) levels in brain of kainate-treated rats and inhibited induced COX activity, demonstrating the ability of this compound to act on COX-2 in CNS. Doses of celecoxib that inhibited brain COX-2 were lower than those needed for anti-inflammatory activity in adjuvant arthritis, demonstrating a potent direct central action of the compound.

Cyclooxygenase-2 in human pathological disease.

To understand the potential role of cyclooxygenase (COX) in normal and inflammatory human diseases, we characterized the expression of COX-1 and COX-2 in biopsies of osteoarthritis, atherosclerosis, and cancer. Tissues were prepared for immunohistochemistry by standard methods, and representative cases assayed via Western blot and quantitative RT-PCR. COX-2 was not detected in normal human tissues with few exceptions. Moderate to marked COX-2 was observed in the macula densa (MD) and thick ascending limb (TAL) in human fetal kidneys, but was not detected in neonatal and adult MD and TALs. Low level, constitutive COX-2 was detected in colonic epithelium, peribronchial glands, and pancreatic ductal epithelium. Low to moderate COX-2 was detected basally in the cortex, hippocampus, hypothalamus, and spinal cord, and in reproductive tissues during ovulation, implantation and labor. No COX-2 was detected in the existing vasculature in normal tissues, and was also not expressed throughout the ductus arteriosus. COX-2 was markedly induced in human tissues of osteoarthritis, atherosclerosis and cancer. COX-2 was prominently expressed in the synovium, fibrocartilage of osteophytes, and in the blood vessels in the osteoarthritic (OA) knee joint. COX-2 was also prominently detected in the macrophages/foam cells in atherosclerotic plaques, and in the endothelium overlying and immediately adjacent to the fibrofatty lesion. Moderate- to intense COX-2 expression was consistently observed in the inflammatory cells, neoplastic lesions, and blood vessels in all epithelial-derived human cancers studied. In contrast, COX-1 was relatively ubiquitously observed in both normal and pathophysiological conditions. These data collectively imply COX-2 plays an important role in mediating a variety of inflammatory diseases, and imply COX-2 inhibitors may be effective in the prevention and/or treatment of OA, heart disease, and epithelial cancers.

The Role of Cyclooxygenase-2 in Inflammation.

Prostaglandins (PGs) can be synthetized via two isoforms of cyclooxygenase (COX). COX-1 is constitutively expressed in normal tissues, and its activity represent the normal physiological output of PGs. In inflammatory states, the newly discovered COX-2 is rapidly induced, and its activity accounts for the large amounts of PGs seen in inflammation. The commercially available nonsteroidal anti-inflammatory drugs (NSAIDs) are nonselective inhibitors of both COX isoforms; therefore, they provide anti-inflammatory activity as well as side effects associated with COX-1 inhibition. Selective inhibition of COX-2 expression explains at least in part the potent anti-inflammatory activity of steroids. Anti-inflammatory activity of newly developed COX-2 inhibitors, such as NS-398 or SC-58125, suggest a new approach of inflammatory diseases with more efficacious NSAIDs essentially devoid of side effects such as stomach ulcers.