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...

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1 The effects of two dihydropyridines, PY 108-068 (PY) and nicardipine (N), and two other calcium antagonists, verapamil (V) and diltiazem (D), on regional blood flow were measured in open-chest cats, anaesthetized with chloralose-urethane. 2 Each substance was infused at 3 different dose rates, each for 10 min. The total doses given were 5 plus 10 plus 35 (total of 50) jig/kg for PY, 10 plus 20 plus 70 (total of 100) jug/kg for N and 100 plus 200 plus 700 (total of 1000) Ag/kg for V and D.3 All substances lowered blood pressure and increased total peripheral conductance. Heart rate was lowered only by V, D and PY. Cardiac output was markedly increased only by the diyhydroypridine derivatives; D had small and V almost no effects. 4 AU substances increased coronary flow and redistributed it in favour of the subepicardial layer. All substances also increased blood flow to the brain. The effects of verapamil were comparatively small. 5 Skeletal muscle flow was increased strongly by the two dihydropyridine derivatives. D and V had negligible effects. 6 Blood flow to stomach and small intestine was only slightly increased. Flow to the kidneys increased slightly in diltiazem-treated animals but did not change with all other treatments. Flow to the liver, the adrenals, and the spleen remained unchanged or showed a tendency to decrease. 7 The organ conductances which reflect the active changes in vascular tone better than blood flow values, showed that there was a tendency towards vasodilatation even in most organs where blood flow tended to decrease.8 Results obtained in an earlier series of experiments with nifedipine were very similar to those described here for N, except that nifedipine was about twice as potent. 9 Calcium antagonists were thus neither general peripheral vasodilators nor did they show a uniform pattern of preferential sites of action. The most important common features were increases in coronary and cerebral blood flow and the most important differences the divergent effects of the dihydropyridines on one side and V and D on the other side on skeletal muscle flow. The size of this vascular bed may help to explain why dihydropyridines appear to be particularly potent as peripheral vasodilators.

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BRL34915 (BRL) is a vasodilator with a novel structure. Its mechanism of action, its effects on depolarization-induced and receptor-mediated blood vessel contraction, and its hemodynamic effects were investigated. In the rat portal vein, BRL inhibited spontaneous mechanical activity [IC50 0.013 +/- 0.001 microM (mean +/- SEM) for (-)-BRL], the initial effect being a reduced frequency of contraction. At higher concentrations, the spontaneous contractions were abolished and 86Rb+ efflux was increased. These results suggest that BRL preferentially acts on the pacemaker cells, the K+ channels in other cells being activated only at higher BRL concentrations in this vessel. In experiments on the rabbit aorta, (-)-BRL shifted the KCl concentration-response curve to the right and noncompetitively inhibited responses to angiotensin II. A concentration of 3 microM (-)-BRL reduced maximal angiotensin II contractions by around 50%, higher concentrations having little further effect. This inhibition of angiotensin II contractions is notably greater than that seen with Ca2+ antagonists in this vessel. In anesthetized rabbits, (-)-BRL was a peripheral vasodilator at doses of 3-30 micrograms/kg, but it had no relevant effects on heart rate and myocardial contractile force. This suggests tissue selectivity of this compound or this mechanism of action. BRL preferentially dilated the coronary, gastrointestinal, and cerebral vessels but not those of the kidneys or skeletal muscle as measured with tracer microspheres. This profile of activity is different from that of calcium antagonists or nonspecific vasodilators like dihydralazine. All effects were stereoselective, the (-)-enantiomer being 100 to 200 times more active than the (+)-enantiomer.