Second generation S1P pathway modulators: Research strategies and clinical developments

Biochimica et Biophysica Acta 1841 (2014) 745–758

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbalip

Review

Second generation S1P pathway modulators: Research strategies and clinical developments☆ Marc Bigaud ⁎, Danilo Guerini, Andreas Billich, Frederic Bassilana, Volker Brinkmann ⁎⁎ Novartis Institutes for Biomedical Research, CH-4056 Basel, Switzerland

a r t i c l e

i n f o

Article history: Received 12 August 2013 Received in revised form 30 October 2013 Accepted 4 November 2013 Available online 12 November 2013 Keywords: Immunomodulator S1P modulator S1P1 Fingolimod

a b s t r a c t Multiple Sclerosis (MS) is a chronic autoimmune disorder affecting the central nervous system (CNS) through demyelination and neurodegeneration. Until recently, major therapeutic treatments have relied on agents requiring injection delivery. In September 2010, fingolimod/FTY720 (Gilenya, Novartis) was approved as the first oral treatment for relapsing forms of MS. Fingolimod causes down-modulation of S1P1 receptors on lymphocytes which prevents the invasion of autoaggressive T cells into the CNS. In astrocytes, down-modulation of S1P1 by the drug reduces astrogliosis, a hallmark of MS, thereby allowing restoration of productive astrocyte communication with other neural cells and the blood brain barrier. Animal data further suggest that the drug directly supports the recovery of nerve conduction and remyelination. In human MS, such mechanisms may explain the significant decrease in the number of inflammatory markers on brain magnetic resonance imaging in recent clinical trials, and the reduction of brain atrophy by the drug. Fingolimod binds to 4 of the 5 known S1P receptor subtypes, and significant efforts were made over the past 5 years to develop next generation S1P receptor modulators and determine the minimal receptor selectivity needed for maximal therapeutic efficacy in MS patients. Other approaches considered were competitive antagonists of the S1P1 receptor, inhibitors of the S1P lyase to prevent S1P degradation, and anti-S1P antibodies. Below we discuss the current status of the field, and the functional properties of the most advanced compounds. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Fingolimod/FTY720 (Gilenya, Novartis) was approved as the first oral treatment for relapsing forms of MS in September 2010. In parallel, significant efforts have been initiated to develop next generation S1P receptor modulators and determine the minimal receptor selectivity needed for maximal therapeutic efficacy. This article aims at giving an overview of the field with particular focus on the most advanced compounds. 2. Biology of sphingosine 1-phosphate and its receptors Many studies have examined the immunomodulatory and proinflammatory actions of S1P, a bioactive sphingolipid mediator [1–4]. S1P concentrations are high in blood (200–900 nM) but low in tissues, and excessive production of the pleiotropic mediator at inflammatory sites may participate in various pathological conditions. A detailed

☆ This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology. ⁎ Corresponding author. Tel.: +41 61 324 35 73. ⁎⁎ Corresponding author. Tel.: +41 61 324 77 30. E-mail addresses: [email protected] (M. Bigaud), [email protected] (V. Brinkmann). 1388-1981/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbalip.2013.11.001

description of S1P and S1P receptor function is beyond the scope of this review and the reader is referred to reviews for details [1,5,6]. A brief summary is given below, and the major activities of S1P and its possible cellular targets are summarized in Table 1. S1P1 mRNA is ubiquitously expressed, and protein expression in adult mouse tissue has been determined by introducing a βgalactosidase reporter gene into the S1P1 locus [7]. S1P1 receptor protein is found in brain N lung = spleen N heart & vasculature N kidney. Genetic deletion of S1P1 in mice indicates a key role in angiogenesis and neurogenesis, as well as the regulation of immune cell trafficking, endothelial barrier function and vascular tone. More recent studies used RT-PCR and immunohistochemistry to localize S1P1 expression in human post-mortem samples of brain and spinal cord [8,9]. S1P1 was restricted to astrocytes and endothelial cells, and receptor protein was strongly expressed in gray, but not white matter of the brain. Strongest expression occurred in the membrane of the astrocytic foot processes of glia limitans, and in astrocytes with radial cytoplasm. In contrast, S1P1 was not found in neurons. In neurological disorders, hypertrophic astrocytes with strong expression of glial fibrillary acidic protein exhibited significantly decreased expression of S1P1, in contrast to its strong expression in astrocytes forming fibrillary gliosis. Conditional deletion of S1P1 in astrocytes reduced gliosis in a murine model of Multiple Sclerosis (MS), suggesting that it may indeed be a target of S1P1-directed therapeutic agents [38].

746

M. Bigaud et al. / Biochimica et Biophysica Acta 1841 (2014) 745–758

Table 1 Major activities of S1P and cellular targets. Potential cellular targets

Induced activity

S1P receptor subtype involved

Reference

Many cell types

Migration

[125]

Many cell types Thymocytes T- and B-cells Leukoytes/monocytes & endothelium Dendritic cells & endothelium Mast cells Eosinophils Endothelial cells

Survival Egress from thymus Egress from lymph nodes Reduced leukocyte/monocyte adhesion to endothelium Reduced dendritic cell migration into lymph nodes Migration, degranulation Migration Angiogenesis Barrier enhancement Barrier decrease Vasodilation, eNOS activation Heart rate reduction Vasoconstriction, blood pressure increase, airway hyperreactivity, myometrial contraction (possible synergies with muscarinic signals and endothelin-1) Embryonic neurogenesis Neurite retraction Astroglial activation and proliferation Inhibition of gap junctional communication between astrocytes, neurons and the blood brain barrier Process retraction

Induced by S1P1 and S1P3 via Gi, inhibited by S1P2 (and S1P3?) via G12/13 S1P1, S1P3 S1P1 S1P1 S1P1 S1P1 S1P1?, S1P2? S1P1?, S1P3? S1P1 S1P1 S1P3 S1P3, S1P1 S1P3, S1P1 S1P2, S1P3 (S1P1?)

[126,127] [28] [28] [128,129] [130,131] [132,133] [134] [135,136] [68,137,69] [68,137] [74,70,75,138] [56,62,55,200] [79,80,82,55,84,85]

S1P1 S1P1? S1P1?, S1P3? S1P1?, S1P3?

[139] [140,141] [142] [143]

S1P5

[53]

Heart atrial myocytes Smooth muscle cells

Neurons Neurons Astrocytes & glia Astrocytes, neurons & endothelial cells Oligodendrocytes

Like S1P1, the S1P2 receptor also shows widespread expression. Although S1P2-deficient mice are born with no apparent anatomical or physiological defects, the mice develop spontaneous, sporadic, and occasionally lethal, seizures between 3 and 7 weeks of age. At a cellular level, loss of the S1P2 receptor leads to an increase in the excitability of neocortical pyramidal neurons, demonstrating that S1P2 plays a role in the development and/or mediation of neuronal excitability [10]. S1P2 is essential for proper functioning of the auditory and vestibular systems, and S1P2 deficiency results in deafness [11,12]. Furthermore, S1P2 functions as a negative regulator of vascular permeability [13]. The S1P3 receptor is highly expressed in heart, lung, spleen, kidney, intestine, diaphragm, and certain cartilaginous regions, but genetic deletion of S1P3 does not result in an obvious phenotype [14]. However, the receptor fine-tunes several cardiovascular functions in the adult. Double deletion of S1P2 and S1P3 receptors in mice often results in perinatal lethality, although double-null survivors lack any obvious phenotype [15], but are more sensitive to myocardial ischemia/reperfusion injury [16]. Triple deletion of S1P1, S1P2 and S1P3 results in embryonic lethality as a consequence of massive hemorrhage/bleeding [17], suggesting cooperative functions of these receptors during embryonic development. S1P4 is predominantly expressed on lymphocytic and hematopoietic cells [18], but its role in immune homeostasis is still poorly understood. S1P4-deficient animals show normal peripheral lymphocyte numbers and a regular architecture of secondary lymphoid organs [19]. Interestingly, S1P4 only marginally affected T-cell function in vivo. On the other hand, lack of S1P4 on dendritic cells (DCs) significantly reduced TH17 cell differentiation, and consistently increased TH2-dominated immune responses [19]. Further, S1P4-deficient mice showed decreased pathology in a model of colitis, suggesting that S1P4 may constitute an interesting target to influence the course of this disease. In rodents, the S1P5 receptor is primarily expressed in the white matter tracts of the CNS, with high levels of mRNA in oligodendrocytes, the myelinating cells of the brain [20,21] and the receptor is identical to the previously described rat nerve growth factor-regulated GPCR NRG-1 [22,23]. S1P5-deficient immature oligodendrocytes display reduced responses to S1P in vitro; however, no deficits in myelination are observed in receptor-deficient mice [53]. In human multiple sclerosis, S1P1 expression was restricted to astrocytes and co-localization studies of S1P5 with the oligodendrocyte marker CNPase established that the receptor is expressed on these cells, and is reduced in demyelinated

lesions [9]. However, its role in inflammation and (re)myelination remains to be elucidated. S1P5 was not found on astrocytes, microglia– macrophages and blood vessels. 3. Fingolimod—1st generation of S1P modulators 3.1. Current understanding of the mode of action in Multiple Sclerosis The original observation that fingolimod acts as potent S1P receptor agonist in vitro [24–26] was in conflict with the finding that S1P1 gene deletion could mimic the therapeutic effects of fingolimod in vivo [27,28]. It was then found that fingolimod causes internalization of S1P1 receptors, thereby acting as “functional antagonist” [28–30], and a similar mode of action has been proposed for other S1P receptor agonists in clinical development [31]. Finally, the discovery of potent S1P1 selective, competitive antagonists confirmed that functional antagonism of S1P1, rather than continuous signaling, is required for therapeutic efficacy of S1P1 ‘agonists’ [32–36]. Down-modulation of lymphocytic S1P1 reduces the egress of lymphocytes from lymph nodes and prevents the invasion of pathogenic T cells into the CNS [28,37]. Further, functional antagonism of S1P1 in astrocytes directly reduces gliosis, a hallmark of MS [38]. Together these effects reduce central inflammation, and this may restore productive interaction of astrocytes with other neuronal cells and the blood brain barrier [1,38], allow remyelination to occur [39,40], restore neuronal function [40] and prevent neuronal/axonal loss and brain atrophy [41] (Fig. 1). In human MS, such mechanisms may explain the significant decrease in the number of inflammatory markers on brain magnetic resonance imaging in recent clinical trials, and the reduction of brain atrophy by fingolimod. A similar functional antagonism of S1P1 receptors by drug may occur in some vascular beds [42] and, as outlined below, this may provide a mechanistic explanation for the individual cases of macular edema and the mild increase in blood pressure of 2 mmHg on average that occurs in fingolimod-treated patients [1]. Furthermore, a transient activation of S1P1 by fingolimod in atrial myocytes, prior to functional antagonism of the receptor, may underlay the transient reduction of heart rate in those patients (Fig. 2). While abundant information on internalization of S1P1 is available, much less is known for the other S1P receptors which may respond differently to agonistic ligands: S1P4 did not readily internalize in response to fingolimod agonism [30,43], and S1P3 had an intermediary

M. Bigaud et al. / Biochimica et Biophysica Acta 1841 (2014) 745–758

747

Fig. 1. Pathological mechanisms of Multiple Sclerosis relevant to fingolimod mode of action. In multiple sclerosis (MS), pathogenic T cells committed to T helper 1 (TH1) and TH17 lineages cross the blood brain barrier (BBB) to invade the CNS. In the CNS, the specific T cells are re-activated by local dendritic cells (DC) presenting the respective autoantigens. The re-activated T cells proliferate, differentiate into effector cells and display direct cytotoxic functions, or secrete the proinflammatory cytokines IFNγ and IL-17 to activate macrophages and astrocytes, respectively, and cause gliosis. The reactive astrocytes release cytokines and chemokines which then cause a ‘second wave’ recruitment of leucocytes to the CNS, resulting in an MS relapse with demyelination, neurodegeneration and axonal loss. Treatment with fingolimod leads to a down-modulation of S1P1 receptors on lymphocytes, thereby preventing their egress from lymph nodes and the invasion into the CNS. In the CNS, down-modulation of S1P1 in astrocytes reduces astrogliosis, and this could improve communication of astrocytes with neural cells and cells of the BBB. Further fingolimod reduces the numbers of activated macrophages in the CNS, but it remains to be determined whether this represents a direct effect of the drug on the macrophages, or whether it is a consequence of the reduced inflammation. Together these protective effects may contribute to the significant decrease in both inflammatory markers on brain magnetic resonance imaging and brain volume loss by fingolimod observed in clinical trials.

phenotype, showing partial internalization [43,44]. Conflicting data exist for S1P5, where the reported agonist-induced internalization [30] could not be reproduced by us (D. Guerini, unpublished observation). So far, data on S1P2 down-modulation by agonists are not available, although S1P2 have been associated, together with S1P3, with Akt and Erk activation and pro-fibrotic responses of fibroblasts in vitro [201]. It was however observed that fingolimod had protective effects against fibrosis in vivo [202,203]. The kinetics of agonist-induced internalization from cell membranes, and the recycling of receptors to the cell membrane after ligand

shedding are critical for the occurrence of “functional antagonism”. In vitro, fingolimod causes complete and persistent internalization of S1P1 within hours [43,45] and the receptor recycles only after ligand washout. In contrast, fingolimod did not produce a complete internalization of S1P3 and, compared to S1P1, the receptor recycled more readily to the cell membranes, presumably as a result of the lower affinity of the drug for S1P3. S1P1 receptors internalized by fingolimod are unable to sense extracellular S1P concentration changes relevant to cell migration, particularly out of lymphoid tissues back into circulation. However, internalized

Fig. 2. Effects of fingolimod treatment on the homeostatic signaling of vascular S1P1, S1P2 and S1P3 by plasma S1P. A high concentration of S1P (200–900 nM) is present in plasma and binds to S1P1–3, suggesting that in vivo all vascular receptors are activated by S1P in the control of vascular tone and permeability. As the affinity of fingolimod-P to S1P2 and S1P3 is markedly lower compared with S1P1, it is unlikely that fingolimod directly targets S1P2 and S1P3. In contrast, as fingolimod has a higher affinity to S1P1, initiating fingolimod transiently activates S1P1, resulting in: 1—Activation of GIRK channels on atrial monocytes and consequently bradycardia; 2—Activation of endothelial eNOS and subsequently a decrease in blood pressure; 3—Increase in endothelial cell barriers protecting from vessel leakage. Following this initial agonism, continuous dosing of fingolimod results in functional antagonism of S1P1 and internalization of S1P1 receptors. Down-modulation of S1P1 may cause a relative increase in the activation of S1P2 and S1P3 by endogenous S1P, causing an activation of Rho-Kinase (ROCK) in vascular smooth muscle cells, which may lead to vasoconstriction and a mild increase in blood pressure, and a decrease in endothelial cell barriers causing isolated cases of macular edema.

748

M. Bigaud et al. / Biochimica et Biophysica Acta 1841 (2014) 745–758

receptors may continue to signal in response to bound ligand for some hours [46] until the ligand is shed and the receptor either recycled to the membrane or degraded via the proteasomal degradation pathway [47,48]. In vivo, the level of S1P receptor down-modulation by drug may further depend on a) the concentration of drug, b) the presence of S1P which competes on the receptors, and c) the level of S1P receptor expression that in turn depends on the inflammatory status of the tissue. 3.2. Design of next generation S1P receptor modulators The modulation of S1P receptors by synthetic agonists and/or antagonists in vivo is critically determined by the natural ligand S1P, produced through phosphorylation of intracellular sphingosine (Sph) by sphingosine kinases (SphKs) mainly in the cardiovascular system (platelets [49], endothelial cells (ECs) [50] and erythrocytes [60]). Hence, variable degree of S1P receptor modulation might be seen in various organs depending on the competition between S1P and the drugs for their receptor binding sites (S1P1–5) [1,4,51]. Fingolimod is a structural analogue of sphingosine and is rapidly phosphorylated by SphK2 to yield fingolimod-P, the bioactive form of the drug [24,52]. In vitro, fingolimod-P binds to S1P1, -3, -4 and -5, but spares S1P2 [24,26]. Recent data suggested that most of its therapeutic activities in MS, as in Experimental Allergic Encephalomyelitis (EAE) models, may relate to a functional antagonism of S1P1 on lymphocytes and astrocytes and, perhaps, modulation of S1P5 on oligodendrocytes [28,38](Fig. 1). On the other hand, modulation of S1P3 receptors in rodents has been associated with the modulation of heart rate and blood pressure. Therefore, most new generation S1P receptor modulators were designed to target S1P1 and spare the cardiovascular receptors S1P2 and S1P3. Below we discuss in more detail the functional role of individual S1P receptor subtypes in cardiovascular regulation, and whether sparing of these receptors by drug could lead to improved tolerability. 3.2.1. S1P receptors and regulation of heart rate In atrial myocytes of the heart, stimulation of S1P receptors by the natural ligand S1P or of muscarinic M2 receptors by acetylcholine (ACh), causes an activation of G-protein-gated inwardly rectifying K+ channels (GIRK; alternatively named IKACh or Kir3) [193]. The GIRK channel comprises a tetramer of two GIRK1 and GIRK4 subunits, each. GIRK channels are membrane spanning-proteins that regulate the resting membrane potential, the pulse frequency of pacemaker cells and the shape and duration of the cardiac action potential [54]. An acute and transient decrease in heart rate can be produced by administration of S1P or fingolimod to mice [55], and the effect is linked to the S1P receptor-dependent activation of the GIRK channels in atrial myocytes [193](Fig. 3). This results in a hyperpolarization of cell membranes and a transient reduction of the excitability of cells [56–58,193].

Accordingly, genetic deletion of GIRK4 in mice completely abolishes heart rate reduction by fingolimod [193]. A short priming of isolated guinea pig atrial myocytes with fingolimod-P ex vivo completely prevents GIRK activation upon restimulation with compound or S1P, but not with ACh [59](Pott and Brinkmann, unpublished observation), confirming that the drug specifically desensitizes S1P receptors after a short activation, and this is consistent with the transient nature of heart rate reduction by fingolimod. Studies in S1P3-deficient mice suggested a dominant role of this receptor in the regulation of heart rate [55]. Later experiments in guinea pig atrial monocytes revealed that GIRK channels could also be activated by the S1P1-selective agonist AUY954 (Pott and Brinkmann, unpublished observation), and that an S1P1-agonist inactive at rodent S1P3, such as KRP-203, could reduce heart rate in rats when injected intravenously [61]. Detailed analysis of human cardiovascular tissue by in situ hybridization and immunohistochemistry then showed that in humans, S1P1 mRNA and protein are strongly expressed in ventricular, septal, and atrial cardiomyocytes, [62], whereas S1P3 is found in the smooth muscle cell layer of aorta and cardiac vessels, but not in cardiomyocytes from both atria and ventricles [62]. Together these data suggested that the S1P1-GIRK axis may play a dominant role in the regulation of atrial myocyte function and heart rate, and that fingolimod may transiently activate atrial S1P1 to cause bradycardia. In line with this hypothesis, the S1P3-sparing agonist siponimod (BAF312) also transiently reduced heart rate in humans [31] (for details see section below on siponimod).

3.2.2. S1P receptors and endothelial barriers Vascular endothelium expresses S1P1, -2 and -3, but not -4 and -5 [1,42], and the maintenance of endothelial barrier function is largely mediated by S1P bound to HDL [64]. Signaling of endothelial S1P1 stabilizes the adherence and tight junctions between cells [65,66] and causes persistent activation of eNOS and the downstream NO target, soluble guanylate cyclase (sGC) [66]. In contrast, activation of S1P2 and/or S1P3 is associated with a disruption of adherens junctions and an increase in paracellular permeability [67,65,68]. Accordingly, administration of a selective S1P1 antagonist to mice produced a loss of capillary integrity [69] and potentiated histamine-induced vascular leakage [13], which could be prevented by co-administration of the S1P2specific antagonist JTE-013 [13]. Together, the data suggest that the balanced signaling, particularly of S1P1 and S1P2, by the high S1P levels in blood significantly contributes to the homeostasis of vascular barriers (Fig. 2). Several studies in animals suggested that fingolimod may increase endothelial barrier function and prevent disruption of permeability barriers through signaling of S1P1. Administration of fingolimod protected mice from VEGF-induced vascular leakage [67] and lipopolysaccharideinduced pulmonary edema [194]. In the respective models, a specific upregulation of endothelial S1P1 may occur as a consequence of the inflammation [70], rendering endothelium more susceptible to S1P1-

Fig. 3. Fingolimod transiently activates S1P1 and S1P3 and temporarily mimics acetylcholine-induced bradycardia via activation of muscarinic type-2 receptors on atrial myocytes. Fingolimod-induced S1P1 receptor internalization by functional antagonism terminates this response. GIRK: G-protein-coupled inwardly rectifying K+; Ach: acetylcholine; S1P: sphingosine-1-phosphate.

M. Bigaud et al. / Biochimica et Biophysica Acta 1841 (2014) 745–758

signaling and barrier enhancement by fingolimod [67], with less pronounced down-modulation of the receptors by drug. In other models, specific competitive S1P1 antagonists blocked S1P1-signaling, reduced endothelial barrier function and caused leakage [69]. Interestingly, administration of S1P1 agonists caused a transient increase in barrier function in vivo, presumably through early signaling at S1P1, but later reduced barriers, presumably as a result of receptor internalization and degradation (Bigaud, unpublished observations). It is therefore possible that the isolated cases of macular edema (ME) observed in some fingolimod-treated patients may relate to a downmodulation of endothelial S1P1, i.e. in the blood-retinal barriers in the eye, thereby reducing eNOS-dependent pathways and tight/adherence junctions between endothelial cells. It is important to note that the development of ME in fingolimod-treated humans apparently require confounding factors, as this complication was an infrequent, reversible event in MS patients, but occurred at higher incidence in renal transplant patients with co-existing (diabetic) retinopathies and preexisting impaired barrier function [2,71]. It is unlikely that, in the cardiovascular system, fingolimod displaces natural S1P from endothelial S1P2 and -3, because the blood concentrations of S1P as well as the affinities of S1P for these receptors are considerably higher compared to fingolimod-P. This holds particularly in humans where the approved daily 0.5-mg dosing results in low nM blood levels of the drug [72], whereas the blood concentrations of S1P are 100-fold higher [73]. It is therefore unlikely that fingolimod directly targets endothelial S1P2 and S1P3 in humans at the therapeutically used dosing regimen (Fig. 2).

3.2.3. S1P receptors and blood pressure In vivo, administration of S1P in mice causes a short and transient reduction of mean blood pressure, which is then followed by an increase over baseline [55,63]. The observed counter-regulation may involve S1P receptors on endothelium and smooth muscle cells [1]. The initial reduction of blood pressure following the administration of S1P may result from a transient S1P1- and/or S1P3-dependent activation of eNOS and the production of nitric oxide (NO) [70,74–76] which relaxes SMCs and causes vasodilation [77,70]. The same pathway maintains endothelial barrier function where activation of S1P1 induces eNOS as well as activity of the downstream NO target, soluble guanylate cyclase (sGC) [66]. NO may also counter-regulate the secretion of endothelin-1 by endothelial cells, thereby antagonizing endothelin-1dependent SMC contraction [78].

749

After the transient reduction of blood pressure, the following increase over base line may involve an S1P2- and/or S1P3-dependent activation of Rho-Kinase (ROCK) in VSMCs and a resulting constriction, as observed in cerebral-, basilar- and mesenteric arteries, and gastric- and airway smooth muscle stimulated by S1P [79–85]. Accordingly, the ROCK inhibitor Y-27632 dramatically corrects hypertension in several hypertensive rat models, and also blocks methacholine-induced contraction and hyperreactivity of human bronchial smooth muscle [85,86]. Like S1P, fingolimod-P may (transiently) activate the endothelial S1P1-eNOS/NO pathway to cause vasodilation [66], and this could explain the transient blood pressure decrease observed in fingolimodtreated MS patients. At later stages, functional antagonism of endothelial S1P1 by drug may reverse the effect, reducing the homeostatic vasodilatory S1P1-eNOS signal. Alternatively, S1P-agonists may directly down-modulate S1P1 on VSMCs, thereby favoring a relative increase of S1P–S1P2-signaling in these cells which, in fingolimod-treated MS patients, may cause a mild constrictor effect resulting in the observed average increase of blood pressure by 2 mmHg [87,88] (Fig. 2). 4. The clinical experience with next generation S1P-modulators Along with the growing clinical success of fingolimod, new drug discovery programs have been initiated to identify next generation S1P receptor agonists. Originally, preclinical observations in rodents suggested that the S1P3 receptor might be responsible for the transient asymptomatic reduction of heart rate provoked by the first dose of fingolimod [55,69,89,90]. Thus, virtually all optimization strategies were oriented towards the design of S1P3-sparing, S1P1-selective agonists. A brief overview of the clinical landscape for S1P receptor agonists was given in 2010 [91] and the various medicinal chemistry approaches were reviewed in detail elsewhere [92–94]. Here we discuss the most advanced molecules to date (see Table 2). 4.1. Siponimod (BAF312, Novartis AG) Siponimod is an orally active S1P receptor modulator that selectively targets S1P1 and S1P5 at low nM potency, but spares S1P2, -3 and -4 [95]. Exploration of such profile is of particular interest as S1P1 and S1P5 are involved in T cell migration to the CNS [2], astrogliosis [38], oligodendocyte process modulation and cell survival [53]. The drug induced long lasting internalization of the S1P1 receptors and demonstrated significant therapeutic efficacy in EAE models of human MS [31]. Like fingolimod, siponimod selectively reduced CD4+ naïve and

Table 2 Most advanced next generation S1P modulators vs fingolimod. Name

S1P-R selectivity

Fingolimod

Structure

Clinical development: Indications

1st dose bradycardia in Human

References

1N5N4N3

Marketed: RR MS

Yes

[1,2]

Siponimod

1=5N4

P-III: SP MS

Yes

[31,63,95–98] www.novartis.com

Ponesimod

1N5N3

P-II RR MS P-II: Psoriasis

Yes

[92,99–104] www.actelion.com

KRP-203

1N4

P-II: scLE, P-II: UC P-I: RR MS P-I: Transplantation P-I: IBD

Yes

[60,61,105–113]

ONO-4641 RPC1063 CS-0777

1=5N4 1N5N4 1N5N3

P-II: RR MS P-II: RR MS P-II: IBD P-II: UC P-II: MS

Yes Yes Yes

[114–116] www.merkserono.com [117–119] www.receptos.com [120–124]

GSK2018682

1N5

P-I: RR MS

Yes

None

Undisclosed Undisclosed

750

M. Bigaud et al. / Biochimica et Biophysica Acta 1841 (2014) 745–758

central memory T cells and B cells in human blood, while sparing effector memory T cells. In healthy volunteers, blood lymphocyte counts returned to normal ranges within a week after cessation of treatment, in line with a shorter half-life of siponimod compared to fingolimod. Positive phase IIb data showed that treatment with siponimod, when compared to placebo, reduced brain MRI lesions up to 80% [96]. Relapses were infrequent and appeared reduced with treatment (ARR for 2 mg 0.20 vs. placebo 0.58; p = 0.044). Siponimod was well-tolerated; most frequent adverse events were headache, transient reduction of heart rate, dizziness and nasopharyngitis. Like fingolimod, siponimod activated GIRK channels in human atrial myocytes in vitro and induced a transient, 1st dose, bradycardia in MS patients [31], suggesting involvement of S1P1 rather than S1P3 receptors. However, the reduction of heart rate was somewhat less pronounced in rats treated with siponimod if compared to fingolimod [200], suggesting an additional (small) S1P3 component in the bradycardia in humans. Importantly, blood pressure regulation in rats by drug was primarily dependent on S1P3 [200], but it remains to be determined whether this involves a direct action of drug at S1P3, or involves a relative increase in the endogenous S1P–S1P3signaling after down-modulation of S1P1 by drug. More clinical trials with siponimod are needed to confirm potential benefits of sparing drug selectivity for S1P3. A recent clinical study showed that ascending dose titration over 9 or 10 days to a siponimod dose of 10 mg successfully attenuates the initial bradycardia observed on day 1 of treatment with the siponiod 10 mg maintenance dose. These results support implementation of an initial dose titration regimen in future siponimod clinical studies. However, such titration regimen may not necessarily be extrapolated to other S1P receptor modulators with a different pharmacological profile or elimination half-life [98]. A phase III program has now been started, exploring the efficacy and safety of siponimod in patients with Secondary Progressive Multiple Sclerosis (SPMS), a chronic form of MS with a particularly high unmet medical need (EXPAND trial, Clinicaltrialsregister.eu code number: CBAF312A2304, ClinicalTrials.gov identifier: NTC01665144) [97].

4.2. Ponesimod (ACT-128800, Actelion Ltd) Ponesimod is an orally active S1P1 agonist (nM range) that exhibits a 10 fold selectivity over S1P5 and a 20 fold selectivity over S1P3, and does not show activity at S1P2 and S1P4 up to 1 μM concentration [92,99]. The compound internalized S1P1 receptors [100] and caused a dose-dependent decrease in blood lymphocyte counts that returned to baseline within 48 h after discontinuation of dosing [99]. Ponesimod prevented inflammatory cell accumulation and cytokine release in the skin of mice with delayed-type hypersensitivity and prevented the increase in paw volume and joint inflammation in rats with adjuvantinduced arthritis [99]. Ponesimod is currently in clinical development for the treatment of psoriasis and relapsing remitting MS. In psoriasis patients, ponesimod administered orally, once daily for 16 weeks, demonstrated significant clinical efficacy vs placebo, as measured by the proportion of patients with at least 75% improvement in Psoriasis Area and Severity Index (PASI) from baseline and by Physician Global Assessment at the end of treatment (Study ACT-128800/ NTC00852670). In MS patients, ponesimod administered orally once daily for 24 weeks (study ACT-058B201 & extension NCT01093326) significantly reduced the cumulative number of new active lesions on monthly magnetic resonance imaging (MRI) brain scans performed from weeks 12 to 24. This was also associated with a smaller overall number of confirmed relapses and a reduction of the annualized relapse rate [101–103]. At initiation of ponesimod treatments, a transient reduction in heart rate and effect on atrioventricular conduction was observed [104].

4.3. KRP-203 (Novartis AG/Kyorin Pharmaceutical Co Ltd) Like fingolimod, KRP-203 is phosphorylated in vivo by sphingosine kinases, and its active phosphorylated form (KRP203-P) is a potent low nM agonist at S1P1 and S1P4, while it does not hit murine S1P3 [105]. KRP203 and its active metabolite share structural similarities with fingolimod and fingolimod-P, and also inhibit lymphocyte egress from lymph nodes [60,105]. In preclinical studies, KRP-203 showed marked efficacy in a wide range of disease models in rodents, including prolongation of skin-, heart- and kidney allograft survival with reduced lymphocyte infiltration into these grafts [60,61,106–108]; prevention of autoimmune myocarditis in rats [109]; protection against Concanavalin A-induced liver injury in the mouse [110]; prevention of autoimmune colitis in IL-10-deficient mice [105]; reduced severity of spontaneous autoimmune lupus-like disease in MRL/lpr mice [111]; prolongation of pancreatic islet allograft survival in the mouse [112] and inhibition of atherosclerosis development in LDL-R−/− mice [113]. KRP-203 has entered phase 2 clinical trials in 2011 to determine its efficacy in patients with subacute cutaneous lupus erythematosus (trial NCT01294774) and moderately active refractory ulcerative colitis (trial NCT01375179). It is further analyzed for clinical efficacy in patients undergoing stem cell transplantation to treat hematological malignancies (trial NCT01830010). 4.4. ONO-4641 (Ono Pharmaceutical Co Ltd) ONO-4641 is a selective S1P1 and S1P5 agonist with low nM potency at both receptors, showing a 50-fold lower activity at S1P4 and no activity at S1P2 and S1P3. Like other S1P1 agonists, the compound caused effective internalization of S1P1 [114], decreased blood lymphocyte counts in a dose-dependent manner and was effective as a prophylactic treatment in EAE [114]. The pharmacokinetic/pharmacodynamics relationship for ONO4641 was studied in the cynomolgus monkey in an attempt to predict its effects in human prior to starting clinical trials [115]. A phase 2b study in relapsing remitting MS (DreaMS; NCT01081782 and extension NCT01226745) showed that, compared to placebo, ONO-4641 reduced brain lesions detected by MRI. As with other S1P1 agonists, a transient bradycardia was observed, as well as individual cases of first- and second-degree atrioventricular block [116]. 4.5. RPC1063 (Receptos Inc) RPC1063 is the first drug resulting from the NIH-sponsored Molecular Library Program (MLP) to be tested in clinical trials [117]. Information concerning this compound was disclosed only recently in few abstracts. They revealed picomolar potency at S1P1, a N200-fold selectivity over S1P5, and a greater than 20,000-fold selectivity over S1P2, S1P3 and S1P4. RPC1063 was effective in rodent models for MS and inflammatory bowel disease (IBD) [118]. Its ability to reduce peripheral lymphocyte counts and to cause a dose-dependent reduction in heart rate, with individual cases of atrioventicular block on day 1, was confirmed in human phase 1 study [119]. Phase 2/3 studies have been initiated to study RPC1063 in patients with relapsing remitting MS and ulcerative colitis (NCT01628393 and NCT01647516). 4.6. CS-0777 (Daiichi Sankyo Co Ltd/Daiichi Sankyo Inc) Like fingolimod and KRP-203, CS-0777 is phosphorylated in vivo, and its active phosphorylated form (CS-0777-P) is a potent low nM agonist at S1P1, with a 19 and 320 fold higher selectivity over S1P5 and S1P3, respectively, and no activity at S1P2 [120]. Oral administration of CS-0777 to rats and monkeys reduced circulating lymphocyte counts and suppressed disease activity in rodent models of EAE [121,122]. In human healthy volunteers, CS-0777 treatments resulted in a dosedependent decrease in circulating lymphocytes and a transient

M. Bigaud et al. / Biochimica et Biophysica Acta 1841 (2014) 745–758

bradycardia [123]. This was confirmed in MS patients during a 12-week, open-label pilot study [124]. CS-0777 is expected to enter phase Il trials in patients with relapsing remitting MS soon. 4.7. GSK2018682 (GlaxoSmithKline) In the absence of published data, GSK2018682 is presented as an agonist at recombinant human S1P1, with some activity at S1P5 receptors but without agonist activity towards human S1P2, S1P3 or S1P4 up to a concentration of 10 μM. Based on orally efficacy in murine EAE models, GSK2018682 entered clinical development for the treatment of relapsing–remitting MS. It showed reasonable pharmacokinetic profile and tolerability following single oral doses of 0.6 mg to 24 mg in healthy volunteers, with dose dependent decrease in circulating lymphocyte counts (NCT01431937, NCT01387217, NCT01466322). Significant acute reductions in heart rate and cases of AV block were also reported. 5. S1P1 antagonists In 2004 it was shown that the binding of fingolimod at S1P1 receptors results in an internalization of the S1P1 receptors and a “functional antagonism” of receptor signaling [28–30]. This immediately suggested that direct competitive S1P1 antagonists might recapitulate the effects of S1P1 receptor agonists on lymphocyte traffic. This hypothesis was also supported by the demonstration that lymphocyte egress from lymphoid organs could be inhibited by selective S1P1 receptor knock-down in T-cells [27,28]. However, some confusion was generated when the first synthesized S1P1 antagonists (e.g. compound W146) were used in vivo. Unexpectedly, W146 failed to inhibit lymphocyte egress from lymphoid tissues while blocking the effects of S1P1 agonists on this biological process [69,165–167]. A “stromal gate model” was therefore developed, suggesting that S1P1 agonism may increase lymphatic endothelial barriers to prevent lymphocytes from egressing the nodes Table 3 Chemical structures of most S1P1 antagonists. Name

Structure

References

W146

[69,165–167,169]

VPC03090

[166]

VPC44116

[179]

CL2

[164]

TASP0277308

[33,35,181,182]

XLS541

[178]

EX26

[187]

Prodrug 14

[32]

NIBR-0213

[36]

751

[69,93,167,168]. However, in 2011, it was realized that W146 presents poor pharmacokinetic properties and therefore produces only a short and transient effect on lymphocyte traffic that had been missed in earlier experiments [169]. In parallel, several Medicinal Chemistry programs resulted in the generation of novel S1P1 antagonists with increased potency, selectivity and pharmacokinetic properties (Table 3). Studies with these improved agents indeed confirmed that competitive S1P1 antagonists mimic the pharmacological effect of S1P1 agonists on lymphocyte redistribution in vivo [32,34–36,170]. Consequently, the model of “functional antagonism” as mode of action for S1P1 agonists, e.g. fingolimod, is now well established. Some differences in S1P1 receptor modulation may however exist between agonists and antagonists. Competitive S1P1 antagonists do not cause receptor internalization and they prevent S1P1 down modulation provoked by S1P1 agonists [36]. Further, downmodulation of S1P1 by agonists involves formation of CD69–S1P1 complexes which does not occur with antagonists [171,172]; by contrast, CD69 is increased after treatment with a S1P1 antagonist [33]. The potential functional consequences of these differences remain to be determined. Unlike S1P receptor agonists, direct competitive antagonists would not cause any (transient) receptor activation, and as outlined below this could result in improved tolerability profiles. So far, S1P1 antagonists have not reached clinical development, but they are effective in various mechanistic and disease models, including EAE. 5.1. Effects of S1P1 antagonists in animal models 5.1.1. Effects in angiogenesis models Early reports indicated that genetic ablation of the S1P1 receptor inhibits endothelial cell migration during embryonic development [173]. Further, S1P1 receptors are involved in Human Umbilical Vein Endothelial Cells (HUVEC) migration and proliferation [174] as well as in angiogenesis and vascular stabilization [175] and this might involve crosstalk between the S1P1 receptors and growth factors that are crucial in vessel formation such as VEGF [70,176]. Accordingly, the anti-angiogenic effect observed with S1P1 agonists [177] also occurred with the S1P1 antagonists TASP0277308 and CL2 [35,164]. Further, selective S1P1 antagonists inhibited cell proliferation in response to S1P, VEGF or βFGF and also inhibited VEGF-driven HUVEC tube formation (TASP0277308 [35]), tumor growth in mice (VPC03090 [166] XLS541 [178]) and migration/invasion of Wilms tumor (VPC44116 [179]). 5.1.2. Effects in arthritis models In a rat model of collagen-induced arthritis, fingolimod reduced the inflammatory cell infiltration and synovial hyperplasia of the joints [180]. Similar disease modifying effects were obtained with the S1P1 antagonist TASP0277308 which reduced clinical arthritis severity score and paw swelling in a mouse model of collagen-induced arthritis [33]. Such a therapeutic effect was largely mediated by a reduction of T cell infiltration into the joints and, perhaps, some modulation of cytokine production, including TGF-β production [181,182]. However, cytokine modulation may result from altered cell trafficking through the inflamed tissue, rather than a direct modulation of gene transcription. 5.1.3. Effects in transplantation models Phase III clinical studies of fingolimod in renal transplant patients [183] were based on a large series of preclinical data demonstrating efficacy in stringent models of kidney-, heart-, liver-, and skin transplantation [184], particularly when combined to other immunosuppressive agents such as cyclosporine or sirolimus [185]. Similar results were recently reported for combinations of a novel S1P1 antagonist (prodrug 14) with sotrastorin or everolimus at non-efficacious doses in a rat model of heart transplantation [32]. As no drug–drug interaction could be detected, those results suggest pharmacological synergies to be further explored.

752

M. Bigaud et al. / Biochimica et Biophysica Acta 1841 (2014) 745–758

5.1.4. Effects in experimental autoimmune encephalomyelitis (EAE) The clinical development of Gilenya in relapsing forms of MS [1,2] was based on the excellent therapeutic efficacy of the drug in EAE, the rodent model of human MS [37,186]. This therapeutic activity in EAE could now be recapitulated with the S1P1 antagonist NIBR-0213 [36] and was confirmed by a second study using the compound Ex26 [187]. Both compounds originated from the same Medicinal Chemistry program and displayed similar effects on lymphocyte traffic. Compound Ex26 had no detectable brain penetration in healthy naive mice; however, it is not excluded that the agent may have reached the brain during EAE when significant perturbation of the blood brain barrier occurs. In contrast to Ex26, NIBR-0213 can be well detected in the CNS after treatment, reaching the 0.5 μM concentration, with a brain/blood ratio of 10% (Quancard et al., unpublished observations). Interestingly, conditional deletion of S1P1 from astrocytes in mice mimics the central effects of fingolimod in EAE, strongly supporting the relevance of S1P1 antagonism in the CNS for drug efficacy in EAE [38]. This is further supported by the high expression of S1P1 receptor protein in astrocytes forming fibrillary gliosis, a hallmark of human MS. 5.2. Differentiation of S1P1 antagonists from S1P1 agonists Receptor pharmacology suggests that a competitive S1P1 antagonist would not cause a transient reduction of heart rate that occurs with agonists as a result of S1P1 receptor signalling in atrial myocytes (see in 2.2.1). This was indeed confirmed in animal models using the potent and selective S1P1 antagonist “prodrug 14” [32,188], but human clinical data are not yet available. On the other hand, both S1P1 agonists and competitive S1P1 antagonists may modulate endothelial barriers by reducing the homeostatic S1P–S1P1 signal [189–192]. Accordingly, all tested S1P1 antagonists weakened endothelial barriers in vitro and in vivo [32,33,35,36,69,169,187], presumably by shifting the endogenous S1P-signalling balance from S1P1 to S1P2 and/or S1P3 (Fig. 3). It remains to be determined whether such barrier effects can be avoided by using dual S1P1,2- or S1P1,3 antagonists like VPC03093 [166]. 6. S1P lyase—a novel target to interfere with lymphocyte recruitment in inflammation Modulation of the S1P–S1P receptor axis can be achieved either by using synthetic ligands of the receptors, or by variation of endogenous ligand concentrations. An increase of tissue S1P occurs when the enzyme S1P lyase (Sgpl1) is inhibited, leading to changes in T cell distribution similar to those achieved with S1P1 agonists or antagonists. While general reviews of Sgpl1 as pharmacological target appeared before [144,145], we here present a brief overview on Sgpl1 role in T cell homeostasis and the protective effect of Sgpl1 inhibitors in models of inflammation that would suggest potential therapeutic use. 6.1. Regulation of tissue S1P levels Sgpl1 is widely expressed in mammalian cells and is engaged in the irreversible degradation of S1P [146]. The enzyme appears as the major

1

control point to regulate tissue S1P concentrations, except for the CNS. In fact, S1P concentrations in Sgpl1 KO mice are normal at birth but when analyzed two weeks later are highly increased, e.g. by 4700-fold in the thymus; in contrast, S1P levels in brain and spinal cord are similar to those in wild-type mice [147]. Partial inhibition of Sgpl1, as observed in mice with inducible Sgpl1 gene deletion [147], in humanized knockin mice [148], and in rodents treated with Sgpl1 inhibitors leads to less pronounced increase of S1P in the range of 10- to 80-fold for thymic S1P. As discussed below, the elevation of S1P in the lymphoid organs is the basis for the altered T cell distribution encountered upon Sgpl1 inhibition. 6.2. S1P lyase inhibitors Besides the genetic mouse models, small-molecule inhibitors have become important tools to explore Sgpl1 as a pharmacological target. Currently known inhibitors fall into two categories: (i) Active-site directed inhibitors—High-throughput screening for S1P lyase inhibitors yielded a class of compounds examplified by structure 1 (Fig. 4); such compounds inhibit enzyme activity of purified Sgpl1 and, as seen by X-ray crystallography, bind to the active site of the enzyme [149]. They induce an increase in S1P in cultured cells [150,151] and in rodents in vivo, where they rapidly induce peripheral lymphopenia. (ii) Indirect inhibitors—These include deoxypyridoxine (DOP), an antagonist of vitamin B6, and 2-acetyl-4-tetrahydroxybutylimidazole (THI), originally identified as a minor constituent of ammonia caramel food coloring causing immunosuppression in rodents [152](2, 3 in Fig. 4); more recently, derivatives of THI, such as LX2931, have been described [153,154]. While these compounds do not block Sgpl1 activity in biochemical and cellular assays [154,150,151], they cause reduction of Sgpl1 activity when administered in vivo, leading to elevated tissue S1P and T cell redistribution [154–156]. These effects are reverted by dietary vitamin B6 [155], and are enhanced by vitamin B6-deficient diet (our unpublished observations). Since Sgpl1 requires pyridoxal phosphate (PLP), the active form of vitamin B6, it is suggested that the compounds, either themselves or after metabolization, compete with PLP as essential co-factor of Sgpl1. 6.3. S1P lyase controls T cell egress S1P is known to regulate the egress of T cells from lymphoid organs [6], and, as discussed above, levels of S1P in the tissues are controlled by Sgpl1. Hence, Sgpl1 can be expected to modulate T cell recirculation. Indeed, RNA-interference-mediated knock-down of Sgpl1 in hematopoietic cells was shown to increase S1P levels and to block T cell egress [155]. Furthermore, pharmacological inhibition or genetic deletion of Sgpl1 in mice leads to a pronounced decrease of circulating T lymphocytes, affecting both CD4 and CD8 positive T cells in blood and spleen; this reduction has been consistently observed in rodents treated with active-site directed or indirect inhibitors, and in fully and partially Sgpl1-deficient mice [148,147,154–157]. The onset of the lymphopenic effect of the pharmacological inhibitors is delayed after administration

DOP 2 Fig. 4. Chemical structures of selected Sgpl1 inhibitors.

THI 3

M. Bigaud et al. / Biochimica et Biophysica Acta 1841 (2014) 745–758

[154,156], but parallels with tissue S1P concentrations. Peripheral lymphopenia induced by the inhibitors is transient, with normalization of blood T cell numbers occurring when tissue S1P levels return to baseline. In contrast to the decrease of peripheral T cells, cell numbers in the lymph nodes of inducible Sgpl1 KO mice, in particular of CD4+ and CD8+ T cells, are significantly elevated [147]. In the thymus, T cell development appears normal, but thymocytes are enriched in mature singlepositive CD4+ and CD8+ cells [147], as also seen in full Sgpl1 KO mice [148] and after treatment of mice with DOP or THI [155]. In summary, even partial reduction of Sgpl1 activity suffices to induce retention of mature T cells in the thymus and lymph nodes, leading to reduced T cell numbers in blood and spleen. An obvious explanation for the T cell retention in the thymus and lymph nodes is the downregulation of S1P1 receptors on the T cells. Indeed, downmodulation of S1P1 expressed on T cells has been demonstrated in mice treated with Sgpl1-directed shRNA or with THI and DOP [155], and on recombinant CHO cells in vitro using active-site directed inhibitors (A. Billich, unpublished). Functionally, lymphocytes from DOP or THI treated mice show reduced ability to migrate to S1P [155]. Taken together, these data identify S1P1 mediated T cell migration as the primary target of Sgpl1 inhibitors. Interestingly, in inducible Sgpl1-deficient mice and in rodents treated with a direct Sgpl1 inhibitor the effect on blood lymphocyte numbers is confined to the T cells, without any effect on B cells [147], while blood B lymphocytes in fully Sgpl1-deficient mice or in mice treated with indirect inhibitors are partially reduced [147,148,155]. Although increased B cell numbers in the lymph nodes of inducible Sgpl1-deficient mice indicate impaired B cell egress [147], the data suggest that overall the migration and distribution of T cells is more strictly controlled by S1P gradients.

753

mice undergoing EAE contained significant numbers of CNS-invading CD3+ T cells, these cells were undetectable in respective tissue preparations from inducible Sgpl1-deficient mice. The protection observed in these models may be the result of T cell retention in the lymph nodes; however, the increased proportion of CD4+ FoxP3+ regulatory T cells in blood and lymphoid organs and the reduced antigenresponsiveness of the T cells may also contribute to the protection. As mentioned earlier, S1P levels are not elevated in brain and spinal cord of the inducible Sgpl1-deficient mice; therefore, protection in EAE may be due to the peripheral effect on T cell numbers, quality, and CNS immigration rather than on central effects of S1P on cells in the CNS as described for fingolimod [38]. Protection in EAE is not only seen in the mouse genetic model, but is also phenocopied in rat EAE when using the direct Sgpl1 inhibitor 1 (Fig. 4). For the indirect inhibitor LX2931 protection in a mouse model of collagen-induced arthritis has been reported by researchers at Lexicon Pharmaceuticals following both prophylactic and therapeutic dosing regimens; this was further supported by efficacy in the rat adjuvantinduced model of rheumatoid arthritis [154]. Since efficacy was observed even in the presence of minimal lymphopenia, these authors speculate about a contributing role of additional mechanisms induced by Sgpl1 inhibition and S1P increase contributing to the antiinflammatory effect. LX2931 was evaluated in a phase 1 clinical trial in healthy human subjects [154]; the compound was efficacious in reducing peripheral T cell numbers by up to ~50% at the highest dose administered of 180 mg, and was well tolerated. However, a phase II study in RA failed to meet its primary endpoint, apparently due to subtherapeutic dosing [160]. Very recently, decreased atherosclerotic lesion development in LDL receptor deficient mice that were transplanted with Sgpl1−/− bone marrow was reported [161].

6.4. S1P lyase and T cell homeostasis 6.6. Safety aspects of S1P lyase inhibition The thymic retention induced by S1P1 modulators such as fingolimod was previously shown to strongly delay the physiological turnover of the peripheral T cell pool and to contribute to an overrepresentation of T cells with a memory phenotype over naïve T cells [158]. Because naïve T cells express the LN homing receptor CD62L and require S1P responsiveness for LN egress, they would be expected to remain more prominently represented in LN than in spleen. Indeed, in partially Sgpl1-deficient mice, the profoundly reduced pools of splenic T cells are strongly skewed towards cells of a memory phenotype, in particular CD4+ effector memory and CD8+ central memory cells [147]. Likewise, the representation of CD4+ effector memory and CD8+ central memory cells in the lymph nodes is increased. Thus, Sgpl1 deficiency induces a shift towards increased proportions of memory T cells. Inhibition of CD4+ T cells expressing the transcription factor FoxP3 constitute a critically important T regulatory cell subpopulation that dampens inflammatory responses and secures immunological tolerance [159]. Partial Sgpl1 deficiency leads to a profound increase in the proportions of FoxP3+ cells in both spleen and LN to more than twice the physiological levels [147]; this resulted in a weaker decline of total FoxP3+ T cells over other T cell subsets in spleen, and it led to a profound gain in absolute FoxP3+ T cell numbers in the lymph nodes. The reasons for these specific distribution effects on CD4+ FoxP3+ cells remain to be determined; they might include differential expression of S1P receptors, and/or differences in sensitivity or signaling pathways in response to S1P. 6.5. S1P lyase downmodulation protects from inflammation Partial Sgpl1 inhibition was shown to be protective in T cell dependent in vivo models, namely in delayed-type hypersensitivity as a classical inflammation model and in EAE [147]. In EAE, almost complete prevention of disease was observed; while spinal cord tissue of control

Development of Sgpl1 inhibitors is at an early stage; therefore, few data on the safety aspects of inhibitors are available, such as the reports on LX2931 mentioned above. However, one may learn about the potential safety liabilities of Sgpl1 inhibition from the genetic models. Fully Sgpl1-deficient mice do not thrive, feature major derailment of lipid metabolism and innate immune functions, such as disturbed neutrophil homeostasis, and die early in life [148,147,162,158,163]; they exhibit a storage disease phenotype with histoalveolar proteinosis, cardiomyopathy, osteopetrosis and urothelial vacuolization [148]. Sgpl1−/− chimeras were reported to display monocytosis and neutrophilia [161]. In contrast, the partially Sgpl1-deficient mice show normal weight gain and survival and feature normal numbers of blood neutrophils and monocytes [147,148] and no elevation of serum cytokines; histopathological findings appear to be largely absent from nonlymphoid tissues in the humanized knock-in mice featuring low levels of Sgpl1 activity [148]. Importantly, the partially Sgpl1-deficient mice exhibit profound reduction of peripheral T cells, similar to the full KO mice. In our view, the partially Sgpl1-deficient may be a more relevant model of treatment with a drug that may be unlikely to achieve complete inhibition of the target enzyme. However, in view of the very severe findings in the full KO mice, it is obvious that early estimation of a safety margin for future Sgpl1 inhibitors is of importance. Interestingly, the extent of S1P increase upon partial Sgpl1 deficiency in mice differs considerably between various tissues; notably, the highest increase was seen in the spleen and lymph nodes [147]. This is in line with data from mice treated with the Sgpl1 inhibitors LX2931 [154], THI and compound 1 (Fig. 4) (A. Billich, unpublished data), which induce the highest S1P increase in the lymphoid organs as well. It remains to be seen if the apparent selectivity of the inhibitors for the lymphoid tissues helps to attain a sufficient safety margin to avoid systemic on-target toxicity.

754

M. Bigaud et al. / Biochimica et Biophysica Acta 1841 (2014) 745–758

In the blood of the inducible Sgpl1-deficient mice and upon treatment with Sgpl1 inhibitors there is a minor increase in S1P, disproportionate to the one seen in tissues, and no increase in the plasma [147]. Therefore, Sgpl1 inhibition is not expected to lead to adverse effects via S1P receptors: Indeed, in telemetry studies in dogs and cynomolgus monkeys, LX2931 did not induce bradycardia [154]. Also, direct inhibitor 1 (Fig. 4) did not induce plasma leakage into rat lungs (M. Bigaud, unpublished data). In conclusion, Sgpl1 inhibitors have now been proposed as novel possible treatments of MS, arthritis, and other inflammatory and autoimmune diseases. However, it is important to note that the increase in pro-inflammatory S1P resulting from an inhibition of Sgpl1 may sustain (rather than inhibit) many inflammatory processes, despite reducing circulating T cells. The approach is therefore strikingly different from using functional- and competitive S1P receptor antagonists which reduce, rather than increase, S1P-S1P receptor signaling. Further studies will need to demonstrate whether Sgpl1 inhibitors offer an advantage in terms of efficacy and safety over other agents interfering with S1Pregulated lymphocyte trafficking. 7. Anti-S1P antibodies in early clinical development A murine monoclonal antibody against S1P (sphingomab, LT1002) was described in 2006 and was shown to bind and neutralize extracellular S1P in tissues at its physiologically relevant concentrations [195]. It also demonstrated strong antiangiogenic and antitumorigenic effects in various in vitro and in vivo models and was then successfully humanized for further development [196]. Its preclinical and clinical development as sonepcizumab or LT1009 has been reviewed in details elsewhere [197]. Briefly, for clinical development, sonepcizumab was formulated into two separate drug candidates that went recently through Phase I trials: 1—ASONEPTM, an oncology formulation assessed in subjects with refractory advanced solid tumors (Trial NCT00661414) [198]; 2—iSONEP™, an ocular formulation assessed in patients with neovascularization secondary to age-related macular degeneration (Trial NCT00767949) [199]. Overall, sonepcizumab is believed to act as a “molecular sponge”, to selectively and specifically absorb S1P from the extracellular fluid in disease-relevant tissues, lowering locally the effective concentration of pro-inflammatory S1P and ongoing trials will be key to evaluate the therapeutic potential of such an approach. 8. Conclusion There is now convincing body of evidence that the therapeutic effects of the prototype S1P receptor modulator fingolimod in MS are largely related to a down-modulation of S1P1 receptors on lymphocytes which prevents the invasion of autoaggressive T cells into the CNS. In astrocytes, down-modulation of S1P1 results in reduced astrogliosis, allowing restoration of productive astrocyte communication with other neural cells and the blood brain barrier. Interruption of the proinflammatory S1P–S1P1-cascade may further explain the recovery of nerve conduction and remyelination observed with therapeutic fingolimod treatment in animal models of MS. It remains to be determined whether additional effects on myelination result from a direct modulation of S1P1 and/or S1P5 on oligodendrocytes. In human MS, such mechanisms may explain the significant decrease in the number of inflammatory markers on brain magnetic resonance imaging in recent clinical trials, and the reduction of brain atrophy by fingolimod. Based on the above, next generation S1P receptor modulators were designed to either target S1P1 only, S1P1 and -5, or S1P1, -4, and -5, but not S1P2 and -3, and they are currently under clinical investigation. Competitive S1P1 antagonists have not been evaluated in humans so far, but may carry a reduced risk for bradycardia; however, they would also affect barrier function due to S1P1 blockade. More data are needed to judge on the potential of Sgpl1 inhibitors in disease, as treatment results in a major increase in pro-inflammatory S1P, whereas the

above mentioned functional- and competitive S1P receptor antagonists rather aim to reduce S1P action at its receptors. Similarly, anti-S1P antibody sphingomab was designed to antagonize, at least in part, the action of endogenous S1P at all the 5 S1P receptor subtypes. More clinical data are needed to conclude about the true potential of these new entities as 2nd generation of S1P-receptor modulators. Acknowledgements Authors are thankful to Dr. Daniela Angst for proof checking and editing the chemical structures. References [1] V. Brinkmann, Sphingosine 1-phosphate receptors in health and disease: mechanistic insights from gene deletion studies and reverse pharmacology, Pharmacol. Ther. 115 (2007) 84–105. [2] V. Brinkmann, A. Billich, T. Baumruker, P. Heining, R. Schmouder, G. Francis, S. Aradhye, P. Burtin, Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis, Nat. Rev. Drug Discov. 9 (2010) 883–897. [3] S. Spiegel, S. Milstien, Sphingosine-1-phosphate: an enigmatic signalling lipid, Nat. Rev. Mol. Cell Biol. 4 (2003) 397–407. [4] S. Spiegel, S. Milstien, The outs and the ins of sphingosine-1-phosphate in immunity, Nat. Rev. Immunol. 11 (2011) 403–415. [5] T. Mutoh, R. Rivera, J. Chun, Insights into the pharmacological relevance of lysophospholipid receptors, Br. J. Pharmacol. 165 (2012) 829–844. [6] J.G. Cyster, S.R. Schwab, Sphingosine-1-phosphate and lymphocyte egress from lymphoid organs, Annu. Rev. Immunol. 30 (2012) 69–94. [7] S.S. Chae, R.L. Proia, T. Hla, Constitutive expression of the S1P1 receptor in adult tissues, Prostaglandins Other Lipid Mediat. 73 (2004) 141–150. [8] H. Nishimura, T. Akiyama, I. Irei, S. Hamazaki, Y. Sadahira, Cellular localization of sphingosine-1-phosphate receptor 1 expression in the human central nervous system, J. Histochem. Cytochem. 58 (2010) 847–856. [9] C. Brana, M.J. Frossard, R. Pescini Gobert, N. Martinier, U. Boschert, T.J. Seabrook, Immunohistochemical detection of sphingosine-1-phosphate receptor 1 and 5 in human multiple sclerosis lesions, Neuropathol. Appl. Neurobiol. (Mar 31 2013), http://dx.doi.org/10.1111/nan.12048(Epub ahead of print). [10] A.J. MacLennan, P.R. Carney, W.J. Zhu, A.H. Chaves, J. Garcia, J.R. Grimes, An essential role for the H218/AGR16/Edg-5/LP(B2) sphingosine 1-phosphate receptor in neuronal excitability, Eur. J. Neurosci. 14 (2001) 203–209. [11] M. Kono, I.A. Belyantseva, A. Skoura, G.I. Frolenkov, M.F. Starost, J.L. Dreier, Deafness and stria vascularis defects in S1P2 receptor null mice, J. Biol. Chem. 282 (2007) 10690–10696. [12] D.R. Herr, N. Grillet, M. Schwander, R. Rivera, U. Muller, J. Chun, Sphingosine 1-phosphate (S1P) signaling is required for maintenance of hair cells mainly via activation of S1P2, J. Neurosci. 27 (2007) 1474–1478. [13] J.F. Lee, S. Gordon, R. Estrada, L. Wang, D.L. Siow, B.W. Wattenberg, D. Limonadze, M.J. Lee, Balance of S1P1 and S1P2 signaling regulates peripheral microvascular permeability in rat cremaster muscle vasculature, Am. J. Physiol. Heart Circ. Physiol. 296 (2009) H33–H42. [14] I. Ishii, B. Friedman, X. Ye, S. Kawamura, C. McGiffert, J.J. Contos, Selective loss of sphingosine 1-phosphate signaling with no obvious phenotypic abnormality in mice lacking its G protein-coupled receptor, LP(B3)/EDG-3, J. Biol. Chem. 276 (2001) 33697–33704. [15] I. Ishii, X. Ye, B. Friedman, S. Kawamura, J.J. Contos, M.A. Kingsbury, Marked perinatal lethality and cellular signaling deficits in mice null for the two sphingosine 1-phosphate (S1P) receptors, S1P(2)/LP(B2)/EDG-5 and S1P(3)/LP(B3)/EDG-3, J. Biol. Chem. 277 (2002) 25152–25159. [16] C.K. Means, C.Y. Xiao, Z. Li, T. Zhang, J.H. Omens, I. Ishii, Sphingosine 1-phosphate S1P2 and S1P3 receptor-mediated Akt activation protects against in vivo myocardial ischemia –reperfusion injury, Am. J. Physiol. Heart Circ. Physiol. 292 (2007) H2944–H2951. [17] M. Kono, Y. Mi, Y. Liu, T. Sasaki, M.L. Allende, Y.P. Wu, The sphingosine-1phosphate receptors S1P1, S1P2, and S1P3 function coordinately during embryonic angiogenesis, J. Biol. Chem. 279 (2004) 29367–29373. [18] M.H. Gräler, G. Bernhardt, M. Lipp, EDG6, a novel G-protein-coupled receptor related to receptors for bioactive lysophospholipids, is specifically expressed in lymphoid tissue, Genomics 53 (1998) 164–169. [19] T. Schulze, S. Golfier, C. Tabeling, K. Räbel, M.H. Gräler, M. Witzenrath, M. Lipp, Sphingosine-1-phospate receptor 4 (S1P₄) deficiency profoundly affects dendritic cell function and TH17-cell differentiation in a murine model, FASEB J. 25 (2011) 4024–4036. [20] D.S. Im, C.E. Heise, N. Ancellin, B.F. O'Dowd, G.J. Shei, R.P. Heavens, Characterization of a novel sphingosine 1-phosphate receptor, Edg-8, J. Biol. Chem. 275 (2000) 14281–14286. [21] K. Terai, T. Soga, M. Takahashi, M. Kamohara, K. Ohno, S. Yatsugi, Edg-8 receptors are preferentially expressed in oligodendrocyte lineage cells of the rat CNS, Neuroscience 116 (2003) 1053–1062. [22] M. Glickman, R.L. Malek, A.E. Kwitek-Black, H.J. Jacob, N.H. Lee, Molecular cloning, tissue-specific expression, and chromosomal localization of a novel nerve growth

M. Bigaud et al. / Biochimica et Biophysica Acta 1841 (2014) 745–758

[23]

[24]

[25]

[26]

[27] [28]

[29] [30] [31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42] [43]

[44]

[45]

[46]

[47]

factor-regulated G-protein coupled receptor, nrg-1, Mol. Cell. Neurosci. 14 (1999) 141–152. R.L. Malek, R.E. Toman, L.C. Edsall, S. Wong, J. Chiu, C.A. Letterle, Nrg-1 belongs to the endothelial differentiation gene family of G protein-coupled sphingosine-1phosphate receptors, J. Biol. Chem. 276 (2001) 5692–5699. V. Brinkmann, M.D. Davis, C.E. Heise, R. Albert, S. Cottens, R. Hof, C. Bruns, E. Prieschl, T. Baumruker, P. Hiestand, C.A. Foster, M. Zollinger, K.R. Lynch, The immune modulator FTY720 targets sphingosine 1-phosphate receptors, J. Biol. Chem. 277 (2002) 21453–21457. V. Brinkmann, K.R. Lynch, FTY720: targeting G-protein-coupled receptors for sphingosine 1-phosphate in transplantation and autoimmunity, Curr. Opin. Immunol. 14 (2002) 569–575. S. Mandala, R. Hajdu, J. Bergstrom, E. Quackenbush, J. Xie, J. Milligan, Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists, Science 296 (2002) 346–349. M.L. Allende, R.L. Proia, Sphingosine-1-phosphate receptors and the development of the vascular system, Biochim. Biophys. Acta 1582 (2002) 222–227. M. Matloubian, C.G. Lo, G. Cinamon, M.J. Lesneski, Y. Xu, V. Brinkmann, M.L. Allende, R.L. Proia, J.G. Cyster, Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1, Nature 427 (2004) 355–360. J.G. Cyster, Chemokines, sphingosine-1-phosphate, and cell migration in secondary lymphoid organs, Annu. Rev. Immunol. 23 (2005) 127–159. M.H. Gräler, E.J. Goetzl, The immunosuppressant FTY720 down-regulates sphingosine 1-phosphate G-protein-coupled receptors, FASEB J. 18 (2004) 551–553. P. Gergely, B. Nuesslein-Hildesheim, D. Guerini, V. Brinkmann, M. Traebert, C. Bruns, S. Pan, N.S. Gray, K. Hinterding, N.G. Cooke, A. Groenewegen, A. Vitaliti, T. Sing, O. Luttringer, J. Yang, A. Gardin, N. Wang, W.J. Crumb Jr., M. Saltzman, M. Rosenberg, E. Wallstrom, The selective sphingosine 1-phosphate receptor modulator BAF312 redirects lymphocyte distribution and has species-specific effects on heart rate, Br. J. Pharmacol. 167 (2012) 1035–1047. D. Angst, P. Janser, J. Quancard, P. Buehlmayer, F. Berst, L. Oberer, C. Beerli, M. Streiff, C. Pally, R. Hersperger, C. Bruns, F. Bassilana, B. Bollbuck, An oral sphingosine 1-phosphate receptor 1 (S1P(1)) antagonist prodrug with efficacy in vivo: discovery, synthesis, and evaluation, J. Med. Chem. 55 (2012) 9722–9734. Y. Fujii, T. Hirayama, H. Ohtake, N. Ono, T. Inoue, T. Sakurai, T. Takayama, K. Matsumoto, N. Tsukahara, S. Hidano, N. Harima, K. Nakazawa, Y. Igarashi, R. Goitsuka, Amelioration of collagen-induced arthritis by a novel S1P1 antagonist with immunomodulatory activities, J. Immunol. 188 (2012) 206–215. Y. Fujii, H. Ohtake, N. Ono, T. Hara, T. Sakurai, S. Takahashi, T. Takayama, Y. Fukasawa, F. Shiozawa, N. Tsukahara, T. Hirayama, Y. Igarashi, R. Goitsuka, Lymphopenia induced by a novel selective S1P(1) antagonist structurally unrelated to S1P, Biochim. Biophys. Acta 1821 (2012) 600–606. Y. Fujii, Y. Ueda, H. Ohtake, N. Ono, T. Takayama, K. Nakazawa, Y. Igarashi, R. Goitsuka, Blocking S1P interaction with S1P(1) receptor by a novel competitive S1P(1)-selective antagonist inhibits angiogenesis, Biochem. Biophys. Res. Commun. 419 (2012) 754–760. J. Quancard, B. Bollbuck, P. Janser, D. Angst, F. Berst, P. Buehlmayer, M. Streiff, C. Beerli, V. Brinkmann, D. Guerini, P.A. Smith, T.J. Seabrook, M. Traebert, K. Seuwen, R. Hersperger, C. Bruns, F. Bassilana, M. Bigaud, A potent and selective S1P(1) antagonist with efficacy in experimental autoimmune encephalomyelitis, Chem. Biol. 19 (2012) 1142–1151. M. Fujino, N. Funeshima, Y. Kitazawa, H. Kimura, H. Amemiya, S. Suzuki, Amelioration of experimental autoimmune encephalomyelitis in Lewis rats by FTY720 treatment, J. Pharmacol. Exp. Ther. 305 (2003) 70–77. J.W. Choi, S.E. Gardell, D.R. Herr, R. Rivera, C.W. Lee, K. Noguchi, S.T. Teo, Y.C. Yung, M. Lu, G. Kennedy, J. Chun, FTY720 (fingolimod) efficacy in an animal model of multiple sclerosis requires astrocyte sphingosine 1-phosphate receptor 1 (S1P1) modulation, Proc. Natl. Acad. Sci. U. S. A. 108 (2011) 751–766. B. Balatoni, M.K. Storch, E.M. Swoboda, V. Schönborn, A. Koziel, G.N. Lambrou, P.C. Hiestand, R. Weissert, C.A. Foster, FTY720 sustains and restores neuronal function in the DA rat model of MOG-induced experimental autoimmune encephalomyelitis, Brain Res. Bull. 74 (2007) 307–316. C.A. Foster, L.M. Howard, A. Schweitzer, E. Persohn, P.C. Hiestand, B. Balatoni, R. Reuschel, C. Beerli, M. Schwartz, A. Billich, Brain penetration of the oral immunomodulatory drug FTY720 and its phosphorylation in the central nervous system during experimental autoimmune encephalomyelitis: consequences for mode of action in multiple sclerosis, J. Pharmacol. Exp. Ther. 323 (2007) 469–475. A. Groves, Y. Kihara, J. Chun, Fingolimod: Direct CNS effects of sphingosine 1-phosphate (S1P) receptor modulation and implications in multiple sclerosis therapy, J. Neurol. Sci. 328 (2013) 9–18. T. Hla, V. Brinkmann, Sphingosine 1-phosphate (S1P): physiology and the effects of S1P receptor modulation, Neurology 76 (2011) S 3–S 8. D. Guerini, R. Movva, T.T.T. Tran, C. Hangartner, The novel immunomodulator FTY720 desensitizes and disrupts endogeneous S1P receptor signaling pathways in vitro, Am. J. Transplant. 4 (2004) 408–409. M. Jongsma, J. Van Unen, P.B. Van Loenen, M.C. Michel, S.L. Peters, A.E. Alewijnse, Different response patterns of several ligands at the sphingosine-1-phosphate receptor subtype 3 (S1P(3)), Br. J. Pharmacol. 156 (2009) 1305–1311. D. Guerini, C. Hangartner, T.T.T. Tran, M. Rao, The immunomodulator FTY720 mediated internalization of the S1P(1) receptor is independent from its Gi signaling properties in vitro, Am. J. Transplant. 5 (2005) 513. F. Mullershausen, F. Zecri, C. Cetin, A. Billich, D. Guerini, K. Seuwen, Persistent signaling induced by FTY720-phosphate is mediated by internalized S1P1 receptors, Nat. Chem. Biol. 5 (2009) 428–434. M.L. Oo, S. Thangada, M.T. Wu, C.H. Liu, K.R. Lynch, C.Y. Lin, Immunosuppressive and anti-angiogenic sphingosine 1-phosphate receptor-1 (S1P1) agonists induce

[48]

[49]

[50]

[51] [52]

[53]

[54] [55]

[56]

[57]

[58] [59] [60]

[61]

[62]

[63]

[64] [65] [66]

[67]

[68]

[69]

[70]

[71]

[72]

755

ubiquitinylation and proteosomal degradation of the receptor, J. Biol. Chem. 282 (2007) 9082–9089. M.L. Oo, S.H. Chang, S. Thangada, M.T. Wu, K. Rezaul, V. Blaho, S.I. Hwang, D.K. Han, T. Hla, Engagement of S1P -degradative mechanisms leads to vascular leak in mice, J. Clin. Invest. 121 (2011) 2290–2300. D. English, Z. Welch, A.T. Kovala, K. Harvey, O.V. Volpert, D.N. Brindley, Sphingosine 1-phosphate released from platelets during clotting accounts for the potent endothelial cell chemotactic activity of blood serum and provides a novel link between hemostasis and angiogenesis, FASEB J. 14 (2000) 2255–2265. N. Ancellin, C. Colmont, J. Su, Q. Li, N. Mittereder, S.S. Chae, S. Stefansson, G. Liau, T. Hla, Extracellular export of sphingosine kinase-1 enzyme, Sphingosine 1-phosphate generation and the induction of angiogenic vascular maturation, J. Biol. Chem. 277 (2002) 6667–6675. T. Hla, K. Venkataraman, J. Michaud, The S1P gradient-cellular sources and biological significance, Biochim. Biophys. Acta 1781 (2008) 477–482. B. Zemann, B. Kinzel, M. Muller, R. Reuschel, D. Mechtcheriakova, N. Urtz, F. Bornancin, T. Baumruker, A. Billich, Sphingosine kinase type 2 is essential for lymphopenia induced by the immunomodulatory drug FTY720, Blood 107 (2006) 1454–1458. C. Jaillard, S. Harrison, B. Stankoff, M.S. Aigrot, A.R. Calver, G. Duddy, Edg8/S1P5: an oligodendroglial receptor with dual function on process retraction and cell survival, J. Neurosci. 25 (2005) 1459–1469. J. Tamargo, R. Caballero, R. Gómez, C. Valenzuela, E. Delpón, Pharmacology of cardiac potassium channels, Cardiovasc. Res. 62 (2004) 9–33. M. Forrest, S.Y. Sun, R. Hajdu, J. Bergstrom, D. Card, G. Doherty, Immune cell regulation and cardiovascular effects of sphingosine 1-phosphate receptor agonists in rodents are mediated via distinct receptor subtypes, J. Pharmacol. Exp. Ther. 309 (2004) 758–768. M. Bunemann, B. Brandts, D.M. zu Heringdorf, C.J. van Koppen, K.H. Jakobs, L. Pott, Activation of muscarinic K+ current in guinea-pig atrial myocytes by sphingosine1-phosphate, J. Physiol. 489 (1995) 701–777. J. Guo, K.L. MacDonell, W.R. Giles, Effects of sphingosine 1-phosphate on pacemaker activity in rabbit sino-atrial node cells, Pflugers Arch. 438 (1999) 642–648. L.Y. Jan, Y.N. Jan, Heartfelt crosstalk: desensitization of the GIRK current, Nat. Cell Biol. 2 (2000) 165–167. M. Yamada, The role of muscarinic K(+) channels in the negative chronotropic effect of a muscarinic agonist, J. Pharmacol. Exp. Ther. 300 (2002) 681–687. C. Bode, S.C. Sensken, U. Peest, G. Beutel, F. Thol, B. Levkau, Z. Li, R. Bittman, T. Huang, M. Tölle, M. van der Giet, M.H. Gräler, Erythrocytes serve as a reservoir for cellular and extracellular sphingosine 1-phosphate, J. Cell. Biochem. 109 (2010) 1232–1243. J. Fujishiro, S. Kudou, S. Iwai, M. Takahashi, Y. Hakamata, M. Kinoshita, Use of sphingosine-1-phosphate 1 receptor agonist, KRP-203, in combination with a subtherapeutic dose of cyclosporine A for rat renal transplantation, Transplantation 82 (2006) 804–812. D. Mazurais, P. Robert, B. Gout, I. Berrebi-Bertrand, M.P. Laville, T. Calmels, Cell type-specific localization of human cardiac S1P receptors, J. Histochem. Cytochem. 50 (2002) 661–670. R.M. Fryer, A. Muthukumarana, P.C. Harrison, S. Nodop Mazurek, R. Rhonda Chen, K.E. Harrington, R.M. Dinallo, J.C. Horan, L. Patnaude, L.K. Modis, G.A. Reinhart, The clinically-tested S1P receptor agonists, FTY720 and BAF312, demonstrate subtype-specific sradycardia (S1P1) and hypertension (S1P3) in rat, PLoS ONE (2012) 7(misssing page number). M. Maceyka, K.B. Harikumar, S. Milstien, S. Spiegel, Sphingosine-1-phosphate signaling and its role in disease, Trends Cell Biol. 22 (2012) 50–60. B.J. McVerry, J.G. Garcia, In vitro and in vivo modulation of vascular barrier integrity by sphingosine 1-phosphate: mechanistic insights, Cell. Signal. 17 (2005) 131–139. B.A. Wilkerson, G.D. Grass, S.B. Wing, W.S. Argraves, K.M. Argraves, Sphingosine 1-phosphate (S1P) carrier-dependent regulation of endothelial barrier: high density lipoprotein (HDL)-S1P prolongs endothelial barrier enhancement as compared with albumin-S1P via effects on levels, trafficking, and signaling of S1P1, J. Biol. Chem. 287 (2012) 44645–44653. T. Sanchez, T. Estrada-Hernandez, J.H. Paik, M.T. Wu, K. Venkataraman, V. Brinkmann, Phosphorylation and action of the immunomodulator FTY720 inhibits vascular endothelial cell growth factor-induced vascular permeability, J. Biol. Chem. 278 (2003) 47281–47290. P.A. Singleton, S.M. Dudek, S.F. Ma, J.G. Garcia, Transactivation of sphingosine1-phosphate receptors is essential for vascular barrier regulation: novel role for hyaluronan and CD44 receptor family, J. Biol. Chem. 281 (2006) 34381–34393. M.G. Sanna, S.K. Wang, P.J. Gonzalez-Cabrera, A. Don, D. Marsolais, M.P. Matheu, S.H. Wei, I. Parker, E. Jo, W.C. Cheng, M.D. Cahalan, C.H. Wong, H. Rosen, Enhancement of capillary leakage and restoration of lymphocyte egress by a chiral S1P1 antagonist in vivo, Nat. Chem. Biol. 2 (2006) 434–441. J. Igarashi, P.A. Erwin, A.P. Dantas, H. Chen, T. Michel, VEGF induces S1P1 receptors in endothelial cells: implications for cross-talk between sphingolipid and growth factor receptors, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 10664–10669. M. Salvadori, K. Budde, B. Charpentier, J. Klempnauer, B. Nashan, L.M. Pallardo, J. Eris, F.P. Schena, U. Eisenberger, L. Rostaing, A. Hmissi, S. Aradhye, FTY720 0124 Study Group. FTY720 versus MMF with cyclosporine in de novo renal transplantation: a 1-year, randomized controlled trial in Europe and Australasia, Am. J. Transplant. 6 (2006) 2912–2921. K. Wu, F. Mercier, O.J. David, R.L. Schmouder, M. Looby, Population pharmacokinetics of fingolimod phosphate in healthy participants, J. Clin. Pharmacol. 52 (2012) 1054–1068.

756

M. Bigaud et al. / Biochimica et Biophysica Acta 1841 (2014) 745–758

[73] F. Okajima, Plasma lipoproteins behave as carriers of extracellular sphingosine 1-phosphate: is this an atherogenic mediator or an anti-atherogenic mediator? Biochim. Biophys. Acta 1582 (2002) 132–137. [74] A.P. Dantas, J. Igarashi, T. Michel, Sphingosine 1-phosphate and control of vascular tone, Am. J. Physiol. Heart Circ. Physiol. 284 (2003) 2045–2052. [75] J.R. Nofer, M. van der Giet, M. Tölle, I. Wolinska, K. von Wnuck Lipinski, H.A. Baba, HDL induces NO-dependent vasorelaxation via the lysophospholipid receptor S1P3, J. Clin. Invest. 113 (2004) 569–581. [76] M. Tölle, B. Levkau, P. Keul, V. Brinkmann, G. Giebing, G. Giebing, Immunomodulator FTY720 induces eNOS-dependent arterial vasodilatation via the lysophospholipid receptor S1P3, Circ. Res. 96 (2005) 913–920. [77] B. Weinberger, D.E. Heck, D.L. Laskin, J.D. Laskin, Nitric oxide in the lung: therapeutic and cellular mechanisms of action, Pharmacol. Ther. 84 (1999) 401–411. [78] F.L. Marasciulo, M. Montagnani, M.A. Potenza, Endothelin-1: the yin and yang on vascular function, Curr. Med. Chem. 13 (2006) 1655–1665. [79] F. Coussin, R.H. Scott, A. Wise, G.F. Nixon, Comparison of sphingosine 1-phosphateinduced intracellular signaling pathways in vascular smooth muscles: differential role in vasoconstriction, Circ. Res. 91 (2002) 151–157. [80] S. Salomone, S. Yoshimura, U. Reuter, M. Foley, S.S. Thomas, M.A. Moskowitz, S1P3 receptors mediate the potent constriction of cerebral arteries by sphingosine-1phosphate, Eur. J. Pharmacol. 469 (2003) 125–134. [81] D.G. Hemmings, Y. Xu, S.T. Davidge, Sphingosine 1-phosphate-induced vasoconstriction is elevated in mesenteric resistance arteries from aged female rats, Br. J. Pharmacol. 143 (2004) 276–284. [82] H. Zhou, K.S. Murthy, Distinctive G protein-dependent signaling in smooth muscle by sphingosine 1-phosphate receptors S1P1 and S1P2, Am. J. Physiol. Cell Physiol. 286 (2004) 1130–1138. [83] K.R. Watterson, P.H. Ratz, S. Spiegel, The role of sphingosine-1-phosphate in smooth muscle contraction, Cell. Signal. 17 (2005) 289–298. [84] W. Hu, S. Mahavadi, J. Huang, F. Li, K.S. Murthy, Characterization of S1P1 and S1P2 receptor function in smooth muscle by receptor silencing and receptor protection, Am. J. Physiol. Gastrointest. Liver Physiol. 291 (2006) 605–610. [85] H. Kume, N. Takeda, T. Oguma, S. Ito, M. Kondo, Y. Ito, Sphingosine 1-phosphate causes airway hyperreactivity by Rho-mediated myosin phosphatase inactivation, J. Pharmacol. Exp. Ther. 320 (2007) 766–773. [86] M. Uehata, T. Ishizaki, H. Satoh, T. Ono, T. Kawahara, T. Morishita, Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension, Nature 389 (1997) 990–994. [87] J.N. Lorenz, L.J. Arend, R. Robitz, R.J. Paul, A.J. MacLennan, Vascular dysfunction in S1P2 sphingosine 1-phosphate receptor knockout mice, Am. J. Physiol. Regul. Integr. Comp. Physiol. 292 (2007) R440–R446. [88] B.F. Peter, D. Lidington, A. Harada, H.J. Bolz, L. Vogel, S. Heximer, S. Spiegel, U. Pohl, S.S. Bolz, Role of sphingosine-1-phosphate phosphohydrolase 1 in the regulation of resistance artery tone, Circ. Res. 103 (2008) 315–324. [89] J.J. Hale, G. Doherty, L. Toth, S.G. Mills, R. Hajdu, C.A. Keohane, M. Rosenbach, J. Milligan, G.J. Shei, G. Chrebet, J. Bergstrom, D. Card, M. Forrest, S.Y. Sun, S. West, H. Xie, N. Nomura, H. Rosen, S. Mandala, Selecting against S1P3 enhances the acute cardiovascular tolerability of 3-(N-benzyl)aminopropyl-phosphonic acid S1P receptor agonists, Bioorg. Med. Chem. Lett. 14 (2004) 3501–3505. [90] S. Salomone, E.M. Potts, S. Tyndall, P.C. Ip, J. Chun, V. Brinkmann, C. Waeber, Analysis of shingosine 1-phosphate receptors involved in constriction of isolated cerebral arteries with receptor null mice and pharmacological tools, Br. J. Pharmacol. 153 (2008) 140–147. [91] K.P. Cusack, R.H. Stoffel, S1P(1) receptor agonists: assessment of selectivity and current clinical activity, Curr. Opin. Drug Discov. Devel. 13 (2010) 481–488. [92] H. Bolli, S. Abele, C. Binkert, R. Bravo, S. Buchmann, D. Bur, J. Gatfield, P. Hess, C. Kohl, C. Mangold, B. Mathys, K. Menyhart, C. Müller, O. Nayler, M. Scherz, G. Schmidt, V. Sippel, B. Steiner, D. Strasser, A. Treiber, T. Weller, 2-Iminothiazolidin-4-one derivatives as potent, orally active S1P1 receptor agonists, J. Med. Chem. 53 (2010) 4198–4211. [93] C.A. Miller, S1P Receptor agonists. The Royal Society of Chemistry, in: J.I. Levin, S. Laufer (Eds.), RSC Drug Discovery Series No.26—Anti-Inflammatory Drug Discovery, 2012, (Chapter 14). [94] A.J. Dyckman, Recent advances in the discovery and development of sphingosine-1-phosphate-1 receptor agonists, Ann. Reports Med. Chem. 47 (2012) 195–208. [95] S. Pan, S.N.S. Gray, W. Gao, Y. Mi, Y. Fan, X. Wang, T. Tuntland, J. Che, S. Lefebvre, Y. Chen, A. Chu, K. Hinterding, A. Gardin, P. End, P. Heining, C. Bruns, N.G. Cooke, B. Nuesslein-Hildesheim, Discovery of BAF312 (Siponimod), a potent and selective S1P receptor modulator, ACS Med. Chem. Lett. 4 (2013) 333–337. [96] K. Selmaj, D.K.B. Li, H.P. Hartung, B. Hemmer, L. Kappos, M.S. Freedman, O. Stüve, P. Rieckmann, X. Montalban, T. Ziemssen, L.Z. Auberson, H. Pohlmann, F. Mercier, F. Dahlke, E. Wallström, Siponimod for patients with relapsing–remitting multiple sclerosis (BOLD): an adaptive, dose-ranging, randomised, phase 2 study, Lancet (2013), http://dx.doi.org/10.1016/S1474-4422(13)70102-9 (Published on line). [97] L. Kappos, A. Bar-Or, B. Cree, R. Fox, G. Giovannoni, R. Gold, P. Vermersch, E. Lam, H. Pohlmann, L. Zhang-Auberson, A. Dandekar, E. Wallstroem, Siponimod (BAF312) for the treatment of secondary progressive multiple sclerosis: design of the phase 3 EXPAND trial, Neurology 1 (2013) P07.126. [98] E. Legangneux, A. Gardin, D. Johns, Dose titration of BAF312 attenuates the initial heart rate reducing effect in healthy subjects, Br. J. Clin. Pharmacol. 75 (2012) 831–841. [99] L. Piali, S. Froidevaux, P. Hess, O. Nayler, M.H. Bolli, E. Schlosser, C. Kohl, B. Steiner, M. Clozel, The selective sphingosine 1-phosphate receptor 1 agonist ponesimod

[100]

[101]

[102]

[103]

[104]

[105]

[106]

[107]

[108]

[109]

[110]

[111]

[112]

[113]

[114]

[115]

[116] [117] [118]

[119]

[120]

[121]

protects against lymphocyte-mediated tissue inflammation, J. Pharmacol. Exp. Ther. 337 (2011) 547–556. J. Gatfield, L. Monnier, O. Nayler, Ponesimod is a potent S1P1 receptor modulator causing efficient receptor internalisation, degradation and functional antagonism, 28th Congress of ECTRIMS, 2012. T. Olsson, A. Boster, O. Fernandez, M.S. Freedman, C. Pozzilli, D. Bach, O. Berkani, M. Mueller, T. Sidorenko, M. Melanson, Efficacy and safety of ponesimod, an oral, selective sphingosine 1-phosphate receptor-1 modulator, in patients with relapsingremitting multiple sclerosis: results from a phase IIb, randomised, double-blind, placebo-controlled trial, Mult. Scler. J. 18 (4 Suppl.) (2012) 152. O. Fernandez, C. Pozzilli, M.S. Freedman, T. Olsson, M. Melanson, D. Bach, O. Berkani, M. Mueller, T. Sidorenko, A. Boster, Pharmacodynamic effect, safety and tolerability of ponesimod, a selective sphingosine 1-phosphate receptor-1 modulator, in patients with relapsing-remitting multiple sclerosis, Mult. Scler. J. 18 (4 Suppl.) (2012) 417(P919). M.S. Freedman, T. Olsson, M. Melanson, O. Fernandez, A. Boster, D. Bach, O. Berkani, M. Mueller, T. Sidorenko, C. Pozzilli, Dose-dependent effect of ponesimod, an oral, selective sphingosine 1-phosphate receptor-1 modulator, on magnetic resonance imaging outcomes in patients with relapsing–remitting multiple sclerosis, Mult. Scler. J. 18 (4 Suppl.) (2012) 417(P923). P. Brossard, H. Derendorf, J. Xu, H. Maatouk, A. Halabi, J. Dingemanse, Pharmacokinetics and pharmacodynamics of Ponesimod, a selective S1P receptor modulator, in the first-in-human study, Br. J. Clin. Pharmacol. 18 (2013) 12129. J. Song, C. Matsuda, Y. Kai, T. Nishida, K. Nakajima, T. Mizushima, M. Kinoshita, T. Yasue, Y. Sawa, T. Ito, A novel sphingosine 1-phosphate receptor agonist, 2-amino2-propanediol hydrochloride (KRP-203), regulates chronic colitis in interleukin-10 gene-deficient mice, J. Pharmacol. Exp. Ther. 324 (2008) 276–283. J. Fujishiro, C. Suzuki, S. Kudou, T. Yasue, Y. Hakamata, M. Takahashi, K. Hashizumi, E. Kobayashi, Change from cyclosporine to combination therapy of mycophenolic acid with the new sphingosine-1-phosphate receptor agonist, KRP-203, prevents host nephrotoxicity and transplant vasculopathy in rats, J. Heart Lung Transplant. 25 (2006) 825–833. C. Suzuki, M. Takahashi, H. Morimoto, A. Izawa, H. Ise, J. Fujihiro, T. Murakami, J. Ishiyama, A. Nakada, J. Nakayama, K. Shimada, U. Ikeda, E. Kobayashi, Efficacy of mycophenolic acid combined with KRP-203, a novel immunomodulator, in a rat heart transplantation model, J. Heart Lung Transplant. 25 (2006) 302–309. M. Takahashi, H. Shimizu, T. Murakami, S. Enosawa, C. Suzuki, Y. Takeno, T. Kaneko, Y. Hakamata, S. Kudou, S. Isawa, T. Yasue, E. Kobayashi, A novel immunomodulator KRP-203 combined with cyclosporine prolonged graft survival and abrogated transplant graft vasculopathy in rat graft allografts, Transplant. Proc. 37 (2005) 143–145. R. Ogawa, M. Takahashi, S. Hirose, H. Morimoto, H. Ise, T. Murakami, T. Yasue, K. Kuriyama, M. Hongo, E. Kobayashi, U. Ikeda, A novel sphingosine-1-phosphate receptor agonist KRP-203 attenuates rat autoimmune myocarditis, Biochem. Biophys. Res. Commun. 361 (2007) 621–628. T. Kaneko, T. Murakami, H. Kawana, M. Takahashi, T. Yasue, E. Kobayashi, Sphingosine-1-phosphate agonists suppress concanavalin A-induced hepatic injury in mice, Biochem. Biophys. Res. Commun. 345 (2006) 85–92. S.E. Wenderfer, S.M. Stepkowski, M.C. Braun, Increased survival and reduced renal injury in MRL/lpr mice treated with a novel sphingosine-1-phosphate receptor agonist, Kidney Int. 74 (2008) 1319–1326. M. Khattar, R. Deng, B.D. Kahan, P.M. Schroder, T. Phan, L.P. Rutzky, S.M. Stepkowski, Novel sphingosine-1-phosphate receptor modulator KRP203 combined with locally delivered regulatory T cells induces permanent acceptance of pancreatic islet allografts, Transplantation 95 (2013) 919–927. F. Poti, F. Gualtieri, S. Sacchi, G. Weissen-Plenz, G. Varga, M. Brodde, C. Weber, M. Simoni, J.R. Nofer, KRP-203, Sphingosine 1-phosphate receptor type 1 agonist, ameliorates atherosclerosisin LDR-R-/- mice, Atherioscler, Thromb. Vasc. Biol. 33 (2013) 1505–1512. T. Komiya, K. Sato, H. Shioya, Y. Inagaki, H. Hagiya, R. Kozaki, M. Imai, Y. Takada, T. Maeda, H. Kurata, M. Kurono, R. Suzuki, K. Otsuki, H. Habashita, S. Nakade, Efficacy and immunomodulatory actions of ONO-4641, a novel selective agonist for sphingosine 1-phosphate receptors 1 and 5, in preclinical models of multiple sclerosis, Clin. Exp. Immunol. 171 (2013) 54–62. T. Ohno, C. Hasegawa, S. Nakade, J. Kitagawa, N. Honda, M. Ogawa, The prediction of human response to ONO-4641, a sphingosine 1-phosphate receptor modulator, from preclinical data based on pharmacokinetic-pharmacodynamic modeling, Biopharm. Drug Dispos. 31 (2010) 396–406. A. Goodman, Novel agent reduces MS lesions in early trial, Neurol. (2012) 40(Today june 7). M. Wadman, Box 1: the molecular libraries program gets the axe, Nat. Biotech. 40 (2012) 309–312. J. Brooks, R. Peach, F. Scott, G. Timony, J. Hartung, M. Boehm, H. Rosen, S. Gujrathi, RPC1063, a potent, selective S1P1 receptor modulator, is active in a therapeutic EAE model and exhibits favourable PK/PD properties in healthy volunteers, 28th Congress of ECTRIMS, 2012. A. Olson, J. Hartung, G. Timony, R. Peach, M. Boehm, H. Rosen, H. Smith, C. Pan, J. Brooks, S. Gujrathi, Safety and tolerability of orally administered RPC1063, a novel S1P1 receptor modulator, in healthy adult volunteers, results of a phase 1 study, Neurology 80 (2013) P01.178. T. Nishi, S. Miyazaki, T. Takemoto, K. Suzuki, Y. Iio, K. Nakajima, T. Ohnuki, Y. Kawase, F. Nara, S. Inaba, T. Izumi, H. Yuita, K. Oshima, H. Doi, R. Inoue, W. Tomisato, T. Kagari, T. Shimozato, Discovery of CS-0777: a potent, selective and orally active S1P1 agonist, ACS Med. Chem. Lett. 2 (2011) 368. T. Shimozato, W. Tomisato, H. Doi, T. Kagari, R. Inoue, H. Yuita, K. Oshima, T. Makino, K. Suzuki, S. Sato, Y. Kawase, K. Yonesu, M. Tamura, F. Nara, Preclinical

M. Bigaud et al. / Biochimica et Biophysica Acta 1841 (2014) 745–758

[122]

[123]

[124]

[125]

[126] [127]

[128]

[129]

[130]

[131]

[132]

[133]

[134]

[135]

[136]

[137]

[138]

[139]

[140]

[141]

[142]

[143]

[144] [145]

evaluation of a selective sphingosine 1-phosphate receptor-1 modulator: in vitro binding selectivity and effects on experimental autoimmune encephalomyelitis in rats, Mult. Scler. J. 15 (2009) S78. S.I. Inaba, K. Tanaka, W. Tomisato, H. Yuita, H. Doi, R. Inoue, K. Oshima, T. Kagari, T. Shimozato, Exposure-response analysis of CS-0777, a sphingosine 1-phosphate receptor modulator, in preclinical models, Mult. Scler. J. 15 (2009) S78. J.B. Moberly, S. Rohatagi, H. Zahir, C. Hsu, R.J. Noveck, K.E. Truitt, Pharmacological modulation of peripheral T and B lymphocytes by a selective sphingosine 1-phosphate receptor-1 modulator, J. Clin. Pharmacol. 52 (2012) 996–1006. J.B. Moberly, D.M. Ford, H. Zahir, S. Chen, T. Mochizuki, K.E. Truitt, T.L. Vollmer, Pharmacological effects of CS-0777, a selective sphingosine 1-phosphate receptor-1modulator: results from a 12-week, open-label pilot study in multiple sclerosis patients, J. NeuroIimmunol. 246 (2012) 100–107. D. Lepley, J.H. Paik, T. Hla, F. Ferrer, The G protein-coupled receptor S1P2 regulates Rho/Rho kinase pathway to inhibit tumor cell migration, Cancer Res. 65 (2005) 3788–3795. C.E. Chalfant, S. Spiegel, Sphingosine 1-phosphate and ceramide 1-phosphate: expanding roles in cell signaling, J. Cell Sci. 118 (2005) 4605–4612. J. Chun, H. Rosen, Lysophospholipid receptors as potential drug targets in tissue transplantation and autoimmune diseases, Curr. Pharm. Des. 12 (2006) 161–171. D.T. Bolick, S. Srinivasan, K.W. Kim, M.E. Hatley, J.J. Clemens, A. Whetzel, N. Ferger, T.L. Macdonald, M.D. Davis, P.S. Tsao, K.R. Lynch, C.C. Hedrick, Sphingosine-1-phosphate prevents tumor necrosis factor-α-mediated monocyte adhesion to aortic endothelium in mice, Arterioscler. Throm. Vasc. Biol. 25 (2005) 976–981. A.M. Whetzel, D.T. Bolick, S. Srinivasan, T.L. Macdonald, M.A. Morris, K. Ley, C.C. Hedrick, Sphingosine-1 phosphate prevents monocyte/endothelial interactions in type 1 diabetic NOD mice through activation of the S1P1 receptor, Circ. Res. 99 (2006) 731–739. Y.Y. Lan, D. Tokita, Z. Wang, H.C. Wang, J. Zhan, V. Brinkmann, A.W. Thomson, Sphingosine 1-phosphate receptor agonism impairs skin dendritic cell migration and homing to secondary lymphoid tissue: association with prolonged allograft survival, Transplant. Immunol. 20 (2008) 88–94. M. Idzko, H. Hammad, M. van Nimwegen, M. Kool, T. Müller, T. Soullié, M.A.M. Willart, D. Hijdra, H.C. Hoogsteden, B.N. Lambrecht, Local application of FTY720 to the lung abrogates experimental asthma by altering dendritic cell function, J. Clin. Invest. 116 (2006) 2935–2944. P.S. Jolly, M. Bektas, A. Olivera, C. Gonzalez-Espinosa, R.L. Proia, J. Rivera, S. Milstien, S. Spiegel, Transactivation of sphingosine-1-phosphate receptors by Fc RI triggering is required for normal mast cell degranulation and chemotaxis, J. Exp. Med. 199 (2004) 959–970. P.S. Jolly, M. Bektas, K.R. Watterson, H. Sankala, S.G. Payne, S. Milstien, S. Spiegel, Expression of SphK1 impairs degranulation and motility of RBL-2H3 mast cells by desensitizing S1P receptors, Blood 105 (2005) 4736–4742. F. Roviezzo, F. Del Galdo, G. Abbate, M. Bucci, B. D'Agostino, E. Antunes, G. De Dominicis, L. Parente, F. Rossi, G. Cirino, R. De Palma, Human eosinophil chemotaxis and selective in vivo recruitment by sphingosine 1-phosphate, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 11170–11175. O.H. Lee, Y.M. Kim, Y.M. Lee, E.J. Moon, D.J. Lee, J.H. Kim, K.W. Kim, Y.G. Kwon, Sphingosine 1-phosphate induces angiogenesis: its angiogenic action and signaling mechanism in human umbilical vein endothelial cells, Biochem. Biophys. Res. Comm. 264 (1999) 743–750. C. McGiffert, J.J.A. Contosb, B. Friedmanb, J. Chun, Embryonic brain expression analysis of lysophospholipid receptor genes suggests roles for s1p1 in neurogenesis and s1p1-3 in angiogenesis, FEBS Lett. 531 (2002) 103–108. Y. Gon, M.R. Wood, W.B. Kiosses, E. Jo, M.G. Sanna, J. Chun, H. Rosen, S1P3 receptor-induced reorganization of epithelial tight junctions compromises lung barrier integrity and is potentiated by TNF, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 9270–9275. G. Theilmeier, C. Schmidt, J. Herrmann, P. Keul, M. Schäfers, I. Herrgott, J. Mersmann, J. Larmann, S. Hermann, J. Stypmann, O. Schober, R. Hildebrand, R. Schulz, G. Heusch, M. Haude, K. von Wnuck Lipinski, C. Herzog, M. Schmitz, R. Erbel, J. Chun, B. Levkau, High-density lipoproteins and their constituent, sphingosine-1-phosphate, directly protect the heart against ischemia/reperfusion injury in vivo via the S1P3 lysophospholipid receptor, Circulation 114 (2006) 1403–1409. K. Mizugishi, T. Yamashita, A. Olivera, G.F. Miller, S. Spiegel, R.L. Proia, Essential role for sphingosinekinases in neural and vascular development, Mol. Cell. Biol. 25 (2005) 11113–11121. F.R. Postma, K. Jalink, T. Hengeveld, W.H. Moolenaar, Sphingosine-1-phosphate rapidly induces Rho-dependent neurite retractation: action through a specific cell receptor, J. EMBO 15 (1996) 2388–2392. K. Sato, H. Tomura, Y. Igarashi, M. Ui, F. Okalima, Exogenous sphingosine 1-phosphate induces neurite retraction possibly through a cell surface receptor in PC12 cells, Biochem. Biophys. Res. Commun. 240 (1997) 329–334. A. Pebay, M. Toutant, J. Premont, C.F. Calvo, L. Venance, J. Cordier, J. Glowinski, M. Tence, Sphingosine-1-phosphate induces proliferation of astrocytes: regulation by intracellular signalling cascades, Eur. J. Neorosci. 13 (2001) 2067–2076. N. Rouach, A. Pébay, W. Même, J. Cordier, P. Ezan, E. Etienne, C. Giaume, M. Tencé, S1P inhibits gap junctions in astrocytes: involvement of G and Rho GTPase/ROCK, Eur. J. Neurosci. 23 (2006) 1453–1464. A. Kumar, J.D. Saba, Lyase to live by: sphingosine phosphate lyase as a therapeutic target, Expert Opin. Ther. Targets 13 (2009) 1013–1025. M. Serra, J.D. Saba, Sphingosine 1-phosphate lyase a key regulator of sphingosine 1-phosphate signaling and function, Adv. Enzyme Regul. 50 (2010) 349–362.

757

[146] P.P. van Veldhoven, G.P. Mannaerts, Sphingosine-phosphate lyase, Adv. Lipid Res. 26 (1993) 69–98. [147] A. Billich, T. Baumruker, C. Beerli, M. Bigaud, C. Bruns, T. Calzascia, A. Isken, B. Kinzel, E. Loetscher, B. Metzler, M. Mueller, B. Nuesslein-Hildesheim, B. Kleylein-Sohn, Partial deficiency of sphingosine-1-phosphate lyase confers protection in experimental autoimmune encephalomyelitis, PLOS One 8 (2013) e59630. [148] P. Vogel, M.S. Donoviel, R. Read, G.M. Hansen, J. Hazlewood, S.J. Anderson, W. Sun, J. Swaffield, T. Oravecz, Incomplete inhibition of sphingosine 1-phosphate lyase modulates immune system function yet prevents early lethality and nonlymphoid lesions, PLoS One 4 (2009) e4112. [149] S. Weiler, N. Braendlin, B. Oberhauser, A. Billich, S. Honnappa, C. Beerli, Orally active 7-substituted (4-benzyl-phthalazin-1-yl)-2-methyl-piperazin-1-yl]nicotinonitriles as active-site inhibitors of sphingosine-1-phosphate lyase for the treatment of multiple sclerosis, 2013. (submitted for publication at J. Med. Chem.). [150] A. Billich, C. Beerli, R. Bergmann, C. Bruns, E. Loetscher, Cellular assay for the characterization of sphingosine-1-phosphate lyase inhibitors, Anal. Biochem. 434 (2013) 247–253. [151] E. Loetscher, K. Schneider, C. Beerli, A. Billich, Assay to measure the secretion of sphingosine-1-phosphate from cells induced by S1P lyase inhibitors, Biochem. Biophys. Res. Commun. 433 (2013) 345–348. [152] R. Gugasyan, A. Coward, L. O'Connor, K. Shortman, R. Scollay, Emigration of mature T cells from the thymus is inhibited by the imidazole-based compound 2-acetyl4-tetrahydroxybutylimidazole, Immunol. 93 (1998) 398–404. [153] J.T. Bagdanoff, M.S. Donoviel, A. Nouraldeen, J. Tarver, Q. Fu, M. Carlsen, T.C. Jessop, H. Zhang, J. Hazelwood, H. Nguyen, S.D.P. Baugh, M. Gardyan, K.M. Terranova, J. Barbosa, J. Yan, M. Bednarz, S. Layek, L.F. Courtney, J. Taylor, A.M. DigeorgeFoushee, S. Gopinathan, D. Bruce, T. Smith, L. Moran, E. O'Neill, J. Kramer, Z. Lai, S.D. Kimball, Q. Liu, W. Sun, S. Yu, J. Swaffield, A. Wilson, A. Main, K.G. Carson, T. Oravecz, D.J. Augeri, Inhibition of sphingosine-1-phosphate lyase for the treatment of autoimmune disorders, J. Med. Chem. 52 (2009) 3941–3953. [154] J.T. Bagdanoff, M.S. Donoviel, A. Nouraldeen, M. Carlsen, C. Jessop, J. Tarver, S. Aleem, L. Dong, H. Zhang, L. Boteju, J. Hazelwood, J. Yan, M. Bednarz, S. Layek, I.B. Owusu, S. Gopinathan, L. Moran, J. Lai Kramer, S.D. Kimball, P. Yalamanchili, W.E. Heydorn, K.S. Frazier, B. Brooks, P. Brown, A. Wilson, W.K. Sonnenburg, A. Main, K.G. Carson, T. Oravecz, D.J. Augeri, Inhibition of sphingosine 1-phosphate lyase for the treatment of rheumatoid arthritis: discovery of (E)-1-(4-((1R2S3R)1234-tetrahydroxybutyl)-1H-imidazol-2-yl)ethanone oxime (LX2931) and (1R2S3R)-1-(2-(isoxazol-3-yl)-1H-imidazol-4-yl)butane-1234-tetraol (LX2932), J. Med. Chem. 253 (2010) 8650–8662. [155] S.R. Schwab, J.P. Pereira, M. Matloubian, Y. Xu, Y. Huang, J.G. Cyster, Lymphocyte sequestration through S1P lyase inhibition and disruption of S1P gradients, Science 309 (2005) 1735–1739. [156] X.Q. Yu, J. Kramer, L. Moran, E. O'Neill, A. Nouraldeen, T. Oravecz, A.G. Wilson, Pharmacokinetic/pharmacodynamic modelling of 2-acetyl-4(5)-tetrahydroxybutyl imidazole-induced peripheral lymphocyte sequestration through increasing lymphoid sphingosine 1-phosphate, Xenobiotica 40 (2010) 350–356. [157] M.L. Allende, J.L. Dreier, S. Mandala, R.L. Proia, Expression of the sphingosine 1-phosphate receptor, S1P1, on T-cells controls thymic emigration, J. Biol. Chem. 279 (2004) 15396–15401; O.K.M.L. Allende, M. Bektas, B.G. Lee, E. Bonifacino, J. Kang, G. Tuymetova, W. Chen, J.D. Saba, R.L. Proia, Sphingosine-1-phosphate lyase deficiency produces a proinflammatory response while impairing neutrophil trafficking, J. Biol. Chem. 286 (2011) 7348–7358. [158] B. Metzler, P. Gfeller, G. Wieczorek, J. Li, B. Nuesslein-Hildesheim, A. Katopodis, M. Mueller, V. Brinkmann, Modulation of T cell homeostasis and alloreactivity under continuous FTY720 exposure, Int. Immunol. 20 (2008) 633–644. [159] S. Sehrawat, T. Rouse, Anti-inflammatory effects of FTY720 against viral-induced immunopathology: role of drug-induced conversion of T cells to become Foxp3+ regulators, J. Immunol. 180 (2008) 7636–7647. [160] R. Fleischmann, Novel small-molecular therapeutics for rheumatoid arthritis, Curr. Opin. Rheumatol. 24 (2012) 335–341. [161] M. Bot, P.P. Van Veldhoven, S.C. de Jager, J. Johnson, N. Nijstad, P.J. Van Santbrink, M.M. Westra, G. Van Der Hoeven, M.J. Gijbels, C. Müller-Tidow, G. Varga, U.J. Tietge, J. Kuiper, T.J. Van Berkel, J.R. Nofer, I. Bot, E.A. Biessen, Hematopoietic sphingosine 1-phosphate lyase deficiency decreases atherosclerotic lesion development in LDL-receptor deficient mice, PLoS One 8 (2013) e63360. [162] M. Bektas, M.L. Allende, B.G. Lee, W. Chen, M.J. Amar, A.T. Remaley, J.D. Saba, R.L. Proia, Sphingosine 1-phosphate lyase deficiency disrupts lipid homeostasis in liver, J. Biol. Chem. 285 (2010) 10880–10889. [163] J. Schmahl, C.S. Raymond, S. Soriano, PDGF signaling specificity is mediated through multiple immediate early genes, Nat. Genet. 39 (2007) 52–60. [164] K. Yonesu, Y. Kawase, T. Inoue, N. Takagi, J. Tsuchida, Y. Takuwa, S. Kumakura, F. Nara, Involvement of sphingosine-1-phosphate and S1P1 in angiogenesis: analyses using a new S1P1 antagonist of non-sphingosine-1-phosphate analog, Biochem. Pharmacol. 77 (2009) 1011–1020. [165] F.W. Foss Jr., A.H. Snyder, M.D. Davis, M. Rouse, M.D. Okusa, K.R. Lynch, T.L. Macdonald, Synthesis and biological evaluation of gamma-aminophosphonates as potent, subtype-selective sphingosine 1-phosphate receptor agonists and antagonists, Bioorg. Med. Chem. 15 (2007) 663–677. [166] P.C. Kennedy, R. Zhu, T. Huang, J.L. Tomsig, T.P. Mathews, M. David, O. Peyruchaud, T.L. Macdonald, K.R. Lynch, Characterization of a sphingosine 1-phosphate receptor antagonist prodrug, J. Pharmacol. Exp. Ther. 338 (2011) 879–889. [167] S.H. Wei, H. Rosen, M.P. Matheu, M.G. Sanna, S.K. Wang, E. Jo, C.H. Wong, I. Parker, M.D. Cahalan, Sphingosine 1-phosphate type 1 receptor agonism

758

[168]

[169]

[170]

[171]

[172]

[173]

[174]

[175]

[176]

[177]

[178]

[179]

[180]

[181]

[182]

[183]

[184]

M. Bigaud et al. / Biochimica et Biophysica Acta 1841 (2014) 745–758 inhibits transendothelial migration of medullary T cells to lymphatic sinuses, Nat. Immunol. 6 (2005) 1228–1235. M.G. Sanna, J. Liao, E. Jo, C. Alfonso, M.Y. Ahn, M.S. Peterson, B. Webb, S. Lefebvre, J. Chun, N. Gray, H. Rosen, Sphingosine 1-phosphate (S1P) receptor subtypes S1P1 and S1P3, respectively, regulate lymphocyte recirculation and heart rate, J. Biol. Chem. 279 (2004) 13839–13848. G. Tarrason, M. Auli, S. Mustafa, V. Dolgachev, M.T. Domenech, N. Prats, M. Dominguez, R. Lopez, N. Aguilar, M. Calbet, M. Pont, G. Milligan, S.L. Kunkel, N. Godessart, The sphingosine-1-phosphate receptor-1 antagonist, W146, causes early and short-lasting peripheral blood lymphopenia in mice, Int. Immunopharmacol. 11 (2011) 1773–1779. W.J. Valentine, G.N. Kiss, J. Liu, E.S.M. Gotoh, K. Murakami-Murofushi, T.C. Pham, D.L. Baker, A.L. Parrill, X. Lu, C. Sun, R. Bittman, N.J. Pyne, G. Tigyi, (S)-FTY720vinylphosphonate, an analogue of the immunosuppressive agent FTY720, is a pan-antagonist of sphingosine 1-phosphate GPCR signaling and inhibits autotaxin activity, Cell. Signal. 22 (2010) 1543–1553. C. Alfonso, M.G. McHeyzer-Williams, H. Rosen, CD69 down-modulation and inhibition of thymic egress by short- and long-term selective chemical agonism of sphingosine 1-phosphate receptors, Eur. J. Immunol. 36 (2006) 149–159. L.R. Shiow, D.B. Rosen, N. Brdickova, Y. Xu, J. An, L.L. Lanier, J.G. Cyster, M. Matloubian, CD69 acts downstream of interferon-alpha/beta to inhibit S1P1 and lymphocyte egress from lymphoid organs, Nature 440 (2006) 540–544. Y. Liu, R. Wada, T. Yamashita, Y. Mi, C.X. Deng, J.P. Hobson, H.M. Rosenfeldt, V.E. Nava, S.S. Chae, M.J. Lee, C.H. Liu, T. Hla, S. Spiegel, R.L. Proia, Edg-1, the G protein-coupled receptor for sphingosine-1-phosphate, is essential for vascular maturation, J. Clin. Invest. 106 (2000) 951–961. F. Wang, J.R. Van Brocklyn, J.P. Hobson, S. Movafagh, Z. Zukowska-Grojec, S. Milstien, S. Spiegel, Sphingosine 1-phosphate stimulates cell migration through a G(i)-coupled cell surface receptor. Potential involvement in angiogenesis, J. Biol. Chem. 274 (1999) 35343–35350. S.S. Chae, J.H. Paik, M.L. Allende, R.L. Proia, T. Hla, Regulation of limb development by the sphingosine 1-phosphate receptor S1p1/EDG-1 occurs via the hypoxia/VEGF axis, Dev. Biol. 268 (2004) 441–447. S.S. Chae, J.H. Paik, H. Furneaux, T. Hla, Requirement for sphingosine 1-phosphate receptor-1 in tumor angiogenesis demonstrated by in vivo RNA interference, J. Clin. Invest. 114 (2004) 1082–1089. K. LaMontagne, A. Littlewood-Evans, C. Schnell, T. O'Reilly, L. Wyder, T. Sanchez, B. Probst, J. Butler, A. Wood, G. Liau, E. Billy, A. Theuer, T. Hla, J. Wood, Antagonism of sphingosine-1-phosphate receptors by FTY720 inhibits angiogenesis and tumor vascularization, Cancer Res. 66 (2006) 221–231. M.A. Ibrahim, H.W.B. Johnson, J. Won Jeong, G.L. Lewis, X. Shi, R.T. Noguchi, M. Williams, J.W. Leahy, J.M. Nuss, J. Woolfrey, M. Banica, F. Bentzien, Y.C. Chou, A. Gibson, N. Heald, P. Lamb, L. Mattheakis, D. Matthews, A. Shipway, X. Wu, W. Zhang, S. Zhou, G. Shankar, Discovery of a novel class of potent and orally bioavailable sphingosine 1-phosphate receptor 1 antagonists, J. Med. Chem. 55 (2012) 1368–1381. M.H. Li, T. Sanchez, H. Yamase, T. Hla, M.L. Oo, A. Pappalardo, K.R. Lynch, C.Y. Lin, F. Ferrer, S1P/S1P1 signaling stimulates cell migration and invasion in Wilms tumor, Cancer Lett. 276 (2009) 171–179. F. Wang, W. Tan, D. Guo, S. He, Reduction of CD4 positive T cells and improvement of pathological changes of collagen-induced arthritis by FTY720, Eur. J. Pharmacol. 573 (2007) 230–240. E. Esplugues, D. Sancho, J. Vega-Ramos, C. Martinez, U. Syrbe, A. Hamann, P. Engel, F. Sanchez-Madrid, P. Lauzurica, Enhanced antitumor immunity in mice deficient in CD69, J. Exp. Med. 197 (2003) 1093–1106. J.E. Hughes, S. Srinivasan, K.R. Lynch, R.L. Proia, P. Ferdek, C.C. Hedrick, Sphingosine-1-phosphate induces an antiinflammatory phenotype in macrophages, Circ. Res. 102 (2008) 950–958. K. Budde, R.L. Schmouder, R. Brunkhorst, B. Nashan, P.W. Lucker, T. Mayer, S. Choudhury, A. Skerjanec, G. Kraus, H.H. Neumayer, First human trial of FTY720, a novel immunomodulator, in stable renal transplant patients, J. Am. Soc. Nephrol. 13 (2002) 1073–1083. V. Brinkmann, D. Pinschewer, K. Chiba, L. Feng, FTY720: a novel transplantation drug that modulates lymphocyte traffic rather than activation, Trends Pharmacol. Sci. 21 (2000) 49–52.

[185] S.M. Stepkowski, M. Wang, X. Qu, J. Yu, M. Okamoto, N. Tejpal, B.D. Kahan, Synergistic interaction of FTY720 with cyclosporine or sirolimus to prolong heart allograft survival, Transplant. Proc. 30 (1998) 2214–2216. [186] L. Kappos, J. Antel, G. Comi, X. Montalban, P. O'Connor, C.H. Polman, T. Haas, A.A. Korn, G. Karlsson, E.W. Radue, Oral fingolimod (FTY720) for relapsing multiple sclerosis, N. Engl. J. Med. 355 (2006) 1124–1140. [187] S.M. Cahalan, P.J. Gonzalez-Cabrera, N. Nguyen, M. Guerrero, E.A. Cisar, N.B. Leaf, S.J. Brown, E. Roberts, H. Rosen, Sphingosine 1-phosphate receptor 1 (S1P(1)) upregulation and amelioration of experimental autoimmune encephalomyelitis by an S1P(1) antagonist, Mol. Pharmacol. 83 (2013) 316–321. [188] H. Obinata, T. Hla, Fine-tuning S1P therapeutics, Chem. Biol. 19 (2012) 1080–1082. [189] C.G. Lo, Y. Xu, R.L. Proia, J.G. Cyster, Cyclical modulation of sphingosine1-phosphate receptor 1 surface expression during lymphocyte recirculation and rela/observed that fingolimod had protectivetionship to lymphoid organ transit, J. Exp. Med. 201 (2005) 291–301. [190] J.G. Garcia, F. Liu, A.D. Verin, A. Birukova, M.A. Dechert, W.T. Gerthoffer, J.R. Bamberg, D. English, Sphingosine 1-phosphate promotes endothelial cell barrier integrity by Edg-dependent cytoskeletal rearrangement, J. Clin. Invest. 108 (2001) 689–701. [191] T. Abbasi, J.G. Garcia, Sphingolipids in lung endothelial biology and regulation of vascular integrity, Handb. Exp. Pharmacol. 216 (2013) 201–226. [192] F.R. Curry, R.H. Adamson, Tonic regulation of vascular permeability, Acta Physiol. (Oxf) 207 (2013) 628–649. [193] L. Koyrakh, M.I. Roman, V. Brinkmann, K. Wickman, The heart rate decrease caused by acute FTY720 administration is mediated by the G protein-gated potassium channel I, Am. J. Transplant. 5 (2005) 529–536. [194] X. Peng, P.M. Hassoun, S. Sammani, B.J. McVerry, M.J. Burne, H. Rabb, D. Pearse, R.M. Tuder, J.G. Garcia, Protective effects of sphingosine-1-phosphate in murine endotoxin-induced inflammatory lung injury, Am. J. Respir. Crit. Care Med. 169 (2004) 1245–1251. [195] B. Visentin, J.A. Vekich, B.J. Sibbald, A.L. Cavalli, K.M. Moreno, R.G. Matteo, W.A. Garland, Y. Lu, S. Yu, H.S. Hall, V. Kundra, G.B. Mills, R.A. Sabbadini, Validation of an anti-sphingosine-1-phosphate antibody as a potential therapeutic in reducing growth, invasion, and angiogenesis in multiple tumor lineages, Cancer Cell 9 (2006) 225–238. [196] N. O'Brien, S.T. Jones, D.G. Williams, H.B. Cunningham, K. Moreno, B. Visentin, A. Gentile, J. Vekich, W. Shestowsky, M. Hiraiwa, R. Matteo, A. Cavalli, D. Grotjahn, M. Grant, G. Hansen, M.A. Campbell, R. Sabbadini, Production and characterization of monoclonal anti-sphingosine-1-phosphate antibodies, J. Lipid Res. 50 (2009) 2245–2257. [197] R.A. Sabbadini, Sphingosine-1-phosphate antibodies as potential agents in the treatment of cancer and age-related macular degeneration, Br. J. Pharmacol. 162 (2011) 1225–1238. [198] M.S. Gordon, R. Just, L.S. Rosen, A. Dorr, A phase 1 study of sonepcizumab (S), a humanized monoclonal antibody to sphingosine-1-phosphate (S1P), in patients with advanced solid tumors, J.Clin.Oncol. 28 (2010) 15S:219s. [199] G.L. Stoller, F. Lapierre-Holme, J. Peterkin, W. Garland, R. Sabbadini, iSONEPTM, an anti-sphingosine-1-phosphate (Anti-S1P) monoclonal antibody for investigation in exudative AMD: results from a phase 1 prospective open-label dose-escalating multi-center study, Invest.Ophthalmol.Vis.Sci. 51 (2010) 1253. [200] R.M. Fryer, A. Muthukumarana, P.C. Harrison, S. Nodop Mazurek, R. Rhonda Chen, K.E. Harrington, R.M. Dinallo, J.C. Horan, L. Patnaude, L.K. Modis, G.A. Reinhart, The Clinically-tested S1P Receptor Agonists, FTY720 and BAF312, Demonstrate Subtype-Specific Bradycardia (S1P1) and Hypertension (S1P3) in Rat, Plos One 7 (2012) e52985. [201] K. Sobel, K. Menyhart, N. Killer, B. Renault, Y. Bauer, R. Studer, B. Steiner, M.H. Bolli, O. Nayler, J. Gatfield, Sphingosine 1-phosphate (S1P) receptor agonists mediate pro-fibrotic responses in normal human lung fibroblasts via S1P2 and S1P 3 receptors and Smad-independent signaling, J. Biol. Chem. 288 (2013) 14839–14851. [202] H. Ni, J. Chen, M. Pan, M. Zhang, J. Zhang, P. Chen, B. Liu, FTY720 prevents progression of renal fibrosis by inhibiting renal microvasculature endothelial dysfunction in a rat model of chronic kidney disease, J. Mol. Histol. 44 (2013) 693–703. [203] H.F. Ni, J.F. Chen, M.H. Zhang, M.M. Pan, J.D. Zhang, H. Liu, R.N. Tang, K.L. Ma, B.C. Liu, FTY720 attenuates tubulointerstitial inflammation and fibrosis in subtotally nephrectomized rats, Ren. Fail. 35 (2013) 996–1004.