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.

Asymptotic analysis of a chemotactic model of bacteria colonies.

An estimate of the distance between spots generated by a bacterial colony model is obtained. The model describes the morphogenesis of a spot pattern in colonies of chemotactic strains of Escherichia coli. Asymptotic methods for other cell-chemotaxis models, which have been successfully used by previous researchers, can be applied also to this model. However the calculations and the result is more complicated for this model. The result is verified by comparing it with the results by numerical computations of solutions of the model.

Excitatory amino acid transporters expressed by synovial fibroblasts in rats with collagen-induced arthritis.

Although previous studies have demonstrated increased levels of the brain neurotransmitter glutamate (Glu) in the synovial fluid from patients with arthritis, not much attention has been paid to the possible role of Glu in joint synovial tissues to date. Constitutive expression of mRNA was for the first time shown with glutamate aspartate transporter, glutamate transporter-1 and excitatory amino acid carrier-1 (EAAC1), in addition to with particular ionotropic and metabotropic Glu receptors, in cultured synovial fibroblasts prepared from knee joints of male Lewis rats. Immunohistochemical analysis revealed high localization of immunoreactive EAAC1 at synovial tissues. The accumulation of [3H]Glu occurred in a temperature- and sodium-dependent manner in cultured synovial fibroblasts, with a Km of 23.1+/-1.1 microM and a Vmax of 237.1+/-31.1 pmol/(mg protein min), respectively. In rats with arthritis induced by immunization to type-II collagen, marked increases were seen in hind paw volume, cytokine mRNA expression and Glu levels in synovial tissues, in addition to histological erosion. In cultured synovial fibroblasts prepared from these arthritic rats, [3H]Glu accumulation was drastically increased with biochemical and pharmacological profiles similar to those seen in normal synovial fibroblasts. The exposure to Glu at 500 microM doubled the incorporation of 5-bromo-2'-deoxyuridine in cultured synovial fibroblasts of arthritic but not normal rats, without significantly affecting mRNA expression of different cytokines in both synovial fibroblasts. These results suggest that Glu may at least in part play a role in mechanisms associated with cellular proliferation through particular transporters functionally expressed by synovium in rheumatoid arthritis.

Prevention of progressive joint destruction in adjuvant induced arthritis in rats by a novel matrix metalloproteinase inhibitor, FR217840.

Matrix metalloproteinase (MMP) has been implicated in joint destruction of chronic arthritis diseases, such as rheumatoid arthritis. FR217840 (2R)-1-([5-(4-fluorophenyl)-2-thienyl]sulfonyl)-N-hydroxy-4-(methylsulfonyl)-2-piperazinecarboxamide is a potent, orally active synthetic MMP inhibitor that inhibits human collagenases (MMP-1, MMP-8 and MMP-13), gelatinases (MMP-2 and MMP-9) and membrane type MMP (MT-MMP) (MT1-MMP/MMP-14). FR217840 also inhibits rat collagenase and gelatinase. We studied the effect of FR217840 on a rat adjuvant induced arthritis model. Although oral administration (days 1-21) of FR217840 (3.2, 10, 32 mg/kg) to adjuvant injected Lewis rats did not affect inflammation, as indicated by both hind paw swelling and histological inflammatory infiltration, FR217840 suppressed both bone destruction and serum pyridinoline content in a dose-dependent manner. Also, FR217840 (32 mg/kg) reduced tartrate-resistant acid phosphatase (TRAP) cell number in the ankle joints of rats with arthritis. These results indicate that FR217840 successfully suppressed joint destruction and suggest that FR217840 may have potential as a novel anti-rheumatic drug.

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

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. 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. FR255031 markedly inhibited cartilage degradation in a dose-dependent manner in the CIA model, but Trocade failed to prevent the degradation. 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. 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.

Differential effects of FK506 and methotrexate on inflammatory cytokine levels in rat adjuvant-induced arthritis.

OBJECTIVE: To investigate the effects of prophylactic and therapeutic treatments with FK506 (tacrolimus), an immunosuppressive drug that specifically inhibits T cell activation, and methotrexate (MTX) on inflammatory cytokines, tumor necrosis factor (TNF)-a, interleukin (IL)-1beta, and IL-6 levels in rat adjuvant-induced arthritis (AIA). METHODS: AIA was induced in female Lewis rats. Arthritis was assessed by hindpaw swelling. TNF-a, IL-1beta, and IL-6 levels in paw extracts were determined by ELISA. To assess the effects on cytokine levels, rats were treated prophylactically with FK506 (3 mg/kg) or MTX (0.1 mg/kg) from day 1 to day 17, and therapeutically with FK506 (5 mg/kg) or MTX (1 mg/kg) from day 15 to day 17 (3-day treatment) or day 15 to 20 (6-day treatment) by oral administration. RESULTS: TNF-a, IL-1beta, and IL-6 levels in paw tissue were found to significantly increase between day 15 and day 21 after adjuvant injection, when the arthritis was in a developed stage. Prophylactic treatment with FK506 and MTX suppressed arthritis and reduced the levels of those inflammatory cytokines. FK506 caused a marked reduction of TNF-a and IL-1beta levels in paw tissue even in short-term (3-day) therapeutic treatment. It reduced all levels of TNF-a, IL-1beta, and IL-6 in paws in 6-day therapeutic treatment. In contrast, therapeutic treatment with MTX affected neither TNF-a or IL-6 levels in paws. MTX reduced IL-1beta levels only in the 6-day treatment. CONCLUSION: FK506 is more effective than MTX in reducing elevated levels of inflammatory cytokines TNF-a, IL-1beta, and IL-6 in established stages of AIA. Our findings suggest that inhibition of T cell activation results in a rapid reduction of inflammatory cytokine levels even after the arthritis is established in AIA.

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.

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

1. 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). 2. 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)-alpha, IL-1beta and PGE(2) levels in paw extracts were determined by ELISA. TNF activity was measured by L929 cell cytotoxicity assay. IL-1beta and cyclooxygenase (COX) mRNA expression in arthritic paws were measured by RT-PCR. 3. Single doses of FK506 and CsA markedly reduced joint hyperalgesia 24 h after drug administration, without affecting inflammation in an advanced stage of AIA. 4. The calcineurin inhibitors partially reduced the elevated level of TNF-alpha in arthritic paws, however, the analgesic effects of these drugs were not associated with the reduction in TNF-alpha level. 5. Moreover, treatment with anti-rat TNF-alpha antibody did not affect the hyperalgesia, when TNF-alpha activity was suppressed in arthritic paws by that treatment. 6. Both calcineurin inhibitors reduced the elevated level of IL-1beta in arthritic paws to a normal level, 24 h after drug administration. 7. FK506 reduced IL-1beta and COX-2 mRNA expression and PGE(2) level in arthritic paws. 8 In conclusion, calcineurin inhibitors rapidly reduce joint hyperalgesia probably by downregulating IL-1beta, but not TNF-alpha, in AIA. Our findings may provide a new strategy for the treatment of pain in 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.