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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.)

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.)

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.)

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.)

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.