FTY720-phosphate is dephosphorylated by lipid phosphate phosphatase 3

FTY720 is a novel immunomodulatory drug efficacious in the treatment of multiple sclerosis. The drug is converted in vivo to the monophosphate, FTY720-P, by sphingosine kinase 2. This conversion is incomplete, suggesting opposing actions of kinase and phosphatase activities. To address which of the known lipid phosphatases might dephosphorylate FTY720-P, we overexpressed the broad specificity lipid phosphatases LPP1-3, and the specific S1P phosphatases (SPP1 and 2) in HEK293 cells, and performed in vitro assays using lysates of transfected cells. Among LPPs, only LPP3 was able to dephosphorylate FTY720-P; among SPPs, only SPP1 showed activity against FTY720-P. On intact cells, LPP3 acted as an ecto-phosphatase or FTY720-P, thus representing the major phosphatase involved in the equilibrium between FTY720 and FTY720-P observed in vivo.     

A novel immunosuppressant FTY720 ameliorates proteinuria and alterations of intrarenal adrenomedullin in rats with autoimmune glomerulonephritis

FTY720 has been originally developed as a new immunosuppressive agent, which prolongs graft survival in organ transplantation. Adrenomedullin (AM) participates in the regulation of sodium homeostasis and has renoprotective effects. The possible involvement of renal AM in the pathophysiology of glomerulonephritis (GN) and the effect of FTY720 has been evaluated in rats. HgCl2 (1 mg/kg body weight) was inoculated subcutaneously 3 times/week for a total of 2 weeks. FTY720 (3 or 10 mg/kg) was inoculated subcutaneously daily. The proteinuria, urinary N-acetyl-beta-D-glucosaminidase (NAG) excretion and serum total cholesterol levels were increased and serum albumin level was reduced in rats with HgCl2-induced GN compared with controls. FTY720 reduced proteinuria (3 mg/kg: -25%; 10 mg/kg: -41%), urinary NAG excretion (-11%; -52%) and total cholesterol level (-21%; -55%) in a dose-dependent manner. Renal AM level and its mRNA expression were increased in rats with GN compared with controls (Peptide Cortex: +69%; Medulla: +82%; mRNA Cortex: +25%). Interestingly, FTY720 additionally increased these levels (Peptide Cortex: +38%; Medulla: +39%; mRNA Cortex: +20%). Renal AM levels correlated with urinary NAG excretion and creatinine clearance. These results suggest that FTY720 suppresses the renal damage in rats with GN and renal AM may participate in the pathophysiology of GN and the renoprotective effects of FTY720.     

Protective Effect of FTY720 Against Sevoflurane-Induced Developmental Neurotoxicity in Rats

Sevoflurane, a common used inhaled anaesthetic, induces neuronal apoptosis in preclinical studies and correlates with functional neurological impairment. We investigated whether FTY720, a known sphingosine-1 phosphate (S1P) receptor agonist, could exert neuroprotective effect against sevoflurane-induced neurotoxicity. Neuroprotective effect of FTY720 was evaluated in vitro in hippocampal neuronal cells from neonatal rats and in vivo in rat pups. In vitro cell apoptosis was determined by flow cytometry after exposure to 3 % sevoflurane for different period of time, or after 6-h exposure to sevoflurane with the presence of FTY720, SEW2871 (selective S1P1 receptor agonist) or combination of FTY720 and VPC23019 (S1P antagonist). Western blot analysis was performed with hippocampal tissue from rat pups exposed to 3 % sevoflurane for 6 h with or without pre-treatment with FTY720 injection. Neurological function tests were also performed with rat pups exposed to 3 % sevoflurane for 6 h with or without pre-treatment with FTY720 injection. FTY720, at nanomolar concentration, significantly prevents sevoflurane-induced neuronal apoptosis. SEW2871 showed similar neuroprotective effect to FTY720, whereas VPC23019 abrogated the neuroprotective effect of FTY720 when given together. Western blots results demonstrated that FTY710 significantly preserved the level of phosphorylated ERK1/2, Bcl-2 and Bax. Although anaesthetic treatment did not affect general health and emotional status, sevoflurane-induced cognitive impairment in rat models. Administration of FTY720 at 1 mg/kg significantly attenuated sevoflurane-induced neurocognitive impairment. Although further studies are needed to evaluate the feasibility of clinical usage of FTY720 as neuroprotective agent, the study provides preclinical experimental evidence for the efficacy of FTY720 against sevoflurane-induced developmental neurotoxicity.     

Antibody against a novel, myriocin (ISP-I)-based immunosuppressant, FTY720

An antibody was prepared by immunizing rabbits with an ovalbumin conjugate of 2-amino-2-(2-(4-(4-mercaptobutyl)phenyl)ethyl)propane-1,3-diol HCl (AMPD-4), which contains the essential structure of the novel immunosuppressant FTY720. As the antibody reacted to not only AMPD-4, but also FTY720, it should be useful for immunoassay of FTY720 in body fluids, tissues and cells.     

Local delivery of FTY720 accelerates cranial allograft incorporation and bone formation

Endogenous stem cell recruitment to the site of skeletal injury is key to enhanced osseous remodeling and neovascularization. To this end, this study utilized a novel bone allograft coating of poly(lactic-co-glycolic acid) (PLAGA) to sustain the release of FTY720, a selective agonist for sphingosine 1-phosphate (S1P) receptors, from calvarial allografts. Uncoated allografts, vehicle-coated, low dose FTY720 in PLAGA (1:200 w:w) and high dose FTY720 in PLAGA (1:40) were implanted into critical size calvarial bone defects. The ability of local FTY720 delivery to promote angiogenesis, maximize osteoinductivity and improve allograft incorporation by recruitment of bone progenitor cells from surrounding soft tissues and microcirculation was evaluated. FTY720 bioactivity after encapsulation and release was confirmed with sphingosine kinase 2 assays. HPLC-MS quantified about 50% loaded FTY720 release of the total encapsulated drug (4.5 μg) after 5 days. Following 2 weeks of defect healing, FTY720 delivery led to statistically significant increases in bone volumes compared to controls, with total bone volume increases for uncoated, coated, low FTY720 and high FTY720 of 5.98, 3.38, 7.2 and 8.9 mm³, respectively. The rate and extent of enhanced bone growth persisted through week 4 but, by week 8, increases in bone formation in FTY720 groups were no longer statistically significant. However, micro-computed tomography (microCT) of contrast enhanced vascular ingrowth (MICROFIL?) and histological analysis showed enhanced integration as well as directed bone growth in both high and low dose FTY720 groups compared to controls.     

FTY720 immunosuppression impairs effector T cell peripheral homing without affecting induction, expansion, and memory

FTY720 (2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol hydrochloride) prolongs survival of solid organ allografts in animal models. Mechanisms of FTY720 immunomodulation were studied in mice infected with lymphocytic choriomeningitis virus (LCMV) to assess T cell responses or with vesicular stomatitis virus to evaluate Ab responses. Oral FTY720 (0.3 mg/kg/day) did not affect LCMV replication and specific CTL and B cells were induced and expanded normally. Moreover, the anti-viral humoral immune responses were normal. However, FTY720 treatment showed first a shift of overall distribution of CTL from the spleen to peripheral lymph nodes and lymphocytopenia was observed. This effect was reversible within 7-21 days. Together with unimpaired T and B cell memory after FTY720 treatment, this finding rendered enhancement of lymphocyte apoptosis by FTY720 in vivo unlikely. Secondly, the delayed-type hypersensitivity reaction to a viral MHC class I-presented peptide was markedly reduced by FTY720. These results were supported by impaired circulation of LCMV specific TCR transgenic effector lymphocytes in the peripheral blood and reduced numbers of tissue infiltrating CTL in response to delayed-type hypersensitivity reaction. Thirdly, in a CD8+ T cell-mediated diabetes model in a transgenic mouse expressing the LCMV glycoprotein in the islets of the pancreas, FTY720 delayed or prevented disease by reducing islet-infiltrating CTL. Thus, FTY720 effectively reduced recirculation of CD8+ effector T cells and their recruitment to peripheral lesions without affecting the induction and expansion of immune responses in secondary lymphoid organs. These properties may offer the potential to treat ongoing organ-specific T cell-mediated immunopathologic disease.     

The immunosuppressant FTY720 inhibits tumor angiogenesis via the sphingosine 1-phosphate receptor 1

FTY720, a sphingosine 1-phosphate (S1P) analog, acts as an immunosuppressant through trapping of T cells in secondary lymphoid tissues. FTY720 was also shown to prevent tumor growth and to inhibit vascular permeability. The MTT proliferation assay illustrated that endothelial cells are more susceptible to the anti-proliferative effect of FTY720 than Lewis lung carcinoma (LLC1) cells. In a spheroid angiogenesis model, FTY720 potently inhibited the sprouting activity of VEGF-A-stimulated endothelial cells even at concentrations that apparently had no anti-proliferative effect. Mechanistically, the anti-angiogenic effect of the general S1P receptor agonist FTY720 was mimicked by the specific S1P1 receptor agonist SEW2871. Moreover, the anti-angiogenic effect of FTY720 was abrogated in the presence of CXCR4-neutralizing antibodies. This indicates that the effect was at least in part mediated by the S1P1 receptor and involved transactivation of the CXCR4 chemokine receptor. Additionally, we could illustrate in a coculture spheroid model, employing endothelial and smooth muscle cells (SMCs), that the latter confer a strong protective effect regarding the action of FTY720 upon the endothelial cells. In a subcutaneous LLC1 tumor model, the anti-angiogenic capacity translated into a reduced tumor size in syngeneic C57BL/6 mice. Consistently, in the Matrigel plug in vivo assay, 10 mg/kg/d FTY720 resulted in a strong inhibition of angiogenesis as demonstrated by a reduced capillary density. Thus, in organ transplant patients, FTY720 may prove efficacious in preventing graft rejection as well as tumor development.     

FTY720, a novel immunosuppressive agent, induces apoptosis in human glioma cells

FTY720, a metabolite from Isaria sinclairii, has been developed to be a potent immunosuppressive drug with induction of apoptosis in T cells and several cell lines. We investigated whether FTY720 induces apoptosis in human glioma cell lines, since they are relatively resistant to multiple apoptotic stimuli. In human glioma cells including T98G, FTY720 induced apoptosiswith ED50 between 1 to 10 microg/ml, while etoposidedid not induce apoptosis at the same doses. Among the caspase family proteases, mainly caspase-6 was activated during the apoptosis by FTY720 but not etoposide. In addition, FTY720 caused tyrosine dephosphorylation of FAK and did not activate a FAK-PI3-kinase survival pathway. This was confirmed also by the observation that orthovanadate prevented FTY720-induced dephosphorylation of FAK and inhibited FTY720-induced cell death. We assumed that FTY720 induced FAK dephosphorylation and cut off the FAK-PI3-kinase pathway resulting in the induction of apoptosis via caspase-6 activation in these glioma cells.     

Phosphorylation of the immunomodulatory drug FTY720 by sphingosine kinases

The immunomodulatory drug FTY720 is phosphorylated in vivo, and the resulting FTY720 phosphate as a ligand for sphingosine-1-phosphate receptors is responsible for the unique biological effects of the compound. So far, phosphorylation of FTY720 by murine sphingosine kinase (SPHK) 1a had been documented. We found that, while FTY720 is also phosphorylated by human SPHK1, the human type 2 isoform phosphorylates the drug 30-fold more efficiently, because of a lower Km of FTY720 for SPHK2. Similarly, murine SPHK2 was more efficient than SPHK1a. Among splice variants of the human SPHKs, an N-terminally extended SPHK2 isoform was even more active than SPHK2 itself. Further SPHK superfamily members, namely ceramide kinase and a "SPHK-like" protein, failed to phosphorylate sphingosine and FTY720. Thus, only SPHK1 and 2 appear to be capable of phosphorylating FTY720. Using selective assay conditions, SPHK1 and 2 activities in murine tissues were measured. While activity of SPHK2 toward sphingosine was generally lower than of SPHK1, FTY720 phosphorylation was higher under conditions favoring SPHK2. In human endothelial cells, while activity of SPHK1 toward sphingosine was 2-fold higher than of SPHK2, FTY720 phosphorylation was 7-fold faster under SPHK2 assay conditions. Finally, FTY720 was poorly phosphorylated in human blood as compared with rodent blood, in line with the low activity of SPHK1 and in particular of SPHK2 in human blood. To conclude, both SPHK1 and 2 are capable of phosphorylating FTY720, but SPHK2 is quantitatively more important than SPHK1.     

FTY720 produces caspase-independent cell death of acute lymphoblastic leukemia cells

Acute lymphoblastic leukemia (ALL), the most common form of childhood cancer, usually responds to chemotherapy but patients who develop disease relapse have a poor prognosis. New agents to treat ALL are urgently required. FTY720 is an immunosuppressive drug that has promising in vitro activity in a number of malignancies, with the proposed mechanism being the reactivation of the protein serine/threonine phosphatase, PP2A. FTY720 reduced the proliferation and viability of Ph(+) and Ph(-) ALL cell lines and patient samples with IC 50 values for viability between 5.3 to 7.9 μM. Cell death was caspase-independent with negligible activation of caspase-3 and no inhibition of FTY720-induced cell death by the caspase inhibitor Z-VAD-FMK. The cytotoxic effects of FTY720 were independent of PP2A reactivation as determined by the lack of effect of the PP2A inhibitor okadaic acid. Features of autophagy, including autophagosomes, LC3II expression and increased autophagic flux, were induced by FTY720. However the phosphorylated form of FTY720 (FTY720-P) similarly induced autophagy but not cell death. This suggests that autophagy is prosurvival in this setting, a finding supported by protection from cell death induced by the cytotoxic agent vincristine. FTY720 also induced reactive oxygen species (ROS) and the antioxidant N-acetyl-cysteine (NAC) partially reversed the cytotoxic effects, demonstrating a role for ROS generation in the cell death mechanism. FTY720 is an active drug in vitro in ALL cell lines and patient samples. Evidence supports a caspase-independent mechanism of cell death with the occurrence of autophagy and necrosis.     

FTY720-enhanced T cell homing is dependent on CCR2, CCR5, CCR7, and CXCR4: evidence for distinct chemokine compartments

FTY720 stimulates CCR7-driven T cell homing to peripheral lymph nodes (LN) by direct activation of sphingosine 1-phosphate receptors, along with the participation of multidrug transporters, 5-lipoxygenase, and G protein-coupled receptors for chemokines. In this study, we demonstrate that FTY720 also directly stimulates in vitro T cell chemotaxis to CCR2-CCL2, but not to a variety of other chemokines, including CCR5-CCL3/4/5 and CXCR4-CXCL12. FTY720 influences CCR2-CCL2-driven migration through activation of the multidrug transporters, Abcb1 and Abcc1, and through 5-lipoxygenase activity. In vivo administration of FTY720 induces chemokine-dependent migration of T cells in the thymus, peripheral blood, LN, and spleen. The CCR7 and CCR2 chemokine ligands are required for both T cell sequestration in LN and thymic T cell egress following FTY720 administration. Furthermore, FTY720 administration uncovers a requirement for CXCR4 ligands for LN homing, but not for thymic egress, and CCR5 for thymic egress, but not LN homing. FTY720-driven splenic and peripheral blood T cell egress are both independent of CCR2, CCR5, CCR7, or CXCR4. These results indicate that FTY720- and sphingosine 1-phosphate receptor-stimulated T cell migration are dependent on the restricted usage of chemokine receptor-ligand pairs within discrete anatomic compartments.     

FTY720 induces cell cycle arrest and apoptosis of rat glomerular mesangial cells

The present study aimed to examine the effect of FTY720, a new immunosuppressive agent, on the proliferation and apoptosis of glomerular mesangial cells (GMC), and investigate the underlying mechanisms. Cultured rat GMC were treated by FTY720, and the cell viability, apoptosis and cell cycle progression were examined. Furthermore, cell cycle related gene expression profile was analyzed by cDNA microarray, and the protein expression of cell cycle related genes as well as Bax and Bcl-2 were examined by Western blot. The results showed that FTY720 inhibited GMC proliferation and induced apoptosis of GMC in a dose- and time-dependent manner, and induced G(1) phase cell cycle arrest in GMC in a dose-dependent manner as well. cDNA microarray analysis revealed that FTY720 regulated the expression of cell cycle-related gene. Western blot analysis showed that FTY720 induced the downregulation of cyclin D1, cyclin E, CDK2, CDK4, Bcl-2 and E2F1 and the upregulation of Kip1/p27, Cip1/p21, Bax and Rb in GMC in a dose-dependent manner. These results demonstrated that FTY720 could inhibit the proliferation of GMC through inducing cell cycle arrest and apoptosis, probably via the regulation of the expression of cell cycle-related genes and Bax/Bcl-2.     

Asymmetric synthesis and biological evaluation of the enantiomeric isomers of the immunosuppressive FTY720-phosphate

A practical asymmetric synthesis of both enantiomers of the immunosuppressive FTY720-phosphate (2) was accomplished, and the enantiomers were pharmacologically evaluated. Several lipases showed considerable activity and enantioselectivity for O-acylation of N-acetyl FTY720 (3) or N-benzyloxycarbonyl FTY720 (7) in combination with vinyl acetate or benzyl vinyl carbonate as the acyl donors. The synthesis using the lipase-catalyzed acylation as the key step produced the enantiomerically pure (>99.5% ee) enantiomers of 2 in multigram quantities. (S)-Isomer of 2 had more potent binding affinities to S1P(1,3,4,5) and inhibitory activity on lymphocyte migration toward S1P than (R)-2, suggesting that (S)-isomer of 2 is responsible for the immunosuppressive activity after administration of 1. Severe bradycardia was observed in anesthetized rats when (S)-2 was administered intravenously, while (R)-2 had no clear effect on heart rate up to 0.3 mg/kg.     

FTY720 produces caspase-independent cell death of acute lymphoblastic leukemia cells

Acute lymphoblastic leukemia (ALL), the most common form of childhood cancer, usually responds to chemotherapy but patients who develop disease relapse have a poor prognosis. New agents to treat ALL are urgently required. FTY720 is an immunosuppressive drug that has promising in vitro activity in a number of malignancies, with the proposed mechanism being the reactivation of the protein serine/threonine phosphatase, PP2A. FTY720 reduced the proliferation and viability of Ph⁺ and Ph^- ALL cell lines and patient samples with IC₅₀ values for viability between 5.3 to 7.9 μM. Cell death was caspase-independent with negligible activation of caspase-3 and no inhibition of FTY720-induced cell death by the caspase inhibitor Z-VAD-FMK. The cytotoxic effects of FTY720 were independent of PP2A reactivation as determined by the lack of effect of the PP2A inhibitor okadaic acid. Features of autophagy, including autophagosomes, LC3II expression and increased autophagic flux, were induced by FTY720. However the phosphorylated form of FTY720 (FTY720-P) similarly induced autophagy but not cell death. This suggests that autophagy is prosurvival in this setting, a finding supported by protection from cell death induced by the cytotoxic agent vincristine. FTY720 also induced reactive oxygen species (ROS) and the antioxidant N-acetyl-cysteine (NAC) partially reversed the cytotoxic effects, demonstrating a role for ROS generation in the cell death mechanism. FTY720 is an active drug in vitro in ALL cell lines and patient samples. Evidence supports a caspase-independent mechanism of cell death with the occurrence of autophagy and necrosis.     

Anti-inflammatory effects of FTY720 against viral-induced immunopathology: role of drug-induced conversion of T cells to become Foxp3+ regulators

FTY720 has been used to control inflammatory lesions, but the mechanisms by which the drug acts in vivo are poorly understood. Such mechanisms may result primarily from effects on lymphocyte and dendritic cell homing to lymphoid and inflammatory sites. We demonstrate that FTY720 may also act by causing the conversion of TCR-stimulated nonregulatory CD4(+) T cells to Foxp3(+)CD4(+) regulatory T cells and by enhancing their suppressive activity. In a model in which mice were ocularly infected with HSV, daily treatment with FTY720 resulted in significantly diminished ocular lesions. The treated animals showed increased frequencies of Foxp3(+) T cells in lymphoid organs and at two inflammatory sites, namely cornea and trigeminal ganglia. In a second series of experiments, immunized DO11.10RAG2(-/-) animals, normally lacking endogenous Foxp3(+) T cells, that were given FTY720 treatment developed high frequencies of Foxp3(+) regulatory T cells in lymph nodes. Some converted cells persisted in treated animals for several weeks after drug administration was discontinued. Finally, FTY720 could effectively induce Foxp3-expressing cells from Foxp3(-) cells in vitro, an effect inhibited by anti-TGF-beta or the proinflammatory cytokine IL-6. Accordingly, the anti-inflammatory effects of FTY720 could be mediated at least in part by its ability to cause the conversion of Ag-stimulated conventional T cells to become Foxp3(+) regulators. The use of FTY720 along with Ag administration could represent a useful therapeutic means to selectively expand Ag-specific regulators, which could be valuable in many clinical situations such as allotransplants, some autoimmunities, as well as with some chronic infections.     

Antinociceptive activity of the S1P-receptor agonist FTY720

FTY720 is a novel immunosuppressive drug that inhibits the egress of lymphocytes from secondary lymphoid tissues and thymus. In its phosphorylated form FTY720 is a potent S1P receptor agonist. Recently it was also shown that FTY720 can reduce prostaglandin synthesis through the direct inhibition of the cytosolic phospholipase A2 (cPLA2). Since prostaglandins are important mediators of nociception, we studied the effects of FTY720 in different models of nociception. We found that intraperitoneal administration of FTY720 reduced dose-dependently the nociceptive behaviour of rats in the formalin assay. Although the antinociceptive doses of FTY720 were too low to alter the lymphocyte count, prostanoid concentrations in the plasma were dramatically reduced. Surprisingly, intrathecally administered FTY720 reduced the nociceptive behaviour in the formalin assay without altering spinal prostaglandin synthesis, indicating that additional antinociceptive mechanisms beside the inhibition of prostaglandin synthesis are involved. Accordingly, FTY720 reduced also the nociceptive behaviour in the spared nerve injury model for neuropathic pain which does not depend on prostaglandin synthesis. In this model the antinociceptive effect of FTY720 was similar to gabapentin, a commonly used drug to treat neuropathic pain. Taken together we show for the first time that FTY720 possesses antinociceptive properties and that FTY720 reduces nociceptive behaviour during neuropathic pain.     

FTY720 ameliorates Th1-mediated colitis in mice by directly affecting the functional activity of CD4+CD25+ regulatory T cells

Following the present concepts, the synthetic sphingosine analog of myriocin FTY720 alters migration and homing of lymphocytes via sphingosine 1-phosphate receptors. However, several studies indicate that the immunosuppressive properties of FTY720 may alternatively be due to tolerogenic activities via modulation of dendritic cell differentiation or based on direct effects on CD4(+)CD25(+) regulatory T cells (Treg). As Treg play an important role for the cure of inflammatory colitis, we used the Th1-mediated 2,4,6-trinitrobenzene sulfonic acid (TNBS) colitis model to address the therapeutic potential of FTY720 in vivo. A rectal enema of TNBS was given to BALB/c mice. FTY720 was administered i.p. from days 0 to 3 or 3 to 5. FTY720 substantially reduced all clinical, histopathologic, macroscopic, and microscopic parameters of colitis analyzed. The therapeutic effects of FTY720 were associated with a down-regulation of IL-12p70 and subsequent Th1 cytokines. Importantly, FTY720 treatment resulted in a prominent up-regulation of FoxP3, IL-10, TGFbeta, and CTLA4. Supporting the hypothesis that FTY720 directly affects functional activity of CD4(+)CD25(+) Treg, we measured a significant increase of CD25 and FoxP3 expression in isolated lamina propria CD4(+) T cells of FTY720-treated mice. The impact of FTY720 on Treg induction was further confirmed by concomitant in vivo blockade of CTLA4 or IL-10R which significantly abrogated its therapeutic activity. In conclusion, our data provide clear evidence that in addition to its well-established effects on migration FTY720 leads to a specific down-regulation of proinflammatory signals while simultaneously inducing functional activity of CD4(+)CD25(+) Treg. Thus, FTY720 may offer a promising new therapeutic strategy for the treatment of IBD.