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Systemic lupus erythematosus (SLE) is an autoimmune disease involving multi-organ systems with a widely heterogeneous clinical presentation. Renal involvement, observed mainly in lupus nephritis (LN), is the most common organ lesion associated with SLE and a determinant of prognosis. However, treatment of LN remains controversial and challenging, prompting the need for novel therapeutic approaches. In particular, development of a clinically relevant LN animal model would greatly facilitate the development of new treatments. Here, we report a novel murine model for LN established by administering polyinosinic-polycytidylic acid (Poly (I:C)) to NZB/W F1 mice. We investigated the effectiveness of administering Poly (I:C) to NZB/W F1 mice for accelerating nephritis onset and explored the optimal conditions under which to enroll mice with nephritis with similar pathology for studying treatment candidates. Gene-expression analysis revealed that activation of macrophages, which are reported to be involved in the progression of LN in patients, was a unique characteristic in this accelerated nephritis model. Evaluation of the therapeutic effect of mycophenolate mofetil (MMF), a recommended first-choice agent for LN, in this novel LN model showed that MMF significantly reduced proteinuria. The cathepsin S (CatS) inhibitor ASP1617, which has been reported to prevent development of lupus-like glomerulonephritis in the spontaneous NZB/W F1 mouse model, also showed marked therapeutic effect in this model. Our novel Poly (I:C) accelerated LN model would thus be very useful for screening clinical candidates for LN, and CatS may be an attractive therapeutic target for the treatment of LN., Competing Interests: Declaration of competing interest All authors are employees of Astellas Pharma, who provided funding for this study., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Non-human primate (NHP) renal transplantation models are widely used vivo models for researching new immunosuppressive therapies including allograft tolerance strategies. To enroll animals into a tolerance study, an immunosuppressive regimen that efficiently establishes stable renal function in NHPs is needed. Here, we assessed the effect of triple therapy comprising 2.0 mg/kg tacrolimus, mycophenolate mofetil and a steroid and its success rate for achieving stable renal function. In addition, to predict the pathophysiological consequences of withdrawing immunosuppressants, an indispensable process after induction of tolerance, we also assessed changes in the stable renal state maintained by triple therapy after drug withdrawal. Six cynomolgus monkeys were used. The median survival time was >176 days over the dosing period and 45 days after drug withdrawal. The triple therapy successfully induced stable graft function without calcineurin inhibitor nephrotoxicity in three of six recipients, although adopting trough-dependent tacrolimus dose adjustment rather than a preset dose regimen could improve on the present strategy. Further, drug withdrawal led to deterioration of renal function, de novo donor specific antibody production and increased the memory/naïve T cell ratio within two weeks post drug withdrawal. We expect that these findings contribute to establish one of the choices for animal model for evaluating future tolerance therapy for renal transplantation., Competing Interests: Declaration of Competing Interest YN, MM, KN, HF, HS, RM, KH, YH, HK, TM were employees of Astellas Pharma Inc. and Astellas Research Technologies Co., Ltd. during the conduct of the study., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the dysregulation of various cell types and immunological pathways. Autoantibodies play an important role in its pathogenesis. The presence of autoantibodies suggests that self-antigen presentation through major histocompatibility complex (MHC) class II on antigen presenting cells is involved in the pathogenesis of autoimmune diseases, including SLE. Cathepsin S (CatS) is a key protease for antigen peptide loading onto lysosomal/endosomal MHC class II molecules through invariant chain degradation to promote antigen presentation. Inhibition of CatS is therefore expected to suppress antigen presentation via MHC class II, T and B cell activation, and antibody production from B cells. Here, we report the pharmacological profile of ASP1617, a novel CatS inhibitor. ASP1617 induced invariant chain accumulation and decreased the expression level of MHC class ΙΙ on the cell surface in both mouse and human B cells. Further, ASP1617 prevented DO11.10 mice T cell proliferation to ovalbumin antigen. We investigated the effects of ASP1617 and mycophenolate mofetil (MMF) on the development of lupus-like nephritis in NZB/W F1 mice, a widely used SLE mouse model. Oral administration of ASP1617 suppressed anti-dsDNA IgG, prevented progression of lupus-like glomerulonephritis, and significantly prevented proteinuria excretion. In contrast, MMF did not suppress anti-dsDNA IgG. Further, we found that plasma and/or urine CatS levels were increased in specimens from NZB/W F1 mice and several SLE patients. These results indicate that CatS may be an attractive therapeutic target for the treatment of SLE., (Copyright © 2022 Elsevier B.V. All rights reserved.)

B cell-targeted therapies have evolved as established therapies for systemic lupus erythematosus (SLE); however, existing approaches still do not thoroughly satisfy clinical requirements due to limited efficacy against memory B cells, autoantibody-producing plasmablasts and disease heterogeneity. To provide a new treatment option for SLE, we created a novel anti-Igβ antibody with enhanced affinity for Fc gamma receptor (FcγR) IIB called ASP2713. ASP2713 cross-reacted with both human and cynomolgus monkey Igβ and showed increased binding affinity for human and monkey FcγRIIB compared to native human IgG1. This binding property allows dominant B cell binding and induction of intrinsic negative feedback signals. In human B cells, ASP2713 significantly and concentration-dependently induced FcγRIIB ITIM phosphorylation, while suppressing proliferation under B cell receptor stimulation. This pharmacological effect was also confirmed in in vitro B cell proliferation and antibody production assays using peripheral B cells isolated from patients with SLE. In a cynomolgus monkey tetanus toxoid-induced antibody production model, ASP2713 almost completely inhibited the increase in antigen-specific antibodies with superior efficacy to rituximab. Additionally, ASP2713 significantly suppressed recall antibody production in response to secondary tetanus toxoid immunization, indicating the memory B cell- and plasmablast-targeting potential of ASP2713. Our results suggest that ASP2713 may have therapeutic potential as a treatment for SLE, where B cells play a pathogenic role., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Phosphoinositide 3-kinases generate lipid-based second messengers that control an array of intracellular signaling pathways. In particular, phosphoinositide 3-kinases delta (PI3Kδ) is expressed primarily in hematopoietic cells and plays an important role in B-cell development and function. B cells play a critical role in autoimmune diseases by producing autoantibodies. Studies have therefore increasingly focused on PI3Kδ as a therapeutic target for the treatment of inflammatory and autoimmune diseases. One such autoimmune disease is systemic lupus erythematosus (SLE). SLE is a chronic systemic autoimmune disease with repeated recurrence and remission, and autoantibodies play an important role in its pathogenesis. Here, we examined the pharmacological profile of the novel PI3Kδ selective inhibitor AS2819899 and investigated its therapeutic potential against SLE in a NZB/W F1 mouse lupus-like nephritis model, a widely-used SLE mouse model. AS2819899 prevented B and T cell activation in vitro, and inhibited antibody production in a T-cell independent de novo antibody production mouse model. In the spontaneous NZB/W F1 mouse model, AS2819899 treatment significantly reduced anti-dsDNA antibody titers and improved kidney dysfunction. Further, AS2819899 inhibited the memory recall reaction in a T-cell dependent antibody production mouse model, suggesting that AS2819899 can potentially maintain remission of SLE. Moreover, we identified a pharmacodynamics marker for AS2819899 that may be useful in clinical studies. These results indicate that AS2819899 may be an attractive therapeutic candidate for SLE, including the maintenance of remission., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Long-term graft survival after organ transplantation is difficult to achieve because of the development of chronic rejection. One cause of chronic rejection arises from antibody-mediated rejection (AMR), which is dependent on the production of donor-specific antibodies (DSA). Current immunosuppression in organ transplantation is effective in preventing acute T cell-mediated rejection, but the risk of DSA production and graft loss due to AMR remains unchanged. Phosphatidylinositol-3-kinase p110δ (PI3Kδ), a member of the family of PI3K lipid kinases, is a key mediator of B cell activation, proliferation and antibody production. AS2541019 is a novel PI3Kδ selective inhibitor that prevents antibody production by inhibiting B cell immunity. The purpose of this study was to evaluate the inhibitory effect of AS2541019 on DSA production in preclinical rodent and non-human primate allotransplant models. Concomitant administration of AS2541019 with tacrolimus and mycophenolate mofetil (MMF) inhibited de novo DSA production in an ACI-to-Lewis rat cardiac allotransplant model. To predict the efficacy of AS2541019 in clinical practice, we evaluated its effects in cynomolgus monkeys. AS2541019 inhibited B cell proliferation and major histocompatibility complex (MHC) class II expression on B cells in cynomolgus monkeys. Oral administration of AS2541019 inhibited MHC class II expression on peripheral B cells and anti-tetanus toxoid antibody production. In cynomolgus monkey renal allotransplant model, concomitant administration of AS2541019 with tacrolimus and MMF significantly inhibited de novo DSA production. Together, our findings indicate that the PI3Kδ selective inhibitor AS2541019 is a potential candidate for preventing AMR development by inhibiting DSA production., (Copyright © 2019. Published by Elsevier B.V.)

Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid that acts through the members of a family of 5 G protein-coupled receptors (S1P 1 to S1P 5 ). Among these, S1P 1 is a major regulator of lymphocyte trafficking. Fingolimod, whose active metabolite, fingolimod phosphate, acts as a nonselective S1P-receptor agonist, exerts its immunomodulatory effect, at least in part, by regulating lymphocyte trafficking via downregulation of S1P 1 expression on lymphocytes. Here, we describe the pharmacologic profile of a novel S1P 1 agonist, ASP1126. ASP1126 preferentially activated S1P 1 compared to S1P 3 in rat and human guanosine-5'-(γ-thio)-triphosphate (GTPγS) assays. Oral single administration of ASP1126 decreased the number of peripheral lymphocytes and repeated dosing showed a cumulative effect on lymphopenia in both rats and monkeys. ASP1126 prolonged allograft survival in a rat heterotopic heart transplantation model in combination with a subtherapeutic dose of tacrolimus that was independent of drug-drug interactions. In addition, in nonhuman primate (NHP) renal transplantation, pretreatment with ASP1126 reduced not only the number of naive T cells and central memory T cells but also effector memory T cells in the peripheral blood, all of which could contribute to acute graft rejection and prolonged allograft survival in combination with tacrolimus. Further, we confirmed that ASP1126 has a broad ranging safety margin with respect to its effect on lung weight in rats and bradycardia in NHPs, which were the adverse events found in clinical studies of fingolimod. ASP1126 with improved safety profile has the potential to be an adjunct therapy in combination with tacrolimus in clinical transplantation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Interleukin (IL)-23 is thought to be critical in the pathogenesis of psoriasis, and anti-IL-23 monoclonal antibodies (mAbs) have been approved for the treatment of psoriasis. We speculated that an anti-IL-23 receptor mAb might have greater efficacy than an anti-IL-23 mAb in the treatment of local inflamed lesions with high IL-23 levels. We previously generated an anti-human IL-23 receptor mAb, AS2762900-00, which potently blocked IL-23-induced cell proliferation, regardless of the concentration of IL-23. Here, we evaluated the therapeutic potential of AS2762900-00 in the treatment of psoriasis. Compared with untreated control, AS2762900-00 significantly reduced the epidermal thickness of lesions in a clinically relevant psoriatic human skin xenograft model. The expression of inflammatory genes including genes downstream of IL-23 signaling in the lesion tended to be lower in the AS2762900-00 group than the untreated group, suggesting that the inhibitory effects of AS2762900-00 in the psoriatic human skin xenograft model might occur via blockade of IL-23 signaling pathways. Further, AS2762900-00 showed an inhibitory effect on signal transducer and activator of transcription 3 (STAT3) phosphorylation as a downstream signal of IL-23 receptor activation in whole blood from patients with psoriasis. We also confirmed that AS2762900-00 inhibited IL-23-induced STAT3 phosphorylation in a concentration-dependent manner using whole blood from healthy donors. These data suggest that AS2762900-00 is a promising drug candidate for the treatment of psoriasis. In addition, STAT3 phosphorylation in whole blood may be a useful biomarker for the evaluation of the pharmacodynamic effects of AS2762900-00 in healthy volunteers in clinical development., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Donor-specific antibodies (DSA) are a major risk factor for antibody-mediated rejection (ABMR) in solid organ transplantation, and ABMR remains a medical challenge. Therefore, effective anti-ABMR therapies are needed to improve overall graft survival. Cathepsin S (Cat S) is an essential protease for antigen peptide loading onto lysosomal/endosomal major histocompatibility complex (MHC) class II molecules to promote antigen presentation. Cat S deficiency produces immuno-deficient phenotypes including a suppressed humoral immune response, and Cat S inhibition reportedly prevents autoimmunity. However, little is known about the effects of Cat S inhibitors on organ transplantation, especially ABMR. Here, we report the pharmacological profile of novel Cat S inhibitors, AS2761325 and AS2863995, and explore their preventive potential on DSA production and acute rejection in a mouse cardiac transplantation model. Cat S inhibitors potently inhibited upregulation of antigen peptide loading MHC class II expression on the surface of splenic B cells and suppressed ovalbumin-induced T cell-dependent antibody production in mice. In a mouse cardiac transplantation model, oral administration of AS2761325 monotherapy inhibited DSA production without affecting graft survival. When combined with a suboptimal dose of tacrolimus, AS2761325 significantly prolonged graft survival. The more potent Cat S inhibitor AS2863995 also prolonged graft survival and almost completely suppressed DSA production. These results suggest that Cat S inhibitors may be promising ABMR prophylaxis drug candidates. Combination therapy comprising a Cat S inhibitor and calcineurin inhibitors may be a more effective immunosuppressive maintenance therapy for controlling both cell-mediated and antibody-mediated rejection., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In renal transplant patients, using mycophenolate mofetil (MMF) with calcineurin inhibitors (CNIs; cyclosporine and tacrolimus [TAC]) has led to a significant improvement in graft survival. However, reducing or withholding MMF due to its gastrointestinal adverse events increases rejection risk. CNI-sparing strategies are important to avoid CNI-related nephrotoxicity in clinical settings. Here, we investigated AS2553627, a JAK inhibitor replacing MMF in combination with a sub-therapeutic dose of TAC to treat allograft rejection in a monkey model. AS2553627 inhibited proliferation of IL-2 stimulated T cells with little species difference between monkeys and humans. In MMF monotherapy, oral administration of 20 or 40 mg/kg/day prolonged graft survival with median survival times (MSTs) of 16.5 days and 33 days, respectively, whereas untreated animals showed MST of 6 days. In MMF/TAC (1 mg/kg/day, p.o.) combination therapy, pharmacokinetic analysis indicated that MMF 20 mg/kg/day achieved the clinical target AUC 0-24h and prolonged renal allograft survival, with MST of 24 days. Oral administration of AS2553627 0.24 mg/kg/day in combination with TAC significantly prolonged renal allograft survival to MST of >90 days with low plasma creatinine levels. Histopathological analysis revealed that acute T cell-mediated rejection events such as vasculitis and interstitial mononuclear cell infiltration were significantly inhibited in AS2553627/TAC-treated allografts compared with MMF/TAC-treated allografts. All AS2553627/TAC-treated monkeys surviving >90 days exhibited less interstitial fibrosis/tubular atrophy than monkeys in the MMF/TAC group. These results suggest that AS2553627 replacing MMF is an attractive CNI-sparing strategy to prevent renal allograft rejection., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Interleukin (IL)-12 and IL-23 share a common subunit (p40) and function in T-helper (Th) 1 and Th17 immunity, respectively. Anti-IL-12/23p40 and specific anti-IL-23 antibodies are currently in clinical use for psoriasis and undergoing trials for autoimmune diseases. Since expression levels of the IL-23 receptor are likely to be much lower than those of IL-23, an anti-IL-23 receptor antibody might offer greater promise in inhibiting the IL-23-IL-17 pathways involved in inflammatory disorders. To our knowledge, no anti-IL-23 receptor antibody has been trialed in clinical studies to date. This study describes the generation and characterization of AS2762900-00, a fully human monoclonal antibody against the IL-23 receptor. AS2762900-00 bound both human and cynomolgus monkey IL-23 receptors. AS2762900-00 showed potent inhibitory effects on IL-23-induced Kit-225 cell proliferation compared to the existing anti-IL-12/23p40 antibody, ustekinumab. In a single dose administration pharmacodynamics study in cynomolgus monkeys, 1 mg/kg of AS2762900-00 significantly inhibited (> 85%) IL-23-induced STAT3 phosphorylation in blood for up to 84 days. Therefore, AS2762900-00 represents a potent novel IL-23-IL-17 pathway inhibitor with the potential to be developed into a new therapy for the treatment of autoimmune diseases., (Copyright © 2018 Elsevier B.V. All rights reserved.)

B cell-mediated antibodies play a critical role in protecting the body from infections; however, excessive antibody production is involved in the pathogenesis of autoimmune diseases and transplanted organ rejection. Regulation of antibody production is therefore crucial for overcoming these complications. Phosphatidylinositol-3-kinase p110δ (PI3Kδ), a member of the family of PI3K lipid kinases, is a key mediator of B cell activation and proliferation, with a small molecule PI3Kδ inhibitor having been approved for the treatment of B cell lymphoma. However, the effect of PI3Kδ inhibitors on B cell-mediated antibody production has not been clearly elucidated. In this study, we investigated the effect of the selective PI3Kδ inhibitor, AS2541019, on B cell immunity and antibody production. Our results show that AS2541019 effectively prevented B cell activation and proliferation in vitro, and that oral administration of AS2541019 resulted in significant inhibition of both T-dependent and T-independent de novo antibody production in peripheral blood. Further, in a hamster to rat concordant xenotransplant model, AS2541019 significantly prolonged graft survival time by inhibiting xenoreactive antibody production. Therefore, our study demonstrates that the selective PI3Kδ inhibitor AS2541019 inhibits antibody production through potent inhibitory effects on B cell activation, and can protect against organ dysfunction., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Experimental colitis studies, including T cell-mediated colitis, indicate that IL-23 rather than IL-12 orchestrates intestinal inflammation in inflammatory bowel disease (IBD). Previous studies have identified the roles of IL-12 and IL-23 using mice deficient for their specific subunits, p35 and p19, respectively. However, these studies do not completely reflect the difference in roles between IL-12 and IL-23, especially since the discovery of novel IL-12 family cytokines, which also include p35 or p19 subunits. Here, to clarify the contribution of IL-12 and IL-23 in T cell-mediated colitis, we compared the efficacy of a monoclonal antibody (mAb) to an IL-23-specific receptor subunit with that of an anti-IL-12/23p40 mAb in a naive CD4 + T cell transfer model of experimental colitis, which is associated with enhanced Th1 and Th17 responses. Both antibodies almost completely prevented the development of colitis and showed reduced associated histological changes, including mucosal hyperplasia, infiltration of inflammatory cells and loss of goblet cells. The anti-IL-23 receptor mAb inhibited not only the systemic Th17-response but also the Th1-response, both of which were up-regulated in this model. These results suggest that IL-23, but not IL-12, signaling is critical for the development of colitis. Blockade of IL-23 signaling is a promising therapeutic approach for IBD., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Janus kinase (JAK) inhibitors are thought to be promising candidates to aid renal transplantation. However, the effectiveness of JAK inhibitors against features of chronic rejection, including interstitial fibrosis/tubular atrophy (IF/TA) and glomerulosclerosis, has not been elucidated. Here, we investigated the effect of AS2553627, a novel JAK inhibitor, on the development of chronic rejection in rat renal transplantation., Methods: Lewis (LEW) to Brown Norway (BN) rat renal transplantation was performed. Tacrolimus (TAC) at 0.1mg/kg was administered intramuscularly once a day for 10 consecutive days starting on the day of transplantation (days 0 to 9) to prevent initial acute rejection. After discontinuation of TAC treatment from days 10 to 28, AS2553627 (1 and 10mg/kg) was orally administered with TAC. At 13weeks after renal transplantation, grafts were harvested for histopathological and mRNA analysis. Creatinine and donor-specific antibodies were measured from plasma samples. Urinary protein and kidney injury markers were also evaluated., Results: AS2553627 in combination with TAC exhibited low plasma creatinine and a marked decrease in urinary protein and kidney injury markers, such as tissue inhibitor of metalloproteinase-1 and kidney injury molecule-1. At 13weeks, histopathological analysis revealed that AS2553627 treatment inhibited glomerulosclerosis and IF/TA. In addition, upregulation of cell surface markers, fibrosis/epithelial-mesenchymal transition and inflammation-related genes were reduced by the combination of AS2553672 and TAC, particularly CD8 and IL-6 mRNAs, indicating that AS2553627 prevented cell infiltration and inflammation in renal allografts., Conclusions: These results indicate the therapeutic potential of JAK inhibitors in chronic rejection progression, and suggest that AS2553627 is a promising agent to improve long-term graft survival after renal transplantation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Janus family kinases (JAKs) are essential molecules for cytokine responses and attractive targets for the treatment of transplant rejection and autoimmune diseases. Several JAK inhibitors have shown demonstrable effects on acute rejection in experimental cardiac transplant models. However, little is known about the potential benefits of JAK inhibitors on chronic rejection outcomes such as vasculopathy and fibrosis. Here, we examined the pharmacological profile of a novel JAK inhibitor, AS2553627, and explored its therapeutic potential in chronic rejection as well as acute rejection in a rat cardiac transplant model. AS2553627 potently inhibited JAK kinases but showed no inhibition of other kinases, including TCR-associated molecules. The compound also suppressed proliferation of IL-2 stimulated human and rat T cells. In a rat cardiac transplant model, oral administration of AS2553627 alone or co-administration with a sub-therapeutic dose of tacrolimus effectively prolonged cardiac allograft survival, suggesting the efficacy in treating acute rejection. To evaluate the effect on chronic rejection, recipient rats were administered a therapeutic dose of tacrolimus for 90 days. In combination with tacrolimus, AS2553627 significantly reduced cardiac allograft vasculopathy and fibrosis that tacrolimus alone did not inhibit. AS2553627 at the effective dose in rat transplantation models did not significantly reduce reticulocyte counts in peripheral whole blood after in vivo erythropoietin administration, indicating a low risk for anemia. These results suggest that AS2553627 may be a therapeutic candidate for the prevention of not only acute but also chronic rejection in cardiac transplantation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

FTY720, a S1P-receptor modulator, has shown to be effective in several transplant and autoimmune disease models, via modulating lymphocyte homing into secondary lymphoid organs (SLOs), and thereby reducing these cells in peripheral blood. ASP0028, a newly developed S1P 1 /S1P 5 -selective agonist, presented comparable efficacy to FTY720 and wider safety margins than FTY720. In this study, we assessed the efficacy and safety of ASP0028 co-administered with suboptimal-dose of tacrolimus in the Cynomolgus monkey renal transplantation model. Seven animals in group-1 or group-2 received mono-tacrolimus 1.0mg/kg once a day (QD), or ASP0028 0.6mg/kg plus tacrolimus 1.0mg/kg QD, respectively. Eight animals in group-3 received ASP0028 1.2mg/kg plus tacrolimus 1.0mg/kg QD. The allograft median survival time (MST) in group-2 and group-3 were significantly extended to 41 and 61.5days, versus that of 28days in group-1 (p=0.036 and 0.001, respectively). ASP0028 administration remarkably reduced absolute numbers of peripheral lymphocytes, particularly subsets of CD4 + / or CD8 + /naive and central memory cells, CD4 + /Treg cells, and to a lesser extent on B cells, but not CD4 + / or CD8 + /effector memory cells and NK cells. These data show ASP0028 combined with suboptimal-dose of tacrolimus effectively prolongs renal allograft survival in nonhuman primates (NHPs) with well tolerated safety, supporting its further investigation to optimize CNI-sparing regimens., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: The Fischer-to-Lewis (LEW) rat model of kidney transplantation is a widely accepted and well-characterized model of chronic rejection. In contrast to transplantation in a clinical setting, however, the absence of treatment with immunosuppressants and only minor mismatch of major histocompatibility complexes (MHCs) are critical discrepancies. Here, we established a rat model of chronic rejection using fully MHC-mismatched strains in which kidney disease progresses even under immunosuppressive therapy., Methods: LEW (RT1(l)) rats were used as donors and Brown Norway (BN, RT1(n)) rats as recipients. Intramuscular administration of 0.1mg/kg of tacrolimus was initiated on the day of transplantation. Post-transplantation, this dose was maintained until Day 9, suspended until Day 28 and then resumed from Day 29. Renal function, histopathology, and levels of donor-specific antibody (DSA) and several biomarkers of renal injury were assessed., Results: On Day 91 post-transplantation, recipients received tacrolimus treatment with short-term suspension exhibited reduced renal function and changes in histology. Those were characteristics of chronic rejection including glomerulosclerosis, interstitial fibrosis, and tubular atrophy in human transplantation recipients. Urinary protein excretion increased in a linear fashion, and elevated levels of several biomarkers of renal injury and DSA were observed even under administration of an immunosuppressant., Conclusions: We established an allograft rejection model with impaired renal function and typical histopathological changes of chronic rejection in fully MHC-mismatched rats by controlling administration of an immunosuppressant. These findings suggest that this model more accurately reflects transplantation in a clinical setting than existing models and enables the evaluation of therapeutic agents., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid that acts through the members of a family of five G protein-coupled receptors (S1P1-S1P5). S1P1 is a major regulator of lymphocyte trafficking, and fingolimod, whose active metabolite fingolimod phosphate acts as a nonselective S1P receptor agonist, exerts its immunomodulatory effect, at least in part, by regulating the lymphocyte trafficking by inducing down regulation of lymphocyte S1P1. Here, we detail the pharmacological profile of 5-{5-[3-(trifluoromethyl)-4-{[(2S)-1,1,1-trifluoropropan-2-yl]oxy}phenyl]-1,2,4-oxadiazol-3-yl}-1H-benzimidazole (ASP4058), a novel next-generation S1P receptor agonist selective for S1P1 and S1P5. ASP4058 preferentially activates S1P1 and S1P5 compared with S1P2, 3, 4 in GTPγS binding assays in vitro. Oral administration of ASP4058 reduced the number of peripheral lymphocytes and inhibited the development of experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Further, ASP4058 prevented relapse of disease in a mouse model of relapsing-remitting EAE. Although these immunomodulatory effects were comparable to those of fingolimod, ASP4058 showed a wider safety margin than fingolimod for bradycardia and bronchoconstriction in rodents. These observations suggest that ASP4058 represents a new therapeutic option for treating multiple sclerosis that is safer than nonselective S1P receptor agonists such as fingolimod.

In the second antibody modeling assessment, we used a semiautomated template-based structure modeling approach for 11 blinded antibody variable region (Fv) targets. The structural modeling method involved several steps, including template selection for framework and canonical structures of complementary determining regions (CDRs), homology modeling, energy minimization, and expert inspection. The submitted models for Fv modeling in Stage 1 had the lowest average backbone root mean square deviation (RMSD) (1.06 Å). Comparison to crystal structures showed the most accurate Fv models were generated for 4 out of 11 targets. We found that the successful modeling in Stage 1 mainly was due to expert-guided template selection for CDRs, especially for CDR-H3, based on our previously proposed empirical method (H3-rules) and the use of position specific scoring matrix-based scoring. Loop refinement using fragment assembly and multicanonical molecular dynamics (McMD) was applied to CDR-H3 loop modeling in Stage 2. Fragment assembly and McMD produced putative structural ensembles with low free energy values that were scored based on the OSCAR all-atom force field and conformation density in principal component analysis space, respectively, as well as the degree of consensus between the two sampling methods. The quality of 8 out of 10 targets improved as compared with Stage 1. For 4 out of 10 Stage-2 targets, our method generated top-scoring models with RMSD values of less than 1 Å. In this article, we discuss the strengths and weaknesses of our approach as well as possible directions for improvement to generate better predictions in the future., (© 2014 Wiley Periodicals, Inc.)

Optimization starting with our lead compound 1 (IC(50)=4.9 nM) led to the identification of pyrrolidinyl phenylurea derivatives. Further modification toward improvement of the bioavailability provided (R)-1-(1-((6-fluoronaphthalen-2-yl)methyl)pyrrolidin-3-yl)-3-(2-(2-hydroxyethoxy)phenyl)urea 32 (IC(50)=1.7 nM), a potent and orally active CCR3 antagonist., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The synthesis and structure-activity relationships of ureas as CCR3 antagonists are described. Optimization starting with lead compound 2 (IC(50)=190 nM) derived from initial screening hit compound 1 (IC(50)=600 nM) led to the identification of (S)-N-((1R,3S,5S)-8-((6-fluoronaphthalen-2-yl)methyl)-8-azabicyclo[3.2.1]octan-3-yl)-N-(2-nitrophenyl)pyrrolidine-1,2-dicarboxamide 27 (IC(50)=4.9 nM) as a potent CCR3 antagonist., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Inosine 5'-monophosphate (IMP) dehydrogenase is a critical target in solid organ transplantation. To this end, the development of mycophenolate mofetil (MMF) represents a major advance in transplant medicine. Here, we investigated the in vitro and in vivo pharmacological effects of a novel IMP dehydrogenase inhibitor, AS2643361, in several immunological and non-immunological models. The in vitro inhibitory activity of AS2643361 on immune cell and endothelial cell proliferation and on antibody production from lipopolysaccharide-stimulated B cells, was significantly more potent than that of mycophenolic acid, the active form of MMF, despite the similar potency of these compounds on IMP dehydrogenase. In a rat heterotopic cardiac transplant model, monotherapy using orally administered AS2643361 at 10 or 20mg/kg/day prolonged the median graft survival time from 6 to 16 and 19days, respectively. In dinitrophenol-lipopolysaccharide stimulated rats, oral administration of AS2643361 at 2.5, 5 or 10mg/kg/day resulted in suppression of antibody production. In vivo antibody production against alloantigen was also suppressed by AS2643361 treatment at 5 or 10mg/kg/day. Furthermore, treatment with AS2543361 effectively inhibited balloon injury induced-intimal thickening, which is a major cause of late allograft loss. Overall, the in vivo activity of AS2643361 was over two-fold more potent than that of MMF. In addition, gastrointestinal toxicity, considered a dose-limiting factor for MMF, was reduced with AS2643361 treatment. These results suggest AS2643361 has higher potency and less toxicity than MMF, making it a potential candidate for treatment of acute and chronic rejection in transplant medicine., (Copyright © 2011. Published by Elsevier B.V.)

Background: We have previously reported operational tolerance in patients receiving human leukocyte antigen-mismatched combined kidney and bone marrow transplantation (CKBMT). We now report on transient multilineage hematopoietic chimerism and lymphocyte recovery in five patients receiving a modified CKBMT protocol and evidence for early donor-specific unresponsiveness in one of these patients., Methods: Five patients with end-stage renal disease received CKBMT from human leukocyte antigen-mismatched, haploidentical living-related donors after modified nonmyeloablative conditioning. Polychromatic flow cytometry was used to assess multilineage chimerism and lymphocyte recovery posttransplant. Limiting dilution analysis was used to assess helper T-lymphocyte reactivity to donor antigens., Results: Transient multilineage mixed chimerism was observed in all patients, but chimerism became undetectable by 2 weeks post-CKBMT. A marked decrease in T- and B-lymphocyte counts immediately after transplant was followed by gradual recovery. Initially, recovering T cells were depleted of CD45RA+/CD45RO(-) "naïve-like" cells, which have shown strong recovery in two patients, and CD4:CD8 ratios increased immediately after transplant but then declined markedly. Natural killer cells were enriched in the peripheral blood of all patients after transplant.For subject 2, a pretransplant limiting dilution assay revealed T helper cells recognizing both donor and third-party peripheral blood mononuclear cells. However, the antidonor response was undetectable by day 24, whereas third-party reactivity persisted., Conclusion: These results characterize the transient multilineage mixed hematopoietic chimerism and recovery of lymphocyte subsets in patients receiving a modified CKBMT protocol. The observations are relevant to the mechanisms of donor-specific tolerance in this patient group.

Chronic allograft nephropathy (CAN) is a major cause of late allograft loss. One morphological characteristic of CAN is renal interstitial fibrosis. Mycophenolate mofetil (MMF), the inosine 5'-monophosphate dehydrogenase (IMPDH) inhibitor, has been reported to attenuate the progression of renal interstitial fibrosis. However, the question of whether the newly synthesized IMPDH inhibitors with structures different from MMF have an antifibrotic effect remains unanswered. We evaluated the antifibrotic effects of BMS-566419, a chemically synthesized IMPDH inhibitor, using an experimental rat model, unilateral ureteral obstruction (UUO), in comparison with those of MMF. Expression levels of monocyte chemoattractant protein-1 (MCP-1) and transforming growth factor-beta1 (TGF-β1), which play important roles in UUO-induced renal fibrosis, were also investigated to determine the mechanism by which BMS-566419 affects the progression of renal fibrosis. After 14 days of UUO, interstitial fibrosis was frequently observed in the renal cortex of rats administered vehicle control. BMS-566419 by oral administration showed a significant and dose-dependent suppressive effect on UUO-induced renal fibrosis in histopathological experiments. BMS-566419 treatment also decreased collagen content, as indicated by hydroxyproline concentration, and the expression of collagen type 1 mRNA. BMS-566419 also decreased the expression of mRNA for both MCP-1 and TGF-β1. The antifibrotic effects of treatment with BMS-566419 at 60 mg/kg seemed comparable to those with MMF at 40 mg/kg. These results suggest that BMS-566419 and other chemically synthesized IMPDH inhibitors have beneficial pharmacological effects similar to those of MMF, and are potential pharmaceutical candidates in the treatment of fibrotic renal disease, including CAN., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Inosine 5'-monophosphate dehydrogenase (IMPDH) inhibition is a critical target in solid organ transplantation, and the development of mycophenolate mofetil (MMF) represents a major advance in transplant medicine. In this study, the in vitro and in vivo pharmacological effects of BMS-566419, a novel chemically synthesized IMPDH inhibitor, were compared to those of mycophenolic acid (MPA) and MMF based on results from several immunological experiments. The in vitro inhibitory activity of BMS-566419 on IMPDH type I/II, immune cell proliferation and antibody production from lipopolysaccharide (LPS)-stimulated B cells was similar, albeit slightly less potent than that of MPA. In a rat heterotopic cardiac transplant model, monotherapy using orally administered BMS-566419 60mg/kg or MMF 40mg/kg prolonged the median survival time (MST) of transplanted grafts in the vehicle group from 5 to 18 and 18.5 days, respectively. In the presence of a sub-therapeutic dose of FK506, BMS-566419 30mg/kg and MMF 20mg/kg showed identical efficacy with an MST of 21.5 days. In dinitrophenol-LPS-stimulated rats in which calcineurin inhibitors failed to inhibit antibody production, in vivo oral administration of BMS-566419 resulted in antibody production suppression with similar efficacy to MMF. The in vivo antibody production against alloantigen was also suppressed by MMF or BMS-566419 treatment. In addition, gastrointestinal toxicity, considered a dose-limiting factor of MMF, was reduced in BMS-566419 treatment. These results suggest that BMS-566419 and other chemically synthesized IMPDH inhibitors have beneficial pharmacological effects similar to those of MMF, and are potential pharmaceutical candidates in transplant indications.

Our laboratory has identified several acrylamide derivatives with potent CCR3 inhibitory activity. In the present study, we evaluated the in vitro metabolic stability (CL(int); mL/min/kg) of these compounds in human liver microsomes (HLMs), and assessed the relationship between their structures and CL(int) values. Among the compounds identified, N-{(3R)-1-[(6-fluoro-2-naphthyl)methyl]pyrrolidin-3-yl}-2-[1-(2-hydroxybenzoyl)piperidin-4-ylidene]acetamide (30j) was found to be a potent inhibitor (IC(50)=8.4nM) with a high metabolic stability against HLMs.

In our previous study on discovering novel types of CCR3 antagonists, we found a fluoronaphthalene derivative (1) that exhibited potent CCR3 inhibitory activity with an IC(50) value of 20 nM. However, compound 1 also inhibited human cytochrome P450 2D6 (CYP2D6) with an IC(50) value of 400 nM. In order to reduce its CYP2D6 inhibitory activity, we performed further systematic structural modifications on 1. In particular, we focused on reducing the number of lipophilic moieties in the biphenyl part of 1, using ClogD(7.4) values as the reference index of lipophilicity. This research led to the identification of N-{(3-exo)-8-[(6-fluoro-2-naphthyl)methyl]-8-azabicyclo[3.2.1]oct-3-yl}-3-(piperidin-1-ylcarbonyl)isonicotinamide 1-oxide (30) which showed comparable CCR3 inhibitory activity (IC(50)=23 nM) with much reduced CYP2D6 inhibitory activity (IC(50)=29,000 nM) compared with 1.

A novel class of potent CCR3 receptor antagonists were designed and synthesized starting from N-{1-[(6-fluoro-2-naphthyl)methyl]piperidin-4-yl}benzamide (1),which was found by subjecting our chemical library to high throughput screening (HTS). The CCR3 inhibitory activity of the synthesized compounds against eotaxin-induced Ca(2+) influx was evaluated using CCR3-expressing preB cells. Systematic chemical modifications of 1 revealed that the 6-fluoro-2-naphthylmethyl moiety was essential for CCR3 inhibitory activity in this new series of CCR3 antagonists. Further structural modifications of the benzamide and piperidine moieties of 1 led to the identification of exo-N-{8-[(6-fluoro-2-naphthyl)methyl]-8-azabicyclo[3.2.1]oct-3- yl}biphenyl-2-carboxamide [corrected] (31) as a potent CCR3 antagonist with an IC(50) value of 0.020 microM.

Eosinophilic chemokines and histamine play distinct but important roles in allergic diseases. Inhibition of both eosinophilic chemokines and histamine, therefore, is an ideal strategy for the treatment of allergic inflammation, such as asthma, allergic rhinitis, and atopic dermatitis. YM-344484 was found to potently inhibit both the CCL11-induced Ca2+ influx in human CCR3-expressing cells (Kb=1.8 nM) and histamine-induced Ca2+ influx in histamine H1 receptor-expressing PC3 cells (Kb=47 nM). YM-344484 also inhibited the CCL11-induced chemotaxis of human CCR3-expressing cells (IC50=6.2 nM) and CCL11-induced eosinophil-derived neurotoxin release from human eosinophils (IC50=19 nM). Orally administered YM-344484 inhibited the increase in histamine-induced vascular permeability in mice (82% inhibition at a dose of 10 mg/kg) and the accumulation of eosinophils in a mouse asthma model (74% at a dose of 300 mg/kg). These results indicate that YM-344484, a novel and functional dual antagonist for chemokine CCR3 receptor and histamine H1 receptor, is an attractive candidate for development as a novel anti-allergic inflammation drug.

T cells play a regulatory role in the pathogenesis of various immune and allergic diseases, including human asthma. Recently, it was reported that a pyrazole derivative, YM-58483 (BTP2), potently inhibits Ca(2+) release-activated Ca(2+) (CRAC) channels and interleukin (IL)-2 production in T cells. We investigated the effects of YM-58483 on T helper type 2 (Th2) cytokine production in vitro and antigen-induced airway asthmatic responses in vivo. YM-58483 inhibited IL-4 and IL-5 production in a conalbumine-stimulated murine Th2 T cell clone (D10.G4.1), and IL-5 production in phytohemagglutinin-stimulated human whole blood cells with IC(50) values comparable to those reported for its CRAC channel inhibition (around 100 nM). YM-58483 inhibited antigen-induced eosinophil infiltration into airways, and decreased IL-4 and cysteinyl-leukotrienes content in inflammatory airways induced in actively sensitized Brown Norway rats. Furthermore, orally administered YM-58483 prevented antigen-induced late phase asthmatic bronchoconstriction and eosinophil infiltration in actively sensitized guinea pigs. These data suggest that the inhibition of Ca(2+) influx through CRAC channel leads to the prevention of antigen-induced airway inflammation, probably via the inhibition of Th2 cytokine production and inflammatory mediators release. YM-58483 may therefore be useful for treating airway inflammation in bronchial asthma.

CC chemokine ligand 11 (CCL11/eotaxin) and other CC chemokine receptor 3 (CCR3) ligands (CCL24/eotaxin-2, CCL26/eotaxin-3, CCL13/monocyte chemotactic protein-4, etc.) play important roles in the chemotaxis and activation of eosinophils and other CCR3-expressing cells (basophils, mast cells, and CD4(+) T helper 2 cells) in allergic inflammation incidents, including asthma and rhinitis. A newly synthesized compound, N-{(3R)-1-[(6-fluoro-2-naphthyl)methyl]pyrrolidin-3-yl}-2-{1-[(5-hydroxy-3-methylpyridin-2-yl)carbonyl]piperidin-4-ylidene}-acetamide hemifumarate (YM-355179), inhibited the binding of CCL11 and CCL5/regulated on activation normal T cell expressed and secreted to CCR3-expressing B300-19 cells with IC(50) values of 7.6 and 24 nM, respectively. In contrast, YM-355179 did not affect the binding of CCL5 to CCR1 or CCR5. In functional assays, YM-355179 inhibited CCL11-induced, intracellular Ca(2+) influx, chemotaxis, and eosinophil degranulation with IC(50) values of 8.0, 24, and 29 nM, respectively. YM-355179 did not, however, affect any CC chemokine receptor (CCR1, CCR2, CCR4, or CCR5)-mediated Ca(2+) influx signals. Furthermore, oral administration of YM-355179 (1 mg/kg) inhibited CCL11-induced shape change of whole blood eosinophils in cynomolgus monkeys. Intravenous injection of YM-355179 (1 mg/kg) also inhibited eosinophil infiltration into airways of cynomolgus monkeys after segmental bronchoprovocation with CCL11. These results indicate that YM-355179 is a novel, selective, and orally available CCR3 antagonist with therapeutic potential for treating eosinophil-related allergic inflammatory diseases.

Eosinophils play a prominent proinflammatory role in a broad range of diseases, including atopic dermatitis and asthma. Eotaxin-1 and its receptor CCR3 are implicated in the recruitment of eosinophils from blood into inflammatory tissues, therefore inhibition of Eotaxin-1/CCR3 interaction may have therapeutic potential for allergic inflammation with eosinophil infiltration. YM-344031, a novel and selective small molecule CCR3 antagonist, potently inhibited ligand binding (IC(50)=3.0nM), ligand-induced Ca(2+) flux (IC(50)=5.4nM), and the chemotaxis of human CCR3-expressing cells (IC(50)=19.9nM). YM-344031 (1-10mg/kg) orally administered to cynomolgus monkeys significantly inhibited Eotaxin-1-induced eosinophil shape change in whole blood. Additionally, orally administered YM-344031 (100mg/kg) prevented both immediate- and late-phase allergic skin reactions in a mouse allergy model. YM-344031 therefore has potential as a novel and orally available compound for the treatment of allergic inflammation, such as atopic dermatitis and asthma.

A newly synthesized compound, YM-90709, 2,3-dimethoxy-6,6-dimethyl-5,6-dihydrobenzo[7,8]indolizino[2,3-b]quinoxaline, was previously reported to specifically inhibit the binding of interleukin-5 (IL-5) to its receptor (R) on human eosinophils. In this study, the intravenous injection of YM-90709 inhibited antigen-induced infiltration of eosinophils into the bronchoalveolar lavage fluid (BALF) of BDF1 mice, with an ED(50) value of 0.050mg/kg. Anti-murine IL-5 monoclonal antibody (mAb) also inhibited the infiltration of eosinophils with an ED(50) value of 0.035mg/kg. These results indicate that YM-90709, which is a novel IL-5R antagonist, inhibits antigen-induced eosinophil recruitment into the airway, the same as anti-IL-5 mAb does.

Interleukin-5 (IL-5) plays an important role in the activation of eosinophils in the allergic inflammation in conditions such as asthma, rhinitis, and atopic dermatitis. A newly synthesized compound, YM-90709 (2,3-dimethoxy-6,6-dimethyl-5,6-dihydrobenzo[7,8]indolizino[2,3-b]quinoxaline), was previously reported to inhibit the binding of IL-5 to its receptor (R) on human eosinophils and eosinophilic HL-60 clone 15 cells. However, it did not inhibit the binding of granulocyte-macrophage colony-stimulating factor (GM-CSF) to its receptor on the same cells. In this study, the intravenous injection of YM-90709 resulted in the inhibition of antigen-induced infiltration of eosinophils and lymphocytes, but not neutrophils or monocytes, into the bronchoalveolar lavage fluid (BALF) of Brown-Norway (BN) rats, with ED50 values of 0.32 mg/kg and 0.12 mg/kg, respectively. Two glucocorticoids, dexamethasone and prednisolone, inhibited neutrophil, eosinophil, and lymphocyte infiltration into the BALF. However, both significantly reduced the number of peripheral blood leukocytes and bone marrow leukocytes. In contrast, YM-90709 did not affect the peripheral blood leukocytes or the bone marrow leukocytes. These results indicate that, in this model, YM-90709, which is a novel IL-5 R antagonist, inhibits antigen-induced eosinophil and lymphocyte recruitment into the airway, without any suppressive effects on peripheral blood leukocytes or bone marrow leukocytes, in contrast to the glucocorticoids., (Copyright 2004 Elsevier B.V.)

Interleukin-5 (IL-5) plays an important role in the activation of eosinophils in allergic inflammation including asthma and atopic dermatitis. A newly synthesized compound, YM-90709, 2,3-dimethoxy-6,6-dimethyl-5,6-dihydrobenzo[7,8]indolizino [2,3-b]quinoxaline, is reported here to inhibit the binding of IL-5 to its receptor on peripheral human eosinophils and butyric acid-treated eosinophilic HL-60 clone 15 cells, with IC50 values of 1.0 and 0.57 microM, respectively. In contrast, YM-90709 did not affect the binding of granulocyte-macrophage colony-stimulating factor (GM-CSF) to its receptor on eosinophils and eosinophilic HL-60 clone 15 cells. In functional assays, YM-90709 inhibited IL-5-prolonged eosinophil survival with an IC50 value of 0.45 microM and did not affect the GM-CSF-prolonged eosinophil survival. Furthermore, YM-90709 inhibited the IL-5-induced but not GM-CSF-induced tyrosine phosphorylation of Janus kinase 2 (JAK2) in eosinophilic HL-60 clone 15 cells. These results indicate that YM-90709 is a novel IL-5 inhibitor which selectively blocks the binding of IL-5 to the IL-5 receptor (IL-5R).

We evaluated the effects of YM976, a selective inhibitor of phosphodiesterase type 4, on antigen-induced eosinophil infiltration into the lungs in rats, mice, and ferrets. In rats, YM976 inhibited the accumulation of eosinophils at an oral ED(50) value of 1.7 mg/kg, and in C57Black/6 mice, exhibited a dose-dependent inhibition at an ED(50) value of 5.8 mg/kg. In the same dose range in the same mouse model, YM976 suppressed interleukin-5 production. We then compared the inhibitory effect of chronic administration with that of single administration in another rat model of eosinophilia induced by repeated antigen exposure. YM976 administered chronically offered more potent inhibition (ED(50) = 0.32 mg/kg p.o.) than a single dose (1.4 mg/kg p.o.). These results indicated that chronic administration is more effective in antigen-induced eosinophilia than a single administration. Emetogenicity is known to be a major adverse effect of phosphodiesterase type 4 inhibitors. We compared the anti-inflammatory activity of YM976 with its emetic activity in ferrets, in which it dose dependently suppressed eosinophil infiltration at an ED(50) value of 1.2 mg/kg, but induced no emesis at 10 mg/kg. This suggested that the compound exhibits a considerable dissociation between its anti-inflammatory and emetic effects. In summary, YM976 inhibited eosinophil infiltration in a dose-dependent manner in rats, mice, and ferrets. In ferrets, it suppressed antigen-induced eosinophil infiltration without emesis. Additionally, we demonstrated that the inhibitory effect on eosinophil infiltration was increased by chronic administration. In conclusion, YM976 is a promising drug for the treatment of diseases involving eosinophil activity, such as asthma.

In the present experiments, costimulatory effects of interleukin 12 (IL-12) and B7-2 on interleukin 2 receptor alpha chain (IL-2R alpha) expression and IL-2-dependent proliferation of naive and memory CD4+ T cells were studied in comparison with the effect of these molecules on Th1 clones. The naive and memory T cells were negatively sorted on the basis of the expression of CD44 and CD45RB, respectively, to diminish the effect of these antibodies on the activation of these cells. When these three T cell populations were stimulated with allogeneic B cells, the addition of IL-12 elicited IL-2R alpha expression and proliferation of all these T cell populations. With anti-B7-2 being included in culture, these responses of naive T cells were abrogated, whereas those of memory T cells and Th1 clones were affected only marginally. When they were stimulated with immobilized anti-CD3, the inclusion of either IL-12 or Chinese hamster ovary cells expressing B7-2 (B7-2CHO) induced IL-2R alpha expression and enhanced IL-2-dependent proliferation of memory T cells, whereas these responses of naive T cells were induced by the inclusion of B7-2CHO and enhanced by the addition of IL-12, although the addition of IL-12 alone did not affect the responses. The responses of Th1 clones were markedly enhanced by IL-12, but not by B7-2CHO, and the enhancement of the proliferation was augmented further by the addition of both IL-12 and B7-2CHO. In IFN-gamma production B7-2 costimulation was required for the enhancing effect of IL-12 on the responses of naive T cells. IL-2 production of all T cell populations was not affected by IL-12 even in the presence of B7-2CHO. These results indicate that IL-12 provides the costimulation for IL-2R alpha expression and IL-2-dependent proliferation of memory T cells and Th1 clones stimulated with TCR ligation, whereas naive T cells require B7-2 costimulation for their response to IL-12. Possible mechanisms of B7-2 costimulation in the response of T cells to IL-12 is discussed.

B cells activated with anti-mu antibody plus interferon (IFN)-gamma exerted strong antigen presentation activity for T cell proliferation. The enhanced antigen presentation function was shown to be due to the increase in B7-2 expression. When B cells were stimulated with anti-mu, expression of MHC major histocompatibility complex class II, heat-stable antigen (HSA), ICAM-1 and B7-2 was increased. The presence of IFN-gamma further augmented the expression of B7-2 on anti-mu-stimulated B cells. B7-1 was not expressed on B cells under these conditions. The participation of B7-2 in the elicitation of the proliferative response of T cells was confirmed by the inclusion of anti-B7-2 antibody in cultures. The enhanced expression of either HSA or ICAM-1 was shown not to play a major role in the increased B cell antigen presentation capacity. The major T cell population responding to this activated B cell antigen presentation was shown to be CD44low naive CD4+ T cells, whereas CD45RBlow memory CD4+ T cells responded only weakly. The difference in proliferative responses between naive and memory CD4+ T cells was explained by the different efficiency in IL-2 production of these cell populations in response to antigen presentation by B cells activated by anti-mu plus IFN-gamma. These results suggest that IFN-gamma plays an important role in recruitment of naive T cells for an immune response.