FTY720: ALTERED LYMPHOCYTE TRAFFIC RESULTS IN ALLOGRAFT PROTECTION (2001)

Current standard drugs used to prevent organ graft rejection all interfere with discrete sites in the T-cell/B-cell activation cascade. They can be classified as inhibitors of cytokine transcription (CsA, FK506), inhibitors of nucleotide synthesis (azathioprine, mycophenolate mofetil, mizoribine, leflunomide), inhibitors of growth factor signal transduction (rapamycin, leflunomide), and inhibitors of differentiation (15-deoxyspergualin) (1). The beneficial effects of these therapeutics in disease relate to their broad immunosuppressive potential; however, generalized immunosuppression may reduce defense against infection and malignancies. As described below, the novel immunomodulator FTY720 protects allografts including islets with remarkable potency, apparently without inducing generalized immunosuppression (2,3). FTY720 is a structural analog of myriocin (Fig. 1), a metabolite of the ascomycete Isaria sinclairii(4–7). However, unlike myriocin, FTY720 does not bind the serine-palmitoyl transferase and does not inhibit the sphingolipid pathway (8). Although much research has yet to be done to completely unravel the mechanism of action of FTY720, recent data suggest that the drug acts as an agonist and increases the intrinsic mobility of lymphocytes, thereby accelerating chemokine-dependent lymphocyte homing to secondary lymphoid tissues (9) and reducing the recruitment of effector lymphocytes into grafted organs and peripheral lesions (10,11). The high efficacy of the drug in animal models (3) combined with the good tolerability in Phase I and II clinical trials (12–14) may relate to the compound’s novel mode of action that has not been observed with any other immunosuppressive agent. Figure 1: Structure of FTY720.MODELS OF SOLID ORGAN TRANSPLANTATION FTY720 prolonged with remarkable potency the survival of allografted rat skin (10,15,16), heart (17,18), liver (19–23), and small bowel (20,24) in strain combinations both incompatible and compatible for the major histocompatibility complex. A dose-dependent linear increase of drug efficacy was observed over a range of 0.1 to 10 mg/kg (16). In rats, a short 2-day pretransplant treatment with FTY720 at 5 mg/kg significantly prolonged survival of liver and heart allografts (19,25), suggesting that administration of “loading” doses before transplant surgery could be beneficial. In the same models, FTY720 given only on days 3 and 4 after grafting also prolonged survival (19,26), suggesting that the drug may have potential to reverse ongoing rejection. In combination with subtherapeutic amounts of CsA, the daily dose of FTY720 could be reduced to 0.1–0.5 mg/kg to protect allografted rat skin (15) and heart (17,20), dog kidney (19,27), and monkey kidney (28) (Table 1). Comparable effects have been observed for combinations of FTY720 with rapamycin (29), RAD (30,31), and FK506 (32,33), indicating that FTY720 synergizes effectively with inhibitors of T-cell activation and proliferation to prevent acute rejection. Table 1: Prolongation of allograft survival by FTY720 alone or in combination with CsAIn transplant patients, only 2%–3% of all allografts are lost during the first year to irreversible acute rejection, whereas a larger number is lost during each subsequent year to chronic rejection (34,35). The major manifestation of chronic rejection in all organs is persistent perivascular inflammation and allograft arteriosclerosis, also termed graft-vessel disease (GVD), which is only partially controlled by classic immunosuppressants. There is now evidence that low doses of FTY720 at 0.1 mg/kg added to a subtherapeutic CsA regimen effectively suppressed all aspects of GVD in a rat carotid artery transplant model, including perivascular inflammation, concentric neointima formation, medial thinning, and necrosis (36), suggesting that this regimen could be used clinically to prevent the development of GVD. MODELS OF AUTOIMMUNE DIABETES AND ISLET TRANSPLANTATION Type 1 diabetes (or insulin dependent diabetes mellitus, and II IDDM) is a common endocrine disease caused by the progressive, autoimmune destruction of the insulin-producing β-cells within the islets of the pancreas (37). At present, daily insulin injections remain the treatment of choice for the majority of diabetic patients. Although insulin therapy sustains a good quality of life, it requires constant monitoring of glucose levels; moreover, it can delay but not prevent secondary complications. Recently, with refinement of immunosuppression and transplantation techniques, single allogeneic pancreatic or simultaneous pancreaticorenal transplantation has become a possibility, in particular for patients with chronic renal failure (38). FTY720 may be an attractive therapeutic in islet transplantation, because the drug effectively protected islet allografts in streptozotocin (STZ)-induced diabetic rats (39) and mice (40), resulting in reversal of hyperglycemia and maintenance of normoglycemia throughout treatment of >100 days. Moreover, the drug suppressed with high potency the autoimmune component of diabetes in murine models; low dose treatment with FTY720 at 0.3 mg/kg per day prevented destruction of islets by autoreactive CD8+ CTL in lymphocytic choriomeningitis virus (LCMV) –infected transgenic mice expressing an LCMV peptide in the islets (2). Ongoing experiments demonstrated similar protective effects of the drug in nonobese diabetic (NOD) mice having received syngeneic or allogeneic islet allografts. In contrast to classic antiproliferative agents, FTY720 displayed no direct toxicity on the islets, but rather acted as an agonist leading to an increased insulin secretion from isolated islets in vitro (40). Taken together, FTY720 may open new therapeutic strategies through suppressing allo- and autoimmunity to transplanted islets, thereby facilitating graft survival in patients with type 1 diabetes. OTHER MODELS OF AUTOIMMUNE DISEASES FTY720 given at low doses of 0.3 mg/kg per day to rats inhibited the development of adjuvant- or collagen-induced arthritis (CIA) with efficacies equal or even better than optimal dosing of mizoribine and prednisolone (41). Efficacy of FTY720 has further been demonstrated in experimental models of autoimmune myocarditis (42), uveoretinitis (43), autoimmune encephalitis (EAE; a model of human multiple sclerosis), and systemic lupus erythematosus (SLE) (K. Chiba, personnel communication). FTY720 was especially active if applied prophylactically, however, in the SLE model FTY720 could reverse established disease and proteinuria by >80%. These data suggest significant potential of FTY720 for the treatment of autoimmune disorders. REDUCED LYMPHOCYTE RECRUITMENT TO GRAFTED ORGANS AND INFLAMMATORY SITES Therapeutic doses of FTY720 sequestered naïve and activated CD4 and CD8 T-cells and B-cells from the blood into the lymph nodes (LN) and Peyer’s patches (PP) (10,16). This process was completely reversible (2) and, in contrast to earlier suggestions (44,45), not related to apoptotic cell death, because fluorescently labeled lymphocytes transferred to mice and depleted from the circulation by FTY720 reappeared in the blood after withdrawal of the drug (2). Moreover, the concentrations of FTY720 that reportedly induced apoptosis in tumor cell lines in vitro (45) are approximately two orders of magnitude greater than the blood concentrations in rats given 1 mg/kg (16), rendering induction of lymphocyte death at clinically relevant doses of less than 0.3 mg/kg unlikely. The FTY720-induced sequestration of lymphocytes from the blood to the LN and PP in rats coincided with a strikingly reduced infiltration of T-cells into skin and heart allografts (10,11). Similar observations were made in autoimmune diabetes in mice, for which low dose treatment with FTY720 at 0.3 mg/kg per day prevented disease by suppressing the infiltration of specific cytotoxic CD8+ T-cells into the islets (2). Moreover, the prevention of SLE in MRL/lpr mice was associated with a suppression of CD3+ T-cell infiltration into the kidneys (K. Chiba, personnel communication). By using a T-cell receptor (TCR) -transgenic mouse model, we could recently show that FTY720 directly suppressed the recirculation of antigen-activated T cells to the blood (Fig. 2 A). This phenomenon may also explain the reduced recruitment of auto- and alloreactive T cells from the site of activation in the LN to peripheral lesions and transplanted organs (10,11). Figure 2: FTY720 does not impair antiviral immunity. A, FTY720 impairs recirculation of CD8 effector T cells to the periphery. C57BL6 mice received 104 spleen cells from transgenic mice expressing a T-cell receptor (TCR) specific for the lymphocytic choriomeningitis virus (LCMV) peptide GP33 on day -2. Mice were immunized on day 0 with LCMV (200 pfu i.v.), and treated daily with an oral dose of FTY720 (0.3 mg/kg) from day -1 to 15 (▪), or were left untreated (□). The number of TCR-transgenic (Vα2+ Vβ8+) T-cells recirculating to the blood was followed using fluorescence activated cell sorting. B, FTY720 does not impair specific antibody responses. Mice were treated daily with FTY720 as in A), and were immunized with LCMV on day 0. Production of specific antibody was followed for 14 days. In drug-treated and untreated mice, maximal immunoglobulin (Ig) M and IgG levels were detectable by enzyme-linked immunosorbent assay on days 8 and 12, respectively, and are given in the figure. C, FTY720 does not impair the generation of cytotoxic T lymphocytes. Mice were treated with FTY720 and immunized with LCMV as in B). On day 8, cells from spleen, inguinal lymph node (ILN), and submandibular LN (SLN) were tested for cytotoxicity against LCMV GP33 peptide-labeled EL4 target cells in primary 51Cr-release assays. (The data in Figure 1 represent control experiments to the publication in Ref. 2).NO IMPAIRMENT OF LYMPHOCYTE ACTIVATION, EXPANSION, AND MEMORY An important feature of FTY720 may be its potential to protect organ grafts without inducing general immunosuppression. In vitro, FTY720 did not inhibit rodent and human T-cell activation and proliferation (18,46), T-helper 1 (TH1) and TH2 cytokine production (10,18,46), and B-cell antibody production (46) at therapeutically relevant concentrations. Accordingly, FTY720 synergized effectively in vivo (but not in vitro) with inhibitors of early T-cell activation, i.e. CsA (see Table 1), and inhibitors of growth factor-driven cell proliferation, i.e., RAD or rapamycin (29,30,46). In vivo in mice, therapeutic doses of FTY720 did not impair primary (Fig. 2 B) and memory (2) antibody responses to LCMV and vesicular stomatitis virus (VSV), and did not affect clearance of the virus (2). It was observed that FTY720 decreased the number of virus-specific cytotoxic CD8 T lymphocytes (CTL) in the spleen, while increasing CTL in the LN (Fig. 2 C), and this phenomenon was related to migration of the cells from blood and spleen to LN (2). In summary, therapeutic dosing of FTY720 seems to modulate lymphocyte circulation without affecting immune cell function, which could be an advantage compared with classic immunosuppressives. In line with these observations, transplant protection by FTY720 in rats did not correlate to a suppression of alloreactivity in the LN (47) but correlated with an inhibition of the recirculation of specific T cells to the periphery and to grafted tissue (2,10,11). MODULATION OF CHEMOKINE-DRIVEN LYMPHOCYTE MIGRATION Lymphocyte sequestration by FTY720 was effectively inhibited by a combination of antibodies to the classic adhesion molecules L-selectin, lymphocyte-function-antigen-1 (LFA-1), and very-late-antigen-4 (VLA-4) (16); however, there is no convincing evidence for direct effects of FTY720 on the adhesion process. It is now clear that the molecular regulation of lymphocyte trafficking and migration is more complex, involving the cellular interactions of not only adhesion molecules such as selectins and integrins, but also of an entire superfamily of chemoattractant cytokines (chemokines) and their G protein-coupled receptors (GPCR) (48). Moreover, cytoskeleton rearrangements required for cell migration involve another GPCR family that interacts with glycero- and sphingolysolipid phosphate ligands (49) sharing some structural homologies with FTY720. The data available to date indicate that FTY720 affects GPCR-dependent, chemokine-driven cell trafficking (9). First, the sequestration of lymphocytes to LN and PP by FTY720 could be antagonized by Bordetella pertussis toxin (PT) (3), an agent that specifically blocks GPCR signaling by preventing receptor coupling to G proteins of the Gi family (48,49). Second, lymphocytes pretreated with nanomolar (nM) concentrations of FTY720 in vitro showed an increased intrinsic mobility and migrated more strongly in response to secondary-lymphoid-chemokine (SLC/6Ckine/Exodus2) (Fig. 3 A), and the EBI1-ligand-chemokine (ELC/CKbeta-11/MIP-3beta) (9). Earlier reports have demonstrated that SLC and ELC are constitutively expressed in the LN and PP, bind to CC-chemokine receptor-7 (CCR7) expressed on naïve and activated T-cells and regulate T-cell recirculation to secondary lymphoid tissues (48). Third, FTY720 was unable to readily deplete blood T-cells in SLC/ELC-deficient plt/plt mutant mice (plt; paucity of lymph node T-cells) but, nevertheless, depleted B-cells in these animals (B-cells use other homing chemokines) (9). This is shown in Figure 3 b, where FTY720 strongly increased the ratio of blood T:B-cells in plt/plt mutant mice, but did not alter the T:B-cell ratio in +/+ wild-type mice. Fourth, FTY720-untreated, CCR7-deficient mice showed impaired homing of T-cells but not B-cells and accumulated T-cells in the blood (50), a phenotype opposite to that of FTY720-treated wild-type mice (16). All together, these data suggest that FTY720 functions by modulating the lymphocyte chemotactic response to chemokines, inducing a homing chemokine-dependent sequestration of lymphocytes to secondary lymphatic organs and, thus, preventing lymphocytes from reacting and migrating to inflammatory chemokines at graft sites. Figure 3: FTY720 affects the responsiveness of lymphocytes to chemokines. A, Increased migration of lymphocytes in vitro. Spleen cells from C57Bl/6 mice were incubated in vitro with FTY720 (0.5 μM) for 6 hr (•) or were left untreated (○). A total of 25 μl of increasing concentrations of secondary lymphoid chemokine (SLC) was placed in the lower wells of a 48-well chemotaxis chamber and separated from 50 μl of cell suspension in the top wells by 5-μm pore-size polyvinylpyrrolidone-free polycarbonate filter. After incubation at 37°C for 2 hr, sedimented cells on the top surface of the filter were wiped off and migrated cells on the undersurface were fixed in methanol and stained using Diff-Quik. Results are expressed as mean cell number per five high-power fields. B, Lack of effects of FTY720 in plt mice. BALB/c-plt/plt mice or wild-type controls received an oral dose of FTY720 (0.3 mg/kg), and blood samples were collected after 6 hr. The relative numbers of CD3+ T-cells versus B220+ B-cells in PBL was analyzed by fluorescence activated cell sorting.POSSIBLE MOLECULAR TARGET(S) OF FTY720 In vitro, FTY720 induced most profound lymphocyte migration in response to the CCR7 ligands SLC and ELC, but to a lesser extent also to chemokines binding to other receptors (9), rendering a specific interaction of the drug with CCR7 unlikely. Consistent with this view, we were unable to demonstrate effects of FTY720 on the expression of homing chemokines and chemokine receptors. However, we found that the drug mobilizes Ca2+ from intracellular stores, activates phosphatidylinositol-3 kinase (PI-3K), and increases intrinsic locomotion of lymphocytes at the absence of chemokines (9), suggesting an activation of the cell mobility machinery such as actin stressfiber formation (49). Ongoing experiments indeed show that FTY720 stimulates actin polymerization in HEK293 cells transfected with the endothelial-cell-differentiation-gene-6 (EDG-6). EDG-6 is a GPCR specifically expressed on leukocytes (51) and belongs to a novel family of membrane receptors that recognize the bioactive lipids sphingosine-1-phosphate (S1P) and lysophosphotidic acid (LPA) as extracellular ligands (49,51). As described for sphingosine (49), the activity of the structurally similar lipid drug FTY720 is stereospecific and not related to a nonspecific association of the amphiphilic molecule with cell membranes, because only the R-enantiomer of two chiral analogues of FTY720 is biologically active, despite that both analogues share the same physicochemical properties (6). Taken together, these data suggest that FTY720 may activate the cell mobility machinery by means of EDG-6 and, thereby, accelerate the response to chemokines. The resulting sequestration of lymphocytes to LN may not relate to a specific effect of the drug on homing chemokine receptors, but rather relate to the high constitutive expression of the homing chemokines SLC and ELC in lymphoid tissues (48). SAFETY PHARMACOLOGY AND CLINICAL TRIALS FTY720 was not mutagenic, clastogenic, or aneugenic in any of the in vitro or in vivo studies performed. Oral bioavailability of FTY720 was high (37%–71%) in rats, dogs, and monkeys. There was no evidence of infection by observation or histology in cynomolgus monkeys treated with up to 10 mg/kg per day for 52 weeks. The dose-limiting toxicity of FTY720 may be effects on lungs, including smooth muscle hypertrophy/hyperplasia and bronchoconstriction observed in some animal species at high doses of drug. However, lung effects have not been observed in clinical trials at doses that are well in the therapeutic range. Administration of FTY720 in combination with CsA did not result in increased toxicity in rats when compared with treatment using the individual compounds as single agents. FTY720 was extensively metabolized in the liver by the mixed function oxidase system before excretion. Studies performed with human liver microsomes suggest that FTY720 is metabolized by enzymes not involved in the metabolism of CsA, rapamycins, and FK506, rendering drug-drug interaction with FTY720 unlikely. Human safety and tolerability trials in stable renal transplant patients explored single oral doses of FTY720 at 0.25 to 3.5 mg, and multiple doses at 0.125 to 5.0 mg given once daily for 28 days. Patients were maintained on Neoral (CsA for microemulsion) plus steroids There have been no and no significant of As in animal models, administration of FTY720 in a of CD4 and CD8 T cells and cells from the blood Although the clearance of FTY720 was the of was resulting in a for a dose in the range of mg, hr, hr, where is the to blood is the is the of the and is the the Similar blood levels were observed in animal species given single oral doses of FTY720 at mg/kg cynomolgus A recently Phase dose tolerability and efficacy of FTY720 mycophenolate with and in renal that FTY720 is in the prevention of acute rejection when used with Neoral and steroids trials are to the of FTY720 in the prevention of acute rejection and graft after renal The clinical are in with the in dogs, and and suggest that the drug be at concentrations that with Current data suggest that the novel immunomodulator FTY720 acts by means of that are not observed with any immunosuppressive FTY720 the lymphocyte chemotactic response to chemokines, inducing a homing chemokine-dependent sequestration of lymphocytes to secondary lymphatic and, thus, preventing lymphocytes from reacting and migrating to inflammatory chemokines at graft this without of immune responses to systemic The high efficacy and good tolerability of combination involving FTY720 and subtherapeutic doses of CsA or RAD in models of transplantation may relate to the completely of action of the FTY720 may the of new therapeutic strategies to prevent acute as well as chronic rejection and, moreover, may have potential for the treatment of autoimmune including type 1 diabetes. for of for the and Chiba, for sharing data from SLE and autoimmune Moreover, we at and for on clinical trials and and for of the