Yasuyuki Higashi

Tacrolimus suppressed the production of cytokines involved in atopic dermatitis by direct stimulation of human PBMC system. (Comparison with steroids).

Tacrolimus (FK506) ointment showed remarkable efficacy against atopic dermatitis in animal models and clinical trials. The suppressive effect of tacrolimus on the production of the cytokines involved in atopic dermatitis (IL-2, IL-3, IL-4, IL-5, IFN-gamma and GM-CSF) from human peripheral blood mononuclear cells (PBMC) was investigated. We constructed a new cytokine production system in which T cells are activated by direct stimulation in vitro with anti-CD3/CD2 or anti-CD3/CD28 antibody combination. Tacrolimus inhibited the production of these cytokines by both stimulations. In a comparative study with steroids (alclometasone dipropionate and betamethason valerate) in anti-CD3/CD2 system, tacrolimus and both steroids inhibited Th1 cytokines (IL-2, IFN-gamma), Th2 cytokines (IL-4, IL-5) and IL-3, GM-CSF (produced by both Th1 and Th2). The suppressive effect of tacrolimus on cytokine production was stronger than that of alclometasone dipropionate and equal to or stronger than that of betamethason valerate. The effective dose of tacrolimus (IC50, 0.02-0.11 ng/ml) is almost the same as for Th1 and Th2 cytokines, and 1 ng/ml of tacrolimus suppressed all cytokines completely. These results suggest that tacrolimus suppresses the allergic cytokines from T cells, and that tacrolimus ointment is effective against atopic dermatitis through the inhibition of cytokine production.

Atopic Dermatitis-Like Skin Lesions Induced by Topical Application of Mite Antigens in NC/Nga Mice

Background: Atopic dermatitis (AD) is a chronic relapsing inflammation usually observed in patients with an individual or a familial history of atopic diseases, precipitated by environmental factors including mite antigens (Ag). However, the exact etiology of AD is unclear. To further explore the pathogenesis and treatment of AD, a suitable animal model is necessary. In this study, we developed a new animal model of AD induced by mite Ag in NC/Nga mice. Methods: We injected the extracts of mite Ag intradermally at the ventral side of the ear of SPF NC/Nga mice on days 0, 2, 4, 7, 9, 11, 14 and 16, and measured the clinical symptoms and the ear thickness. On day 18, we collected blood and submandibular lymph nodes (LN) of the immunized ear to perform a histochemical analysis, and to measure the plasma immunoglobulins and cytokines. Results: The NC/Nga mice immunized with mite Ag suffered from AD-like skin lesions including erythema followed by edema, excoriation and scaling. The histological and immunohistochemical examinations of the affected skin showed epidermal hyperplasia with hyperkeratosis, severe infiltration of CD4+ T lymphocytes, eosinophils and macrophages, and degranulation of mast cells. The total plasma IgE level was markedly elevated in mite Ag-treated mice. LN cells of mice immunized with mite Ag synthesized IgE in an Ag-dependent manner and secreted interleukin-4 (IL-4) and IL-5 but not interferon-γ. Conclusions: NC/Nga mice treated with mite Ag manifest clinical and immunological aspects similar to patients with AD, suggesting that this model is suitable for exploring the pathogenesis of human AD.

FK506 suppresses neutrophil chemoattractant production by peripheral blood mononuclear cells

To understand the mechanism of action of FK506 (Tacrolimus) on neutrophil chemotaxis, we examined its effect on human neutrophil chemotaxis and neutrophil chemoattractant production by peripheral blood mononuclear cells. FK506 and cyclosporin A had no direct suppressive effect on neutrophil chemotaxis induced by interleukin-8, leukotriene B(4), complement 5a (C5a), zymosan-activated serum and formyl-Met-Leu-Phe (fMLP). FK506 and cyclosporin A only slightly suppressed the chemotactic activity of platelet-activating factor (PAF). Dexamethasone did not inhibit the chemotactic activity of any chemoattractant. The supernatant of peripheral blood mononuclear cells stimulated with anti-CD3 and CD2 antibodies induced neutrophil chemotaxis. FK506 and cyclosporin A suppressed the chemotactic activity of the supernatant in parallel to the suppression of interleukin-8 production by peripheral blood mononuclear cells. Anti-interleukin-8 antibody completely suppressed the chemotactic activity of the supernatant without drugs. These studies indicate that FK506 may exert a beneficial effect on human inflammatory diseases by suppressing neutrophil chemotaxis secondary to inhibition of chemoattractant (for example, interleukin-8) production by leukocytes.

Topical Application of FK506 (Tacrolimus) Ointment Inhibits Mite Antigen-Induced Dermatitis by Local Action in NC/Nga Mice

Background: FK506 ointment (tacrolimus ointment, protopic) is a new drug therapeutically effective for patients with atopic dermatitis (AD). However, the mechanism of action of FK506 ointment on AD is not fully understood. Methods: We examined the effect of FK506 ointment on mite antigen-induced dermatitis in NC/Nga mice. Clinical symptoms and ear thickness were recorded, and histopathological studies and in vitro analyses were performed. Results: Topical application of FK506 ointment (0.03–0.3%) suppressed the development of dermatitis. In the lesional skin, both interleukin (IL)-4 and interferon (IFN)-γ were detected, even though the IL-4+/IFN-γ– T helper 2 (Th2) population was predominant in the regional lymph nodes (LNs). Topical application of FK506 treatment reduced the elevated level of both IL-4 and IFN-γ in the skin, but did not decrease the expansion of the Th2 population in the LNs. Conclusions: Topical application of FK506 ointment suppresses dermatitis by inhibiting the activation of inflammatory cells locally, without systemic immune suppression, in this AD model.

Role of Inhibition of Penicillin Binding Proteins and Cell Wall Cross-Linking by Beta-Lactam Antibiotics in Low- and High-Level Methicillin Resistance of Staphylococcus aureus

We have investigated the relationships between the minimum inhibitory concentrations (MICs) of eight β-lactams and their binding affinities of penicillin binding proteins in low- and high-level methicillin-resistant Staphylococcus aureus (MRSA). For high-level MRSA (H-MRSA), a significant correlation was found between IC50 for PBP2A and MIC, whilst no clear relationship was apparent for low-level MRSA (L-MRSA). However, the product of the IC50s for PBP2A and PBP4 significantly correlated with the MIC in L-MRSA. Since PBP4 is thought to mediate secondary cross-linking of the staphylococcal cell wall, we analyzed the effect on cell wall cross-linking of L- and H-MRSA of two representative drugs (cefoselis and flomoxef). Interestingly, the decrease in cell wall cross-linking, which was clearly observed in L-MRSA, was diminished with H-MRSA. It is concluded that for L-MRSA, a reduction in cell wall cross-linking caused by inhibiton of PBP4 may contribute to the antimicrobial activity of β-lactam antibiotics, while for H-MRSA, inhibition of PBP2A mainly determines the antimicrobial activity. Since neither alteration of expression nor binding affinity of PBP4 were observed in these studies, unknown factors must operate to diminish the effect of PBP4 inhibition and contribute to the mechanism of high level resistance of MRSA.

Biochemical and pharmacological characterization of FK706, a novel elastase inhibitor

FK706, sodium 2-[4-[[(S)-1-[[(S)-2-[[(RS)-3, 3, 3-trifluoro-1-isopropyl-2-oxopropyl]aminocarbonyl]pyrrolidin -1-yl]carbonyl]-2-methylpropyl] aminocarbonyl] benzoylamino] acetate, C26H32F3N4NaO7, is a synthetic water-soluble inhibitor of human neutrophil elastase. This compound demonstrated a competitive and slow-binding inhibition of human neutrophil elastase with a Ki of 4.2 nM. In studies using synthetic substrates, FK706 inhibited human neutrophil elastase activity and porcine pancreatic elastase activity with respective IC50 values of 83 and 100 nM. FK706, however, inhibited more weakly, (IC50 values > 340 microM) other serine proteinases such as human pancreatic alpha-chymotrypsin, human pancreatic trypsin and human leukocyte cathepsin G. FK706 also effectively inhibited the hydrolysis of bovine neck ligament elastin (2 mg/ml final concentration) by human neutrophil elastase (4 microg/ml final concentration) with an IC50 value of 230 nM. FK706 protected animals against human neutrophil elastase (50 microg/animal)-induced lung hemorrhage with ED50 values of 2.4 microg/animal by intratracheal administration and 36.5 mg/kg by intravenous administration, respectively. Subcutaneous administration of FK706 significantly suppressed human neutrophil elastase (20 microg/paw)-induced paw edema in mice in a dose-dependent manner (47% inhibition at a dose of 100 mg/kg). These results suggest that FK706 would be a useful tool for investigating the role of human neutrophil elastase in inflammatory disorders associated with an excess of elastase, such as pulmonary emphysema, adult respiratory distress syndrome, septic shock, cystic fibrosis, chronic bronchitis and rheumatoid arthritis.

Discovery of 3,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2(1H)-one derivatives as novel JAK inhibitors.

Because Janus kinases (JAKs) play a crucial role in cytokine-mediated signal transduction, JAKs are an attractive target for the treatment of organ transplant rejection and autoimmune diseases such as rheumatoid arthritis (RA). To identify JAK inhibitors, we focused on the 1H-pyrrolo[2,3-b]pyridine derivative 3, which exhibited moderate JAK3 and JAK1 inhibitory activities. Optimization of 3 identified the tricyclic imidazo-pyrrolopyridinone derivative 19, which exhibited potent JAK3 and JAK1 inhibitory activities (IC50=1.1 nM, 1.5 nM, respectively) with favorable metabolic stability.

The Effect of ASP2409, a Novel CD86-Selective Variant of CTLA4-Ig, on Renal Allograft Rejection in Nonhuman Primates.

Background Blockade of CD28-mediated T cell costimulation by a modified cytotoxic T lymphocyte-associated antigen 4 (CTLA4-Ig), belatacept, is a clinically effective immunosuppressive therapy for the prevention of renal allograft rejection. Use of belatacept-based calcineurin inhibitor-free immunosuppression, however, has demonstrated an increased frequency of cellular rejection episodes and immunosuppression-related safety issues relative to conventional regimens. Furthermore, belatacept typically requires infusion for its administration chronically, which may present an inconvenience to patients. To address these issues, a novel CTLA4-Ig variant, ASP2409, with improved CD86 binding selectivity and affinity relative to belatacept was created using DNA shuffling directed evolution methods. Methods We evaluated the immunosuppressive effect of ASP2409 on in vitro alloimmune T cell responses, in vivo tetanus toxoid (TTx)-induced immunological responses and renal transplantation in cynomolgus monkeys. Results ASP2409 had 6.1-fold higher and 2.1-fold lower binding affinity to monkey CD86 and CD80 relative to belatacept, respectively. ASP2409 was 18-fold more potent in suppressing in vitro alloimmune T cell responses relative to belatacept. In a cynomolgus monkey TTx immunization model, ASP2409 inhibited anti-TTx immune responses at a 10-fold lower dose level than belatacept. In a cynomolgus monkey renal transplantation model, subcutaneous injection of 1 mg/kg ASP2409 prevented allograft rejection through complete CD86 and partial CD80 receptor occupancies and dramatically prolonged renal allograft survival in combination with tacrolimus or mycophenolate mofetil/methylprednisolone. Conclusions These results support the potential of ASP2409 as an improved CTLA4-Ig for maintenance immunosuppression in organ transplantation.

Anti-inflammatory effect and selectivity profile of AS1940477, a novel and potent p38 mitogen-activated protein kinase inhibitor

Given the key role p38 mitogen-activated protein kinase (MAPK) plays in inflammatory responses through the production of cytokines and inflammatory mediators, its inhibition is considered a promising therapeutic strategy for chronic inflammatory diseases such as rheumatoid arthritis, psoriasis, inflammatory bowel disease, and chronic obstructive pulmonary disease. Here, we evaluated the anti-inflammatory effect and selectivity profile of the novel p38 MAPK inhibitor AS1940477. AS1940477 inhibited the enzymatic activity of recombinant p38α and β isoforms but showed no effect against other 100 protein kinases including p38γ and δ isoforms. We also confirmed the selectivity of AS1940477 in the intracellular signaling pathway. In human peripheral blood mononuclear cells, AS1940477 inhibited lipopolysaccharide (LPS)- or phytohemagglutinin A (PHA)-induced production of proinflammatory cytokines, including TNFα, IL-1β, and IL-6 at low concentrations (LPS/TNFα, IC(50)=0.45n M; PHA/TNFα, IC(50)=0.40 nM). In addition, equivalent concentrations of AS1940477 that inhibited cytokine production also inhibited TNFα- and IL-1 β-induced production of IL-6, PGE(2), and MMP-3 in human synovial stromal cells. AS1940477 was also found to potently inhibit TNF production in whole blood (IC(50)=12 nM) and effectively inhibited TNFα production induced by systemically administered LPS in rats at less than 0.1mg/kg (ED(50)=0.053 mg/kg) with an anti-inflammatory effect lasting for 20h after oral administration. Overall, this study demonstrated that AS1940477 is a novel and potent p38 MAPK inhibitor and may be useful as a promising anti-inflammatory agent for treating inflammatory disorders.

Effect of novel PKCθ selective inhibitor AS2521780 on acute rejection in rat and non-human primate models of transplantation.

Selective inhibition of protein kinase Cθ (PKCθ) may be useful in inducing T cell-specific immunosuppression with a reduced rate of side effects. To our knowledge, however, no reports have been published regarding the selective inhibition of PKCθ by small-molecule compounds in animal models of allograft rejection. Here, we investigated the effect of the newly synthesized PKCθ selective inhibitor AS2521780 in mono- and combination therapies on acute rejection in ACI-to-Lewis rat cardiac and non-human primate (NHP) renal transplantation models. In the rat cardiac transplantation model, AS2521780 significantly prolonged graft survival to 14days at 10mg/kg twice daily (b.i.d.) and to 20days at 30mg/kg b.i.d. In contrast, acute rejection occurred in all recipients in the non-treated group by Days 5 or 6 post-transplantation. Significant improvements (P<0.001) in graft survival were observed following treatment with a combination of AS2521780 at 3mg/kg b.i.d. and a suboptimal dose of tacrolimus (0.02mg/kg) or mycophenolate mofetil (15mg/kg). In the NHP renal transplantation model, AS2521780 at 3mg/kg b.i.d. and tacrolimus at 1mg/kg (suboptimal dose) significantly improved graft survival compared to tacrolimus alone (P<0.05). The present study of AS2521780 in rat cardiac and NHP renal transplantation models demonstrates the potential of PKCθ as a novel drug target for organ transplantation. As AS2521780 was well tolerated and the dose of tacrolimus or mycophenolate mofetil can be reduced when used in combination with this drug, immunosuppressive regimens containing selective inhibitors of PKCθ might have good safety profiles.