Treatment or prevention of ischemic stroke reperfusion injury

Abstract

The present invention relates to salts and compositions for use in the treatment of ischemic stroke reperfusion (IR) injury. In particular, the present invention relates to a salt of formula (I) (wherein X, Y, n, m, Z, A, and B are as defined herein) for use in the treatment or prevention of ischemic stroke reperfusion injury. JPEG2023505365000006.jpg5076 formula (I)

Description

[Technical Field]

[0001] The present invention relates to salts for use in the treatment or prevention of ischemic stroke reperfusion (IR) injury. [Background technology]

[0002] Stroke is a major health problem worldwide. Recent estimates (Feigin VL et al. Global and regional burden of stroke during 1990-2010: findings from the Global Burden of Disease Study 2010. Lancet. 2014;383(9913):245-254) indicate that the global stroke prevalence is 5 per 1,000 people per year, corresponding to 33 million people surviving a stroke. In 2010, 5.9 million people suffered stroke-related deaths, and stroke resulted in the loss of more than 102 million disability-adjusted life years (DALYs), which is equivalent to the combined total of years of health lost due to disability and premature death.

[0003] In recent decades, there has been little progress in stroke treatment options and correspondingly little progress in reducing mortality / morbidity.

[0004] Currently, there is no specific treatment for ischemic stroke other than halting the ischemic attack via thrombectomy or thrombolysis. Minimizing ischemic time is important for salvaging ischemic tissue.

[0005] However, any treatment that interferes with clotting cannot be used in the emergency treatment of ischemic stroke because such treatment would be harmful in the case of hemorrhagic stroke.

[0006] Therefore, patients suspected of having an ischemic stroke must first undergo imaging, for example, using MRI or CT imaging, to determine the type of stroke, after which they can be administered medications effective in treating stroke, such as clopidogrel / ticagrelor or aspirin, or thrombolytic drugs.

[0007] Thus, valuable time is lost due to the need for urgent imaging, and ischemic time is prolonged.

[0008] Once the ischemic insult is halted, reperfusion of blood into the ischemic tissue is itself damaging, resulting in the ischemia / reperfusion (IR) injury of stroke.

[0009] There is a clear unmet clinical need for drugs that reduce the extent of IR injury after stroke. Summary of the Invention

[0010] In view of the above, there is a need to develop compounds and compositions for use in the treatment and prevention of ischemic stroke reperfusion injury in cases of hemorrhagic stroke that are not harmful to the patient. Such compounds and compositions can be administered as acute stroke medications (i.e., before diagnosis of the type of stroke) and thus improve the clinical outcome of ischemic stroke patients by reducing the damage caused by reperfusion after the ischemic attack has stopped.

[0011] There is also a general need for improved compounds and compositions useful for treating or preventing ischemic stroke reperfusion injury. Ideally, such improved compounds and compositions could be administered before removing the ischemic event to reduce the injury caused when reperfusion begins. Ideally, such compounds and compositions could also be administered in conjunction with treatments aimed at chemically or mechanically removing the ischemic event, e.g., a thrombus.

[0012] The present invention provides a compound of formula (I): [ka] Formula (I) [In the formula, X is a negative charge, H, or C1-C 12 alkyl; Y is H, OH, or C1-C 12 alkyl; n is 0 or 1; m is 0 or 1; Z is one or more pharmaceutically acceptable cations, and A and B are independently any integer, and the net charge of the salt is 0.] The present invention relates to a salt represented by the formula:

[0013] The present invention also relates to the salts of formula (I) as defined above for use as emergency medication after a stroke is suspected, prior to diagnosis of the type of stroke.

[0014] The present invention also relates to a composition for use in treating or preventing ischemic stroke reperfusion injury, comprising a salt of formula (I) in combination with one or more pharmaceutically acceptable excipients, carriers or diluents.

[0015] Preferably, the salt of formula (I) above is a salt of formula (II) defined below, more preferably a salt of malonic acid. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 shows the effect of a representative compound, disodium malonate, on infarct volume following ischemic stroke compared to saline control.

[0017] [Figure 2]FIG. 2 shows the effect of a representative compound, disodium malonate, on cerebral blood flow (CBF) at various time points during ischemic stroke and reperfusion compared to saline control.

[0018] [Figure 3] FIG. 3 shows the effect of the comparative compound dimethyl malonate on infarct volume following ischemic stroke compared to saline control.

[0019] [Figure 4] FIG. 4 shows the in vitro uptake by tissues and cells for a representative compound, disodium malonate.

[0020] [Figure 5] FIG. 5 shows the in vivo uptake by mouse tissues for a representative compound, disodium malonate.

[0021] [Figure 6] FIG. 6 shows an in vivo mouse model of acute ischemic stroke.

[0022] [Figure 7] FIG. 7 shows MALDI images of succinate levels in acute middle cerebral artery (MCA) occlusion.

[0023] [Figure 8] FIG. 8 shows succinate accumulation during stroke in mouse brain.

[0024] [Figure 9] FIG. 9 shows succinate accumulation during stroke in the human brain.

[0025] [Figure 10] FIG. 10 shows ischemic succinate levels in brain tissue of a mouse stroke model.

[0026] [Figure 11] FIG. 11 shows the activity of complex I in brain tissue from a mouse stroke model.

[0027] [Figure 12] FIG. 12 shows the levels of malonate in mouse brain tissue after administration of disodium malonate from 5 minutes before to 5 minutes after reperfusion.

[0028] [Figure 13] FIG. 13 shows the levels of malonate in mouse cerebrospinal fluid (CSF) after administration of disodium malonate from 5 minutes before to 5 minutes after reperfusion.

[0029] [Figure 14] FIG. 14 shows the effect of disodium malonate on infarct size in a mouse model of acute ischemic stroke.

[0030] [Figure 15] Figure 15 shows the brain used to generate the data shown in Figure 14. The lightly colored brain areas represent the infarcted areas.

[0031] [Figure 16] FIG. 16 shows succinate plasma levels in venous blood from patients undergoing thrombolysis for acute ischemic stroke. DETAILED DESCRIPTION OF THE INVENTION

[0032] Timely reperfusion following ischemic stroke is important for salvaging ischemic tissue. However, paradoxically, reperfusion of blood into ischemic tissue can be injurious in itself, contributing to the ischemia / reperfusion (IR) injury of the stroke.

[0033] The current best clinical approach is to perform rapid reperfusion to minimize ischemic time, but this can result in extensive IR injury, and there is a clear unmet clinical need for agents that reduce the extent of IR injury after stroke.

[0034] Historically, IR injury was conceived as a random, disorganized series of damaging events that resulted in the formation of reactive oxygen species (ROS) from reperfused ischemic tissue.

[0035] Recently, metabolic mechanisms within mitochondria have been shown to be central to IR injury. Succinate, a metabolite of the citric acid cycle, accumulates in tissues during ischemia. During reperfusion, this succinate is rapidly oxidized by succinate dehydrogenase (SDH) and transported to mitochondrial complex I. 2 This ROS pulse, along with calcium dysregulation and a decrease in ATP, initiates a series of damaging events that lead to reperfusion injury. This reperfusion results in damage beyond that caused by ischemia alone. Therefore, developing agents that reduce reperfusion injury after ischemic stroke offers a therapeutic avenue for reducing organ damage.

[0036] SDH is a key enzyme in succinate formation during ischemia and its oxidation during reperfusion. Ischemic stroke reperfusion injury could be ameliorated by altering succinate metabolism, either by preventing its accumulation during ischemia (thus reducing succinate available for oxidation during reperfusion) or by directly blocking its oxidation during reperfusion (e.g., by inhibiting SDH).

[0037] Dimethyl malonate (DMM) has previously been shown to be protective when administered before and during ischemia, as described in WO2016 / 001686, but is ineffective when administered at or just before reperfusion of the stroke, as shown in Figure 3.

[0038] The present invention arises from the surprising discovery that malonate salts and related compounds are particularly effective in treating and preventing ischemic stroke reperfusion injury. This discovery is particularly surprising because such salts were previously thought to be cell-impermeable in vivo. However, as illustrated in Figures 4 and 5, such salts are surprisingly able to penetrate a variety of cells and tissues, including brain tissue.

[0039] In particular, the present invention provides a compound of formula (I): [ka] Formula (I) The present invention relates to a salt represented by the formula:

[0040] In the above formula, X represents a negative charge, H, or C1-C 12 Preferably, X is selected from the group consisting of a negative charge, H, or a C1-C 10 More preferably, X is selected from a negative charge, H, or C1-C5 alkyl.

[0041] In the above formula, Y is H, OH, or C1-C 12 Preferably, Y is selected from H, OH, or C-C alkyl. 10 More preferably, Y is selected from H, OH, or C1-C5 alkyl.

[0042] In the above formula, n is 0 or 1.

[0043] In the above formula, m is 0 or 1.

[0044] In the above formula, Z is one or more pharmaceutically acceptable cations. Each Z is a pharmaceutically acceptable cation, provided that the net charge of the salt of formula (I) is 0. For example, Z may include a cation having a +1 charge, a +2 charge, or a +3 charge. Preferably, Z is Li + , Na + , K. + , Rb + , Cs + , Mg 2+ , Ca 2+ , Sr 2+ , Ba 2+ , Cu + , Cu 2+ , Fe 2+ , Fe 3+ , Pb 2+ , Ni 2+ , Ag + , Sn 2+ , Cr 3+ , Zn 2+ , Al 3+ , and / or NH4 + cations independently selected from: + , Na + , K. + , Rb + , Cs + , Mg 2+ , Ca 2+ , Sr 2+ , Ba 2+ , Pb 2+ , Ni 2+ , Ag + , Sn 2+ , Cr 3+ , Zn 2+ , Al 3+ , and / or NH4 + cations independently selected from: + , Na + , K. + , Rb + , Cs + , Mg 2+ , Ca 2+ , Sr 2+ , Ba 2+ , Pb 2+ , Ni 2+ , Sn 2+ , Cr 3+ , Zn2+ , and / or NH4 + cations independently selected from: + , Na + , K. + , Mg 2+ , Ca 2+ , Zn 2+ , and / or NH4 + cations independently selected from

[0045] A and B can independently be any integer (e.g., independently 1, 2, or 3), provided that the net charge of the salt of Formula (I) is 0. For example, if Z is a +3 cation (positive ion) and the anion is a −2 anion (negative ion), then A is 3 and B is 2, thereby causing the net charge of the salt to be 0. In one embodiment, A and B are each independently selected from 1 and 2, e.g., A is 1 and B is 1 or 2.

[0046] Z is a single ion, e.g., Na + ion, or multiple ions, e.g., Na + and K. + Therefore, salts with multiple cations (e.g., sodium potassium malonate) are included in formula (I).

[0047] In a preferred embodiment, the salt of formula (I) has a value of n of 0 and a value of m of 0. In this case, the salt has a value of formula (II): [ka] Formula (II) wherein A, B, Z, Y, and X are as defined above. It is a salt represented by the formula:

[0048] The salt of formula (I) for use in the treatment or prevention of ischemic stroke reperfusion injury may preferably be a salt of malonic acid, in which case the salt is represented by formula (II) and Y is an H atom.

[0049] For example, the salt of formula (II) can be disodium malonate, monosodium malonate, dipotassium malonate, monopotassium malonate, dilithium malonate, monolithium malonate, calcium malonate, magnesium malonate, ammonium malonate, aluminum malonate, or zinc malonate. Optionally, the salt of formula (II) can be disodium malonate.

[0050] The use of salts of the present invention, such as malonates, overcomes many barriers to clinical translation. The salts of the present invention, particularly malonates, can enter mitochondria via endogenous transport mechanisms, thus allowing compounds to reach target sites in a timely manner (as illustrated by FIG. 5). Furthermore, the salts of the present invention, particularly malonates, have limited toxicity, well-established metabolism, and are used as excipients in pharmaceutical development.

[0051] The salt for use in treating or preventing ischemic stroke reperfusion injury may alternatively be a salt of formula (II) wherein Y is a C1-C5 alkyl, more preferably a butyl group.

[0052] A salt of formula (I) can be administered at any time after a suspected ischemic stroke, for example, after observation of one or more stroke symptoms selected from sudden numbness or weakness in the face, arms, or legs; sudden confusion, difficulty speaking, or difficulty understanding speech; sudden visual impairment in one or both eyes; sudden trouble walking; dizziness, loss of balance, or lack of coordination; sudden severe headache; complete paralysis on one side of the body; difficulty swallowing (dysphagia); and loss of consciousness.

[0053] As used herein, reperfusion refers to the point at which blood flow to ischemic tissue begins after a stroke. Reperfusion can occur naturally (such as after a transient ischemic attack or a minor stroke) or can be initiated. When reperfusion is initiated, it can be initiated by any method known in the art that can relieve the blockage of blood flow, such as by either mechanical or chemical means. Preferably, reperfusion is initiated by thrombolysis (e.g., by administering an anticoagulant or by applying a lytic agent to the patient) and / or by thrombectomy.

[0054] Suitable anticoagulants may be selected from Coumadin™ (warfarin); Pradaxa™ (dabigatran); Xareito™ (rivaroxaban) and Eliquis™ (apixaban), fondaparinux, low molecular weight heparins including but not limited to unfractionated heparin, enoxaparin and dalteparin, thrombolytic agents including but not limited to streptokinase (SK), urokinase, lanoteplase, reteplase, staphylokinase, tenecteplase and alteplase, or antiplatelet agents such as aspirin, clopidogrel or ticagrelor.

[0055] The salt of formula (I) may be initially administered to a patient after ischemic stroke, prior to reperfusion. Preferably, administration of the salt of formula (I) is then continued until reperfusion is established, optionally during reperfusion, and optionally until reperfusion is terminated.

[0056] Administering the salt of formula (I) for a period of time before the onset or onset of reperfusion ensures that the salt of formula (I) can accumulate at a concentration sufficient to minimize ROS generation during reperfusion, thereby protecting the ischemic tissue from IR injury. Therefore, it is preferable to initially administer the salt of formula (I) to the patient as soon as possible after the onset of stroke symptoms. Therefore, the salt of formula (I) is preferably administered at least at the onset or onset of reperfusion, preferably at least about 5 minutes, preferably 10 minutes, preferably 15 minutes, and preferably 20 minutes or more before the onset or onset of reperfusion.

[0057] Alternatively, the salt of Formula (I) may be initially administered to a patient following the onset or occurrence of reperfusion. In this case, the salt of Formula (I) is preferably initially administered to a patient as soon as possible after the onset of reperfusion to minimize IR injury. After the salt of Formula (I) is initially administered to a patient, administration is preferably continued until reperfusion is established, optionally throughout the reperfusion, and optionally until reperfusion is terminated.

[0058] Alternatively, the salt of formula (I) may be initially administered to the patient at the time of reperfusion, and preferably, administration of the salt of formula (I) is then continued until reperfusion is complete.

[0059] The salt of formula (I) may be administered to a patient in combination with a treatment used or intended to remove a blockage in blood flow, i.e., a treatment used or intended to initiate reperfusion. This may be done simultaneously or separately, for example, when the salt of formula (I) is administered to a patient at the time of reperfusion, for example, the salt of formula (I) is administered after stroke ischemia but before reperfusion, and treatment to initiate reperfusion is initiated thereafter (during or after the administration of the salt of formula (I) has ceased). Preferably, the salt of formula (I) is administered to a patient in combination with thrombolytic therapy and / or thrombectomy. Preferably, the thrombolytic therapy is selected from the administration of an anticoagulant or a lytic agent to a patient, as defined above.

[0060] As mentioned above, the important advantage of the present invention is that the salt of formula (I) is beneficial for the treatment and prevention of reperfusion injury in ischemic stroke, but is not harmful in the case of hemorrhagic stroke.Therefore, the salt of formula (I) can be administered to patients suspected of having suffered from stroke before the type of stroke is diagnosed.Therefore, the salt of formula (I) can be used as an emergency medicine before the type of stroke is diagnosed, thereby minimizing the damage caused by reperfusion.

[0061] Thus, in one embodiment, the present invention relates to a salt of formula (I) as defined herein for use as an emergency medication after a suspected stroke, prior to diagnosis of the type of stroke. Preferably, the salt is a salt of formula (II), more preferably a salt of malonic acid as defined herein.

[0062] When administered as an emergency medication, the salt of formula (I) is preferably administered to a patient suspected of having suffered a stroke any time before diagnosis of the type of stroke, preferably within about 4 hours of the onset of stroke symptoms, preferably within about 3 hours of the onset of stroke symptoms, preferably within about 2 hours of the onset of stroke symptoms, more preferably within about 1 hour of the onset of stroke symptoms.

[0063] The salt of formula (I) may be administered, for example, in a hospital ward, in a situation where treatment can begin very quickly after the onset of stroke symptoms. Thus, administration of the salt of formula (I) can begin within about 50 minutes of the onset of stroke symptoms, preferably within about 40 minutes of the onset of stroke symptoms, preferably within about 30 minutes of the onset of stroke symptoms, preferably within about 20 minutes of the onset of stroke symptoms, and most preferably within about 15 minutes of the onset of stroke symptoms.

[0064] Regardless of when the initial administration of the salt of Formula (I) is performed, it is preferable to continue administering the salt of Formula (I) (e.g., continuously or periodically) until reperfusion is established or, if a suspected ischemic stroke has been treated, until ischemic stroke has been ruled out. In some cases, initiating reperfusion using chemical or mechanical means is not feasible. For example, the patient may have a contraindication to thrombolytic therapy, such as an increased risk of bleeding if thrombolytic therapy is initiated. Alternatively, thrombectomy may not be available at the time of the patient's examination. In these cases, the salt of Formula (I) may be administered continuously until reperfusion occurs spontaneously. Thus, the salt of Formula (I) may be administered continuously for up to 6 hours from the time of the initial administration. Preferably, the total administration time of the salt of Formula (I) is longer than about 2 minutes, preferably longer than about 5 minutes, preferably longer than about 10 minutes, and most preferably longer than about 15 minutes. Preferably, the total administration time of the salt of formula (I) is less than about 5 hours, preferably less than about 4 hours, preferably less than about 3 hours, and most preferably less than about 2 hours.

[0065] The salts of formula (I) can be administered as part of a combination therapy to prevent tissue damage, for example, the salts of formula (I) can be administered with other drugs or procedures that target ischemia-reperfusion injury.

[0066] The salt of formula (I) can be administered by any method known in the art.Preferably, the salt of formula (I) is administered orally, topically, subcutaneously, parenterally, intramuscularly, intraperitoneally, intraocularly, intranasally, intraarterially or intravenously.Most preferably, the salt of formula (I) is administered intravenously.

[0067] The salt of formula (I) can be administered at a dose ranging from about 0.1 mg / kg to about 500 mg / kg body weight. Preferably, the salt of formula (I) is administered at a dose greater than about 0.2 mg / kg body weight, preferably greater than about 0.3 mg / kg body weight, preferably greater than about 0.4 mg / kg body weight, and preferably greater than about 0.5 mg / kg body weight. Preferably, the salt of formula (I) is administered at a dose less than about 450 mg / kg body weight, preferably less than about 400 mg / kg body weight, preferably less than about 350 mg / kg body weight, and preferably less than about 300 mg / kg body weight.

[0068] The salt of formula (I), preferably the salt of formula (II), more preferably the salt of malonic acid, can be formulated into a composition for use in treating or preventing ischemic stroke reperfusion injury in a subject in need thereof and / or for use as an emergency medication after a suspected stroke prior to diagnosis of the type of stroke. The composition comprises the salt of formula (I), preferably the salt of formula (II), more preferably the salt of malonic acid, and one or more pharmaceutically acceptable excipients, carriers, or diluents.

[0069] Suitable excipients, carriers, and diluents can be found in standard pharmaceutical literature, see, for example, Handbook for Pharmaceutical Additives, 3rd Edition (eds. M. Ash and I. Ash), 2007 (Synapse Information Resources, Inc., Endicott, New York, USA), and Remington: The Science and Practice of Pharmacy, 2nd Edition (ed. D.B. Troy) 2006 (Lippincott, Williams and Wilkins, Philadelphia, USA).

[0070] Excipients for use in the compositions of the present invention include, but are not limited to, microcrystalline cellulose, sodium citrate, calcium carbonate, dicalcium phosphate, and glycine, along with various disintegrating agents such as starch (preferably corn, potato, or tapioca starch), alginic acid, and certain silicate complexes, as well as granulating binders such as polyvinylpyrrolidone, sucrose, gelatin, and acacia. Additionally, lubricants such as magnesium stearate, sodium lauryl sulfate, and talc are often very useful for tableting purposes. Solid compositions of a similar type may also be used as fillers for gelatin capsules; preferred materials in this regard include lactose or milk sugar and high molecular weight polyethylene glycols. When aqueous suspensions and / or elixirs are desired for oral administration, the active ingredient may be combined with various sweeteners or flavoring agents, coloring agents, or dyes, and, if desired, emulsifying and / or suspending agents, along with various diluents such as water, ethanol, propylene glycol, glycerin, and combinations thereof.

[0071] Pharmaceutical carriers include solid diluents or fillers, sterile aqueous media, and various non-toxic organic solvents, etc.

[0072] Pharmaceutically acceptable carriers include gum, starch, sugar, cellulose-based material and their mixtures.The compound can be administered to the subject by, for example, subcutaneously implanting pellets.The preparation can also be administered by intravenous, intraarterial or intramuscular injection of liquid preparation, by oral administration of liquid or solid preparation, or by topical application.The administration can also be carried out by using rectal suppository or urethral suppository.

[0073] Furthermore, as used herein, "pharmaceutically acceptable carriers" are well known to those skilled in the art and include, but are not limited to, 0.01-0.1M, preferably 0.05M, phosphate buffer, or 0.9% saline. Furthermore, such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffer media.

[0074] Pharmaceutically acceptable parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives, such as antimicrobials, antioxidants, modifiers, inert gases, and the like, may also be present.

[0075] Pharmaceutically acceptable carriers for administrable controlled- or sustained-release compositions according to the invention include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). Also encompassed by the invention are particulate compositions coated with polymers (e.g., poloxamers or poloxamines), and compounds conjugated to antibodies against tissue-specific receptors, ligands, or antigens, or compounds conjugated to ligands of tissue-specific receptors.

[0076] Pharmaceutically acceptable carriers include compounds modified by the covalent attachment of water-soluble polymers, such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, or polyproline. These compounds are known to exhibit substantially longer half-lives in the blood after intravenous injection than the corresponding unmodified compounds (Abuchowski and Davis, Soluble Polymer-Enzyme Adducts, Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, NY, (1981), pp. 367-383). Such modifications may also increase the compound's solubility in aqueous solution, eliminate aggregation, increase the compound's physical and chemical stability, and significantly reduce the compound's immunogenicity and reactivity. As a result, desired in vivo biological activity can be achieved by administering such polymer compound derivatives less frequently or in lower doses than the unmodified compound.

[0077] The present invention also relates to a method for treating or preventing ischemic stroke reperfusion injury in a subject, comprising administering to the subject a salt of formula (I) as defined herein or a composition as defined herein. The present invention also relates to a method for the emergency treatment of suspected stroke in a subject, prior to diagnosis of the type of stroke, comprising administering to the subject a salt of formula (I) as defined herein or a composition as defined herein. Preferably, the salt is a salt of formula (II), more preferably a malonate salt as defined herein. Preferably, the method comprises administering to the subject said salt, as further defined herein.

[0078] The present invention also relates to the use of a salt of formula (I) as defined herein, or a composition as defined herein, for the manufacture of a medicament for treating or preventing ischemic stroke reperfusion injury. The present invention also relates to the use of a salt of formula (I) as defined herein, or a composition as defined herein, for the manufacture of a medicament for use as an emergency medication after a stroke is suspected, prior to diagnosis of the type of stroke. Preferably, the salt is a salt of formula (II), more preferably a malonate salt as defined herein. Preferably, the salt is for the manufacture of a medicament for preventing ischemic stroke reperfusion injury, as further defined herein.

[0079] As used herein, "C1-C n The term "alkyl" generally refers to straight-chain and branched saturated hydrocarbon groups having 1 to n carbon atoms. Examples of alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, pent-1-yl, pent-2-yl, pent-3-yl, 3-methylbut-1-yl, 3-methylbut-2-yl, 2-methylbut-2-yl, 2,2,2-trimethyleth-1-yl, and the like.

[0080] As used herein, the terms "drug," "drug substance," "active pharmaceutical ingredient," and the like refer to a chemical compound that can be used to treat a subject in need of such treatment.

[0081] As used herein, the term "excipient" means any substance that can affect the bioavailability of a drug but is otherwise pharmacologically inactive.

[0082] As used herein, the term "pharmaceutically acceptable" means a chemical species that is within the scope of sound medical judgment suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit-to-risk ratio, and that is effective for its intended use.

[0083] As used herein, the term "pharmaceutical composition" refers to a combination of one or more drug substances and one or more excipients.

[0084] As used herein, the term "subject" means a human or non-human mammal.

[0085] Examples of non-human mammals include livestock such as sheep, horses, cattle, pigs, goats, rabbits, and deer, and companion animals such as cats, dogs, rodents, and horses.

[0086] As used herein, the term "body" refers to the body of a subject as defined above.

[0087] As used herein, the term "therapeutically effective amount" of a drug refers to an amount of a drug or composition effective to treat a subject and thereby produce the desired therapeutic, ameliorative, suppressive, or preventive effect. The therapeutically effective amount may depend, among other things, on the weight and age of the subject and the route of administration.

[0088] As used herein, the term "treating" means reversing, alleviating, inhibiting or preventing the progression of the disorder, disease or condition to which such term applies, or reversing, alleviating, inhibiting or preventing the progression of one or more symptoms of such disorder, disease or condition.

[0089] The term "treatment", as used herein, refers to the act of treating, as "treating" is defined above.

[0090] As used herein, the term "preventing" refers to reducing the risk of contracting a given disease or disorder, or reducing the severity of symptoms of a given disease or disorder if the disease or disorder is contracted after preventative measures. Thus, "preventing" refers to prophylactic treatment for a subject in need thereof. Prophylactic treatment can be achieved by administering an appropriate dose of a therapeutic agent to a subject who has a predisposition to or is at risk of developing a disorder, even if the symptoms of the disorder are absent or minimal, thereby substantially avoiding the onset of the disorder or substantially reducing the severity of symptoms of the disorder if the disorder is contracted after preventative measures.

[0091] As used herein, the term "succinate dehydrogenase inhibitor" or "SDHi" refers to a chemical species that inhibits the action of succinate dehydrogenase.

[0092] As used herein, the term "thrombolysis" means the removal of a blockage in blood flow, such as a blood clot, using chemical means, for example, by the use of a thrombolytic agent.

[0093] As used herein, the term "thrombectomy" means the removal of a blockage to blood flow, such as a blood clot, using mechanical means.

[0094] As used herein, the term "comprising" means "consisting at least in part of." When interpreting statements herein that include the term "comprising," other functionality than or presupposed by that term may also be present. Related terms such as "comprise" and "comprises" should be interpreted in the same manner. [Example]

[0095] Example 1. Transient middle cerebral artery occlusion (MCAO) model Six- to eight-week-old male C57Bl6J mice were anesthetized with 3% isoflurane and maintained at 1.5–2% isoflurane. A Doppler flowmeter probe (Perimed, Sweden) was attached to the skull to continuously monitor cerebral blood flow. The left common carotid artery (CCA) and left external carotid artery were exposed and permanently ligated. The left internal common carotid artery was temporarily clamped. Next, the CCA was incised, and a filament with a silicon tip (0.22 mm diameter, 2–3 mm length, Doccol, USA) was inserted and guided anteriorly into the internal carotid artery until it reached the middle cerebral artery (MCA). MCA occlusion was confirmed by demonstrating at least a 70% decrease in cerebral blood flow. After 45 minutes of ischemia, the filament was withdrawn and reperfusion was allowed for 2 hours. Reperfusion was confirmed when cerebral blood flow reached 80% of baseline.

[0096] Either disodium malonate (DSM) or vehicle control was administered intravenously for 20 minutes starting just before reperfusion.

[0097] At the end of reperfusion, mice were sacrificed by cervical dislocation. Brains were collected, sliced ​​into 2 mm thick slices, and stained with 2% triphenyltetrazolium chloride in saline at 37°C for 10–15 minutes. Brain sections were then fixed overnight in 4% paraformaldehyde and imaged using a scanner. The healthy tissue area (stained red) in both hemispheres was measured using ImageJ. The percent infarct volume was calculated by first calculating the healthy tissue volume in each hemisphere (Σ area of ​​each section × thickness of section), and then using the following formula: ((volume of healthy tissue in the intact hemisphere – volume of healthy tissue in the injured hemisphere) ÷ volume of healthy tissue in the intact hemisphere) × 100.

[0098] As shown in Figure 1, when DSM was given for 20 minutes starting just before reperfusion, the volume of necrotic cerebral infarction was reduced.

[0099] Cerebral blood flow was significantly reduced during transient middle cerebral artery occlusion (MCAO), as shown in Figure 2. Administration of DSM for 20 min starting just before reperfusion did not significantly alter blood flow during the procedure, indicating that DSM targets reperfusion injury rather than cerebral blood flow.

[0100] Comparative Example 1 The method of Example 1 was performed, except that dimethyl malonate (DMM) was administered instead of DSM. As shown in Figure 3, when DMM was administered for 20 minutes starting just before reperfusion, there was no significant difference in the volume of necrotic cerebral infarction.

[0101] Example 2. In vitro malonate uptake We first investigated whether malonate could be taken up by cells in culture. C2C12 mouse myoblasts were plated at 300,000 cells / well and allowed to adhere overnight. The following day, cells were treated with DSM (a - 0.25 mM; b - 0.25, 1, or 5 mM) or left untreated for 0–240 min. Afterwards, plates were cooled on ice, quickly washed four times with ice-cold PBS, and placed on dry ice. For mass spectrometry analysis, 1 nmol of malonate was used. 13 Cells were extracted with 500 μl of extraction buffer (50% methanol, 30% acetonitrile, and 20% water) with C-malonate internal standard and centrifuged to remove insoluble debris.

[0102] This demonstrated rapid uptake of malonate into cells, reaching intracellular levels of approximately 0.2–0.4 nmol / mg protein when incubated with 250 μM disodium malonate (Figure 4a). Malonate uptake was both dose- and time-dependent (Figure 4b), with 5 mM DSM achieving high levels of intracellular malonate (~2 nmol / mg protein) after 5 min. Taking into account a cell volume of 260 mg protein / ml, the intracellular malonate concentrations after 5 min of incubation with 250 μM or 5 mM malonate are approximately 50 μM and 500 μM, respectively.

[0103] Example 3. Malonate uptake in vivo To confirm whether this uptake also occurs in vivo, we injected disodium malonate into normoxic mice via a tail vein bolus and measured tissue malonate levels 5 minutes later. C57 / BL6 mice were injected with DSM (160 mg / kg) via the tail vein. Five minutes after injection, they were cervically dislocated, and tissues were harvested and clamp-frozen in liquid nitrogen. For tissue analysis, tissues were weighed, extracted with 25 μl / mg of extraction buffer containing an internal standard, and homogenized in a Precellys 24 homogenizer. The homogenates were centrifuged to remove insoluble debris. Extracts were analyzed by LC-MS / MS, and malonate levels were assessed by interpolation of a malonate standard curve.

[0104] This revealed tissue levels of malonate that were particularly high in the kidney, with significant amounts in the heart and liver, and also showed uptake in the brain (Fig. 5), consistent with tissue uptake of malonate.

[0105] Example 4 animal Male C57Bl6J mice were purchased from Charles River Laboratories and housed in a room with a 12-hour light / dark cycle, with free access to food and water. Mice were allowed to acclimate for one week before use in experiments. All experimental procedures were carried out in accordance with the UK Animals Act 1986 and the University of Cambridge Animal Welfare Policy and were approved by the Home Office (Project Licenses 70 / 8238 and 70 / 08840).

[0106] Warm cerebral ischemia in humans and mice Eight- to ten-week-old mice were sacrificed by cervical dislocation, and brains were clamp-frozen immediately or after 5, 15, 60, or 120 minutes of warm ischemia at 37°C. Human brain biopsies were collected from patients undergoing brain tumor surgery in collaboration with Dr. Richard Mair of the Cambridge Department of Neurosurgery. Biopsies (20-80 mg) were rapidly cut into 3-5 pieces depending on tissue size. One piece was immediately frozen (dissection-to-freezing time: 30 seconds to 2 minutes), while the other pieces were incubated in warm ischemia at 37°C for the indicated time (5 minutes to 120 minutes) and then clamp-frozen.

[0107] Metabolite extraction Approximately 20 mg of mouse brain tissue and 5–20 mg of human brain tissue were weighed on dry ice and placed in pre-chilled Precellys tubes (CK28-R, Bertin Instruments, France). Then, 25 μl / mg of dry ice-cold extraction buffer (50% [v / v] methanol, 30% [v / v] acetonitrile, and 20% [v / v] HO) was added to 1 nmol of [ 13 C4]-succinate (Sigma-Aldrich, UK) was added to the tube, and the tissue was homogenized using a Precellys24 tissue homogenizer (6,500 rpm, 15 seconds; Bertin Instruments, France). After 5 minutes of incubation on dry ice, the homogenization procedure was repeated. The sample was then centrifuged at 17,000 rpm at 4°C, and the supernatant was collected and incubated in a -20°C freezer for 1 hour. The centrifugation step was repeated twice, and the supernatant was transferred to a pre-chilled MS vial and stored at -80°C until succinate analysis by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0108] LC-MS / MS for succinate quantification LC-MS / MS analysis of succinate salts was performed using an LCMS-8060 mass spectrometer (Shimadzu, UK) and a Nexera X2 UHPLC system (Shimadzu, UK). Samples were stored in a refrigerated autosampler (4 °C) using 5 μl injections into a 15 μl flow-through needle. Separation was performed using a SeQuant™ ZIC™-HILIC column (3.5 μm, 100 Å, 150 × 2.1 mm, column temperature 30 °C; Merck Millipore, UK) and a ZIC™-HILIC guard column (200 Å, 1 × 5 mm). A flow rate of 200 μl / min was used with a mobile phase of A) 10 mM ammonium bicarbonate and B) 100% acetonitrile. The gradient used was: 0-0.1 min, 80% MS buffer B; 0.1-4 min, 80%-20% B; 4-10 min, 20% B; 10-11 min, 20%-80% B; and 11-15 min, 80% B. The mass spectrometer was operated in negative ion mode with multiple reaction monitoring (MRM). Labsolutions software (Shimadzu, UK) was used to acquire spectra and calculate compound amounts from the relevant standard curves of MS extraction buffer [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 29 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 13 C4]-succinic acid internal standard.

[0109] Measurement of mitochondrial complex I activity Frozen mouse brain weighing 7–10 mg was lysed in 400 ml of ice-cold 50 mM KH2PO4 (KPi buffer) using a Precellys CK14 tube and a Precellys tissue homogenizer (Bertin Instruments) at 6500 rpm for 15 seconds. The homogenate was quickly aliquoted, frozen in dry-ice-cooled tubes, and stored at -80°C until further processing. Protein concentration was measured using a standard BCA assay. A fresh aliquot of the sample was diluted with KPi buffer containing 0.05% dodecyl maltoside (DDM) to obtain 100 μl of buffer for the complex I activity assay, containing 5 μg of total protein. In a 96-well plate, 40 μl of assay buffer (200 μM KCN ​​and 0.3 μM antimycin A in KPi buffer) was added to each well, followed by 5 μl of ethanol plus 100 μM decylubiquinone or 5 μl of ethanol plus 0.5 μM rotenone. The assay was initiated by adding 50 μl of freshly prepared 0.8 mM NADH. NADH oxidation was measured on a plate reader by monitoring absorbance at λ = 340 and 380 nm (8-10 s intervals) for 30 min. Samples were run in duplicate. The maximum linear rate of NADH oxidation was calculated by subtracting the absorbance between 340 and 380 nm, and background rates were removed by subtracting the rate of samples containing rotenone. The NADH concentration is calculated using the Beer-Lambert law and the extinction coefficient ε340-380 = 4.81 mM -1 cm -1 was determined using.

[0110] Middle cerebral artery occlusion (MCAO) non-recovery model of stroke Mice aged 8–12 weeks, weighing 22–30 grams, were anesthetized with isoflurane (3% induction, 1.5–2% maintenance) delivered in 100% oxygen. A Doppler (PeriFlux System 5000, Perimed, Sweden) flow measurement probe was attached to the ipsilateral skull to monitor and record blood circulation in the MCA territory. Next, the left common carotid artery (CCA) and external carotid artery were exposed and permanently ligated. After temporarily occluding the left internal carotid artery with a clamp, the CCA was incised and a 6-0 suture with a silicone-coated tip (602223PK10RE, Doccol, USA) was inserted. The clamp was removed, and the suture was inserted anteriorly until a decrease in blood flow was observed by Doppler, confirming MCAO. The ischemia duration was 30 or 45 minutes, after which the suture was removed to allow reperfusion. For metabolite analysis, mice were sacrificed at the designated time points after ischemia or reperfusion by cervical dislocation, and the ipsilateral and contralateral brain regions were rapidly dissected and clamp-frozen. For infarct measurement, mice were kept under anesthesia for 2 hours of reperfusion and then sacrificed by cervical dislocation. Brains were dissected and freshly sliced ​​for staining and infarct size measurement. Mice were excluded from the study if the reduction in blood flow after MCAO exceeded 30% of baseline or for surgical reasons, such as excessive bleeding. No exclusions were performed after endpoint measurements.

[0111] Matrix-assisted laser desorption / ionization (MALDI) imaging of succinate Mice underwent MCAO surgery (30 min ischemia ± 5 min reperfusion) as described above, and brains were immediately dissected and frozen in liquid nitrogen-cooled isopentane. Coronal sections were cut at 20 μm thickness, mounted on slides (Superfrost Plus, Thermo Scientific, UK), rapidly dried on a 60 °C heat pad, and stored at -80 °C until analysis. Two sections per brain at the level of the striatum (MCA region) were used for MALDI. Matrix solution (1,5-diaminonaphthalene in 80:20 MeOH:HO (v / v), 10 mg / ml) was sprayed onto the sections (20 layers) using a spray nozzle (Suncollect MALDI spotter; KR Analytical, Cheshire, UK). Imaging was performed using a MALDI LTQ Orbitrap XL (Thermo Fisher Scientific, Hemel Hempstead, UK). Spectra were acquired in negative ion mode for mitochondrial metabolites. Metabolite identities were obtained by comparing observed values ​​with theoretical m / z values. Succinate was detected at 117.0166 m / z. Images were reconstructed using ImageQuest (Thermofisher).

[0112] Collection of cerebrospinal fluid (CSF) after MCAO Mice underwent non-recovery MCAO surgery and were sacrificed by an overdose of sodium pentobarbital at the indicated time points. Mice were then perfused with ice-cold PBS via the left ventricle at a rate of 2 ml / min for 2.5 min. CSF was collected from the cisterna magna using a glass capillary.

[0113] Disodium Malonate Treatment Ten minutes before reperfusion, non-recovery mice were treated intravenously (iv) with disodium malonate (DSM) or vehicle (phosphate-buffered saline, PBS) at a rate of 5 μl / min. The total volume of the injection was 100 μl. Randomization was not performed in non-recovery surgeries, but the researchers who measured the infarcts were blinded to the treatment groups. For recovery surgeries, researchers randomly labeled the Eppendorf tubes containing DSM or PBS with ID numbers. The surgeon randomly selected the tube for treatment. Therefore, the surgeon and researchers analyzing behavior and infarct volume were blinded to the treatment throughout the experiment and measurements. Mice weighing 22–28 (24.7 ± 1.4; mean ± SD) grams were administered 100 μl of PBS containing 4 mg of DSM (162.2 ± 9.1 mg / kg; mean ± SD) or PBS alone, infused intravenously at a rate of 10 μl / min, starting 5 min before reperfusion.

[0114] Infarct volume measurement after non-recovery MCAO The brains of mice that underwent non-recovery surgery were immediately sliced ​​into 2 mm thick slices and stained with 2% triphenyltetrazolium chloride (TTC) at 37°C for 10 minutes to identify the infarcted area (see Figure 15). The sections were fixed overnight in 4% paraformaldehyde and imaged using a scanner. The healthy area (stained red) was measured in the two hemispheres. The infarcted area was calculated as (contralateral healthy area - ipsilateral healthy area). The infarct volume was calculated as the sum of (infarct area x slice thickness) and expressed as a percentage of the healthy hemisphere volume.

[0115] statistics A nonparametric Mann-Whitney test was performed for infarct size comparing two groups. For multiple group comparisons (blood flow and succinate volume), one-way or two-way ANOVA with Tukey or Sidak post-hoc tests was performed. All statistical analyses were performed using Graphpad Prism version 7.0e.

[0116] Example 5. Metabolites in venous blood of stroke patients Venous blood samples were collected from patients with acute ischemic stroke at five time points before and after thrombolysis (A - before initiation of thrombolysis; B - 5 minutes, C - 15 minutes, D - 30 minutes, and E - 60 minutes after initiation of thrombolysis). Blood samples were analyzed for plasma succinate levels. Healthy controls were age- and sex-matched. Results are shown in Figure 16. No increase in succinate was observed in the venous blood of patients undergoing thrombolysis.

[0117] While specific embodiments of the present invention have been described above, it will be understood that the invention may be practiced otherwise than as described. The above description is intended to be illustrative and not limiting. Thus, it will be apparent to those skilled in the art that modifications can be made to the invention as described without departing from the scope of the claims set forth below.

Claims

1. Formula (II): 【Chemistry 1】 Formula (II) [In the formula, X is a negative charge or H; Y is H; Z is one or more pharmaceutically acceptable cations: and A and B are independently any integer, and the net charge of the salt of malonic acid is 0. A therapeutic or prophylactic agent for treating ischemic stroke reperfusion injury, comprising a salt of malonic acid represented by the formula:

2. 2. The therapeutic or prophylactic agent for treating ischemic stroke reperfusion injury according to claim 1, wherein X is a negative charge.

3. Each Z is Li ＋ , Na ＋ , K. ＋ , Rb ＋ , Cs ＋ , Mg ２＋ , Ca ２＋ , Sr ２＋ , Ba ２＋ , Cu ＋ , Cu ２＋ , Fe ２＋ , Fe ３＋ , Pb ２＋ , Ni ２＋ , Ag ＋ , Sn ２＋ , Cr ３＋ , Zn ２＋ , Al ３＋ , and / or NH ４ ＋ 3. The therapeutic or prophylactic agent for treating ischemic stroke reperfusion injury according to claim 1 or claim 2, wherein the therapeutic or prophylactic agent is independently selected from:

4. 2. The therapeutic or prophylactic agent for treating ischemic stroke reperfusion injury according to claim 1, wherein the salt is disodium malonate, monosodium malonate, dipotassium malonate, monopotassium malonate, dilithium malonate, monolithium malonate, calcium malonate, magnesium malonate, ammonium malonate, aluminum malonate, or zinc malonate.

5. The therapeutic or prophylactic agent for treating ischemic stroke reperfusion injury according to any one of claims 1 to 4, wherein the salt is administered orally, topically, subcutaneously, parenterally, intramuscularly, intraperitoneally, intraocularly, intranasally, intraarterially, or intravenously.

6. The therapeutic or prophylactic agent for treating reperfusion injury following ischemic stroke according to any one of claims 1 to 5, wherein the salt is administered intravenously.

7. The therapeutic or preventive agent for treating ischemic stroke reperfusion injury according to any one of claims 1 to 6, wherein administration of the salt is initiated after stroke ischemia and before reperfusion.

8. 8. The therapeutic or prophylactic agent for treating ischemic stroke reperfusion injury according to claim 7, wherein the salt is administered at least 10 minutes before the onset of reperfusion.

9. The therapeutic or prophylactic agent for treating reperfusion injury following ischemic stroke according to any one of claims 1 to 8, wherein the salt is administered during reperfusion.

10. The therapeutic or prophylactic agent for treating ischemic stroke reperfusion injury according to any one of claims 1 to 9, wherein the salt is administered as an emergency medication before diagnosis of the type of stroke.

11. The therapeutic or prophylactic agent for treating ischemic stroke reperfusion injury according to any one of claims 1 to 10, wherein the salt is administered within 1 hour from the onset of stroke symptoms.

12. The therapeutic or prophylactic agent for treating ischemic stroke reperfusion injury according to any one of claims 1 to 11, wherein the salt is administered for a total time period ranging from about 10 minutes to about 2 hours.

13. The therapeutic or prophylactic agent for treating ischemic stroke reperfusion injury according to any one of claims 1 to 12, wherein the salt is administered in combination with a therapy used or intended to remove blockage of blood flow.

14. 14. The therapeutic or prophylactic agent for the treatment of ischemic stroke reperfusion injury according to claim 13, wherein the therapy used or intended to remove the blockage of blood flow is selected from thrombolysis by application of an anticoagulant or a dissolving agent, and / or thrombectomy.

15. The therapeutic or prophylactic agent for treating ischemic stroke reperfusion injury according to any one of claims 1 to 14, wherein the salt is administered at a dose ranging from about 0.1 mg / kg to about 500 mg / kg of body weight.

16. The therapeutic or prophylactic agent for treating ischemic stroke reperfusion injury according to any one of claims 1 to 15, wherein the salt is administered at a dose ranging from about 0.5 mg / kg to about 100 mg / kg of body weight.

17. The therapeutic or prophylactic agent for treating ischemic stroke reperfusion injury according to any one of claims 1 to 16, wherein the salt is administered as a combination therapy to prevent tissue damage.

18. A therapeutic or prophylactic agent for the treatment of ischemic stroke reperfusion injury, comprising a composition comprising a salt of malonic acid of formula (II) according to any one of claims 1 to 4 in combination with one or more pharmaceutically acceptable excipients, carriers or diluents, and performing the treatment according to any one of claims 5 to 17.

19. Use of a malonic acid salt of formula (II) according to any one of claims 1 to 4 for the manufacture of a medicament for treating or preventing ischemic stroke reperfusion injury.

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