Evidence that FTY720 induces T cell apoptosis in vivo

The immunosuppressant FTY720 induces a drastic decrease in blood lymphocytes, especially T cells; a decrease which is assumed to be the immunosuppressive mechanism of this drug. FTY720 causes cell death in vitro in lymphocytes and leukemia cells. However, the deletion mechanism of blood lymphocytes in vivo remains unclear. We investigated whether administration of FTY720 induced lymphocyte apoptosis in blood circulation. A marked decrease in the number of blood lymphocytes was observed within an hour after a single oral administration of FTY720 at doses of 5-10 mg/kg in rats and mice. Experiments using fluorescein isothiocyanate (FITC)-Annexin V and APO-BRDU methods revealed that FTY720 induced blood lymphocyte apoptosis in a dose-dependent manner. On the other hand, lymphocyte homing to Peyer's patches was proposed as the mechanism underlying the blood lymphocyte decrease at these doses. However, similar results were obtained when using aly/aly mice, which lack Peyer's patches and lymph nodes. Thus, we concluded that apoptosis of blood lymphocytes was induced immediately after administration of FTY720, and the cells could be immediately scavenged by phagocytes or the reticuloendothelial system in addition to Peyer's patches homing. We also concluded that T cells were highly sensitive to FTY720, which induced apoptosis in vivo.     

FTY720对免疫性肝损伤保护作用的研究

目的　应用免疫性肝损伤模型研究FTY72 0对肝脏的保护作用并对其作用机制进行初步探讨。方法　分别以BCG +LPS和ConA诱导两种免疫性肝损伤模型并通过测定血清中ALT和AST水平的变化检测FTY72 0对两种肝损伤的保护作用。以ELISA方法测定血清中细胞因子IFN γ及IL 4水平的变化并应用淋巴细胞增殖实验检测FTY72 0对淋巴细胞增殖能力的影响。结果　FTY72 0可以使两种肝损伤模型中升高的血清ALT和AST水平下降 ,并可以降低肝损伤时血清中IFN γ及IL 4的水平 ,实验还证明FTY72 0可以抑制淋巴细胞的增殖。结论　FTY72 0预防应用对免疫性肝损伤有保护作用 ,其保肝作用机制可能和抑制淋巴细胞的增殖 ,进一步抑制细胞因子的产生有关     

FTY720 preferentially depletes naive T cells from peripheral and lymphoid organs

The sphingosine-1-phosphate receptor agonist FTY720 induces lymphopenia by inhibiting lymphocyte egress from thymus and lymph nodes. The immediate effect of the drug on T cells in blood and lymphoid tissues is well documented, however effects on peripheral T cell sub-populations have not been studied. We therefore analyzed the changes in T cell subset compositions in liver, lung, kidney, spleen, lymph nodes and blood induced by FTY720-treatment using 9-parameter flow cytometry. In untreated mice, naive T cells were present in all peripheral organs. Naive T cells were depleted from peripheral organs within 3 days by FTY720, and with slower kinetics from lymphoid organs. Antigen-experienced T cell subsets were less affected by FTY720-treatment and substantial numbers were retained in the periphery. The proportion of CD8(+)CD44(+)CD43(+) Gr-1(+) effector memory cells increased after FTY720-treatment, while that of CD8(+)CD44(+)CD62L(+) central memory cells was unchanged. Our data demonstrate that naive T cells pass peripheral tissues as part of their default recirculation pathway. FTY720 treatment primarily affects the recirculation of naive and central memory cells, both of which re-circulate through lymph nodes on a regular basis, but does not influence effector memory cells. This suggests that treatment with FTY720 may not interfere with immune functions mediated locally by tissue-resident peripheral effector/memory T cells.     

Update on pharmacokinetic/pharmacodynamic studies with FTY720 and sirolimus

Expanding the cytokine paradigm beyond the use of calcineurin inhibitors as baseline therapy provides new strategies in immunosuppression. Drugs such as FTY720 alter the sensitivity of lymphocytes to homing chemokines, and agents such as sirolimus (SRL) disrupt downstream cytokine signal transduction. Confirming studies in rodents and nonhuman primates, administration of either FTY720 or both of these drugs afford synergistic interactions with cyclosporine to renal transplant patients to rapidly and dramatically deplete peripheral blood lymphocytes (PBL) but neither granulocytes nor monocytes. Present information suggests that FTY720 facilitates lymphocyte homing mechanisms, leading to T and B cell sequestration in secondary lymphoid structures. Interestingly, FTY720 displays pharmacokinetic characteristics suggesting that therapeutic drug monitoring (TDM) will not be essential for clinical applications. In contrast, SRL is a critical-dose drug that requires TDM. SRL disrupts costimulatory and cytokine-stimulated T cell activation by inhibiting a multifunctional kinase, mammalian target of sirolimus (mTOR). Two pivotal trials including more than 1,300 patients demonstrated that addition of SRL to a CsA-based regimen reduces the incidence, time to onset, and severity of acute rejection episodes. When used alone, SRL seems therapeutically equivalent to CsA. In the coming decade, SRL is likely to be used in a variety of drug combination regimens both simultaneously and sequentially, not only to avert acute rejection episodes, but also to forestall chronic nephropathic processes. These two new agents are likely to usher in a new era of transplant therapy.     

Overview of FTY720 clinical pharmacokinetics and pharmacology

Drug discovery programs are actively exploring for therapeutic agents targeting enzymes and receptors regulating sphingolipid metabolism and biologic functions. FTY720 is a close structural analogue of sphingosine with immunomodulatory properties. After oral administration, FTY720 is phosphorylated by sphingosine kinase to form the active moiety FTY720-phosphate, which subsequently binds to the sphingosine-1-phosphate receptor. In characterizing the safety and pharmacological effects of FTY720, detailed clinical pharmacology studies in healthy subjects and renal transplant recipients have focused on cardiac responses and lymphocyte trafficking. After the first dose, FTY720 causes a mild, transient decrease in heart rate that returns to baseline in approximately 1 to 2 weeks despite continued administration of the drug. FTY720 elicits a prompt and dose-dependent decrease in peripheral blood lymphocytes by redirecting them from the circulation to the lymph nodes without impairing lymphocyte functions. An association among FTY720 blood concentration, decrease in lymphocyte counts, and freedom from acute rejection episodes has been observed in early clinical development trials in de novo kidney transplantation.     

FTY720: a most promising immunosuppressant modulating immune cell functions

FTY720, the pharmacological analog of S1P, acts as an agonist of sphingosine-1-phosphate receptors, resulting in the inhibition of lymphocyte egress from secondary lymphoid tissues and thymocytes from the thymus, peripheral lymphopenia and interfering with normal functions of several other cell types. FTY720 has been clinically tried for transplantation and multiple sclerosis, showing promising protective effects. This review will summarize potential applications and effects of FTY720.     

Activation of the lung S1P1 receptor reduces allergen-induced plasma leakage in mice

Background and purpose:

It has been suggested that intratracheal administration of the immunomodulator, FTY720, could have anti-inflammatory effects without causing a decrease in blood lymphocyte counts. However, the receptor responsible for this effect has not been defined.

Experimental approach:

We have described, in a mouse model of allergen-induced inflammation, the use of proton magnetic resonance imaging to non-invasively assess lung fluid accumulation and inflammation. Here, we used this model to investigate the sphingosine-1-phosphate (S1P) receptor responsible for the anti-inflammatory effect of FTY720.

Key results:

When given intranasally, FTY720 (3 and 10 µg·kg⁻¹) inhibited by approximately 50% the allergen-induced accumulation of fluid in the lung detected by magnetic resonance imaging, but had no effect on the cellular inflammation in the airway space or on circulating blood lymphocytes. Inhibition of the infiltration of inflammatory cells into the airways was only observed at a dose of FTY720 that induced lymphopenia (100 µg·kg⁻¹). Similar results were observed in S1P₃-deficient mice. The effect of FTY720 was mimicked by intranasal treatment of wild-type mice with a S1P₁-specific agonist, AUY954.

Conclusions and implications:

Thus, in contrast to previously published work, our results suggest that systemic exposure of FTY720 is necessary to obtain an airway anti-inflammatory effect. On the contrary, inhibition of the allergen-induced accumulation of fluid in the lung, via activation of the S1P₁ receptor, is obtainable without systemic effects.     

Tacrolimus in combination with FTY720--an analysis of renal and blood parameters

Calcineurin inhibitors (CNIs) are routinely used in immunosuppressive therapy and both Cyclosporine (CsA) and Tacrolimus (FK506) show similar efficacies to prevent rejection and death within the first year after organ transplantation. However, their use is limited by side effects such as kidney damage, hypertension, onset of diabetes and hyperlipidemia. It is a consensus that compared with CsA, FK506 causes less changes in blood pressures, serum lipids and renal function. Nevertheless, FK506 use is associated with a higher incidence of post-transplant diabetes mellitus (PTDM). FTY720 is a new compound that has shown a protective effect in animal models with respect to rejection in transplantation, ischemia-reperfusion injury, autoimmune diseases and tumor development. FTY720 acts by altering lymphocytes homing from blood to peripheral lymphoid organs. In mice, FTY720 administered in combination with CsA during 21 days has prolonged skin allograft survival without causing significant renal changes. In a model of CsA-induced chronic nephropathy in rats, FTY720 administration prevented renal injury suggesting benefit from using a combination of these drugs. In a canine kidney allograft model, FTY720 in combination with low doses of CsA or FK506 showed an addictive anti-rejection effect without causing critical adverse effects. We therefore, investigated whether 21 days of FTY720 administration in association with FK506 could prevent renal damage and development of diabetes in mice. Mice receiving FK506 alone or FTY720 + FK506 during 21 days showed changes in kidney function and structure besides an increase in blood glucose and lymphopenia. The FTY720 + FK506 combination requires further investigation with an aim toward understanding the mechanisms involved with respect to side effects.     

FTY720 in combination with cyclosporine--an analysis of skin allograft survival and renal function

Acute and chronic nephrotoxicity caused by CsA continuous administration impair kidney allograft survival. Several clinical and experimental protocols have shown benefits to the kidney after decreasing CsA dose, withdrawing the drug or delaying its introduction after transplantation. FTY720 is a new compound that has immunosuppressive characteristics and increase allograft survival in animal models without causing the side effects of calcineurin inhibitors (CNIs). FTY720 described mechanism of action that consists to alter the lymphocyte migration pattern without impairment of the immune system response against pathogens. In our mice model, FTY720 administered alone or in combination with CsA during 21 days increased skin allograft survival in a fully mismatched strain combination and did not cause significant changes in renal function. Moreover, renal structure was normal in all groups suggesting that at low doses (10 mg/kg/day) CsA can be associated during short-term period to other immunosuppressive drugs, i.e. FTY720 without affecting the kidney. Combination of immunosuppressive compounds with FTY720 and/or delayed introduction of low cyclosporine dose could prevent graft rejection and avoid nephrotoxicity.     

Tacrolimus in combination with FTY720 — an analysis of renal and blood parameters

Calcineurin inhibitors (CNIs) are routinely used in immunosuppressive therapy and both Cyclosporine (CsA) and Tacrolimus (FK506) show similar efficacies to prevent rejection and death within the first year after organ transplantation. However, their use is limited by side effects such as kidney damage, hypertension, onset of diabetes and hyperlipidemia. It is a consensus that compared with CsA, FK506 causes less changes in blood pressures, serum lipids and renal function. Nevertheless, FK506 use is associated with a higher incidence of post-transplant diabetes mellitus (PTDM). FTY720 is a new compound that has shown a protective effect in animal models with respect to rejection in transplantation, ischemia–reperfusion injury, autoimmune diseases and tumor development. FTY720 acts by altering lymphocytes homing from blood to peripheral lymphoid organs. In mice, FTY720 administered in combination with CsA during 21 days has prolonged skin allograft survival without causing significant renal changes. In a model of CsA-induced chronic nephropathy in rats, FTY720 administration prevented renal injury suggesting benefit from using a combination of these drugs. In a canine kidney allograft model, FTY720 in combination with low doses of CsA or FK506 showed an addictive anti-rejection effect without causing critical adverse effects. We therefore, investigated whether 21 days of FTY720 administration in association with FK506 could prevent renal damage and development of diabetes in mice. Mice receiving FK506 alone or FTY720 + FK506 during 21 days showed changes in kidney function and structure besides an increase in blood glucose and lymphopenia. The FTY720 + FK506 combination requires further investigation with an aim toward understanding the mechanisms involved with respect to side effects.     

FTY720 in combination with cyclosporine—an analysis of skin allograft survival and renal function

Acute and chronic nephrotoxicity caused by CsA continuous administration impair kidney allograft survival. Several clinical and experimental protocols have shown benefits to the kidney after decreasing CsA dose, withdrawing the drug or delaying its introduction after transplantation.FTY720 is a new compound that has immunosuppressive characteristics and increase allograft survival in animal models without causing the side effects of calcineurin inhibitors (CNIs). FTY720 described mechanism of action that consists to alter the lymphocyte migration pattern without impairment of the immune system response against pathogens.In our mice model, FTY720 administered alone or in combination with CsA during 21 days increased skin allograft survival in a fully mismatched strain combination and did not cause significant changes in renal function. Moreover, renal structure was normal in all groups suggesting that at low doses (10 mg/kg/day) CsA can be associated during short-term period to other immunosuppressive drugs, i.e. FTY720 without affecting the kidney.Combination of immunosuppressive compounds with FTY720 and/or delayed introduction of low cyclosporine dose could prevent graft rejection and avoid nephrotoxicity.     

新型免疫抑制剂FTY720的研究进展

FTY720是一种来源于子囊菌冬虫夏草的新型免疫抑制剂,与常规免疫抑制剂环孢素、他克莫司、西罗莫司等相比具有不同的作用机制,其主要通过致淋巴细胞凋亡及归巢效应等起到免疫抑制的作用,在多种动物模型的同种异体移植中具有抑制移植物排斥作用,在人体肾脏移植中也呈较强的免疫抑制活性,故在临床器官移植及自身免疫性疾病的治疗等方面具有广泛的应用前景.     

Single-dose FTY720 pharmacokinetics, food effect, and pharmacological responses in healthy subjects

AIMS: FTY720 is a sphingosine-1-phosphate receptor agonist that redirects lymphocytes from the circulation to lymph nodes without impairing lymphocyte function. It is being developed as an immunomodulator for the prevention of acute rejection after organ transplantation. This study was performed to provide guidance on administration with respect to meals and to measure pharmacologic responses in healthy subjects. METHODS: In this randomized, two-period, crossover study, 14 healthy subjects received placebo on day -1 of each period with baseline circadian measurements of lymphocyte count and heart rate. Subjects subsequently received a single 1 mg oral dose of FTY720 on day 1 under fasting conditions and after a high fat meal. Blood FTY720 concentrations, lymphocyte count, and supine heart rate were assessed over an 8 day period after each FTY720 dose. The effect of food on FTY720 pharmacokinetics was assessed by standard bioequivalence testing. RESULTS: Both the peak concentration (0.65 +/- 0.17 vs 0.64 +/- 0.18 ng ml(-1)) and total exposure (AUC 149 +/- 65 vs 139 +/- 43 ng ml(-1) h) did not differ significantly between fasting and fed states, respectively. The corresponding fed/fasting ratios and 90% confidence intervals were 1.00 (0.86, 1.17) for Cmax and 0.98 (0.86, 1.11) for AUC. Under both treatment conditions peripheral blood lymphocyte count decreased from baseline by 38 +/- 9% over the first 2 days postdose and then increased towards predose values over the subsequent week. Whereas a circadian rhythm in supine heart rate was preserved in the presence of FTY720, the heart rate vs time curve was shifted downwards by 10% over the first day postdose and then recovered to prestudy values by days 3-5 postdose. These changes were asymptomatic. CONCLUSIONS: Single 1 mg doses of FTY720 were well tolerated in healthy subjects and elicited a moderate decrease in peripheral blood lymphocyte count and a transient decrease in heart rate consistent with its pharmacological mode of action. FTY720 may be administered without regard to the timing of meals or their fat content.     

芬戈莫德干预大鼠抗Thy-1系膜增生性肾炎的研究

目的:观察芬戈莫德(FTY720)对淋巴细胞亚群的影响,并藉此在体内发挥抑制大鼠系膜增生和系膜基质扩张的作用。方法:通过一次性注射抗Thy-1抗体制备大鼠抗Thy-1系膜增生性肾炎模型,同时给予FTY720灌胃干预,观察注射后1,3,7 d大鼠外周血淋巴细胞计数和亚群分类、尿蛋白定量、肾小球系膜增生程度。结果:在注射抗体1 d后,模型组即出现蛋白尿,3 d后可见系膜细胞和基质增生,7 d增生最明显。FTY720干预组在1,3,7 d后均能显著减少外周血淋巴细胞和各淋巴细胞亚群数量;在注射后3 d和7 d,FTY720可以显著减少模型大鼠蛋白尿,减轻肾小球系膜增生程度和细胞外基质扩张。结论:FTY720可以影响外周血淋巴细胞数量和淋巴细胞亚群分布,显著减少模型大鼠蛋白尿,减轻模型大鼠系膜增生和系膜基质扩张,干预抗Thy-1系膜增生性肾炎的发生。     

Depletion of T lymphocytes ameliorates cardiac fibrosis in streptozotocin-induced diabetic cardiomyopathy

T cell infiltration has been associated with increased coronary heart disease risk in patients with diabetes mellitus. Effect of modulation of T cell trafficking on diabetes-induced cardiac fibrosis has yet to be determined. Therefore, our aim was to investigate the circulatory T cell depletion-mediated cardioprotection in streptozotocin-induced diabetic cardiomyopathy. Fingolimod (FTY720), an immunomodulatory drug, was tested in wild-type (WT) C57BL/6 and recombination activating gene 1 (Rag1) knockout (KO) mice without mature lymphocytes in streptozotocin-induced type 1 diabetic model. FTY720 (0.3 mg/kg/day) was administered intraperitoneally daily for the first 4 weeks with interim 3 weeks then resumed for another 4 weeks in 11 weeks study period. T lymphocyte counts, cardiac histology, function, and fibrosis were examined in diabetic both WT and KO mice. FTY720 reduced both CD4⁺ and CD8⁺ T cells in diabetic WT mice. FTY720-treated diabetic WT mouse myocardium showed reduction in CD3 T cell infiltration and decreased expression of S1P₁ and TGF-β1 in cardiac tissue. Fibrosis was reduced after FTY720 treatment in diabetic WT mice. Rag1 KO mice exhibited no CD4⁺ and CD8⁺ T cells in the blood and CD3 T cells in the heart. Diabetic Rag1 KO mouse hearts appeared no fibrosis and exhibited preserved myocardial contractility. FTY720-induced antifibrosis was abolished in diabetic Rag1 KO mice. These findings demonstrate that chronic administration with FTY720 induces lymphopenia and protects diabetic hearts in WT mice whereas FTY720 increases cardiac fibrosis and myocardial dysfunction in diabetic Rag1 KO mice without mature lymphocytes.     

The FTY720 story

The chemical 2-amino-2[2-(4-octylphenyl)ethyl]-1,3,propane diol is one of a class of small-molecule immunosuppressive agents. Better known as FTY720, this compound was chemically synthesized in an effort to minimize the toxic in vivo properties of a structurally related and highly potent immunosuppressive agent, myriocin. FTY720's mechanism of action, although not fully characterized, appears to be unique among immunosuppressants. Whereas the most well known biochemical characteristic of myriocin is its ability to inhibit serine palmitoyl transferase, the enzyme that initiates the biosynthetic pathway that leads to sphingosine, FTY720 is ineffective in this regard. In vivo, FTY720 induces a significant reduction in the number of circulating lymphocytes. It is thought to act by altering lymphocyte trafficking/homing patterns through modulation of cell surface adhesion receptors and ligands in a manner that has yet to be elucidated. Although much research has yet to be done to unravel the nature of the mechanism of action of FTY720, its efficacy has been sufficiently proven in numerous animal models, especially when administered in combination with cyclosporine. The agent is now progressing through human clinical trials, with the results of phase 1 clinical trials showing safety and tolerability in adult recipients of renal transplants. It is hoped that FTY720 will eventually prove to be an efficacious new weapon in the immunosuppressive armamentarium.     

Fingolimod (FTY720), sphingosine 1-phosphate receptor modulator, shows superior efficacy as compared with interferon-β in mouse experimental autoimmune encephalomyelitis

Fingolimod (FTY720), a sphingosine 1-phosphate (S1P) receptor modulator, inhibits S1P-dependent lymphocyte egress from secondary lymphoid organs and is highly effective in experimental autoimmune encephalomyelitis (EAE) in mice. In this study, we directly compared the therapeutic effects of FTY720 and recombinant mouse interferon (rm-IFN)-β on relapse and progression of EAE in mice. When FTY720 at oral dose of 0.03 to 1 mg/kg was administered daily after establishment of EAE induced by myelin proteolipid protein (PLP) in SJL/J mice, relapse of EAE was significantly inhibited during administration period. Subcutaneous injection of rm-IFN-β (10,000 IU/mouse) also inhibited the relapse of EAE at early period; however EAE was relapsed in all the mice within administration period. Therapeutic administration of FTY720 (0.03 to 1 mg/kg) significantly improved the symptoms of chronic EAE induced by myelin oligodendrocyte glycoprotein in C57BL/6 mice whereas rm-IFN-β (10,000 IU/mouse) showed no clear effect. These results indicate that FTY720 is more efficacious in mouse EAE as compared with rm-IFN-β. FTY720 markedly reduced the frequency of PLP-specific Th17 and Th1 cells in the spinal cord of EAE mice. On the contrary, FTY720 increased the frequency of PLP-specific Th17 and Th1 cells in the inguinal lymph nodes, suggesting inhibition of egress of myelin antigen-specific Th cells from draining lymph nodes. From these results, the ameliorating effects of FTY720 on EAE are likely due to reduction of infiltration of myelin antigen-specific Th17 and Th1 cells into the central nervous system.     

一种新的免疫抑制剂FTY720对小鼠心脏移植的救治作用(英文)

AIM: FTY720 is a new synthetic immunosuppressive agent which has a unique mechanism of action and induces long-term graft acceptance in rat and dog allotransplantation as prophylactic administration. The present study investigated whether FTY720 was able to rescue ongoing acute rejection of solid organ transplants in a mouse heterotopic cardiac transplantation model. METHODS: BALB/c hearts were heterotopically grafted in C57BL/6 mice. FTY720, at the doses of 0.5, 1, and 5 mg·kg~(-1)·d~(-1) or vehicle was administered to recipients once daily by oral gavage from d 3 to d 7 after transplantation. Histological changes of grafts, and the lymphocyte number in the peripheral blood and the peripheral lymph nodes were determined on d 5 after transplantation. RESULTS: FRY720 prolonged the median graft survival time dose-dependently and significantly. Histological evaluation revealed less lymphocytic infiltration in cardiac allografts treated with FTY720. Moreover, FTY720 remarkably lowered the number of periph     

A novel immunosuppressive agent FTY720 induced Akt dephosphorylation in leukemia cells

1. Our previous studies revealed that the immunosuppressive agent, FTY720, mainly induces mitochondria-involved apoptosis in some types of cancer cells, since Bcl-2 overexpression prevents the FTY720-induction of apoptotic stimuli. Furthermore, FTY720 induces G0/G1 cell cycle arrest. The present study further examines the correlation between intracellular signaling kinases with FTY720-induced mitochondria-involved apoptosis. 2. Human T cell leukemia Jurkat was exposed to FTY720. Dephosphorylation of Akt occurred in a time- and concentration-dependent manner. FTY720 also induced Bad (Ser(136)) and ribosomal p70S6 kinase (p70(S6k)) (Thr(389)) dephosphorylation. 3. FTY720-induced Akt dephosphorylation was not because of Akt upstream phosphatidylinositol 3'-kinase (PI 3-kinase) pathway inhibition. 4. FTY720 also induced Akt dephosphorylation in human B cell leukemia BALL-1. BALL-1 cells were resistant to FTY720-induced apoptosis. 5. Okadaic acid (OA) inhibited the FTY720-induced dephosphorylation of Akt and p70(S6k), suggesting that FTY720 promotes Ser/Thr protein phosphatase (PP) activity. 6. OA partially inhibited FTY720-induced caspase-3 activation. 7. PP2A or PP2A-like phosphatase was temporarily activated in cells exposed to FTY720. In addition, FTY720 activated purified PP2A (ABC). 8. Overall, the results suggest that FTY720 activated PP2A or PP2A-like phosphatase and dephosphorylated Akt pathway factors resulting in the enhancement of apoptosis via mitochondria.