Blockade of EP4 by ASP7657 Modulates Myeloid Cell Differentiation In Vivo and Enhances the Antitumor Effect of Radiotherapy.

The tumor microenvironment (TME) is thought to influence the antitumor efficacy of immuno-oncology agents through various products of both tumor and stromal cells. One immune-suppressive factor is prostaglandin E2 (PGE2), a lipid mediator whose biosynthesis is regulated by ubiquitously expressed cyclooxygenase- (COX-) 1 and inducible COX-2. By activating its receptors, PGE2 induces immune suppression to modulate differentiation of myeloid cells into myeloid-derived suppressor cells (MDSCs) rather than dendritic cells (DCs). Pharmacological blockade of prostaglandin E receptor 4 (EP4) causes a decrease in MDSCs, reprogramming of macrophage polarization, and increase in tumor-infiltrated T cells, leading to enhancement of antitumor immunity in preclinical models. Here, we report the effects of the highly potent EP4 antagonist ASP7657 on the DC population in tumor and antitumor immune activation in an immunocompetent mouse tumor model. Oral administration of ASP7657 inhibited tumor growth, which was accompanied by an increase in intratumor DC and CD8+ T cell populations and a decrease in the M-MDSC population in a CT26 immunocompetent mouse model. The antitumor activity of ASP7657 was dependent on CD8+ T cells and enhanced when combined with an antiprogrammed cell death-1 (PD-1) antibody. Notably, ASP7657 also significantly enhanced the antitumor efficacy of radiotherapy in an anti-PD-1 antibody refractory model. These results indicate that the therapeutic potential of ASP7657 arises via upregulation of DCs and subsequent CD8+ T cell activation in addition to suppression of MDSCs in mouse models and that combining EP4 antagonists with radiotherapy or an anti-PD-1 antibody can improve antitumor efficacy.

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.

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.

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.

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.

Inhibition of antigen-induced airway inflammation and hyperresponsiveness in guinea pigs by a selective antagonist of "chemoattractant receptor homologous molecule expressed on Th2 cells" (CRTH2).

Chemoattractant receptor homologous molecule expressed on T helper type 2 cells (CRTH2) is a PGD2 receptor found on eosinophils, basophils, and Th2 type T cells which exhibits chemotaxis and functions in activation cascades. However, while a number of CRTH2 antagonists, including ramatroban, are known to exert activity in certain animal models, activity in a guinea pig model of EA-induced airway hyperresponsiveness has not been demonstrated. The newly developed CRTH2 antagonist ASP5642 has shown antagonistic activity against human and guinea pig CRTH2 in previous studies and has also been found effective in treating guinea pig models of airway inflammation and airway hyperresponsiveness. While previous studies have used animals such as rats and mice to evaluate CRTH2 antagonist effects, ours is the first attempt to evaluate CRTH2 function in a guinea pig asthma model, which may prove useful in evaluating the compound's effects in humans, given the comparable airway function between the two species taken together, these data from the present study strongly suggest the utility of ASP5642 in investigating the role of CRTH2 in inflammatory responses and as a drug treatment for human asthma.

Greenhouse gas reduction and improved sustainability of animal husbandry using amino acids in swine, poultry feeds.

In Annex 1 countries, nitrous oxide (N2 O) emissions from swine and poultry excreta have been calculated and the N2 O reduction potential of each country by using amino acids in feed could also be calculated, then a comparison made among the countries. The N2 O reduction rates were approximately 25% for these Annex 1 countries and amino acids were able to make a large contribution to that reduction. Greenhouse gases (GHG) which are N2 O combined with methane (CH4 ) were estimated to reduce by 24.8% in Japan when amino acids were introduced into the feed, but only a 7.2% reduction was estimated in France. Purification, which is mainly used for manure treatment in Japan, emits much more N2 O and less CH4 , whereas the liquid system which is mainly used in France emits more CH4 and less N2 O based on the emission factors from the United Nations Framework Convention on Climate Change data base. Changing the French manure treatment system to the Japanese style with amino acids in feed would reduce GHG emissions by 23.4%. Reduction of the arable land use in Japan by changing crop formulations supported by adding amino acids to feed was also quantified as about 10% and led to an increase in the production of meat using the same arable land area.

Anti-inflammatory activity of non-nucleoside adenosine deaminase inhibitor FR234938.

Adenosine has anti-inflammatory activity. Adenosine deaminase (EC 3.5.4.4) metabolizes extracellular adenosine, resulting in an exacerbation of inflammation. Consequently, it was hypothesized that adenosine deaminase inhibitors produce anti-inflammatory activity by increasing extracellular adenosine concentration. This group recently developed a non-nucleoside adenosine deaminase inhibitor, FR234938, by using rational structure-based drug design. FR234938 inhibits recombinant human adenosine deaminase enzyme competitively. FR234938 inhibits interleukin (IL)-6-dependent immunoglobulin (Ig) M production by SKW6.4 cells, in the presence of adenosine. Inhibitory effect of FR234938/adenosine combination is blocked by an A2a adenosine receptor antagonist. FR234938 also inhibits anti-type II collagen delayed type hypersensitivity (DTH) in a dose-dependent manner, both in the presence and absence of recombinant human adenosine deaminase. Moreover, FR234938 inhibits tumor necrosis factor (TNF)-alpha and IL-10 production in a lipopolysaccharide (LPS)-induced cytokine production model in mice. These results indicate that FR234938 has potential anti-inflammatory activity. Non-nucleoside adenosine deaminase inhibitor FR234938 has good potential as a new type of anti-rheumatic and anti-inflammatory drug, by modulating host-defense concentrations of adenosine.

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

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

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

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