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Current treatments for patients with Crohn's disease (CD) are based on recent advances in elucidating the pathophysiology of the disease. A satisfactory therapeutic strategy has not been well established. A new sphingosine 1-phosphate (S1P) receptor agonist, 2-amino-2-propanediol hydrochloride (KRP-203), has been developed for immunomodulation in autoimmune diseases and organ transplantation. We aimed to evaluate the efficacy and potency of KRP-203 on the treatment of chronic colitis in an interleukin (IL)-10 gene-deficient (IL-10(-/-)) mouse model. KRP-203 agonistic activity on S1P receptor was assessed in vitro. KRP-203 was administered for 1 or 4 weeks to IL-10(-/-) mice with clinical signs of colitis. The histological appearance of the colon and the numbers, phenotype, and cytokine production of lymphocytes were compared with a control group. KRP-203 treatment was effective in preventing body weight loss in the IL-10(-/-) colitis model. One-week administration resulted in the sequestration of circulating lymphocytes within the secondary lymphoid tissues. After 4 weeks of treatment, highly significant reductions were observed in number of CD4(+) T cell and B220(+) B cell subpopulations in the lamina propria of the colon and peripheral blood. KRP-203 obviously inhibited the production of interferon-gamma, IL-12, and tumor necrosis factor-alpha by the colonic lymphocytes, but had no influence on IL-4 production. KRP-203 significantly inhibits ongoing IL-10(-/-) colitis in part through decreasing the infiltration of lymphocytes at inflammatory sites and by blocking T-helper 1 cytokine production in the colonic mucosa. Therefore, the possibility arises that KRP-203 plays a potential role in control of chronic colitis.

Sphingosine-1-phosphate (S1P) is an active sphingolipid metabolite that exerts important biological effects. Recently, we demonstrated that KRP-203 is a novel S1P receptor agonist that can alter lymphocyte homing and act as an immunomodulating agent. We investigated the efficacy of KRP-203 in the treatment of rat experimental autoimmune myocarditis. KRP-203 significantly attenuated the inflammation area, heart weight/body weight ratio, and left ventricular function. Immunohistochemical analysis and RT-PCR revealed that KRP-203 significantly decreased the infiltration of macrophages and CD4 T cells in the myocardium and the expression of inflammatory cytokines. Flow cytometric analysis revealed that treatment with KRP-203 effectively reduced the number of peripheral CD4 and CD8 T cells but not that of B cells and granulocytes. Further, late KRP-203 treatment was effective even against established EAM. These results demonstrate the therapeutic potential of KRP-203 for the treatment of human myocarditis and provide new insights into the pathogenesis of this disease.

Background: We demonstrate the long-term effectiveness of KRP-203 treatment in combination with a subtherapeutic dose of cyclosporine A (CsA) on rat renal allografts., Methods: We tested the effect of KRP-203 in combination with CsA using a rat skin allograft model. The Pharmacokinetic interaction between CsA and KRP-203 was evaluated. The selectivity of KRP-203 for sphingosine-1-phosphate (S1P)1 and S1P3 receptors were investigated in vitro. Heart rate alteration following bolus injection of phosphorylated KRP-203 (KRP-203-P) or FTY720 (FTY720-P) was also monitored in rats. Finally, the long-term effectiveness of KRP-203 in conjunction with a low dose of CsA was investigated in a rat renal transplantation model., Results: Administration of KRP-203 with CsA prolonged skin allograft survival. KRP-203 and CsA had no effect on the pharmacokinetics of the other. While FTY720-P activated both S1P1 and S1P3 receptors, KRP-203-P selectively activated S1P1, but not the S1P3 receptor (EC50:>1000 nM). Compared to FTY720-P, a tenfold higher dose of KRP-203-P was necessary to induce transient bradycardia. With a low dose of CsA (1 mg/kg/day), KRP-203 (0.3 mg/kg/day) significantly prolonged renal allograft survival (P<0.05, survival time: 9.8 days (CsA) vs. >27.4 days (CsA+KRP)). Although a higher dose of CsA (3 mg/kg/day) alone kept recipients alive, this caused severe renal graft dysfunction. Use of KRP-203 (3 mg/kg/day) in conjunction with CsA markedly improved graft function (P<0.05, creatinine clearance: 0.41+/-0.25 ml/min [CsA] vs. 1.15+/-0.16 ml/min [CsA+KRP])., Conclusions: The selectivity of KRP-203 for S1P1 reduces the risk of bradycardia, and the combination therapy of KRP-203 with CsA represents a safe and effective strategy for use in renal transplantation.

Background: Replacement of calcineurin inhibitor (CI) with anti-metabolic agents in transplant patients with CI-induced nephrotoxicity is performed clinically and improves renal function, but increases the risk of rejection. We investigated whether the change from cyclosporine (CsA) to a limited dose of mycophenolic acid (MPA) together with a new sphingosine-1-phosphate (S1P) receptor agonist, KRP-203, is sufficient to prevent both transplant vasculopathy and CsA-induced nephrotoxicity., Methods: Orthotopic aortic transplantation was conducted in a high-responder rat combination of Dark Agouti (DA; major histocompatibility complex [MHC] haplotype RT-1a) to Lewis (RT-1(l)). After CsA administration (15 mg/kg/day) for 2 weeks, the recipients were divided into the following treatment groups for 6 weeks: MPA (10 mg/kg); KRP-203 (KRP; 1 mg/kg); and MPA + KRP. Serum creatinine (Cr), arteriolar hyalinosis and expression of transforming growth factor (TGF)-beta1 in the recipient kidney were examined as parameters indicating nephrotoxicity. Intimal hyperplasia was assessed by vascular occlusion, and graft-infiltrated cells were semi-quantitatively evaluated histologically and then characterized immunohistochemically., Results: Continuous CsA treatment attenuated intimal hyperplasia and cell infiltration (2.9 +/- 0.3% and 0.4 +/- 0.1; p < 0.01 vs vehicle), but increased Cr and hyalinosis (0.43 +/- 0.03 mg/dl and 57.2 +/- 0.4%; p < 0.01) with upregulated TGF-beta1. Replacement of CsA by MPA or KRP treatment alone improved nephrotoxicity, but worsened intimal hyperplasia and cell infiltration. Conversion to MPA + KRP treatment prevented nephrotoxicity (Cr, 0.32 +/- 0.02 mg/dl; hyalinosis, 5.6 +/- 1.3%; p < 0.01 vs CsA) and markedly suppressed intimal hyperplasia and cell infiltration (3.6 +/- 1.2% and 1.0 +/- 0.3; p = not significant vs CsA), with reduced T-cell infiltrates in the graft., Conclusions: Changing from CsA to a combined therapy of MMF with S1P agonist is a promising strategy in clinical transplantation to overcome CI-induced nephrotoxicity and chronic rejection.

T cell-mediated immune responses play a critical role in a variety of liver injuries including autoimmune hepatitis. Injection of concanavalin A (Con A) into mice mimics the histological and pathological phenotype of T cell-mediated hepatitis. Recent advances in host immune control of organ transplantation include the development of sphingosine-1-phosphate (S1P) receptor agonists such as FTY720, which alter lymphocyte homing but do not suppress host general immunity. Herein we examined the effect of the new S1P receptor agonist KRP-203 on the Con A-induced liver damage model. In normal liver lymphocytes of BALB/c mice, both FTY720 and KRP203 promoted lymphocyte sequestering from the liver to secondary lymph nodes and significantly reduced the number of liver lymphocytes (p<0.05). Based on this observation, KRP203 was employed in the Con A-induced hepatitis model. KRP203 markedly reduced the number of CD4(+) lymphocytes that infiltrate Con A-treated liver (p<0.05) and successfully reduced serum transaminase elevation (p=0.017), therefore protecting mice from Con A-induced liver injury. Interestingly this homing modulation less occurs in natural hepatic T cell homing through the chemokine receptor, CXCR4. Therefore, S1P receptor agonists preferentially target CXCR4(+)CD4(+) peripheral blood T lymphocytes and suppress the occurrence of Con A-induced hepatitis, suggesting their therapeutic usefulness against T cell-mediated hepatic injury.

Background: A novel immunomodulator, KRP-203, the molecular structure of which has some similarity to FTY720, has been developed for use in organ transplantation. The present study was designed to investigate the potency and safety of KRP-203 on allograft survival against both acute and chronic rejection in rat skin and heart transplantation., Methods and Results: KRP-203 significantly prolonged skin or heart allograft survival of a minor histocompatibility complex (mHC)-disparate (LEW to F344) rat combination. Histopathological and immunohistochemical analysis at 100 days after mHC-disparate rat heart transplantation revealed that KRP-203 treatment significantly inhibited infiltration of inflammatory cells, including macrophages and T cells; expression of endothelin-1 and transforming growth factor-beta1; and IgG deposition and eventually attenuated neointimal formation and myocardial fibrosis. KRP-203 also prolonged heart allograft survival in a major histocompatibility complex (MHC)-incompatible (DA to LEW) rat combination, but the efficacy was not as significant. However, KRP-203 combined with a subtherapeutic dose of cyclosporin A synergistically prolonged the heart allograft survival. Flow cytometric analysis demonstrated that KRP-203 reduced the number of peripheral blood mononuclear cells (lymphocytes and monocytes) but not granulocytes and enhanced lymphocyte homing into peripheral lymph nodes. The influence of KRP-203 on heart rate changes in Hartley guinea pigs was examined. KRP-203 had less of a tendency to cause bradycardia than FTY720., Conclusions: These findings demonstrated that KRP-203 prolonged skin and heart allograft survival and significantly attenuated chronic rejection and bradycardia as an adverse effect. Therefore, KRP-203 offers considerable potential as a novel therapeutic immunosuppressant in patients with organ transplantation.

To find more effective and less toxic immunosuppressive strategies in long-term treatment for organ transplantation patients, we examined the effects on rat heart allograft survival of a novel sphigosine-1-phosphate receptor agonist, KRP-203, combined with a subtherapeutic dose of cyclosporine (CsA). Rat heart transplantation was performed across a major histocompatibility complex-incompatible (DA to LEW) rat combination. KRP-203 alone showed little or no effect on heart allograft survival. In contrast, KRP-203 combined with a subtherapeutic dose of CsA led to prolonged allograft survival. Histologic analyses showed that the combination completely suppressed acute rejection, as characterized by allograft vasculopathy, mononuclear cell infiltration, and myocardial necrosis in the heart allografts. RT-PCR analysis showed that the allografts treated with CsA or KRP-203 alone showed no suppression of IL-10, IFN-gamma, and TNF-alpha mRNA expression, but when combined with a subtherapeutic dose of CsA it completely suppressed their mRNA expressions. Furthermore, the combination treatment reduced donor-specific antibody production. KRP-203 combined with a subtherapeutic dose of CsA synergistically prolonged rat heart allograft survival. The combination of CsA with KRP-203 may provide an option to prevent allograft rejection and reduce adverse effects.