Fusako Nishigaki

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.

Effects of FK317, a novel anti-cancer agent, on survival of mice bearing B16BL6 melanoma and Lewis lung carcinoma

The effects of FK317 (11-acetyl-8-carbamoyloxymethyl-4-formyl-6-methoxy-14- oxa-1,11-diazatraacylo[7.4.1.0(2.7).0(10.2)]-tetradeca-2,4,6-trien-9-yl acetate), a novel anti-cancer agent, and mitomycin C (MMC) on survival time of mice bearing B16BL6 melanoma and Lewis lung carcinoma (LLC), induced by intravenous inoculation of the tumor, were investigated. Treatment with FK317 resulted in a significant prolongation of survival time in both tumor models. Four of ten mice bearing B16BL6 were disease-free following FK317 treatment. In contrast, MMC was not effective in prolonging survival time. Overall, this study demonstrated that FK317 shows more potent survival extension in mice bearing B16BL6 and LLC than MMC, suggesting that FK317 may have therapeutic utility for cancer chemotherapy.

FR177391, a new anti-hyperlipidemic agent from Serratia. II. Pharmacological activity of FR177391.

The pharmacological effect of FR177391, isolated from Serratia liquefaciens No. 1821, was studied in normal animals and various types of animal models of hypertriglyceridemia. Treatment of normal mice with FR177391 resulted in an increase in heparin-releasable lipoprotein lipase (LPL) activity in the blood and epididymal fat tissue. FR177391 treatment decreased triglyceride (TG) and increased high-density lipoprotein cholesterol in the blood in normal rats following 7 days treatment, suggesting potent LPL activating properties of FR177391. Both Triton WR1339-induced severe and fructose-induced mild hypertriglyceridemia in rats were attenuated by FR177391 treatment. Severely elevated levels of TG in db/db mice, an insulin resistant diabetic animal model, also significantly decreased from 14 days of treatment with FR177391. FR177391 treatment for 9 days caused a decrease in the elevated levels of TG in mice induced by intraperitoneal inoculation of murine lymphoma EL-4. Overall, this study demonstrated that FR177391 can be possibly a LPL activating agent and that FR177391 treatment improved hypertriglyceridemia in various rat and mouse animal models. These results suggest that FR177391 is a promising candidate compound for the management of hypertriglyceridemia.

AS1069562, the (+)-isomer of indeloxazine, exerts analgesic effects in rat models of nociceptive pain

Abstract Objectives: The (+)-isomer of indeloxazine AS1069562 has multiple pharmacological actions, such as serotonin (5-HIT) and norepinephrine (NE) reuptake inhibition and analgesic effects in animal models of neuropathic pain. Here, we investigated the analgesic effects of AS1069562 in rat models of inflammatory and noninflammatory nociceptive pain. Methods: Adjuvant-induced arthritis (AIA) and bradykinin-induced knee joint pain were used as rat models of inflammatory pain. The chronic phase of monoiodoacetate-induced arthritis (MIA) was used as a rat model of noninflammatory pain. Analgesic effects were evaluated by weight-bearing deficit in the AIA and MIA models and by pain response in the bradykinin-induced knee joint pain model. Results: In the AIA model and the bradykinin-induced knee joint pain model, AS1069562 significantly ameliorated the pain-related behavior of weight-bearing deficit and the pain response, respectively. AS1069562 also significantly improved the pain-related behavior of weight-bearing deficit in the chronic phase of the MIA model. Further, following monoiodoacetate injection, repeated administration of AS1069562 or duloxetine significantly improved weight-bearing deficit in the MIA model. Interestingly, the analgesic effect of AS1069562 was sustained for 24 hours after the last administration, although the plasma concentration of AS1069562 was reduced to undetectable levels. In contrast, the analgesic effect of duloxetine did not continue after treatment discontinuation. Discussion: AS1069562 exerts analgesic effects on inflammatory and noninflammatory nociceptive pain in rat models of arthritis pain, and repeated administration of AS1069562 exerts a more persistent analgesic effect on arthritis pain than duloxetine. These findings suggest that AS1069562 has an attractive analgesic profile for the treatment of nociceptive pain.

Human resource development contributes to the creation of outstanding regenerative medicine products

Regenerative medicine is currently the focus of global attention. Countries all around the world are actively working to create new regenerative treatment modalities through pioneering research and novel technologies. This is wonderful news for patients who could not be treated with existing medical options. New venture businesses and companies are being established in regenerative medicine and their rapid industrialization is anticipated. However, to ensure high-quality products, human resources qualified in research and development and the manufacturing of these products are essential. The Forum for Innovative Regenerative Medicine (FIRM) conducted a questionnaire of its industry members to examine the training and hiring of people in research and development, product creation, manufacturing, and more. Regenerative medicine is a brand new field; thus, many different businesses will need to cooperate together. People with a broad range of technical skills, abilities, and knowledge will be in demand, with various levels of expertise, from basic to advanced.