WO2012031057A1 - Bms- 582949 for the treatment of resistant rheumatic disease - Google Patents

Abstract

The present invention generally relates to a method of treating resistant rheumatic disease, such as refractory rheumatoid arthritis, with a therapeutically effective amount of a dual action p38 inhibitor that is safe an well - tolerated. A dual action p38 kinase inhibitor is a compound that inhibits both activation of p38 kinase and p38 kinase activity in cells, in particular the compound BMS - 582949. The present invention contemplates the treatment of rheumatoid arhtritis patients with an inadequate response to methotrexate.

Description

BMS - 582949 FOR THE TREATMENT OF RESISTANT RHEUMATIC DISEASE

CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims the benefit of U.S. Provisional Application Serial No. 61/379,001 , filed September I , 2010, the disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention generally relates to a method of treating resistant rheumatic disease, such as refractory rheumatoid arthritis, with a therapeutically effective amount of a dual action p38 inhibitor that is safe and well-tolerated. A dual action p38 kinase inhibitor is a compound that inhibits both activation of p38 kinase and p38 kinase activity in cells. BACKGROUND OF THE INVENTION

[0003] A large number of cytokines participate in the inflammatory response, including IL- 1 , IL-6, IL-8 and TNF-a. Overproduction of cytokines such as IL-1 and TNF-a are implicated in a wide variety of diseases, including inflammatory bowel disease, rheumatoid arthritis, psoriasis, multiple sclerosis, endotoxin shock, osteoporosis, Alzheimer's disease, and congestive heart failure, among others. See e.g., Henry et al., Drugs Fut. , 24: 1345- 1354 ( 1999); Salituro et al., Curr. Med. Ckem., 6:807-823 (1999)]. Important mediators of proinflammatory cytokines such as TNFct and IL-1 β,. as well as cellular responses to such cytokines production, are the mitogen-activated protein (MAP) kinases, and in particular, p38 kinase. See e.g., Schieven, G.L., "The biology of p38 kinase: a central role in inflammation", Current Topics in Medicinal Chemistry, 5 :921 - 928 (2005). Accordingly, modulation of p38 kinase may be useful in the treatment of inflammatory disease including rheumatic diseases such as rheumatoid arthritis (RA).

[0004] Compounds that reportedly inhibit p38 kinase and cytokines such as IL-1 and TNF-a for use in treating inflammatory diseases are disclosed in U.S. Patent Nos.

6,277,989 and 6, 130,235 to Scios, Inc; U.S. Patent. Nos. 6, 147,080 and 5,945,41 8 to Vertex Pharmaceuticals Inc; U.S. Patent Nos. 6,251 ,914, 5,977, 103 and 5,658,903 to Smith-Kline Beecham Corp.; U.S. Patent Nos. 5,932,576 and 6,087,496 to G.D. Searle & Co.; WO 00/56738 and WO 01 /27089 to Astra Zeneca; WO 01/34605 to Johnson & Johnson; WO 00/12497 (quinazoHne derivatives as p38 kinase inhibitors); WO 00/56738 (pyridine and pyrimidine derivatives for the same purpose); WO 00/12497 (discusses the relationship between p38 kinase inhibitors); and WO 00/12074 (piperazine and piperidine compounds useful as p38 inhibitors). Other compounds that inhibit p38 kinase are pyrrolotriazine aniline compounds, information on these compounds is disclosed in U.S. Patent Nos. 6,670,357; 6,867,300; 7,034, 151 ; 7, 160,883; 7,21 1,666; 7,253, 167; and U.S. Publication Nos. 2003/023283 1 (published Dec. 18, 2003); 2004/0229877 (published Nov. 1 8, 2004); 2005/0043306 (published Feb. 24, 2005; 2006/0003967 (published Jan. 5, 2006); 2006/0030708 (published Feb. 9, 2006); 2006/0041 124 (published Feb. 23, 2006); 2006/0229449 (published Oct. 12, 2006); 2006/0235020 (published Oct. 19, 2006); and 2007/0213300 (published Sept 13, 2007).

[0005] In particular, WO 2003/090912 (U.S. Patent Nos. 7, 160,883, 7,388,009, p38 inhibitor, BMS-582949 (Example 7,

including processes of making and uses thereof.

[0006] Many present treatments for rheumatic disease involve the administration of nonspecific cytotoxic immunosuppressive drugs, such as methotrexate, and

cyclophosphamide, and other immunosuppressive drugs such as azathiopnne, cyclosporin A and TNFa blockers or antagonists, which suppress the immune system of the subject. Long-term use may result in a host of negative effects including increased risk of infection, malignancies, kidney failure, bone marrow suppression, pulmonary fibrosis, malignancy, diabetes and liver function disorders. In addition, these and other drugs (Disease Modifying Antirheumatic Drugs or DMARDs) can be ineffective against resistant rheumatic disease (see Kelley's Textbook of Rheumatology, 6th Edition, pp.

1001 - 1022). As used herein a resistant rheumatic disease is a disease that eventuaily fails to respond to at least one DMARD. This specifically includes rheumatoid arthritis patients who are methotrexate-inadequate responders [0007] Many ρ38α kinase inhibitors have exhibited safety issues such as liver toxicity or CNS toxicity. See e.g., Genovese, M.C., "Inhibition of p38: has the fat lady sung?", Arthritis Rheum., 60:317-320 (2009).; Hammalcer, D. et al., "Go upstream, young man: lessons learned from the p38 saga", Annals of the Rheumatic Diseases 69 (Suppl. I ): i77- 82; and Cohen, P,, "Targeting protein kinases for the development of anti-inflammatory drugs", Curr, Opin. Cell, Biol, 21 :317-324 (2009).Recently, more selective p38a inhibitors such as SCIO-469, VX-702, and pamipimod, have reported improved clinical safety profiles, but exhibit only transient efficacy in rheumatoid arthritis clinical trials. See Genovese et al., "Evaluation of the efficacy and safety of pamapimod, a p38 MAP kinase inhibitor, in a double-blind, methotrexate-controlled study of patients with active rheumatoid arthritis", Arthritis Rheum. 60:335-344 (2009); and Damjanov, N. et al., "Efficacy, pharmacodynamics, and safety of VX-702, a novel p38 MAPK. inhibitor, in rheumatoid arthritis: results of two randomized, double-blind, placebo-controlled clinical studies", Arthritis Rheum. 60: 1232- 1241 (2009).

[0008] Accordingly, there exists a need to provide a safe and effective means for treating resistant rheumatic disease, particularly refractory rheumatoid arthritis wherein the subject has fai led at least one DMARD.

SUMMARY OF THE INVENTION

[0009] The present invention describes a method of treating a resistant rheumatic disease, such as refractory rheumatoid arthritis, with a therapeutically effective amount of a dual action p38 kinase inhibitor that is safe and well-tolerated. A dual action p38 kinase inhibitor is a compound that inhibits both activation of p38 kinase and p38 kinase activity in cells. Safety is indicated by such an inhibitor that is tested in clinical trials having a rate of adverse events (AEs) and discontinuation due to AEs substantially similar to treatment with placebo plus a DMARD such as methotrexate.

BRIEF DESCRIPTION OF THE DRAWINGS

[001.0] Figure 1 (a), Inhibition of p38 phosphorylation and kinase activity in cells. Immunoblot analysis of levels of phospho- and total p38 and Hsp27 (red) compared to actin (green) from cells without LPS or treated with LPS and varying concentrations of BMS-582949. Quantitation is shown in Figure 1 (c). Blots are representative of 4 independent experiments,

[0011] Figure 1 (b). BMS-582949 reverses p38 activation in cells. Immunoblot analysis of phospho- and total p38a levels in cells without stimulation or in cells stimulated with LPS and then treated with 10 μΜ BMS-582949 at various times.

[0012] Figure 1 (c). Quantitation of inhibition of phosphorylation of p38 (triangles) and Hsp27 (squares) by SCI0469 (filled symbols) compared to BMS-582949 (open symbols), as detected by immunoblot analysis as in Figure 1 (a). Levels of signaling proteins were normalized to actin from the same blots and then the ratio of each phosphoprotein relative to its total protein counterpart was calculated as the measure of phosphorylation response.

[0013] Figure 1 (d). Quantitation of inhibition of phosphorylation of p38 (triangles) and Hsp27 (squares) by VX-702 compared to BMS-582949, as detected by immunoblot analysis as in Figures 1 (a) and 1 (c).

[0014) Figure 2. Structure of p38a docked to MKK3 compared to structures with inhibitors bound, (a) The structure of p38a (gold) bound to BMS-582949 is

superimposed on the structure of p38a (blue) docked to M K3 peptide (purple), (b) The structure of p38a (pink) bound to SCIO-469 is superimposed on the structure of p38a (blue) docked to MK 3 peptide (purple).

[0015] Figure 3. Regulation of expression of p38 MAPK signaling pathway components by p38 inhibitors, (a, b) Upregulation of MKK6 and MKK3 gene expression in the TC5 hepatic eel! line treated with p38 inhibitors; signal intensity for Affymetrix probesets 205698_s_at/MKK6 and (a) 215498_s_at/MKX3 and (b) is shown for biological replicate samples obtained from three independent treatment groups, indicated by symbol. This data is also displayed as the heatmap rows for MK 3 and MKK6 in (c) transcriptional regulation of p38 MAPK pathway components whose expression in the THLE celi line is consistently regulated by the five p38 inhibitors (P < 0.05). Expression is shown as average of fold-change in signal relative vehicle, (d) Pathway diagram showing the relative positions and roies for the components of the p38 MAPK pathway (as diagrammed in Johnson, G.L., "p38 MAPK Pathway", Sci. Signal. (Connections Map in the Database of Cell Signal ing, as seen 30 July 2010),

http://stke.sciencemag.org/cgi/cm/stkecm;CMP_I 0958), whose expression is consistently regulated (P < 0.05) by five p38 inhibitors (c and e), plus MEKK4 (grey) which is included for I^'inkage of upstream factors. Components regulated in the THLE hepatic cell line are outlined in bold, (e) Pathway components regulated by p38 inhibition in the THP 1 monocytic cell line /LPS model system. The fold-change for [LPS+vehicle] treatment is relative to vehicle treatment, while fold-change for [LPS+p38 inhibitor]- treated samples is relative to treatment with [LPS+vehicle], The genes are ordered to correspond with the pathway component diagram in d. Color scale saturates at 1 .5-fold for (c) and (e).

[0016] Figure 4(a). inhibition of p38 dependent responses in b!ood of human subjects treated with 300 mg BMS-582949. Total and pHsp27 concentrations in the blood without stimulation from samples collected at various times after the first dose on d 1 and the last dose on d 28 were determined by ELISA, and the ratio of phospho- to total Hsp27 was calculated as a measure of p38 signaling in the blood cells. The percent change in the ratio is plotted with the scale on the left axis. The concentration of BMS- 582949 in the same samples is plotted with the scale on the right axis.

[0017] Figure 4(b). Blood samples collected at the same times and were stimulated ex vivo with LPS and TNFa production after 6 h as determined by ELISA. The percent change in TNFa production is plotted with the scale on the left axis on the right axis and the BMS-582949 concentration in the samples is plotted with the scale on the right axis.

[0018] Figure 5a. Efficacy of BMS-582949 in RA patients as analyzed by baseline CRP and BMS-582949 trough levels. Patients were treated with 300 mg BMS-582949 for 85 days. DAS28(ESR) and ACR scores were determined on days 0, 15, 29, 57, 85 and 1 13. Trough concentrations (minimum observed concentration, Cmin) were determined on days 1 5, 29, 57, and 85. The mean trough concentration over those days was calculated for each subject. Sufficient data was not available to calculate mean trough concentrations for 7 subjects treated with BMS-582949. ACR20 and ACR50 response rates are plotted for placebo subjects (n = 61 , open bars), BMS-582949 subjects with average Cmin of 1 1 - 56 ng/mL (n = 26, grey bars) and BMS-582949 subjects with average Cmin of 57 - 257 ng mL (n = 27, black bars).

[0019] Figure 5b. DAS28(ESR) scores for the 30 BMS-582949 and 24 placebo group subjects with baseline CRP concentrations greater than 10 mg/L. The last day of dosing is shown by the arrow. *, p<0.05 vs. placebo. [0020] Figure 5c. DAS28(ESR) scores for the 30 B S-582949 and the 37 placebo group subjects with baseline CRP concentrations are < 10 mg/L.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The features and advantages of the invention may be more readily understood by those of ordinary skill in the art upon reading the following detailed description. It is to be appreciated that certain features of the invention that are, for clarity reasons, described above and below in the context of separate embodiments, may also be combined to form a single embodiment. Conversely, various features of the invention that are, for brevity reasons, described in the context of a single embodiment, may also be combined so as to form sub-combinations thereof.

[0022] As discussed above, p38 kinase plays a central role inflammation, regulating both the production of proinflammatory cytokines such as TNF and IL- 1 β, as well as cellular responses to such cytokines. However, many earl ier p38 compounds dropped out of cl inical development due to safety issues such as liver toxicity. Though more selective p38 inhibitors have improved clinical safety profiles they have exhibited only transient efficacy in RA clinical trials, with little or no benefit observed in 3 month studies.

[0023] The present invention describes a method of treating a resistant rheumatic disease, such as refractory rheumatoid arthritis, with a therapeutically effective amount of a dual action p38 kinase inhibitor that is safe and well-tolerated. A dual action p38 kinase inhibitor is a compound that inhibits both activation of p38 kinase and p38 kinase activity in cells. Safety is indicated by an inhibitor that is tested in clinical trials in combination with a D ARD such as methotrexate having a rate of adverse events (AEs) and discontinuation due to AEs substantially similar to treatment with placebo plus the same DMARD (see Table 1 ).

[0024] Accordingly, embodiment I of the present invention provides a method of treating a subject having resistant rheumatic disease, comprising administering an effective amount of a dual action p38 inhibitor having cellular p38 activation

Embodiment 2 of the present invention provides the method of embodiment 1 wherein the p38 inhibitor is B S-582949,

[0025] Embodiment 3 of the present invention provides the method of embodiments 1 -2 wherein the subject has failed at least one of methotrexate, cyclophosphamide, azathiprine, chloroquine, cyclosporine A, tumor necrosis factor-alpha (TNF-a) blocker or antagonists, costimulation blocker such as abatacept, or rituximab.

[0026] Embodiment 4 of the present invention provides the method of embodiments 1 -3, wherein the resistant rheumatic disease is refractory rheumatoid arthritis.

[0027] Embodiment 5 of the present invention provides the method of embodiments 1 -4, for reducing a symptom of rheumatoid arthritis.

[0028] Embodiment 6 of the present invention provides the method of embodiment 5 wherein the symptom is selected from the group consisting of joint swelling, joint tenderness, inflammation, morning stiffness, pain, structural damage.

[0029] Embodiment 7 of the present invention provides the method of embodiment 6 wherein the structural damage is bone or joint erosion.

[0030] Embod iment 8 of the present invention provides the method of embodiments 1 -7.

[0031 ] Embodiment 9 of the present invention provides the method of embodiments 1 -8 wherein administration of the p38 inhibitor results in a Cmin (minimum concentration in the course of 24 hours) or trough level in the blood greater than 56 ng/ml.

Embodiment 1 0 of the present invention provides the method of embodiments 1 -9 wherein administration of the p38 inhibitor results in a Cmin or trough level in the blood from about 57 ng/ml to about 250 ng/mi.

[0032] Embod iment 1 1 of the present invention provides the method of embodiments

1 - 10 wherein the administration of the dual action p38 inhibitor improves the difference in ACR 20 and ACR 50 response rates compared to placebo by at least 20% and at least

5%, respectively.

[0033] Embodiment 12 of the present invention provides the method of embodiments 1 - 1 1 wherein the adm inistration of the dual action p38 inhibitor improves the difference in ACR 20 and ACR 50 response rates compared to placebo by at least 34% and at least 9%, respectively.

[0034] Embodiment 1 3of the present invention provides the method of embodiments 1 - 12 wherein the administration of the dual action p38 inhibitor improves the

DAS28(ESR) score in a subject with baseline C reactive protein level greater than 10 mg/l by at least 1 ,5. [0035] A particular dual inhibitor, BMS-582949, is a potent p38 kinase inhibitor, displaying an IC50 of 1 3 n for p38a, 5-fold selectivity over ρ38β, and greater than 2000-fold selectivity over ρ38γ and δ. See Chunj ian Liu, J.L. et at., Journal of Medicinal Chemistry, in press. BMS-582949 is highly selective over a diverse panel of other kinases, and d isplays strong activity in human cellular assays and animal models of inflammation.

[0036] Cel lular resistance mechanisms characteristic of resistant rheumatic disease, particularly refractory rheumatoid arthritis, and the use of dual inhibitors, such as BMS- 582949, are identified herein that demonstrate the inhibition of p38 activation in cells and durable inhibition of p38 signaling in human subjects. These dual inhibitors provide significant safety and durable efficacy in treating resistant rheumatic disease, such as refractory rheumatoid arthritis, as measured in clinical studies described herein.

Blockade of p38a Activation in Cells

[0037] The effect of BMS-582949 on p38 cellular signal transduction utilizing normal human PBMC stimulated with LPS was investigated. BMS-582949 inhibited phosphorylation of Hsp27, which is downstream of the p38 substrate MAPKAP K2 (Fig la). Notably, BMS-582949 also inhibited the increase in p38 phosphorylation at Thrl 80/Tyrl 82 following LPS stimulation.

[0038] BMS-582949 also reverses p38 activation. Stimulation of cells with LPS for 1 5 min resu lted in p38 activation as shown by Tyrl 80Thrl 82 phosphorylation (Fig l b). When vehicle was then added for 5 m in, p38 phosphorylation was maintained. Treatment of the activated cel ls for 5 min with BMS-582949 reversed p38 activation to basal levels. The reversal of activation was maintained over the 75 m in time for the return of vehicle treated cell p38 to return to basal levels of phosphorylation.

[0039] Thus, we have discovered that BMS-582949 is a dual action inhibitor, blocking the activation of p38a in cells as well as the enzymatic activity of the activated kinase.

[0040] in addition, BMS-582949 binds with high affinity to both activated and unactivated p38a. This was confirmed by direct measurement of BMS-582949 binding by surface plasmon resonance and isothermal calorimetry, each of which gave very similar D values of 13.7 - 21 nM thai agree within error between activated and unactivated p38a. See Table 1 below.

Table 1

* D values and kinetic parameters of BMS-582949 binding to both unactivated and activated forms of p38a as determined by surface plasmori resonance (SPR) and isothermal titration calorimetry (ITC). Values as shown represent mean +/- 1 standard deviation. [0041 ] In comparison to BMS-582949, other compounds that have reported tachyphylaxis in RA trials do not substantially inhibit p38a phosphorylation. SCIO-469 did not demonstrate consistent dose response for inhibition of p38a phosphorylation, in contrast to the clear dose response observed for inhibition of Hsp27 phosphorylation (Fig lc). However, in the same experiment, BMS-582949 inhibited both Hsp27

phosphorylation and p38 phosphorylation with a clear dose response, achieving 80% inhibition of p38 phosphorylation at higher concentrations. VX-702 also showed no consistent dose response for inhibition of p38 phosphorylation, in contrast to its effects on Hsp27 phosphorylation and the effects of BMS-582949 in the same experiment (Fig. Id). The p38 inhibitor pamipimod has also been reported not to affect p38 phosphorylation in ceils. See H ill, R.J. et al ., "Painapimod, a novel p38 mitogen-activated protein kinase inhibitor: preclinical analysis of efficacy and selectivity", The Journal of Pharmacology and Experimental Therapeutics, 327:610-61 (2008). Thus all 3 compounds that exhibit tachyphylaxis in the clinic fail to substantial ly inhibit the activating phosphorylation of p38 in cells. [0042] The x~ray crystal structures of p38a bound to BMS-582949 and SCIO-469 were compared to the structure reported for p38a with the docking site for the upstream kinase M K3 occupied (see Chang, C.l . et al., "Crystal structures of MAP kinase p38 compiexed to the docking sites on its nuclear substrate MEF2A and activator M 3b", Molecular Cell, 9: 1 241 - 1249 (2002)), The structure of p38a with BMS-582949 bound showed a substantial conformational change in the position of the activation loop of p38a, including the DFG sequence, compared to its location in the structure of p38a ■ without inhibitor bound to M K3 peptide (Fig 2a). By contrast, the p38a structure with SCO-469 bound showed the activation loop, including the DFG sequence, in a very sim ilar conformation to that observed in the structure of p38a bound to M K3 peptide (Fig 2b). BMS-582949 appeared to induce a conformational change that may make the phosphorylation sites less accessible to docked upstream kinases such as MK 3, more accessible to regulatory phosphatases such as MK.P-1 , or both. Resistance Mechanisms

[0043] The effect of p38 inhibition in THLE-5 ceils, an SV40-immortalized human liver l ine that retains normal expression of detoxification systems and most Phase II metabolizing enzymes (see U .S. Patent No. 7,041 ,501 to O. Flint et a!.), was investigated to determine the effect on gene expression in hepatocytes. Cells were treated with 0.5μΜ BMS-582949 for 8 hours and transcript levels from over 12,000 genes then measured using Affymetrix HG-U 133A arrays. Only eight transcripts were regulated more than 1 .2-fold (PO.01 ) relative to paired control samples. These include M K6 and MK 3, the primary kinases that activate p38a (Fig. 3a, b),

[0044] To ex lore whether such changes in gene expression regulating p38 activation and signal transduction were a general property of p38a inhibition, four additional inhibitors (B!RB-796, RO3201 1 95, SB-503280 and VX-745) with diverse structures and off-target activities, such that shared effects would strongly indicate an on -target activity were investigated. Concentrations equal to 10 times the IC50 value for inhibition of LPS- T Fa production were employed to correct for differences in compound potency whi le providing virtual ly complete inhibition of p38 . It was discovered that transcripts of six pathway components are signi ficantly regulated (PO.05) by p38 inhibitors as a class (Fig. 3c), out of 34 components of the p38 MAP pathway represented on the HG- U l 33 A array {see Table 2) among the 36 cited as important for p38 signal transduction (Fig. 3d) Joh nson, G. L., "p38 MAPK Pathway", Set Signal. (Connections Map in the Database of Cel l Signal ing, as seen 30 July 2010),

http://stke.sciencemag.org/cgi/cm/stkecm ;CMP_10958.

Table 2

*Reguiation in THLE-5 cells for the 76 Affymetrix probesets that correspond to 41 loci/34 components from a canonical p38 MAPK pathway diagram (Johnson, G.L., "p38 MAPK Pathway", Sci. Signal. (Connections Map in the Database of Cell Signaling, as seen 30 July 2010), http://stke.sciencemag.org/cgi/cm/stkecm;CMP_10958). The top 8 probesets are regulated with PO.05 (in bold text) in a paired t-

test of DMSO treatment versus p38-inhibitor treatment with process block as random factor, and are ordered by relative position in the canonical p38 MAPK. pathway diagram. Data for probesets with P<0.05 is presented as a heatmap in the body of the manuscript, with the exception of two redundant probesets noted in grey.

[0045] Upstream of p38, MKK3, M K6 and GADD45 showed consistently increased expression, as does p38a itself, whereas MKPl , the phosphatase primarily responsible for deaciivation of p38, showed decreased expression. Taken together, these changes would al l be expected to drive p38 signal ing more strongly by increasing activation wh i le decreasing inactivation. MKK4 shows consistently decreased expression. Although KK4 can phosphorylate p38a in biochemical assays, it is primarily regarded as a regulator of the Jnk kinases.

[0046] The effects of the p38 inhibitors on expression of the 34 com onents of the p38 MAPK pathway represented on the HG-U 1 33A array (Supplementary Table 3) in the TH P- i monocyte cell l ine following LPS stimulation was explored. In this model 1 8 out of the 34 pathway components are significantly regulated (Fig. 3e), with effects observed both upstream and downstream of p38 (Fig. 3d), Compared to LPS plus vehicle, the combination of LPS plus p38 inhibitors often completely counteracted the transcriptional effects of LPS, including down regulation of p38 and its downstream kinases and upregu!ation of MKP l that provide feedback mechanisms to reduce further signaling (Fig, 3e), See Table 3, below.

Table 3

Regulation in a THP 1/LPS mode! of inflammation for the 76 Affymetrix probesets that correspond to 41 loci/34 components from a canonical p38 MAPK pathway diagram (Johnson, G.L., "p38 MAPK Pathway", Sci. Signal, (Connections Map in the Database of Cell Signaling, as seen 30 July 2010), http://stke.sciencemag.Org/cgi/cm/stkecm:CMP__10958). The top 25 probesets are regulated with O.05

(in bold text) in a paired /-test of LPS treatment versus [p38-inhibitor plus LPS treatment] with process ordered by the relative position of their gene products in the canonical p38 MAPK pathway diagram.

10047] Thus we have discovered that cells responding to p38 inhibition by changes in gene expression that would tend to predominantly drive activation more strongly, suggesting a potential cellular resistance mechanism to p38 inhibitors. Durable inhibition of p38 Signal Transduction

[0048] The signaling of p38 as measured by phosphorylation of Hsp27 in the blood in the absence of any exogenous stimulation was inhibited by over 90% at earlier time points after the first 300 mg dose, and showed substantial inhibition at trough of 57% (Fig 4a). On d 28 for the 300 mg dose, pHsp27 is already inhibited some as a trough effect from the previous dose, and again inhibited over 90% at earlier time points.

Substantial inhibition of 43% is observed at trough as for the d 1 dose. The

pharmacokinetic profile of B S-582949 is similar at days 1 and 28. The similar inhibition observed on days I and 28 indicates that once a day dosing of 300 mg BMS- 582949 durably maintains inhibition of p38 signal iransduction in human subjects.

It has been suggested that resistance to p38 inhibitors might theoretically involve compensation that would bypass a need for p38 signal transduction. See e.g., Genovese, .C., "Inhibition of p38: has the fat lady sung?" Arthritis Rheum., 60:317-320 (2009); Hammaker, D. et al., "Go upstream, young man: lessons learned from the p38 saga", Annals of the Rheumatic Diseases, 69 (Suppl. l):i77-82; and Gaestel, . et al.,

"Targeting innate immunity protein kinase signalling in inflammation", Nat. Rev. Drug Discov., 8:480-499 (2009). The ability of the 300 mgdose of BMS-582949 to inhibit LPS-induced TNPa production ex vivo in blood samples from the 28 day study. Doses on day 1 and day 28 gave over 90% inhibition of LPS-induced TNFa production at earlier times, and 67% inhibition at trough (Fig.4b). Accordingly it was discovered that no resistance developed over the 28 d of dosing in human subjects that was able to bypass the inhibition of LPS-induced TNFa production by BMS-582949.

Safety and Efficacy in RA Patients

[0049] BMS-582949 was examined in a 12 week multicenter randomized parailel group, double blind, placebo-controiled double blind clinical trial in RA patients with an inadequate response to methotrexate (MTX), comparing treatment with 300 mg BMS- 582949 administered orally once daily plus methotrexate (MTX) to treatment with placebo plus TX. It was discovered that B S-582949 is general ly safe and well tolerated in the study. The rates of adverse events (AEs) and discontinuation due to AEs were sim ilar for the BMS-582949 and placebo groups (Table 4, below). Two subjects treated with BMS-582949 discontinued (diarrhea and fatigue). Most AEs were mild to moderate in intensity.

Table 4

Safety Outcomes from 3 Month RA Clinical Study

[0050] Several AEs and serum chemistry abnormalities were of specific interest based on previous reports on p38 inhibitors. Infection-related AEs and rash-related AEs occurred in a sim i lar percentage of subjects in the BMS-582949 and placebo groups (Table 1 ). Rates of dizziness increased in the BMS-582949 group, but none of the reports of dizziness led to dose modification or discontinuation, and all were assessed as transient and mi ld or moderate in intensity. The rates of ALT and AST elevations were sim ilar for the BMS-582949 and placebo groups (Table 1 ), in contrast to elevations reported for many other p38 inhibitors. See Genovese, M.C., "Inhibition of p38: has the fat lady sung?", Arthritis Rheum. , 60:3 17-320 (2009) and Dambach, D.M, "Potential adverse effects associated with inhibition of p38alpha/beta MAP kinases", Current Topics in Medicinal Chemistry, 5 :929-939 (2005).

[0051 ] Overal l, it was discovered that BMS-582949 treatment gave ACR20 responses of 53% vs. 33% for placebo, ACR50 responses of 15% vs. 10% respectively, and ACR70 responses of 2% vs. 7% respectively. The difference in ACR20 response was statistically signi ficant (p< 0.05). This efficacy is substantially greater than reported for any previous p38 inhibitor (see id., Genovese et aL). BMS-58949 average trough exposures varied over 20-fold between subjects with the lowest and highest exposures. In order to determ ine whether BMS-582949 patients achieving higher exposures displayed greater efficacy, the treated subjects were divided into two equal groups based on their average trough exposure over the course of the study and compared their outcomes in a post hoc analysis (Fig, 5a). The BMS-582949 group in the lower exposure range showed little additional response compared to placebo. However, the BMS-582949 group with higher exposures showed marked ly higher ACR20 response rates of 67% vs. 33% for placebo (p<0.0 1 ) and ACR50 response rates of 1 9% vs. 1 0% for placebo.

[0052] The baseline C-reactive protein (CRP) levels among the subjects varied over 100-fold. Since CRP is a biomarker of systemic inflammation, we sought to examine whether patients with higher CRP levels showed any di fference in response in a post hoc analysis. We selected a baseline CRP level of greater than 10 mg/L because that has been an inclusion criterion for a variety of other RA cl inical trials and it divided the BMS- 582949 subjects into two equal sized groups. To avoid a direct impact of CRP on the efficacy score we uti lized the DAS28(ESR) score since it lacks CRP as a component. Among the higher baseline CRP patients, BMS-582949 showed rapid onset of action, achieving significant efficacy by the DAS28 score relative to piacebo in 2 weeks that further improved through the last day of dosing (Fig. 5b). By contrast, among the lower basel ine CRP patients both the placebo and BMS-582949 groups showed very simi lar reductions in the DAS28 score over the first 30 d, but at later times the two groups diverged, with the BMS-582949 group showing continuing improvement whi le the placebo group response decreased (Fig. 5c). Notably, the DAS28 scores regressed after the cessation of treatment for the BMS-582949 subjects in both CRP categories, consistent with the changes being due to the treatment. There was no evidence for tachyphylaxis among patients in either the higher or lower CRP groups, as the DAS28 scores decreased throughout the course of the treatment.

UTILITY

[0053] The present invention thus provides methods for treating resistant rheumatic disease, particularly refractory rheumatoid arthritis, comprising administering to a subject in need thereof an effective amount of BMS-582949 or a salt thereof. The methods of treating p38 kinase-associated conditions may comprise administering BMS-582949 alone or in combination with each other and/or other s itable therapeutic agents useful in treating such conditions. Exemplary of such other therapeutic agents include

corticosteroids, rolipram, calphostin, CSAIDs, 4-substituted imidazo [I,2-A]quinoxalines as disclosed in U.S. Patent No.4,200,750; InterSeukin-10, glucocorticoids, salicylates, nitric oxide, and other immunosuppressants; nuclear translocation inhibitors, such as deoxyspergualin (DSG); non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen, celecoxib and rofecoxib; steroids such as prednisone or dexameihasone;

antiviral agents such as abacavir; antiproliferative agents such as methotrexate, leflunomide, FK506 (tacrolimus, PROGRAF®); cytotoxic drugs such as azathiprine and cyclophosphamide; TNF-a inhibitors such as tenidap, anti-TNF antibodies or soluble TNF receptor, abatacept, belatacept or other blockers of costimulation, anti-IL6 antibodies, anti-IL-6 receptor antibodies such as tociiizumab, and rapamycin (sirolimus or RAPA UNE®) or derivatives thereof.

[0054] The above other therapeutic agents, when employed in combination with the compounds of the present invention, may be used, for example, in those amounts indicated in the Physicians' Desk Reference (PDR) or as otherwise determined by one of ordinary skill in the art. In the methods of the present invention, such other therapeutic agent(s) may be administered prior to, simultaneously with, or following the

administration of the inventive compounds.

[0055] The present invention also provides pharmaceutical compositions capable of treating p38-kinase associated conditions, including TNF-a, IL-l, and/or IL-8 mediated conditions, as described above. The inventive compositions may contain other therapeutic agents as described above and may be formulated, for example, by employing conventional solid or liquid vehicles or diluents, as well as pharmaceutical additives of a type appropriate to the mode of desired administration (e.g., excipients, binders, preservatives, stabilizers, flavors, etc.) according to techniques such as those well known in the art of pharmaceutical formulation.

[0056] BMS-582949 may be administered by any means suitable for the condition to be treated, which may depend on the need for site-specific treatment or quantity of drug to be delivered. Topical administration is generally preferred for skin-related diseases, and systematic treatment preferred for cancerous or pre-cancerous conditions, although - other modes of delivery are contemplated. For example, the compounds may be

delivered orally, such as in the form of tablets, capsules, granules, powders, or liquid formulations including syrups; topically, such as in the form of solutions, suspensions, gels or ointments; subl ingual ly; bucal!y; parenterally, such as by subcutaneous, intravenous, intramuscular or inirasternal injection or infusion techniques (e.g., as sterile injectable aq. or non-aq. solutions or suspensions); nasally such as by inhalation spray; topically, such as i n the form of a cream or ointment; rectally such as in the form of suppositories; or liposomal ly. Dosage unit formulations containing non-toxic, pharmaceutically acceptable vehicles or diluents may be administered, The compounds may be administered in a form suitable for immediate release or extended release.

Immediate release or extended release may be achieved with suitable pharmaceutical compositions or, particularly in the case of extended release, with devices such as subcutaneous implants or osmotic pumps.

[0057] Exemplary compositions for topical adm inistration include a topical carrier such as PLAST1BASE© (mineral oil gel led with polyethylene).

[0058] Exemplary compositions for oral administration include suspensions which may contain, for example, microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methyiceilu!ose as a viscosity enhancer, and sweeteners or flavoring agents such as those known in the art; and immediate release tablets which may contain, for example, m icrocrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and/or lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants such as those known in the art. BMS-582949 may also be orally del ivered by sublingual and/or buccal adm inistration, e.g. , with molded, compressed, or freeze-dried tablets. Exemplary compositions may include fast-dissolving diluents such as mannitol, lactose, sucrose, and/or cyclodextrins. Also included in such formulations may be high molecular weight excipients such as cel luloses (AVICEL®) or polyethylene glycols (PEG); an excipient to aid mucosal adhesion such as hydroxypropyl cellulose (HPC), hydroxypropyl methyl cel lulose (HPMC), sodium carboxymethyl cell ulose (SC C), and/or maleic anhydride copolymer (e.g. , GANTREZ®); and agents to control release such as polyacrylic copolymer (e.g. , CARBOPOL 934®). Lubricants, glidants, flavors, coloring agents and stabilizers may also be added for ease of fabrication and use.

[0059] Exemplary compositions for nasal aerosol or inhalation administration include solutions which may contain, for example, benzyl alcohol or other suitable preservatives, absorption promoters to enhance absorption and/or bioavailabil ity, and/or other solubi lizing or dispersing agents such as those known in the art.

[0060] Exemplary compositions for parenteral administration include injectable solutions or suspensions which may contain, for example, suitable non-toxic, parenterally acceptable diluents or solvents, such as mannitol, 1 ,3-butanediol, water, Ringer's solution, an isotonic sodium chloride solution, or other suitable dispersing or wetting and suspend ing agents, including synthetic mono- or diglycerides, and fatty acids, including oleic acid .

[0061 ] Exemplary compositions for rectal administration include suppositories which may contain, for example, suitable non-irritating excipients, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary

temperatures but l iquefy and/or dissolve in the rectal cavity to release the drug.

[0062] The effective amount of BMS-582949 may be determined by one of ordinary skil l in the art, and includes exemplary dosage amounts for a mammal of from about 0.05 to 1 00 mg/kg of body weight of active compound per day, which may be administered in a single dose or in the form of individual divided doses, such as from 1 to 4 times per day. It wil l be understood that the specific dose level and frequency of dosage for any particular subject may be varied and wi ll depend upon a variety of factors, including the activity of the specific compound employed, the metabolic stability and length of action of that com pound, the species, age, body weight, general health, sex and diet of the subject, the mode and time of administration, rate of excretion, drug combination, and severity of the particular condition. Preferred subjects for treatment include animals, most preferably mammalian species such as humans, and domestic animals such as dogs, cats, horses, and the like. Thus, when the term "patient" is used herein, this term is intended to include al i subjects, most preferably mammalian species, that are affected by mediation of p38 kinase activity or enzyme activation levels.

[0063] The phrase "other anti-inf!ammaiory agent" includes any known agent useful for treating a resistant rheumatic disease, preferably refractory rheumatoid^' arthritis. The other anti-inflammatory agent may have the same or different mechanism of action than the primary therapeutic agent, it may be especially useful to employ anti-inflammatory drug combinations wherein the two or more drugs being administered act in different manners or in d ifferent phases of the cell cycle, and/or where the drugs being combined each has a demonstrated efficacy in treating the particular disease state manifested by the patient.

[0064] The invention herein further comprises use of B S-582949 in preparing medicaments for the treatment of inflammatory-associated disorders, and/or it comprises the packaging of BMS-582949 herein together with instructions that it is to be used in combination with other anti-inflammatory agents and treatments for the treatment of inflammation-associated diseases.

10065] The present invention further comprises combinations of BMS-582949 and one or more additional agents in kit form, e.g., where they are packaged together or pSaced in separate packages to be sold together as a kit, or where they are packaged to be formulated together.

[0066] Add itional ly, BMS-582949 can be formulated or co-administered with other therapeutic agents that are selected for their particular usefulness in addressing side effects associated with the aforementioned conditions.

[0067] The above other therapeutic agents, when employed in combination with BMS-582949, can be used, for example, in those amounts indicated in the Physicians' Desk Reference (PDR) or as otherwise determined by one of ordinary skill in the art.

[0068] In one embodiment, BMS-582949 is used to treat refractory rheumatoid arthritis.

[0069] Any pharmaceutical composition contemplated herein can, for example, be delivered orally via any acceptable and su itable oral preparations, Exemplary oral preparations, include, but are not limited to, for example, tablets; troches; lozenges; aqueous or oily suspensions; dispersible powders or granules; emulsions; hard or soft capsules; syrups; and elixirs. Pharmaceutical compositions intended for oral

administration can be prepared according to methods known in the art and can contain at least one agent selected from sweetening agents, flavoring agents, coloring agents, demulcents, antioxidants, and preserving agents.

[0070] Exemplary excipients include, but are not limited to, for example, inert diluents, such as, for example, calcium carbonate, sodium carbonate, lactose, calcium phosphate, and sodium phosphate; granulating and disintegrating agents, such as, for example, microcrystalline cellulose, sodium croscarmel!ose, corn starch, and aiginic acid; binding agents, such as, for example, starch, gelatin, polyvinyl-pyrroiidone, and acacia; and lubricating agents, such as, for example, magnesium stearate, stearic acid, and talc. {0071] An aqueous suspension can be prepared, for example, by admixing BMS- 582949 with at least one excipient suitable for the manufacture of an aqueous suspension. Exemplary excipients suitable for the manufacture of an aqueous suspension, include, but are not limited to, for example, suspending agents, such as, for example, sodium carboxymethylce!lulose or methylcellulose. Oily suspensions can, for example, be prepared by suspending BMS-582949 in either a vegetable oil, such as, for example, arachis oil; olive oil; sesame oil; and coconut oil; or in mineral oil, such as, for example, liquid paraffin.

[0072] Any pharmaceutical composition contemplated herein can, for example, also be delivered intravenously, subcutaneously, and/or intramuscularly via any

pharmaceutically acceptable and suitable injectable form. Exemplary injectable forms include, but are not limited to, for example, sterile aqueous solutions comprising acceptable vehicles and solvents, such as, for example, water, Ringer's solution, and isotonic sodium chloride solution; sterile oil-in-water microemulsions; and aqueous or oleaginous suspensions.

[0073] A sterile injectable oil-in-water microemulsion can, for example, be prepared by 1) dissolving BMS-582949 in an oi!y phase, such as, for example, a mixture of soybean oil and lecithin; 2) combining BMS-582949 containing oil phase with a water and glycerol mixture; and 3) processing the combination to form a microemulsion.

[0074] Any pharmaceutical composition contemplated herein can, for example, further be administered via any acceptable and suitable rectal preparation, including, but not limited to, for example, a suppository. A suppository can be prepared by mixing BMS-582949 with at least one suitable non-irritating excipient that is liquid at rectal temperatures but sol id at a temperature below rectal temperature.

{0075] Any pharmaceutical composition contemplated herein can, for example, be administered via any acceptable and suitable topical preparations including, but not limited to* for example, creams; ointments; jellies; solutions; suspensions, transdermal patches; and intranasal inhalers. For purposes of this appl ication, topical preparations include mouth washes and gargles.

[0076] Exemplary compositions for nasal aerosol or inhalation administration include solutions that may contain, for example, benzyl alcohol or other suitable preservatives, absorption promoters to enhance absorption and/or bioavailability, and/or other solubi !izing or d ispersing agents such as those known in the art,

j0077] An "effective amount" of BMS-582949 may be determ ined by one of ordinary skil l in the art, and includes exemplary dosage amounts for a mamma! of from about 0.05 to about 300 mg/kg/day, preferably less than about 200 mg/kg/day, in a single dose or in 2 to 4 divided doses. The specific dose level and frequency of dosage for any particular subject, however, may be varied and generally depends on a variety of factors, including, but not l imited to, for example, the bioavailability of BMS-582949 in the administered form; metabol ic stabi l ity and length of action of BMS-582949; species, age, body weight, general health, sex, and diet of the subject; mode and time of administration; rate of excretion; drug combination; and severity of the particular condition.

[0078] in one embodiment, the patient is an animal.

[0079] In one embodiment, the patient is a human.

In yet another embodiment, the patient is a mammalian species including, but not limited to, for example, humans and domestic animals, such as, for example, dogs, cats, and horses.

METHODS

Cell Treatment and A ffymetrix Gene Chip Analysis

[0080] p38 inhibitors were dosed at 10-fold their IC50 for inhibition of TNF-alpha production in the THP 1 monocytic cell line /LPS model system, i.e., BMS-582949, 0.5μΜ; BIRB 0.4μΜ; RO 1 .4μΜ; SB-203580 3.3μΜ; Vx-745 4.2μΜ. Vehicle concentration was 0.1 % DMSO in all treatments. For each treatment, three biological replicate samples were handled in separate processing groups. Procedures for handling the THLE-5 cell line are described in {patent citation]; briefly THLE-5 cells were seeded on fibronectin/collagen coated 1 0cm petrs dishes at 8000 cells/cm² in modified PMFR4 (Biofluids, P99- 1 0-000) and grown for two days. Media was changed 1 6 hours prior to treatment with vehicle or compounds (final 0.5% DIvlSO) for a further eight hours. The THP 1 cell line was obtained from ATCC® (TIB-202; lot 2305952) and frozen stocks made after 5 passages. One frozen stock was grown and inoculated at 1 .7 x 10⁶ ceils/mi in Gibco RPMI 1 640 with GLUTAMAX®, 1 0% FBS (CELLGRO®) 16 hours prior to addition of vehicle or compounds. After 40 minutes pre-incubation, LPS ( l OOng/ml) was added and cells were incubated for a further 5 hours, then pelleted (1 000 r.p.m. for 5 minutes at room temperature). Stipernatants were retained for evaluation of TNF-a content by EL1SA. For RNA extraction, media was removed by aspiration, and the THLE-5 or THP 1 cel ls were Sysed in 1 ml RLT1 buffer and frozen in a dry ice/ethanol bath. For expression profi l ing, manufacturer's protocols were used to prepare RNA using the RNeasy Min i Kit (Qiagen), to evaluate integrity on a Bioanalyzer 2100 (Agilent Technologies), to process 2.5 of total RNA for hybrid ization, and to scan the HG- U 133A arrays with a GCS3000 scanner (Affymeirix). Efficiency of cDNA synthesis was confirmed from ratio of 5' and 3' probesets from the GAPDH and Actin transcripts; hybrid ization efficiency was confirmed using labeled RNA for four Escherichia cols genes (BioB at 1 .5 pM, BioC at 5 , BioD at 25pM and CreX at 1 OOpM) added to the hybridization sol ution. We normalized the ΤΉΡ1 and THLE-5 datasets separately with the RMA algorithm [Irizarry et al reference]. The 22,283 probesets on the HG- 133A array were mapped to loci against human genome releasewhich contains 46427 named loci. The 36 components named in the STKE p38 MAPK pathway diagram were manually mapped to 45 loci (Tables 2 and 3).

Statistical Analysis

[00811 For expression profil ing data, the RMA intensity values were used to perform paired /-tests with the processing block as a random factor, using the GLM function in ArrayStudio (Om icSoft). See Irizarry, R.A. et al., "Summaries of Affymeirix GeneChip probe level data", Nucleic Acids Res, 3 1 :e l 5 (2003). [0082] Clinical studies. Safety and efficacy in RA patients were evaluated in a Phase Ila, 1 week, multi-center, randomized, parallel-group, double-blind, placebo-controlled, multiple oral dose study in subjects who have been diagnosed with RA for at least 6 months and have an inadequate response to MTX. 121 subjects who were on background therapy with MTX were random ized and dosed to BMS-582949 300 mg qd (n = 60) or matching placebo (n = 61 ). Patient demographics are described in Table 5, beiow. Safety analysis was performed on the treated subject population that consisted of all subjects who received at least one dose of double-bl ind study medication.

Table 5

Summary of Patient Demographics for 3 Month Clinical Study in Subjects with RA

[0083] Statistical Analysis. Efficacy analysis was performed on the Intent-to-treat (ITT) analysis population that included all randomized subjects who took at least one dose of double-bl ind study medication. A continuity corrected Chi-square test was used to compare the ACR20 responses of the BMS-582949 300 mg qd group and the placebo group at 1 2 weeks. Subjects who discontinued the study prior to week 4 or discontinued the study due to lack of efficacy (i.e. , worsening RA) were considered ACR20 non- responders at all subsequent time points. For ail subjects who discontinued for other reasons, their last ACR20 response was carried forward provided they completed at least 4 weeks of dosing. ACR50 and ACR70 responses were analyzed using the same statistical methodology as described for the ACR20 response. Changes from baseline in the DAS28 were assessed using an analysis of covanance (ANCOVA) model with treatment as the main factor and baseline values as a covariate. For subjects who discontinued the study, their last post baseline DAS28 score was carried forward for all subsequent time points, EXAMPLES

[0084] The fol lowing Examples illustrate the present invention, and are not intended to limit the scope of the claims. Example 1

Clinical Studies

[0085] Safety and efficacy in refractory RA patients was evaluated in a Phase Ha, 1 6 week, multi-center, random ized, parallel-group, double-blind, placebo-control led, multiple oral dose study in subjects who have been diagnosed with RA for at least 6 months and have an inadequate response to MTX. 121 subjects who were on background therapy with MTX were randomized and dosed to BMS-582949 300 mg qd (n = 60) or ^' match ing placebo (n = 61 ). Safety analysis was performed on the treated subject population that consisted of al l subjects who received at least one dose of double-blind study medication. Efficacy analysis was performed on the Intent-to-treat (ITT) analysis population that included al l randomized subjects who took at least one dose of double- bl ind study medication. A continuity corrected Chi-square test was used to compare the ACR20 responses of the BMS-582949 300 mg qd group and the placebo group at 12 weeks. Subjects who discontinued the study prior to week 4 or discontinued the study due to lack of efficacy (i.e., worsening RA) were considered ACR20 non-responders at all subsequent time points. For all subjects who discontinued for other reasons, their last

ACR20 response was carried forward provided they completed at least 4 weeks of dosing. ACR50 and ACR70 responses were analyzed using the same statistical methodology as described for the ACR20 response. Changes from baseline in the DAS28 were assessed using an analysis of covariance (ANCOVA) model with treatment as the main factor and baseline values as a covariate. For subjects who discontinued the study, their last post baseline DAS28 score was carried forward for all subsequent time points.

Example 2

Pyrroiotriazine Data

[0086] p38a Kinase Assay. The assays were performed in V-bottomed 96-wel! plates. The final assay volume was 60 μΐ, which was from three 20-μΕ additions of enzyme, substrates (myelin basic protein (MBP) and ATP) and test compounds in assay buffer (50 niM Tris pH 7.5, 10 mM MgCI_2s 50 mM NaCI and 1 mM DTT). Bacterially expressed, activated p38 was pre-incubated with test compounds for 10 min prior to the initiation of reaction by adding substrates. The plates were incubated at room temperature for 45 min. The reaction was terminated by adding 5 μΕ of 0.5 M EDTA to each well. The reaction mixture was aspirated onto a pre-wet fiitermat using a Skatron M:cro96 Ceil Harvester (Skatron) and washed with PBS. The fiitermat was dried in a microwave oven for 1 min, coated with a layer of MELT1LEX© A scintillation wax (PerkinElmer), and counted on a ICROBETA® scintillation counter (Model 1450, PerkinElmer). The data were analyzed using the Prizm nonlinear least-squares regression (GraphPad Software). The final concentrations of reagents in the assays were [ATP], 1 μΜ; [[γ- Ρ]ΑΤΡ]], 3 nM, [MBP] (Sigma,M18 1),2 μ ΛνβΙΙ; [p38], 15 ng/weli; [DMSO], 0.3%.

[00871 LPS-Induced TNF Production in Human PBMC. Human PBMCs were isolated from whole blood collected from healthy donors. Blood was diluted into RPMI 1640 (Life Technologies) containing 2.5 mM EDTA (Life Technologies), 10 g/mL polymyxin (Sigma) then underlaid with ficolt (Accurate Scientific Co.) and centrifuged at 600 Xg for 25 minutes. The interface was collected and cells were washed twice and resuspended in RPMI, 10% FBS. Cells are then distributed (200 μίΛνεΙΙ) into 96 well tissue culture treated plates (FALCON®) at 1 x 106 cells/mL in RPMI, 10% FBS. Test compounds were added to appropriate wells and incubated with cells for 30 minutes. Cells were then stimulated by the addition of lipopotysaccharide (LPS, BioWhittaker), with a final concentration of 25 ng/mL, and incubated for 6 h at 37 °C, 5% C0₂. The cell supernatants were removed and assayed for TNF by ELISA (R&D Systems). [0088] Inhibition of TNFa Release in Mice (Mouse P -PD). BALB\c female mice, 6-8 weeks of age, were obtained from Harlan Laboratories and maintained ad libitum on water and standard rodent chow (HARLAN TE LAD©). Mice were acclimated to ambient conditions for at least one week prior to use. For oral dosing, the compounds were prepared in a solution of 100% polyethylene glycol (mw 300) and a dosing volume of 0.2 inL per mouse was adm inistered by gavage at various times prior to LPS injection (0.5 in L of LPS suspended at 2 μg ml in PBS, administered ip). Blood samples were obtained 90 m in after LPS injection. Serum was separated from clotted blood samples by centrifugation (5 min, 5000 Xg, room temperature) and analyzed for levels of TNFa by ELISA assay (R&D Systems) according to the manufacturer's directions. A compound is described as active in this study when statistically significant inhibition of TNFa levels is observed relative to vehicle treated animals (Student's t Test, n = 8 mice per treatment group). Al l procedures involving animals were reviewed and approved by the

Institutional Animal Care and Use Committee.

[0089] Rat Adjuvant Arthritis. Male Lewis rats (Harlan, 175-200 g) were immunized sc at the base of the tai l with 0. 1 mL complete Freund's adjuvant containing 10 mg/mL Mycobacterium butyricum . Seven days later, baseline (predisease) measurements of hind paw. volume were determ ined by volume displacement p!ethysmometry (Ugo Basile, Italy). Compounds were administered orally in I mL PEG 300 beginning on day i I . Paw volume measurements were repeated 3 times/week for the remainder of the study. Data are presented as the summed increase in volume (expressed in mL) above basel ine for each rat's two hind paws. A compound is described as active in this study when statistical ly significant inhibition of baseline paw swelling is observed relative to vehicle treated animals (Student's t Test, n = 8 rats per treatment group).

[0090] Caco-2 Cell Permeability. Caco-2 cells were seeded onto a collagen coated polycarbonate filter membrane at a density of 60,000 cells/cm. Permeability studies were conducted with cell monolayers after 21 days in culture. The transport medium was modified Hank's balanced salt solution containing 10 mM N-2-hydroxyethylpiperazine- N'-2-ethanesulfonic acid (HEPES) (pH 7.4 for both apical and basolateral sides, respectively), For the bi-directional permeability studies compound (50 μΜ) was added to either the apical or basolateral side of the monolayer and then incubated for 2 hours at 37 °C. Samples were collected from both the apical and basolateral compartments, and analyzed for test compound using an HPLC/UV assay.

Table 6

vitro Rat AA

* Active = statistically significant inhibition relative to vehicle control

Table 7

24h Pre-dose Comparison - Prolonged Efficacy

Whi le both compounds were efficacious in the rat AA model, BMS-582949 displayed a more prolonged effect in the LPS challenge model at a lower dose.

Table 8

Permeability

* Active = statistical ly significant inhibition relative to vehicle control

Whi le both compounds were efficacious in the mouse TNF model, BMS-949 displays higher cell permeabi l ity (—5-fold) as measured in the Caco-2 assay.

In vitro Permeability

Claims

WHAT I S CLA IM ED I S :

1 . A method of treating a subject having resistant rheumatic disease, comprising admin istering an effective amount of a dual action p38 inhibitor having cellular p38 activation.

2. The method of claim 1 wherein the p38 inhibitor is B S-582949 having the followi ng formula

3. The method of claims 1 -2 wherein the subject has fai led at least one of methotrexate, cyclophosphamide, azathiprine, chioroquine, cyclosporine A, a tumor necrosis factor-alpha (TNF-a) blocker or antagonists, costimulation blockade, anti-IL-6 receptor antibody, or rituximab.

4. The method of claims 1 -3, wherein the resistant rheumatic d isease is refractory rheumatoid arthritis.

5. The method of any one of claims 1 -4, for reducing a symptom of rheumatoid arthritis.

6. The method of claim 5 wherein the symptom is selected from the group consisting of joint swel l i ng, joint tenderness, inflammation, morn ing stiffness, pain, structural damage

7 The method of claim 6 wherei n the structural damage is bone orjoint erosion.

8. The method of claims 1-7 wherein the p38 inhibitor is administered in a dose of at least 300 mg per day.

9. The method of claims 1 -9 wherein administration of the p38 inhibitor resuits in a Cmin or trough level in the blood greater than 56 ng/ml.

10. ' The method of claims 1-9 wherein administration of the p38 inhibitor resuits in a Cmin or trough level in the blood from about 57 ng/ml to about 250 ng/ml.

11. The method of claims 1-10 wherein the administration of the dual action p38 inhibitor improves the difference in ACR 20 and ACR 50 response rates compared to placebo by at least 20% and at least 5%, respectively.

12. The method of claims 1-11 wherein the administration of the dual action p38 inhibitor improves the difference in ACR 20 and ACR 50 response rates compared to placebo by at least 34% and at least 9%, respectively.

13. The method of claims 1-12 wherein the administration of the dual action P38 inhibitor improves the DAS28(ESR) score in a subject with baseline C reactive protein level greater than 10 mg/1, by at least 1.5.