Compounds and methods for treating neurological and cardiovascular conditions

Abstract

To provide compounds and methods for treating neurological and cardiovascular conditions.SOLUTION: The present invention relates to compounds and methods of use thereof for treatment of certain disorders and conditions, e.g., brain injuries such as a stroke or traumatic brain injury. For example, the present invention provides a method of treating an injury, disease or condition selected from a traumatic brain injury (TBI), a stroke, a neurodegenerative condition, and a heart or cardiovascular disease, the method comprising administering to a patient in need thereof an effective amount of an agonist of an A3 adenosine receptor (A3R).SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Application No. 62 / 325,860, filed on April 21, 2016, the entire disclosure of which is incorporated herein by reference.

[0002] Field of the Invention The present invention relates to compounds and methods of using the same for the treatment, alleviation, or promotion of recovery from certain conditions of the brain, central nervous system (CNS), or cardiovascular system, such as brain injury, neurodegenerative conditions, or myocardial ischemia.

[0003] Statement of Government Assistance This invention was made with government support under Grant No. NS093756 awarded by the National Institutes of Health. The government has certain rights in this invention.

Background Art

[0004] Background of the Invention Brain injury is a common and very painful medical condition and is one of the leading causes of morbidity and mortality worldwide. In particular, the brain is vulnerable to injury because neurons have limited repair capabilities. When an individual is born, the brain already has essentially all of the neurons it will have throughout its life. Neurons, unlike other cells in the body, stop regenerating shortly after birth. These cells cannot be replaced if they are damaged or die, and often cause impairments in human cognitive and sensorimotor abilities, mainly deteriorating irreversibly. Conditions that result in nerve cell death and damage range from ischemic episodes (such as stroke) and trauma to degenerative disorders (such as Alzheimer's disease).

[0005] Injuries to the central nervous system (CNS) are a substantial cause of death and disability worldwide. For example, according to the CDC, approximately 1.7 million people suffer traumatic brain injury (TBI) annually, imposing costs exceeding $60 billion annually on the US economy in terms of medical expenses and lost productivity (Finkelstein, E; Corso, P; Miller, T, The Incidence and Economic Burden of Injuries in the United States, Oxford University Press: New York, 2006). Furthermore, stroke, a major cause of disability, is the third leading cause of death in the US, with an estimated 795,000 cases annually, imposing costs exceeding $34 billion annually on the US economy (NINDS, 2014; stroke.nih.gov; and Mozaffarian D, Benjamin EJ, Go AS et al., "Heart disease and stroke statistics - 2015 update: a report from the American Heart Association", Circulation. 2015; pp. e29 - 322).

[0006] In acute situations, the opportunity to treat patients within 24 hours can limit the extent of injury. Immediately following an ischemic or hemorrhagic stroke, the injured site in the brain typically contains a core of irreversibly damaged tissue, as well as a tissue region called the penumbra, which is viable but at risk. During this period, if the supply of oxygen and glucose to brain cells is insufficient, additional secondary injury occurs in the penumbra. When oxygen and glucose are lacking, energy production by cell mitochondria is reduced. The immediate effect of this energy depletion is the failure of ion pumps, which causes depolarization waves that repeatedly spread within the brain tissue due to an increase in extracellular potassium (K + ) ions. At the same time, sodium (Na + ) ions flow into the cells, followed by chloride (Cl -)The influx of ions causes cell swelling due to increased osmotic pressure, compressing adjacent neurons and their processes, ultimately resulting in lysis (cell rupture) and an inflammatory response. Generally, the disruption of this ion homeostasis leads to excitotoxicity, cell swelling, and cell death, which damage adjacent tissues and expand the lesion through secondary mechanisms. To protect stressed brain cells, effective treatment is necessary within the first 24 hours. The spread of brain injury in stroke is similar to that observed in other forms of brain injury, such as trauma and concussion.

[0007] Effective astrocyte function plays a very important role not only in acute treatment but also in more extensive nerve repair over 24 to 96 hours after brain injury, in patients with neurodegeneration such as Alzheimer's disease, or most commonly in elderly individuals over several months to years. When brain cells can no longer regenerate, the remaining intact brain tissue needs to be reorganized to recover any lost function. This potential for nerve reorganization decreases in older individuals.

[0008] GPCR receptors have been suggested to mediate cardioprotective effects. Therefore, there is a potential to treat heart and cardiovascular conditions through a similar mechanism of action via modulation of these receptors. The urgent medical need to more effectively treat brain injury, CNS injury, heart disease and cardiovascular disease, and related conditions, and to promote nerve repair in patients with neurodegenerative conditions such as Alzheimer's disease, remains unmet.

Prior Art Documents

Non-Patent Documents

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Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Means for Solving the Problems

[0010] Summary of the Invention In one aspect, the present invention is a method for treating an injury, disease, or condition selected from traumatic brain injury (TBI), stroke, neurodegenerative condition, or heart disease or cardiovascular disease, the method comprising administering to a patient in need thereof an effective amount of an agonist of the A3 adenosine receptor (A 3 R).

[0011] In one aspect, the present invention is a method for treating an injury, disease, or condition selected from traumatic brain injury (TBI), stroke, neurodegenerative condition, or heart disease or cardiovascular disease, the method comprising administering to a patient in need thereof

Chemical Formula

[0012] In another aspect, the present invention is a method for treating an injury or condition of the brain or central nervous system (CNS) selected from traumatic brain injury (TBI) or stroke, the method comprising administering to a patient in need thereof

Chemical formula

[0013] In another aspect, the present invention

Chemical formula

Chemical formula

[0014] In another aspect, the present invention is a method for treating or alleviating traumatic brain injury (TBI), radiation injury, stroke, migraine, heart disease or cardiovascular disease, or neurodegenerative disorders, the method comprising administering to a patient in need thereof an effective amount of the disclosed compound.

[0015] In another aspect, the present invention is a method for treating or alleviating traumatic brain injury (TBI), radiation injury, stroke, migraine, heart disease or cardiovascular disease, or neurodegenerative disorders, comprising administering to a patient in need thereof adenosine, ADP, 2-methylthio-ADP trisodium salt, ATP, ATP disodium salt, α,β-methylene ATP, α,β-methylene adenosine 5'-triphosphate trisodium salt, 2-methylthioadenosine triphosphate tetrasodium salt, 2-MeSATP, BzATP triethylammonium salt, inosine, cytidine, acylated cytidine, cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), CDP-choline, CMP-choline, denufosol, denufosol tetrasodium, GTP, ITP, MRS541, MRS542, MRS1760, MRS2179, MRS2279, MRS2341, MRS2365, MRS2500, MRS2690, MRS2698, MRS3558, MRS4322, MRS5151, MRS5676, MRS5678, MRS5697, MRS5698, MRS5923, MRS5930, benzyl-NECA, IB-MECA, Cl-IB-MECA, LJ529, DPMA, CCPA, DBXRM, HEMADO, PEMADO, HENECA, PENECA, CP608,039, CP532,903, CGS21680, AR132, VT72, VT158, VT160, VT163, PSB0474, uridine 5'-diphosphate (UDP), UDP-glucose, uridine β-thiodiphosphate (UDPβS), uridine 5'-triphosphate (UTP), uridine γ-thiophosphate (UTPγS), 2-thio UTP tetrasodium salt, UTPγS trisodium salt, uridine-5'-diphosphoglucose, dipyridamole analogs, diadenosine tetraphosphate Ap 4 U, Ap 4A method is provided that includes administering an effective amount of a compound selected from INS365, INS37217, or INS48823, wherein each sugar may be replaced with a methanocarba sugar in a North or South conformation, or each sugar may be replaced with D-ribose, or a pharmaceutically acceptable salt thereof. In certain embodiments, for example, the following items are provided. (Item 1) A method of treating an injury, disease, or condition selected from traumatic brain injury (TBI), stroke, neurodegenerative condition, or heart or cardiovascular disease, the method comprising administering to a patient in need thereof an effective amount of an agonist of the adenosine receptor (A 3 R). 3 (Item 2) The compound is TIFF2025084908000005.tif36102 or a pharmaceutically acceptable salt thereof, the method according to item 1. (Item 3) The compound is TIFF2025084908000006.tif35101 or a pharmaceutically acceptable salt thereof, the method according to item 1. (Item 4) The method according to any one of items 1 to 3, wherein the injury, disease, or condition is TBI. (Item 5) The method according to item 4, wherein the TBI is selected from concussion, blast injury, combat-related injury, or mild, moderate, or severe impact to the head. (Item 6) The method according to any one of items 1 to 3, wherein the injury, disease, or condition is a stroke selected from ischemic stroke, hemorrhagic stroke, subarachnoid hemorrhage, cerebral vasospasm, or transient ischemic attack (TIA). (Item 7) The method according to any one of items 4 to 6, wherein the neuroprotection or neurorepair of the patient is increased compared to an untreated patient. (Item 8) The method according to any one of items 1 to 3, wherein the neurodegenerative disease is selected from Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), chronic traumatic encephalopathy (CTE), or a neurodegenerative condition caused by a virus, alcohol dependence, a tumor, a toxin, or repetitive brain injury. (Item 9) The method according to item 8, wherein the neurodegenerative disease is Parkinson's disease. (Item 10) The method according to item 8, wherein the injury, disease, or condition is a neurological side effect associated with Alzheimer's disease, migraine, brain surgery, or cancer chemotherapy. (Item 11) The method according to any one of items 1 to 7, wherein the recovery period after the TBI, stroke, myocardial ischemia, or myocardial infarction is shortened compared to an untreated patient. (Item 12) The method according to any one of items 1 to 3, wherein the heart disease or cardiovascular disease is selected from myocardial ischemia, myocardial infarction, cardiomyopathy, coronary artery disease, arrhythmia, myocarditis, pericarditis, angina, hypertensive heart disease, endocarditis, rheumatic heart disease, congenital heart disease, or atherosclerosis. (Item 13) The method according to item 12, wherein the heart disease or cardiovascular disease is myocardial ischemia or myocardial infarction. (Item 14) The method according to any one of items 1 to 3, wherein the compound is administered chronically during the period of recovery of the injury after the injury has occurred for treating a stroke, myocardial ischemia, or myocardial infarction. (Item 15) Said A 3 The method according to any one of items 1 to 14, wherein R is partially agonized. (Item 16) A method for treating an injury, disease, or condition selected from traumatic brain injury (TBI), stroke, or a neurodegenerative condition, comprising administering to a patient in need thereof A 3 adenosine receptor (A 3administering an agonist in an effective amount of R), wherein said A 3 R is another A 3 R-mediated pathway activation is accompanied by little or no preferential activation of intracellular calcium mobilization, or by preferential activation of Gq11-mediated intracellular calcium mobilization, Gi-mediated modulation of cAMP production, or Gi-mediated phosphorylation of ERK1 / 2 and Akt, said A 3 agonized in a manner biased towards the neuroprotective function of the R receptor, method. (Item 17) The method according to item 16, wherein the injury, disease, or condition is TBI. (Item 18) The method according to item 17, wherein the TBI is selected from concussion, blast injury, combat-related injury, or mild, moderate, or severe impact to the head. (Item 19) The method according to item 16, wherein the injury, disease, or condition is a stroke selected from ischemic stroke, hemorrhagic stroke, subarachnoid hemorrhage, cerebral vasospasm, or transient ischemic attack (TIA). (Item 20) The method according to item 16, wherein the neurodegenerative disease is selected from Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), chronic traumatic encephalopathy (CTE), or a neurodegenerative condition caused by virus, alcohol dependence, tumor, toxin, or repetitive brain injury. (Item 21) The method according to item 20, wherein the neurodegenerative disease is Parkinson's disease. (Item 22) The method according to item 20, wherein the injury, disease, or condition is Alzheimer's disease, migraine, brain surgery, or neurological side effects associated with cancer chemotherapy. (Item 23) The method according to any one of items 16 to 22, wherein the neuroprotection or nerve repair of the patient is increased compared to an untreated patient. (Item 24) said A3 R is a complete A 3 The method according to any one of items 16 to 23, which is partially agonized with an improved neuroprotective function or nerve repair function compared to an R agonist. (Item 25) A method for treating a heart disease or a cardiovascular disease, comprising administering to a patient in need thereof an agonist in an effective amount of an adenosine receptor (A 3 R), wherein the agonist is the following A 3 R-mediated pathway: activation of Gq11-mediated intracellular calcium mobilization, Gi-mediated modulation of cAMP production, Gi-mediated phosphorylation of ERK1 / 2 and Akt, modulation of ATP-sensitive potassium channels, or modulation of beta-arrestin activation, by one or more preferential activations of a complete A 3 R agonist has an improved cardioprotective function compared to an A 3 R biased agonist, method. 3 The method according to any one of items 25 to 28, wherein the compound is a partial agonist of A (Item 26) The method according to item 25, wherein the patient is suffering from myocardial ischemia or myocardial infarction. (Item 27) The method according to item 25, which increases the cardioprotection of the patient or the regeneration of damaged heart tissue. (Item 28) The method according to any one of items 25 to 27, wherein the recovery period after TBI, stroke, myocardial ischemia, or myocardial infarction is shortened compared to an untreated patient. (Item 29) The compound is a complete A 3 R agonist has an improved cardioprotective function compared to an A 3 R partial agonist, method.

Brief Description of the Drawings

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[0036] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION 1. Brain, CNS, Cardiovascular, and Other Injuries and Conditions In some embodiments, the present invention provides new methods for preventing and / or treating brain injuries associated with acute traumatic brain injury, as well as long-term diseases of the brain and CNS, and heart and cardiovascular diseases and conditions. In one aspect, the present invention utilizes the neuroprotective and neurorestorative effects mediated by astrocytes, which are currently understood as very important natural caregiver cells of neurons, and astrocyte mitochondria that supply a significant portion of the brain's energy, to provide a method for treating such injuries, diseases, and conditions. In another aspect, the present invention is A 3Provided are methods of treating such injuries, diseases, and conditions by the cardioprotective and regenerative effects mediated by R receptors. With regard to the neuroprotective and neurorestorative effects, without being bound by theory, A 3 R and / or P2Y 1 By selectively enhancing the energy metabolism of astrocytes mediated by receptors, the functions of astrocytes as caregivers, such as their neuroprotective and neurorestorative functions, are promoted, thereby enhancing the resistance of neurons and other cells to both acute injury and chronic stress. In some cases, A 3 R and / or P2Y 1 It may be advantageous to bias, i.e., selectively or preferentially achieve, one or more pathways mediated by receptors, where one or more unwanted pathways are not activated or are activated to a relatively low degree. In addition to or as an alternative to astrocytes, the neuroprotective or neurorestorative functions of glia, microglia, neurons, endothelial cells, and other brain and / or CNS cell types can be activated. Thus, in one aspect, the present invention provides a compound for treating, alleviating, or promoting recovery from certain conditions of the brain or central nervous system (CNS), such as brain injury, by increasing the neuroprotective and / or neurorestorative effects mediated by, for example, astrocytes, glia, microglia, neurons, endothelial cells, or other cells of the brain and / or CNS, and a method of using the same, the method comprising administering to a patient in need thereof an effective amount of the disclosed compound.

[0037] Astrocytes play a very important role in assisting and protecting neurons and critically influence the outcome of brain injuries, such as those caused by ischemic injury. The central role played by astrocyte mitochondria themselves in these brain functions is not fully understood. For example, when astrocyte mitochondria are inhibited, swelling increases and leads to necrotic cell death. Neurons are permanently damaged by repetitive spreading depolarization only when astrocyte mitochondria do not function, and astrocyte mitochondria are necessary to reduce the pathological increase in extracellular K + and thereby reduce the spread of depolarization. Activation of purinergic receptors on astrocytes increases mitochondrial Ca 2+ and mitochondrial Ca 2+ enhances mitochondrial citric acid cycle function and increases respiration and ATP production. Thus, in one aspect, the present invention relates to the discovery that activation of purinergic receptors in astrocytes enhances the brain cell survival signaling pathway and enables both astrocytes and neurons to survive during oxidative stress. Furthermore, activated astrocytes produce and supply reduced glutathione, a very important antioxidant that contributes to the resistance of both astrocytes and neurons to oxidative stress. Thus, in one aspect, the present invention provides a method of modulating purinergic receptors in astrocytes to promote the survival and survival rate of one or more cell types in the brain of a patient after oxidative stress, such as that caused by brain injury, ischemia-reperfusion, or neurodegenerative conditions, the method comprising administering to a patient in need thereof a disclosed compound.

[0038] In some embodiments, activation of astrocytes is achieved by contacting one or more purinergic receptors, such as adenosine receptors (AR), that are associated with or expressed by astrocytes, with a disclosed compound, thereby modulating the activity of one or more receptors. In some embodiments, the compound is an adenosine receptor on astrocytes, such as A 1 、A 2A 、A2B and A 3 activate astrocytes by the effects of, and treat one or more of the disclosed diseases or conditions. In some embodiments, the disclosed compounds, after administration to a patient in need thereof, affect one or more functions such as glutamate uptake, reactive gliosis, swelling, and the release of neurotrophic and neurotoxic factors that affect metabolic stress, and thus, as a result, treat one or more diseases or conditions. In some embodiments, the compound is an AR agonist. In some embodiments, the purinergic receptor is A 3 adenosine receptor (A 3 R). In some embodiments, the compound is A 3 R agonist. In some embodiments, the compound is A 3 receptor (A 3 R), e.g., a partial agonist or a biased agonist or a biased partial agonist at the human A 3 receptor (hA 3 R). In some embodiments, the compound is a biased antagonist at the A 3 receptor. In some embodiments, the compound is MRS4322 or MRS1873 or a pharmaceutically acceptable salt thereof.

[0039] P2Y receptors are G protein-coupled receptors, and various subtypes of these receptors play important roles in processes such as synaptic communication, cell differentiation, ion flux, vasodilation, blood-brain barrier permeability, platelet aggregation, and neuromodulation. Characterized members of the purinergic P2Y receptor family include mammalian P2Y that bind to adenine nucleotides 1 , P2Y 11 , P2Y 12 and P2Y 13 receptors, P2Y 4 , P2Y 6 , and P2Y 14 receptors that bind to uracil nucleotides, and P2Y 2 and rodent P2Y 4A receptor is included. In some embodiments, activation of astrocytes is achieved by contacting one or more purinergic receptors, such as P2Y receptors, e.g., receptors associated with or expressed by astrocytes, with the disclosed compounds, thereby modulating the activity of one or more receptors. In some embodiments, the compound is associated with or expressed by astrocytes and the P2Y receptor, e.g., P2Y 1 , P2Y 11 , P2Y 12 and P2Y 13 receptors to activate astrocytes and treat one or more of the disclosed diseases or conditions. In some embodiments, the P2Y receptor is the P2Y 1 receptor. In some embodiments, the P2Y 1 receptor is located on the intracellular mitochondrial membrane. In some embodiments, the compound is a P2Y agonist. In some embodiments, the compound is, for example, a P2Y 1 agonist at the human P2Y 1 receptor. In some embodiments, the compound is a biased agonist, partial agonist, or biased partial agonist at the P2Y 1 receptor, e.g., the human P2Y 1 receptor. In some embodiments, the compound is a biased antagonist at the P2Y 1 receptor. In some embodiments, the compound is MRS4322 or a pharmaceutically acceptable salt thereof.

[0040] In another aspect, the present invention provides a method of treating or reducing brain injury, e.g., brain injury resulting from TBI or a progressive neurodegenerative disorder, in a patient in need thereof, the method comprising administering to the patient an effective amount of the disclosed compound. In some embodiments, the subject is suffering from TBI, concussion, stroke, partial or complete spinal cord transection, or nutritional disorder. In other embodiments, the subject is suffering from toxic neuropathy, meningoencephalopathies, neurodegeneration caused by genetic disorders, age-related neurodegeneration, or vascular disease suffering from, or having another disease disclosed in US8,691,775 incorporated herein by reference. In some embodiments, the present invention is a method for treating or reducing brain injury in a patient in need thereof, such as brain injury resulting from TBI or a progressive neurodegenerative disorder, the method comprising administering to the patient an effective amount of A 3 R agonist. In other embodiments, the present invention is a method for treating or reducing brain injury in a patient in need thereof, such as brain injury resulting from TBI or a progressive neurodegenerative disorder, the method comprising administering to the patient an effective amount of P2Y 1 agonist. In some embodiments, the compound is a biased agonist, partial agonist, or biased partial agonist at the A 3 receptor. In some embodiments, the compound is a biased agonist, partial agonist, or biased partial agonist or antagonist at the P2Y 1 receptor. In some embodiments, the compound is MRS4322 or MRS1873 or a pharmaceutically acceptable salt thereof.

[0041] In another aspect, the present invention provides a method for promoting astrocyte-mediated neuroprotection or neurorepair in a patient in need thereof, the method comprising administering to the patient an effective amount of the disclosed compound. In some embodiments, the present invention is a method for promoting astrocyte-mediated neuroprotection or neurorepair in a patient in need thereof, the method comprising administering to the patient an effective amount of A 3 R agonist. In some embodiments, the present invention is a method for promoting astrocyte-mediated neuroprotection or neurorepair in a patient in need thereof, the method comprising administering to the patient an effective amount of P2Y 1 agonist. In some embodiments, the compound is a biased agonist, partial agonist, or biased partial agonist or antagonist at the A 3 receptor. In some embodiments, the compound is a biased agonist, partial agonist, or biased partial agonist or antagonist at the P2Y 1It is a biased agonist, partial agonist, or biased partial agonist or antagonist at the receptor. In some embodiments, the compound is MRS4322 or MRS1873 or a pharmaceutically acceptable salt thereof.

[0042] In another aspect, the present invention provides a method of promoting the survival of neurons, glial cells, endothelial cells or other brain cells, such as brain cells in the ischemic periphery, in a patient in need thereof, the method comprising administering to the patient an effective amount of a compound disclosed herein. In some embodiments, the present invention provides a method of promoting the survival of neurons, glial cells, endothelial cells or other brain cells, such as brain cells in the ischemic periphery, in a patient in need thereof, the method comprising administering to the patient an effective amount of 3 an A 1 R agonist. In some embodiments, the present invention provides a method of promoting the survival of neurons, glial cells or other brain cells, such as brain cells in the ischemic periphery, in a patient in need thereof, the method comprising administering to the patient an effective amount of a 3 P2Y 1 agonist. In some embodiments, the compound is a biased agonist, partial agonist, or biased partial agonist or antagonist at the receptor. In some embodiments, the compound is a biased agonist, partial agonist, or biased partial agonist or antagonist at the P2Y

[0043] In further embodiments, the patient has or is at risk of having a brain injury, such as the following brain injuries. Accordingly, methods of treating the conditions discussed below are also provided. Traumatic brain injury

[0044] Traumatic brain injury (TBI) is a common and painful medical condition that is expected to be the third leading cause of morbidity and mortality worldwide by 2020. There are no approved treatments for TBI, and most TBI patients are discharged without pharmacological treatment (Witt, 2006). Repeated TBI, such as concussion, can induce age-related neurodegeneration that results in various symptoms and disabilities over decades (McKee, 2013). TBI can be caused by sports-related injuries, motor vehicle accidents, falls, blast injuries, physical assaults, etc. Injuries vary widely in their complexity and severity, ranging from "mild" concussions with short-term changes in mental state, cognitive difficulties, or loss of consciousness, to "severe" cases with prolonged post-injury unconsciousness and / or memory loss. In the United States, approximately 1.7 million people sustain an injury that results in TBI each year and seek medical intervention (USCSF and CDC), and the CDC estimates that an additional 1.6 to 3.8 million concussions occur each year in sports and other recreational activities that do not result in a visit to a hospital or emergency department (CDC; Langlois, 2006). Approximately 5-10% of athletes experience a concussion each sports season (Sports Concussion Institute, 2012). Football is the sport with the highest risk of concussion in males (75% likelihood of concussion), and soccer is the sport with the highest risk of concussion in females (50% likelihood of concussion). TBI is a leading cause of death and disability in children and young adults (CDC), and the majority of these cases have generally been military-related injuries. Approximately 20% of U.S. military personnel deployed since 2003 have had at least one sustained TBI (Chronic Effects of Neurotrauma Consortium (CENC); Warden, 2006; Scholten, 2012; Taylor, 2012; Gavett, 2011; Guskiewicz, 2005; Omalu, 20 In 2005, the total direct and indirect medical costs associated with TBI are estimated to be $77 billion annually (UCSF and CDC). At least 5 million Americans require ongoing daily assistance to perform activities as a result of TBI (CDC and Thurman in 1999).

[0045] Activation of astrocytes according to the present invention provides a new treatment option for such conditions. Thus, in one aspect of the present specification, there is provided a method of treating TBI or promoting recovery from TBI, the method comprising administering to a patient in need thereof an effective amount of the disclosed compound. In some embodiments, the TBI is selected from trauma to the brain (e.g., concussion, blast injury, combat-related injury) or trauma to the spinal cord (e.g., partial or complete spinal cord transection). In some embodiments, the TBI results from a mild, moderate or severe impact to the head, includes an open or closed head wound, or results from a penetrating or non-penetrating impact to the head. In some embodiments, the present invention provides a method of treating TBI or promoting recovery from TBI, the method comprising administering to a patient in need thereof an effective amount of A 3 R agonist. In some embodiments, the present invention provides a method of treating TBI or promoting recovery from TBI, the method comprising administering to a patient in need thereof an effective amount of P2Y 1 agonist. In some embodiments, the compound is a biased agonist, partial agonist, or biased partial agonist or antagonist at the A 3 receptor. In some embodiments, the compound is a biased agonist, partial agonist, or biased partial agonist or antagonist at the P2Y 1 receptor. In some embodiments, the compound is MRS4322 or MRS1873 or a pharmaceutically acceptable salt thereof. Stroke

[0046] A stroke occurs when the blood vessels that transport oxygen and nutrients to the brain are disrupted due to ischemic blockage or hemorrhagic rupture of the blood vessels within the brain, killing neurons, glia, and endothelial cells within the damaged area of the brain. The outcome of a stroke varies depending on the location and extent of the damage, and the effects of that damage are observed in the body functions regulated by the damaged brain regions. A stroke can cause unilateral or bilateral paralysis, speech and language disorders, memory loss, behavioral changes, and even death. Stroke is the fourth leading cause of death in the United States and the leading cause of disability in adults. Each year, approximately 800,000 people experience a new or recurrent stroke. Every day, more than 2,000 Americans have a stroke, and over 400 of these occurrences result in death. Stroke accounted for approximately 1 in 19 deaths in the United States in 2010. An estimated 6.8 million Americans aged ≥20 years had a stroke (AHA and Go 2014). As of 2010, the direct and indirect annual costs of stroke were estimated to be $36.5 billion. Within minutes of a stroke occurring, blood flow becomes insufficient, permanently damaging the core of the brain tissue. Between this damaged core and the normal brain tissue is a tissue region known as the penumbra, which undergoes progressive stress due to reduced blood flow and some disruption of energy metabolism. Over the first 24 - 48 hours following a stroke, the stress on the penumbral neurons and glial cells is resolved by either some recovery or further cell death.

[0047] In one aspect, the present invention provides a method for neuroprotective treatment of a stroke patient, the method comprising administering to a patient in need thereof an effective amount of the disclosed compound. In some embodiments, such treatment salvages as much of the penumbra as possible and / or limits further acute tissue damage and / or promotes neuronal recovery. In another aspect, there is provided a method for treating a stroke or promoting recovery from a stroke, the method comprising administering to a patient in need thereof an effective amount of the disclosed compound. In another aspect, there is provided a method for treating a stroke or promoting recovery from a stroke, the method comprising administering to a patient in need thereof an effective amount of A 3 R agonist. In some embodiments, the present invention provides a method for treating a stroke or promoting recovery from a stroke, the method comprising administering to a patient in need thereof an effective amount of P2Y 1 agonist. In some embodiments, the compound is a biased agonist, partial agonist, or biased partial agonist or antagonist at the A 3 receptor. In some embodiments, the compound is a biased agonist, partial agonist, or biased partial agonist or antagonist at the P2Y 1 receptor. In some embodiments, the compound is MRS4322 or a pharmaceutically acceptable salt thereof.

[0048] In some embodiments, the stroke is selected from ischemic stroke, hemorrhagic stroke, subarachnoid hemorrhage, cerebral vasospasm, or transient ischemic attack (TIA). In some embodiments, the stroke is ischemic. In some embodiments, the stroke is hemorrhagic. In some embodiments, the compound is administered within 48 hours after the stroke. In some embodiments, the compound is administered within 24 hours after the stroke. In some embodiments, the compound is administered within 16 hours after the stroke. In some embodiments, the compound is administered within 8 hours, 4 hours, 2 hours, or 1 hour after the stroke. In some embodiments, the compound is administered at least within the first 1 - 72 hours after the stroke. In some embodiments, the compound is administered at least within the first 8 - 52 hours after the stroke. In some embodiments, the compound is administered at least within the first 8 - 48 hours after the stroke. In some embodiments, the compound is administered at least within the first 24 - 48 hours after the stroke. In some embodiments, the compound is chronically administered to treat a stroke if a stroke occurs. In some embodiments, the compound is chronically administered to treat a transient ischemic attack (TIA).

[0049] In some embodiments, the compound is chronically administered to treat ischemic stroke, hemorrhagic stroke, subarachnoid hemorrhage, cerebral vasospasm, transient ischemic attack (TIA), or to treat patients at high risk of stroke, such as patients who have had a stroke in the past, and patients at risk of further stroke, such as patients over 40, 45, 50, 55, 60, 65, 70, 75 or 80 years of age.

[0050] In some embodiments, the compound treats ischemia-reperfusion injury caused by a stroke. Neurodegenerative disease

[0051] Neurodegenerative diseases are incurable, progressive, and ultimately debilitating syndromes resulting from the progressive degeneration and / or death of neurons in the brain and spinal cord. Neurodegeneration leads to motor disorders (ataxia) and / or cognitive impairment (dementia), including a wide variety of diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and chronic traumatic encephalopathy (CTE). Many neurodegenerative diseases are primarily of genetic origin, and other causes include viruses, alcohol dependence, tumors, or toxins, and as currently revealed, repetitive brain injury may also be included.

[0052] Neurons accumulate cell damage over time due to the above factors, which is generally considered the reason why many neurodegenerative diseases associated with persistent cell stress, such as Alzheimer's disease and Parkinson's disease, occur in elderly individuals. Dementia is the main outcome of neurodegenerative diseases with AD, corresponding to approximately 60 - 70% of cases (Kandale 20 13). As discussed previously, activation of neuroprotective and nerve repair mechanisms can reduce the progression of one or more neurodegenerative diseases. Thus, in one aspect, the present invention provides a method for treating neurodegenerative diseases or promoting the recovery of neurodegenerative diseases, comprising administering an effective amount of the disclosed compound to a patient in need thereof.

[0053] In one aspect, the present invention provides a method for promoting neuroprotection or nerve repair in a patient suffering from a neurodegenerative disease, comprising administering an effective amount of the disclosed compound to the patient. In some embodiments, a method for promoting neuroprotection or nerve repair in a patient suffering from a neurodegenerative disease, comprising administering an effective amount of A 3 R agonist is provided. In other embodiments, a method for promoting neuroprotection or nerve repair in a patient suffering from a neurodegenerative disease, comprising administering an effective amount of P2Y 1A method is provided that includes the step of administering an agonist. In some embodiments, the compound is a biased agonist, partial agonist, or biased partial agonist or antagonist at the A 3 receptor. In some embodiments, the compound is a biased agonist, partial agonist, or biased partial agonist or antagonist at the P2Y 1 receptor. In some embodiments, the compound is MRS4322 or a pharmaceutically acceptable salt thereof. Alzheimer's disease (AD)

[0054] In 2014, an estimated 5.2 million Americans of all ages had AD. 11% of the population aged 65 and older had AD (Alzheimer's Association). By 2050, the number of people aged 65 and older with AD is estimated to nearly triple to an estimated 13.8 million. In the United States, the cost of caring for AD patients is approximately $214 billion per year, and 70% of this cost is borne by Medicare and Medicaid. At current trends, these costs could reach $1.2 trillion per year by 2050.

[0055] Activation of astrocytes according to the present invention, as well as promotion of neuroprotection and neurorepair, represents a new treatment option for AD. Thus, in one aspect of the present specification, there is provided a method of treating AD or promoting neuroprotection or neurorepair in a patient suffering from AD, the method comprising administering to the patient an effective amount of a compound disclosed herein. In some embodiments, the present invention is a method of treating AD or promoting neuroprotection or neurorestoration in a patient suffering from AD, the method comprising administering to the patient an effective amount of A 3 R agonist. In some embodiments, the present invention is a method of treating AD or promoting neuroprotection or neurorestoration in a patient suffering from AD, the method comprising administering to the patient an effective amount of P2Y 1Provided is a method comprising the step of administering an agonist. In some embodiments, the compound is a biased agonist, partial agonist, or biased partial agonist or antagonist at the A 3 receptor. In some embodiments, the compound is a biased agonist, partial agonist, or biased partial agonist or antagonist at the P2Y 1 receptor. In some embodiments, the compound is MRS4322 or a pharmaceutically acceptable salt thereof. Parkinson's disease (PD)

[0056] One million Americans live with PD, and approximately 60,000 Americans are newly diagnosed each year, although this does not include the thousands of cases that go undetected (Parkinson's Disease Foundation). The total cost of PD, including direct and indirect costs such as medical treatment, social security costs, and lost benefits, is estimated to be nearly $25 billion annually in the United States (Parkinson's Disease Foundation and Huse 2005).

[0057] The neuroprotective and neurorestorative activation according to the present invention is a new treatment option for PD. Thus, in one aspect of the present specification, provided is a method of treating PD or promoting neuroprotection or neurorestoration in a patient suffering from PD, the method comprising the step of administering to the patient an effective amount of the disclosed compound. In some embodiments, the present invention is a method of treating PD or promoting neuroprotection or neurorestoration in a patient suffering from PD, the method comprising the step of administering to the patient an effective amount of an A 3 R agonist. In some embodiments, the present invention is a method of treating PD or promoting neuroprotection or neurorestoration in a patient suffering from PD, the method comprising the step of administering to the patient an effective amount of a P2Y 1 agonist. In some embodiments, the compound is an A 3It is a biased agonist, partial agonist, or biased partial agonist or antagonist at the receptor. In some embodiments, the compound is P2Y 1 It is a biased agonist, partial agonist, or biased partial agonist or antagonist at the receptor. In some embodiments, the compound is MRS4322 or MRS1873 or a pharmaceutically acceptable salt thereof. Multiple sclerosis (MS)

[0058] In the United States, over 400,000 people have MS. In young adults, MS is the most common disease of the central nervous system (Multiple Sclerosis Foundation). Astrocytes have the potential ability to reverse the destruction of neuronal myelin coating caused by MS through their nerve repair effects and promote the healing of the damaged CNS in MS patients.

[0059] Therefore, the neuroprotection and activation of nerve repair of the CNS according to the present invention are new treatment options for MS. Thus, in one aspect of the present specification, there is provided a method for treating MS or promoting neuroprotection or nerve repair in a patient suffering from MS, the method comprising administering to the patient an effective amount of the disclosed compound. In some embodiments, the present invention is a method for treating MS or promoting neuroprotection or nerve recovery in a patient suffering from MS, the method comprising administering to the patient an effective amount of A 3 R agonist. In some embodiments, the present invention is a method for treating MS or promoting neuroprotection or nerve recovery in a patient suffering from MS, the method comprising administering to the patient an effective amount of P2Y 1 agonist. In some embodiments, the compound is A 3 It is a biased agonist, partial agonist, or biased partial agonist or antagonist at the receptor. In some embodiments, the compound is P2Y 1It is a biased agonist, partial agonist, or biased partial agonist or antagonist at the receptor. In some embodiments, the compound is MRS4322 or MRS1873 or a pharmaceutically acceptable salt thereof. Amyotrophic lateral sclerosis (ALS) / Lou Gehrig's disease

[0060] In the United States, approximately 5,600 people are diagnosed with ALS each year, and as many as 30,000 Americans may have the disease simultaneously (ALS Association). Activation of astrocytes can stimulate neuron recovery and repair, as well as neuron binding, in ALS patients.

[0061] Accordingly, in one aspect of the present specification, there is provided a method of treating ALS or promoting neuroprotection or neurorepair in a patient suffering from ALS, the method comprising administering to the patient an effective amount of the disclosed compound. In other embodiments, there is provided a method of stimulating neuron recovery and repair and neuron binding in an ALS patient, the method comprising administering to the patient an effective amount of the compound disclosed herein. In some embodiments, the invention is a method of treating ALS or promoting neuroprotection or neurorecovery in a patient suffering from ALS, the method comprising administering to the patient an effective amount of A 3 R agonist. In some embodiments, the invention is a method of treating ALS or promoting neuroprotection or neurorecovery in a patient suffering from ALS, the method comprising administering to the patient an effective amount of P2Y 1 agonist. In some embodiments, the compound is A 3 It is a biased agonist, partial agonist, or biased partial agonist or antagonist at the receptor. In some embodiments, the compound is P2Y 1It is a biased agonist, partial agonist, or biased partial agonist or antagonist at the receptor. In some embodiments, the compound is MRS4322 or a pharmaceutically acceptable salt thereof. Chronic traumatic encephalopathy (CTE)

[0062] CTE (a form of tauopathy) is a progressive neurodegenerative disease found in individuals who have received one or more (often multiple or repeated over time) severe impacts to the head. CTE is most often diagnosed in professional athletes in American football, soccer, hockey, professional wrestling, stunts, bull riding and rodeo, motocross, and other contact sports who have experienced repeated brain injuries and / or concussions. A subset of humans affected by CTE have chronic traumatic encephalomyopathy (CTEM) characterized by motor neuron disease symptoms similar to ALS. Progressive muscle weakness, as well as movement and gait abnormalities, are thought to be early signs of CTEM. The first stage symptoms of CTE include progressive attention deficit, disorientation, floating dizziness, and headache. The second stage symptoms include memory loss, social instability, restlessness, and impaired judgment. In the third and fourth stages, the patient suffers from progressive dementia, slow movement, tremors, hypomimia, rotary dizziness, speech disorders, hearing loss, and suicidal tendencies, and may further include dysarthria, dysphagia, and eye abnormalities such as ptosis. vertigo, speech disorders, hearing loss, and suicidal tendencies, and may further include dysarthria, dysphagia, and eye abnormalities such as ptosis.

[0063] Accordingly, in one aspect of the present specification, there is provided a method for treating or preventing CTE in a patient suffering from CTE, or promoting neuroprotection or nerve repair, the method comprising administering to the patient an effective amount of the disclosed compound. In other embodiments, there is provided a method for stimulating the recovery and repair of neurons and the connection of neurons in a CTE patient, the method comprising administering to the patient an effective amount of the disclosed compound. In some embodiments, the compound treats one or more symptoms of the first, second, third, or fourth stage of CTE. In some embodiments, the present invention is a method for treating CTE in a patient suffering from CTE, or promoting neuroprotection or nerve repair, the method comprising administering to the patient an effective amount of A 3 R agonist. In some embodiments, the present invention is a method for treating CTE in a patient suffering from CTE, or promoting neuroprotection or nerve repair, the method comprising administering to the patient an effective amount of P2Y 1 agonist. In some embodiments, the compound is a biased agonist, partial agonist, or biased partial agonist or antagonist at the A 3 receptor. In some embodiments, the compound is a biased agonist, partial agonist, or biased partial agonist or antagonist at the P2Y 1 receptor. In some embodiments, the compound is MRS4322 or MRS1873 or a pharmaceutically acceptable salt thereof.

[0064] At the microscopic scale, this pathology includes neuronal death, accumulation of tau, accumulation of TAR DNA-binding protein 43 (TDP43) beta-amyloid, white matter changes, and other abnormalities. The accumulation of tau includes an increase in the presence of high-density neurofibrillary tangles (NFTs), neurites, and glial condensates composed of astrocytes and other glial cells. Thus, in some embodiments, the method treats, enhances the elimination of, or prevents neuronal death, accumulation of tau, accumulation of TAR DNA-binding protein 43 (TDP43) beta-amyloid, white matter changes, and other abnormalities associated with CTE.

[0065] In some embodiments, the present invention provides for the long-term administration of a compound disclosed herein, such as A, for treating neurodegenerative diseases, such as the diseases discussed above and below. 3 a biased agonist, partial agonist or biased partial agonist of R, or a biased agonist, partial agonist or biased partial agonist of P2Y 1 Cardiovascular diseases

[0066] The disclosed compounds are also useful for treating various cardiovascular diseases and conditions. In some embodiments, the present invention provides a method for treating a heart disease or a cardiovascular disease, such as myocardial ischemia, myocardial infarction, cardiomyopathy, coronary artery disease, arrhythmia, myocarditis, pericarditis, angina, hypertensive heart disease, endocarditis, rheumatic heart disease, congenital heart disease, or atherosclerosis, the method comprising administering to a patient in need thereof an effective amount of a disclosed compound, such as MRS4322 or MRS1873 or a pharmaceutically acceptable salt thereof. In some embodiments, the disclosed compounds provide modulation of ATP-sensitive potassium channels by, for example, biased agonism, partial agonism, or biased partial agonism of the A 3 R receptor.

[0067] In some embodiments, the heart disease or cardiovascular disease is myocardial ischemia or myocardial infarction. Other diseases

[0068] ​ For example, compounds that modulate beneficial effects such as neuroprotection by increasing the activity of astrocyte mitochondria also have the potential to treat a variety of other diseases. For example, due to the role of astrocytes in neuroprotection disclosed in the present invention, for example, A 3 R and / or P2Y 1 Activation of astrocytes by modulation of receptors can be useful for the treatment of various diseases and conditions discussed below. Thus, in some embodiments, the present invention provides a method of treating or promoting neuroprotection or neuroregeneration in a patient suffering from a disease or condition, the method comprising administering to the patient an effective amount of a disclosed compound, such as MRS4322 or a pharmaceutically acceptable salt thereof. In some embodiments, the disease or condition is selected from autoimmune diseases, allergic diseases, and / or graft rejection and graft-versus-host disease (for the use of certain nucleoside and nucleotide compounds in the treatment of these conditions, see, for example, WO2007 / 20018, which is incorporated herein by reference). In other embodiments, the disease or condition is selected from ocular hypertension and / or glaucoma (for the use of certain nucleoside and nucleotide compounds in the treatment of these conditions, see, for example, WO2011 / 77435, which is incorporated herein by reference). In other embodiments, the disease or condition is selected from smell sensitivity and / or olfactory disorders (for the use of certain nucleoside and nucleotide compounds in the treatment of these conditions, see, for example, EP1624753, which is incorporated herein by reference). In other embodiments, the disease or condition is selected from type 2 diabetes and / or pain management (for the use of certain nucleoside and nucleotide compounds in the treatment of these conditions, see, for example, US2010 / 0256086, which is incorporated herein by reference).

[0069] In other embodiments, the disease or condition is selected from respiratory diseases and / or cardiovascular (CV) diseases (for the use of certain nucleoside and nucleotide compounds in the treatment of these conditions, see, for example, FASEB J., incorporated herein by reference (2013) 27:1118.4 (abstract).). In other embodiments, the disease or condition is selected from CNS dysfunction, learning disorders and / or cognitive deficits (for the use of certain nucleoside and nucleotide compounds in the treatment of these conditions, see, for example, Neuropsychopharmacology. January 2015; 40(2):305-14. doi: 10.1038 / npp.2014.173. Epub July 15, 2014, "Impaired cognition after stimulation of a P2Y 1 receptor in the rat medial prefrontal cortex", Koch, H. et al., PMID: See also 25027332. In other embodiments, the disease or condition is selected from neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, prion disease, and / or amyotrophic lateral sclerosis (see, e.g., US8,691,775, which is incorporated herein by reference for the use of certain nucleoside and nucleotide compounds in the treatment of these conditions). In other embodiments, the disease or condition is selected from ear disorders, Meniere's disease, endolymphatic hydrops, progressive hearing loss, floating dizziness, rotational dizziness, tinnitus, radiation-induced brain injury associated with cancer radiotherapy, and / or migraine treatment (see, e.g., US2009 / 0306225, UY31779, and US8,399,018, each of which is incorporated herein by reference for the use of certain nucleoside and nucleotide compounds in the treatment of these conditions). In other embodiments, the disease or condition is selected from pathological sleep disorders, depression, sleep disorders in the elderly, Parkinson's disease, Alzheimer's disease, epilepsy, schizophrenia, and / or symptoms experienced during recovery from alcohol dependence (see, e.g., US2014 / 0241990, which is incorporated herein by reference for the use of certain nucleoside and nucleotide compounds in the treatment of these conditions). In other embodiments, the disease or condition is selected from damage to neurons or nerves of the peripheral nervous system during surgery (see, e.g., US8,685,372, which is incorporated herein by reference for the use of certain nucleoside and nucleotide compounds in the treatment of these conditions). In other embodiments, the disease or condition is cancer, such as prostate cancer (see, e.g., Biochem Pharmacol. Aug 15, 2011;82(4):418 - 425, doi:10.1016 / j.bcp.2011.05.013. "Activation of the P2Y1 Receptor Induces", which is incorporated herein by reference for the use of certain nucleoside and nucleotide compounds in the treatment of these conditions). pp, doi:10.1016 / j.bcp.2011.05.013. "Activation of the P2Y1 Receptor Induces "Apoptosis and Inhibits Proliferation of Prostate Cancer Cells", Qiang See Wei et al.). In other embodiments, the disease or condition is one or more gastric intestinal conditions, such as constipation and / or diarrhea (for the use of certain nucleoside and nucleotide compounds in the treatment of these conditions, see, for example, Acta Physiol (Oxf). December 2014; 212 (4): 293-305, doi: 10.1111 / apha.12408. "Differential functional role of purinergic and nitrergic inhibitory cotransmitters in human colonic relaxation", Mane N1, Gil V, Martinez-Cutillas M, Clave P, Gallego D, Jimenez M.; and Neurogastroenterol. Motil. January 2014; 26(1 (1): 115-23, doi: 10.1111 / nmo.12240. Epub October 8, 2013, "Calcium responses in subserosal interstitial cells of the guinea-pig proximal colon", Tamada H., Hashitani H. PMID: 24329947). Other embodiments, the disease or condition is pain mediated by the CNS, such as neuropathic pain, inflammatory pain, and / or acute pain (for the use of certain nucleoside and nucleotide compounds in the treatment of these conditions, see, for example, Br J Pharmacol. March 2010; 159(5 ): pp. 1106 - 17, doi:10.1111 / j.1476 - 5381.2009.00596.x. Epub Feb 5, 2010, "A comparative analysis of the activity of ligands acting at P2X and P2Y receptor subtypes in models of neuropathic, acute and inflammatory pain." Ando RD1, Mehesz B, Gyires K, Illes P, Sperlagh B. PMID: 20136836). See also

[0070] In other embodiments, the disease or condition is selected from brain cancer, such as glioblastoma (for the use of certain nucleoside and nucleotide compounds in the treatment of these conditions, see, for example, Purinergic Signal. 2015 Sep;11(3):331 - 46, doi: 10.1007 / s11302 - 015 - 9454 - 7. Epub May 15, 2015, "Potentiation of temozolomide antitumor effect by purine receptor ligands able to restrain the in vitro growth of human glioblastoma stem cells." D'Alimonte, I. et al., PMID: 25976165). In other embodiments, the disease or condition is pain (for the use of certain nucleoside and nucleotide compounds in the treatment of pain, see, for example, each of which is incorporated herein by reference, Pharmacol Biochem Behav. Jan 2015;128:23 - 32, doi: 10.1016 / j.pbb.2014.11.001, Epub Nov 6 2014, "Participation of peripheral P2Y 1 , P2Y 6 and P2Y11 receptors in formalin-induced inflammatory pain in rats.」 Barragan-Iglesias P. et al., PMID: 25449358; and Neuropharmacology. April 2014; 79: 368-79, doi: 10.1016 / j.neuropharm.2013.12.005, Epub December 12, 2013, 「Blockade of peripheral P2Y 1 receptors prevents the induction of thermal hyperalgesia via modulation of TRPV1 expression in carrageenan-induced inflammatory pain rats: involvement of p38 MAPK phosphorylation in DRGs.」 Kwon SG, Roh DH, Yoon SY, Moon JY, Choi SR, Choi HS, Kang SY, Han HJ, Beitz AJ , Lee JH. See PMID: 24333674). In other embodiments, the disease or condition is , a gastrointestinal disorder, such as diarrhea (for the use of certain nucleoside and nucleotide compounds in the treatment of these conditions, see, for example, Acta Physiol (Oxf). December 2014; 212(4): 293 ~305, doi: 10.1111 / apha.12408. See "Differential functional role of purinergic and nitrergic inhibitory cotransmitters in human colonic relaxation", Mane N., Gil V, Martinez-Cutillas M, Clave P, Gallego D, Jimenez M.). In other embodiments, the disease or condition is a cognitive disorder (for the use of certain nucleoside and nucleotide compounds in the treatment of this condition, see, for example, Neuropsychopharmacology. January 2015; 40(2): 305-14, doi: 10.1038 / npp.2014.173, Epub July 15, 2014, "Impaired cognition after stimulation of P2Y 1 receptors in the rat medial prefrontal cortex", Koch H, Bespalov A, Drescher K, Franke H, Krugel U. PMID: 25027332).

[0071] In some embodiments, the present invention provides a method of treating a disease or condition associated with a brain injury or neurodegenerative condition, such as epilepsy, migraine, radiation-induced brain injury associated with cancer radiotherapy, depression, mood or behavioral changes, dementia, restlessness, suicidal tendencies, tremors, Huntington's disease, loss of motor coordination, hearing loss, aphasia, dry eye, hypomania, attention deficit, memory loss, cognitive difficulties, vertigo, dysarthria, dysphagia, eye abnormalities, or disorientation, the method comprising administering to a patient in need thereof an effective amount of the disclosed compound. In some embodiments, the compound is an A 3 R agonist. In some embodiments, the compound is a P2Y 1 agonist. In some embodiments, the compound is an A 3It is a biased agonist, partial agonist, or biased partial agonist or antagonist at the receptor. In some embodiments, the compound is P2Y 1 It is a biased agonist, partial agonist, or biased partial agonist or antagonist at the receptor. In some embodiments, the compound is MRS4322 or MRS1873 or a pharmaceutically acceptable salt thereof.

[0072] In a further embodiment, the present invention provides a method of treating a neurodegenerative disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis, and prion disease in a patient in need thereof, the method comprising administering an effective amount of the disclosed compound. In some embodiments, the compound is an A 3 R agonist. In some embodiments, the compound is a P2Y 1 agonist. In some embodiments, the compound is an A 3 It is a biased agonist, partial agonist, or biased partial agonist or antagonist at the receptor. In some embodiments, the compound is a P2Y 1 It is a biased agonist, partial agonist, or biased partial agonist or antagonist at the receptor. In some embodiments, the compound is MRS4322 or MRS1873 or a pharmaceutically acceptable salt thereof.

[0073] In some embodiments, the improvement in cognitive or neural function is measured as an increase in score between about 1% and 20% in the language recall task delay of the Revised Wechsler Memory Scale. For example, the improvement in cognitive function can be measured as an increase in score between about 1% and 10%, or between about 1% and 5%. 2. Description of certain compounds of the present invention

[0074] In one aspect, the present invention provides compounds useful for the treatment, alleviation or promotion of recovery of certain conditions of the brain or central nervous system (CNS), such as brain injury or neurodegenerative conditions. In some embodiments, the disclosed compounds increase astrocyte-mediated neuroprotection and neuroregeneration, thereby treating, alleviating or promoting recovery of the condition. In some embodiments, the compound is 3 selective for the 3 receptor, for example at least 10-fold selective for the 3 receptor compared to other adenosine receptors, or more than 25-fold, 50-fold, 100-fold, 500-fold or 1000-fold selective compared to other adenosine receptors. In some embodiments, the compound selectively modulates the 3 receptor. In some embodiments, the compound is a selective agonist at the 3 receptor. In some embodiments, the compound is a selective partial agonist at the 3 receptor. In some embodiments, the compound is a biased full or biased partial agonist. In some embodiments, the compound is a biased full or biased partial antagonist.

[0075] In further embodiments, the compound is selective for the 1 receptor, for example at least 10-fold selective for the 1 receptor compared to other P2Y receptors, or more than 25-fold, 50-fold, 100-fold, 500-fold or 1000-fold selective compared to other P2Y receptors. In some embodiments, the compound selectively modulates the 1 receptor. In some embodiments, the compound is a selective agonist at the 1 receptor. In some embodiments, the compound is a selective partial agonist at the 1 receptor. In some embodiments, the compound is a biased full or biased partial agonist. In some embodiments, the compound is a biased full or biased partial antagonist.

[0076] The term "biased" refers to a compound that preferentially modulates, activates, agonizes, or antagonizes one or more, but not all, of the pathways associated with a receptor.

[0077] Without being bound by theory, selective modulation of one or more of the pathways associated with the A 3 or P2Y 1 receptor by biased full or biased partial agonism or biased antagonism may enable improved treatment of a disease or condition and avoidance of unwanted modulation of the pathway (which may cause side effects). Selective modulation, as disclosed herein, can preferentially activate astrocytes to treat, for example, brain injury, or a neurodegenerative disease or condition. Thus, in some embodiments, the disclosed compounds are biased full or biased partial agonists or antagonists of one or more G-coupled or G-independent pathways associated with the adenosine A 3 receptor or P2Y 1 receptor. In some embodiments, the compound selectively modulates a pathway mediated by the A 3 or P2Y 1 receptor, such as beta-arrestin activation, intracellular calcium mobilization, cAMP modulation, ATP-dependent potassium channel activation, or ERK1 / 2 phosphorylation, or other downstream cellular activities associated with such pathways. In some embodiments, the pathway is related to increasing or being associated with neuroprotection or neurorepair, or cardioprotection or cardiac regeneration. In some embodiments, the compound is selected from (N)-methylcarbamoyl nucleosides, such as MRS4322 or a pharmaceutically acceptable salt thereof.

[0078] As used herein, the term "methanocarba nucleoside" refers to a nucleoside analog in which the oxygen present in the tetrahydrofuran ring of ribose sugar is replaced by a methylene unit, and the resulting carbocyclic ring is fused to a cyclopropyl ring to form a bicyclo[3.1.0]hexane, e.g., the structure

Chem.

Chem.

Chem.

Chem.

Chem.

[0079] In some embodiments, the compound is A 3 or P2Y 1 functionally selective at the receptor, i.e., modulates one or more pathways, for example, and not others, or activates one or more pathways and inactivates one or more other pathways, thereby selectively identifying the pathway mediated by A 3 or P2Y 1 receptor. In some embodiments, the compound is an antagonist as measured by cAMP signaling, but a partial agonist for β-arrestin recruitment. In other embodiments, the compound is an agonist for Gq / 11-mediated Ca 2+ mobilization and a partial agonist or antagonist for arrestin recruitment. In some embodiments, the present invention provides a method of treating a brain injury, or a neurodegenerative disease or condition, by biased or functionally selective A 3 receptor modulation (e.g., by selective agonism or antagonism in a pathway such as those listed above), comprising administering to a patient in need thereof an effective amount of the disclosed compound. In some embodiments, the compound is selected from DMPA, CCPA, MRS1760, or MRS542 (see Verzijl D et al., "Functional selectivity of adenosine receptor ligands", Purinergic Signaling 7:171-192 (2011)). In some embodiments, the compound is DBXRM. In some embodiments, the compound is selected from (N)-methanocarba nucleoside, such as MRS4322 or a pharmaceutically acceptable salt thereof.

[0080] Surprisingly, the monophosphate, diphosphate, and triphosphate forms of certain purine nucleosides, such as those detailed herein, have been found to be rapidly dephosphorylated in vivo by ectonucleotidases, enzymes involved in the dephosphorylation of nucleotides, which are present in both the cell membrane surface and circulation in blood and plasma (see Ziganshin et al., Pflugers Arch. (1995) 429:412-418). It is often extremely difficult to predict which nucleotide analogs are substrates for ectonucleotidases and, thus, are predicted to be dephosphorylated in vivo. In some embodiments, the dephosphorylated compounds are involved in therapeutic efficacy. Thus, in some embodiments, the corresponding phosphorylated monophosphate, diphosphate, or triphosphate forms, or phosphate esters, such as their alkyl or phenyl esters, are prodrugs or precursors of agents involved in therapeutic effects.

[0081] In some embodiments, the compounds of the invention can cross the blood-brain barrier (BBB). The term "blood-brain barrier" or "BBB" as used herein refers to the BBB in the strict sense as well as the blood-spinal cord barrier. The blood-brain barrier, consisting of the endothelium, basement membrane, and glial cells of the blood vessels in the brain, acts to limit the permeation of substances into the brain. In some embodiments, the brain / plasma ratio of the total drug is at least approximately 0.01 after administration to a patient (e.g., orally or intravenously). In some embodiments, the brain / plasma ratio of the total drug is at least approximately 0.03. In some embodiments, the brain / plasma ratio of the total drug is at least approximately 0.06. In some embodiments, the brain / plasma ratio of the total drug is at least approximately 0.1. In some embodiments, the brain / plasma ratio of the total drug is at least approximately 0.2.

[0082] Prototype adenosine A 3Agonists, such as Cl-IB-MECA and MRS5698, are low solubility lipophilic compounds with cLogP values typically >2. This lipophilicity contributes to the high plasma protein binding and high brain binding of these compounds, and is a major factor reducing the free fraction of drug available to interact with the A 3 receptor in the brain. In some embodiments, the physicochemical properties of the compounds of the present invention, such as MRS4322 and MRS1873, for example in neurological and neurodegenerative conditions, are substantially different. These compounds and related compounds are hydrophilic compounds with cLogP <0, increasing solubility, reducing plasma and brain binding, and A 3 increasing the concentration of unbound drug available to interact with the A receptor.

[0083] Thus, in some embodiments, the compound has a cLogP of less than about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, about 0.1, about 0.05, about 0.01, or about 0.005. In some embodiments, the compound has a cLogP of less than about 0, such as about -0.1, -0.2, -0.3, -0.4, -0.5, -0.6, -0.7, -0.8, or -0.9 or less. In some embodiments, the compound has an unbound fraction in plasma of about 0.5 to 0.9. In some embodiments, the compound has an unbound fraction in plasma of about 0.6 to 0.85, 0.7 to 0.8, or about 0.75. In some embodiments, the compound has an unbound fraction in the brain of at least about 0.02, or at least about 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.12, 0.15 or 0.17 or more. In some embodiments, the compound has an unbound fraction in plasma of about 0.6 to 0.85, 0.7 to 0.8 or about 0.75, and / or has an unbound fraction in the brain of at least 0.08.

[0084] The compounds of the present invention can be prepared using methods known in the art and only routine experimental methods. For example, certain compounds of the present invention can be prepared according to the procedures presented in U.S. Patent No. 7,087,589 (and the references cited therein), which is incorporated herein by reference.

[0085] In some embodiments, the compounds are adenosine, ADP, 2-methylthio-ADP trisodium salt, ATP, ATP disodium salt, α,β-methylene ATP, α,β-methylene adenosine 5'-triphosphate trisodium salt, 2-methylthioadenosine triphosphate tetrasodium salt, 2-MeSATP, BzATP triethylammonium salt, inosine, cytidine, acylated cytidine, cytidine-monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), CDP-choline, CMP-choline, denufosol, denufosol tetrasodium, GTP, ITP, MRS541, MRS542, MRS1760, MRS2179, MRS2279, MRS2341, MRS2365, MRS2500, MRS2690, MRS2698, MRS3558, MRS4322, MRS5151, MRS5676, MRS5678, MRS5697, MRS5698, MRS5923, MRS5930, benzyl-NECA, IB-MECA, Cl-IB-MECA, LJ529, DPMA, CCPA, DBXRM, HEMADO, PEMADO, HENECA, PENECA, CP608,039, CP532,903, CGS21680, AR132, VT72, VT158, VT160, VT163, PSB0474, uridine 5'-diphosphate (UDP), UDP-glucose, uridine β-thiodiphosphate (UDPβS), uridine 5'-triphosphate (UTP), uridine γ-thiophosphate (UTPγS), 2-thio UTP tetrasodium salt, UTPγS trisodium salt, uridine-5'-diphosphoglucose, dipyridamole analogs, diadenosine tetraphosphate Ap 4 U, Ap4 A, INS365, INS37217 or INS48823 (wherein each sugar may be replaced with a methanocarba sugar in the North or South conformation, or each sugar may be replaced with D-ribose), or a pharmaceutically acceptable salt thereof.

[0086] In some embodiments, 2-methylthio-ADP or a pharmaceutically acceptable salt thereof is useful in the methods of the invention. Without being bound by theory, 2-MeS ADP is thought to be rapidly hydrolyzed in vivo to 2-methylthioadenosine, where it is thought to become a biased agonist, partial agonist, or biased partial agonist of A3R. 2-Methylthioadenosine is thought to have receptor data very similar to that of MRS4322.

[0087] In some embodiments, the compound is A 3 R agonist, such as N 6 -benzyladenosine-5'-N-methyluronamide, such as N 6 -(3-iodobenzyl)-adenosine-5'-N-methyluronamide (also known as IB-MECA or Can-Fite CF-101) or 2-chloro-N 6-(3-Iodobenzyl)-adenosine-5'-N-methyluronamide (also known as 2-Cl-IB-MECA or Can-Fite CF-102), (N)-methanocarba nucleoside, for example (1R,2R,3S,4R)-4-(2-chloro-6-((3-chlorobenzyl)amino)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-1-carboxamide (also known as CF502, Can-Fite Biopharma, MA), (2S,3S,4R,5R)-3-amino-5-[6-(2,5-dichlorobenzylamino)purin-9-yl]-4-hydroxy-tetrahydrofuran-2-carboxylic acid methylamide (also known as CP532,903), (1’S,2’R,3’S,4’R,5’S)-4-(2-chloro-6-(3-chlorobenzylamino)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-1-carboxamide (also known as MRS3558), 2-(1-hexynyl)-N-methyladenosine, (1S,2R,3S,4R)-2,3-dihydroxy-4-(6-((3-iodobenzyl)amino)-4H-purin-9(5H)-yl)-N-methylcyclopentanecarboxamide (also known as CF101, Can-Fite), (1S,2R,3S,4R)-4-(2-chloro-6-((3-iodobenzyl)amino)-4H-purin-9(5H)-yl)-2,3-dihydroxy-N-methylcyclopentanecarboxamide (also known as CF102, Can-Fite), (1’R,2’R,3’S,4’R,5’S)-4-{2-chloro-6-[(3-iodophenylmethyl)amino]purin-9-yl-}-1-(methylaminocarbonyl)-bicyclo[3.1.0]hexane-2,3-diol (also known as MRS1898) or a 2-dialkynyl derivative of (N)-methanocarba nucleoside, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is selected from IB-MECA (also known as CF101) or Cl-IB-MECA (also known as CF102), or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is selected from (N)-methanocarba nucleosides such as those disclosed above or pharmaceutically acceptable salts thereof.

[0088] A that enhances receptor activity in the presence of a natural ligand 3 R allosteric modulators, such as 2-cyclohexyl-N-(3,4-dichlorophenyl)-1H-imidazo[4,5-c]quinolin-4-amine (CF602, also known as Can-Fite), are also included. However, the A 3 R agonists are not at all exclusive, and such other agonists can also be used. A covalently bound to a polymer 3 Administration of an R agonist is also contemplated. For example, A 3 R agonists can be administered in the form of conjugates in which the agonist is bound to a polyamidoamine (PAMAM) dendrimer.

[0089] Without being bound by theory, selective modulation of one or more pathways can be achieved by full or partial agonism, including biased agonism by certain uridine analogs, to improve the treatment of the disclosed diseases or conditions and avoid modulation of undesirable pathways (which may lead to side effects). In some embodiments, the selective modulation preferentially activates astrocytes or other glial cells, such as microglia and oligodendrocytes, to treat the disclosed brain injuries or neurodegenerative diseases or conditions. Certain uridine analog compounds suitable for use in the present invention are disclosed in WO2014 / 160502, which is hereby incorporated by reference in its entirety. In some embodiments, the compound is an A 3 R agonist. In some embodiments, the compound is a P2Y 1 agonist. In some embodiments, the compound is an adenosine receptor, such as A 1 A 2A A 2B or A 3It is a biased agonist at the receptor. In some embodiments, the compound is A 3 It is a biased agonist, partial agonist, or biased partial agonist at the receptor. In some embodiments, the compound is P2Y 1 It is a biased agonist, partial agonist, or biased partial agonist at the receptor. In some embodiments, the compound is

Chemical formula

Chemical formula

Chemical formula

[0090] In some embodiments, the compound is selected from the following

Chemical formula

Chemical formula

Chem.

Chem.

Chem.

Chem.

Chem.

[0091] In some embodiments, the compound is

Chemical formula

Chemical formula

Chemical formula

[0092] In some embodiments, the compound is

Chemical formula

Chemical formula

[0093] In some embodiments, the compound is

Chemical formula

Chemical formula

Chemical formula

[0094] In some embodiments, the compound is

Chemical formula

[0095] In some embodiments, the compound is

Chemical formula

[0096] In some embodiments, the compound is

Chemical formula

[0097] In some embodiments, the compound is [Chemical formula] or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is [Chemical formula] or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is [Chemical formula] or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is [Chemical formula] or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is [Chemical formula] is.

[0098] In one aspect, the present invention provides a pharmaceutical composition comprising the disclosed compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient. In some embodiments, the compound is [Chemical formula] or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is [Chemical formula] or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is [Chemical formula] or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is [Chemical formula] .

[0099] In some embodiments, the compound is selected from the compounds described in FIG. 15 or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is [Chemical formula] [Chemical formula] (MRS1898) or MRS1873 or a pharmaceutically acceptable salt thereof.

[0100] In one aspect, the present invention provides a method for treating a brain or central nervous system (CNS) injury or condition selected from traumatic brain injury (TBI) or stroke, the method comprising administering to a patient in need thereof [Chemical formula] an effective amount of a compound selected from, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable composition comprising the same.

[0101] In some embodiments, the compound is [Chemical formula] or a pharmaceutically acceptable salt thereof.

[0102] In some embodiments, the compound is [Chemical formula] or a pharmaceutically acceptable salt thereof.

[0103] In some embodiments, the brain or central nervous system (CNS) injury or condition is TBI.

[0104] In some embodiments, the TBI is selected from concussion, blast injury, combat-related injury, or mild, moderate, or severe impact to the head.

[0105] In some embodiments, the compound is administered within 24 hours after TBI or stroke.

[0106] In some embodiments, the compound is administered within 8 hours after TBI or stroke.

[0107] In some embodiments, the compound is administered at least during the first 8 - 48 hours after TBI or stroke.

[0108] In some embodiments, the brain or central nervous system (CNS) injury or condition is stroke.

[0109] In some embodiments, the compound is administered chronically for treating stroke during the period of stroke recovery after stroke has occurred.

[0110] In some embodiments, the neuroprotection or neurorepair of the patient is increased compared to an untreated patient.

[0111] In some embodiments, the compound is human A 3 an adenosine receptor (A 3 R) biased partial agonist.

[0112] In some embodiments, A 3 R is partially agonized in a manner biased towards the neuroprotective function of the A 3 R receptor.

[0113] In some embodiments, the compound is administered orally, intravenously, or parenterally.

[0114] In one aspect, the present invention is a method of enhancing neuroprotection or neurorepair in a patient suffering from TBI or stroke, the method comprising administering to a patient in need thereof

Chemical formula

[0115] In some embodiments, the compound is

Chemical formula

[0116] In some embodiments, the compound is

Chemical formula

[0117] In some embodiments, the neuroprotection or neurorepair shortens the recovery period after TBI or stroke as compared to an untreated patient.

[0118] In some embodiments, the compound is a biased partial agonist at the human A 3 adenosine receptor (A 3 R), and the A 3 R is partially agonized in a manner biased towards the neuroprotective function of the A 3 R receptor.

[0119] In some embodiments, the compound is administered orally, intravenously, or parenterally.

[0120] In some embodiments, the compound or a pharmaceutically acceptable salt thereof has a non-bound fraction of at least 0.7 in plasma, or at least 0.08 in the brain, or both.

[0121] In some embodiments, the compound or a pharmaceutically acceptable salt thereof has a non-bound fraction of at least 0.7 in plasma, or at least 0.08 in the brain, or both.

[0122] In one aspect, the present invention is a method for treating an injury, disease, or condition selected from traumatic brain injury (TBI), stroke, neurodegenerative conditions, or heart or cardiovascular disease, the method comprising administering to a patient in need thereof

Chemical formula

[0123] In some embodiments, the compound is

Chemical formula

[0124] In some embodiments, the compound is

Chemical formula

[0125] In some embodiments, the injury, disease, or condition is TBI.

[0126] In some embodiments, the TBI is selected from concussion, blast injury, combat-related injury, or mild, moderate, or severe impact to the head.

[0127] In some embodiments, the injury, disease, or condition is a stroke selected from ischemic stroke, hemorrhagic stroke, subarachnoid hemorrhage, cerebral vasospasm, or transient ischemic attack (TIA).

[0128] In some embodiments, the neurodegenerative disease is selected from Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), chronic traumatic encephalopathy (CTE), or a neurodegenerative condition caused by a virus, alcohol dependence, tumor, toxin, or repetitive brain injury.

[0129] In some embodiments, the injury, disease, or condition is Parkinson's disease.

[0130] In some embodiments, the injury, disease, or condition is a neurological side effect associated with Alzheimer's disease, migraine, brain surgery, or cancer chemotherapy.

[0131] In some embodiments, the heart disease or cardiovascular disease is selected from myocardial ischemia, myocardial infarction, cardiomyopathy, coronary artery disease, arrhythmia, myocarditis, pericarditis, angina, hypertensive heart disease, endocarditis, rheumatic heart disease, congenital heart disease, or atherosclerosis.

[0132] In some embodiments, the heart disease or cardiovascular disease is myocardial ischemia or myocardial infarction.

[0133] In some embodiments, the compound is administered chronically during the period of recovery from the injury after the injury has occurred to treat a stroke, myocardial ischemia, or myocardial infarction.

[0134] In some embodiments, the neuroprotection or neurorepair of the patient is increased compared to an untreated patient.

[0135] In some embodiments, A 3 R is another A 3By preferentially activating intracellular calcium mobilization with little or no activation of the R-mediated pathway, or by preferentially activating Gq11-mediated intracellular calcium mobilization, Gi-mediated modulation of cAMP production, or Gi-mediated phosphorylation of ERK1 / 2 and Akt, A 3 is agonized in a manner biased towards the neuroprotective function of the R receptor.

[0136] In some embodiments, A 3 R is a different A 3 By preferentially activating intracellular calcium mobilization with little or no activation of the R-mediated pathway, or by preferentially activating Gq11-mediated intracellular calcium mobilization, Gi-mediated modulation of cAMP production, or Gi-mediated phosphorylation of ERK1 / 2 and Akt, A 3 is partially agonized in a manner biased towards the cardioprotective function of the R receptor.

[0137] In some embodiments, the method increases neuroprotection or neurorepair in patients suffering from a neurological condition associated with or resulting from cancer chemotherapy or brain surgery.

[0138] In some embodiments, the compound is administered orally.

[0139] In one aspect, the present invention provides a method of increasing neuroprotection or neurorepair in a patient suffering from TBI or stroke, thereby treating TBI or stroke, the method comprising administering to a patient in need thereof

Chemical formula

[0140] In one aspect, the present invention is a method for treating myocardial ischemia or myocardial infarction by enhancing the cardiac protection or regeneration of damaged heart tissue in a patient suffering from myocardial ischemia or myocardial infarction, the method comprising administering to a patient in need thereof,

Chemical formula

[0141] In some embodiments, the compound is

Chemical formula

[0142] In some embodiments, the compound is

Chemical formula

[0143] In some embodiments, the recovery period after TBI, stroke, myocardial ischemia, or myocardial infarction is shortened compared to an untreated patient.

[0144] In some embodiments, A 3 R is partially agonized in a manner biased towards the neuroprotective function of the A 3 R receptor.

[0145] In some embodiments, A 3 R is partially agonized in a manner biased towards the cardioprotective function of the A 3 R receptor.

[0146] In some embodiments, the compound is administered orally.

[0147] In some embodiments, the compound is a complete A 3 A having an improved cardioprotective function compared to a full A 3 biased agonist of R.

[0148] In some embodiments, the compound is the following A 3 R-mediated pathway: activation of Gq11-mediated intracellular calcium mobilization, Gi-mediated modulation of cAMP production, Gi-mediated phosphorylation of ERK1 / 2 and Akt, or modulation of beta-arrestin activation, one or more preferential activations of a complete A 3 A having an improved cardioprotective function compared to a full A 3 biased agonist of R.

[0149] In some embodiments, the compound is other A 3 Preferential activation of intracellular calcium mobilization with little or no activation of other R-mediated pathways, a complete A 3 A having an improved cardioprotective function compared to a full A 3 biased agonist of R.

[0150] In some embodiments, the compound is a complete A 3 A having an improved cardioprotective function compared to a full A 3 partial agonist of R.

[0151] The amount of the disclosed compound (i.e., the active agent) to be present in the composition for use in the disclosed method or the disclosed pharmaceutical composition is generally a therapeutically effective amount. A "therapeutically effective amount" or dose (or "effective amount") refers to an amount of the active agent sufficient to produce a desired therapeutic result, such as neuroprotection, activation of nerve regeneration, and / or improvement of cognitive or neurological function. The toxicity and therapeutic efficacy of the compositions of the active agents are determined by procedures known in the art, such as LD 50 (the dose lethal to 50% of the test group) and ED 50It can be determined in cell culture or experimental animals to determine the therapeutically effective dose (in 50% of the test group). The dose ratio of toxicity to therapeutic effect is the therapeutic index and can be expressed as the LD 50 / ED 50 ratio. Compositions showing a high therapeutic index are advantageous. Data obtained from cell culture assays and animal tests can be used in determining a range of dosages for use in humans. In some embodiments, the dosage of such a composition is within a range of circulating concentrations that include an ED 50 with little or no toxicity. The dosage can vary within this range depending on the dosage form used and the route of administration utilized.

[0152] In some embodiments, the effective dose and / or desired therapeutic outcome is established by comparing the cognitive function or another parameter of the test subject or patient for at least two measurements, although more than two measurements may be used. An initial cognitive function measurement establishes the initial baseline of the test subject or patient. Cognitive function can be measured using established cognitive tests, such as the language recall task delay of the Revised Wechsler Memory Scale. After treatment, a second measurement is established by further testing using a cognitive test. The effective amount is established when it is demonstrated that the second measurement is improved by at least about 1% compared to the first measurement. In some embodiments, the improvement in cognitive function measured by the language recall task delay of the Revised Wechsler Memory Scale is between about 1% and 20%. In some embodiments, the improvement is between about 1% and 10%. In some embodiments, the improvement is between about 1% and 5%. One of ordinary skill in the art should understand that other methods of determining improvement in cognitive function are equally applicable unless they measure the stage of dementia.

[0153] Accordingly, the present invention encompasses a method of improving cognitive function or neurological function by administering to a subject in need thereof an effective amount of the disclosed compound, wherein the enhancement of neurological function and cognitive function is measured as an increase in score between 1% and 20% in the delay of the language recall task score of the Revised Wechsler Memory Scale.

[0154] The disclosed treatment methods may optionally include administration of the disclosed compounds to obtain a desired therapeutic effect. The compositions may be administered as necessary to maintain the desired therapeutic effect. In some embodiments, the compound is administered for about 1 month to 12 months. In some embodiments, the compound is administered for 1 month to 6 months. In some embodiments, the compound is administered for 1 month to 3 months.

[0155] In one aspect of the invention, the disclosed compounds are administered in an amount between about 5 mg / day and 10 g / day. In some embodiments, each dose of the compound is in an amount between about 5 mg / dose and 10 g / dose. For example, satisfactory results are obtained by orally administering the compounds of the present invention disclosed herein at a dose between about 0.05 and 10 mg / kg / day, between about 0.1 and 7.5 mg / kg / day, between about 0.1 and 2 mg / kg / day, or at a dose of 0.5 mg / kg / day, once daily or in 2 to 4 divided doses. For example, for parenteral administration by i.v. drip or injection, doses between about 0.01 and 5 mg / kg / day, between about 0.05 and 1.0 mg / kg / day, and between about 0.1 and 1.0 mg / kg / day can be used. Thus, a daily dose suitable for a patient is between about 2.5 and 500 mg p.o., between about 5 and 250 mg p.o., between about 5 and 100 mg p.o., or between about 0.5 and 250 mg i.v., between about 2.5 and 125 mg i.v. and between about 2.5 and 50 mg i.v. 3. Use, Formulation and Administration Pharmaceutically Acceptable Compositions

[0156] According to another embodiment, the present invention provides a composition comprising the disclosed compound, and a pharmaceutically acceptable carrier, adjuvant or vehicle. In certain embodiments, the compositions of the present invention are formulated for administration to a patient in need of such a composition. In some embodiments, the compositions of the present invention are formulated for oral administration to a patient.

[0157] As used herein, the term "patient" means an animal, preferably a mammal, most preferably a human.

[0158] The term "pharmaceutically acceptable carrier, adjuvant, or vehicle" refers to a non-toxic carrier, adjuvant or vehicle that does not destroy the pharmacological activity of the compound formulated therewith. Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffers such as phosphate, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and lanolin.

[0159] "Pharmaceutically acceptable derivative" means any non-toxic salt, ester, salt of an ester or other derivative of a compound of the present invention that, upon administration to a recipient, can directly or indirectly provide the compound of the present invention, or a metabolite or residue thereof that is inhibitorily active.

[0160] The compositions of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or by an implantable reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intramedullary, intraliver, intralesional, and intracranial injection or infusion techniques. In some embodiments, the composition is administered orally, intraperitoneally, or intravenously. Injectable sterile forms of the compositions of the present invention can be aqueous or oily suspensions. These suspensions can be formulated according to techniques known in the art using appropriate dispersing or wetting agents and suspending agents. Injectable sterile preparations can be, for example, injectable sterile solutions or suspensions in a non-toxic parenterally acceptable diluent or solvent such as a 1,3-butanediol solution. Acceptable vehicles and solvents that can be used include water, Ringer's solution, and isotonic saline. Additionally, conventionally, sterile fixed oils have been used as a solvent or suspending medium.

[0161] For this purpose, any non-irritating fixed oil containing synthetic monoglycerides or diglycerides can be used. Natural pharmaceutically acceptable oils such as olive oil or castor oil, particularly those that are polyoxyethylenated, as well as fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables. These oily solutions or suspensions can also contain long-chain alcohol diluents or dispersing agents such as carboxymethylcellulose or similar dispersing agents commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants such as Tweens, Spans, and other emulsifying agents or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for formulation purposes.

[0162] The pharmaceutically acceptable compositions of the present invention can be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, generally used carriers include lactose and corn starch. Lubricants such as magnesium stearate are also typically added. Diluents useful for oral administration in capsule form include lactose and dried corn starch. When an aqueous suspension is required for oral use, the active ingredient is combined with emulsifying and suspending agents. Optionally, certain sweetening, flavoring or coloring agents may also be added.

[0163] Alternatively, the pharmaceutically acceptable compositions of the present invention can be administered in the form of suppositories for rectal administration. These can be prepared by mixing the drug with suitable non-irritating excipients that are solid at room temperature but liquid at rectal temperature and thus melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycol.

[0164] The pharmaceutically acceptable compositions of the present invention can also be administered topically, particularly when the treatment target includes areas or organs including diseases of the eye, skin, or lower intestinal tract that are readily accessible by topical application. Suitable topical formulations are readily prepared for each of these areas or organs.

[0165] Topical application for the lower intestinal tract can be effected with rectal suppository formulations (see above) or suitable enema formulations. Topical transdermal patches can also be used.

[0166] In local applications, the pharmaceutically acceptable compositions provided can be formulated as suitable ointments containing the active ingredient(s) suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of the present invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax, and water. Alternatively, the pharmaceutically acceptable compositions provided can be formulated as suitable lotions or creams containing the active ingredient(s) suspended or dissolved in one or more carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water.

[0167] For ophthalmic use, the pharmaceutically acceptable compositions provided can be formulated as a micronized suspension in pH-adjusted isotonic sterile saline, with or without a preservative such as benzylalkonium chloride, or as a solution in pH-adjusted isotonic sterile saline. Alternatively, for ophthalmic use, the pharmaceutically acceptable compositions can be formulated as an ointment, such as petrolatum.

[0168] The pharmaceutically acceptable compositions of the present invention can also be administered by nasal aerosol or inhalation. Such compositions can be prepared according to techniques well known in the pharmaceutical art and can be prepared as an aqueous saline solution using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents.

[0169] In some embodiments, the pharmaceutically acceptable compositions of the present invention are formulated for oral administration. Such formulations can be administered with or without food. In some embodiments, the pharmaceutically acceptable compositions of the present invention are administered without food. In other embodiments, the pharmaceutically acceptable compositions of the present invention are administered with food.

[0170] In other embodiments, the pharmaceutically acceptable compositions of the invention are formulated for intravenous (IV) administration.

[0171] The amount of the compounds of the invention that can be combined with a carrier material to produce a single dosage form composition will vary depending on the host being treated and the particular method of administration. Preferably, the compositions provided should be formulated so that they can administer to a patient receiving these compositions an inhibitor at a dosage between 0.01 and 100 mg per kg of body weight per day.

[0172] It should also be understood that the specific dosage and treatment regimen for any particular patient will vary depending on a variety of factors including the activity of the specific compound being used, age, weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated. The amount of the compound of the invention in the composition will also vary depending on the particular compound in the composition. Use of the Compounds and Pharmaceutically Acceptable Compositions

[0173] The compounds and compositions described herein are generally useful for treating a variety of diseases and conditions such as brain injury and neurodegenerative conditions, as well as the various methods disclosed herein.

[0174] The activity of the compounds utilized in the present invention can be assayed in vitro, in vivo or in cell lines. In vitro assays include assays that determine protein modulation or binding to proteins. Detailed conditions for assaying the compounds are described in the following examples.

[0175] As used herein, the terms "treatment", "treating" and "treatment" refer to ameliorating, alleviating, delaying the onset of, or inhibiting the progression of, a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment can be administered after one or more symptoms have developed. In other embodiments, treatment can be administered even in the absence of symptoms. For example, treatment can be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). Treatment can be continued even after symptoms have resolved, for example, to prevent or delay their recurrence.

[0176] The compounds and compositions according to the methods of the invention can be administered using any amount and any route of administration effective in treating or reducing the severity of the disclosed diseases or conditions, or related conditions or syndromes. The exact amount required will vary from subject to subject depending on the species, age, and general condition of the subject, the severity of the disease or condition, the particular agent, its method of administration, etc. The compounds of the invention are preferably formulated in unit dosage form in order to facilitate administration and ensure uniform dosage. The expression "unit dosage form" as used herein refers to physically discrete units suitable for the patient to be treated. However, it will be understood that the total daily usage of the compounds and compositions of the invention will be determined by the attending physician within the scope of sound medical judgment. The specific effective dosage level for any particular patient or organism will vary depending on a variety of factors including the disorder being treated and the severity of the disorder, the activity of the specific compound used, the specific composition used, the age, weight, general health, sex and diet of the patient, the time of administration, route of administration and rate of excretion of the specific compound used, the duration of the treatment, drugs used in combination with or concurrently with the specific compound used, and like factors well known in the medical arts. The term "patient" as used herein means an animal and in some embodiments means a mammal or in certain other embodiments means a human.

[0177] The pharmaceutically acceptable compositions of the present invention can be administered to humans and other animals orally, sublingually, rectally, parenterally, intracapsularly, vaginally, intraperitoneally, topically (as powders, ointments, or drops), intravitreally (e.g., as eye drops), buccally in the mouth, or as oral or nasal sprays, etc., depending on the severity of the disease or condition to be treated. In certain embodiments, the compounds of the present invention can be administered orally or parenterally once or multiple times a day at a dosage level of about 0.01 mg to about 50 mg or about 1 mg to about 25 mg per kg of body weight of the subject per day to obtain the desired therapeutic effect.

[0178] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. The liquid dosage forms may contain, in addition to the active compound, inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 - butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof, etc. Oral compositions may also contain, in addition to the inert diluent, adjuvants such as wetting agents, emulsifying agents and suspending agents, sweetening agents, flavoring agents, and perfuming agents.

[0179] Injectable preparations, for example, injectable sterile aqueous or oily suspensions, can be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents. Injectable sterile preparations can be, for example, injectable sterile solutions, suspensions or emulsions in a non-toxic parenterally acceptable diluent or solvent such as a 1,3-butanediol solution. Acceptable vehicles and solvents that can be used include water, Ringer's solution, U.S.P. and isotonic saline. Further, conventionally, sterile fixed oils have been used as solvents or suspending media. For this purpose, any non-irritating fixed oil containing synthetic monoglycerides or diglycerides can be used. Further, in the preparation of injectables, fatty acids such as oleic acid are used.

[0180] Injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other injectable sterile media before use.

[0181] To prolong the effect of the compounds of the present invention, it is often desirable to slow the absorption of the compounds from subcutaneous or intramuscular injections. This can be achieved by using suspensions of crystalline or amorphous materials with low water solubility. Next, the absorption rate of the compound varies according to its dissolution rate, which can vary according to the crystal size and crystal form. Alternatively, delayed absorption of the parenterally administered compound form is achieved by dissolving or suspending the compound in an oily vehicle. Injectable depot forms are prepared by forming a microencapsulation matrix of the compound in a biodegradable polymer such as polylactide-polyglycolide. The release rate of the compound can be controlled according to the ratio of the compound to the polymer and the nature of the specific polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

[0182] Compositions for rectal or vaginal administration are preferably suppositories prepared by mixing a compound of the invention with a suitable non-irritating excipient or carrier which is solid at ambient temperature but liquid at body temperature and thus melts in the rectal or vaginal cavity to release the active compound, such as cocoa butter, polyethylene glycol or suppository wax.

[0183] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate, and / or a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) dissolution retarders such as paraffin, f) absorption promoters such as quaternary ammonium compounds, g) wetting agents such as cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also contain buffering agents.

[0184] Similar types of solid compositions can also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycol. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells, such as enteric coatings and other coatings well-known in the pharmaceutical formulation art. These solid dosage forms can optionally contain opacifiers and can be compositions in which the active ingredient(s) are released only in the intestinal tract or in a specific part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Similar types of solid compositions can be used as excipients such as lactose or milk sugar and high molecular weight polyethylene (polethylene) glycol as fillers in soft and hard-filled gelatin capsules. can also be used as fillers in soft and hard-filled gelatin capsules using excipients.

[0185] The active compound(s) may, as described above, be in microencapsulated form using one or more excipients. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells, such as enteric coatings, controlled release coatings and other coatings well-known in the pharmaceutical formulation art. In such solid dosage forms, the active compound(s) can be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms can also contain, in the usual routine, additional substances other than inert diluents, such as tableting lubricants and other tableting aids such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage form can also contain buffering agents. Such dosage forms can optionally contain opacifiers and can be compositions in which the active ingredient(s) are released only in the intestinal tract or in a specific part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0186] Dosage forms for topical or transdermal administration of the compounds of the present invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active ingredients are mixed under aseptic conditions with pharmaceutically acceptable carriers and any necessary preservatives or buffers if required. Ophthalmic formulations, ear drops, and eye drops are also intended to be included within the scope of the present invention. Further, the present invention contemplates the use of transdermal patches, which have the additional advantage of being able to control the delivery of the compound to the body. Such dosage forms can be prepared by dissolving or suspending the compound in a suitable medium. Absorption enhancers can also be used to increase the compound flux through the skin. The absorption rate can be controlled by providing a rate-limiting membrane or by dispersing the compound in a polymeric matrix or gel.

[0187] Depending on the particular condition or disease being treated, additional therapeutic agents that are normally administered to treat that condition can also be present in the compositions of the present invention. As used herein, additional therapeutic agents that are normally administered to treat a particular disease or condition are known to be "suitable for the disease or condition being treated".

[0188] As shown in the following examples, in certain exemplary embodiments, the compounds are prepared and used according to the following general procedures. Although the synthesis of certain specific compounds of the present invention is shown by general methods, it will be understood that the following general methods and other methods known to those skilled in the art can be applied to all compounds, as well as to each subclass and species of these compounds, as described herein.

[0189] The content of each document described herein is hereby incorporated by reference in its entirety.

Examples

[0190] (Example 1) Pharmacokinetics of MRS4322 after Intraperitoneal Administration to Mice Objective This study was designed to determine the plasma and brain concentrations of MRS4322 after intraperitoneal administration of the doses used in mouse models of photothrombosis and traumatic brain injury. Methods

[0191] Chemicals: MRS4322 was obtained through the kindness of Dr. Ken Jacobson at the National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD). Analytical grade tolbutamide was obtained from a commercial supply from Seventh Wave Laboratories (Maryland Heights, MO). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).

[0192] Animals: Female C576BL / 6J mice weighing approximately 0.02 kg, supplied by the University of Texas Health Science Center (San Antonio, TX), were used in this study. All studies were conducted under an approved University of Texas Health Science Center IACUC protocol.

[0193] Drug administration: MRS4322 was solubilized in DMSO and then diluted with saline to prepare the administration solution. The final concentration of MRS4322 in the administration solution was 100 μM. An administration solution with a volume of 100 μL per 20 grams of body weight was intraperitoneally administered to each mouse, and MRS4322 was intraperitoneally administered at 0.16 mg / kg or 0.5 μmol / kg. Three mice were administered MRS4322 at each sampling time point.

[0194] Tissue sample collection: Blood and brain samples were obtained at 0 hours, 0.083 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, and 8 hours after administration. At each time point, mice (3 per time point) were euthanized in a carbon monoxide chamber. Whole blood was obtained by cardiac puncture, placed in a Microtainer tube containing heparin, immediately centrifuged to prepare plasma, and the plasma was stored at -80°C. At each time point, whole brain samples were obtained by decapitation, rinsed with ice-cold phosphate-buffered saline, and weighed. The brain samples were then immediately snap-frozen in liquid nitrogen and stored at -80°C. Bioanalysis

[0195] The plasma and brain concentrations of MRS4322 were determined by LC-MS / MS using tolbutamide as the internal standard. The following table summarizes the LC and MS / MS conditions used.

Table 1-1

Table 1-2

[0196] Standard curves were generated for each tissue matrix and the LLOQ / ULOQ concentrations were determined. The calibration range of the standard curve for the plasma concentration of MRS4322 was 2.42 - 242 ng / mL. The calibration range of the brain concentration of MRS4322 was 2.41 - 233 ng / mL.

[0197] For the bioanalysis of the brain concentration of MRS4322, brain samples were homogenized at a 4-fold dilution in ice-cold phosphate-buffered saline. An aliquot of the resulting diluted brain homogenate was treated with acetonitrile and analyzed by LC-MS / MS. Due to the 4-fold homogenate dilution, the calibration range of the brain standard curve for MRS4322 was converted to 9.64 - 932 ng / gm. In some samples, MRS4322 was detectable and the brain LLOQ decreased below 9.64 ng / gm, but exceeded the background. In these cases, the finally recorded brain concentration was extrapolated based on the height of the MS peak. Results

[0198] After intraperitoneal administration to mice, the concentration of MRS4322 was detectable in both plasma and brain samples (Figure 1A and Table 2). It should be noted that, as described in Example 11 below and shown in Figures 1B and 16, the concentration of MRS4322 was detectable in the plasma and brain samples of neonatal pigs. After intravenous administration to neonatal pigs, the concentration of MRS4322 was detectable in plasma, brain, extracellular brain fluid, and cerebrospinal fluid samples (Figure 1B and Figure 16, Table 13).

Table 2

Table 3

[0199] Brain concentrations were detectable, but the data were insufficient to estimate half-life or other pharmacokinetic parameters other than Cmax and Tmax. However, based on the available plasma and brain data, the brain / plasma ratio of the total drug was estimated to be approximately 0.06 based on the mean Cmax concentrations in plasma and brain.

[0200] These results demonstrate that the circulating plasma concentration of MRS4322 is detectable after intraperitoneal administration to mice under the dosing conditions used in the models of photothrombosis and traumatic brain injury, and that MRS4322 is confirmed to distribute to the brain under these dosing conditions. (Example 2) Pharmacokinetics of MRS4322 after Intraperitoneal Administration of MRS2365 to Mice Objective

[0201] This study was designed to determine the plasma and brain concentrations of MRS4322 after intraperitoneal administration of MRS2365, a P2Y 1 agonist, at the doses used in the mouse models of photothrombosis and traumatic brain injury. Methods

[0202] The chemical MRS4322 was obtained through the kindness of Dr. Ken Jacobson at the National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD). MRS2365 was obtained from Tocris Biosciences (Bristol, UK). Analytical grade tolbutamide was obtained from a commercial supply from Seventh Wave Laboratories (Maryland Heights, MO). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).

[0203] Animals: Female C576BL / 6J mice weighing approximately 0.02 Kg, supplied by the University of Texas Health Science Center (San Antonio, TX), were used in this study. All studies were conducted under an approved University of Texas Health Science Center IACUC protocol.

[0204] Drug administration: MRS2365 was solubilized in phosphate buffered saline and then diluted with phosphate buffered saline to prepare the administration solution. The final concentration of MRS2365 in the administration solution was 100 μM. An administration solution with a volume of 100 μL per 20 grams of body weight was intraperitoneally administered to each mouse, and MRS2365 was intraperitoneally administered at 0.5 μmol / kg or 0.24 mg / kg. MRS2365 was administered to 3 mice at each sampling time point.

[0205] Tissue sample collection: Blood and brain samples were obtained at 0 hours, 0.083 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, and 8 hours after administration. At each time point, the mice (3 per time point) were euthanized in a carbon monoxide chamber. Whole blood was obtained by cardiac puncture, placed in a Microtainer tube containing heparin, immediately centrifuged to prepare plasma, and the plasma was stored at -80 °C. At each time point, whole brain samples were obtained by decapitation, rinsed with ice-cold phosphate buffered saline, and weighed. Next, the brain samples were immediately snap-frozen in liquid nitrogen and stored at -80 °C.

[0206] Bioanalysis: In past studies, it has been demonstrated that detectable circulating and brain concentrations of MRS2365 are not observed after intraperitoneal or intravenous administration. Therefore, this study focused only on the detection and quantification of its metabolite, MRS4322. Plasma and brain concentrations of MRS4322 were determined by LC-MS / MS using tolbutamide as the internal standard. The following table summarizes the LC and MS / MS conditions used.

Table 4

[0207] Standard curves were generated for each tissue matrix and the LLOQ / ULOQ concentrations were determined. The calibration range of the standard curve for the plasma concentration of MRS4322 was 2.26 - 241 ng / mL. The calibration range of the brain concentration of MRS4322 was 2.35 - 242 ng / mL.

[0208] For the bioanalysis of the brain concentration of MRS4322, brain samples were homogenized at a 4-fold dilution in ice-cold phosphate-buffered saline. An aliquot of the resulting diluted brain homogenate was treated with acetonitrile and analyzed by LC-MS / MS. Due to the 4-fold homogenate dilution, the calibration range of the brain standard curve for MRS4322 was converted to 9.40 - 968 ng / gm. In some samples, MRS4322 was detectable and the brain LLOQ decreased below 9.40 ng / gm, but exceeded the background. In these cases, the finally recorded brain concentrations were extrapolated based on the height of the MS peak. Results

[0209] After intraperitoneal administration of MRS2365 to mice, concentrations of MRS4322 were detectable in both plasma and brain samples (Figure 2 and Table 5).

Table 5

Table 6

[0210] Brain concentrations were detectable, but the data were insufficient to estimate half-life or other pharmacokinetic parameters other than Cmax and Tmax. However, based on the available plasma and brain data, the brain / plasma ratio of the total drug was estimated to be approximately 0.10 based on the mean Cmax concentrations in plasma and brain.

[0211] These results indicate that the circulating plasma concentration of MRS4322 was detectable under the dosing conditions used in the models of photothrombosis and traumatic brain injury, and MRS4322 was confirmed to distribute to the brain under these dosing conditions. 1 after intraperitoneal administration of the P2Y

[0212] The plasma and brain concentrations of MRS4322 were determined in two different studies after intraperitoneal administration of MRS4322 itself (see Example 1) and after intraperitoneal administration of the P2Y 1 agonist MRS2365. In both of these studies, MRS4322 or MRS2365 was administered at an equimolar dose of 0.5 μmol / kg. Comparing the results of both studies, the observed plasma concentrations of MRS4322 were virtually identical and the brain concentrations of MRS4322 were very similar (compare Figures 1 and 2). There were no statistically significant differences in the half-life and AUC values of MRS4322 after administration of equimolar doses of MRS4322 or MRS2365. These data indicate that MRS2365 is rapidly and completely metabolized to MRS4322 after intraperitoneal administration to mice, and the plasma and brain pharmacokinetics of MRS4322 are very similar to the pharmacokinetics after intraperitoneal administration of MRS4322 itself. (Example 3) Plasma and Brain Binding of MRS4322 in Mice Purpose

[0213] This study was designed to determine the plasma and brain free fractions of MRS4322 in mice and compare the free fraction with that of MRS5698, a prototypical adenosine A 3 receptor agonist. MRS5698 has the following chemical structure. [Chemical formula] Methods

[0214] Chemicals: MRS4322 was obtained through the kindness of Dr. Ken Jacobson at the National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD). MRS5698 was obtained from Tocris Biosciences (Bristol, UK). Analytical grade sulfamethoxazole and warfarin were obtained from commercial supplies from Seventh Wave Laboratories (Maryland Heights, MO). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).

[0215] Animals and tissue preparation: Plasma from male CD-1 mice was obtained from BioreclamationIVT (Westbury, NY) and stored at -80 °C until use. Brains from male CD-1 mice were obtained from BioreclamationIVT (Westbury, NY).

[0216] Blank samples of plasma ultrafiltrate were prepared by thawing the frozen plasma and then pre-warming the plasma at 37 °C for 60 minutes in a humidified 5% CO 2 chamber. An aliquot of 800 μL was transferred to a Centrifree centrifugal filter (Ultracel regenerated cellulose (NMWL 30,000 amu) Lot R5JA31736) and centrifuged at 2900 RPM at 37 °C for 10 minutes. The filtrate of plasma water was collected and used for the preparation of standards, blanks, and QC standards.

[0217] The brain was weighed and homogenized with 1:9 phosphate buffered saline, pH 7.4 using an Omni tissue homogenizer. Brains from 4 mice were homogenized, pooled, and mixed to form one sample.

[0218] Plasma binding determination: MRS4322, MRS5698, sulfamethaxazole and warfarin were solubilized in DMSO and then diluted with 1:1 acetonitrile: water to prepare 100 μM dialysis stock solutions. Sulfamethaxazole and warfarin were utilized as research standards with known plasma binding values. Plasma samples were pre-warmed in a humidified 5% CO 2 incubator at 37 °C for 60 minutes. To 3 mL aliquots of pre-warmed plasma, 100 μM stock solutions of each compound were used to spike MRS4322, MRS5698, sulfamethaxazole or warfarin respectively to a final test concentration of 1 μM. The spiked plasma samples were incubated in a humidified 5% CO 2 chamber on a rotary mixer for at least 60 minutes at 37 °C. After 60 minutes, 800 μL aliquots of each sample were added to Centrifree centrifugal filters. The filters were centrifuged at 2900 rpm for 10 minutes at 37 °C. 100 μL aliquots of the remaining plasma were collected together with the ultrafiltrate for bioanalysis.

[0219] Brain binding determination: MRS4322, MRS5698, sulfamethaxazole and warfarin were solubilized in DMSO and diluted with 1:1 acetonitrile: water to prepare 100 μM dialysis stock solutions. The pooled homogenized brains were pre-warmed in a humidified 5% CO 2 incubator at 37 °C for 60 minutes. To 3 mL aliquots of the brain homogenate, 100 μM stock solutions of each compound were used to spike MRS4322, MRS5698, sulfamethaxazole or warfarin respectively to a final spiking concentration of 1 μM. The pooled spiked brain homogenates were incubated in a humidified 5% CO 2It was placed on a Nutator mixer in an incubator for 60 minutes. After 60 minutes, three aliquots of 800 μL of each sample were added to Centrifree centrifugal filters. The filters were centrifuged at 2900 rpm at 37 °C for 10 minutes. Aliquots of the remaining brain homogenate and ultrafiltrate were collected for bioanalysis. Bioanalysis

[0220] The plasma and brain concentrations of MRS4322 and MRS5698 in spiked plasma, brain homogenate and related ultrafiltrate were determined by LC-MS / MS using tolbutamide as the internal standard. The related concentrations of sulfamethoxazole and warfarin were also determined by LC-MS / MS using standard conditions (data not shown). The following tables summarize the LC and MS / MS conditions used (Tables 7 and 8). The bioanalytical method was the same for all matrices. The statistics of the standard curves (e.g., fit, intercept, slope, correlation coefficient) were determined for each matrix but there were no significant differences and therefore are not shown for each matrix.

Table 7

Table 8

[0221] Standard curves were generated and LLOQ / ULOQ concentrations were determined for each tissue matrix. The calibration ranges of the standard curves for the plasma concentrations of MRS4322 and MRS5698 were 400 - 1200 nM. The calibration ranges of the standard curves for the plasma ultrafiltrate of MRS4322 and MRS5698 were 100 - 1200 nM. The calibration ranges of the standard curves for the brain homogenate and brain homogenate ultrafiltrate of MRS4322 were 400 - 1200 nM and 100 - 1200 nM, respectively. The calibration ranges of the standard curves for the brain homogenate and brain homogenate ultrafiltrate of MRS5698 were 400 - 1200 nM and 1 - 500 nM, respectively. Results

[0222] The plasma-bound and free fractions were determined for MRS4322 and MRS5698 using plasma ultrafiltration. The plasma binding was 25.8% for MRS4322, and the associated free fraction was 0.742 (Table 9).

[0223] The concentration of MRS5698 was not detected in the plasma ultrafiltrate. MRS5698 was completely recovered in the spiked residual plasma samples obtained from the donor side of the Centrifree device (data not shown). This indicates that the low concentration of MRS5698 in the ultrafiltrate was not due to low recovery for compound analysis. Overall, these data are consistent with the high protein binding (99.88%) of MRS5698 in mouse plasma and tissues reported in the literature (Tosh, D.K. et al., Purinergic Signalling (2015) 11:371-387). The binding of the study standards sulfamethoxazole and warfarin was consistent with the literature values.

Table 9

[0224] The brain-bound and free fractions were determined for MRS4322 and MRS5698 using brain homogenate ultrafiltration. The brain binding was 87% for MRS4322, and the associated free fraction was 0.13 (Table 10).

[0225] The concentration of MRS5698 was not detected in the brain homogenate ultrafiltrate. For estimation purposes, the LLOQ of the brain homogenate ultrafiltrate of MRS5698 was used to calculate the brain binding value. The obtained brain binding value was 99.99%. Overall, these data are consistent with the high protein binding (99.88%) of MRS5698 in mouse plasma and tissues reported in the literature (Tosh, D.K. et al., Purinergic Signalling (2015 (Volume 11: Pages 371 - 387). The binding of the sulfamethoxazole and warfarin, which are research reference substances, was consistent with the values in the literature.

Table 10

[0226] Overall, these data indicate that in both plasma and brain, MRS4322 has a substantially higher free fraction and lower protein binding than MRS5698, which is an adenosine A 3 agonist. These data indicate that for a given total plasma or brain concentration, a substantially higher concentration of MRS4322 than the concentration that could be available for MRS5698 could be available to interact with the effector site. (Example 4) In vitro Stability and Metabolism of MRS2365 in Mouse and Human Blood and Plasma Objective

[0227] This study was designed to determine the in vitro stability and metabolic fate of MRS2365, a P2Y 1 agonist, in mouse and human blood and plasma. Method

[0228] Chemical substances: MRS2365 was obtained from Tocris Biosciences (Bristol, UK). MRS4322 was obtained through the kindness of Dr. Ken Jacobson at the National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD). All other chemical substances were obtained from Sigma - Aldrich (St. Louis, MO). Enalapril and procaine were used as reference substances for mouse and human plasma and blood stability, respectively.

[0229] Preparation of tissues: Plasma from male CD-1 mice and humans was obtained from BioreclamationIVT (Westbury, NY) and stored at -80 °C until use. Whole blood was obtained from male CD-1 mice and human volunteers at Seventh Wave Laboratories (Maryland Heights, MO). Plasma and blood samples were prepared using either EDTA (1 mM) or lithium heparin as anticoagulants.

[0230] Determination of plasma stability: MRS2365, enalapril, and procain were solubilized in phosphate-buffered saline, pH 7.4. Plasma samples (prepared from blood using either EDTA or lithium heparin as anticoagulants) were pre-warmed in a humidified 5% CO 2 incubator at 37 °C for 60 minutes. Incubation for stability was initiated by adding MRS2365 (final concentration 1 μM). For the initial assessment of stability in plasma prepared with EDTA, incubation time points of 0, 10, 30, 60, 120, and 240 minutes were utilized (Figures 3 and 4). In subsequent studies comparing plasma prepared with EDTA and plasma prepared with heparin, incubation time points of 0, 1, 2.5, 5, 7.5, 10, and 30 minutes were utilized (Figure 7). Additional incubations for plasma stability comparing plasma prepared with EDTA and plasma prepared with heparin were performed using time points of 0, 5, 10, 20, 30, 45, 60, and 90 seconds (Figure 8). For exploratory analysis of metabolites, MRS2365 was incubated at a concentration of 100 μM for 10 or 30 minutes in heparinized human plasma (Figure 9). In all studies, plasma was immediately placed into microtainer tubes, frozen on dry ice, and stored at -80 °C until analysis.

[0231] Determination of blood stability: MRS2365, enalapril, and procain were solubilized in phosphate-buffered saline, pH 7.4. Blood samples (treated with EDTA or lithium heparin) were maintained in a humidified 5% CO 2It was pre-warmed in an incubator for 60 minutes. Incubation for stability was initiated by adding MRS2365 (final concentration 1 μM) (Figs. 5 and 6). Aliquots of blood samples were obtained at 0, 1, 2.5, 7.5, 10, and 30 minutes and placed in microtainer tubes. For exploratory analysis of metabolites, MRS2365 was incubated in heparinized human whole blood at a concentration of 100 μM for 10 or 30 minutes (Fig. 10). In all studies, plasma was immediately prepared by centrifugation at 4 °C, placed in microtainer tubes, frozen on dry ice, and stored at -80 °C until analysis. Bioanalysis

[0232] The plasma and blood concentrations of MRS2365 were determined by LC-MS / MS using tolbutamide as the internal standard. The LC and MS / MS conditions used are summarized in the following table (Table 11). The bioanalytical method was the same for all matrices. The statistics of the standard curves (e.g., fit, intercept, slope, correlation coefficient) were determined for each matrix but there were no significant differences, and thus are not shown for each matrix.

Table 11

[0233] Standard curves were generated for each tissue matrix and the LLOQ / ULOQ concentrations were determined. The calibration ranges of the standard curves for the plasma and blood concentrations of MRS2365 were 5 - 5000 ng / mL. Samples for quality control were used at concentrations of 32 ng / mL, 160 ng / mL, 800 ng / mL, and 4000 ng / mL.

[0234] Plasma and blood samples were qualitatively analyzed for MRS2365 metabolites using an LC-MS / MS-based "metabolite discovery" protocol. By reviewing the metabolic pathways of other nucleotides (e.g., ATP, ADP), a hypothesis was established that the most likely metabolites of MRS2365 would be the dephosphorylated (i.e., monophosphate) metabolite (MRS2347, mw = 403.07) and / or the fully dephosphorylated riboside metabolite (MRS4322, mw = 323.10). Since phosphorylated compounds should preferentially produce negative ions and MRS4322, which is a riboside, should preferentially produce positive ions, blood and plasma samples were analyzed by both positive and negative ion LC / MS-MS for the parent compound and metabolites. To search for MRS4322 and MRS2347 respectively, in the positive ion mode, masses in the ranges of 323.7 - 324.7 and 403.7 - 404.7 were monitored by the extracted ion chromatogram. Any ion peaks in these ranges were further analyzed to generate product ion spectra for these ion chromatogram peaks. To search for MRS4322, MRS2347 and MRS2365 respectively, in the negative ion mode, masses in the ranges of 321.70 - 322.70, 401.70 - 402.70 and 481.70 - 482.70 were monitored by the extracted ion chromatogram. The peaks were further analyzed to generate product ion spectra. Since authentic standards for MRS4322 and MRS2347 were not available, the height / area of the ion peaks could not be compared due to potential differences in ionization efficiency, but only qualitative identification of the metabolites could be performed. Results

[0235] To evaluate the plasma stability of MRS2365 in mouse and human plasma, two time-course protocols were utilized. Preliminary data were generated over a 240-minute time course with plasma prepared using EDTA. In subsequent studies, time points from 1 minute to 30 minutes post-incubation were utilized, and in the final study, a shorter time course from 5 seconds to 90 seconds post-incubation was utilized. In all protocols, plasma stability was analyzed in plasma prepared from blood using either EDTA or lithium heparin as an anticoagulant. In vitro stability data for enalapril (mouse) and procain (human) in plasma and whole blood were consistent with published values (data not shown).

[0236] In the plasma stability study using plasma prepared with EDTA, MRS2365 was essentially stable over the incubation period up to 240 minutes post-incubation (Figures 3 and 4). Half-life values could not be calculated over the incubation time course and were thus >240 minutes. In subsequent exploratory analysis for metabolites, neither MRS2347 nor MRS4322 could be detected. These data suggested that MRS2365 was stable in vitro in mouse and human plasma.

[0237] Similar observations were obtained when evaluating the stability of MRS2365 in mouse and human whole blood treated with EDTA as an anticoagulant (Figures 5 and 6). In those studies, the half-life of MRS2365 in mouse whole blood treated with EDTA was 47 minutes, suggesting that EDTA did not completely inhibit the clearance of MRS2365 in this matrix. However, MRS2365 was also completely stable in human whole blood treated with EDTA, with an estimated half-life of >240 minutes. No metabolites were detected in these whole blood studies.

[0238] These data are consistent with the initial in vivo pharmacokinetic evaluation of MRS2365 in mice using heparin as a blood anticoagulant for plasma preparation, where MRS2365 appeared to be rapidly cleared at a rate approximating cardiac output, suggesting extensive extrahepatic clearance of the compound. EDTA is known to chelate divalent cations required for the enzymatic activity of ectonucleotidase, an enzyme involved in the dephosphorylation of nucleotides present on the cell membrane surface as well as in both blood and plasma circulation (see Ziganshin et al., Pflugers Arch. (1995) 429:412-418). Therefore, the major metabolic pathway of the P2Y 1 agonist nucleotide analog MRS2365 may have been completely inhibited by the EDTA used to prepare plasma and whole blood in the above study.

[0239] To investigate this possibility, additional stability studies were performed to compare the stability of MRS2365 in plasma made with either EDTA or lithium heparin, an anticoagulant for which no inhibitory effects have been reported against ectonucleotidases. The studies were first performed over a time course of 0 - 30 minutes using heparinized plasma and whole blood. Quantification of the MRS2365 concentration revealed extremely low concentrations of MRS2365 at all time points, but the data were insufficient to calculate an in vitro half-life (data not shown). Low / undetectable MRS2365 concentrations were observed even at the shortest time points (0 and 1 minute) of this study, so the in vitro stability in both EDTA-treated and heparin-treated plasma was compared, and the study was repeated using a significantly shorter time course (0 - 90 seconds) to attempt to calculate an in vitro half-life. In this shorter-time study, MRS2365 was relatively stable in EDTA-treated mouse and human plasma (Figures 5 and 6). The variability in the MRS2365 concentration was likely due to incomplete mixing at the start of the short time course and variability at multiple sample collection time points over the 90-second incubation time course. However, the MRS2365 concentration in heparin-treated plasma was extremely low even at the shortest (0 and 5 seconds) time points. MRS2365 was completely stable when incubated in phosphate-buffered saline.

[0240] Overall, these data suggest that MRS2365 is inherently stable in solution but is rapidly degraded in mouse and human plasma and blood by a process inhibited by EDTA. Considering that MRS2365 is a nucleotide analog and EDTA is a known inhibitor of ectonucleotidases that dephosphorylate nucleotides, these data strongly suggest that MRS2365 is susceptible to rapid dephosphorylation in plasma and whole blood, which is consistent with the in vivo pharmacokinetic data generated for MRS2365 in mice.

[0241] To further investigate the potential of dephosphorylation of MRS2365 as a cause of instability in mouse and human plasma and whole blood, MRS2365 was incubated at 100 μM for 10 or 30 minutes, and then metabolite search was carried out in heparin-treated human plasma and blood. To ensure detection of any metabolites formed, higher substrate concentrations were used. Representative ion chromatograms and product ion spectra under positive ionization conditions are shown (Figs. 7 and 8). Similar results were obtained by negative ionization chromatograms and product ion spectra (data not shown). Since no chemical standards were present, the absolute concentration of the metabolites could not be determined, but after 10 - 30 minutes of incubation, the relative abundance of metabolite M1 increased compared to metabolite M2. The parent ion chromatograms of metabolites M2 and M1, as well as the product ion spectra obtained under both ionization conditions, were consistent with the masses and structures of MRS2347, a dephosphorylated monophosphate metabolite, and MRS4322, a fully dephosphorylated riboside metabolite, respectively (Fig. 9). Detection of both metabolites and the increase in the relative abundance of metabolite M1 (MRS4322) compared to metabolite M2 (MRS2347) suggested that MRS2365 was stepwise dephosphorylated in human plasma and blood, and that the dephosphorylation proceeded via MRS2347, a monophosphorylated intermediate, and then MRS2347 was further dephosphorylated to MRS4322, a non-phosphorylated riboside.

[0242] Overall, these data support the hypothesis that MRS2365 is rapidly metabolized in plasma and blood by circulating ectonucleotidases that dephosphorylate the parent compound, ultimately yielding MRS4322, a non-phosphorylated riboside. This process is inhibited by EDTA, an agent known to chelate divalent cations required for ectonucleotidase activity. (Example 5) Neuroprotective efficacy of MRS4322 after TBI in mice Objective

[0243] This study was designed to determine the neuroprotective efficacy of MRS4322 in mice with traumatic brain injury (TBI), and to compare it with mice without traumatic brain injury treated with Cl-IB-MECA, a full agonist of the MRS2365 and adenosine A 3 receptors. Methods

[0244] Chemicals: MRS4322 was obtained through the kindness of Dr. Ken Jacobson at the National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD). Cl-IB-MECA is commercially available from Tocris Biosciences (Bristol, UK) and several other vendors. All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).

[0245] Animals and traumatic brain injury (TBI): TBI was induced as described by Talley-Watts et al., 2012 (J. Neurotrauma 30, 55-66) using a controlled closed head injury model . Using the method described herein, a moderate TBI was created using an air impact device, leaving the skull and dura intact. To achieve this, C57BL / 6 mice were anesthetized with isoflurane (3% induction, 1% maintenance) in 100% oxygen. A heating surgical table was used under temperature control to maintain a body temperature of 37°C. A small midline incision was made in the scalp using aseptic surgical technique. A 5-mm stainless steel disk was placed on the skull and fixed using a strong instant adhesive on the right parietal bone between bregma and lambda overlying the somatosensory cortex. The mouse was then placed on the table directly under the air impact tip. A calibrated impact was delivered at 4.5 m / s with a depth of 2 mm, thereby causing moderate injury to the mouse. The scalp incision was sutured using 4-0 nylon braided thread, and an antibiotic ointment was applied to the incision. The mouse was placed in a Thermo-Intensive Care Unit (Braintree Scientific model FV-1; 37°C; 27% O 2) were placed in it and monitored until they were fully awake and able to move around freely. Thirty minutes after injury or sham (uninjured), the mice were treated with either vehicle (saline) or drug (MRS4322, Cl-IB-MECA or MRS2365). The doses of MRS4322, Cl-IB-MECA and MRS2365 were 0.16 mg / kg, 0.24 mg / kg and 0.2 mg / kg, respectively, and were equivalent to equimolar doses of approximately 0.5 μmol / kg each.

[0246] Western blot analysis for GFAP: At the selected survival time, the mice were anesthetized with isoflurane and sacrificed. The brains were removed and placed on ice to cut them into the impacted and non-impacted cerebral hemispheres. The isolated tissues were rapidly homogenized in cold homogenization buffer (0.32 M sucrose, 1 mM EDTA, 1 M Tris-HCL, pH = 7.8) using a Wheaton glass Dounce homogenizer on ice (20 reciprocations). The homogenate was transferred to 2 mL tubes and centrifuged at 1000 g for 10 minutes at 4 °C, and the supernatant was collected and analyzed. The protein concentration was determined by BCA assay using a 1:50 dilution. 100 μg of protein was taken out as a fixed amount for each sample, Laemmli buffer containing β-mercaptoethanol was added, and the sample was placed in a heat block at 95 °C for 3 minutes. The samples were loaded onto a 12% gel and electrophoresed at 80 V for 20 minutes and then at 130 V for 40 minutes. The samples were transferred to a nitrocellulose membrane at 100 V for 1 hour. The membrane was blocked with 5% milk in TBS-T for 30 minutes. GFAP (1:1000 - Imgenex IMG-5083-A) was added and left at 4 °C overnight. The membrane was washed 3 times with TBS-T for 10 minutes. The secondary antibody for GFAP (conjugated donkey anti-rabbit HRP (ImmunoJackson Laboratories; 711-035-152; 1:20000)) was applied at room temperature for 1 hour. The membrane was washed with TBS-T for 15 minutes (3 times) and developed using the Western Lightning Plus-ECL kit (PerkinElmer, Inc.) according to the manufacturer's instructions. Results

[0247] MRS4322 reduces GFAP expression in the mouse brain after TBI. Glial fibrillary acidic protein (GFAP) expression was used as a biomarker for reactive gliosis after TBI (Talley-Watts et al., 2012; Sofroniew, 2005). The present inventors performed Western blot analysis for GFAP expression in sham, TBI or re-treated TBI (MRS4322 or MRS2365) mice sacrificed on day 7 after injury. First, Western blot analysis confirmed that TBI induced a significant increase in GFAP expression in both the ipsilateral (the side where the impact was given centrally) and contralateral sides of the brain on day 7 after injury (Figure 12A). GFAP expression was significantly lower in blots from mice treated with MRS4322 or MRS2365 injected within 30 minutes of the initial trauma (Figure 12A). For the loading control, a beta-actin Western blot was used and is shown below each lane. The Western blots shown in Figure 12A were all obtained from representative experiments and were run on the same gel. Data showing the relative change in the GFAP / actin ratio (band intensity measured with Image J software) averaged from three separate experiments are presented as mean + / - SEM in Figure 12B. To integrate the data, the values were normalized to the TBI level on day 7 (100%). The total number of mice for a given experimental treatment is indicated by N in Figure 12B.

[0248] MRS4322 and adenosine A3 receptor agonist reduce GFAP expression in mouse plasma after TBI. GFAP levels in plasma are also used as a biomarker for TBI because the blood-brain barrier (BBB) breaks down after trauma. Consequently, the present inventors collected plasma samples from TBI mice on day 7. The inventors found that GFAP levels were easily detected by Western blot analysis on day 7, similar to brain tissue (Figure 12C). More importantly, the inventors found that MRS4322 or Cl-IB-MECA (adenosine A 3We found that Western blot analysis of plasma obtained from TBI mice treated with either MRS4322 (a low affinity (4900 nM) agonist of the A3 receptor in mice) or Cl-IB-MECA (a high affinity (0.18 nM) agonist in mice, with a ~25,000-fold difference in affinity between these two compounds) showed a significant decrease in GFAP levels compared to actin (Figure 12C). A histogram plot of the mean GFAP / actin ratio for each experimental condition is presented in Figure 12D. The total number of mice for each experimental treatment is indicated by the N value.

[0249] MRS4322 is a low affinity (4900 nM) agonist of the A3 receptor in mice. In contrast, Cl-IB-MECA is a high affinity (0.18 nM) agonist in mice, and the difference in affinity between these two compounds is approximately 25,000-fold. However, in mouse models of photothrombotic stroke and TBI, MRS4322 shows significant efficacy, which is blocked by the A3 antagonist MRS1523, while Cl-IB-MECA is either inactive (in stroke) or has weak activity (in TBI, Figure 12). This is clearly a non-trivial result from the perspective of receptor affinity. The present inventors' current explanation for this finding is based on their unique ADME / PK data generated for MRS4322 and Cl-IB-MECA. Cl-IB-MECA is a lipophilic compound (cLogP ~2.5) that binds highly to plasma proteins (free fraction 0.002) and non-specifically highly to brain tissue (free fraction 0.002). MRS4322 is a very hydrophilic compound (cLogP <0) with very high unbound fractions in plasma (0.74) and brain (0.13). Only unbound drug is available for membrane permeation distribution and interaction with receptors. Thus, despite the lower receptor affinity of MRS4322, the fraction of MRS4322 available for interaction with the A3 receptor in these mouse models is at least 1000-fold greater than that of Cl-IB-MECA. The inventors propose that these significant differences in the physicochemical properties and ADME / PK characteristics of the compounds result in Cl-IB-MECA (and another lipophilic, highly bound / high affinity full A 3It is considered to contribute to the non-trivial efficacy of MRS4322 compared to MRS5698, which is an R agonist.

[0250] Biased agonism. Adenosine A 3 receptor is a pleiotropic receptor that is coupled to G proteins, i.e., agonism of this receptor may potentially activate multiple downstream pathways via multiple G proteins as well as beta-arrestin. A 3 The pathways activated by receptor agonism have been currently identified, but cannot be limited to Gq11-mediated intracellular calcium mobilization, Gi-mediated modulation of cAMP production, and Gi-mediated phosphorylation of ERK1 / 2 and Akt. One aspect of the inventors' discovery is the calcium-mediated mobilization of intracellular calcium that results in the promotion of mitochondrial ATP production in astrocytes. A 3 mediated mobilization.

[0251] A new concept in receptor pharmacology is biased agonism. This concept presents that for pleiotropic receptors, there are actually different classes of agonists, some of which can activate all downstream pathways, while others show bias in the activation of a subset of downstream pathways. In drug discovery and receptor pharmacology, biased agonism introduces the possibility of increasing the specificity of pathway activation while suppressing off-target effects, i.e., side effects. A 3 There is evidence regarding the biased agonism of the receptor. However, prototype high-affinity agonists, such as Cl-IB-MECA and MRS5698, are full agonists that do not show bias in the aforementioned downstream pathway activation. Therefore, without being bound by any particular theory, MRS4322 preferentially activates intracellular calcium mobilization, but other 3The mediating pathway is thought to be a biased agonist that is little / almost not activated. This finding provides an explanation for the observation of the efficacy of MRS4322 in mouse models of stroke and TBI, as compared to the full / non-biased agonists Cl-IB-MECA and MRS5698. (Example 6) Neuroprotective efficacy of MRS4322 after stroke in mice Objective

[0252] This study was designed to determine the neuroprotective efficacy of MRS4322 in mice that had a stroke, and to compare mice treated with MRS2365 with A 3 in the presence and absence of the receptor antagonist MRS1523, in comparison with the full A3R agonists MRS5698 and Cl-IB-MECA. MRS1523 has the following chemical structure. [Chemical formula] Methods

[0253] Chemicals: MRS4322 was obtained through the kindness of Dr. Ken Jacobson of the National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD). Cl-IB-MECA, MRS5698, and MRS2365 are commercially available from Tocris Bioscience (Bristol, UK) and several other vendors. All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).

[0254] Photothrombosis-induced stroke: Photothrombosis was induced as described by Zheng et al., 2010 (PloS One 5(12):e14401). Briefly, Rose Bengal is a fluorescent dye that, when injected into the vasculature and excited, generates singlet oxygen that damages the endothelial wall and induces local thrombosis (blood clot). Using this technique, mice were injected with 0.1 mL of sterile Rose Bengal (RB, Sigma, U.S.A.) in artificial cerebrospinal fluid (aCSF) via the tail vein. The RB concentration was 20 mg / mL. The cortical region was placed at the center of the imaging field, and a green laser (543 nm, 5 mW) was irradiated using a 40x water immersion lens with 0.8-NA (Nikon, Tokyo). Blood clot formation was monitored in real time until the target blood vessel or downstream capillaries were completely blocked. Subsequently, stable blood clots were identified by the demarcation of non-fluorescent blood vessels ending in highly fluorescent regions. In control experiments, no blood clots were formed by either laser irradiation or Rose Bengal itself. As a treatment, MRS4322 (0.16 mg / kg; 0.5 μmol / kg) or MRS2365 (0.24 mg / kg; 0.5 μmol / kg) was introduced by intraperitoneal injection (i.p.). A 3 In the experiment using the receptor antagonist MRS1523, to ensure receptor antagonism throughout the study, mice were administered by intraperitoneal injection (2 mg / kg) at time points 0 h and 2 h.

[0255] Animals and photothrombosis-induced stroke: Stroke was induced as described by Zheng et al., 2010 (PloS One 5(12):e14401). In this study, female C57Bl / 6 mice (4 - 6 months old) were used. By the method of this manuscript, mice were anesthetized with 3% isoflurane with 100% oxygen via a nose cone and then maintained with 1% isoflurane. Anesthetic depth was monitored and adjusted according to vital signs, pinch withdrawal from the tail, and blinking. Body temperature was maintained with feedback-controlled heating pa Maintained at 37°C by a Gaymar T / Pump. Vital signs including oxygen saturation, respiratory rate, and heart rate were continuously monitored by using a MouseOx system (STARR Life Sciences). The hair on the head of each mouse was shaved, and the scalp was incised slightly to expose the skull. A custom stainless-steel plate was adhered to the skull using VetBond tissue adhesive (3M, St. Paul, MN). A cranial imaging window with a reduced skull thickness was created over the right primary somatosensory cortex (about 1.5 mm posterior to bregma and 2 mm lateral to the midline) according to the experiment. Briefly, a wide area of the skull was first thinned with an electric drill and then further thinned with a surgical blade. The final thickness of the thinned skull was approximately 50 μm. After the cranial imaging window was created, the mouse was transferred to the microscope stage and used for photothrombosis or imaging experiments. In repeated imaging experiments, the plate was carefully detached from the skull and the scalp was sutured (6-0 silk (sild) suture from Ethicon). After each experiment, the mouse was returned to the cage until the next time point or until sacrifice. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Texas Health Science Center at San Antonio. Thirty minutes after stroke or sham (uninjured), the mice were treated with either vehicle (saline) or drug (MRS4322, MRS2365, MRS5698, or Cl-IBMECA).

[0256] Evaluation after photothrombotic infarction. The size of the cerebral infarction was evaluated using 2,3,5-triphenyltetrazolium chloride (TTC) staining as described by Zheng et al. in 2010 (PloS One 5(12):e14401). Briefly, RB-induced lesions in brain slices were stained with TTC. TTC is a colorless dye that stains healthy brain tissue red when reduced by the mitochondrial enzyme succinate dehydrogenase (Bederson JB et al., 1986). Next, the absence of staining in necrotic tissue was used to demarcate the cerebral infarction area. Mice were sacrificed by cervical dislocation, their brains were removed, and then placed in ice-cold HBSS for 3 minutes. Subsequently, the brains were transferred to a brain mold (KOPF), sliced into 1-mm sections, and immersed in 2% TTC at 37°C for 5 minutes. The sections were fixed overnight at 4°C in a 10% buffered formaldehyde solution. The slices were imaged at 1200 dpi using a flatbed scanner (HP scanjet 8300) for analysis of lesion size. Results

[0257] Treatment with MRS4322 reduces post-stroke cerebral infarction. A multifocal photothrombotic stroke was induced in mice as described above by injecting RB via the tail vein. Within 30 minutes after blood clot formation, mice were injected intraperitoneally with either vehicle (saline control), MRS4322 (0.16 mg / kg; 0.5 μmol / kg), or MRS2365 (0.24 mg / kg; 0.5 μmol / kg). Twenty-four hours after the initial stroke occurred, the size of the cerebral infarction was evaluated using TTC staining as described above. Representative brain slices stained with TTC are presented in Figure 13A. The inventors found that the size of the cerebral infarction was significantly reduced by both MRS4322 and MRS2365. A histogram plot of the mean size of cerebral infarctions in mice treated with vehicle, MRS4322, MRS2365, MRS5698, or Cl-IBMECA is presented in Figure 13C. These data were pooled from two independent experiments. N refers to the total number of mice tested.

[0258] A 3The receptor antagonist MRS1523 inhibits the neuroprotection by treatment with MRS4322 and MRS2365 after stroke. Focal photothrombotic stroke was induced in mice as described above. However, in this experiment, to ensure receptor antagonism, mice were treated intraperitoneally with A at time points 0 h and 2 h 3 with the receptor antagonist MRS1523 (2 mg / kg). Next, mice were injected with either vehicle, MRS4322, MRS2365, MRS5698 or Cl-IBMECA at the aforementioned concentrations within 30 minutes after blood clot formation. After 24 h, the size of the cerebral infarct was evaluated using TTC staining. Representative brain slices stained with TTC are presented in Fig. 13B. The inventors found that the size of the cerebral infarct in mice pretreated with MRS1523 was not reduced by treatment with any of MRS4322 or MRS2365 or MRS5698. Histogram plots of the mean size of cerebral infarcts in these experiments are presented in Fig. 13D. The data were pooled from two independent experiments. N refers to the total number of mice tested. (Example 7) Neuroprotective efficacy of MRS1873 after stroke in mice Objective

[0259] This study was designed to determine the neuroprotective efficacy of MRS1873, the corresponding 2-chloro analog of MRS4322. Experiments were performed in mice that had suffered a stroke, comparing mice treated with MRS4322 and mice treated with vehicle. Methods

[0260] Chemicals: MRS1873 and MRS4322 were obtained through the kindness of Dr. Ken Jacobson at the National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD). MRS1873 has the following structure.

Chemical formula

[0261] Photothrombosis-induced stroke: Photothrombosis was induced as described by Zheng et al. in 2010 (PloS One 5(12): e14401). Briefly, Rose Bengal is a fluorescent dye that, when injected into the vasculature and excited, produces singlet oxygen that damages the endothelial wall and induces local thrombosis (blood clot). Using this technique, mice were injected with 0.1 ml of sterile Rose Bengal (RB, Sigma, U.S.A.) in artificial cerebrospinal fluid (aCSF) via the tail vein. The RB concentration was 20 mg / ml. The cortical area was placed at the center of the imaging field and irradiated with a green laser (543 nm, 5 mW) using a 40x water immersion lens with 0.8-NA (Nikon, Tokyo). Blood clot formation was monitored in real time until the target vessel or downstream capillaries were firmly occluded. Subsequently, stable blood clots were identified by the demarcation of non-fluorescent vessels ending in highly fluorescent areas. In control experiments, no blood clots were formed by either laser irradiation or Rose Bengal itself. As a treatment, MRS1873 (100 ul of 100 uM) or MRS4322 (100 ul of 100 uM) was introduced by intraperitoneal injection (i.p.).

[0262] Animals and Photothrombotic Stroke: Stroke was induced as described by Zheng et al., 2010 (PloS One 5(12):e14401). Female C57Bl / 6 mice (4 - 6 months old) were used in this study. By the method of this manuscript, mice were anesthetized with 3% isoflurane via a nose cone with 100% oxygen and then maintained with 1% isoflurane. Anesthetic depth was monitored and adjusted according to vital signs, withdrawal response from pinching, and blinking. Body temperature was maintained at 37 °C with a feedback-controlled heating pad (Gaymar T / Pump). Vital signs including oxygen saturation, respiratory rate, and heart beat were continuously monitored by using the MouseOx system (STARR Life Sciences). The hair on the head of the mice was shaved and the scalp was incised slightly to expose the skull. A custom stainless-steel plate was adhered to the skull using VetBond tissue adhesive (3M, St. Paul, MN). A cranial imaging window with a thinner skull was created over the right primary somatosensory cortex (about 1.5 mm posterior to bregma and 2 mm lateral to the midline) according to the experiment. Briefly, a wide area of the skull was first thinned with an electric drill and then further thinned with a surgical blade. The final thickness of the thinned skull was approximately 50 μm. After the cranial imaging window was created, the mice were transferred to the microscope stage and used for photothrombosis or imaging experiments. In repeated imaging experiments, the plate was carefully detached from the skull and the scalp was sutured (6-0 silk suture from Ethicon). After each experiment, the mice were returned to their cages until the next time point or until sacrifice. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Texas Health Science Center at San Antonio. Thirty minutes after stroke or sham (uninjured), the mice were treated with either vehicle (saline) or drug (MRS4322 or MRS2365).

[0263] Evaluation after photothrombotic infarction. The size of the cerebral infarction was evaluated using 2,3,5-triphenyltetrazolium chloride (TTC) staining as described by Zheng et al., 2010 (PloS One 5(12):e14401). Briefly, RB-induced lesions in brain slices were stained with TTC. TTC is a colorless dye that stains healthy brain tissue red when reduced by the mitochondrial enzyme succinate dehydrogenase (Bederson JB et al., 1986). Next, the absence of staining in necrotic tissue was used to demarcate the cerebral infarction area. Mice were sacrificed by cervical dislocation, their brains were removed, and then placed in ice-cold HBSS for 3 minutes. Subsequently, the brains were transferred to a brain mold (KOPF), sliced into 1 mm sections, and immersed in 2% TTC at 37°C for 5 minutes. The sections were fixed overnight at 4°C in a 10% buffered formaldehyde solution. The slices were imaged at 1200 dpi using a flatbed scanner (HP scanjet 8300) for analysis of lesion size. Results

[0264] Treatment with MRS1873 reduces cerebral infarction after stroke. A multifocal photothrombotic stroke was induced in mice as described above by injecting RB via the tail vein. Within 30 minutes after blood clot formation, mice were injected intraperitoneally with either vehicle (saline control), MRS1873 (100 μl of 100 μM), or MRS4322 (100 μl of 100 μM). Twenty-four hours after the initial stroke occurred, the size of the cerebral infarction was evaluated using TTC staining as described above. Representative brain slices stained with TTC are presented in Figure 14B. The inventors found that the size of the cerebral infarction was significantly reduced by both MRS1873 and MRS4322. Histogram plots of the mean size of cerebral infarctions in mice treated with vehicle, MRS1873, and MRS4322 are presented in Figure 14B. These data were pooled from three independent experiments. N refers to the total number of mice tested. Pharmacokinetics and theoretical basis for efficacy

[0265] MRS1873 is a 2-Cl analog of MRS4322 and is also an adenosine A3 agonist. The physicochemical properties of MRS1873 are identical to those of MRS4322 with respect to low molecular weight, hydrophilicity (cLogP < 0), and topological polar surface area. Since ADME / PK parameters, such as plasma and brain binding, clearance, and volume of distribution, are determined by these physicochemical properties, the inventors demonstrated similar pharmacokinetics for MRS4322 and MRS1873. Furthermore, the inventors demonstrated similar efficacy for MRS4322 and MRS1873 in a photothrombotic mouse model of stroke. (Example 8) A 3 Experimental protocol for determining the biased agonism of a compound at an adenosine receptor (A 3 R)

[0266] The following assay can be used to determine whether a disclosed compound, such as MRS4322 or MRS1873, exhibits biased agonism (also known as functional selectivity or agonist trafficking) at the A 3 receptor.

[0267] Materials. Fluo-4, Dulbecco's Modified Eagle Medium (DMEM), and penicillin-streptomycin can be purchased from Invitrogen (Carlsbad, CA). Adenosine deaminase (ADA) and hygromycin-B can be purchased from Roche (Basel, Switzerland). Fetal bovine serum (FBS) can be purchased from ThermoTrace (Melbourne, Australia). AlphaScreen SureFire extracellular signal-regulated kinase 1 and 2 (ERK1 / 2), Akt1 / 2 / 3, and cAMP kits can be obtained from PerkinElmer (Boston, MA). All compounds designated by the initials MRS can be synthesized as previously described (Tosh et al., 2012a, b). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO).

[0268] Cell culture. Human A 3 The sequence of R can be cloned into the Gateway entry vector pDONR201 using the method already described (Stewart et al., 2009) and then transferred to the Gateway destination vector pEF5 / FRT / V5-dest. A -FlpIn-CHO cells can be prepared using the method already described (May et al., 2007) and maintained at 37°C in DMEM supplemented with 10% FBS and the selective antibiotic hygromycin-B (500 μg / ml) in a humidified incubator containing 5% CO 3 For cell survival, ERK1 / 2 phosphorylation, Akt1 / 2 / 3 phosphorylation, and calcium mobilization assays, cells can be seeded into 96-well culture plates at a density of 4×104 cells / well. After 6 hours, the cells are washed with serum-free DMEM and maintained in serum-free DMEM at 37°C in 5% CO2 for 12 - 18 hours before assay. For the cAMP assay, cells are seeded into 96-well culture plates at a density of 2×104 cells / well and can be assayed after incubation overnight at 37°C in 5% CO 2 in a humidified incubator containing 5% CO in a humidified incubator containing 5% CO2. They can be maintained at 37°C in DMEM supplemented with 10% FBS and the selective antibiotic hygromycin-B (500 μg / ml). For cell survival, ERK1 / 2 phosphorylation, Akt1 / 2 / 3 phosphorylation, and calcium mobilization assays, cells can be seeded into 96-well culture plates at a density of 4×104 cells / well. After 6 hours, the cells are washed with serum-free DMEM and maintained in serum-free DMEM at 37°C in 5% CO2 for 12 - 18 hours before assay. For the cAMP assay, cells are seeded into 96-well culture plates at a density of 2×104 cells / well and can be assayed after incubation overnight at 37°C in 5% CO 2 2. They can be assayed after incubation overnight at 37°C in 5% CO2.

[0269] Cell survival assay. Remove the medium and A 3 in the absence and presence of the R ligand, in HEPES-buffered saline solution (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 146 mM NaCl, 10 mM D-glucose, 5 mM KCl, 1 mM MgSO 4 4, 1.3 mM CaCl 2 2, and 1.5 mM NaHCO 3, replace it with pH 7.45). Next, after maintaining the plate in a humidified incubator at 37 °C for 24 hours, add 5 mg / ml propidium iodide to the cells. Next, the plate can be read by an EnVision plate reader (PerkinElmer) with the excitation and emission set at 320 nm and 615 nm, respectively. Normalize the data to 100% cell survival and 0% cell survival, determined at t = 0 hours in HEPES buffer and t = 24 hours in Milli-Q water, respectively.

[0270] ERK1 / 2 and Akt1 / 2 / 3 phosphorylation assays. Concentration-response curves for ERK1 / 2 and Akt1 / 2 / 3 phosphorylation for each ligand can be obtained in serum-free DMEM containing 1 U / ml ADA (exposure at 37 °C for 5 minutes). Stimulation by the agonist can be terminated by removing the medium and adding 100 μl of SureFire lysis buffer to each well. The plate is then vortexed for 5 minutes. For the detection of pERK1 / 2, a 80:20:120:1:1 v / v / v / v / v dilution of lysate:activation buffer:reaction buffer:AlphaScreen acceptor beads:AlphaScreen donor beads in a total volume of 11 μl in a 384-well ProxiPlate can be used. After incubating the plate at 37 °C for 1 hour in the dark, fluorescence measurements can be performed using an EnVision plate reader (PerkinElmer) with excitation and emission set at 630 nm and 520 - 620 nm, respectively. For the detection of Akt1 / 2 / 3 phosphorylation, a 40:9.8:39.2:1 v / v / v / v dilution of lysate:activation buffer:reaction buffer:AlphaScreen acceptor beads in a total volume of 9 μl in a 384-well ProxiPlate can be used. After incubating the plate at room temperature for 2 hours in the dark, a 19:1 v / v dilution of dilution buffer:AlphaScreen donor beads in a total volume of 11 μl can be added. After incubating the plate at room temperature for an additional 2 hours, fluorescence measurements can be performed using an EnVision plate reader (PerkinElmer) with excitation and emission set at 630 nm and 520 - 620 nm, respectively. The concentration-response curve of the agonist is normalized to phosphorylation mediated by 10% FBS (stimulation for 5 minutes).

[0271] Calcium mobilization assay. The medium can be removed from the 96-well plate and replaced with HEPES-buffered saline aqueous solution containing 1 U / ml of ADA, 2.5 mM of probenecid, 0.5% bovine serum albumin (BSA), and 1 M of Fluo4. The plate can be incubated at 37 °C for 1 hour in a humidified incubator in the dark. Using a FlexStation plate reader (Molecular Devices, Sunnyvale, CA), HEPES-buffered saline aqueous solution can be added in the absence and presence of an agonist, and fluorescence can be measured every 1.52 seconds for 75 seconds (excitation, 485 nm; emission, 520 nm). The difference between the peak fluorescence and the baseline fluorescence can be measured as a marker of intracellular Ca 2+ mobilization. A 3 concentration-response curve of the A

[0272] agonist can be normalized to the response mediated by 100 μM of ATP to account for differences in cell number and loading efficiency. 4 cAMP accumulation inhibition assay. The medium can be replaced with stimulation buffer (140 mM of NaCl, 5 mM of KCl, 0.8 M of MgSO 2 4, 0.2 mM of Na 4 2HPO 2 4, 0.44 mM of KH 4 2PO 2 4, 1.3 mM of CaCl 3 2, 5.6 mM of D-glucose, 5 mM of HEPES, 0.1% BSA, 1 U / ml of ADA, and 10 μM of rolipram, pH 7.45) and incubated at 37 °C for 1 hour. Inhibition of cAMP accumulation is determined in A 3It can be evaluated by pre-incubating with an R agonist for 10 minutes and then adding 3 μM forskolin and incubating for an additional 30 minutes. The reaction can be terminated by quickly removing the buffer and adding 50 μl of ice-cold 100% ethanol. After evaporating the ethanol, 50 μl of detection buffer (0.1% BSA, 0.3% Tween-20, 5 mM HEPES, pH 7.45) is added. After mixing the plate for 10 minutes, 10 μl of the lysate is transferred to a 384-well Optiplate. For detection, the addition of 5 μl of a 1:49 v / v dilution of AlphaScreen acceptor beads: stimulation buffer can be used. Subsequently, 15 μl of a 1:146:3 v / v / v dilution of AlphaScreen donor beads: detection buffer: 3.3 U / μl biotinylated cAMP is added to form a total volume of 30 μl. The donor beads / biotinylated cAMP mixture can be equilibrated for 30 minutes before addition. After incubating the plate overnight at room temperature in the dark, fluorescence measurements can be performed using an EnVision plate reader (PerkinElmer) with the excitation and emission wavelengths set at 630 nm and 520 - 620 nm, respectively. The concentration-response curve of the agonist can be normalized to the response mediated by 3 μM forskolin (0%) or buffer (100%) alone.

[0273] Molecular modeling. Docking simulations can be performed for all compounds investigated in this study using the human A 3 R homology model. In particular, the following three models that have already been reported can be used. The agonist-bound hA 2A AR crystal structure-based model (PDB ID: 3QAK), the hybrid A 2A AR-β2 adrenergic receptor template-based model, and the hybrid A 2A AR-opsin template-based model (β2 adrenergic receptor X-ray structure PDB ID: 3SN6; opsin crystal X-ray structure PDB ID: 3DQB) (Tosh et al., 2012a). The hybrid template-based model is A 2AShows the movement of TM2 outwards compared to the AR-based model. A 3 The structure of the R ligand can be constructed and prepared for docking using the Builder and LigPrep tools implemented in the Schrodinger suite (Schrodinger Release 2013-3, Schrodinger, LLC, New York, NY, 2013). A 3 Molecular docking of the ligand in the R model can be carried out by using the Glide package part of the Schrodinger suite. In particular, the Glide Grid can be placed at the center of gravity of several very important residues in the binding pocket of the adenosine receptor, namely, the centroid of Phe(EL2), Asn(6.55), Trp(6.48), and His(7.43). The Glide Grid can be constructed using an inner box of 14 Å × 14 Å × 14 Å (a box at the midpoint of the ligand diameter) and an outer box expanded 25 Å in each direction from the inner box (a box in which all ligand atoms must be contained). Docking of the ligand can be carried out at the rigid binding site using the XP (extra precision) procedure. Visual inspection and analysis of the protein-ligand interaction can be performed on the top-score docking conformation for each ligand to select the proposed binding conformation that is consistent with the experimental data.

[0274] Data analysis. Statistical analysis and curve fitting can be performed using Prism 6 (GraphPad Software, San Diego, CA). To quantify signaling bias, agonist concentration-response curves can be analyzed by non-linear regression using the induction of the Black-Leff operational model of agonism as previously described (Kenakin et al., 2012; Wootten et al., 2013; van der Westhuizen et al., 2014). The bias agonism can be quantified using the conversion coefficient, τ / KA [expressed as Log(τ / KA)]. To account for cell-dependent effects on agonist responses, the conversion rate can be normalized to the values obtained for the reference agonist, IB-MECA, to generate ALog(τ / KA). To determine the bias for each agonist in different signaling pathways, ALog(τ / KA) can be normalized to the reference pathway, pERK1 / 2, to generate AALog(τ / KA). The bias can be defined as 10 AALog(τ / KA) and can be obtained such that in the absence of bias, values that do not statistically differ from 1 or, when expressed as a logarithm, do not statistically differ from 0 are obtained. All results can be expressed as mean ± S.E.M. Statistical analysis can include an F-test or one-way analysis of variance, with Tukey's or Dunnett's post hoc tests, depending on the statistical significance determined as P < 0.05. (Example 9) Synthetic route of MRS4322

[0275] MRS4322 and similar compounds, such as MRS1873, can be prepared according to methods known in the art. For example, MRS4322 can be prepared from D-ribose by the following routes described in Choi, W. J. et al., J. Org. Chem. 2004, 69, 2634-2636; Tosh, D. K. et al., Purinergic Signalling 2015, 11, 371-387; and Chem. Eur. J., 2009, 15, 6244-6257. The following Schemes 1 and 2 show the synthetic routes.

Chem.

[0276] Zhan catalyst-1B has the following structure.

Chem.

[0277] Scheme 2 shows the rest of the synthesis.

Chem.

[0278] The inventors have demonstrated a very rapid quantitative dephosphorylation of P2Y1 agonists to adenosine A 3 agonists such as MRS4322. These adenosine A 3 agonists have surprisingly been found to be effective in several mouse models of stroke and traumatic brain injury as described herein. Without being bound by any particular theory, A3 agonists such as MRS4322 and MRS1873 described herein are thought to be effective as cardioprotective agents.

[0279] Our data showing rapid quantitative dephosphorylation of MRS2365 and related phosphorylated nucleosides to compounds such as MRS4322 and MRS1873 support our claim that the cardioprotective efficacy of these alleged P2X4 agonists is actually due to the adenosine A3 agonism of their dephosphorylated metabolites. Indeed, we have demonstrated that MRS1873 is an effective adenosine A3 agonist in stroke and traumatic brain injury models. (Example 11) Pharmacokinetics and Binding of MRS4322 after Intravenous Administration to Neonatal Piglets Purpose

[0280] This study was designed to determine the plasma, brain, and CSF concentrations of MRS4322 after intravenous administration to neonatal piglets. Methods

[0281] Chemical. MRS4322 was obtained through the kindness of Dr. Ken Jacobson at the National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD).

[0282] Animals. In this study, four-week-old female neonatal piglets weighing approximately 7.5 Kg, supplied by the Department of Bioengineering at the University of Pennsylvania (Philadelphia, PA), were used. During the study, the animals were equipped with a brain microdialysis probe to obtain extracellular fluid samples of the drug for determination of drug concentration. All studies were conducted under an approved University of Pennsylvania IACUC protocol.

[0283] Drug administration: MRS4322 was solubilized in DMSO and then diluted with saline to prepare the administration solution. A 10 mL volume of the administration solution was administered to each neonatal piglet (n = 3) by intravenous bolus injection.

[0284] Tissue sample collection: Blood samples were obtained at 0.25, 0.5, 1, 2, 4, and 6 hours after administration. Brain extracellular fluid samples were obtained from implanted microdialysis probes at 1, 4, and 6 hours after administration. Whole blood (1 mL) was obtained at each time point, placed in a Vacutainer tube containing heparin, immediately centrifuged to prepare plasma, and the plasma was stored at -80°C. Brain extracellular and cerebrospinal fluid samples were stored at -80°C. At the time of euthanasia (6 hours after administration), cerebrospinal fluid samples were obtained and frozen, while brain samples from the cortex and hippocampus were obtained by decapitation, rinsed with ice-cold phosphate-buffered saline, and weighed. Next, the brain samples were immediately snap-frozen in liquid nitrogen and stored at -80°C. Bioanalysis

[0285] The concentrations of MRS4322 in plasma, brain, brain extracellular fluid, and cerebrospinal fluid were determined by LC-MS / MS using tolbutamide as the internal standard. The following table summarizes the LC and MS / MS conditions used. [Table 12]

[0286] Standard curves were generated for each tissue matrix, and the LLOQ / ULOQ concentrations were determined. All calibration ranges of the standard curves for the matrix of MRS4322 were 0.1 - 1000 ng / mL.

[0287] For the bioanalysis of the brain concentration of MRS4322, brain samples were homogenized at a 4-fold dilution in ice-cold phosphate-buffered saline. A fixed volume of the resulting diluted brain homogenate was treated with acetonitrile and analyzed by LC-MS / MS. Results

[0288] After intravenous administration to neonatal pigs, the concentration of MRS4322 was detectable in plasma, brain, brain extracellular fluid, and cerebrospinal fluid samples (Figure 1B and Figure 16, Table 13). [Table 13]

Table 14

[0289] Initial estimates of Tmax, Cmax, plasma clearance, volume of distribution, half-life, and AUC could be obtained based on the plasma concentration (Table 14).

[0290] The concentrations of MRS4322 in the brain, extracellular fluid of the brain, and cerebrospinal fluid were detectable, but the data were insufficient to estimate pharmacokinetic parameters other than the half-life, or Cmax and Tmax. However, based on the available plasma and brain data at 6 hours post-administration for which samples were obtained for all matrices, the brain / plasma ratio of the total drug was estimated to be approximately 0.3 based on the mean concentrations in plasma and brain.

[0291] These results confirm that the circulating plasma concentration of MRS4322 is detectable after intraperitoneal administration to neonatal pigs, and that MRS4322 is sufficiently distributed to the brain under these dosing conditions. (Example 12) Plasma and Brain Binding of MRS4322 in Neonatal Pigs Objective

[0292] This study was designed to determine the plasma and brain free fractions of MRS4322 in neonatal pigs. Methods

[0293] Chemical substances. MRS4322 was obtained through the kindness of Dr. Ken Jacobson at the National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD). Analytical grade sulfamethoxazole and warfarin were obtained from commercial supplies from Seventh Wave Laboratories (Maryland Heights, MO). All other chemical substances were obtained from Sigma-Aldrich (St. Louis, MO).

[0294] Preparation of animals and tissues. Plasma and brain samples from female piglets were obtained from the University of Pennsylvania and stored at -80 °C until use.

[0295] Blank samples of plasma ultrafiltrate were prepared by thawing the frozen plasma and then pre-warming the plasma at 37 °C for 60 minutes in a humidified 5% CO2 chamber. An aliquot of 800 μl was transferred to a Centrifree Centrifugal filter (Ultracel regenerated cellulose (NMWL 30,000 amu) Lot R5JA31736) and centrifuged at 2900 RPM at 37 °C for 10 minutes. The filtrate of plasma water was collected and used for the preparation of standards, blanks, and QC standards.

[0296] The brain was weighed and homogenized with 1:9 phosphate buffered saline, pH 7.4 using an Omni tissue homogenizer. Brains from 4 mice were homogenized, pooled, and mixed to form one sample.

[0297] Plasma binding determination. MRS4322, sulfamethoxazole, and warfarin were solubilized in DMSO and then diluted with 1:1 acetonitrile:water to prepare 100 μM stock solutions for dialysis. Sulfamethoxazole and warfarin were utilized as research standards with known plasma binding values. Plasma samples were pre-warmed in a humidified 5% CO 2 incubator at 37 °C for 60 minutes. To 3 ml aliquots of pre-warmed plasma, 100 μM stock solutions of each compound were used to spike MRS4322, sulfamethoxazole, or warfarin respectively to a final test concentration of 1 μM. The spiked plasma samples were incubated in a humidified 5% CO 2 chamber at 37 °C on a rotating mixer for at least 60 minutes. After 60 minutes, three 800 μl aliquots of each sample were added to Centrifree centrifugal filters. The filters were centrifuged at 2900 rpm at 37 °C for 10 minutes. Three 100 μl aliquots of the residual plasma were collected together with the ultrafiltrate for bioanalysis.

[0298] Brain Binding Determination: MRS4322, sulfamethoxazole, and warfarin were solubilized in DMSO and diluted with 1:1 acetonitrile:water to prepare a 100 μM dialysis stock solution. Pooled and homogenized brains were pre-warmed in a humidified 5% CO 2 incubator at 37 °C for 60 minutes. To 3 ml aliquots of the brain homogenate, 100 μM stock solutions of each compound were used to spike in MRS4322, sulfamethoxazole, or warfarin, respectively, to a final spiking concentration of 1 μM. The pooled and spiked brain homogenates were placed on a Nutator mixer in a 37 °C humidified 5% CO 2 incubator for 60 minutes. After 60 minutes, 800 μl aliquots of each sample were added to Centrifree centrifugal filters. The filters were centrifuged at 2900 rpm at 37 °C for 10 minutes. Aliquots of the remaining brain homogenate and the ultrafiltrate were collected for bioanalysis. Bioanalysis

[0299] The plasma and brain concentrations of MRS4322 in spiked plasma, brain homogenates, and related ultrafiltrates were determined by LC-MS / MS using tolbutamide as the internal standard. The related concentrations of sulfamethoxazole and warfarin were also determined by LC-MS / MS using standard conditions (data not shown). The following tables summarize the LC and MS / MS conditions used (Tables 15 and 16). The bioanalytical method was the same for all matrices. Statistical analysis of the standard curves (e.g., fit, intercept, slope, correlation coefficient) was determined for each matrix but showed no significant differences and thus is not shown for each matrix.

Table 15

[0300] Standard curves were generated for each tissue matrix and the LLOQ / ULOQ concentrations were determined. The calibration ranges of the standard curves for the plasma concentration of MRS4322 were 5 - 1000 nM. The calibration ranges of the standard curves for the ultrafiltrate of MRS4322 in plasma were 5 - 1000 nM. The calibration ranges of the standard curves for the brain homogenate and the ultrafiltrate of the brain homogenate of MRS4322 were 5 - 1000 nM and 5 - 1000 nM, respectively. Results

[0301] Plasma binding and free fractions for MRS4322 were determined using plasma ultrafiltration. The plasma binding for MRS4322 was 21.6% and the associated free fraction was 0.784 (Table 16). The binding of the study standards sulfamethoxazole and warfarin was consistent with the literature values. [Table 16]

[0302] Brain binding and free fractions for MRS4322 were determined using brain homogenate ultrafiltration. The brain binding for MRS4322 was 74.7% and the associated free fraction was 0.253 (Table 17). The binding of the study standards sulfamethoxazole and warfarin was consistent with the literature values. [Table 17]

[0303] These data indicate that for a given total plasma or brain concentration, the substantial unbound drug concentration of MRS4322 is available in the brain for interaction with the adenosine A3 receptor. These findings are also consistent with those observed in mice. (Example 13) Pharmacological Characterization of MRS4322

[0304] Compound MRS4322 was investigated in competitive binding studies at human and mouse A 3 adenosine receptors recombinantly expressed in Chinese hamster ovary (CHO) cells using cell membrane preparations. 3 [H]NECA was used as the A 3 agonist radioligand. Since CHO cells do not naturally express adenosine receptors, the non-selective agonist NECA could be used. The concentration-dependent displacement of the radioligand by MR4322 was determined.

[0305] Furthermore, cAMP experiments were performed in CHO cells recombinantly expressing either human A 3 or mouse A 3 adenosine receptors. The non-selective agonist NECA was used as a control. Results

[0306] In radioligand binding studies, MRS4322 showed a K 3 value of 1490 ± 410 nM for the human A 3 receptor against [H]NECA and a K i value of 4940 ± 974 nM for the mouse A 3 receptor. i In functional cAMP accumulation experiments in CHO cells expressing the A

[0307] adenosine receptor, MRS4322 showed agonist activity with an EC 3 value of 3630 ± 370 nM for the human A 3 receptor and an EC 50 value of 759 ± 170 nM for the mouse A 3 receptor. The EC 50 value of an agonist at a GPCR is determined by the receptor expression level. The human A 50 receptor cell line appeared to have a lower expression level than the cell line containing the mouse A 3 receptor as higher EC 50 values were observed, and this was also true for the control agonist NECA. 3 This was also true for the control agonist NECA.

Table 18

Table 19

[0308] Figure 17 shows the competitive binding experiment of MRS4322 against [3H]NECA (10 nM) for the human A receptor expressed in CHO cells. The K value calculated for MRS4322 was 1490 ± 410 nM. 3 receptor, A 3 agonist radioligand 3 Figure 17 shows the competitive binding experiment of MRS4322 against [3H]NECA (10 nM) for the human A receptor expressed in CHO cells. The K value calculated for MRS4322 was 1490 ± 410 nM. i value was 1490 ± 410 nM.

[0309] Figure 18 shows the competitive binding experiment of MRS4322 against [3H]NECA (10 nM) for the mouse A receptor expressed in CHO cells. 3 receptor, A 3 agonist radioligand 3 Figure 18 shows the competitive binding experiment of MRS4322 against [3H]NECA (10 nM) for the mouse A receptor expressed in CHO cells. The K value calculated for MRS4322 was 4940 ± 974 nM. Figure 19 shows the cAMP accumulation experiment of MRS4322 and NECA for the human A receptor expressed in CHO cells. i value was 4940 ± 974 nM. Figure 19 shows the cAMP accumulation experiment of MRS4322 and NECA for the human A receptor expressed in CHO cells. 3 receptor, MRS4322 and NECA 50 value was 3630 ± 370 nM, and for NECA, the EC 50 value was determined to be 41.8 ± 6.3 nM. Figure 20 shows the cAMP accumulation experiment of MRS4322 and NECA for the mouse A receptor expressed in CHO cells. 3 receptor, MRS4322 and NECA 50 value was 759 ± 170 nM, and for NECA, the EC 50 value was determined to be 6.85 ± 0.88 nM.

[0310] These results are lower than the known binding data for MRS1873 but are related to that data. The human A, published by Gao, Z-G et al. in J. Med. Chem. 2002, Vol. 45: pp. 4471-4484, "Structural Determinants of A3 Adenosine Receptor Activation: Nucleoside Ligands at the Agonist / Antagonist Boundary" 3 , Ki data: MRS1873 Ki = 353 nM; EC50 = 803 nM.

[0311] In earlier documents, the Ki of human A 3 is reported to be 85 nM. Bioorganic and Medicinal Chemistry Letters 2001, Vol. 11: pp. 1333-1337, "Ring-Constrained (N)-Methanocarba Nucleosides as Adenosine Receptor Agonists: Independent 5'-Uronamide and 2'-Deoxy Modifications", Lee, K. et al.

[0312] Although several embodiments of the invention are described, it is understood that the above specific examples can be varied using routine experimental methods to provide other embodiments that utilize the compounds and methods of the invention. Accordingly, it should be understood that the scope of the invention should be defined not by the specific embodiments provided, but only by the following claims.

Claims

[Claim 1] The invention described in this specification.

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