Stabilization of alcohol intoxication-induced cardiovascular instability

The present invention concerns use of an agent that increases sphingosine-1-phosphate (S1P) receptor activity, such as S1P, or an S1P receptor agonist, for treatment of alcohol-intoxicated subjects will help stabilize blood pressure and improve resuscitation efforts in alcohol-intoxicated subjects, including alcohol-intoxicated trauma patients. Another aspect concerns a kit for stabilization of alcohol-intoxicated subjects, comprising an agent that increases S1P receptor activity.

BACKGROUND OF THE INVENTION

Acute alcohol intoxication is a significant health problem in the United States and contributes to an increased risk of traumatic injury, accounting for the majority of alcohol-related disorders encountered in emergency rooms [17,41]. Frequent drinkers, reported as those individuals who drink on a weekly basis, are six times more likely to be admitted to the emergency room than individuals who drink infrequently [7]. Nearly 40% of injured patients admitted to the ER have intoxicating BAC greater than 80 mg/dL [7,22,38,41]. Intoxicated trauma patients often present with increased initial injury severity compared to non-intoxicated patients [18,38]. In addition, these patients typically require a significantly greater frequency of interventions in the hospital such as endotracheal intubations, placement of intracranial monitoring devices, and greater use of diagnostic peritoneal lavage [18].

One particularly problematic outcome that can occur with acute alcohol intoxication is aggravated hemodynamic instability following hemorrhage [33]. Intoxicated trauma patients are significantly more hypotensive upon arrival to the emergency department compared to non-intoxicated patients. Clinical studies have demonstrated that this low mean arterial blood pressure (MABP) in alcohol-intoxicated patients is a predictor of poor outcomes [3,16,41]. In addition, these patients require significantly greater volumes of intravenous crystalloid resuscitative fluids and blood products, despite the fact that there is no increase in severity of injury or difference in the international normalized ratio (used in determining the time blood takes to clot) compared to non-intoxicated patients [3,12,33]. Several studies have postulated that an impaired baroreceptor reflex or associated neural, endocrine, and metabolic mechanisms that control vascular tone may be responsible for this increased hypotension [14,24,26,27]. This is supported by the findings that: 1) significantly less blood from alcohol-intoxicated rats is required to produce the same hypotensive pressure during a fixed-pressure hemorrhage; and 2) with fixed-volume hemorrhage, alcohol-intoxicated rats are significantly more hypotensive following equal volumes of removed blood [24,27]. The elevations in plasma levels of epinephrine, norepinephrine, and arginine vasopressin to fixed-pressure hemorrhage are all significantly blunted in alcohol intoxicated rats compared to non-intoxicated animals [26]. However, more recent studies have shown that sympathetic control of blood pressure and acetylcholine-induced vasodilation are not impaired by alcohol intoxication [23,25]. Intracerebroventricular administration of choline, a precursor of the preganglionic neurotransmitter acetylcholine, is unable to improve hemodynamic and neuroendocrine counter-responses to hemorrhagic shock in alcohol-intoxicated rats [23]. Furthermore, studies using isolated aorta and mesenteric arteries of intoxicated and non-intoxicated rats show that this impaired hemodynamic counter-regulation to hemorrhage is not due to a decrease in responsiveness of blood vessels to vasoconstrictors like phenylephrine, or vasodilators like acetylcholine [25].

BRIEF SUMMARY OF THE INVENTION

Traumatic injuries often are associated with alcohol intoxication, which causes a complex, altered physiologic state. A hallmark feature is hypotension (low blood pressure) in the cardiovascular system, which can significantly worsen outcomes for trauma patients due to poor tissue perfusion. Improving alcohol-induced hypotension is a goal to improve outcomes for these patients. Several studies have investigated the impact of alcohol on the baroreceptor reflex, neuroendocrine control, and local control of arterial tone, which are altered but do not fully explain the alcohol-induced hypotension. In recent years, the inventors discovered that alcohol impacts the microcirculation and lymphatics, which also have a key role in maintaining central blood volume. It was observed that alcohol intoxication increases microvascular leakage of albumin in the rat mesentery, which can produce edema. Second, alcohol causes mesenteric lymphatics to dilate and reduces their pump activity, which would cause these vessels to act more like a second capacitance vessel system than as a delivery system to return plasma proteins and fluids to the central circulation. Moreover, the inventors have found that alcohol worsens tissue blood flow and significantly elevates microvascular leakage in the rat mesentery following a two-hit model of combined alcohol intoxication and hemorrhagic shock. The inventors have determined that administration of the bioactive lipid sphingosine-1-phosphate (S1P) can improve mean arterial blood pressure and blood flow within the gut following acute alcohol intoxication in rats. S1P also reduces alcohol-induced leakage of plasma proteins. Using endothelial cell monolayer models, empirical evidence was developed showing that the barrier enhancing effects of S1P can be mediated by canonical S1P receptors, but may also involve non-receptor mediated effects. S1P is also reported to improve tone of collecting lymphatic vessels. Without being limited by theory, it is proposed that administration of agents that increase S1P activity such as S1P, or other S1P receptor agonists, attenuate alcohol-induced microvascular hyperpermeability, helping to stabilize their blood pressure and improve resuscitation efforts in alcohol-intoxicated subjects. In some embodiments, the subjects are alcohol-intoxicated trauma patients. In some embodiments, the trauma patients are hemorrhage trauma patients. In some embodiments, the alcohol-intoxicated patients are not trauma patients (not currently suffering from an acute trauma).

DETAILED DESCRIPTION OF THE INVENTION

Traumatic injury is the number one killer of healthy individuals. Alcohol intoxication is typically found in trauma patients. Alcohol-induced hypotension is a significant clinical problem, particularly when patients are in shock or have lost blood. Conventional fluid resuscitation typically is not very effective in alcohol-intoxicated patients.

The inventors investigated whether alcohol impacts vascular mechanisms other than those that focus on vascular tone. One such potential mechanism behind alcohol-induced hypotension following hemorrhage is a reduction in central fluid volume due to elevated microvascular permeability. The inventors recently demonstrated that alcohol intoxication causes microvascular leakage in the mesentery [10]. The inventors have now investigated whether alcohol-induced microvascular hyperpermeability contributes to the hypotension.

Because hypotension is a strong predictor of poor outcomes, the inventors have developed a new strategy to ameliorate alcohol-induced hypotension based on their findings. The invention involves activation of sphingosine-1-phosphate (S1P) receptors, either by its endogenous agonist S1P, or by other exogenous receptor agonists. S1P receptors are ubiquitous throughout the body, and the effects of S1P throughout the body can collectively stabilize blood pressure. The agent that increases S1P receptor activity may stabilize blood pressure, for example, by causing a reduction (partially or fully resolving) alcohol-induced endothelial barrier dysfunction. Stabilization of blood pressure may be determined using methods known in the art such as by measurement of transepithelial electrical resistance (TER). For example, the agent may reduce the recovery time for TER to a reestablished normal or baseline for the subject.

Typically, the extent of intoxication is one resulting in alcohol-induced hypotension in the subject. Thus, the methods of the invention also include amelioration of alcohol-induced hypotension in the subject by administration of an effective amount of an agent that increases sphingosine-1-phosphate (S1P) receptor activity in the subject. In some embodiments, administration of the one or more agents causes improvement in endothelial barrier integrity (at least to a level that is a medically healthy level or normal for that subject). In some embodiments, administration of the one or more agents causes sustained improvement of endothelial barrier integrity (at least to a level that is a medically healthy level or normal for that subject) for a period of time, e.g., sixty minutes or longer.

Optionally, the subject may be identified as an alcohol-intoxicated subject prior to administration of the agent to the subject. In some embodiments, the methods of the invention include measuring the extent of alcohol intoxication in the subject (e.g., by measuring blood alcohol concentration (BAC), ethanol in saliva, or large osmolar gap (gap between measured and calculated plasma osmolality)), or otherwise determining whether the individual is suffering from alcohol intoxication (e.g., by observable symptoms of intoxication), before, during, and/or after administration of the agent. BAC measurement may be done directly by blood analysis or by breath testing, for example.

In some embodiments, the methods of the invention include measuring the subject's blood pressure before, during, and/or after administration of the agent to the subject. This may be useful in detecting or monitoring hypotension in the subject. Various methods for measuring blood pressure may be utilized, including a variety of techniques and devices, such as those described in Pickering TG et al.,Circulation,2005, 111:697-716, which is incorporated herein by reference in its entirety. Useful techniques include, for example, auscultatory, oscillometric, palpation, and use of obliteration of the pulse wave on a pulse oximeter. Mercury, aneroid, or hybrid sphygmomanometers may be used. Other techniques that may be used include the finger cuff method of Penaz, ultrasound, and tonometry. Manual or automated systems may be used.

Accordingly, in some embodiments, the methods of the invention include administering the agent to a subject in which blood pressure has been measured, extent of intoxication has been determined, or both.

In some embodiments, the method further comprises evaluating one or more times whether S1P receptor activity has increased in the subject after administration of the agent.

In some embodiments, the method further comprises evaluating one or more times following administration of the agent whether endothelial barrier enhancement actions on the microcirculation has occurred (to reduce endothelial hyper permeability and subsequent loss of central fluid volume to the extravascular space as occurred).

Agents that may be administered to a subject to increase S1P activity include, but are not limited to, S1P or an S1P agonist.

In some embodiments, S1P is delivered to the subject in its natural state. Optionally, the S1P can include one or more modifications at one or both ends of the molecule, and/or internally.

In some embodiments, the agent that increases S1P receptor activity is an agonist of one or more S1P receptors. Selective S1P1receptor agonists are compounds which preferentially activate the human S1P1receptor sub-type from among the S1P1, S1P2, S1P3, S1P4, and S1P5family members of sphingosine-1-phosphate-sensitive human G-protein coupled receptors. S1P receptor agonists decrease the number of circulating lymphocytes in peripheral blood in humans or animals after e.g., oral administration, therefore they have therapeutic potential in a variety of diseases associated with a dysregulated immune system. For example, the S1P receptor agonist FTY720 has been found to reduce the rate of clinical relapses in multiple sclerosis patients (Kappos L et al.,N Engl J Med.,2006 Sep. 14, 355(11): 1124-40).

S1P receptor agonists useful in the invention may be identified on the basis of their ability to produce endothelial barrier enhancement actions on the microcirculation to reduce endothelial hyperpermeability and subsequent loss of central fluid volume to the extravascular space. Typically, the agonists have greater affinity for the S1P receptor 1 (S1PR1), as this is the receptor responsible for the barrier enhancing effects of S1P. Permeability assays may be used in vitro to assess the effectiveness of these compounds on permeability in cultured cells and isolated vessels. These include transendothelial electrical resistance measurements and transwell assays. In some embodiments, the agonists have no affinity for S1PR2, or little affinity for S1PR2 relative to the affinity for S1PR1. The S1PR2 receptor does not become activated until S1P levels are much higher than the reported physiological concentration (0.5-4 μM) and activated RhoA pathways which are barrier disruptive. Treatments that act as a negative inotrope or negative chronotrope can be detrimental in trauma patients, particularly hemorrhage patients.

In some embodiments, the agent that increases S1P receptor activity is FTY720 or an FTY720 analog or derivative thereof. FTY720 is a compound structurally similar to S1P with demonstrated barrier-enhancing activity, similar to S1P. See Adachi et al.,Perspect Medicin Chem,2007, 1:11-23; and Camp et al.J Pharmacol Exp Ther,2009, 331:54. FTY720, FTY, and Fingolimod are used interchangeably, referring to 2-amino-2-(4-octylphenethyl)propane-1,3-diol (Fingolimod). FTY720 is clinically approved for treating multiple sclerosis, in which it acts as an anti-inflammatory. The drug is a S1P receptor agonist selectively activating S1P receptor 1 (S1PR1). This receptor is selective for S1P and produces the desired endothelial barrier enhancement actions of S1P. In some embodiments, the S1P receptor agonist is phosphorylated or unphosphorylated FTY720. In some embodiments, the S1P receptor agonist comprises FTY720-phosphate (FTY720P). In some embodiments, the S1P receptor agonist comprises FTY720 (S)-phosphonate.

As used herein, the term “FTY720 derivative or analog” refers to a class of compounds, natural or synthetic, that are structurally similar to FTY720 suitable for use in the instant invention but the term does not encompass FTY720 (2-amino-2-(4-octylphenethyl)propane-1,3-diol; Fingolimod).

FTY720 derivatives or analogs include, without limitation, the (R) or (S) enantiomer of FTY720-phosphonate, the (R) or (S) enantiomer of FTY720-enephosphonate, and the (R) or (S) enantiomer of FTY720 regioisomer (3-(aminomethyl)-5-(4-octylphenyl)pentane-1,3-diol), or pharmaceutically acceptable salts thereof. In some embodiments, the FTY720 derivative or analog has one or more phosphate or phosphonate groups.

In some embodiments, the FTY720 analog or derivative is an FTY720-phosphonate, including the (R) and (S) enantiomers of FTY720-phosphonate, i.e., enantiomerically enriched or purified preparations of (R)- and (S)-3-amino-3-(hydroxymethyl)-5-(4-octylphenyl)pentylphosphonic acid, and the (R) and (S) enantiomer of FTY720-enephosphonate, i.e., enantiomerically enriched or purified preparations of (R)- and (S)-3-amino-3 -(hydroxymethyl)-5-(4-octylphenyl)pent-1-enylphosphonic acid. In some embodiments, the FTY720 analog or derivative is FTY720-phosphonate, including the (R) and (S) enantiomers of FTY720-phosphonate, i.e., enantiomerically enriched or purified preparations of (R)- or (S)-3-amino-3-(hydroxymethyl)-5-(4-octylphenyl)pentylphosphonic acid. In some embodiments, the FTY720 analog or derivative is (S)-FTY720-phosphonate. In some embodiments, the FTY720 analog or derivative does not include FTY720-phosphate (p-FTY720, FTY720P, or FTY720-P). Many S1P agonists are commercially available, such as CS 2100, CYM 50260, CYM 50308, CYM 5442 hydrochloride, CYM 5520, CYM 5541, RP 001 hydrochloride, SEW 2871, TC-G 1006, and TC-SP 14 (Tocris Bioscience).

SEW2871 (5-[4-phenyl-5-(trifluoromethyl)-2-thienyl]-3-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazole), CAS # 256414-75-2, is also obtainable from Cayman Chemical (Ann Arbor, Mich.)). SEW2871 is a selective S1P receptor agonist that is an immunosuppressant.

Selective S1P1receptor agonists also include those disclosed in the published PCT applications WO 2005/054215, WO 2005/123677, WO 2006/010544, WO 2006/100635, WO 2006/100633, WO 2006/100631, WO 2006/137019, WO 2007/060626, WO 2007/086001, WO 2007/080542, WO 2008/029371, WO 2008/029370, WO 2008/029306, WO 2008/035239, WO 2008/114157, WO 2009/024905, and WO 2009/025767, and are incorporated herein by reference in their entirety.

In some embodiments, an S1P1agonist based on the 2-imino-thiazolidin-4-one scaffold is used, such as the S1P1agonist in Bolli MH et al.,J Med Chem,2010, 53(10):4198-4211, which are incorporated herein by reference.

U.S. Pat. No. 7,838,562 (Hla T et al.) describes agonists of vascular endothelial SP1 receptors, and are incorporated herein by reference in their entirety.

U.S. Patent application publication 20140100195 (Caldwell RD et al.) describes compounds that have agonist activity at one or more S1P receptors, and are incorporated herein by reference in their entirety.

U.S. Patent application publication 20140309190 (Jermaine T et al.) describes bicyclic aryl compounds that have agonist activity at one or more of the S1P receptors.

In a further aspect, the invention provides therapeutic methods that use an S1P-receptor agonist, which refers to any molecule that induces S1P receptor biological activity, including downstream pathways mediated by S1P signaling; such as receptor binding and elicitation of a cellular response to S1P.

The S1P receptor agonists may be in the form of a pharmaceutically acceptable salt. The term “salt” refers to a pharmaceutically acceptable and relatively non-toxic, inorganic or organic acid addition salt of a compound of the present invention. Pharmaceutical salt formation consists in pairing an acidic, basic or zwitterionic drug molecule with a counterion to create a salt version of the drug. A wide variety of chemical species can be used in a neutralization reaction. Pharmaceutically acceptable salts of the invention thus include those obtained by reacting the main compound, functioning as a base, with an inorganic or organic acid to form a salt, for example, salts of acetic acid, nitric acid, tartric acid, hydrochloric acid, sulfuric acid, phosphoric acid, methane sulfonic acid, camphor sulfonic acid, oxalic acid, maleic acid, succinic acid or citric acid. Pharmaceutically acceptable salts of the invention also include those in which the main compound functions as an acid and is reacted with an appropriate base to form, e.g., sodium, potassium, calcium, magnesium, ammonium, or choline salts. Though most of salts of a given active principle are bioequivalents, some may have, among others, increased solubility or bioavailability properties. Salt selection is now a common standard operation in the process of drug development as taught by H. Stahl and C. G Wermuth in their handbook (Stahl H & Wermuth C G (2011) Pharmaceutical salts: Properties, selection, and use, 2nd ed. (Wiley-VCH, ed.).

In a preferred embodiment, the designation of a compound is intended to designate the compound per se, as well as any pharmaceutically acceptable salt, hydrate, isomer, or racemate thereof

The term “agonist” implies no specific mechanism of biological action, and is deemed to expressly include and encompass all possible pharmacological, physiological, and biochemical interactions with the S1P receptor and its consequences which can be achieved by a variety of different, and chemically divergent, compositions. In one aspect, the S1P receptor agonists are sphingosine and S1P structural analogs which bind to an S1P receptor under physiological conditions in vitro or in vivo, wherein the binding exerts an agonistic effect on the S1P receptor. Agonists of all types of S1P receptors, such as S1P(1-5) are acceptable, but those of S1P1 are preferred. S1P structural analogs are believed to bind to the S1P binding site of S1P receptors. However, agonists may bind at sites other than the S1P binding site, provided such binding results in an agonistic effect. Typically, the agonists have greater affinity for the S1P receptor 1 (S1PR1), as this is the receptor responsible for the barrier enhancing effects of S1P. Permeability assays may be used in vitro to assess the effectiveness of these compounds on permeability in cultured cells and isolated vessels. These include transendothelial electrical resistance measurements and transwell assays. In some embodiments, the agonists have no affinity for S1PR2, or little affinity for S1PR2 relative to the affinity for S1PR1.

In aspects where the S1P receptor agonist is a small molecule, a small molecule can have a molecular weight of about any of 100 to 20,000 daltons, 500 to 15,000 daltons, or 1000 to 10,000 daltons. Libraries of small molecules are commercially available. The small molecules can be administered using any means known in the art, including inhalation, intraperitoneally, intravenously, intramuscularly, subcutaneously, intrathecally, intraventricularly, orally, enterally, parenterally, intranasally, or dermally. Usually, when the

S1P receptor agonist according to the invention is a small molecule, it will be administered at the rate of 0.1 to 300 mg/kg of the weight of the patient divided into one to three or more doses. For an adult patient of normal weight, doses ranging from 1 mg to 5 g per dose can be administered.

Kits Comprising Blood Pressure Stabilizing Agents of the Invention

The invention also provides kits comprising agents of the invention, which may be used, for example, for stabilizing alcohol-intoxicated patients. The kits may be used for any of the methods described herein, including, for example, to treat a subject with alcohol-induced intoxication. The kits may optionally provide additional components such as, buffers and instructions for use of an agent in any of the methods described herein. In some aspects, the kits include instructions for treating pain. In some aspects, the kit comprises an agent described herein and instructions for stabilizing blood pressure in an alcohol-intoxicated subject.

The kits of the invention are useful for storing and dispensing the agents that increase

S1P activity described herein. In addition to such S1P activity increasing agents, the kit may further include one or more other components, including but not limited to, agents useful for treating or stabilizing alcohol intoxicated individuals. In some embodiments, the agent that increases S1P activity is S1P or an agonist of the S1P receptor. The kit comprises an agent that increases S1P activity in a subject; and one or more agents selected from the group consisting of: glucose, thiamine (vitamin B1), intravenous access device, intravenous fluid comprising normal saline (e.g., sodium chloride), benzodiazepine (e.g., such as lorazepam (Ativan), diazepam (Valium), or chlordiazepoxide (Librium)), or pentobarbital. The kits comprise one or more doses of the S1P receptor activity-increasing agent, and one or more additional components. In one embodiment, the kit comprises two of the foregoing additional components. In another embodiment, the kit comprises three of the foregoing additional components. In another embodiment, the kit comprises four of the foregoing additional components. In other embodiments, the agent is in solid (e.g., powder), liquid, or semi-solid form. The kit may include printed instructions for carrying out one or more steps of the methods described herein.

It should be appreciated that the kits of the invention are not limited to any particular container configuration. The container(s) can be constructed and arranged, and the agent can be prepared (e.g., solid or liquid), stored, and dispensed, in any of numerous ways within the scope of the invention. For example, in some embodiments, the kit includes a tray or other housing with one or more cavities of desirable geometries for receiving one or more components of the kit. Containers for holding components of the kit can be rigid (such as canisters) or soft (such as bags or pouches), as needed or desired. Materials for constructing containers for medicaments in various physical states are known in the art. In some embodiments, the container(s) and housing is plastic, such as polypropylene. The kit is preferably packaged for ease of handling and use by the subject to be treated or by medical personnel. The kit is preferably sealed and sterilized.

The kit may contain one or more containers containing an S1P receptor activity-increasing agent and, optionally, other agents, contained in the same container or provided separately in individual containers. Each container may contain one or more doses of the S1P receptor activity-increasing agent.

As used herein, the term “S1P” refers to Sphingosine-1-Phosphate. An “S1P receptor”, also referred to as “S1PR”, refers to a polypeptide that is bound by or activated by S1P. S1P receptors include those of any mammalian species, including, but not limited to, human, canine, feline, equine, primate, or bovine. This definition includes S1P receptor subtypes S1P, S1P2, S1P3, S1P4 and S1P5, also known as S1PR1, S1PR2, S1PR3, S1PR4, and S1PR5, respectively.

“Biological activity” of S1P generally refers to the ability to bind S1P receptors and activate S1P receptor signaling pathways, including but not limited to S1P1 receptor signaling pathways. Biologically active fragments and variants of S1P (i.e., those with biological activity) may be administered to the subject. Without limitation, a biological activity includes any one or more of the following: the ability to bind an S1P receptor, the ability to promote S1P receptor dimerization and phosphorylation; the ability to activate an S1P receptor signaling pathway; and the ability to produce endothelial barrier enhancement actions on the microcirculation to reduce endothelial hyperpermeability and subsequent loss of central fluid volume to the extravascular space. Typically, biologically active fragments and variants have greater affinity for the S1P receptor 1 (S1PR1), as this is the receptor responsible for the barrier enhancing effects of S1P. Permeability assays may be used in vitro to assess the effectiveness of fragments and variants on permeability in cultured cells and isolated vessels. These include transendothelial electrical resistance measurements and transwell assays.

As used herein, “substantiality pure” refers to material which is at least 50% pure (i.e., free from contaminants), more preferably at least 90% pure, more preferably at least 95% pure, more preferably at least 98% pure, more preferably at least 99% pure.

The subject is a mammal, such as a human. In some embodiments, the subject is an alcohol-intoxicated trauma patient. For example, the subject may have endured acute trauma. For example, the subject may be undergoing treatment in an emergency room (ER) setting. In some embodiments, the subject is a hemorrhage trauma patient. In other embodiments, the subject is not one suffering acute trauma at the time of administration. In some embodiments, the subject is not suffering from chronic alcoholism.

In some embodiments, the method further comprises identifying the subject as being alcohol-intoxicated and/or suffering from alcohol-intoxicated trauma prior to administering the agent that increases S1P receptor activity in the subject. In some embodiments, the subject is identified as an alcohol-intoxicated hemorrhage trauma patient prior to administration of the agent. Subjects may be identified as alcohol-intoxicated, or having alcohol-intoxicated trauma, and/or having hemorrhage by a qualified medical care provider (e.g., physician or nurse) by evaluating signs and/or symptoms known in the art.

As used herein, a “pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein (e.g., an agent that increases S1P receptor activity) with other components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of an agent to the subject.

As used herein, the term “active ingredient” refers to the agent accountable for the intended biological effect (e.g., increase in S1P receptor activity). In some embodiments, the agent is S1P or a biologically active fragment or variant thereof In some embodiments, the agent is an agonist of the S1P receptor. As used herein, the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier,” which may be used interchangeably, refer to a carrier or a diluent that does not cause significant irritation to the subject and does not abrogate the biological activity and properties of the administered agent.

As used herein, the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in the latest edition of “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., which is herein fully incorporated by reference.

Suitable routes of administration for agents that increase S1P receptor activity (e.g., S1P and S1P agonists) and the pharmaceutical compositions containing them may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal, or parenteral delivery, including intramuscular, subcutaneous, and intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.

Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a subject.

For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated may be used in the formulation. Such penetrants are generally known in the art.

For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane, or carbon dioxide. In the case of a pressurized aerosol, the dosage may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base, such as lactose or starch.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., a sterile, pyrogen-free, water-based solution, before use.

The pharmaceutical composition of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, for example, conventional suppository bases such as cocoa butter or other glycerides.

Pharmaceutical compositions suitable for use in the context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, an “effective amount” means an amount of active ingredients (e.g., agent that increases S1P receptor activity) effective to prevent, delay onset of, alleviate, or ameliorate symptoms of a disorder (e.g., alcohol-induced microvascular hyperpermeability).

Determination of an effective amount is well within the capability of those skilled in the art in light of the detailed disclosure provided herein.

For any preparation used in the methods of the invention, the dosage or the effective amount can be estimated initially from in vitro and cell culture assays (e.g., permeability assays sucha s transendothelial electrical resistance measurements and transwell assays, and as further described herein below). For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

Exemplified Embodiments Of The Invention

Embodiment 1. A method for stabilizing blood pressure in an alcohol-intoxicated subject, comprising administering an effective amount of an agent that increases sphingosine-1-phosphate (S1P) receptor activity in the subject.

Embodiment 2. The method of embodiment 1, wherein the agent is S1P, or an agonist of the S1P receptor.

Embodiment 3. The method of embodiment 1 or 2, wherein the subject is suffering from acute physical trauma.

Embodiment 4. The method of embodiment 1 or 2, wherein the subject is not suffering from acute physical trauma. Embodiment 5. The method of any preceding embodiment, further comprising identifying the subject as being alcohol-intoxicated at the time of said administering.

Embodiment 6. The method of any preceding embodiment, further comprising measuring the extent of alcohol-intoxication in the subject before, during, and/or after said administering.

Embodiment 7. The method of any preceding embodiment, further comprising measuring the subject's blood pressure before, during, and/or after said administering.

Embodiment 8. The method of embodiment 1, wherein the agent comprises phosphorylated or unphosphorylated 2-amino-2-[2-(4-octaphenyl)ethyl]propane-1,3 diol (FTY720; Fingolimod).

Embodiment 9. The method of embodiment 1, wherein the agent comprises FTY720-phosphate (FTY720P) or FTY720 (S)-phosphonate.

Embodiment 10. The method of any preceding embodiment, wherein an S1P receptor agonist is administered to the subject, and wherein the agonist is selected from the group consisting of:

or a combination of two or more of the foregoing agonists.

Embodiment 11. The method of embodiment 2, wherein the S1P receptor agonist is a selective agonist for the S1P1 receptor.

Embodiment 12. The method of embodiment 1 or 2, wherein the S1P receptor agonist is selected from the group consisting of Fingolimod, BAF312, ACT-128800, ONO-4641, CS-0777, KRP-203, PF-991, W146, and SEW2871, or a combination of two or more of the foregoing agonists.

Embodiment 13. The method of embodiment 1, wherein the agent is an agonist of the S1P receptor and has one or more phosphate or phosphonate groups.

Embodiment 14. The method of any preceding embodiment, wherein the subject has alcohol-induced hypotension, and wherein said administering ameliorates the alcohol-induced hypotension.

Embodiment 15. A kit comprising an agent that increases sphingosine-1-phosphate (S1P) receptor activity in a subject; and one or more agents selected from the group consisting of:

intravenous access device,

intravenous fluid comprising normal saline (e.g., sodium chloride),

benzodiazepine (e.g., such as lorazepam (Ativan), diazepam (Valium), or chlordiazepoxide (Librium)), or pentobarbital. Embodiment 16. The kit of embodiment 15, wherein the agent is S1P or an agonist of the S1P receptor.

Embodiment 17. The kit of embodiment 15, wherein the agent comprises phosphorylated or unphosphorylated 2-amino-2-[2-(4-octaphenyl)ethyl]propane-1,3 diol (FTY720; Fingolimod).

Embodiment 18. The kit of embodiment 15, wherein the agent comprises FTY720-phosphate (FTY720P) or FTY720 (S)-phosphonate.

Embodiment 19. The kit of embodiment 15, wherein agent is selected from the group consisting of:

or a combination of two or more of the foregoing agonists.

Embodiment 19. The kit of embodiment 15, wherein the agent is a selective agonist for the S1P1 receptor.

Embodiment 20. The kit of embodiment 15, wherein agent is selected from the group consisting of Fingolimod, BAF312, ACT-128800, ONO-4641, CS-0777, KRP-203, PF-991, W146, and SEW2871, or a combination of two or more of the foregoing agonists.

Embodiment 21. The kit of embodiment 15, wherein the agent is an agonist of the S1P receptor, and has one or more phosphate or phosphonate groups.

All patents, patent applications, provisional applications, and publications referred to or cited herein, supra or infra, are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

Acute Alcohol Intoxication Stimulated Microvascular Hyperpermeability In Vivo

Administration of a 2.5-g/kg dose of alcohol was performed via an intragastric catheter that was implanted 2-3 days before the day of experiment. The animals were allowed to roam freely for 30 minutes after administration. This dosage of alcohol produced intoxicating BAC within 30 minutes and was significantly higher than those animals in the time-matched, water-treated groups (FIG. 1). Sixty minutes after the start of IVM, alcohol-treated animals had noticeable extravasation of FITC-albumin from the mesenteric microcirculation compared to controls (FIGS. 2A and 2B). There is marked fluorescence in the surrounding interstitium in the alcohol rats as opposed to the water rats where the fluorescence remains within the vessels of the arterioles, capillaries, and venules. In addition, there is accumulation of FITC-Albumin within and just outside the vessel walls resulting in intense areas of fluorescence, or hotspots of FITC-Albumin extravasation. This extravasation was quantified by calculating the maximum change in IOI from baseline. The mean maximal change in IOI in the extravascular space adjacent to postcapillary venules was significantly higher in the alcohol-treated rats compared to control rats (FIG. 2C). Mean arteriolar diameter of third-order arterioles was also assessed to determine whether changes in blood flow may contribute to the overall permeability response. There was no significant difference in mean arteriolar diameter between the alcohol and control groups, supporting the notion that increased permeability of the venular endothelium primarily contributed to the observed macromolecule leakage (FIG. 2D).

These experiments demonstrate that acute alcohol intoxication in naive Sprague-Dawley rats can induce microvascular hyperpermeability to FITC-albumin in the mesenteric microcirculation compared to water-treated controls. In addition, the increase in hyperpermeability is not due to increases in the diameter of the arterioles, suggesting that increased filtration forces from an increase in capillary hydrostatic pressure is not responsible for the increased permeability of the microvessels.

Acute Alcohol Intoxication Significantly Decreased the Barrier Function of Cultured Endothelial Cells

The direct impact of alcohol on endothelial barrier function was tested using cultured HUVEC or HMVEC-c confluent monolayers grown on gold-plated electrodes. Thirty minutes of baseline transepithelial electrical resistance (TER) measurements were recorded before the cells were treated with alcohol at final bath concentrations of 20, 50 or 100 mM. Alcohol was added directly into the MEDIA within the wells. For both HUVEC (FIG. 3A) and HMVEC-c (FIG. 3B), all concentrations of alcohol tested caused an immediate decline in the TER of the monolayer. The decrease in TER was alcohol concentration-dependent with high alcohol concentrations eliciting a larger drop in TER. The maximal decline in TER from baseline was significant for the 50 and 100 mM concentrations in both HUVEC (FIG. 3C) and HMVEC-c (FIG. 3D). These experiments demonstrated that acute alcohol intoxication alone had a direct effect on the endothelial monolayer and caused barrier compromise.

Acute Alcohol Intoxication Significantly Decreased Baseline MAP and Blunted Recovery of MAP During Resuscitation Following Fix-Pressure Hemorrhage

The impact of alcohol intoxication combined with traumatic injury on microvascular hyperpermeability was assessed. A fixed-pressure hemorrhage and resuscitation (HSR) model previously described by Molina and colleagues was employed to achieve injury [23-27,41]. Animals had catheters implanted in the carotid artery, jugular vein, and an indwelling gastric catheter 3-4 days before the experiment. The animals were administered a 2.5-g/kg dose of alcohol or isovolumic and allowed to roam freely for 30 minutes. After 30 minutes, baseline MAP measurements were recorded for 30 minutes before the start of hemorrhage. Blood was shed to achieve a fixed-pressure of 40-60 mm Hg for 60 minutes and followed by resuscitation with lactated Ringer's solution for 60 minutes. MAP was monitored and recorded in real-time for the duration of the HSR protocol (FIG. 4A). Alcohol intoxicated animals presented with significantly lower baseline MAP compared with water-treated animals. In addition, recovery of MAP during the resuscitation phase was significantly blunted in alcohol-treated animals compared to water. The alcohol-exacerbated hypotension during hemorrhage is further illustrated by examining total blood volume removed (TBR) (FIG. 4B). Alcohol animals required significantly less of their total blood volume to be shed to achieve the 40-60 mm Hg target MAP compared to water-treated animals.

These results confirm the data seen by Molina and colleagues as well as the clinic in that alcohol intoxication induces hemodynamic instability and increased hypotension [4,23-27,33,41]. Alcohol intoxication induces hypotension in Sprague-Dawley rats during baseline and blunts recovery of MAP during hemorrhage. Further, the volume of blood removed during hemorrhage is significantly reduced with alcohol intoxication.

Acute Alcohol Intoxication Combined with HSR Significantly Enhanced Microvascular Hyperpermeability In Vivo

Immediately following completion of the HSR protocol the animal was anesthetized and prepped for IVM. Sixty minutes after the start of IVM, alcohol-sham animals had noticeable extravasation of FITC-albumin from the mesenteric microcirculation compared to water shams (FIGS. 5A and 5B). As seen before with animals receiving only alcohol or water, there is marked fluorescence in the surrounding interstitium in the alcohol rats as opposed to the water rats where the fluorescence remains within the vessels of the arterioles, capillaries, and venules. In addition, there is accumulation of FITC-Albumin within and just outside the vessel walls resulting in intense areas of fluorescence, or hotspots of FITC-Albumin extravasation. Those animals treated with water combined with HSR also display fluorescence in the extravascular regions of the surrounding interstitium and hotspots of accumulation in the vessel walls consistent with results seen by fellow investigators [34-37] (FIG. 5C). Finally, animals that were administered alcohol even greater fluorescence in the surrounding interstitium that extravasates even further from the vasculature than seen in the alcohol-shams and water-HSR groups (FIG. 5D). This extravasation was quantified by calculating the mean 60-minute RM. The mean 60-minute IOI in the extravascular space adjacent to postcapillary venules was significantly higher in the alcohol-sham rats compared to water-sham rats while RN was significantly higher in alcohol-HSR rats compared to water-HSR rats (FIG. 5E).

These results demonstrate that alcohol has an exacerbating effect on HSR-induced microvascular hyperpermeability. This supports the inventors' hypothesis that the hemodynamic instability and hypotension seen in intoxicated patients following hemorrhage is due to loss of central fluid volume due to alcohol-induced microvascular hyperpermeability.

S1P significantly reduced recovery time of TER following alcohol treatment

The inventors next set out to determine if S1P could similarly reduce the recovery time for TER to be reestablished to baseline in HUVEC pretreated with alcohol. Following 30 minutes of baseline TER measurements, alcohol was added to the wells to a final concentration of 50 mM for 5 minutes before treatment with 1 μM S1P or vehicle. Treatment with S1P following alcohol caused the TER to rapidly rise to the previously established baseline levels, while a slow recovery occurred in vehicle groups (FIG. 6A). The recovery time was significantly reduced in the S1P-treated groups post-alcohol, reestablishing baseline TER about 30 minutes faster than cells treated with vehicle (FIG. 6B).

These data demonstrate that exogenous S1P could resolve alcohol-induced endothelial barrier dysfunction significantly faster similar to 8-CPT.

Infusion of S1P Significantly Attenuated Alcohol-Induced Microvascular Hyperpermeability In Vivo

S1P has been used to reduce the permeability of rat microvessels to BSA, however, only one study has been performed in intact animals [1,2,8,9,30]. Using the inventors' established method of observing hyperpermeability in the rat mesentery, the ability of S1P to abolish or attenuate the enhanced extravasation of FITC-albumin caused by acute alcohol intoxication was examined. After receiving a 2.5 g/kg bolus of alcohol and allowed free roaming for 30 minutes, rats were prepared for IVM and administered S1P to a final concentration of 2 μM within the FITC-albumin infusion. This provided an estimated final plasma concentration of S1P in the rats of 0.2 μM. Rats that did not receive S1P in the FITC-albumin infusion displayed extravasation of FITC-albumin from the microcirculation into the surrounding tissues (white circles) as well as accumulation of FITC-Albumin within and just outside of the vessel walls (white arrows) resulting in intense areas of fluorescence, or hotspots of FITC-Albumin (FIG. 7A). On the other hand, rats that received 0.2 μM S1P in the infusion had a marked reduction in the extravasation of FITC-albumin (FIG. 7B). The mean IOI of the rats receiving S1P in the FITC-albumin infusion was significantly lower than those rats receiving FITC-albumin infusion alone (FIG. 7C).

These data demonstrate that exogenous STP infusion at physiological concentrations is capable of attenuating the microvascular hyperpermeability caused by acute alcohol intoxication significantly. This is evidenced by reduced extravasation of FITC-albumin into the surrounding interstitium of alcohol-intoxicated rats treated with STP compared to vehicle treated rats.

Administration of S1P within Resuscitation Fluid Does not Prevent Blunted Recovery of Map During Resuscitation in Alcohol-Intoxicated Animals

The impact of STP infusion within the resuscitation fluid on the recovery of MAP following hemorrhage was assessed in alcohol-intoxicated rats. The HSR model previously described was employed with the addition of STP within the lactated Ringer's resuscitation fluid to a final dose of 0.1 mg/kg. The animals were administered a 2.5-g/kg dose of alcohol or isovolumic and allowed to roam freely for 30 minutes. After 30 minutes, baseline MAP measurements were recorded for 30 minutes before the start of hemorrhage. Blood was shed to achieve a fixed-pressure of 40-60 mm Hg for 60 minutes and followed by resuscitation containing S1P for 60 minutes. MAP was monitored and recorded in real-time for the duration of the HSR protocol (FIG. 8). Alcohol intoxicated animals receiving 0.1 mg/kg S1P within the resuscitation fluid presented with the same impaired recovery of MAP as animals that did not receive STP during resuscitation previously shown (FIGS. 4A and 4B). These results suggest that S1P is unable to restore MAP in alcohol-intoxicated animals compared to water-treated animals in the immediate phase of resuscitation.

S1P-Infused Sprague-Dawley Rats have Improved MAP During IVM Following HSR

As before, immediately following completion of the HSR protocol the animal was anesthetized and prepped for IVM. During IVM, MAP is continually monitored. Animals receiving 0.1 mg/kg S1P demonstrated improved MAP during this later stage of the protocol (FIG. 9). Alcohol-intoxicated animals receiving S1P during resuscitation had MAP between 20-25 mm Hg higher than the alcohol-intoxicated animals that received standard fluids. This improved MAP was significant at the 5, 20, and 25 minute time points for 30 minutes IVM performed. At the time of writing this, only 3 IVM experiments were performed with alcohol-intoxicated animals that received S1P during resuscitation.

These results demonstrate that while infusion of S1P within the resuscitation fluid does not help with the recovery of MAP in alcohol-intoxicated rats during the resuscitation phase, MAP is improved during the later stage of IVM compared to animals that received standard fluids. These results and those demonstrating S1P infusion attenuating alcohol-induced microvascular permeability suggests that S1P within the resuscitation fluid is restoring MAP by reducing microvascular hyperpermeability and subsequent loss of central fluid volume.

S1P acts on at least five receptors to elicit diverse cellular responses in multiple tissues. Current evidence suggests that activation of S1P1R promotes endothelial barrier integrity, while S1P2R activation disrupts barrier integrity [20,32]. The S1P mimetic FTY720 (fingolimod) has the advantage that it activates all S1P receptors except S1P2R. FTY720 is phosphorylated by sphingosine kinase, to form FTY720-phosphate (FTY720-P), which in turn activates the S1P receptors [6,31]. FTY720 enhances endothelial barrier function in vitro [11,39] and reduces VEGF- and histamine-induced microvascular hyperpermeability in vivo [19,31]. FTY720 is currently FDA-approved for treatment of multiple sclerosis, and has been tested in the clinic to determine effectiveness for reducing inflammation after intracerebral hemorrhage [13,21,28]. A key finding supporting this aim is that FTY720 reduced hypotension and lung injury caused by combined hemorrhage and trauma (laparotomy) in anesthetized rats [5]. In a swine model of uncontrolled hemorrhage, FTY720 also improved survival, which was attributed to sequestration of lymphocytes into the mesenteric lymph nodes and spleen and reducing neutrophil-driven inflammation [15]. FTY720 has also been named a functional antagonist because it can cause proteasome-mediated turnover of S1P1R, which may account for some of its pleotropic effects. A novel analog, FTY720 (S)-phosphonate, limits this S1P1R turnover and produces longer-lasting endothelial barrier enhancement [40]. One potential concern is a report that 0.5 mg/kg FTY720 reduced baseline blood pressure in rats [29]. However, this study examined blood pressure several hours after FTY720 was administered. The rats were also anesthetized in that study, which typically facilitates decreases in MAP. Their observation may also be due to turnover of S1P1R over time. The novel compound FTY720 (S)-phosphonate can prevent this problem [40].

The data inFIG. 8show that FTY720 and its analogs expedite the restoration of barrier integrity of cultured EC monolayers exposed to alcohol.FIGS. 10A-Cshow TER data. TER is an index of the barrier function of endothelial cells. After alcohol initially decreased barrier function, FTY720 accelerated its restoration to the baseline level observed in time-matched control EC monolayers, compared to alcohol alone (FIG. 10A). Unlike S1P, which restored the barrier in about 5 min (seeFIG. 6A), FTY720 restored barrier function occurred gradually (FIG. 10A). This was probably due to the fact that FTY720 must be phosphorylated by sphingosine kinase before it can exert its activity, as application of phosphorylated FTY720 (FTY720-P) to the monolayers (FIG. 10B) resulted in a much more rapid elevation in barrier function. FTY720-P caused an overshoot over the original baseline, followed by a gradual drop over time. In comparison, the novel analog FTY720 (S)-phosphonate, applied after alcohol (FIG. 10C), also caused a rapid rescue of barrier function, rising above baseline levels. However, FTY720 (S)-phosphonate also caused a sustained enhancement of barrier integrity over time. Collectively, these data show the feasibility of using S1P1R agonists to rescue endothelial barrier function in the presence of alcohol, including the sustained improvement in endothelial barrier integrity with FTY720 (S)-phosphonate.

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