Differential MHC class II presentation of a pathogenic autoantigen during health and disease

Glucose-6-phosphate isomerase (GPI) is the target autoantigen recognized by KRN T cells in the K/BxN model of rheumatoid arthritis. T cell reactivity to this ubiquitous Ag results in the recruitment of anti-GPI B cells and subsequent immune complex-mediated arthritis. Because all APCs have the capacity to process and present this autoantigen, it is unclear why systemic autoimmunity with polyclonal B cell activation does not ensue. To this end, we examined how GPI is presented by B cells relative to other immunologically relevant APCs such as dendritic cells (DCs) and macrophages in the steady state, during different phases of arthritis development, and after TLR stimulation. Although all APCs can process and present the GPI:I-A(g7) complex, they do so with different efficiencies. DCs are the most potent at baseline and become progressively more potent with disease development correlating with immune complex uptake. Interestingly, in vivo and in vitro maturation of DCs did not enhance GPI presentation, suggesting that DCs use mechanisms to regulate the presentation of self-peptides. Non-GPI-specific B cells are the weakest APCs (100-fold less potent than DCs) and fail to productively engage KRN T cells at steady state and during arthritis. However, the ability to stimulate KRN T cells is strongly enhanced in B cells after TLR ligation and provides a mechanism whereby polyclonal B cells may be activated in the wake of an acute infection.     

Discovery of imidazo[1,2-b]pyridazines as IKKβ inhibitors. Part 3: exploration of effective compounds in arthritis models

We have discovered imidazo[1,2-b]pyridazine derivatives that show suppressive activity of inflammation in arthritis models. We optimized the substructures of imidazo[1,2-b]pyridazine derivatives to combine potent IKKβ inhibitory activity, TNFα inhibitory activity invivo and excellent pharmacokinetics. The compound we have acquired, which had both potent activities and good pharmacokinetic profiles based on improved physicochemical properties, demonstrated efficacy on collagen-induced arthritis models in mice and rats.     

Novel p38 inhibitors with potent oral efficacy in several models of rheumatoid arthritis

A library of trisubstituted oxazoles, thiazoles, imidazoles (1,2,4- and 2,4,5-substituted) and imidazo[1,2-b]pyridines was prepared and evaluated in vitro as p38alpha inhibitors and in vivo in several models of rheumatoid arthritis. Four structures--32, 37, 45 and 59--were identified as potent inhibitors of p38alpha with high efficacy in the LPS induced TNFalpha release model in the mouse, the adjuvant induced arthritis and the collagen induced arthritis in the rat with ED50s between 1.0 and 9.5 mg/kg p.o.     

Effects of disease-modifying anti-rheumatic drugs (DMARDs) on the activities of rheumatoid arthritis-associated cathepsins K and S

Rheumatoid arthritis is an inflammatory and disabling joint disease affecting 0.5-1.5% of the population. Although various anti-inflammatory (NSAIDs) and disease-modifying (DMARDs) drugs are in clinical use, their precise mechanisms of action are not always defined. In this report, we discuss the effects of widely used DMARDs such as gold derivatives and chloroquine on cathepsins K and S, which have been implicated as critical mediators of inflammation and joint erosion in rheumatoid arthritis. We demonstrate that clinically potent gold derivatives inhibit cathepsins K and S in in vitro and cell-based assays. An X-ray analysis of the gold thiomalate/cathepsin K complex reveals that the inhibitor is bound to the active-site cysteine residue of the protease. Chloroquine, a lysosomotropic agent of lower clinical potency than gold derivatives, inhibits neutral pH-labile cathepsins intracellularly, but does not affect the neutral pH-stable cathepsin S. The potent inhibition of cathepsins implicated in the pathogenesis of rheumatoid arthritis by gold derivatives may explain the therapeutic efficacy of these drugs.     

Suppression of the effector phase of inflammatory arthritis by double-stranded RNA is mediated by type I IFNs

Innate immune receptors that recognize nucleic acids, such as TLRs and RNA helicases, are potent activators of innate immunity that have been implicated in the induction and exacerbation of autoimmunity and inflammatory arthritis. Polyriboinosine-polyribocytidylic acid sodium salt (poly(IC)) is a mimic of dsRNA and viral infection that activates TLR3 and the RNA helicases retinoic acid-induced gene-1 and melanoma differentiation-associated gene-5, and strongly induces type I IFN production. We analyzed the effects of systemic delivery of poly(IC) on the inflammatory effector phase of arthritis using the collagen Ab-induced and KRN TCR-transgenic mouse serum-induced models of immune complex-mediated experimental arthritis. Surprisingly, poly(IC) suppressed arthritis, and suppression was dependent on type I IFNs that inhibited synovial cell proliferation and inflammatory cytokine production. Administration of exogenous type I IFNs was sufficient to suppress arthritis. These results suggest a regulatory role for innate immune receptors for dsRNA in modulating inflammatory arthritis and provide additional support for an anti-inflammatory function of type I IFNs in arthritis that directly contrasts with a pathogenic role in promoting autoimmunity in systemic lupus.     

Synthesis and pharmacological characterization of a potent,orally active p38 kinase inhibitor

Inhibitors of the MAP kinase p38 provide a novel approach for the treatment of osteoporosis, inflammatory disorders, and cancer. We have identified N-(3-tert-butyl-1-methyl-5-pyrazolyl)-N'-(4-(4-pyridinylmethyl)phenyl)urea as a potent and selective p38 kinase inhibitor in biochemical and cellular assays. This compound is orally active in two acute models of cytokine release (TNF-induced IL-6 and LPS-induced TNF) and a chronic model of arthritis (20-day murine collagen-induced arthritis).     

Arylsulphonyl hydroxamic acids: potent and selective matrix metalloproteinase inhibitors

A series of novel matrix metalloproteinase inhibitors is described in which selectivity between MMP and 'sheddase' activity has been achieved and which demonstrate potent in vivo activity in models of arthritis and cancer.     

Inhibition of HDAC activity by ITF2357 ameliorates joint inflammation and prevents cartilage and bone destruction in experimental arthritis

Inhibition of histone deacetylases (HDAC) has been shown to modulate gene expression and cytokine production after stimulation with several stimuli. In the present study, the antiinflammatory effect of a potent HDACi, ITF2357, was explored in different experimental models of arthritis. In addition, the bone protective effect of ITF2357 was investigated in vitro. Treatment of acute arthritis (Streptococcus pyogenes cell wall [SCW] arthritis) with ITF2357 showed that joint swelling and cell influx into the joint cavity were reduced. Furthermore, the chondrocyte metabolic function was improved by treatment of ITF2357. The production of proinflammatory cytokines by synovial tissue was reduced after ITF2357 treatment. To examine the effect of HDAC inhibition on joint destruction, ITF2357 was applied to both rat adjuvant arthritis and mouse collagen type II arthritis. ITF2357 treatment both ameliorates the severity scores in arthritis models and prevents bone destruction. In an in vitro bone destruction assay, ITF2357 was highly effective at a dose of 100 nmol/L. In conclusion, inhibition of HDAC prevents joint inflammation and cartilage and bone destruction in experimental arthritis.     

Synthesis and anti-arthritic activity of a series of 1-aryl-3-dimethylamino-1,4-dihydroisoquinolines

1-Aryl-3-dimethylamino-1,4-dihydroisoquinoline (1) were synthesised from 1-aryl-1,4-dihydroisoquinol-3-ones (2) by heating in dimethylcarbamyl chloride. Compounds bearing electron withdrawing substituents on the 1-aryl ring were active in inhibiting polyarthritis in the rat adjuvant arthritis model, when administered orally. Several compounds were also potent inhibitors of inflammatory cell accumulation. The most potent compound of the series, overall, was the 3′-chloro analogue, 10.     

Suppressive effects of QFGJS, a preparation from an anti-arthritic herbal formula, on rat experimental adjuvant-induced arthritis

To analyze the anti-arthritic effects of QFGJS (a pharmaceutical preparation from herbs) on rheumatoid arthritis, adjuvant-induced arthritis (AIA) was established in male SD rats, and two administration protocols, i.e., oral treatment with different doses of QFGJS on the day of arthritis induction or on the day when visible clinical signs of arthritis occurred, were initiated and continued until day 30. Treatments with QFGJS using both administration protocols significantly suppressed the incidence and severity of arthritis in a dose-dependent manner, showing dramatic reduction of paw swelling and ESR throughout the disease progression of AIA. Radiological and histopathological examinations showed markedly decreased tissue and bone destruction of ankle joints in the QFGJS-treated rats. The serum levels of TNF-alpha, IL-1beta, and IL-6 were significantly decreased in the QFGJS-treated rats. QFGJS demonstrates pronounced anti-arthritic effects on AIA, indicating that this herbal preparation would be a potent candidate as a novel botanical drug for further investigation.     

Discovery of novel FMS kinase inhibitors as anti-inflammatory agents

The optimization of the arylamide lead 2 resulted in identification of a highly potent series of 2,4-disubstituted arylamides. Compound 8 (FMS kinase IC(50)=0.0008 microM) served as a proof-of-concept candidate in a collagen-induced model of arthritis in mice.     

Identification of SD-0006, a potent diaryl pyrazole inhibitor of p38 MAP kinase

Starting from an initial HTS screening lead, a novel series of C(5)-substituted diaryl pyrazoles were developed that showed potent inhibition of p38α kinase. Key to this outcome was the switch from a pyridyl to pyrimidine at the C(4)-position leading to analogs that were potent in human whole blood based cell assay as well as in a number of animal efficacy models for rheumatoid arthritis. Ultimately, we identified a clinical candidate from this substrate; SD-0006.     

Pyrazoloheteroaryls: novel p38alpha MAP kinase inhibiting scaffolds with oral activity

A test library with three novel p38alpha inhibitory scaffolds and a narrow set of substituents was prepared. Appropriate combination of substituent and scaffold generated potent p38alpha inhibitors, for example, pyrazolo[3,4-b]pyridine 9, pyrazolo[3,4-d]pyrimidine 18a and pyrazolo[3,4-b]pyrazine 23b with potent in vivo activity upon oral administration in animal models of rheumatoid arthritis.     

p38 kinase inhibitors for the treatment of arthritis and osteoporosis: thienyl, furyl, and pyrrolyl ureas

Inhibitors of the MAP kinase p38 are potentially useful for the treatment for osteoporosis, arthritis, and other inflammatory diseases. A series of thienyl, furyl, and pyrrolyl ureas has been identified as potent p38 inhibitors, displaying in vitro activity in the nanomolar range.     

Structure activity studies of a novel cytotoxic benzodiazepine

Analogues of Bz-423, a pro-apoptotic 1,4-benzodiazepine with potent activity in animal models of systemic lupus erythematosus and rheumatoid arthritis, have been designed, synthesized, and evaluated in cell-culture assays. The results of these experiments have defined the structural elements of this new cytotoxic agent required for activity.     

Acetylenic TACE inhibitors. Part 2: SAR of six-membered cyclic sulfonamide hydroxamates

The SAR of a series of potent sulfonamide hydroxamate TACE inhibitors bearing a butynyloxy P1' group was explored. In particular, compound 5k has excellent in vitro potency against TACE enzyme and in cells, and oral activity in an in vivo model of TNF-alpha production and a collagen-induced arthritis model.     

Novel DMARDs on the basis of a new concept of dual cytokine regulation, TNF-alpha suppression and IL-10 augmentation

A series of arylpiperazine derivatives was synthesized to obtain agents showing apparent therapeutic effects in a chronic inflammatory animal model, starting from a lead possessing potent dual cytokine regulatory activity in vivo. We found a pyrimidylpiperazine derivative 17c showing the dual regulatory activity and an excellent therapeutic effect in an adjuvant-induced arthritis model.     

Gastrin-releasing peptide,substance P and cytokines in rheumatoid arthritis

Many studies have shown that modulation of cytokine function is effective in ameliorating symptoms of rheumatoid arthritis. Neuropeptides have recently been shown to have powerful effects on the production and release of cytokines and have also been shown to exert potent proinflammatory and anti-inflammatory effects in animal models of inflammatory diseases. An analysis of cytokine and neuropeptide content of synovial fluid from patients with rheumatoid arthritis has revealed a significant correlation between two neuropeptides, bombesin/gastrin-releasing peptide and substance P, and the proinflammatory cytokine interleukin-6 as well as the erythrocyte sedimentation rate. These findings provide further evidence for a role of neuropeptides and cytokines in the pathophysiology of rheumatoid arthritis, as well as suggesting additional approaches for the development of novel therapeutic interventions.     

Copper chelation with tetrathiomolybdate suppresses adjuvant-induced arthritis and inflammation-associated cachexia in rats

Tetrathiomolybdate (TM), a drug developed for Wilson's disease, produces an anti-angiogenic and anti-inflammatory effect by reducing systemic copper levels. TM therapy has proved effective in inhibiting the growth of tumors in animal tumor models and in cancer patients. We have hypothesized that TM may be used for the therapy of rheumatoid arthritis and have examined the efficacy of TM on adjuvant-induced arthritis in the rat, which is a model of acute inflammatory arthritis and inflammatory cachexia. TM delayed the onset of and suppressed the severity of clinical arthritis on both paw volume and the arthritis score. Histological examination demonstrated that TM significantly reduces the synovial hyperplasia and inflammatory cell invasion in joint tissues. Interestingly, TM can inhibit the expression of vascular endothelial growth factor in serum synovial tissues, especially in endothelial cells and macrophages. Moreover, the extent of pannus formation, which leads to bone destruction, is correlated with the content of vascular endothelial growth factor in the serum. There was no mortality in TM-treated rat abnormalities. TM also suppressed inflammatory cachexia. We suggest that copper deficiency induced by TM is a potent approach both to inhibit the progression of rheumatoid arthritis with minimal adverse effects and to improve the well-being of rheumatoid arthritis patients.     

Relevance of posttranslational modifications for the arthritogenicity of type II collagen

To establish the role of posttranslational modification in modulating the immune response to collagen, recombinant human type II collagen (rCII) was produced using a yeast expression system (rCII(pic)) and a baculovirus expression system (rCII(bac)). The biosynthesis of CII requires extensive posttranslational modification including the hydroxylation of prolyl and lysyl residues and glycosylation of selected hydroxylysyl residues. Amino acid analyses indicated that the rCII(bac) was adequately hydroxylated at prolyl residues but underhydroxylated at lysyl residues and underglycosylated compared with tissue-derived CII, whereas rCII(pic) was adequately hydroxylated at prolyl residues but unhydroxylated at lysyl residues and had no glycosylation. When DBA/1 mice were immunized with rCII, rCII(pic) induced a lower incidence of arthritis than tissue-derived CII, whereas rCII(bac) induced an intermediate level of arthritis. The severity of the arthritis was significantly lower in mice immunized with rCII(pic) compared with mice immunized with tissue-derived CII, whereas that of rCII(bac) was intermediate. These data indicate that the degree of lysine hydroxylation and glycosylation plays a role in the induction of arthritis. The recombinant collagens were then compared with tissue-derived CII when given as i.v. or oral tolerogens to suppress arthritis. Both recombinant collagens were less potent than tissue-derived CII, and this decrease in arthritis was associated with a decrease in Ab response to CII. These data suggest that the degree of glysosylation affects the immune response to CII, so that underglycosylated CII is less effective in the induction of arthritis and in its ability to suppress collagen-induced arthritis.