Immunosuppressant drugs such as cyclosporin have allowed widespread organ transplantation, but their utility remains limited by toxicities, and they are ineffective in chronic management of autoimmune diseases such as multiple sclerosis. In contrast, the immune modulating drug FTY720 is efficacious in a variety of transplant and autoimmune models without inducing a generalized immunosuppressed state and is effective in human kidney transplantation. FTY720 elicits a lymphopenia resulting from a reversible redistribution of lymphocytes from circulation to secondary lymphoid tissues by unknown mechanisms. Using FTY720 and several analogs, we show now that FTY720 is phosphorylated by sphingosine kinase; the phosphorylated compound is a potent agonist at four sphingosine 1-phosphate receptors and represents the therapeutic principle in a rodent model of multiple sclerosis. Our results suggest that FTY720, after phosphorylation, acts through sphingosine 1-phosphate signaling pathways to modulate chemotactic responses and lymphocyte trafficking.FTY720 is derived from ISP-1 (myriocin), a fungal metabolite that is an eternal youth nostrum in traditional Chinese herbal medicine (1). The compound (2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol) is a novel, high potency immune modulating agent that is remarkably effective in a variety of autoimmune and transplant models including islet transplantation (2) and has recently proven to be effective in renal transplantation in man (3). Unlike the currently used immunosuppressive agents (e.g. the calcineurin inhibitors cyclosporin and tacrolimus), FTY720 does not inhibit T cell activation and proliferation and in rodent models does not impair immunity to systemic viral infection (4). If confirmed in man, the latter property provides a striking advantage over current immunosuppressive therapies. FTY720 apparently sequesters lymphocytes from circulation to secondary lymph tissue compartments (5) with concomitant reduction of specific effector T cells recirculating from the lymph nodes to inflamed peripheral tissues (4) and graft sites (6). FTY720 does not act via the lymphocytehoming chemokine receptor CCR-7 because FTY720 is active both in CCR-7-deficient mice and plt (paucity of lymph node T cells) mice, which lack CCR-7 ligands (CCL-19 and CCL-21) (7).FTY720-induced lymphocyte homing is sensitive to suppression by pertussis toxin (6 -8), which suggests that the molecular target of the drug is a G protein-coupled receptor (GPCR) 1 interacting with heterotrimeric G proteins of the ␣ i/o type. The affected GPCR(s) is on the lymphocyte since fluorescently labeled lymphocytes treated with pertussis toxin ex vivo and transferred to mice are not depleted by FTY720 in vivo (8). The structural similarity of FTY720 and sphingosine has prompted speculation that the drug might act via the sphingosine 1-phosphate (S1P) receptor S1P 4 (formerly 2 that is known to be expressed by lymphocytes (9). S1P is a pleiotropic lysophospholipid mediator; the prominent cellular responses to applied S...

FTY720[2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol hydrochloride] is an oral sphingosine-1-phosphate receptor modulator under development for the treatment of multiple sclerosis (MS). The drug is phosphorylated in vivo by sphingosine kinase 2 to its bioactive form, FTY720-P. Although treatment with FTY720 is accompanied by a reduction of the peripheral lymphocyte count, its efficacy in MS and experimental autoimmune encephalomyelitis (EAE) may be due to additional, direct effects in the central nervous system (CNS). We now show that FTY720 localizes to the CNS white matter, preferentially along myelin sheaths. Brain trough levels of FTY720 and FTY720-P in rat EAE are of the same magnitude and dose dependently increase; they are in the range of 40 to 540 ng/g in the brain tissue at efficacious doses and exceed blood concentrations severalfold. In a rat model of chronic EAE, prolonged treatment with 0.03 mg/kg was efficacious, but limiting the dosing period failed to prevent EAE despite a significant decrease in blood lymphocytes. FTY720 effectiveness is likely due to a culmination of mechanisms involving reduction of autoreactive T cells, neuroprotective influence of FTY720-P in the CNS, and inhibition of inflammatory mediators in the brain.FTY720 is an oral sphingosine-1-phosphate (S1P) receptor modulator (Baumruker et al., 2007) under development for the treatment of multiple sclerosis (MS), representing the first of a new class of immunomodulatory agents. Promising results in phase II trials with relapsing MS patients mirror the striking efficacy of FTY720 in MS models of experimental autoimmune encephalomyelitis (EAE), shown by preventive and therapeutic treatment (Brinkmann et al., 2002;Fujino et al., 2003;Webb et al., 2004;Kataoka et al., 2005;Balatoni et al., 2007). FTY720 is converted in vivo to its biologically active phosphate ester metabolite (FTY720-P), which acts as a high-affinity agonist for four of the five known G-protein-coupled S1P receptors, namely S1P 1 and S1P 3-5 (Brinkmann et al., 2002;Mandala et al., 2002). Sphingosine kinase (SPHK) 2 is the primary enzyme required for FTY720-P formation, as we and others subsequently confirmed in SPHK2 knockout mice (Kharel et al., 2005;Zemann et al., 2006). The fact that SPHK1 null mice become lymphopenic after FTY720 administration further supports the view that SPHK2 is sufficient for the functional activation of FTY720 (Allende et al., 2004).Emerging evidence suggests that the effectiveness of FTY720 in the central nervous system (CNS) extends beyond immunomodulation to encompass other aspects of MS pathophysiology, including an influence on the blood-brain barrier and glial repair mechanisms that could ultimately contribute to restoration of nerve function (Baumruker et al., 2007; This work was supported by Novartis Pharma AG. 1

FK 506 and cyclosporin A are potent immunosuppressive compounds that inhibit T-cell activation by interfering with signal transduction. In vitro, FK 506 binds and inhibits the activity of FK 506-binding protein (FKBP), a peptidylprolyl rotamase (cis-rans isomerase). Cyclosporin A acts similarly on a different proline rotamase, cyclophilin. Experiments described here demonstrate genetically that FKBP is a target for FK 506 in vivo. We have isolated the gene encoding the FKBP proline rotamase (FPRI) from Saccharomyces cerevisiae. The encoded yeast protein is highly homologous with bovine and human FKBP and shares no homology with cyclophilin. Disruption of FPRI and CPRI (encoding cyclophilin) individually or in combination is not lethal; thus, either enzymatic proline rotamerization is not essential for life or an unknown proline rotamase can substitute for the missing enzymes. Overexpression or disruption of FPRI confers resistance to growth inhibition by FK 506, suggesting that FKBP is a target for FK 506 in yeast. However, FKBP is only one of at least two targets because strains lacking FKBP are only partially resistant to FK 506. FK 506, a macrolide antibiotic produced by Streptomyces tsukubaensis (1, 2), is a potent immunosuppressive compound (3-5) that inhibits graft rejection after organ transplantation (6-8). Human clinical trials have indicated that FK 506 is effective against graft rejection at lower doses and with a different spectrum of side effects compared with the widely prescribed immunosuppresive drug cyclosporin A (CsA) (9, 10). In addition, both FK 506 and CsA have potential therapeutic indications in the treatment of immune and autoimmune disorders (12, 13).Although structurally unrelated, FK 506 and CsA appear to suppress the human immune system by similar means. In human T cells, antigen presentation to the T-cell receptor triggers a signal transduction cascade resulting in increased gene expression and T-cell activation (14, 15). FK 506 and CsA do not inhibit the first steps in this pathway (inositol 1,4,5-trisphosphate/diacylgylcerol production or Ca2" release from the endoplasmic reticulum) (5, 15-17) but rather block a subsequent step required for synthesis or activation of the transcription factors (NF-AT, AP-3, and NF-KB), which promote expression of lymphokine genes (such as interleukin 2) (14,(18)(19)(20)(21) (27). These findings suggest that proline rotamases play a role in T-cell signal transduction, perhaps by some posttranslational regulatory mechanism involving folding of key substrates. Consistent with this, a component of the Drosophila visual transduction system is a cyclophilin homolog (28, 29). Because cyclophilin and FKBP differ in substrate specificity in vitro (30), each may fold different proteins in vivo, only some of which need participate in signal transduction.To determine the role of proline rotamases in cellular physiology and to define a possible intracellular target of FK 506, we have taken a genetic approach with the unicellular eukaryotic yeast, Sacchar...

SDZ ASM 981, a novel ascomycin macrolactam derivative, has high anti-inflammatory activity in animal models of allergic contact dermatitis and shows clinical efficacy in atopic dermatitis, allergic contact dermatitis and psoriasis, after topical application. Here we report on the in vitro activities of this promising new drug. SDZ ASM 981 inhibits the proliferation of human T cells after antigen-specific or non-specific stimulation. It downregulates the production of Th1 [interleukin (IL)-2, interferon-gamma] and Th2 (IL-4, IL-10) type cytokines after antigen-specific stimulation of a human T-helper cell clone isolated from the skin of an atopic dermatitis patient. SDZ ASM 981 inhibits the phorbol myristate acetate/phytohaemagglutinin-stimulated transcription of a reporter gene coupled to the human IL-2 promoter in the human T-cell line Jurkat and the IgE/antigen-mediated transcription of a reporter gene coupled to the human tumour necrosis factor (TNF)-alpha promoter in the murine mast-cell line CPII. It does not, however, affect the human TNF-alpha promoter controlled transcription of a reporter gene in a murine dendritic cell line (DC18 RGA) after stimulation via the FcgammaRIII receptor. SDZ ASM 981 also prevents the release of preformed pro-inflammatory mediators from mast cells, as shown in the murine cell line CPII after stimulation with IgE/antigen. In summary, these results demonstrate that SDZ ASM 981 is a specific inhibitor of the production of pro-inflammatory cytokines from T cells and mast cells in vitro.

(Me-Ile-4)cyclosporin (SDZ NIM 811) is a 4-substituted cyclosporin which is devoid of immunosuppressive activity but retains full capacity for binding to cyclophilin and exhibits potent anti-human immunodeficiency virus type 1 (HIV-1) activity. SDZ NIM 811 selectively inhibits HIV-1 replication in T4 lymphocyte cell lines, in a monocytic cell line, and in HeLa T4 cells. Furthermore, its antiviral activity against laboratory strains and against clinical isolates from geographically distinct regions in primary T4 lymphocytes and in primary monocytes (50%o inhibitory concentration = 0.011 to 0.057 ,g/ml) was demonstrated. SDZ NIM 811 does not inhibit proviral gene expression or virus-specific enzyme functions, either free or bound to cyclophilin. The compound does not influence CD4 expression or inhibit fusion between virus-infected and uninfected cells. SDZ NIM 811 was, however, found to block formation of infectious particles from chronically infected cells. Oral administration to mice, rats, dogs, and monkeys resulted in levels in blood considerably exceeding the drug concentration, which completely blocked virus replication in primary cells. SDZ NIM 811 caused changes of toxicity parameters in rats to a smaller degree than cyclosporine (formerly cyclosporin A). Thus, the potent and selective anti-HIV-1 activity of SDZ NIM 811 and its favorable pharmacokinetic behavior together with its lower nephrotoxicity than that of cyclosporine make this compound a promising candidate for development as an anti-HIV drug.