A Selective p 38 Mitogen-Activated Protein Kinase Inhibitor Reverses Cartilage and Bone Destruction in Mice with Collagen-Induced Arthritis

Destruction of cartilage and bone is a poorly managed hallmark of human rheumatoid arthritis (RA). p38 Mitogen-activated protein kinase (MAPK) has been shown to regulate key proinflammatory pathways in RA, including tumor necrosis factor , interleukin (IL)-1 , and cyclooxygenase-2, as well as the process of osteoclast differentiation. Therefore, we evaluated whether a p38 MAPK inhibitor, indole-5-carboxamide (SD282), could modulate cartilage and bone destruction in a mouse model of RA induced with bovine type II collagen [collageninduced arthritis (CIA)]. In mice with early disease, SD-282 treatment significantly improved clinical severity scores, reduced bone and cartilage loss, and reduced mRNA levels of proinflammatory genes in paw tissue, including IL-1 , IL-6, and cyclooxygenase-2. Notably, SD-282 treatment of mice with advanced disease resulted in significant improvement in clinical severity scoring and paw swelling, a reversal in bone and cartilage destruction as assessed by histology, bone volume fraction and thickness, and three-dimensional image analysis. These changes were accompanied by reduced osteoclast number and lowered levels of serum cartilage oligomeric matrix protein, a marker of cartilage breakdown. Thus, in a model of experimental arthritis associated with significant osteolysis, p38 MAPK inhibition not only attenuates disease progression but also reverses cartilage and bone destruction in mice with advanced CIA disease. Cartilage and bone destruction is the hallmark of rheumatoid arthritic (RA) joints that are poorly managed by current therapeutic modalities. The therapeutic effectiveness of drugs that target either inflammatory cytokines, such as TNFand IL-1 or cyclooxygenase (COX)-2, have demonstrated the importance of these pathways on the pathology of the disease. p38 Mitogen-activated protein kinase (MAPK), an intracellular signaling protein, has been shown to regulate the expression of TNF, IL-1 , and COX-2, and inhibitors of this kinase have demonstrated beneficial effects in animal models of arthritis (Kumar et al., 2003; Nishikawa et al., 2003). The roles of p38 MAPK on bone and cartilage function suggest that inhibitors of this kinase may offer an important therapeutic benefit by modulating tissue destruction in RA patients. In this article, we describe the effect of SD-282, an -specific inhibitor of p38 MAPK, on bone and cartilage in a mouse model of collagen-induced arthritis (CIA). CIA in the mouse reproduces many aspects of the human disease, including joint inflammation, cartilage and bone destruction, and pannus accumulation (Williams et al., 1992). Furthermore, inhibition of TNFor IL-1 with antibodies has demonstrated beneficial effects in this preclinical model (Joosten et al., 1999a) as well as in studies with RA patients. Osteolysis in mice with CIA is associated with increased joint osteoclasts (Suzuki et al., 1998), and p38 MAPK plays an important role in osteolytic processes. p38 MAPK regulates osteoclast difArticle, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.105.098020. ABBREVIATIONS: RA, rheumatoid arthritis; TNF, tumor necrosis factor; IL, interleukin; COX, cyclooxygenase; MAPK, mitogen-activated protein kinase; SD-282, indole-5-carboxamide (ATP-competitive inhibitor of p38 kinase), CIA, collagen-induced arthritis; PGE2, prostaglandin E2; FR167653, pyridinyl imidazole compound (specific inhibitor of p38 pathway); LPS, lipopolysaccharide; ELISA, enzyme-linked immunosorbent assay; SB202190, (4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole; COMP, cartilage oligomeric matrix protein; 3-D, threedimensional; micro-CT, CT, microcomputed tomography. 0022-3565/06/3181-132–141$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 318, No. 1 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 98020/3120166 JPET 318:132–141, 2006 Printed in U.S.A. 132 at A PE T Jornals on Sptem er 3, 2017 jpet.asjournals.org D ow nladed from ferentiation induced by a wide variety of factors including PGE2, TNF, granulocyte cell-stimulating factor, and RANK ligand (Li et al., 2002). In addition, p38 MAPK inhibition blocks bone resorption in fetal rat long bone cultures in vitro (Badger et al., 1996; Kumar et al., 2001) and prevents bone loss in isolated osteoblasts and bone marrow cells (Li et al., 2002). FR167653 is an inhibitor of p38 MAPK that, when used prophylactically, inhibits osteoclast numbers in rats with CIA (Nishikawa et al., 2003). None of the studies addressed the role of specific or nonspecific p38 MAPK inhibitors on bone destruction in mice with an advanced state of CIA disease. Therefore, the effect of SD-282 on the osteolytic nature of joint lesions was evaluated by measuring bone destruction and osteoclast number in mice with advanced CIA disease. Materials and Methods Animals. Male DBA-1 Lac/J mice were obtained from The Jackson Laboratory (Bar Harbor, ME), and the study was initiated when mice were 8 to 9 weeks of age. Materials. Mouse diet (Lab Diet-5015) was from Deans Animal Feeds (San Carlos, CA). Complete Freund’s adjuvant, incomplete Freund’s adjuvant, and Mycobacterium tuberculosis (strain H37Ra) were obtained from Difco Laboratories (Detroit, MI). Lipopolysaccharide (LPS) from Escherichia coli (0111:84) and acetic acid were purchased from Sigma (St. Louis, MO). Bovine type II collagen was purchased from the University of Utah (Salt Lake City, UT). Protease inhibitor cocktail was purchased from Roche (Indianapolis, IN), phosphatase inhibitor cocktail was from Calbiochem (San Diego, CA), BCA assay kit was from Pierce (Rockford, IL), antibodies for p38 MAPK were from Santa Cruz Biotechnology (Santa Cruz, CA), antibody for phospho-p38 MAPK was from Cell Signaling Technologies (Beverly, MA), and chemiluminescence ECL kit was from Amersham (Piscataway, NJ). Chemical Description of SD-282. SD-282 is an indole-5-carboxamide, ATP-competitive inhibitor of p38 kinase. It is a small-molecule, orally active inhibitor of p38 MAPK. SD-282 Potency and Specificity. IC50 of SD-282 against the human p38 MAPKs ( , , , and isoforms) were performed in duplicate using ELISA assays adapted from published methods (Goedert et al., 1997; Kumar et al., 1997; Clerk and Sugden, 1998; Hale et al., 1999). Purified human recombinant (E. coli) p38 MAPK isoforms were incubated with substrate (10.75 g/ml myelin basic protein) in the presence of inhibitor or control in a buffer composed of 50 mM HEPES, 20 mM MgCl2, 0.2 mM Na3VO4, and 1 mM dithiothreitol, pH 7.4, for 60 min at 25°C. The readout was ELISA quantitation of incorporation of ATP into myelin basic protein (MDS Pharma, Tampa, FL). Enzyme concentrations were as follows: 75 ng/ml p38 and 250 ng/ml p38 , p38 , and p38 . ATP concentration has been previously optimized for each assay and was 100 M in the p38 assay, 10 M in the p38 assay, 1 M in the p38 assay, and 3 M in the p38 assay. The control for the and isoforms was SB 202190, and for the and isoforms, the control was staurosporine. The IC50 values of SD-282 for p38 MAPK were derived from two studies. As reported previously (Li et al., 2004; Sweitzer et al., 2004), SD-282 demonstrates 14.3-fold selectivity for p38 MAPK (IC50 values of 0.0016 and 0.0011 M) compared with p38 MAPK (IC50 values of 0.023 and 0.022 M), whereas inhibition of p38 MAPK and p38 MAPK was less than 50%, even at concentrations of 10 M (Table 1). When tested in vitro at a concentration of 10 M, SD-282 demonstrated no inhibitory activity against a panel of other kinases, including extracellular signal-regulated kinase 2, c-Jun NH2-terminal kinase-1, and mitogen-activated protein kinase-activated protein kinase-2. In addition, SD-282 demonstrates no effect on the activity of purified COX-1 or COX-2 enzymes. Induction of Arthritis in Mice. Experimental arthritis was induced in mice with collagen and LPS treatments by the modified procedure of Joosten et al. (1994, 1997). Mice were immunized intradermally with bovine collagen II (100 g) emulsified with Mycobacterium butyricum in complete Freund’s adjuvant (Difco Laboratories). On day 21, the animals were boosted with an intradermal injection of 100 g of collagen II dissolved in incomplete Freund’s adjuvant. On day 23, animals received 50 g of LPS i.p. Clinical Assessment of Arthritis in Mice. In this and subsequent studies, assessments were made by observers blinded to specimen identity and treatment and to time of treatment. Mice paws were examined for disease severity, and macroscopic scores were assigned as described previously (Williams et al., 1992). The clinical severity of arthritis was graded on a scale of 0 to 3 for each paw, according to changes in redness and swelling (0, normal; 1, slight swelling and erythema; 2, pronounced edema; and 3, joint rigidity). Each limb was graded, resulting in a maximal clinical severity score of 12 per animal. The clinical severity score, number of paws affected, and body weight were monitored daily during the entire study period. Paw swelling/volume was measured with reference to its baseline by plethysmography (Buxco, Wilmington, NC). Paws were extracted for protein, and p38 MAPK and phospho-p38 MAPK were detected by Western blot. Relative quantization of gene expression was performed as described previously in Li et al., 2004. Treatment of Animals with Early Stage Disease. Two independent studies examined the effects of SD-282 treatment on the disease progression in animals with early stage CIA for a period of 10 days. In the first experiment, observation ended on day 10. In the second experiment, SD-282 treatment was withdrawn on day 10, and clinical severity scores were monitored for an additional 10 days. Mice with early signs of CIA (clinical severity score between 1 and 2) on day 24 were treated twice daily by oral gavage (0.25 ml/animal, n 15 per treatment group) with 30 or 90 mg/kg SD-282 or