Susumu Miyata

Inhibition of arterial thrombosis by a protease-activated receptor 1 antagonist, FR171113, in the guinea pig.

The antiplatelet and antithrombotic effects of FR171113, 3-(4-chlorophenyl)-2-(2,4-dichlorobenzoylimino)-5-(methoxycarbonyl methylene)-1,3-thiazolidin-4-one, a non-peptide protease-activated receptor 1 (PAR1) antagonist, were evaluated in guinea pigs. FR171113 inhibited Ser-Phe-Leu-Leu-Arg-Asn-NH2 (a synthetic PAR1 agonist peptide)-induced and thrombin-induced aggregation of guinea pig platelets in a concentration-dependent manner in vitro (IC50=1.5 and 0.35 microM, respectively). Subcutaneous administration of FR171113 (0.1-3.2 mg/kg) produced a dose-dependent inhibition of platelet aggregation ex vivo. The ED50 value of FR171113 for platelet aggregation was 0.49 mg/kg s.c. However, FR171113 did not have an inhibitory effect on ADP- or collagen-induced platelet aggregation in vitro and ex vivo. One hour after FR171113 treatment at 1.0 mg/kg s.c., significant inhibition of arterial thrombosis without a prolongation of thrombin time or coagulation time was seen in the FeCl3-induced carotid artery thrombosis model in guinea pigs. Furthermore, FR171113 did not prolong bleeding time even at 32 mg/kg s.c., which is a much higher dose than that required in the thrombosis model. These observations indicate that FR171113 has desirable antiplatelet effects both in vitro and in vivo and that its in vivo antithrombotic activity is efficacious without causing a prolongation of bleeding time.

FK506 potently inhibits T cell activation induced TNF-α and IL-1β production in vitro by human peripheral blood mononuclear cells

The aim of this study was to elucidate the in vitro inhibitory potency of FK506 on production of the inflammatory cytokines, tumour necrosis factor (TNF)‐α and interleukin (IL)‐1β, with a view to assessing this immunosuppressive agent as a potential anti‐rheumatic drug. We employed an in vitro model which produces TNF‐α and IL‐1β through T cell activation. Human peripheral blood mononuclear cells (PBMC) were cultured with immobilized anti‐CD3/CD28 monoclonal antibody in this model. FK506 inhibited anti‐CD3/CD28 induced TNF‐α and IL‐1β production at concentrations less than 1 ng ml−1. Flow cytometric analysis of intracellular TNF‐α and IL‐1β positive cells showed that FK506 potently suppresses inflammatory cytokine production from CD14+ monocytes as well as from T cells. Cyclosporin A (CsA) and dexamethasone (DEX) also inhibited the anti‐CD3/CD28 induced cytokine production, but were less potent than FK506. FK506 and CsA, but not DEX, specifically inhibited anti‐CD3/CD28 induced inflammatory cytokine production without affecting the lipopolysaccaride (LPS) induced effect. Methotrexate (MTX) was completely inactive for suppressing cytokine production under either condition. Anti‐CD3/CD28 stimulated PBMC culture supernatants were found to enhance the expression of adhesion molecules in human vascular endothelial cells. FK506, CsA and DEX led to the suppression of adhesion molecule expression probably by inhibiting cytokine production from PBMC. The inhibitory potency of agents on TNF‐α and IL‐1β production was compared with cytotoxicity and FK506 was not cytotoxic at concentrations several orders of magnitude greater than those required for cytokine inhibition. These results strongly suggest that FK506 may be most effective to specifically prevent T cell activation mediated inflammatory cytokine production in a clinical setting.

Prevention of progressive joint destruction in collagen‐induced arthritis in rats by a novel matrix metalloproteinase inhibitor, FR255031

1 FR255031 (2‐[(7S)‐7‐[5‐(4‐ethylphenyl)‐2‐thienyl]‐1,1‐dioxido‐4‐(2‐pyridinylcarbonyl)hexahydro‐1,4‐thiazepin‐7‐yl]‐N‐hydroxyacetamide) is a novel synthetic matrix metalloproteinase (MMP) inhibitor that inhibits human collagenases (MMP‐1, MMP‐8 and MMP‐13), gelatinases (MMP‐2 and MMP‐9) and membrane type 1 MMP (MT1‐MMP/MMP‐14). FR255031 also inhibits rat collagenase and gelatinase. We studied the effect of FR255031 and Trocade, an inhibitor of collagenase and MMP‐14, on a rat collagen‐induced arthritis (CIA) model. 2 Rat CIA was induced by intradermal injection of type II collagen (IIC) and oral administration of FR255031 or Trocade was performed for 28 days. Body weight loss, hind paw swelling, elevation of serum anti‐IIC antibody, and histological and radiographic scores were evaluated. 3 FR255031 markedly inhibited cartilage degradation in a dose‐dependent manner in the CIA model, but Trocade failed to prevent the degradation. 4 FR255031 at a dose of 100 mg kg−1 also had statistically significant effects on bone destruction and pannus formation and on the recovery of body weight loss on day 28. 5 These results indicate that FR255031 is effective for rat CIA, especially on joint cartilage destruction. These data suggest that as well as collagenases or MT‐MMP, gelatinases are also involved in joint destruction in arthritis.

Effects of FK506 and other immunosuppressive anti-rheumatic agents on T cell activation mediated IL-6 and IgM production in vitro

The objective of this study was to investigate the therapeutic potential of FK506 and other immunosuppressive agents for the treatment of rheumatoid arthritis (RA), focusing on the effects on in vitro IL-6 production and IL-6-mediated immune response. We employed an in vitro model producing IL-6 via T cell activation in human PBMC, based on the hypothesis that T cells play a central role in the pathogenesis of RA. FK506 potently inhibited IL-6 production from PBMC stimulated with anti-CD3 and anti-CD28 monoclonal antibody (anti-CD3/CD28). Cyclosporin A (CsA) also inhibited the anti-CD3/CD28 induced IL-6 production but was about 100 times less potent than FK506. Dexamethasone (DEX) inhibited both anti-CD3/CD28 and LPS induced IL-6 production at almost the same concentration. Methotrexate (MTX) did not affect cytokine production. Anti-CD3/CD28 stimulated PBMC culture supernatants were found to enhance IgM production in SKW6.4 cells. The effects of anti-CD3/CD28 stimulated culture supernatants in the presence of agents on IgM production in SKW6.4 cells were investigated. FK506 and CsA led to suppression of IgM production induced by culture supernatants probably via inhibition of IgM inducible cytokine production from PBMC. DEX profoundly enhanced IgM production, although IL-6 production from PBMC was strongly inhibited by the agent. MTX decreased IgM production although it has no inhibitory effect on IL-6 production. The present study suggests that FK506 is the most effective among the four agents for the suppression of IL-6 production and IL-6-mediated autoantibody production in T cell activation related autoimmune diseases such as RA.

FK506 induces chondrogenic differentiation of clonal mouse embryonic carcinoma cells, ATDC5

FK506 (Tacrolimus) and cyclosporin A exert their immunosuppressive effects via a common mechanism, calcineurin inhibition, after binding to intracellular proteins termed immunophilins: FK506-binding protein (FKBP) and cyclophilin. In this study, FK506 was found to induce chondrogenic differentiation of ATDC5 cells (clonal mouse embryonal carcinoma cells) in a concentration-dependent manner (0.1-1000 ng/ml). Immunohistochemical staining showed that ATDC5 cells induced to differentiate by FK506 produced proteoglycan and type II collagen, main components of the extracellular matrix of cartilage. Rapamycin, an immunosuppressant that binds to FKBP, antagonized the effect of FK506. Cyclosporin A did not induce chondrogenesis at concentrations up to 1000 ng/ml. Taken together, these results suggest that FK506 induces chondrogenic differentiation of ATDC5 cells via a calcineurin-independent mechanism, after binding to FKBP.

Calcineurin inhibitors exert rapid reduction of inflammatory pain in rat adjuvant‐induced arthritis

FK506 and cyclosporin A (CsA) are immunosuppressive drugs, that specifically inhibit T‐cell activation via calcineurin inhibition. This study was undertaken to investigate whether calcineurin inhibitors exert analgesic actions in rat adjuvant‐induced arthritis (AIA), an animal model of rheumatoid arthritis (RA). AIA was induced in female Lewis rats. Single doses of FK506 and CsA were orally administered to arthritic rats 17 days after arthritis induction. Intensity of hyperalgesia was assessed by measuring the pain threshold of hind paws. Tumor necrosis factor (TNF)‐α, IL‐1β and PGE2 levels in paw extracts were determined by ELISA. TNF activity was measured by L929 cell cytotoxicity assay. IL‐1β and cyclooxygenase (COX) mRNA expression in arthritic paws were measured by RT–PCR. Single doses of FK506 and CsA markedly reduced joint hyperalgesia 24 h after drug administration, without affecting inflammation in an advanced stage of AIA. The calcineurin inhibitors partially reduced the elevated level of TNF‐α in arthritic paws, however, the analgesic effects of these drugs were not associated with the reduction in TNF‐α level. Moreover, treatment with anti‐rat TNF‐α antibody did not affect the hyperalgesia, when TNF‐α activity was suppressed in arthritic paws by that treatment. Both calcineurin inhibitors reduced the elevated level of IL‐1β in arthritic paws to a normal level, 24 h after drug administration. FK506 reduced IL‐1β and COX‐2 mRNA expression and PGE2 level in arthritic paws. In conclusion, calcineurin inhibitors rapidly reduce joint hyperalgesia probably by downregulating IL‐1β, but not TNF‐α, in AIA. Our findings may provide a new strategy for the treatment of pain in RA.

Pharmacological profile of FK881(ASP6537), a novel potent and selective cyclooxygenase-1 inhibitor.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are now understood to fall into one of two agent classes in clinical use. Traditional NSAIDs inhibit both cyclooxygenases-1 and 2 (COX-1, 2), which act as key enzymes catalyzing the same reaction in the production of prostaglandins (PGs), while the second class of NSAIDs selectively inhibit COX-2. Inhibition of the inducible COX-2 isoform is believed to be responsible for the therapeutic effects of NSAIDs, such as anti-inflammatory, analgesic, and antipyretic effects, while COX-1 inhibition results in side-effects on the gastrointestinal (GI) system. In the present study, however, we changed this notion that inhibiting only COX-1 causes adverse effects. We discovered FK881, a specific COX-1 inhibitor which exhibits a 650-fold ratio for human whole blood COX-1/COX-2 and rats in vivo. In rats, FK881 dose dependently inhibited carrageenan-induced paw edema (ED30: 22 mg/kg; diclofenac ED30: 3.6 mg/kg, rofecoxib ED30: 26 mg/kg) and paw swelling associated with adjuvant arthritis (ED50: 17 mg/kg; diclofenac ED50: 1.4 mg/kg, rofecoxib ED50: 1.8 mg/kg). Further, FK881 dose dependently inhibited acetic acid-induced writhing in mice (ED50: 19 mg/kg; diclofenac ED50: 14 mg/kg, rofecoxib ED50: >100mg/kg) and adjuvant arthritis hyperalgesia in rats (ED50: 1.8 mg/kg; diclofenac ED50: 1.0mg/kg, rofecoxib ED50: 0.8mg/kg). However, unlike traditional NSAIDs, GI tolerability was improved, although the antipyretic effect of FK881 was weak (NOEL: >320 mg/kg; diclofenac NOEL: <1mg/kg, rofecoxib NOEL: 100 mg/kg). These results suggest that FK881 may be useful in treating symptoms of rheumatoid arthritis and osteoarthritis.