FIELD OF THE INVENTION:

The present invention is in the field of medicine, in particular vascular diseases of the central nervous system (CNS).

BACKGROUND OF THE INVENTION:

The functional status of the cerebral microvasculature is a critical determinant of brain health ¹ _’ ² . Endothelial cells (ECs) play a particularly important role in cerebrovascular function by coordinating tissue perfusion, blood-brain barrier (BBB) permeability and leukocyte extravasation ³ . Common cardiovascular risk factors impair microvascular function, and vascular dysfunction may in turn exacerbate the risk and impact of acute cardiovascular events including stroke, and potentially contribute to Alzheimer’s disease and related dementia ² ’ ^{4 7} . Conversely, methods to stimulate microvascular function may improve the outcome of stroke and other acute cardiovascular events and delay the onset and progression of dementia by improving tissue perfusion and reducing inflammation and tau phosphorylation.

Elucidation of endogenous protective mechanisms may identify therapeutic targets for vascular protection. ⁸ Ischemic tolerance, a phenomenon by which a preconditioning insult mitigates tissue damage from a major and sometimes unrelated insult, has been explored to reveal such mechanisms. ⁸ _’ ⁹ Sphingosine kinases (Sphks) 1&2, which convert sphingosine to the bioactive lipid mediator sphingosine- 1 -phosphate (SIP), are essential for preconditioning effects in experimental models of myocardial infarction and stroke. ^{10 12} SIP is a signaling lipid with critical roles in vascular and immune homeostasis exerted by five cognate G protein- coupled receptors (GPCRs), SIP1-5. ^13,14 SlPi is one of the most abundant EC GPCRs, and selective constitutive or temporal deletion of Slprl in mouse ECs impairs embryonic and postnatal angiogenesis, vascular integrity, and flow-mediated vasodilation, and increases susceptibility to the development of atherosclerosis. ^{15 20} Mechanistically, loss of EC SlPi is associated with destabilization of adherens junctions, reduction in eNOS activity, and increased expression of leukocyte adhesion molecules. ¹⁷ _’ ^{19 21} SlPi thus plays a critical role in sustaining hallmark endothelial functions, properties that it also confers to its main plasma chaperone high density lipoprotein (HDL). ²⁰ SlPi also plays an important yet subtle role in the brain vasculature, where EC Slprl deletion leads to selective permeability to small (<10kD) molecules. ²² When induced in hematopoietic cells, SlPi deficiency blocks the egress of lymphocytes from lymphoid organs, resulting in profound lymphopenia. ²³ While multiple sources of SIP sustain embryonic SIPi signaling, postnatal lymphopenia and loss of lung vascular integrity are both replicated by loss of circulating SIP pools. ^24,25 Plasma SIP is highly abundant, carried by HDL and albumin, and provided primarily by erythrocytes. ^26,27 Platelets also store SIP that can be released locally upon activation. ²⁸ Blood ECs have the capacity to produce and export SIP, but the functional relevance of this source is unclear as specific deletion does not impact lung vascular integrity or plasma SIP levels. ^25,29 Thus, S IP is a critical regulator of vascular and immune homeostasis and if targeted selectively, its receptor SIPi could potentially constitute a useful target for the stimulation of cerebrovascular function.

The capacity of the brain vasculature to rapidly respond to stress is particularly pertinent to ischemic stroke, which is one of the most prevalent causes of death and morbidity worldwide and represents an enormous societal and economic burden (https://www.who.int). ⁸ Despite numerous clinical trials with neuroprotective therapeutics, treatment options remain limited to thrombolysis and surgical recanalization (thrombectomy). ^{30,3 1} Clot removal can be effective, but is only performed in subset of patients due to the short window of efficacy, risk of bleeding, technical demands, and cost. ^31"33 Novel safe and affordable adjunct treatment strategies are therefore warranted. In ischemic stroke, thromboembolic occlusion of a large cerebral artery - typically the middle cerebral artery (MCA) - rapidly results in the creation of a non-perfused necrotic core immediately downstream, surrounded by a hypoperfused “penumbra” that can be salvaged if perfusion is recovered within the first few hours. ^8,32,34 The size of the penumbra decreases with time and depends both on the extent of occlusion and the efficiency of retrograde perfusion of the area at risk through pre-existing collateral anastomoses within and between cerebral arterial trees and branches. ^35,36 Without spontaneous, mechanical, or thrombolytic removal of the original thrombus, the efficiency of blood rerouting through these collaterals is the principle determinant of infarct size. Governed primarily by the number and size of existing anastomoses, collateral rerouting is also influenced by the dilatory capacity, integrity, and patency of the regional vasculature. A therapeutic opportunity therefore exists in optimizing vascular function and patency so as to improve collateral rerouting even in the absence of reperfusion. Improving local microvascular function may also improve drug access and the efficacy and safety of anterograde reperfusion. Strategies explored to achieve this without increasing bleeding risk include the stimulation of endothelial cell (EC) function to mediate local redistribution of blood flow, reinforce the blood-brain-barrier (BBB), and counteract inflammation, coagulation and their cross-talk in affected territories. ^36,37

The multiple sclerosis drug fmgolimod is a functional antagonist of SIPi that also activates S1P _3,4&5 and has shown promise for treatment of ischemic and hemorrhagic stroke in experimental models and small-scale clinical trials. ³⁸⁴² Similar efficacy of SIPi-selective agonists argues SIPi dependence. ^37,43 Correlation between lymphopenia and infarct reduction and loss of efficacy in lymphocyte-deficient mice both suggested that a key action of SIPi modulators is to desensitize lymphocyte SIPi, ^43,44 and experimental and clinical trials involving SIPi modulators have mostly been designed with the goal of targeting lymphocyte receptors to reduce inflammation and inflammatory thrombosis. Improvement of BBB function has also been reported although not reproduced in all studies. ^37,39,45,46 While this improvement could be secondary to reduced inflammation, it may also suggest that SIPi activation in the NVU has the potential to directly stimulate endothelial function

In order to explore the function of EC SIPi at the BBB, its mechanism of activation, and its potential as a therapeutic target for the stimulation of cerebrovascular function, the inventors studied SIPi expression and signaling at the BBB and addressed the impact of tissue specific deficiency in SIPi and select sources of activating ligand on cerebrovascular anatomy and function under homeostasis and after transient and permanent middle cerebral artery (MCA) occlusion in the presence or absence of SIPi agonists in mice. This revealed both an important role for EC SIPi in cerebrovascular homeostasis and a critical protective role for EC- autonomous engagement of SIPi signaling during cerebral ischemia. The need for EC- autonomous SIP provision was explained by polarization of SIPi to the abluminal surface of most endothelial cells at the blood-neural barrier, an observation that has important implications for drug targeting. Addressing the capacity of SIPi-selective agonists to activate SIPi on the brain endothelium and stimulate its function in ischemic stroke, they demonstrated that EC SIPi engagement at the blood-neural interface requires efficient BBB penetration. A BBB- penetrating SIPI agonist provided considerable protection against cortical infarct expansion in mouse models of ischemic stroke, protection that depended on EC SIPi and was not reproduced with an agonist that provided similar lymphopenia but did not cross the BBB. Collectively, these observations illustrated the therapeutic potential of targeting SIPi for stimulation of cerebrovascular function and revealed the need for BBB penetration for such targeting to be effective.

SUMMARY OF THE INVENTION:

The present invention relates to the use of BBB-penetrating SIPi agonists as a means to stimulate cerebrovascular function at the blood-neural barrier. Improved cerebrovascular function may in turn improve local blood flow, blood-neural barrier integrity and reduce inflammation and thrombosis. This strategy may have utility for the treatment of ischemic stroke, intracerebral haemorrhage, retinopathies, vascular dementia, Alzheimer’s disease and other CNS disorders. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

As critical regulators of blood-brain barrier (BBB) function and tissue perfusion, endothelial cells (EC) are vital for neurovascular adaptation to stress and thus for neuronal survival. The circulating lipid mediator sphingosine-1 -phosphate (SIP) sustains both EC functions and lymphocyte egress through the SIPi receptor. The inventors report that EC SIPi signaling becomes restricted to arterioles upon maturation of the blood-neural barrier coincident with gradual receptor polarization away from blood, shielding SIPi from circulating endogenous and synthetic ligands in the majority of ECs in the mature mouse brain. Restricted EC SIPi signaling under homeostasis is nevertheless sufficient to sustain cerebral blood flow, BBB function, and anti-inflammatory properties in naive mice. During cerebral ischemia, EC SIPi signaling expands with the recruitment of EC-autonomous SIP provision. EC-selective deficiency in SIP production, export, or signaling exacerbates the outcome of transient and permanent middle cerebral artery (MCA) occlusion. Exaggerated vasogenic edema and impaired blood flow recovery suggested that SIPi supports BBB function and the rerouting of blood to hypoperfused brain tissues through collateral anastomoses. Loss of hematopoietic SIPi confers mild protection, consistent with the capacity of the SIPi modulator fmgolimod to reduce lymphocyte-driven thromboinflammation. Dual targeting of EC and lymphocyte SIPi with BBB-penetrating agonists limits cortical infarct expansion with or without reperfusion when applied up to 6 hours after MCA occlusion. By contrast, a non-BBB penetrating agonist that provided similar lymphopenia did not provide neuroprotection after permanent MCA occlusion. Thus, SIPi is critical for cerebrovascular function and can be harnessed in ischemic disease of the brain with the use of BBB-penetrating agonists.

Accordingly, the first object of the present invention relates to a method for stimulating cerebrovascular function in a patient in need thereof comprising administering to the subject a therapeutically effective amount of a BBB-penetrating SIPi agonist

As used herein, the term “cerebrovascular function” refers primarily to critical functions of the brain endothelium, which include the maintenance of the blood-brain barrier, the control of vascular tone, and the inhibition of inflammation and microvascular coagulation and thrombosis. As used herein, the term “endothelium” refers to the layer of cells that line the interior surface of blood vessels and act as a selective barrier between the vessel lumen and surrounding tissue, by controlling the transit of fluids, materials and cells into and out of the bloodstream. Excessive or prolonged dysfunction of endothelial cell barrier leads to tissue oedema/swelling. Besides its critical role as a component of the barrier that prevents the passive crossing of fluids, solutes and cells from blood to the brain parenchyma, the endothelium also plays a key role in regulating local blood perfusion in the brain in response to neuronal, hormonal and mechanical stimuli and in maintaining vascular patency and limiting brain infiltration of inflammatory cells. The endothelium thus plays a critical role in cerebrovascular function and thereby in neuronal health and survival both under homeostasis and in response to stress. Stimulating cerebrovascular function may therefore alleviate both acute disease to the brain such as thromboembolic or hemorrhagic stroke as well as chronic neuropathies such as vascular dementia and Alzheimer’s disease. The inventors have discovered that SIPi is critical for sustaining all major endothelial functions in the brain and that BBB-penetration is critical for the capacity of synthetic agonists to efficiently engage the receptor on the brain endothelium. The use of BBB penetrating SIPi agonists herein disclosed is therefore a particularly suitable means of stimulating cerebrovascular function at the blood-brain barrier because SIPi controls several key endothelial protective mechanisms and because BBB penetration is required to efficiently reach this receptor on the endothelium at the blood-neural barrier, where the receptor is polarized to the abluminal domain of most endothelial cells.

Thus, in some embodiments, the patient suffers from ischemic stroke, haemorrhagic stroke, subarachnoid haemorrhage, retinopathies, vascular dementia, Alzheimer’s disease or from other CNS disorders.

In some embodiments, the patient suffers from cerebral ischemia. As used herein, the term “cerebral ischemia,” refers to any condition in which there is insufficient blood flow to at least a portion of the brain due to occlusion or insufficiency of one or more arteries that supply blood to the brain or cardiac arrest. The definition encompasses both focal and global ischemia. The term encompasses all medical causes of insufficient blood flow, for example, cerebral hypoxia, traumatic brain injury, stroke (including thrombosis, cerebrovascular embolism, ischemic stroke, perinatal stroke, and cerebral infarction), and cardiac arrest.

In some embodiments, the method of the present invention is thus particularly suitable for the treatment of ischemic stroke. As used herein, the term “ischemic stroke” refers to those patients having or at risk for “definite ischemic cerebrovascular syndrome (ICS)” as defined by the diagnostic criteria of Kidwell et al. “Acute Ischemic Cerebrovascular Syndrome: Diagnostic Criteria,” Stroke, 2003, 34, pp. 2995-2998 (incorporated herein by reference). Accordingly, ischemic stroke refers to an onset of neurologic dysfunction of any severity consistent with focal brain ischemia. In some embodiments, the BBB-penetrating SIPi agonist of the present invention is particularly suitable for preventing cortical infarct expansion during stroke.

In some embodiments, the BBB-penetrating SIPi agonist of the present invention is particularly suitable for promoting retrograde perfusion of the peri-infarct area during stroke.

In some embodiments, the BBB-penetrating SIPi agonist of the present invention is particularly suitable for improving vascular patency and/or blood perfusion during stroke.

In some embodiments, the BBB-penetrating SIPi agonist of the present invention is particularly suitable for preventing no-reflow.

As used herein, the term “no-reflow” has been increasingly used in published medical reports to describe microvascular obstruction and reduced flow after opening an occluded artery. In its broadest meaning, the term “preventing no-reflow” or “prevention of no-reflow” refers to reducing or avoiding the no-reflow.

In some embodiments, the BBB-penetrating SIPi agonist of the present invention is particularly suitable for preventing cerebral ischemia-reperfusion injuries.

As used herein, the term “reperfusion” refers to the restoration of blood flow to a tissue following ischemia. Accordingly, the term "ischemia reperfusion" is thus intended to encompass an event wherein an episode of ischemia is followed by an episode of reperfusion and the term “ischemia reperfusion injury” refers to the tissue damage caused by an ischemia reperfusion event.

In particular, the method of the present invention is performed sequentially or concomitantly with a standard method for treating ischemic conditions, or alone in patients not eligible for or without access to standard methods. Standard methods include reperfusion of the ischemic organ by angioplasty, thrombolysis, or surgically thrombectomy. The term "thrombolysis" means the administration of thrombolytic agents. Typically, thrombolysis involves the use of tissue-type plasminogen activator (t-PA). The term includes native t-PA and recombinant t-PA, as well as modified forms of t-PA that retain the enzymatic or fibrinolytic activities of native t-PA. Recombinant t-PA has been described extensively in the prior art and is known to the person of skill. t-PA is commercially available as alteplase (Activase® or Actilyse®). Modified forms of t-PA ("modified t-PA") have been characterized and are known to those skilled in the art. Modified t-PAs include, but are not limited to, variants having deleted or substituted amino acids or domains, variants conjugated to or fused with other molecules, and variants having chemical modifications, such as modified glycosylation. Several modified t-PAs have been described in PCT Publication No. W093/24635; EP 352,119; EP382174. In some embodiments, the modified form of t-PA is Tenecteplase. As used herein, the term “tenecteplase,” also known as TNK-t-PA or TNKASE™ brand of tissue-plasminogen activator variant, refers to a t-PA variant designated T103N, N117Q, K296A, H297A, R298A, R299A t- PA available from Genentech, Inc. (South San Francisco Calif.) wherein Thrl03 of wild-type t-PA is changed to Asn (T103N), Asn 117 of wild-type t-PA is changed to Gin (N117Q), and Lys-His-Arg-Arg 296-299 of wild-type t-PA is changed to Ala-Ala-Ala-Ala (KHRR296- 299AAAA). Tenecteplase is a genetically engineered variant of human t-PA cloned and expressed in Chinese hamster ovary cells (see Keyt et al., Proc. Natl. Acad. Sci USA, 91: 3670- 3674 (1994) and Verstraete, Am. J. Med, 109: 52-58 (2000) for an overview of third-generation thrombolytic drugs in general). Tenecteplase was engineered to have increased fibrin specificity and an increased half-life compared to alteplase.

In some embodiments, the present invention thus relates to a method of treating cerebral ischemia in a patient in need thereof comprising the steps consisting of i) restoring blood supply in the ischemic tissue, and ii) maintaining endothelial function of said ischemic tissue by administering to said patient a therapeutically effective amount of a BBB-penetrating SIPi agonist.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

As used herein, the term “SIP receptor’ or “S1PR” refers to a receptor that is bound by or activated by sphingosine-1 -phosphate (SIP). SIP receptors are cell surface receptors which include known receptor subtypes 1, 2, 3, 4, 5 and are regarded herein as SIP receptors. As used herein, the term SIPi refers to the SIP receptor- 1.

As used herein, the term “sphingosine-1 phosphate receptor-1 agonist” or “SIPi agonist” refers to a substance that has itself a function of acting on a sphingosine-1 phosphate receptor-1 (SIPi). The binding of an agonist to SIPi, for example, result in the dissociation of intracellular heterotrimeric G-proteins into Ga-GTP and Gpy-GTP, and/or the increased phosphorylation of the agonist-occupied receptor, and/or the activation of downstream signaling pathways. Thus, the SIPi agonists are known in the prior art and those skilled in the art have sufficient general knowledge to identify compounds that are agonists of SIPi. To do this, they can carry out an in vitro functional ³⁵ S-GTPyS binding test. They may, for example, refer to the publication D S. Im et al., Mol. Pharmacol. 2000; 57-753. In this functional test, the binding between the GTPyS and the G proteins, mediated by the ligand, is measured in a GTP binding buffer (in mM: 50 HEPES; 100 NaCl, 10 MgCk, pH 7.5) using 25 pg of a membrane prepared from transiently transfected HEK293 cells. The ligand is added to the membranes in the presence of 10 mM of GDP and of 0.1 nM of ³⁵ S-GTPyS (1200 Ci/mmol) and incubated at 30° C. for 30 minutes. The bound GTPyS is separated from the unbound GTPyS using a Brandel collector (Gaithersburg, Md ), and counted with a liquid scintillant counter.

As used herein, the term “BBB-penetrating SIPi agonist” refers to a SIPi agonist that is able to cross/penetrate the BBB. As used herein, the term “blood-brain barrier” or “BBB” denotes the physiological barrier between the peripheral circulation and the brain and spinal cord which is formed by tight junctions within the brain capillary and arteriolar endothelial plasma membranes, creating a tight barrier that restricts the transport of molecules into the brain. Methods for assaying the ability of a compound to cross the BBB are well known in the art and typically include those described in:

- Ulrich Bickel, « How to measure drug transport across the blood-brain barrier. », NeuroRx, vol. 2, 2005, p. 15-26 - M. Gumbleton et K. L. Audus, « Progress and limitations in the use of in vitro cell cultures to serve as a permeability screen for the blood-brain barrier. », J. Pharm. Sci., vol. 90, 2001, p. 1681-1698

A. Reichel, D. J. Begley et N. J. Abbott, « An overview of in vitro techniques for blood-brain barrier studies. », Methods Mol. Med., vol. 89, 2003, p. 307-324

- R. Cecchelli, B. Dehouck, L. Descamps, L. Fenart, V. V. Buee-Scherrer, C Duhem, S. Lundquist, M. Rentfel, G. Torpier et M. P. Dehouck, « In vitro model for evaluating drug transport across the blood-brain barrier. », Adv. Drug Deliv. Rev., vol. 36, 1999, p. 165-178

- William H. Oldendorf, « Measurement of brain uptake of radiolabeled substances using a tritiated water internal standard. », Brain Res., Elsevier, vol. 24, 1970, p. 372-376

J. D. Fenstermacher, R. G. Blasberg et C. S. Patlak, « Methods for quantifying the transport of drugs across the blood-brain system. », Pharmacol. Ther., vol. 14, 1981, p. 217-248

Ikumi Tamai et Akira Tsuji, « Drug delivery through the blood-brain barrier. », Adv. Drug Deliv. Rev., vol. 19, 1996, p. 401-424

In addition, BBB penetration of the SIPi agonist may be assessed using SIPi signaling reporter mice as described in EXAMPLE. These mice were described in the following papers:

- Kono, M., E.G. Conlon, S.Y. Lux, K. Yanagida, T. Hla, and R.L. Proia. 2017. Bioluminescence imaging of G protein-coupled receptor activation in living mice. Nature communications 8: 1163.

Kono, M., A.E. Tucker, J. Tran, J.B. Bergner, E.M. Turner, and R.L. Proia. 2014. Sphingosine-1 -phosphate receptor 1 reporter mice reveal receptor activation sites in vivo. The Journal of clinical investigation 124:2076-2086.

In particular, the BBB -penetrating SIPi agonist of the present invention is a low- molecular weight agonist, for example, a small (possibly natural) organic molecule. The term “small organic molecule” refers to a molecule, which is possibly natural, having a size comparable to that of the organic molecules generally used as medicaments. This term excludes macromolecules (for example proteins, nucleic acid molecules, etc ). Preferred small organic molecules have a size of at most 10000 Da, preferably of at most 5000 Da, more preferentially of at most 2000 Da, and even more preferentially of at most 1000 Da.

In some embodiments, the BBB-penetrating SIPi agonist of the present invention is not selected for its ability to induce a sustained lymphopenia. Accordingly, the BBB-penetrating S1P1 agonist of the present invention is not fmgolimod. As used herein, the term “fingolimod” refers to 2-amino-2-(2-[4-octylphenyl]ethyl)-l, 3 -propanediol) that is also named FTY-720.

In some embodiments, the BBB-penetrating S1P1 agonist of the present invention is CYM-5442 ((+/-)-2-((4-(5-(3,4,diethoxyphenyl)-l,2,4-oxadiazol-3-yl)-2 ,3-dihydro-lH-inden- l-yl)amino)ethanol) that has the formula of:

In some embodiments, the BBB-penetrating S1P1 agonist of the present invention is SEW2871 (5-[4-phenyl-5-(trifluoromethyl)-2-thienyl]-3-[3-(trifluorom ethyl)phenyl]- 1,2,4- oxadiazole) that has the formula of:

In some embodiments, the BBB-penetrating S1P1 agonist of the present invention is ozanimod (5-(3-{(l S)-l-[(2-Hydroxyethyl)amino]-2, 3 -dihydro- 177-inden-4-yl}- 1,2,4- oxadiazol-5-yl)-2-isopropoxybenzonitrile) that the formula of:

According to the invention, the SIPi agonist is administered to the patient in a therapeutically effective amount. By a "therapeutically effective amount" is meant a sufficient amount of the active ingredient for treating or reducing the symptoms at reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination with the active ingredients; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

Typically the active ingredient of the present invention (e.g. SIPi agonist) is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. The term "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. In the pharmaceutical compositions of the present invention, the active ingredients of the invention can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. Endothelial cell autonomous SIPi signaling is protective in ischemic stroke. A. Infarct volumes 24 hours after permanent MCAO in male mice lacking SIPi signaling selectively in endothelial cells ( Slprl ^ECK0 ) relative to littermate controls. B. Infarct volumes 24 hours after permanent MCAO in male mice lacking SIP production endothelial cells (Sphkl &2 ^eck0 ) relative to littermate controls. Bar graphs show mean ± SD. Statistical significance assessed by Mann- Whitney test.

Figure 2. A BBB penetrating SIPi agonist limits cortical infarct expansion in ischemic stroke. A. Effects of CYM-5442, RP-001 and FTY720 at indicated concentrations on lymphocyte counts 3 and 24 hours after bolus administration. Values normalized to pre-bleed. Statistical significance in comparison to vehicle control at indicated time points. B. Effect of CYM-5442 (3 mg/kg i.p. 0-6 hours after occlusion) on infarct size 24 hours after pMCAO in wild-type males. C, D. Effect of CYM-5442 (3 mg/kg i.p. immediately after occlusion) on the impact of EC Sphkl&2 (C) and SIPi (D) deficiency on infarct size 24 hours after pMCAO. E Effect of CYM-5442 (3 mg/kg i.p. immediately before reperfusion) on infarct size 24 hours after 60 minutes tMCAO (total and regional infarct size). F. Effect of RP-001 (0.6 mg/kg i.p. immediately after occlusion-on infarct size 24 hours after pMCAO. Bar graphs show mean ± SD. Statistical significance assessed by one-way ANOVA (A), Kruskall-Wallis test with Dunn’s multiple comparisons test (E) or Mann- Whitney test (all other). EXAMPLE:

Results

Endothelial SIPi plays a critical protective role during cerebral ischemia. In order to define endogenous roles of EC and hematopoietic cell (HC) SIPi in ischemic stroke, we generated EC- and HC-selective Slprl knockout (KO) mice with Cd¾5-iCreERT2 or Pdgfb- iCreERT2 ( Slprl ^ECK0 ) and Mxl-Cre or Vavl-Cre ( Slprl ^HCK0 ) as described ^17,25,47 and subjected these to transient (t) and permanent (p) MCA occlusion. In a filament-based 60 minutes tMCAO model, infarct volumes measured at 24 hours were more than twice as large and neurological deficits significantly exacerbated in Slprl ^ECK0 mice (data not shown). Small infarct volumes and modest neurological affection in controls was attributed to documented protective effects of tamoxifen, ^48,49 which was administered thrice up to 10 days prior to experimentation to induce deletion of EC Slprl. To avoid confounding effects of tamoxifen and potential implication of SIPi for tamoxifen protection, EC Slprl deletion was induced neonatally in all subsequent experiments. When neonatally-induced mice were subjected to a severe model where reperfusion was delayed to 90 minutes, infarcts were not significantly larger in Slprl ^ECK0 mice (data not shown). However, infarcts covered a substantial portion of the MCA territory in controls, and Slprl ^ECK0 mice exhibited a significant increase in post reperfusion mortality (68% vs. 32%, respectively, p=0.022), manifesting between 8 and 16 hours after occlusion (data not shown). Assigning the maximal observed infarct volume to all mice that died in this period under the assumption that death reflects upon the extent of brain injury suggested that brain injury was greater in Slprl ^ECK0 mice also in this model (data not shown). This notion is further explored by magnetic resonance imaging below. When we next deleted Slprl in all hematopoietic lineages, we observed profound lymphopenia and a modest reduction in 24 hours infarct volumes after 60 minutes tMCAO (data not shown). In an electrocoagulation-based pMCAO model, in which dissection of the MCA distal to the lenticulo-striate arteries yields smaller and cortically confined infarcts with no evident neurological deficits, ^50,51 the mean infarct volume was 71 % larger in Slprl ^ECK0 males and 75 % larger in Slprl ^ECKO females than in controls (Figure 1A and data not shown). The relative increase was greater 3 days after pMCAO with 148 % larger infarcts in Slprl ^ECK0 males, suggesting an effect also on infarct resolution (data not shown). The /Vq/¾-iCreERT2 allele used for neonatal EC-selective gene deletion was reported to excise in megakaryocytes, ⁵² and SIPi proposed to play a role in platelet aggregation. ⁵³ Yet this did not explain the phenotype, as it was reproduced in Slprl ^ECK0 males generated with neonatal Gi¾5-iCreERT2- mediated deletion (data not shown), but not in mice in which Slprl was selectively deleted in megakaryocytes with Pf4-Cre (57/;/7 ^MKKO ; data not shown). ⁴⁷ Hematopoietic SIPi deficiency did not impact outcome in the pMCAO model (data not shown). Endothelial SIPi thus plays an important vascular protective role after both transient and permanent MCA occlusion in mice, while leukocyte SIPi plays a net disruptive role only in the transient model, presumably by sustaining lymphocyte egress and thromboinflammation (data not shown). ⁴⁴

Endothelial protective functions of SIPi are sustained by cell-autonomous SIP production and export. Lymphocyte egress and lung vascular integrity are both sustained by circulating SIP. ^24,26 Surprisingly, however, impairing SIP provision to plasma and lymph by postnatal deletion of Sphkl&2 in Mxl- Cre sensitive cells ^25,26,47 did not significantly impact infarct volumes in either MCAO model (data not shown). This could reflect broad targeting with MXI-CXQ and compound effects of loss of circulating SIP on the activation of SIPi and other SIP receptors in different cellular compartments. ⁵⁴ Yet even if SIP2 contributes to injury in the tMCAO model, ⁵⁵ neither SIP2 nor SIP3 deficiency impacted outcome in the pMCAO model (data not shown). Selective impairment of SIP release from platelets, which distribute SIP to albumin and may thus favor SIP2 activation, ²⁵ also did not impact infarct size in the tMCAO model (data not shown). Instead, consistent with expression of the necessary machinery for de novo SIP synthesis by the brain endothelium, ⁵⁶ impairment of both SIP production ( Sphkl&2 ^ECK0 ; Figure IB) and export ( Spns2 ^ECK0 ; data not shown) from ECs significantly increased infarct size in both models. In order to confirm the need for EC- autonomous SIP provision to SIPi and to address where signaling is engaged after MCAO, we generated SIPi GFP signaling (S1P1GS) mice - which leave a nuclear GFP signal after SlPi- b-arrestin coupling ⁵⁷ - with or without the capacity for EC SIP production ( Slprl ^w+ :H2B - Gfp ^Tg/+: Sphlcl &2 ^ecwt/k0 ). Surprisingly, SIPi signaling was highly restricted to arteriolar ECs in the cerebral cortex (data not shown) despite widespread SIPi expression in both ECs and astrocytes (data not shown). SIPi-independent H2B-GFP reporter activation was also observed in perivascular cells of arterioles and venules but not ECs (data not shown). After pMCAO, SIPi signaling was also induced in capillary and venous ECs in the infarct region (data not shown). Although redundant sources or S IP-independent activation ¹⁷ sustained homeostatic signaling in arterioles, EC-autonomous SIP production was required for most of the signaling increase after MCAO (data not shown). Thus, while circulating SIP sustains lymphocyte trafficking ²⁶ and may contribute to homeostatic signaling in arterioles, SlPi- dependent neuroprotection is driven primarily by the recruitment of local SIP provision from EC (data not shown). Postnatal impairment of EC-autonomous SIP signaling does not impact cerebrovascular anatomy. Although recombination of loxP-flanked alleles in neonates avoided confounding effects of tamoxifen protection on stroke outcome, it introduced potential confounding effects of developmental phenotypes. As reported for E2//?5 - i C re E R T2 - m ed i ate d neonatal deletion of Slprl, ¹¹ /Vg/¾-iCreERT2-mediated deletion induced vascular hyper- sprouting and delayed outgrowth of the retinal vasculature (data not shown). However, this phenotype was not replicated with Pdgfb-\ C re E R T2 - m ed i ated deletion of Sphkl&2 (data not shown), suggesting that redundant SIP sources sustain postnatal as well as embryonic ²⁵ angiogenesis, and thus, that vascular patterning is normal in Sphkl &2 ^ECK0 mice. Consistent with prenatal development of the cerebral vasculature, we also did not observe differences in the number of collateral connections between the MCA and branches of anterior cerebral artery (ACA) extending laterally from the midline between Slprl ^ECK0 and littermate controls (data not shown). Vascular density in the cortex was also unaltered by /Vg/¾-iCreERT2-mediated Slprl excision as reported for ( Hh5 - i C reE RT2- m edi ated deletion (data not shown) ²² Thus, underlying differences in vascular anatomy do not explain sensitivity to MCAO in Slprl ^ECK0 mice.

SIPi maintains microvascular patency in the ischemic penumbra. SIPi maintains the anti-inflammatory status of the aortic endothelium, and leukocyte adhesion contributes to downstream microvascular thrombosis in cortical venules after MCAO. ^20,58 We also observed a significant increase in ICAM-1 in homogenates of cerebral cortex from naive Slprl ^ECK0 mice (data not shown), attributable to increased expression in postcapillary venules (data not shown). This increase was not replicated by selective impairment of SIP production in either endothelial or hematopoietic cells (data not shown), suggesting redundancy or a systemic origin. At a whole hemisphere level, the relative increase in ICAM-1 protein in Slprl ^ECK0 was overcome in the acute phase after pMCAO, with increased expression in the ipsilateral hemisphere of controls but not Slprl ^ECK0 mice at 2.5 hours (data not shown). We nevertheless observed an increase in myeloperoxidase in the ipsilateral hemisphere of Slprl ^ECK0 (data not shown), albeit not beyond the increase in infarct size. We next stained for erythrocytes, neutrophils, platelets, and fibrin(ogen) in sections of brains perfused transcardially with heparinized saline three hours after pMCAO. Plasma serotonin was slightly increased at this time independent of genotype, arguing against a significant difference in platelet activation (data not shown). We nevertheless observed a striking reduction in the penetration of tomato lectin, infused 15 minutes prior to transcardial perfusion, into the MCA territory superior/distal to the core in Slprl ^ECK0 mice (data not shown). Platelets and fibrin(ogen) were observed within both perfused and non-perfused capillaries only superior to the core and correlated with more intense staining for PECAM1/CD31 (data not shown). Fibrin deposition was more widespread and significantly more abundant in Slprl ^ECK0 mice (data not shown). Occasional neutrophils were observed within capillaries on both sides of the infarct core at similar frequency in both genotypes (data not shown). These observations suggest that although EC SIPi signaling does not have evident effects on neutrophil recruitment and platelet activation in the acute phase of ischemic stroke, it plays critical roles in promoting microvascular patency and retrograde perfusion of the in the ischemic penumbra.

Endothelial SIPi maintains BBB function in the ischemic brain. Both strategies of EC Slprl deletion used in this study result in constitutive vascular leak in lung that can be replicated by plasma but not by EC SIP deficiency. ^25,59 /Vq/¾-iCreERT2-mediated Slprl deletion also resulted in a modest accumulation of low molecular weight (4 kD) dextran in the brain similar to that reported with Slprl deletion (data not shown). ²² Endotoxin challenge (10 mg/kg, 24 hours) increased dextran accumulation to the same degree in Slprl ^ECK0 and littermate controls (data not shown). Thus, EC SIPi deficiency does not critically impair inter-EC junctional stability. Intriguingly, even if Slprl ^ECK0 mice do not show increased paracellular permeability to dextrans >10 kD, ²² the cortex of naive Slprl ^ECK0 mice did show increased permeability to Evans Blue/albumin, which crosses the BBB by transcellular transport (data not shown). ⁶⁰ Neither basal phenotype was replicated in mice lacking EC SIP production (data not shown), again suggesting source redundancy or SIP-indepence ¹⁷ of homeostatic signaling. Twenty- four hours after pMCAO, Evans Blue/albumin accumulation in the ipsilateral hemisphere exceeded the relative increase in infarct size in Slprl ^ECK0 mice with a more diffuse and widespread appearance, and was also significantly higher in the contralateral hemisphere, consistent with results in niave mice (data not shown). In the acute phase after 90 minutes tMCAO - which is associated with high mortality of Slprl ^ECK0 mice (data not shown) - full T2 weighted magnetic resonance imaging (MRI) revealed severe edema in Slprl ^ECK0 mice as early as 2.5 hours after reperfusion with a clear shift in the midline 2 hours later (data not shown). Image analysis confirmed significantly larger T2 lesions 4.5 hours after reperfusion, demonstrating a clear impact of EC SIPi deficiency also after 90 minutes of tMCAO despite no significant difference in infarct volumes in survivors at 24 hours (data not shown). Thus, SIPi preserves BBB liinction in ischemic stroke, possibly by restraining transcellular vesicular transport, which underlies BBB dysfunction in the acute phase of ischemic stroke. ⁶⁰ SIPi supports cerebral vasoreactivity and promotes tissue perfusion after MCA occlusion. We next addressed if EC SIP signaling regulates vessel diameter and thereby the redistribution of blood to the ischemic penumbra through existing collateral anastomoses. As previously reported, ¹⁹ flow-mediated dilation was blunted in both cerebral and mesenteric artery segments from Slprl ^ECK0 mice (data not shown). A similar phenotype in mesenteric arteries from Sphkl&2 ^ECK0 mice (data not shown) suggested that shear forces engage EC SIPi through cell-autonomous SIP release rather than by ligand-independent mechanisms. ¹⁷ While basal brain perfusion was normal in Slprl ^ECK0 mice as assessed by arterial spin labeling MRI (data not shown), ⁶¹ significantly blunted blood flow responses assessed by Doppler ultrasonography in the somatosensory cortex in response to acetylcholine superfusion (data not shown) and in the basilar trunk (BT) in response to CO2 inhalation (data not shown) argued an important role for EC SIP also in cerebral blood flow regulation. ⁶² Unaltered blood flow responses in somatosensory cortex in response to whisker stimulation confirmed normal neurovascular coupling (data not shown). We next monitored mean blood flow velocities (mBFVs) in the left and right internal carotid arteries (ICA) and in the basilar trunk (BT) (data not shown) before, 50 and 120 minutes after pMCAO in Slprl ^ECK0 and littermate controls. No genotype-dependent difference was observed in baseline mBFVs (data not shown). Permanent left MCAO decreased mBFVs in the left ICA to 77 % of pre-occlusion values 50 minutes after occlusion in both Slprl ^ECK0 and controls (data not shown), with no significant change in mBFVs in the BT or the right ICA (data not shown). A subsequent recovery to near 90 % of pre-occlusion values at 120 minutes after occlusion in controls was interpreted to reflect upon changes in peri-infarct reflow through branches of the MCA originating upstream from the occlusion and through the distal branches of the ACA and posterior cerebral artery (PCA) (data not shown). This recovery was absent Slprl ^ECK0 mice (data not shown), and a significant inverse correlation was found between infarct volume and blood flow recovery (data not shown). No genotype-dependent difference in blood flow reduction at 50 minutes argued a lag time before protective effects of SIPi signaling establish, possibly because of the need to engage EC-autonomous SIP production. Accordingly, when the occlusion time was limited to 35 minutes in the tMCAO model, we observed no difference in infarct size (data not shown). This also supports a mechanism involving collateral recruitment, as lack of collateral connections in the lenticulo striate arteries render the striatum more affected than the cortex when the MCA is reperfused early. ⁶³ To address perfusion of the affected cortex in the acute phase after pMCAO in real time, we visualized red blood cell flux in the area of collateral anastomoses in the leptomeningeal arteries between the MCA and ACA by sidestream dark field imaging (data not shown). Two hours after pMCAO, unidirectional flow towards the MCA territory was observed in all ACA-MCA collaterals, allowing perfusion of the territory normally supplied by branches of the MCA downstream of, but distal to the occlusion site (data not shown). Blood moving retrograde switched anterograde up other MCA branches when encountering coagulated blood at the ischemic core (data not shown). While the same general pattern was observed in mice of both genotypes, microvascular perfusion in the peri-infarct area was significantly reduced in Slprl ECKO mice (data not shown). Collectively, these observations are consistent with EC S lPi promoting retrograde perfusion of the peri-infarct area via collateral anastomoses in the acute phase of stroke by supporting local vasodilation.

Receptor polarization restricts SIPi signaling and ligand access at the blood-neural barrier. The need for EC SIP production and export suggested either loss of circulating ligand, as reported in myocardial infarction and systemic inflammation ^{25 ,4} ’ ⁵ , or restricted ligand access to SIPi at the BBB. Plasma and platelet SIP levels remained unchanged in the early acute phase of stroke with or without reperfusion (data not shown). We therefore addressed if SIPi is polarized at the blood-neural barrier. In the developing retina, SIPi was expressed primarily on the luminal surface of capillaries, where it co-localized with ICAM-2 (data not shown). In the mature retina, however, expression became restricted to the abluminal surface of capillaries - occasional luminal staining was confined to organelle-like structures (data not shown) - while only a subset of arteriolar ECs retained luminal expression (data not shown). In the mature brain, EC SIPi expression also appeared to be luminally excluded, although abluminal expression was difficult to distinguish from astrocyte end feet (data not shown). This was overcome by deleting Slprl in astrocytes ( Slprl ^ACKO ), revealing abluminal polarization of SIPi also in brain capillaries (data not shown). Further attesting to specificity, the antibody recognized only astrocytes in Slprl ^ECK0 mice (data not shown). Gradual receptor polarization in all but a small subset of arterial ECs with maturation of the blood-neural barrier provides an explanation for why EC-autonomous SIP provision becomes critical for local SIP provision in stroke (data not shown) although it is dispensable for retinal angiogenesis (data not shown) and homeostatic signaling in arterioles (data not shown). The absence of signaling in most ECs in the naive brain (data not shown) also argues that they do not constitutively secrete SIP, and could thus explain the delay in SIPi engagement after MCAO (data not shown)

To confirm that SIPi is indeed functionally expressed but luminally excluded in capillaries and venules of the cerebral cortex, we again made use of the SIPi signaling reporter mouse. In naive S1P1GS mice, hepatocytes show no nuclear GFP although they express high levels of SIPi (data not shown). ⁵⁷ Systemic administration of the potent SIPi-selective agonist RP-001 (0.6 mg/kg) ^57,66 induced SIPi signaling in hepatocytes and EC of skeletal muscle and lung, but not brain (data not shown). In striking contrast, when injected directly into the brain parenchyma at a 1/10 dose, RP-001 substantially increased SIPi signaling not only in arteries but also in capillaries and veins (data not shown). Astrocytes remained GFP negative despite SIPi expression (data not shown) ⁵⁶ , suggesting together with mostly punctate staining (data not shown) that the receptor is not expressed on the astrocyte surface under homeostasis. Surprisingly, induction of SIPi signaling in brain capillaries was also minimal after two consecutive injections of high dose (5 mg/kg) fmgolimod, despite strong activation of the SIPi reporter in other organs and sustained lymphopenia (data not shown). It should be noted that although fmgolimod is known to cross the BBB and desensitize SIPi on brain endothelium, this has been demonstrated with high doses and over extended time. ^22,67 Further attesting to polarization and a role in blood flow regulation, we observed a significant EC SIPi-dependent increase in cerebral blood flow when RP-001 was administered directly into the cerebrospinal fluid for paravascular access ⁶⁸ but not systemically (data not shown). CYM-5442 is an SIPi selective agonist that distributes rapidly and preferentially to the brain after systemic injection while inducing only transient lymphopenia. ⁶⁹ Accordingly, at a dosage required to induce SIPi signaling equivalent to RP-001 (0.6 mg/kg) and fmgolimod (2x5 mg kg) in hepatocytes after systemic administration, CYM-5442 (10 mg kg) also induced substantial signaling in brain capillaries (data not shown), unlike the other two agonists (data not shown).

Collectively, these observations argue that abluminal polarization shields SIPi in capillary, venous, and most but not all arterial ECs from circulating endogenous and synthetic ligand once the blood-neural barrier is established.

A BBB-penetrating SIPi-selective agonist prevents cortical infarct expansion after both permanent and transient MCA occlusion. Our results so far argue that the optimal SlPi- targeting drug for stroke therapy would transiently suppress lymphocyte trafficking and activate but not desensitize EC SIPi, and that CYM-5442 has a more suitable profile than RP-001 and fmgolimod. In accordance with the literature, CYM-5442 induced transient lymphopenia that was evident at 1 mg/kg and as profound as RP-001 (0.6 mg/kg) and fmgolimod (1 mg/kg) at 3 mg kg 3 hours after administration. ⁶⁹ Only fmgolimod-induced lymphopenia was sustained at 24 hours (Figure 2A). CYM-5442 modestly reduced infarct size 24 hours after pMCAO at the 1 mg/kg dose (data not shown), and provided substantial benefit when administered both immediately and up to 6 hours after occlusion at the 3 mg/kg dose (Figure 2B), with a 65% reduction in infarct volumes observed when administered 2 hours after pMCAO (Figure 2B). In a modified pMCAO model that included permanent ligation of the ipsilateral CCA, infarct volumes were also reduced at 7 days with daily CYM-5442 (3 mg/kg) administration (data not shown). Consistent with EC SIPi engagement, CYM-5442 (3 mg/kg) fully reversed sensitivity to stroke in Sphkl&2 ^ECK0 mice (Figure 2C) but not Slprl ^ECK0 mice (Figure 2D). CYM-5442 (3 mg/kg) also afforded significant protection when administered with reperfusion 60 minutes after MCAO (Figure 2E). Intriguingly and consistent with a mechanism dependent on collateral supply, ⁵¹ protection in tMCAO was delimited to the cortex, where an infarct reduction of 70% mirrored protection achieved in the cortically restricted pMCAO model. RP-001, which did not efficiently cross the BBB, did not provide protection in the pMCAO model (Figure 2F) despite inducing equivalent lymphopenia and strong SIPi signaling in other organs (data not shown). Thus, optimal SIPI targeting for ischemic stroke requires BBB penetration for engagement of endothelial receptors and can provide substantial protection against cortical infarct expansion independent of reperfusion and in a therapeutically relevant time window.

Discussion;

A number of recent studies have shown that SIPi modulators can provide benefit in experimental and human stroke, ³⁷ Yet as outcomes are variable and mechanisms of action not fully defined, the optimal targeting strategy remains unclear. Thus far, protective functions have mostly been attributed to immunosuppression. ³⁷ ’ ⁴³ ’ ⁴⁴ Employing a variety of genetic and experimental murine models to address endogenous roles of SIPi signaling, we confirm a role for lymphocyte SIPi but also reveal a critical role for EC SIPi in cerebral blood flow regulation and BBB integrity, and thereby in endogenous protection against ischemic brain damage. Addressing mechanism ofSIPi engagement at the BBB, we uncover a need for EC-autonomous ligand provision arising from SIPi polarization, which shields the receptor from natural and synthetic ligands at the BBB. This argues optimal benefit by dual targeting of EC and lymphocyte receptor pools with minimally desensitizing BBB penetrating SIPi agonists, a strategy that proved efficacious in limiting cortical infarct expansion in mouse models of ischemic stroke. While the protective capacity of SIPi agonists remains to be addressed in the context of co-morbidities, efficacy in other disease models argues that SIPi remains functional in both diabetes and hypertension. ¹⁹ _’ ⁷⁰ Together with sustained EC expression of SIPi in brains of older male and female individuals (www.proteinatlas.org), ⁷¹ extensive experience with side effects of SIPi modulation in multiple sclerosis, ^{72 74} and promise and safety of small-scale clinical trials with fmgolimod in ischemic as well as hemorrhagic stroke, ³⁷ this study attests to the potential of SIPi as a versatile and safe target for stroke therapy. Strong and early sensitization to MCAO in mice with selective deficiency of SIPi in blood endothelium argues an important functional role of the endothelium in the acute phase of stroke that is supported by SIPi. A functional role for EC SIPi was suggested by the observations that 1) neonatal deletion of SIPi did not substantially alter vascular anatomy in the adult brain, 2) exacerbation of infarct size was also observed with adult-induced EC SIPi deficiency, 3) EC-selective deficiency in SIP production induced sensitization to MCAO equivalent to EC SIPi deficiency even if it did not reproduce defects in retinal angiogenesis or subtle defects in BBB integrity and anti-inflammatory properties of brain EC observed in Slprl ^ECK0 mice, and 4) the SIPi-selective agonist CYM-5442 fully reversed sensitization to MCAO in mice lacking SIP production in ECs. Thus, endogenous EC SIPi signaling acts dynamically to limit the expansion of ischemia after MCA occlusion. SIPi supports key roles of the endothelium in mediating smooth muscle relaxation, maintaining endothelial barrier function, and maintaining vascular quiescence; ¹³ we provide evidence that SIPi exerts all these functions in the naive and ischemic brain.

A role for EC SIPi in control of local blood flow was suggested by 1) impaired acetylcholine- and hypercapnia-induced blood flow responses in naive Slprl ^ECK0 mice, 2) the capacity of an SIPi agonist to increase cerebral blood flow in naive wild-type but not Slprl ^ECK0 mice, 3) impaired upstream blood flow recovery and downstream microvascular perfusion in Slprl ^ECK0 in the acute phase after pMCAO, 4) lack of EC S lPi dependence of infarct size when the MCA was reperfused before the time at which SIPi contributed to blood flow recovery in the pMCAO model, and 5) the impact of SIPi activation on injury to the cortex, in which MCA braches are well connected to contiguous vascular territories, relative to the striatum, in which they are not. ^51,63 ’ ⁷⁵ ’ ⁷⁶ After MCAO, the efficiency of collateral blood rerouting depends on the pressure differential between the affected territory and contiguous vascular beds, the efficiency of flow-mediated dilatation, and the patency of the affected vasculature. ⁷⁷ SIPi coordinates developmental angiogenesis by promoting perfusion of the nascent vasculature, ¹⁷ and flow- mediated dilatation of mesenteric arteries depends on SIPi. ^19,78 Endothelial NOS, which is important for the vasoactive functions of SIPi, ^17,19 promotes the early establishment of collateral supply in ischemic stroke, and eNOS deficient mice display larger infarcts after MCAO. ^{79 81} While the implication of eNOS and other effectors remains to be defined, our results argue that EC SIPi limits ischemic expansion in part by supporting retrograde perfusion of the affected MCA territory through collateral anastomoses with contiguous arterial branches.

Although defects observed in BBB integrity in naive Slprl ^ECK0 mice were more subtle than what has been described for lung vascular integrity, ^25,82 dramatic and early edema after MCAO argues an important role for this receptor in supporting BBB function during ischemia. Evans Blue/albumin accumulation was more than doubled in Slprl ^ECK0 24 hours after pMCAO, and MRI analysis revealed profound edema with compression of the contralateral hemisphere of Slprl ^ECK0 mice only hours after tMCAO. Acute edema in ischemic stroke is known to involve increased transcellular transport, while tight junction impairment is only observed 2 days after occlusion. ⁶⁰ Early BBB dysfunction therefore suggested a role for SIPi in restricting vesicular transport. Minimal impact on 4 kDa dextran leak in a model of septic encephalopathy further argued against a critical role in regulating paracellular permeability. Neutrophils contribute to full BBB breakdown in ischemic stroke in the context of reperfusion injury, and SIPi regulated both ICAM-1 expression and neutrophil influx. However, neither was strongly exacerbated in Slprl ^ECK0 brains, and we found no evidence that hemorrhagic transformation was increased in Slprl ^ECK0 mice. This is consistent with observations from other disease models in which loss of EC SIPi signaling increases protein and tracer extravasation but does not induce hemorrhage or leukocyte infiltration unless accompanied by vascular remodeling. ^24,25 We thus conclude that SIPi limits edema formation in ischemic stroke most likely by suppressing transcellular vesicular transport. A role in maintaining BBB function suggests that the capacity of SIPi agonists to reduce edema formation in ischemic and hemorrhagic stroke may be direct and not secondary to immune suppression. ^37,45,46

EC SIPi deficiency was associated with increased venous ICAM-1 expression in the naive brain, suggestive of a role for SIPi in suppressing endothelial activation and leukocyte adhesion. However, expression was not further increased in the context of ischemia, and we observed no significant difference in the early recruitment of neutrophils to the ischemic penumbra in Slprl ^ECK0 mice, nor in hemorrhagic transformation, which depends on leukocyte- mediated BBB destruction at later stages. Circulating markers of platelet activation and platelet recruitment to ischemic capillaries were also not substantially affected by EC SIPi deficiency On the other hand, the deposition of fibrin, which extended well beyond the boundary of no perfusion 3 hours after pMCAO, reached significantly further into distal MCA territories in Slprl ^ECKO mice than in littermate controls. This suggests that microvascular coagulation contributes to rapid deterioration of collateral supply in Slprl ^ECK0 mice. Whether this reflects the loss of direct actions of SIPi signaling on the anticoagulant or profibrinolytic status of the endothelium or is a consequence of reduced perfusion remains to be determined. Regardless, it highlights the critical dynamic role of the endothelium in maintaining microvascular patency. Thus, while our data point to a direct vasoactive role of SIPi, increased edema and loss of vascular patency may also contribute to reduced blood flow and infarct expansion in Slprl ^ECK0 mice.77

Highly restricted SIPi signaling in the naive cerebral cortex and the need for EC autonomous SIP provision during cerebral ischemia both argue that SIP signaling is tightly controlled at the BBB. Characterization of S1P1GS mice suggests that SIPi remains silent in most ECs and astrocytes after maturation of the blood-neural barrier. Our results argue that EC signaling is restricted by polarization of SIPi away from circulating SIP in all but a subset of arteriolar endothelial cells in the mature brain. High expression of lipid phosphate phosphatase- 3 in pericytes, smooth muscle cells, and astrocytes of the neurovascular unit suggest that local SIP availability is also regulated by local dephosphorylation. ^{83 56} Limiting SIP availability may serve both to maintain EC SIPi sensitivity to stress and to prevent S IP-mediated activation of astrocytes, ^{83 85} which appear to be subject to the added safeguard of intracellular SIPi storage. Limited signaling also in peripheral organs argues that polarization is not unique to the brain, but has greater impact on synthetic ligand access in the context of the BBB. Restricted EC signaling also suggests that brain ECs only export SIP when stressed. Although EC SIPi sustained critical SIPi signaling during ischemia, homeostatic functions of SIPi in the naive brain did not depend on SIP production in EC. In the resting state, most brain endothelial cells express Spns2 and SIPi, but not Sphkl. ⁵⁶ We thus speculate that Sphkl induction - a known response to hypoxia ⁸⁶ - could be a trigger for EC SIPi activation in stroke, explaining the slight delay observed in SIPi-dependence. Thus, homeostatic SIPi signaling appears to be maintained by a limited subset of primarily arteriolar ECs with access to circulating ligand, while broader receptor engagement in ischemic stroke depends on the activation of EC SIP production, possibly through the induction of Sphkl expression.

Fingolimod and other SIPi modulators show promise for stroke therapy. ^{38 40} _’ ⁸⁷ Our study sheds new light on their mechanism of action that may inform on future trial design. Loss of fingolimod protection in lymphocyte deficient mice provided convincing evidence for a dominant role for its capacity to trigger lymphocyte retention and a resulting reduction in thromboinflammation ⁴⁴ The capacity of fingolimod and other SIPi modulators to promote BBB function has been inconsistent and sometimes viewed as secondary to immune suppression. ^39,44 ’ ⁴⁶ ’ ⁸⁷ Our findings nevertheless highlight significant therapeutic potential in targeting EC SIPi and suggest that lack of consideration for the capacity of SIPi agonists to cross the BBB and their propensity to desensitize SIPi and hamper endogenous protection may explain lack of efficacy in some studies. Although our results with the S1P1GS mouse suggest that fingolimod and RP-001 do not efficiently engage EC SIPi signaling at the BBB in a relevant time-frame, it should also be noted that the model may under-represent S lPi activation because of sensitivity and bias in reporting b-arrestin coupling. ⁵⁷ Collectively, our results argue that an optimal SIPi-directed therapeutic for stroke will efficiently cross the BBB to engage and minimally desensitize EC SIPi while inducing only transient immunosuppression to decrease the risk of post-stroke infections. Fingolimod has prolonged actions, is poorly BBB penetrating and induces long lasting lymphopenia. Although also desensitizing at high doses, CYM-5442 rapidly and preferentially distributes to brain, triggers only transient immunosuppression, and is mostly washed out from plasma and brain 24 hours after administration. ⁶⁹ Other second-generation S1PR modulators such as the FDA approved SIP1&5 agonist Ozanimod may share these properties. ^45,73

In conclusion, our observations argue a key role for EC autonomous SIP production, export, and signaling in the regulation of endothelial function and vascular reactivity in the brain that becomes critical in the context of cerebral ischemia. Although SIPi signaling is partially sustained by endogenous SIP delivery in young, healthy mice, it can be further boosted with pharmacological agonists. Joint targeting of lymphocyte SIPi - which is highly sensitive to desensitization - and endothelial SIPi with BBB penetrating SIPi agonists is feasible and provides substantial protection in transient and permanent ischemic stroke models up to 6 hours after MCA occlusion. Mechanistically, joint vascular and immune SIPi targeting may provide protection by promoting blood flow, sustaining BBB integrity, and limiting microvascular thrombosis, ⁴⁴ thus improving vascular patency and blood perfusion independent of the prolonged immunosuppression induced by some SIPi modulators. This is consistent with observed efficacy of FTY720 on downstream microvascular perfusion even in patients with failed recanalization to altapase, and suggests that SIPi agonists may preserve microvascular function and the recruitment of the collateral circulation even when recanalization is either not possible or not successful. This therapeutic strategy could therefore be envisioned in patients as soon as stroke is diagnosed, without waiting for the outcome of thrombolysis, which provides measurable benefit in less than 1/3 of patients treated. Sustained SIPi expression during ageing in mice and humans (data not shown), and efficacy of SIPi agonist on vascular parameters in murine models of diabetes and hypertension as well as in small scale clinical trials of stroke suggest that SIPi remains a viable target in the typical stroke patient. Existing evidence also suggests that SIPi targeting is efficacious and safe in conjunction with thrombolysis and in hemorrhagic stroke, ^{37,88,89,90,91} and SIPi polarization argues that minimally desensitizing BBB penetrating agonists will be preferred also for these indications. The critical protective roles for endothelial SIPi and its mechanisms of engagement observed in the brain may be relevant to vascular dementia as well as to ischemic disease of other organs.

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