Development of a novel Poly (I:C)-induced murine model with accelerated lupus nephritis and examination of the therapeutic effects of mycophenolate mofetil and a cathepsin S inhibitor.

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.

Lit-LAMP-DNA-vaccine for shrimp allergy prevents anaphylactic symptoms in a murine model.

Allergen-specific immunotherapy (AIT) is a promising therapeutic approach to food allergy but requires optimization in terms of both efficacy and safety due to the risk of undesired anaphylactic reactions. Here, we investigated the potential of a single DNA plasmid vaccine (Lit-LAMP-DNA-vaccine) encoding multivalent shrimp antigens (Lit v (Litopenaeus vannamei; Whiteleg shrimp) 1, Lit v4, and Lit v3) and a lysosomal-associated membrane protein (LAMP) as the next generation of AIT for patients with allergy. We first confirmed the expression of the LAMP-1-Lit v1-Lit v4-Lit v3 fusion protein in human cells transfected with the Lit-LAMP-DNA-vaccine, and the induction of anti-Lit v1, Lit v3, and Lit v4 IgG2a antibody production as well as Th1 response in Lit-LAMP-DNA-vaccine-treated mice. Next, we established an anaphylaxis model in mice epicutaneously sensitized with a crude shrimp protein extract (SPE) and investigated both the efficacy of Lit-LAMP-DNA-vaccine, and the difference in the mechanism of action (MOA) from oral immunotherapy (OIT). In the mouse shrimp allergy model, Lit-LAMP-DNA-vaccine potently suppressed anaphylactic reactions and mast cell activation with robust antigen-specific IgG2a production. The IgG1:IgG2a ratio was significantly lower than that of OIT. This suppressive effect was also confirmed by plasma transfer from mice previously vaccinated with the Lit-LAMP-DNA-vaccine. These results suggest that this Lit-LAMP-DNA-vaccine may represent a promising therapeutic strategy for human shrimp allergy which acts via the efficient induction of antigen-specific IgG with antagonism.

Impacts of dosing and drug withdrawal period on tacrolimus-based triple therapy in a non-human primate renal transplantation model.

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.

Potential benefit of the cathepsin S inhibitor, ASP1617, as a treatment for systemic lupus erythematosus.

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.

Effects of AS2819899, a novel selective PI3Kδ inhibitor, in a NZB/W F1 mouse lupus-like nephritis model.

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.

In vitro pharmacological effects of peficitinib on lymphocyte activation: a potential treatment for systemic sclerosis with JAK inhibitors.

OBJECTIVES: Peficitinib, a novel Janus kinase (JAK) inhibitor, demonstrated promising results in treating RA in phase 3 clinical trials. This in vitro study was undertaken to characterize the pharmacological properties of peficitinib and investigate the involvement of JAK and signal transducer and activator of transcription (STAT) pathways in the pathological processes of SSc, which is also an autoimmune disease. METHODS: Phosphorylation levels of STAT molecules were assessed in peripheral blood mononuclear cells collected from patients with RA or SSc and healthy subjects, and in skin specimens obtained from 19 patients with SSc. In vitro inhibition of STAT phosphorylation and cytokine/chemokine production by peficitinib, tofacitinib and baricitinib were also characterized. RESULTS: Higher spontaneous STAT1 or STAT3 phosphorylation was observed in peripheral T-cells and monocytes from patients with RA and SSc compared with healthy subjects. In skin sections from patients with SSc, phosphorylated STAT3-positive cells were found in almost all cases, irrespective of disease subtype or patient characteristics. Conversely, phosphorylated STAT1-positive cells were observed only in samples from untreated patients with diffuse disease of short duration. Peficitinib inhibited STAT phosphorylation induced by various cytokines, with comparable efficacy to tofacitinib and baricitinib. Peficitinib also suppressed cytokine and chemokine production by peripheral blood mononuclear cells and skin fibroblasts. CONCLUSION: Our results suggest that JAK/STAT pathways are constitutively activated in SSc and RA, and that the JAK inhibitor may represent a novel therapeutic option for SSc.

The PI3Kδ selective inhibitor AS2541019 suppresses donor-specific antibody production in rat cardiac and non-human primate renal allotransplant models.

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.

Optimization of 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidines to generate a highly selective PI3Kδ inhibitor.

Chemical optimization of the 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine (THPP) scaffold was conducted with a focus on cellular potency while maintaining high selectivity against PI3K isoforms. Compound 11f was identified as a potent, highly selective and orally available PI3Kδ inhibitor. In addition, 11f exhibited efficacy in an in vivo antibody production model. The desirable drug-like properties and in vivo efficacy of 11f suggest its potential as a drug candidate for the treatment of autoimmune diseases and leukocyte malignancies.

Replacement of mycophenolate mofetil with a JAK inhibitor, AS2553627, in combination with low-dose tacrolimus, for renal allograft rejection in non-human primates.

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 AUC0-24h 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.

Effective suppression of donor specific antibody production by Cathepsin S inhibitors in a mouse transplantation model.

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.

Optimization and in vivo evaluation of pyrazolopyridines as a potent and selective PI3Kδ inhibitor.

Chemical optimization of pyrazolopyridine 1, focused on cellular potency, isoform selectivity and microsomal stability, led to the discovery of the potent, selective and orally available PI3Kδ inhibitor 5d. On the basis of its desirable potency, selectivity and pharmacokinetic profiles, 5d was tested in the trinitrophenylated aminoethylcarboxymethyl-Ficoll (TNP-Ficoll)-induced antibody production model, and showed higher antibody inhibition than a 4-fold oral dose of the starting compound 1. These excellent results suggest that 5d is a potential candidate for further studies in the treatment of autoimmune diseases and leukocyte malignancies.

Discovery and biological evaluation of novel pyrazolopyridine derivatives as potent and orally available PI3Kδ inhibitors.

Phosphatidylinositol-3-kinase (PI3K)δ inhibition is one of the most attractive approaches to the treatment of autoimmune diseases and leukocyte malignancies. Through the exploration of pyrazolopyridine derivatives as potential PI3Kδ inhibitors, compound 12a was identified as a potent PI3Kδ inhibitor but suffered from poor oral exposure in mice. With a modified amide linkage group, compound 15a was developed as an orally available PI3Kδ inhibitor with reduced selectivity against other PI3Ks. To improve the trade-off between selectivity and PK profile, structure-activity relationship (SAR) studies of terminal substituents on the pyrolidine ring were conducted. As a result, we developed potent PI3Kδ inhibitors with good oral availability. In particular, the representative compound 15j showed excellent selectivity for PI3Kδ over other PI3Ks with good oral exposure in mice.

Effects of AS2541019, a novel selective PI3Kδ inhibitor, on antibody production and hamster to rat xenotransplantation.

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.

Prevention of chronic renal allograft rejection by AS2553627, a novel JAK inhibitor, in a rat transplantation model.

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.

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.

A chronic renal rejection model with a fully MHC-mismatched rat strain combination under immunosuppressive therapy.

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.

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.

Effect of AS2521780, a novel PKCθ selective inhibitor, on T cell-mediated immunity.

T cell-mediated immunity is central to the pathogenesis of autoimmune diseases, and is a target in the development of alternative therapeutic strategies with reduced adverse effects on other cell types and organs. Protein kinase C (PKC) is a family of serine/threonine kinases, with knockout of the PKCθ isoform in mice resulting in defective T cell activation. However, the effects of selective inhibition of PKCθ by small-molecule compounds on T cell signaling are still unknown. Here, we evaluated the effect of the novel PKCθ inhibitor AS2521780 on T cell activation and joint inflammation in a rat model of arthritis. AS2521780 exerted potent inhibition of recombinant human PKCθ enzyme activity (IC50=0.48 nM), which was more than 30-fold higher than that of other PKC isoforms. Further, AS2521780 exerted little or no inhibition on other protein kinases. AS2521780 suppressed CD3/CD28-induced Interleukin-2 (IL-2) gene transcription in Jurkat T cells and proliferation of human primary T cells. AS2521780 also suppressed concanavalin A-induced cytokine production by rat splenocytes and monkey peripheral blood mononuclear cells with similar potency. Moreover, AS2521780 significantly reduced paw swelling in a dose-dependent manner in a rat model of adjuvant-induced arthritis. These results indicate that PKCθ is an attractive drug target and AS2521780 is a potential immunosuppressant for T cell-mediated autoimmune diseases.

Efficacy of oral treatment with tacrolimus in the renal transplant model in cynomolgus monkeys.

Many studies have examined the efficacy of tacrolimus in rats and dogs, but few have reported its evaluation in cynomolgus monkeys. The aim of this study was to clarify the efficacy of tacrolimus in a cynomolgus monkey renal transplant model based on the efficacy of various doses. Monkeys that had undergone renal transplant were treated with a vehicle or 0.5, 1.0, or 2.0 mg/kg of tacrolimus by oral administration. Tacrolimus administration prolonged animal survival in a dose-dependent manner, and the median survival time (MST) was 11, 21, and >90 days for the 0.5, 1.0, and 2.0 mg/kg tacrolimus groups, respectively. The MST of the vehicle group was 6 days. Histopathological analyses of all transplanted kidneys were also performed. Typical pathological findings of acute rejection were observed in both the vehicle and tacrolimus (0.5 and 1.0 mg/kg)-treated groups. Only limited mononuclear cell infiltration and hemorrhage were present in the tacrolimus (2.0 mg/kg)-treated group. In conclusion, 2.0 mg/kg was considered to be a therapeutic dose in this model, and 0.5 or 1.0 mg/kg could be used for a study when efficacy of a new compound is evaluated in a combination therapy with tacrolimus.