Etoricoxib (MK-0663): preclinical profile and comparison with other agents that selectively inhibit cyclooxygenase-2.

We report here the preclinical profile of etoricoxib (MK-0663) [5-chloro-2-(6-methylpyridin-3-yl)-3-(4-methylsulfonylphenyl) pyridine], a novel orally active agent that selectively inhibits cyclooxygenase-2 (COX-2), that has been developed for high selectivity in vitro using whole blood assays and sensitive COX-1 enzyme assays at low substrate concentration. Etoricoxib selectively inhibited COX-2 in human whole blood assays in vitro, with an IC(50) value of 1.1 +/- 0.1 microM for COX-2 (LPS-induced prostaglandin E2 synthesis), compared with an IC(50) value of 116 +/- 8 microM for COX-1 (serum thromboxane B2 generation after clotting of the blood). Using the ratio of IC(50) values (COX-1/COX-2), the selectivity ratio for the inhibition of COX-2 by etoricoxib in the human whole blood assay was 106, compared with values of 35, 30, 7.6, 7.3, 2.4, and 2.0 for rofecoxib, valdecoxib, celecoxib, nimesulide, etodolac, and meloxicam, respectively. Etoricoxib did not inhibit platelet or human recombinant COX-1 under most assay conditions (IC(50) > 100 microM). In a highly sensitive assay for COX-1 with U937 microsomes where the arachidonic acid concentration was lowered to 0.1 microM, IC(50) values of 12, 2, 0.25, and 0.05 microM were obtained for etoricoxib, rofecoxib, valdecoxib, and celecoxib, respectively. These differences in potency were in agreement with the dissociation constants (K(i)) for binding to COX-1 as estimated from an assay based on the ability of the compounds to delay the time-dependent inhibition by indomethacin. Etoricoxib was a potent inhibitor in models of carrageenan-induced paw edema (ID(50) = 0.64 mg/kg), carrageenan-induced paw hyperalgesia (ID(50) = 0.34 mg/kg), LPS-induced pyresis (ID(50) = 0.88 mg/kg), and adjuvant-induced arthritis (ID(50) = 0.6 mg/kg/day) in rats, without effects on gastrointestinal permeability up to a dose of 200 mg/kg/day for 10 days. In squirrel monkeys, etoricoxib reversed LPS-induced pyresis by 81% within 2 h of administration at a dose of 3 mg/kg and showed no effect in a fecal 51Cr excretion model of gastropathy at 100 mg/kg/day for 5 days, in contrast to lower doses of diclofenac or naproxen. In summary, etoricoxib represents a novel agent that selectively inhibits COX-2 with 106-fold selectivity in human whole blood assays in vitro and with the lowest potency of inhibition of COX-1 compared with other reported selective agents.

2,3-Diarylcyclopentenones as orally active, highly selective cyclooxygenase-2 inhibitors.

Cyclopentenones containing a 4-(methylsulfonyl)phenyl group in the 3-position and a phenyl ring in the 2-position are selective inhibitors of cyclooxygenase-2 (COX-2). The selectivity for COX-2 over COX-1 is dramatically improved by substituting the 2-phenyl group with halogens in the meta position or by replacing the phenyl ring with a 2- or 3-pyridyl ring. Thus the 3,5-difluorophenyl derivative 7 (L-776,967) and the 3-pyridyl derivative 13 (L-784,506) are particularly interesting as potential antiinflammatory agents with reduced side-effect profiles. Both exhibit good oral bioavailability and are potent in standard models of pain, fever, and inflammation yet have a much reduced effect on the GI integrity of rats compared to standard nonsteroidal antiflammatory drugs.

Therapeutic administration of a selective inhibitor of nitric oxide synthase does not ameliorate the chronic inflammation and tissue damage associated with adjuvant-induced arthritis in rats.

Up-regulation of the inducible isoform of nitric oxide synthase (iNOS) was determined during the development of adjuvant-induced arthritis in the rat. iNOS enzymatic activity, measured in spleen tissue, appeared and increased coincidentally with the appearance and degree of paw swelling and joint destruction in this arthritis model, when measured on days 0 through 21 subsequent to inoculation of the rats with adjuvant. The increase in enzymatic activity was paralleled by an increase in the plasma nitrite/nitrate (NOx) level and the appearance of immunoreactive iNOS, as measured by Western immunoblot, in the spleens of these rats. Prophylactic administration of N-iminoethyl-L-lysine (L-NIL) completely abolished iNOS activity (plasma NOx elevation) and effectively reduced both the swelling and radiographic changes in the joint tissues of the noninjected paw measured on day 21. However, therapeutic administration of L-NIL beginning on day 14 had no effect on the inflammatory or arthritic changes measured on day 21, even though plasma NOx levels were reduced to that of the naive controls. These results suggest that iNOS may be involved with the initial stages of the immune response to adjuvant injection, but its product, NO, does not mediate the chronic inflammation and joint destruction which occur during the later phase in this model.

Pyrroles and other heterocycles as inhibitors of p38 kinase.

Investigation of furans, pyrroles and pyrazolones identified 3-pyridyl-2,5-diaryl-pyrroles as potent, orally bioavailable inhibitors of p38 kinase. 3-(4-pyridyl-2-(4-fluoro-phenyl)-5-(4-methylsulfinylphenyl)-pyrrol e (L-167307) reduces secondary paw swelling in the rat adjuvant arthritis model: ID50 = 7.4 mg/kg/b.i.d.

Inhibition of stromelysin-1 (MMP-3) by P1'-biphenylylethyl carboxyalkyl dipeptides.

Carboxyalkyl peptides containing a biphenylylethyl group at the P1' position were found to be potent inhibitors of stromelysin-1 (MMP-3) and gelatinase A (MMP-2), in the range of 10-50 nM, but poor inhibitors of collagenase (MMP-1). Combination of a biphenylylethyl moiety at P1', a tert-butyl group at P2', and a methyl group at P3' produced orally bioavailable inhibitors as measured by an in vivo model of MMP-3 degradation of radiolabeled transferrin in the mouse pleural cavity. The X-ray structure of a complex of a P1-biphenyl inhibitor and the catalytic domain of MMP-3 is described. Inhibitors that contained halogenated biphenylylethyl residues at P1' proved to be superior in terms of enzyme potency and oral activity with 2(R)-[2-(4'-fluoro-4-biphenylyl)ethyl]-4(S)-n-butyl-1,5-pentane dioic acid 1-(alpha(S)-tert-butylglycine methylamide) amide (L-758,354, 26) having a Ki of 10 nM against MMP-3 and an ED50 of 11 mg/kg po in the mouse pleural cavity assay. This compound was evaluated in acute (MMP-3 and IL-1 beta injection in the rabbit) and chronic (rat adjuvant-induced arthritis and mouse collagen-induced arthritis) models of cartilage destruction but showed activity only in the MMP-3 injection model (ED50 = 6 mg/kg iv).

Aggrecanase and metalloproteinase-specific aggrecan neo-epitopes are induced in the articular cartilage of mice with collagen II-induced arthritis.

OBJECTIVE: To analyze the roles of two classes of proteinases, 'aggrecanase', and matrix metalloproteinases (MMPs), in chondrodestruction during murine collagen-induced arthritis (CIA). METHODS: Generation of the 'aggrecanase' neo-epitope (NITEGE373), and the MMP neo-epitope (VDIPEN341) within aggrecan was studied by immunoperoxidase microscopy using specific anti-peptide antibodies in normal and stromelysin-1 (SLN-1) deficient knockout mice with CIA. RESULTS: High levels of NITEGE373 and VDIPEN341 neo-epitopes were observed in foci within CIA paw articular cartilage exhibiting depletion of glycosaminoglycans, in advance of significant cartilage erosion. The highest concentrations of NITEGE373 and VDIPEN341 labeling were observed and often co-distributed in the chondrocyte pericellular matrix, suggesting that stimulated chondrocytes can synthesize and/or activate both enzymes. Other regions of the cartilage frequently exhibited either NITEGE373 or VDIPEN341 labeling, but not both neo-epitopes simultaneously, suggesting that 'aggrecanase' and MMP cleavages of aggrecan may be generated independently. No detectable differences were observed in expression or distribution of either neo-epitope in SLN-1 knockout versus wild-type mice. In addition, in vitro digestion of joint sections with SLN-1 did not alter the expression of cartilage NITEGE373, while markedly increasing VDIPEN341 labeling. Peripheral nerves and brains of naive mice also exhibited intense anti-NITEGE373 labeling. CONCLUSIONS: These data indicate that NITEGE373 and VDIPEN341 aggrecan neo-epitopes are sensitive and specific markers of early joint pathology, and are consistent with the hypothesis that SLN-1 does not have 'aggrecanase' activity, and that 'aggrecanase' is distinct from the MMPs which cleave aggrecan at the MMP site.

Experimental osteoarthritis in dogs: a comparison of the Pond-Nuki and medial arthrotomy methods.

Lesions induced by transecting the cranial cruciate ligament in two surgical models of osteoarthritis (OA) in mature, male, cross-bred dogs were compared by using an established grading system and alternatives. Previously, we relied on evaluations of lesions in articular cartilage on femurs alone. No statistically significant differences were found between grades for lesions in cartilage when either treated or control joints were compared by surgical method. Because the Pond-Nuki method yielded statistically significant differences between grades for lesions affecting treated and control femurs or tibias, and for some parameters indicative of synovitis, we preferred this method of surgery. Although by using the medial arthrotomy method of surgery, we were able to destabilize the joint in a more consistent manner, significant differences between treated and control joints were found for lesions on tibias, but not femurs, a frequent site for OA in humans. Suggestions are made for enhancing the surgical models and for a more holistic approach to evaluating joints morphologically.