JP2018138592A - Composition for reducing nervous system injury and method for producing the same and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composition containing amiloride and/or an amiloride analog usable to reduce nerve injury or nervous system injury in a subject, and a preparation thereof.SOLUTION: The present invention provides a method for treating nerve injury or nervous system injury by administering to a subject a therapeutic effective amount of a pharmaceutical composition containing amiloride, an amiloride analog or their pharmaceutically acceptable salt.SELECTED DRAWING: Figure 20

Description

The present application relates to the field of neurology. In particular, this application relates to compositions comprising amiloride and / or amiloride analogs that can be used to reduce nerve damage or nervous system damage in a subject.

Nerve damage can result from many symptoms, such as chemical and physical damage to the nervous system. Many types of nerve injury result in a change in ion flux into the nerve cell, thereby causing nerve cell death. Thus, various ion channels are candidates for mediating this change in ion flow, and as a result, the degree of nerve damage can be reduced.

One aspect of the present application relates to a method for reducing nerve damage in a subject. The method includes administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an active ingredient selected from the group consisting of amiloride and amiloride analogs. In some embodiments, the pharmaceutical composition is administered intravenously, intrathecally or intraventricularly.

In some embodiments, the active ingredient comprises amiloride or a pharmaceutically acceptable salt thereof.

In other embodiments, the active ingredient comprises an amiloride analog or a pharmaceutically acceptable salt thereof. In related embodiments, the amiloride analog is benzamyl, phenamyl, 5- (N-ethyl-N-isobutyl) -amylolide (EIPA), bepridil, KB-R7943, 5- (N-methyl-N- Isobutyl) amiloride, 5- (N, N-hexamethylene) amiloride and 5- (N, N-dimethyl) amiloride hydrochloride. In another related embodiment, the amiloride analog is benzamyl. In another related embodiment, the amiloride analog is a methylated analog of benzamyl. In another related embodiment, the amiloride analog comprises one ring formed on a guanidine group. In another related embodiment, the amiloride analog comprises an acyl guanidino group. In another related embodiment, the amiloride analog comprises a water soluble group formed on the guanidine group, which is a Ν, Ν-dimethylamino group or a sugar group.

In some embodiments, amiloride, amiloride analog or a pharmaceutically acceptable salt thereof is given in a dosage range of 0.1 mg to 10 mg per kg body weight.

In some embodiments, the pharmaceutical composition is administered within 1 hour of onset of ischemic event, within 5 hours of onset of ischemic event, or within 1 to 5 hours of onset of ischemic event.

In some embodiments, the nerve injury is brain injury.

Another aspect of the present application relates to a method for treating brain injury in a subject. The method includes administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising amiloride, an amiloride analog, or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical composition is administered intravenously, intrathecally or intraventricularly.

In some embodiments, the amiloride analog is benzamyl, phenamyl, EIPA bepridil, KB-R7943, 5- (N-methyl-N-isobutyl) amylolide, 5- (N, N-hexamethylene) amiloride. And 5- (N, N-dimethyl) amiloride hydrochloride. In a related embodiment, the amiloride analog is benzamyl. In other embodiments, the amiloride analog is formed on a methylated analog of benzamyl, an amiloride analog containing one ring formed on a guanidine group, an amiloride analog containing an acyl guanidino group, and a guanidine group. Wherein the water-soluble group is selected from the group consisting of amiloride analogs that are Ν, Ν-dimethylamino groups or sugar groups.

Another aspect of the present application relates to a method for reducing nervous system damage caused by changes in ion flux into neurons. The method includes administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising amiloride, an amiloride analog, or a pharmaceutically acceptable salt thereof.

In some embodiments, the pharmaceutical composition is administered intravenously, intrathecally, intraventricularly or intramuscularly.

In other embodiments, the amiloride analog is benzamyl, phenamyl, EIPA bepridil, KB-R7943, 5- (N-methyl-N-isobutyl) amylolide, 5- (N, N-hexamethylene) amiloride and It is selected from the group consisting of 5- (N, N-dimethyl) amiloride hydrochloride. In a related embodiment, the amiloride analog is benzamyl. In other embodiments, the amiloride analog is formed on a methylated analog of benzamyl, an amiloride analog containing one ring formed on a guanidine group, an amiloride analog containing an acyl guanidino group, and a guanidine group. Wherein the water-soluble group is selected from the group consisting of amiloride analogs that are Ν, Ν-dimethylamino groups or sugar groups.

Another aspect of the present application relates to a method for reducing nervous system damage. The method includes administering to a subject in need of such treatment a therapeutically effective amount of a pharmaceutical composition comprising amiloride, an amiloride analog, or a pharmaceutically acceptable salt thereof.

In some embodiments, the pharmaceutical composition is administered intravenously, intrathecally, intraventricularly or intramuscularly.

In other embodiments, the amiloride analog is benzamyl, phenamyl, EIPA bepridil, KB-R7943, 5- (N-methyl-N-isobutyl) amylolide, 5- (N, N-hexamethylene) amiloride and It is selected from the group consisting of 5- (N, N-dimethyl) amiloride hydrochloride. In a related embodiment, the amiloride analog is benzamyl. In other embodiments, the amiloride analog is formed on a methylated analog of benzamyl, an amiloride analog containing one ring formed on a guanidine group, an amiloride analog containing an acyl guanidino group, and a guanidine group. Wherein the water-soluble group is selected from the group consisting of amiloride analogs that are Ν, Ν-dimethylamino groups or sugar groups.

Another aspect of the present application relates to a pharmaceutical composition for reducing nervous system damage. The pharmaceutical composition comprises an effective amount of amiloride, an amiloride analog or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, and is formulated for intravenous, intrathecal or intracerebroventricular injection. Has been.

In some embodiments, the pharmaceutical composition comprises an amiloride analog or a pharmaceutically acceptable salt thereof, wherein the amiloride analog is benzamyl, phenamyl, EIPA bepridil, KB-R7943, 5- Selected from the group consisting of (N-methyl-N-isobutyl) amiloride, 5- (N, N-hexamethylene) amiloride and 5- (N, N-dimethyl) amiloride hydrochloride.

In other embodiments, the pharmaceutical composition comprises an amiloride analog or a pharmaceutically acceptable salt thereof, wherein the amiloride analog is a methylated analog of benzamyl, a single guanidine group formed on the guanidine group. Amiloride analogs containing rings, amiloride analogs containing acyl guanidino groups, and water-soluble groups formed on guanidine groups, the water-soluble groups being Ν, Ν-dimethylamino groups or sugar groups Selected from the group consisting of bodies.

Another aspect of the present application relates to a pharmaceutical composition for reducing nervous system damage. The pharmaceutical composition comprises an effective amount of an amiloride analog or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition comprises an effective amount of amiloride, an amiloride analog or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, intravenous, intrathecal, Formulated for intraventricular or intramuscular injection.

In one embodiment, the amiloride analog is a methylated analog of benzamyl. In another embodiment, the amiloride analog comprises one ring formed on a guanidine group. In another embodiment, the amiloride analog comprises an acyl guanidino group. In yet another embodiment, the amiloride analog comprises a water soluble group formed on the guanidine group, which is a Ν, Ν-dimethylamino group or a sugar group.

FIG. 1 is a flowchart diagram illustrating an exemplary method for reducing nerve damage in a subject in an ischemic state. FIG. 2 is a flowchart diagram illustrating an exemplary method of identifying a drug for treating nerve damage associated with an ischemic condition. FIG. 3 is a series of graphs representing typical data relating to electrophysiology and pharmacology of acid-sensing ion channel (ASIC) proteins in cultured mouse cortical neurons. FIG. 4 is a further series of graphs representing typical data regarding electrophysiology and pharmacology of ASIC proteins in cultured mouse cortical neurons. FIG. 5 is a combination of graphs and traces representing exemplary data showing that an ischemic model can promote ASIC protein activity in accordance with aspects of the present teachings. 6 and 7, the ASIC proteins in cortical neurons may be ^{Ca 2+} permeability, and, in combination with graphs and trace represents a typical data showing ^{that Ca 2+} permeability may be ASIC1a dependent is there. Same as above. FIG. 8 is a series of graphs representing exemplary data showing that acid incubation can cause glutamate receptor independent nerve damage that is protected by inhibiting ASIC. FIG. 9 is a series of graphs representing exemplary data showing that ASIC1a may be involved in acid-induced damage in vitro. FIG. 10 is a series of graphs showing, together with data, neuroprotection in cerebral ischemia in vivo by inhibition of ASIC1a and by knockout of the ASIC1 gene. FIG. 11 is a graph plotting exemplary data showing the percentage of ischemic damage caused by stroke in an animal model system as a function of treatment time and type. FIG. 12 is a diagram of the primary amino acid sequence of a typical cystine knot peptide, PcTx1, along with various exemplary peptides that have been characterized. FIG. 13 is a comparative diagram that aligns the cystine-knot peptide of FIG. 12 with various typical deletion derivatives of this peptide. FIG. 14 is a typical graph plotting the magnitude of calcium current measured in cells as a function of ASIC family members expressed in the cells. FIG. 15 is a graph depicting exemplary data relating to the effectiveness of nasal administration of PcTx toxin in reducing ischemic damage in an animal model system. FIG. 16 shows representative ASIC1a current traces in CHO cells treated with benzamyl (panel A) or 5- (N-ethyl-N-isopropyl) amiloride (EIPA) (panel B), and amiloride and amiloride analogs. (Panel C) is a composite diagram showing the dose-dependent inhibition of ASIC1a current expressed in CHO cells. FIG. 17 is a representative ASIC2 current trace in CHO cells treated with benzamyl (panel A) or 5-amylolide (panel B) and is expressed in CHO cells by amiloride and amiloride analogs (panel C). FIG. 2 is a composite diagram showing dose-dependent inhibition of ASIC2 current. FIG. 18 is a graph showing the reduction of infarct volume in mice due to intracerebroventricular injection of amiloride or amiloride analog. FIG. 19 is a composite diagram showing the reduction of infarct volume in mouse cortical tissue by intravenous infusion of saline or amiloride 60 minutes after MCAO. FIG. 20 is a composite diagram showing the reduction of infarct volume in cortical tissue of mice by intravenous infusion of saline or amiloride 3 or 5 hours after MCAO. FIG. 21 shows structure-activity relationships (SAR) for hydrophobic amiloride analogs in various channels.

DETAILED DESCRIPTION The present application provides methods and compositions for reducing nerve damage. Nerve damage can result from degenerative nervous system disease, stroke, ischemia, trauma, chemical and physical damage to the nervous system. As used herein, the term “central nervous system” includes both the central nervous system and the peripheral nervous system. The term “nervous system” or “CNS” includes all cells and tissues of the vertebrate brain and spine. The term "peripheral nervous system" includes parts of the nervous system outside the brain and spine, such as the motor nerves that mediate voluntary movement, the sympathetic nervous system and the parasympathetic nervous system, and the autonomous nervous system that controls involuntary functions, and It refers to all cells and tissues such as the enteric nervous system that controls the digestive tract system. That is, the term “nervous system” includes, but is not limited to, nerve cells, glial cells, astrocytes, cells in the cerebrospinal fluid (CSF), cells in the interstitial space, cells in the spinal cord protective coating, epidural Cells (i.e. cells outside the dura mater), cells in non-neural tissues that are adjacent to or in contact with or distributed in neural tissue, epithelial membrane, perineurium, inner lining, cord Includes cells such as bundles.

In some embodiments, the nerve injury is a nervous system injury. In other embodiments, the nerve injury is brain injury. In some embodiments, the nerve injury is a nervous system injury caused by a change in ion flux into the nerve cell. For example, stroke / cerebral ischemia is a major cause of morbidity and mortality. Post-synaptic glutamate receptor overactivation and subsequent Ca2 + toxicity play an important role in ischemic brain injury. The present application shows that the activation of Ca2 + permeable acid-sensing ion channel (ASIC) is involved in acidosis-induced, glutamate receptor-independent ischemic brain injury and targets ASIC family members (ASIC) And provide new directions for neuroprotection. The present application further provides novel inhibitors of ASIC with increased potency against homomeric ASIC channels and increased water solubility. In some embodiments, the present application provides pharmaceutical compositions and methods for reducing nerve damage by inhibiting ASIC1a channels.

One aspect of the present application relates to a method for reducing nerve damage in a subject. The method includes administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an active ingredient selected from the group consisting of amiloride and amiloride analogs. In some embodiments, the pharmaceutical composition is administered intravenously, intrathecally or intraventricularly.

In some embodiments, the active ingredient comprises amiloride or a pharmaceutically acceptable salt thereof. In other embodiments, the active ingredient comprises an amiloride analog or a pharmaceutically acceptable salt thereof. In related embodiments, the amiloride analog is benzamyl, phenamyl, 5- (N-ethyl-N-isobutyl) -amylolide (EIPA), bepridil, KB-R7943, 5- (N-methyl-N- Isobutyl) amiloride, 5- (N, N-hexamethylene) amiloride and 5- (N, N-dimethyl) amiloride hydrochloride. In another related embodiment, the amiloride analog is benzamyl. In another related embodiment, the amiloride analog is a methylated analog of benzamyl. In another related embodiment, the amiloride analog comprises one ring formed on a guanidine group. In another related embodiment, the amiloride analog comprises an acyl guanidino group. In another related embodiment, the amiloride analog comprises a water soluble group formed on a guanidine group, which is a Ν, Ν-dimethylamino group or a sugar group.

In some embodiments, amiloride, amiloride analog or a pharmaceutically acceptable salt thereof is given in a dosage range of 0.1 mg to 10 mg per kg body weight. In other embodiments, the pharmaceutical composition is administered within 1 hour of onset of ischemic event, within 5 hours of onset of ischemic event, or within 1 to 5 hours of onset of ischemic event.

Another aspect of the present application relates to a method for treating brain injury in a subject. The method includes administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising amiloride, an amiloride analog, or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical composition is administered intravenously, intrathecally or intraventricularly.

In some embodiments, the amiloride analog is benzamyl, phenamyl, EIPA bepridil, KB-R7943, 5- (N-methyl-N-isobutyl) amylolide, 5- (N, N-hexamethylene) amiloride. And 5- (N, N-dimethyl) amiloride hydrochloride. In a related embodiment, the amiloride analog is benzamyl. In other embodiments, the amiloride analog comprises a methylated analog of benzamyl, an amiloride analog that includes one ring formed on a guanidine group, an amiloride analog that includes an acyl guanidino group, and a water-soluble group. , Ν-dimethylamino group or sugar group, selected from the group consisting of an amiloride analog containing one water-soluble group formed on a guanidine group.

Another aspect of the present application relates to a method for reducing nervous system damage caused by changes in ion influx into nerve cells. The method includes administering to a subject in need of such treatment a therapeutically effective amount of a pharmaceutical composition comprising amiloride, an amiloride analog, or a pharmaceutically acceptable salt thereof.

Another aspect of the present application provides a composition for treating ischemia or reducing damage caused by ischemia. The method includes administering a therapeutically effective amount of an active ingredient selected from the group consisting of amiloride, an amiloride analog, and salts thereof, intravenously or intrathecally in a subject in need of such treatment. The present method may provide one or more advantages compared to other methods of ischemic treatment. These benefits include, among others, (1) less ischemia-induced damage, (2) fewer treatment side effects (eg, by selecting a more specific therapeutic target), and / or (3) effective Such as a longer time window of the correct treatment.

FIG. 1 shows a flowchart 20 having exemplary steps 22, 24 that can be performed in a manner that reduces nerve damage in a subject under ischemia. These steps may be performed any suitable number of times and in any suitable combination. In the method, an ischemic subject (or subjects) may be selected for treatment (shown in step 22). Next, an ASIC selective inhibitor may be administered to the ischemic subject (shown in step 24). Administration of an inhibitor to an ischemic subject can be performed in a therapeutically effective amount to reduce ischemia-induced damage to the subject, for example, an amount that reduces the amount of brain damage resulting from stroke.

A possible explanation for the effectiveness of the ischemic treatment of FIG. 1 may be provided by the data of the present teachings (see, eg, Example 1). In particular, the damage effect of ischemia may not be equivalent to acidosis, ie tissue / cell acidification due to ischemia may not be sufficient to cause ischemia-induced damage. Rather, ischemia-induced damage can in many cases be caused by calcium influx into cells mediated by ASIC family members, particularly ASIC1a. Thus, selective inhibition of ASIC1a channel activity may reduce this detrimental calcium influx, thereby reducing ischemia-induced damage.

FIG. 2 shows a flowchart 30 having exemplary steps 32, 34 that may be performed in a method for identifying drugs for ischemia treatment. These steps may be performed any suitable number of times and in any suitable combination. In this method, one or more ASIC selective inhibitors can be obtained (shown in step 32). Inhibitors can then be tested in ischemic subjects for effects on ischemia-induced damage (shown in step 34).

TECHNICAL FIELD This application relates to pharmaceutical compositions and methods for reducing nerve damage in a subject. As used herein, the term “nerve damage” refers to vascular pharmacological damage such as physical interaction or trauma, bruise or compression or surgical trauma, hemorrhagic or ischemic damage, or nerve Means acute or chronic damage or adverse symptoms to nervous system tissue or cells resulting from degenerative or any other neurological disease, or any other factor that causes damage or adverse symptoms of nervous system tissue or cells. In some embodiments, the nerve damage is caused by a cognitive disorder, a mental disorder, a neurotransmitter mediated disorder or a neurological disorder. Nerve damage includes damage to the nervous system (ie, nervous system damage) and brain damage.

As used herein, the term “cognitive impairment” is believed to be associated with or associated with a progressive loss of neuronal structure and / or function, eg, death of a neuronal cell. Refers to, and is intended to be, or related to the diseases and symptoms. In this case, the central feature of the disease may be cognitive (eg memory, attention, cognition and / or thinking) impairment. These diseases include cognitive impairments caused by pathogens, such as HIV-related cognitive impairment or Lyme disease-related cognitive impairment. In some embodiments, the cognitive disorder is a degenerative cognitive disorder. Examples of degenerative cognitive impairment include Alzheimer's disease, Huntington's chorea, Parkinson's disease, amyotrophic lateral sclerosis (ALS), autism, mild cognitive impairment (MCI), stroke, traumatic brain injury (TBI) , Age-related memory impairment (AAMI) and epilepsy.

As used herein, the term “mental disorder” refers to and is intended to refer to a mental illness that is believed or caused to cause abnormal thinking and cognition. Psychiatric disorders are characterized by a loss of reality, delusions, hallucinations (external, with the perception of reality in the sense that they are clear, substantive and located in an external objective space May be accompanied by a change of personality and / or disorganized thinking. Other common symptoms include abnormal or strange behavior, as well as difficulties in social interactions and obstacles when performing activities of daily living. Typical mental disorders are schizophrenia, bipolar disorder, mental disorders, anxiety, depression and chronic pain.

As used herein, the term “neurotransmitter mediated disorder” refers to abnormal levels of neurotransmitters such as histamine, glutamate, serotonin, dopamine, norepinephrine, or aminergic G protein-coupled receptors. It refers to, and is intended to mean, a disease or condition in which dysfunction is believed to be involved or associated or involved or associated. Typical neurotransmitter-mediated disorders include aging protection such as spinal cord injury, diabetic neuropathy, allergic diseases, and aging hair loss (alopecia), age-related weight loss and age-related visual impairment (cataract) ( a disease in which the activity is related. Abnormal neurotransmitter levels are associated with various diseases, including but not limited to Alzheimer's disease, Parkinson's disease, autism, Guillain-Barre syndrome, mild cognitive impairment, schizophrenia, anxiety , Multiple sclerosis, stroke, traumatic brain injury, spinal cord injury, diabetic neuropathy, fibromyalgia, bipolar disorder, mental disorders, depression and various allergic diseases.

As used herein, the term “neurological disorder” refers to a disease that is believed to be associated with or associated with neuronal cell death and / or impaired neuronal function or decreased neuronal function. Or a symptom, intended for them. Typical neurological indications include Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, canine cognitive impairment syndrome (CCDS), Lewy body disease, Menkes disease, Wilson disease, Neurodegenerative diseases and symptoms such as Crozfeldt-Jakob disease, Farle disease, acute or chronic diseases related to cerebral circulation such as ischemic or hemorrhagic stroke, or other damage caused by cerebral hyperemia, age-related memory impairment (AAMI), Mild cognitive impairment (MCI), traumatic-related mild (MCI), post-concussion syndrome, traumatic stress syndrome, adjuvant chemotherapy, traumatic brain injury (TBI), ocular disease mediated by neuronal cell death, macular degeneration, old age Macular degeneration, autism (including autism spectrum syndrome), Asperger syndrome, and Rett syndrome, exfoliation injury (avulsi) n injury), spinal cord injury, myasthenia gravis, Guillain-Barre syndrome, multiple sclerosis, diabetic neuropathy, fibromyalgia, spinal cord injury-related neuropathy, schizophrenia, bipolar disorder, mental disorder, anxiety or depression And chronic pain.

In some embodiments, the nerve injury or nervous system damage is caused by a change in ion flux into a nerve cell or nervous system tissue. As used herein, the term “nervous tissue” refers to animal tissue comprising nerve cells, neural networks, glial cells, neuroinflammatory cells, and endothelial cells in contact with “nervous tissue”. . A “neural cell” can be any type of neural cell known to those skilled in the art, such as, but not limited to, a neuron. As used herein, the term “neuron” refers to a cell of ectodermal embryo origin derived from any part of an animal's nervous system. Nerve cells express well-characterized neuron-specific markers such as the neurofiber protein NeuN (neuronal nuclear marker), MAP2, and class III tubulin. Included as neurons are, for example, hippocampus, cortex, dopaminergic midbrain, spinal motor nerve, sensory, enteric nerve, sympathetic nerve, parasympathetic nerve, cholinergic septum, central nervous system and cerebellar nerve It is a cell. “Glial cells” useful in the present invention include, but are not limited to, astrocytes, Schwann cells, and oligodendritic cells. “Neuroinflammatory cells” useful in the present invention include, but are not limited to, cells of myeloid origin such as macrophages and microglia.

In some embodiments, the pharmaceutical compositions and methods of the present application relate to reducing nerve damage caused by ischemia or ischemia related symptoms. Ischemia, as used herein, is a condition in which blood flow to an organ and / or tissue is reduced. Reduced blood flow is due to a number of mechanisms such as partial or complete inhibition (occlusion), stenosis (compression), and / or leakage / rupture of one or more blood vessels that provide blood flow to organs and / or tissues. Can be caused. Ischemia can be caused by thrombosis, embolism, atherosclerosis, hypertension, bleeding, aneurysms, surgery, trauma, drugs, and the like. Thus, decreased blood flow can be chronic, transient, acute or sporadic.

Any organ or tissue experienced decreased blood flow and required treatment for ischemia. Typical organs and / or tissues include, but are not limited to, the brain, arteries, heart, intestines and eyes (eg, optic nerve). Ischemia-induced damage (ie, diseases and / or damage caused by various types of ischemia) include, but are not limited to, ischemic myelopathy, ischemic optic neuropathy, ischemic colitis, coronary artery Heart diseases and / or heart diseases (eg, angina pectoris, heart attacks, etc.) can be mentioned. Thus, ischemia-induced damage can damage and / or kill cells and / or tissues at affected sites in the body. In particular, for example, necrotic (infarct) tissue generation, inflammation, and / or tissue remodeling can be mentioned. Treatment according to aspects of the present application may reduce the incidence, extent, and / or severity of this injury.

An ischemia related symptom can be any result of ischemia. This result may be substantially simultaneous with the onset of ischemia (eg, a direct effect of ischemia) and / or may occur substantially after the onset of ischemia and / or Or it may occur after ischemia is over (eg, non-direct downstream effects of ischemia, tissue reperfusion at the end of ischemia, etc.). Typical ischemia-related symptoms can include any combination of the symptoms (and / or symptoms) listed above. Alternatively or additionally, the symptoms may include local and / or systemic acidosis (pH reduction), hypoxia (hypoxia), free radical generation, and the like.

In some embodiments, the ischemia-related condition is stroke. A stroke, as used herein, is cerebral ischemia caused by a decrease in blood supply to part (or all) of the brain. Symptoms caused by a stroke can be sudden (eg, loss of consciousness, etc.) or can have a gradual onset over hours or days. Furthermore, the stroke may in particular be a major ischemic stroke (complete stroke) or a less severe transient ischemic stroke. Symptoms caused by stroke include, for example, hemiplegia, hemiplegia, unilateral numbness, unilateral weakness, unilateral paralysis, temporary limb weakening, limb aching, confusion, difficulty in speaking, Difficulty in understanding conversation, difficulty in seeing with one or both eyes, blurred vision, vision loss, difficulty walking, dizziness, tendency to fall, loss of body coordination, sudden severe headache, wheezing, and / or Loss of consciousness can be mentioned. Alternatively or additionally, this symptom may be caused by tests and / or devices such as ischemic blood tests (eg, testing for mutated albumin, specific protein isoforms, damaged proteins, etc.), electrocardiogram, electroencephalogram, exercise It can be detected more easily or solely by stress tests, brain CT or MRI scans.

Acid-base balance is important for biological systems. Normal brain function relies on complete oxidation of glucose, with CO ₂ and H ₂ O being the end products because of its energy requirements. During ischemia, increased anaerobic glycolysis results in the accumulation of lactic acid due to lack of oxygen supply. Lactic acid accumulation is accompanied by an increase in H ⁺ release from ATP hydrolysis, causing a decrease in pH in the tissue. The extracellular pH (pH ₀ ) usually drops to 6.5 during ischemia and can be lowered below 6.0 under severe ischemia or hyperglycemic conditions.

Subjects of Nerve Injury The methods and pharmaceutical compositions of the present application have significant potential to develop nerve damage having or having a history of nerve damage and / or after treatment has begun and during which treatment is still effective. Can be used in any subject having In some embodiments, the subject is an ischemic subject. Ischemic subject, as used herein, has ischemia, ischemia-related symptoms, ischemic history, and / or develops nerve damage after treatment has begun and during which treatment is still effective Any human (human subject) or animal (animal subject) with a significant potential to

The subject may be an animal. The term “animal” as used herein refers to any animal that is not a human. Typical animals that may be suitable include any animal with blood flow, including rodents (mouse, rat, etc.), dogs, cats, birds, sheep, goats, non-human primates, etc. . The animal may be treated for its own purposes, eg for veterinary purposes (eg as a pet treatment). Alternatively, animal models of neurological damage such as ischemia can be used to facilitate testing drug candidates for human use, such as examining drug candidate titers, efficacy time windows, side effects, etc. May be provided.

The ischemic subject for treatment may be selected according to any suitable criteria. Typical criteria include any detectable ischemic symptom, ischemic history, events that increase (or induce ischemia) risk of ischemia (eg, surgical procedures, trauma, drug administration, etc.) ) And the like. The history of ischemia can be accompanied by one or more prior ischemic episodes. In some examples, the subject selected for treatment has an ischemia that has occurred at least about 1, 2, or 3 hours prior to initiating treatment, or from about 1 day, 12 hours or 6 hours initiating treatment. Multiple previous ischemic episodes (eg, transient ischemic attacks) may have occurred.

ASIC Inhibitors, Amiloride and Amiloride Analogs As used herein, an inhibitor of an ASIC family member is an ASIC family, ie, in particular, one or more of ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, and ASIC4. A substance that reduces (partially, substantially or completely inhibits) the activity of a member. In some instances, the inhibitor is a cell membrane ability to pass ions (eg, in particular, sodium, calcium, and / or potassium) across the cell membrane (intracellular and / or extracellular). One or more members may decrease channel activity. The substance may be a compound (small molecule, peptide, nucleic acid, lipid, etc. less than about 10 kDa), a complex and / or a mixture of two or more compounds. In addition, the substance may inhibit ASIC family members by any suitable mechanism, particularly competitive, non-competitive, non-competitive, and / or mixed inhibition.

The inhibitor can be an ASIC1a inhibitor that inhibits acid-sensing ion channel 1a (ASIC1a). ASIC1a, as used herein, refers to an ASIC1a protein or channel from any species. For example, a typical human ASIC1a protein / channel is available from Waldmann, R .; , Et al. 1997, Nature 386, pp. 173-177, which is hereby incorporated by reference.

The expression “ASIC1a inhibitor” refers to a product that is potentially or practically pharmaceutically useful as ASIC1a, within the scope of appropriate pharmacological judgment, and includes pharmaceutically active species, Includes reference to substances described, encouraged or approved as ASIC1a inhibitors.

ASIC1a inhibitors may be selective within the ASIC family. Selective inhibition of ASIC1a, as used herein, relates to another ADIC family member when compared (eg, in cultured cells) after each exposure to the same (submaximal) concentration of inhibitor. It is a substantially stronger inhibition in ASIC1a than in the case. The inhibitor may be ASIC1a as compared to at least one other ASIC family member (ASIC1b, ASIC2a, ASIC2b, ASIC3, ASIC4, etc.) and / or as compared to all other ASIC family members. Can be selectively inhibited. The strength of inhibition of a selective inhibitor is determined by the inhibitor concentration at which inhibition occurs (eg, IC ₅₀ (inhibitor concentration that produces 50% of maximum inhibition)) or Ki value (inhibition constant) when compared to different ASIC family members. Or dissociation constant). ASIC1a selective inhibitors are at least about 2-fold, 4-fold, or 10-fold lower concentrations (1/2, 4 or 4) than the concentration for inhibition of at least one other ASIC family member or all other ASIC family members. ASIC1a activity can be inhibited at a concentration of 1 or 10 or less). Thus, an ASIC1a selective inhibitor is at least about 2-fold, 4-fold, or 10-fold lower (2 minutes) than the concentration for inhibition of at least one other ASIC family member and / or all other ASIC family members. 1 of 1,4 min, or 10 min a density of 1 or less) of may have an IC50 and / or _{K i} for ASIC1a inhibition at.

In addition to being selective, an ASIC1a selective inhibitor may also be specific for ASIC1a. ASIC1a-specific inhibition, as used herein, is an inhibition that is substantially exclusive to ASIC1a compared to all other ASIC family members such as ASIC2a and ASIC3a. An ASIC1a-specific inhibitor may inhibit ASIC1a at an inhibitor concentration that is at least about 20 times lower (5% or less) compared to inhibition of all other ASIC family members. Thus, an ASIC1a-specific inhibitor is at least about 20 times lower (5% or less), for example, such that inhibition of other ASIC family members is at least substantially (or completely) undetectable. Compared to all other members of the ASIC family, it may have an IC ₅₀ and / or K _i for ASIC1a inhibition. ASIC1a can be inhibited at inhibitor concentrations. In some embodiments, the ASIC1a selective inhibitor has high potency against a homomeric ASIC1a channel and is a commercially available amiloride-related ASIC1a inhibitor such as amiloride benzamyl, phenamyl, 5- (N -Ethyl-N-isobutyl) amylolide (EIPA), bepridil, KB-R7943, 5- (N-methyl-N-isobutyl) amylolide, 5- (N, N-hexamethylene) amylolide and 5- (N, N- Higher water solubility than dimethyl) amiloride hydrochloride and the like.

Any suitable ASIC inhibitor or combination of inhibitors can be used. For example, a subject may be treated with an ASIC1a selective inhibitor and a non-selective ASIC inhibitor, or with an inhibitor against a non-ASIC channel protein such as an ASIC1a selective inhibitor and a non-ASIC calcium channel. it can. In some examples, the subject can be treated with inhibitors of NMDA receptors, such as ASIC1a selective inhibitors and glutamate antagonists.

The inhibitor may be a peptide or may include a peptide. A peptide can have any suitable number of amino acid subunits, usually at least about 10 to less than about 1000 subunits. In some examples, the peptide can have a cystine knot motif. A cystine knot, as used herein, typically comprises a sequence of 6 or more cysteines. These cysteine-containing peptides form a “knot” that includes (1) a ring formed by two disulfide bonds and their linking skeleton, and (2) a third disulfide bond that passes through the ring. Yes. In some examples, the peptide can be a conotoxin from a arachnid and / or conical snail. For example, the peptide can be a poison PcTx1 (psalmotoxin 1) from tarantula (Psalmopoeus cambridgei (Pc)).

In some examples, the peptide may be structurally related to PcTx1, such that the peptide and PcTx1 differ by at least one deletion, insertion, and / or substitution of one or more amino acids. is there. For example, a peptide can have at least about 25% or at least about 50% sequence identity and / or at least about 25% or at least about 50% sequence similarity to PcTx1 (see below). Further embodiments of peptides that may be suitable as inhibitors are described in Example 3 below.

Methods for aligning amino acid sequences for comparison and creation of amino acid sequence identity and similarity scores are well known in the art. Typical alignment methods that may be suitable include Smith and Waterman “Best Fit”, Needleman and Wunsch homology alignment algorithms “GAP”, Pearson and Lipman similarity methods “Tfasta and Fasta”, and the like. Computer algorithms for these and other methods that may be suitable include CLUSTAL, GAP, BESTFIT, BLASTP, FASTA, and TFASTA.

As used herein, “sequence identity” or “identity” in the context of two peptides relates to the percentage of residues in the corresponding peptide sequence that are the same when aligned for maximum matching. . In some examples, non-identical peptide residue positions may differ from conservative amino acid substitutions, in which case the amino acid residue has other chemical properties that have similar chemical properties (eg, charged or hydrophobic). It is expected that a substitution with an amino acid residue will result in a smaller effect (or no effect) with respect to the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to give sequence “similarity”, thereby modifying the conservative features of the substitution. For example, each conservative substitution may be scored as partial rather than as a complete mismatch, thereby correcting the percent sequence identity to provide a similarity score. Conservative substitution scoring methods for obtaining similarity scores are well known in the art and are performed in any suitable method, such as the program PC / GENE (Intelligenetics, Mountain View, Calif, USA). Meyers and Miller, Computer Applic. Biol Sci. , 4: 11-17 (1988).

Amiloride is a pyrazine derivative containing a guanidine group and is used for the treatment of mild hypertension, with few reported side effects. Amiloride acts by directly inhibiting epithelial sodium channels (ENaC), thereby inhibiting sodium reabsorption at the distal tubular tubules, connecting ducts, and collecting ducts of the kidney. This also promotes the loss of sodium and water from the body, but no potassium is lost. As used herein, the term “amiloride” refers to both amiloride and a salt of amiloride, such as amiloride hydrochloride.

An amiloride analog, as used herein, refers to a compound that has a slightly altered chemical structure but has the same biological activity as amiloride. Examples of amiloride analogs include, but are not limited to, benzamyl, phenamyl, 5- (N-ethyl-N-isobutyl) amiloride (EIPA), bepridil, KB-R7943, 5- (N-methyl-N- Isobutyl) amiloride, 5- (N, N-hexamethylene) amiloride and 5- (N, N-dimethyl) amilolide hydrochloride. As other examples, as shown in FIG. 21, an amiloride analog having a hydrophobic substituent at the C ₅ -NH ₂ position and / or a guanidino group, and a methylated analog of benzamyl, formed on a guanidine group An amiloride analog containing one ring, an amiloride analog containing an acyl guanidino group, and a water-soluble group formed on the guanidine group, wherein the water-soluble group is an N, N-dimethylamino group or a sugar group And amiloride analogs. In some embodiments, the amiloride analog does not inhibit a human Na ⁺ / Ca ²⁺ ion exchanger. In other embodiments, the amiloride analog is a weak inhibitor of the Na ⁺ / Ca ²⁺ ion exchanger and helps keep intracellular Ca ²⁺ levels low. In other embodiments, the amiloride analog is a very weak inhibitor of the Na ⁺ / Ca ²⁺ ion exchanger with an IC ₅₀ value of 1.1 mM or less. In some other embodiments, the amiloride analog does not inhibit ASIC2a and / or ASIC3 channels. In one embodiment, the amiloride analog exhibits a high selectivity for ASIC1a compared to the ASIC3 channel and / or ASIC2 channel.

As used herein, the term “amiloride analog” refers to both an amiloride analog and a salt of an amiloride analog, such as 5- (N, N-dimethyl) amylolide hydrochloride.

In some embodiments, amiloride and / or amiloride analogs are used with other ASIC inhibitors such as PcTx1 and its derivatives.

Administration of Inhibitor Administration (or administering), as used herein, includes any suitable condition and any route of a subject that is exposed to the inhibitor at any suitable number of times. The administration may be self-administration or administration by another person such as a medical staff (for example, a doctor, a nurse, etc.). Administration can be by infusion (especially intravenous, intramuscular, subcutaneous, intracerebral, intracerebroventricular, and / or intrathecal), ingestion (eg, using capsules, troches, liquid compositions, etc.), inhalation (eg, Aerosols (average droplet diameter less than about 10 microns) inhaled from the nose and / or mouth, absorption from the skin (eg, by skin patches), and / or transmucosal (particularly, eg, oral, nasal) And / or pulmonary mucosa). Mucosal administration can be accomplished, for example, by spray (eg, nasal spray), inhalation aerosol, and the like. The spray may be a surface spray (droplets having an average diameter greater than about 50 microns) and / or a spatial spray (droplets having an average diameter of about 10-50 microns). . In some examples, ischemia results in a change in the blood brain barrier of the ischemic subject, and thus inhibitors introduced into the subject's bloodstream (eg, by infusion and / or absorption) reach the brain. Efficiency can increase. Administration can occur as a single dose or as a treatment. Thus, administration may occur before ischemia is detected (eg, prophylactically), after a mild ischemic episode, during chronic ischemia, after a full stroke, and the like. In some embodiments, amiloride or amiloride analog is administered intravenously. In other embodiments, the amiloride or amiloride analog is administered into the brain. In other embodiments, the amiloride or amiloride analog is administered intraventricularly. In other embodiments, the amiloride or amiloride analog is administered intramuscularly. In other embodiments, amiloride or an amiloride analog is administered intrathecally.

A therapeutically effective amount (or simply “effective amount”) of the inhibitor may be administered. A therapeutically effective amount or an effective amount of an inhibitor, as used herein, when administered to a subject, in a significant number of subjects, the extent, incidence, and / or rate of ischemia-induced damage in the subject. Or any amount of inhibitor that reduces the range. Thus, a therapeutically effective amount can be determined, for example, in a clinical trial in which varying amounts of an inhibitor are administered to a test subject (typically compared to a control subject group). A therapeutically effective amount of inhibitor or inhibitors may be given in a single dose or multiple doses of 0.1-50 ml per dose.

In some embodiments, the inhibitor is amiloride, an amiloride analog or a salt thereof, 0.01-30 mg / kg body weight, 0.01-10 mg / kg body weight, 0.01-3 mg / kg body weight, 0.01-1 mg / kg body weight, 0.01-0.3 mg / kg body weight, 0.01-0.1 mg / kg body weight, 0.01-0.03 mg / kg body weight, 0.03-30 mg / kg body weight 0.03 to 10 mg per kg body weight, 0.03 to 3 mg per kg body weight, 0.03 to 1 mg per kg body weight, 0.03 to 0.3 mg per kg body weight, 0.03 to 0.1 mg per kg body weight, 0.1-30 mg / kg body weight, 0.1-10 mg / kg body weight, 0.1-3 mg / kg body weight, body 0.1-1 mg / kg body weight, 0.1-0.3 mg / kg body weight, 0.3-30 mg / kg body weight, 0.3-10 mg / kg body weight, 0.3-3 mg / kg body weight, 0 / kg body weight 3 to 1 mg, 1 to 30 mg / kg body weight, 1 to 10 mg / kg body weight, 1 to 3 mg / kg body weight, 3 to 30 mg / kg body weight, 3 to 10 mg / kg body weight, or 10 to 30 mg / kg body weight It is given in daily doses (single or multiple doses). In one embodiment, the amiloride analog is benzamyl, phenamyl, EIPA, bepridil, KB-R7943, 5- (N-methyl-N-isobutyl) -amylolide, 5- (N, N-hexamethylene) -amylolide and It is selected from the group consisting of 5- (N, N-dimethyl) amiloride hydrochloride. In another embodiment, amiloride analogues, having a hydrophobic substituent at C ₅ -NH _2-position and / or on the guanidino group. In another embodiment, an amiloride analog is formed on a methylated analog of benzamyl, an amiloride analog containing one ring formed on a guanidine group, an amiloride analog containing an acyl guanidino group, and a guanidine group. Selected from the group consisting of amiloride analogs, wherein the water-soluble groups are N, N-dimethylamino groups or sugar groups.

In other embodiments, the inhibitor is amiloride, an amiloride analog or a salt thereof, 0.1-1000 mg / dose, 0.1-300 mg / dose, 0.1-100 mg / dose, 0.1- 30 mg / dose, 0.1-10 mg / dose, 0.1-3 mg / dose, 0.1-1 mg / dose, 0.1-0.3 mg / dose, 0.3-1000 mg / dose, 0.3- 300 mg / dose, 0.3-100 mg / dose, 0.3-30 mg / dose, 0.3-10 mg / dose, 0.3-3 mg / dose, 0.3-1 mg / dose, 1-1000 mg / dose, 1-300 mg / dose, 1-100 mg / dose, 1-30 mg / dose, 1-10 mg / dose, 1-3 mg / dose, 3-1000 mg / dose, 3-300 mg / dose, 3-100 mg / dose, 3- 30mg Dose, 3-10 mg / dose, 10-1000 mg / dose, 10-300 mg / dose, 10-100 mg / dose, 10-30 mg / dose, 30-1000 mg / dose, 30-300 mg / dose, 30-100 mg / dose, Administered as a pharmaceutical composition formulated as a single dose ranging from 100-1000 mg / dose, 100-300 mg / dose, or 300-1000 mg / dose. In one embodiment, the amiloride analog is benzamyl, phenamyl, EIPA, bepridil, KB-R7943, 5- (N-methyl-N-isobutyl) -amylolide, 5- (N, N-hexamethylene) -amylolide and It is selected from the group consisting of 5- (N, N-dimethyl) amiloride hydrochloride. In another embodiment, amiloride analogues, having a hydrophobic substituent at C ₅ -NH _2-position and / or on the guanidino group. In another embodiment, an amiloride analog is formed on a methylated analog of benzamyl, an amiloride analog containing one ring formed on a guanidine group, an amiloride analog containing an acyl guanidino group, and a guanidine group. Selected from the group consisting of amiloride analogs, wherein the water-soluble groups are N, N-dimethylamino groups or sugar groups. In some embodiments, the pharmaceutical composition is formulated for intravenous injection, intracerebral injection, intraventricular injection, intrathecal injection or intramuscular injection.

The inhibitor may be administered to the subject in any suitable form and in any suitable composition. In some examples, the inhibitor may be configured as a pharmaceutically acceptable salt. The composition may be formulated to contain, for example, a liquid carrier / solvent (vehicle), a preservative, one or more excipients, colorants, flavorings, salts, antifoaming agents, and the like. The inhibitor may be present in the vehicle at a concentration that when administered to an ischemic subject provides a therapeutically effective amount of the inhibitor for ischemic treatment.

In some embodiments, amiloride analogs with higher water solubility or fat solubility are produced. In certain embodiments, the amiloride analog includes a water-soluble group in the guanidino group, such as Ν, Ν-dimethylamino group, or a sugar to improve water solubility (Formulas 13-16, FIG. 24). In some embodiments, the amiloride analog has a water solubility of 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM or more. In other embodiments, the amiloride analog is administered intravenously at a dose of 10 mg, 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, or 500 mg in a single 10 ml human dose. It has the solubility that makes it possible. In yet other embodiments, the amiloride analog is administered intraventricularly at a dose of 10 mg, 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, or 500 mg in a single 2 ml human dose. It has the solubility that can be done.

Synthesis and screening of amiloride analogues Another aspect of the present application relates to the synthesis and screening of novel amiloride analogues. The synthetic route for amiloride analogs is designed based on the structure of the desired analog. Next, newly synthesized amiloride analogs are screened for inhibitory effects on ASIC family members, eg, ASIC1a and ASIC2a. One or more ASIC inhibitors may be obtained, in particular the ASIC1a inhibitors described above. The inhibitors can be obtained by any suitable method, in particular by screening candidate inhibitor combinations (eg a library of two or more compounds) and / or by rational design. sell.

Screening may include any suitable assay system that measures the interaction between the ASIC protein and a series of candidate inhibitors. A typical assay system includes a biochemically performed assay (eg, a binding assay), particularly using cells grown in culture (“cultured cells”) and / or using an organism. May be included.

In some embodiments, cell-based assays are used to examine the effect of each candidate inhibitor on intracellular ion flux, eg, acid-sensitive ion flux. In some embodiments, the ion flow rate is a calcium and / or sodium flow rate. In some embodiments, the assay system determines the selectivity of each inhibitor for these family members, eg, an ASIC family member, eg, a cell expressing ASIC1a or ASIC2a, or two or more distinguishable ASICs. Two or more distinct sets of cells expressing a family member, eg, ASIC1a and another ASIC family member are used. The cell may express each ASIC family member endogenously or by introduction of a foreign nucleic acid. In some examples, the assay system may be electrophysiologically using acid sensitive or membrane potential sensitive dyes (eg, calcium sensitive dyes such as Fura-2) or membrane potential and / or intracellular ions. The ion flux can be measured using a reporter system based on a gene that is sensitive to changes in (eg calcium) concentration. The assay system may be used to test candidate inhibitors for selective and / or specific inhibition of ASIC family members, particularly ASIC1a.

One or more ASIC inhibitors may be administered to a subject with nerve injury, eg, a subject in an ischemic state, to test the effectiveness of the inhibitor for the treatment of nerve injury. The subject in the ischemic state may be a human or an animal. In some instances, a subject that is ischemic may provide an animal model system that is ischemic and / or stroked.
Typical animal model systems include rodents (especially mice and / or rats) in which ischemic conditions have been experimentally induced. The ischemic condition may be induced in particular mechanically (eg surgically) and / or by administration of a drug. In some examples, the ischemic condition can be induced by vascular occlusion, for example, by constriction of the middle cerebral artery.

Another aspect of the present application relates to a pharmaceutical composition for reducing nervous system damage. The pharmaceutical composition comprises an effective amount of amiloride, amiloride analog, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, the pharmaceutical composition comprising intravenous, intrathecal or Formulated for intracerebroventricular injection.

In some embodiments, the pharmaceutical composition comprises an amiloride analog or a pharmaceutically acceptable salt thereof, wherein the amiloride analog is benzamyl, phenamyl, EIPA bepridil, KB-7794, 5- (N -Methyl-N-isobutyl) amiloride, 5- (N, N-hexamethylene) amiloride and 5- (N, N-dimethyl) amiloride hydrochloride.

In other embodiments, the pharmaceutical composition comprises an amiloride analog or a pharmaceutically acceptable salt thereof, wherein the amiloride analog has a ring formed on the methylated analog of benzamyl, a guanidine group. An amiloride analogue comprising an acylguanidino group, and an amiloride analogue comprising a water-soluble group formed on a guanidine group, wherein the water-soluble group is an N, N-dimethylamino group or a sugar group Selected from the group.

In other embodiments, the pharmaceutical composition further comprises one or more other ASIC inhibitors. In one embodiment, the one or more other ASIC inhibitors comprise PcTx1 or a PcTx1 derivative.

Another aspect of the present application relates to a pharmaceutical composition for reducing nervous system damage. The pharmaceutical composition comprises an effective amount of an amiloride analog or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition is formulated for intravenous, intrathecal, intraventricular or intramuscular administration.

In one embodiment, the amiloride analog is a methylated analog of benzamyl. In another embodiment, the amiloride analog comprises one ring formed on a guanidine group. In another embodiment, the amiloride analog comprises an acyl guanidino group. In yet another embodiment, the amiloride analog comprises a water soluble group formed on the guanidine group, which is an N, N-dimethylamino group or a sugar group.

In other embodiments, the pharmaceutical composition further comprises one or more other ASIC inhibitors. In one embodiment, the one or more other ASIC inhibitors comprise PcTx1 or a PcTx1 derivative.

As used herein, “pharmaceutically acceptable carrier” refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, sweetening agents and the like. Including. Pharmaceutically acceptable carriers include a wide variety of substances including, but not limited to, flavoring agents, sweetening agents, and various substances such as buffers and absorbents that may be required to prepare a particular therapeutic composition. Can be prepared from various materials. The use of such media and agents in combination with pharmaceutically active substances is well known in the art. Except where any conventional medium or agent is incompatible with the active ingredient, its use in a therapeutic composition is contemplated. Optionally, the amiloride and / or amiloride analog may be mixed together in a pharmaceutical composition comprising supplementary active ingredients that are not contraindicated with the aforementioned amiloride and / or amiloride analog.

In some embodiments, the pharmaceutical composition is formulated for intravenous infusion. In other embodiments, the pharmaceutical composition comprises amiloride and / or an amiloride analog formulated for intravenous infusion. In other embodiments, the pharmaceutical composition comprises amiloride and / or an amiloride analog formulated for intracerebroventricular injection. In other embodiments, the pharmaceutical composition comprises amiloride and / or an amiloride analog formulated for intrathecal injection. In other embodiments, the pharmaceutical composition comprises amiloride and / or an amiloride analog formulated for intramuscular injection. Pharmaceutical compositions suitable for infusion use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile infusion solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL ^™ (BASF, Parsippany, NJ) or fluids that allow easy needle passage. Injectable compositions must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the activity of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating amiloride and / or an amiloride analog in the required amount in a suitable solvent and then sterile filtered. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization, which gives the active ingredient and any additional ingredients from the previously sterile filtered solution. It is done.

In some embodiments, the pharmaceutical composition is provided in a dry form and is formulated into a tablet or capsule form. Tablets may be formulated according to conventional methods using solid carriers well known in the art. The hard and soft capsules used in the present invention can be made from any pharmaceutically acceptable material, such as gelatin or cellulose derivatives.

In certain embodiments, the pharmaceutical composition is formulated for immediate release, sustained release, delayed release, or a combination thereof. Sustained release, also known as extended release, time release or period release, modified release (CR), modified release (MR), continuous release (CR or Contin), dissolves slowly and releases the active ingredient over time Therefore, it is a mechanism used in tablets or capsules that are pharmaceuticals. The advantage of sustained release tablets or capsules is that they can often be taken less frequently than immediate release formulations of the same drug, and they maintain more stable drug levels in the bloodstream, resulting in a duration of drug action Is the point of extension.

In one embodiment, the pharmaceutical composition is formulated for sustained release by embedding the active ingredient in a matrix of insoluble material such as acrylic or chitin. Sustained release forms are designed to release the active ingredient at a predetermined rate by maintaining a constant drug level for a specified period of time. This can be achieved by various formulations, including, but not limited to, drug-polymer conjugates such as liposomes and hydrogels.

In another embodiment, the pharmaceutical composition is formulated for delayed release such that the active ingredient is not released immediately upon administration. A non-limiting example of a delayed release vehicle is an enteric coated oral drug that dissolves in the intestine rather than in the stomach.

In other embodiments, the pharmaceutical composition is formulated for immediate release of a portion of the active ingredient followed by the remaining sustained release of the active ingredient. In one embodiment, the pharmaceutical composition is formulated as a powder so that the active ingredient released immediately can be ingested. In another embodiment, the pharmaceutical composition is formulated in liquid, gel, suspension or emulsion form. Such a liquid, gel, suspension or emulsion may be ingested by the subject in the form as it is or encapsulated.

In yet another embodiment, the pharmaceutical composition may be provided as a skin or transdermal patch for topical administration of a controlled and / or sustained amount of the active ingredient.

The following examples describe selected aspects and embodiments of the present teachings, in particular, data showing the in vitro and in vivo effects of ASIC inhibition and typical cystine knot peptides for use as inhibitors. Is described. These examples are intended for illustrative purposes and should not be construed to limit the scope of the present teachings.

Example 1: Neuroprotection in ischemia by inhibiting calcium permeable acid-sensing ion channels This example demonstrates the role of ASIC1a mediating ischemic damage and the ability of ASIC1a inhibitors to reduce ischemic damage The experiment which shows is described (refer FIGS. 2-10). Ca2 + toxicity may play a central role in ischemic brain injury. The mechanism by toxic Ca 2+ loading in cells occurring in the ischemic brain has failed to show effective neuroprotection in stroke in multiple human trials of glutamate antagonists. It became ambiguous. Acidosis is a common feature of ischemia and plays an important role in brain injury. This example shows that acidosis can activate Ca2 + permeable acid-sensing ion channel (ASIC), thereby inducing glutamate receptor-independent, Ca2 + -dependent nerve damage. Thus, cells lacking endogenous ASIC may be resistant to acid damage, whereas transfection of Ca2 + permeable ASIC1a may establish sensitivity. In focal ischemia, intracerebroventricular injection of an ASIC1a inhibitor or knockout of the ASIC1a gene can lead to an antagonist effect.

The normal brain requires complete oxidation of glucose to meet its energy requirements. During ischemia, oxygen depletion converts the brain to anaerobic glycolysis. Lactic acid as a byproduct of glycolysis and accumulation of protons generated by ATP hydrolysis lowers the pH in the ischemic brain and exacerbates ischemic brain damage.

Acid-sensing ion channels (ASICs) are a class of ligand-gated channels that are expressed throughout the nerves of the mammalian central and peripheral nervous systems. To date, six types of ASIC subunits have been cloned. Four of these subunits form functional homomultimeric channels activated by acidic pH to conduct sodium-selective, amiloride-selective cation currents. Two of the ASIC subunits, ASIC1a and ASIC2a subunits, have been shown to be abundant in the brain.

Experimental Procedure Nerve cell culture After anesthesia with halothane, cerebral cortex was excised from E16 Swiss mice or PI ASIC1 ^{+ / +} and ASIC1 ^{− / −} mice and 0.05% trypsin-EDTA for 10 minutes at 37 ° C. Incubate. The tissue is then minced with a fired glass pipette and poly-L-ornithine coated 24-well plate or 25 × 25 mm glass coverslips at a density of 2.5 × 10 ⁵ cells per well or 10 ⁶ cells per coverslip. Plated. Neurons were cultured in MEM supplemented with 10% horse serum (for E16 culture) or Neurobasal medium supplemented with B27 (for PI culture) and used for electrophysiology and toxicity studies after 12 days. Glial cell proliferation was inhibited by the addition of 5-fluoro-2-deoxyuridine and uridine, resulting in cultured cells with 90% neuronal cells as measured by NeuN and GFAP staining (data not shown).

Electrophysiology ASIC currents were recorded using whole cell patch clamp and rapid perfusion techniques. Normal extracellular solution (ECF) (unit: mM) 140 NaCl, 5.4 KCl, 25 HEPES, 20 glucose, 1.3 CaCl ₂ , 1.0 MgCl ₂ , 0.0005 TTX (pH 7.4). 320 to 335 mOsm. For low pH solutions, various amounts of HCl were added. For solutions with pH <6.0, MES was used instead of HEPES for a more reliable pH buffer effect. The patch electrode contained (unit: mM) 140 CsF, 2.0 MgCl ₂ , 1.0 CaC ₂ , 10 HEPES, 11 EGTA, 4 MgATP (pH 7.3) at 300 mOsm. The Na ⁺ -free solution consisted of 10 mM CaCl ₂ , 25 mM HEPES containing isotonic NMDG or sucrose instead of NaCl (Chu et al., 2002). A multi-barrel perfusion system (SF-77B, Warner Instrument Co.) was used for rapid exchange of solutions.

Cell injury assay-LDH measurement Cells were washed 3 times with ECF and randomly divided into treatment groups. MK801 (10 μM), CNQX (20 μM), and nimodipine (5 μM) were added to all groups to eliminate potential secondary activation of glutamate receptors and voltage-dependent Ca ²⁺ channels. Following acid incubation, neurons were washed and incubated at 37 ° C. in Neurobasal medium. LDH release was measured in culture medium using an LDH assay kit (Roche Molecular 1 Biochemicals). Medium (100 μL) was transferred from the culture wells to a 96 well plate and mixed with 100 μL of the reaction solution provided by the kit. Optical density was measured after 30 minutes at 492 nm using a microplate reader (Spectra Max Plus, Molecular Devices). The background absorption at 620 was subtracted. At the end of each experiment, maximally releasable LDH was obtained in each well by incubating with 1% Triton X-100 for 15 minutes.

Ca ²⁺ imaging Cortical neurons grown on 25 × 25 mm glass coverslips were washed 3 times with ECF, incubated with 5 μΜ Fura-2-acetoxymethyl ester for 40 minutes at 22 ° C., washed 3 times, Incubated in ECF for 30 minutes. The coverslip was transferred to a perfusion chamber on an inverted microscope (Nikon TE300). Cells were irradiated using a xenon lamp and observed using a 40X UV fluorescent oil immersion objective, and video images were acquired using a cooled CCD camera (Sensys KAF 1401, Photometries). Digitized images were acquired on a PC controlled by Axon Imaging Working Software (Axon Instruments) and analyzed. The shutter and filter wheel (Lambda 10-2) were controlled by software that allowed the cells to be irradiated at the appropriate time with an excitation wavelength of 340 or 380 nm. Fura-2 fluorescence was detected at an emission wavelength of 510 nm. Ratio images (340/380) were analyzed by averaging the pixel ratio values of the localized area of cells in the field of view. Values were extrapolated to SigmaPlot for further analysis.

Fluorescein-diacetate staining and propidium iodide uptake Cells were incubated in ECF containing fluorescein-diacetate (FDA) (5 μ （) and propidium iodide (PI) (2 μΜ) for 30 minutes, then dye-free Washed with the included ECF. Live cells (FDA positive) and dead cells (PI positive) were observed and counted with a microscope (Zeiss) equipped with 580/630 nm excitation / emission for PI and 500/550 nm epifluorescence for FDA. . Images were collected using an Optronics DEI-730 camera equipped with a BQ 8000 sVGA frame holder and analyzed using computer software (Bioquant, TN).

Transfection of COS-7 cells COS-7 cells were cultured in MEM (GIBCO) containing 10% HS and 1% PenStrep. At approximately 50% confluency, cells were co-transfected with the cDNA for ASIC and GFP in pc ^DNA3 vector using FuGENE6 transfection reagent (Roche Molecular Biochemicals). DNA for ASIC (0.75 ng) and 0.25 μg of DNA for GFP were used for each 35 mm dish. GFP positive cells were selected for patch clamp recording 48 hours after transfection. For stable transfection of ASIC1a, one week after transfection, 500 μg / mL G418 was added to the culture medium. Surviving G418 resistant cells were further plated and passaged for more than 5 generations in the presence of G418. Cells were then examined for expression of ASIC1a using patch clamp and immunofluorescence.

Oxygen - glucose deprivation nerve cells were washed 3 _times, with _{85% N 2, 10% H} 2, and 5% _CO 2, an anaerobic chamber with an atmosphere of 35 ℃ (Model 1025, Forma Scientific ) within, pH 7.4 Alternatively, it was incubated with 6.0 glucose-free ECF. Oxygen-glucose depletion (OGD) was terminated after 1 hour by replacing glucose-free ECF with Neurobasal medium and incubating the culture in a normal cell culture incubator. Using HEPES buffered ECF, 1hr OGD slightly reduced pH from 7.38 to 7.28 (n = 3) and 6.0 to 5.96 (n = 4) did.

Local ischemia Transient focal ischemia is induced in male rats anesthetized with 1.5% isoflurane, 70% N ₂ O, and 28.5% O ₂ by intubation and evacuation (SD, 250- 300 g) and mice (having a gene-like C57B 16 background, approximately 25 g) were induced by suture occlusion of the middle cerebral artery (MCAO). Rectal and temporal muscle temperatures were maintained at 37 ° C. + 0.5 ° C. using a thermally stable controlled heating pad and lamp. Cerebral blood flow was monitored with a transcranial laser Doppler. Animals whose blood flow did not decrease by more than 20% were excluded.

The animals were killed with chloral hydrate 24 hours after ischemia. The brain is quickly removed and resected coronally at 1 mm (mouse) or 2 mm (rat) intervals and immersed in vital pigment (2%) 2,3,5-triphenyltetrazolium hydrochloride (TTC) Stain. The infarct area was calculated by subtracting the normal area stained with TTC in the ischemic hemisphere area from the non-ischemic hemisphere area. Infarct volume was calculated by summing the infarct area of all parts and multiplying by the thickness of the section. Injection into the rat ventricle is stereotaxic using a microsyringe pump with a cannula inserted stereotaxically at the 0.8 mm posterior side of the cross suture, 1.5 mm lateral side of the midline, and 3.8 mm abdomen of the dura mater. Made by fixation technique. All manipulations and analyzes were performed by individuals who were not informed about the treatment group.

Results (a) Acidosis activates ASIC in mouse cortical neurons FIGS. 3 and 4 show typical data on electrophysiology and pharmacology of ASIC in cultured mouse cortical neurons. 3A and 3B are graphs showing the pH dependence of the ASIC current activated when the pH is lowered from 7.4 to the indicated pH value. Dose-response curves were fitted to the Hill formula using an average pH _0.5 (n = 10) of 6.18 + 0.06. 3C and 3D are graphs showing the current-voltage relationship of ASIC (n = 5). The magnitude of the ASIC current at various voltages was normalized to the magnitude recorded at -60 mV. 4A and 4B are graphs showing dose-dependent inhibition of ASIC current by amiloride. IC50 = 16.4 + 4.1 μΜ, N = 8. 4C and 4D are graphs showing inhibition of ASIC current by PcTX poison. ^** p <0.01.

ASIC currents in cultured mouse cortical neurons were recorded (see FIG. 3). At a maintenance potential of −60 mV, a rapid drop in extracellular pH (pHe) to a value below 7.0 causes a large transient inward current in many neurons with a small steady state component. (FIG. 3A). The magnitude of the inward current increased in a sigmoidal manner as pHe increased, resulting in a pH _{0.5 of} 6.18 + 0.06 (n = 10, Figure 3B). A reversal approximating the linear IV relationship and Na ⁺ equilibrium potential was obtained (n = 6, FIGS. 3C and 3D). These data indicate that a decrease in pHe can activate typical ASICs in mouse cortical neurons.

The effect of amiloride, a non-specific inhibitor of ASIC, on the acid-activated current was tested (see FIG. 4). As shown in FIG. 4, amiloride dose-dependently inhibited ASIC current in cortical neurons with an IC ₅₀ of 16.4 + 4.1 μM (n = 8, FIGS. 4A and 4B). The effect of PcTX venom on acid-activated current in cortical neurons is shown in FIGS. 4C and 4D. At 100 ng / mL, PcTX toxin reversibly inhibits the peak magnitude of the ASIC current to 47% + 7% (n = 15, FIGS. 4C and 4D), thereby acid activation of the homomeric ASIC1a A significant contribution to the overall current was shown. Increasing PcTX concentration did not induce a further decrease in ASIC current magnitude in the majority of cortical neurons (n = 8, data not shown). This indicates that PcTX-insensitive ASICs (eg, heteromeric ASIC1a / 2a) are present together in these neurons.

(B) ASIC response enhanced by modeled ischemia FIG. 5 shows exemplary data representing that modeled ischemia can increase the activity of the ASIC. FIG. 5A is a series of exemplary traces showing an increase in ASIC current magnitude and a decrease in sensitivity suppression after 1 hour of OGD. FIG. 5B is a graph of summary data showing the increase in ASIC current magnitude in OGD neurons. N = 40 and 44, ^* p <0.05. FIG. 5C is a series of exemplary traces and summary data showing reduced ASIC current sensitivity suppression in OGD neurons. N = 6, ^** p <0.01. FIG. 5D is a pair of exemplary traces showing a lack of acid-activated current at pH 6.0 in ASIC1 ^{− / −} neurons after 1 hour of OGD under control conditions (n = 12 and 13). .

Since acidosis could be a central feature of cerebral ischemia, it was decided to test whether ASIC could be activated under ischemic conditions and whether ischemia could modify the characteristics of these channels ( (See FIG. 5). The ASIC current in neurons was recorded 1 hour after oxygen glucose depletion (OGD). Briefly, a set of cultured cells was washed three times with glucose-free cell fluid (ECF) and subjected to OGD, whereas a control cultured cell was washed with ECF containing glucose. And subjected to incubation in a normal cell culture incubator. OGD was terminated after 1 hour by replacing ECF without glucose with Neurobasal medium and incubating the culture in a normal incubator. Next, if no change in the morphology of the neuron occurred, the ASIC current was recorded 1 hour after OGD. OGD treatment induced a slight increase in the magnitude of the ASIC current (1520 + 138 pA, N = 44 in the control group; 1886 + 185 pA, N = 40, p <0.05 in the neurons after 1 hour of ODA, FIG. 5A and 5B). More importantly, OGD induced a dramatic decrease in ASIC sensitivity suppression indicated by an increase in the time constant of current reduction (814.7 + 58.9 ms in control neurons, N = 6; after OGD 1928.9 + 315.7 ms, N = 6, p <0.01, FIGS. 5A and 5C). In cortical neurons cultured from ASIC1 ^{− / −} mice, the decrease in pH from 7.4 to 6.0 is similar to that of previous studies in hippocampal neurons (Wemmie et al., 2002). The current in the direction was also not activated (n = 52). In these neurons, 1 hour OGD did not activate or increase acid-induced responses (FIG. 5D, n = 12 and 13).

(C) acidosis, 6 and 7 to induce glutamate-independent ^{Ca 2+} entering through ASIC1a may be ASIC in cortical neurons may be ^{Ca 2+} permeability, and, ^{Ca 2+} permeability ASIC1a dependent Typical data suggesting that can be FIG. 6A shows a typical trace obtained using Na ⁺ -free ECF containing 10 mM Ca ²⁺ as the only charge carrier. The inward current was recorded at pH 6.0. The average reversal potential is about -17 mV after correction of the liquid boundary potential (n = 5). FIG. 6B shows representative traces and summary data representing inhibition of Ca ²⁺ mediated currents by amiloride and PcTX poisons. The peak current magnitude mediated by Ca ²⁺ was reduced to 26% + 2% of the control value by 100 μM amiloride (n = 6, p <0.01) and 22% by 100 ng / mL PcTX toxin. + Reduced to 0.9% (n = 5, p <0.01). FIG. 7A shows a typical 340/380 nm ratio as a function of pH, representing an increase in [Ca ²⁺ ] _i with decreasing pH to 6.0. Nerve cells contain 1.3 mM CaCl ₂ with inhibitors for voltage-dependent Ca ²⁺ channels (5 μm nimodipine and 1 μm ω-conotoxin MVIIC) and glutamate receptors (10 μm MK801 and 20 μm CNQX) Soaked in normal ECF. The inset of FIG. 7A shows typical inhibition of acid-induced [Ca ²⁺ ] _i increase by 100 μM amiloride. FIG. 7B shows exemplary summary data representing inhibition of acid-induced increase in [Ca ²⁺ ] _i by amiloride and PcTX toxin. Compared to N = 6-8, ^** p <0.01 pH 6.0 group. FIG. 7C shows a typical 340/380 nm ratio as a function of pH and the presence or absence of NMDA, representing the lack of acid-induced [Ca ²⁺ ] _i increase in ASIC1 ^{− / −} neurons, This represents a normal reaction to NMDA (n = 8).

ASIC Ca ²⁺ permeability in cortical neurons was determined by standard ion replacement protocol (Jia et al, Neuron, 1996, 17: 945-956) and Fura-2 fluorescent Ca ²⁺ imaging technique (Chu et al, 2002, J Neurophysiol.87: 2555-2561). Using an immersion solution containing 10 mM Ca ²⁺ as the only charge carrier (not containing Na ⁺ and K ⁺ ), 15 out of 18 neurons with an inward direction greater than 50 pA at a holding potential of −60 mV Current was recorded. This shows a significant Ca ²⁺ permeability of ASIC in most cortical neurons (FIG. 6A). Consistent with activation of the homomeric ASIC1a channel, the current produced by 10 mM Ca ²⁺ was greatly inhibited by both the non-specific ASIC inhibitor amiloride and the ASIC1a-specific inhibitor PcTX toxin (FIG. 6B). . The peak current magnitude mediated by Ca ²⁺ was reduced to 26% + 2% of the control value by 100 μM amiloride (n = 6, p <0.01) and 22% by 100 ng / mL PcTX toxin. + Reduced to 0.9% (n = 5, p <0.01). Presence of other major Ca ²⁺ entry pathway inhibitors (MK801 10 μΜ and CNQX 20 μΜ for glutamate receptors; nimodipine 5 μΜ and ω-contoxin MVIIC 1 μΜ for voltage-dependent Ca ²⁺ channels) by Ca ²⁺ imaging Below, 18 of the 20 neurons responded to a pH drop with a detectable increase in intracellular Ca ²⁺ ([Ca ²⁺ ] _i ) concentration (FIG. 7A). In general, [Ca ²⁺ ] _i remains elevated during long perfusions of low pH solutions. In some cells, the [Ca ²⁺ ] _i increase lasted much longer than the period of acid perfusion (FIG. 7A). Long-lasting Ca ²⁺ responses can result in intracellular ASIC responses that are less sensitive to suppression than responses in whole-cell recordings, or Ca ²⁺ invasion via ASICs, resulting in intracellular This suggests that Ca ²⁺ is released from the storage location. Preincubation of neurons with 1 μΜ thapsigargin partially inhibited the sustained component of Ca ²⁺ increase. This indicates that release of Ca ²⁺ from intracellular storage locations can also contribute to acid-induced intracellular Ca ²⁺ accumulation (n = 6, data not shown). Similar to the current provided by Ca ²⁺ ions (Fig. ^6B), both in ^[Ca _{2+] i} peak and sustained increase, the involvement of homomeric of ASIC1a in induced acid ^[Ca _{2+] i} increase Consistently, it is greatly inhibited by amiloride and PcTX toxin (FIGS. 7A and 7B, n = 6-8). ASCI gene knockout eliminated acid-induced [Ca ²⁺ ] _i increases in all neurons without affecting the NMDA receptor-mediated Ca ²⁺ response (FIG. 7C, n = 8). ). Patch clamp recordings showed a lack of acid-activated current in 52 of 52 ASIC1 ^{− / −} neurons at pH 6.0, consistent with the absence of the ASIC1a subunit. However, decreasing the pH to 5.0 or 4.0 activated currents detectable in 24 of 52 ASIC1 ^{− / −} neurons. This suggests the presence of the ASIC2a subunit in these neurons (FIG. 7D). Further electrophysiological studies have shown that ASIC1 ^{− / −} neurons have normal responses to various voltage-dependent channels and NMDA, GABA receptor-dependent channels (data not shown).

(D) ASIC inhibition protects against glutamate-independent nerve damage induced by acidosis. FIG. 8 shows that incubation with acid induces glutamate receptor-independent nerve damage protected by ASIC inhibition. Shows typical data suggesting. Figures 8A and 8B show the time induced by 1 hour (Figure 8A) or 24 hour (Figure 8B) incubation of cortical neurons in ECF at pH 7.4 (black bars) or 6.0 (white bars). Figure 2 shows a graph representing typical data for dependent LDH release. Comparison with N = 20-25 wells, ^* p <0.05, and ^** p <0.01, pH 7.4 at the same time point (acid-induced nerve using fluorescein diacetate (FDA) Injury was also analyzed by staining of the cell bodies of living neurons, as well as propidium iodide (PI) staining of the nuclei of dead neurons, Figure 8C is due to 100 μ ア ミ amiloride or 100 ng / mL PcTX toxin. , Shows graphs showing inhibition of acid-induced LDH release (n = 20-27, ^* p <0.05, and ^** p <0.01) MK801, CNQX, and nimodipine are the ECF in all experiments. (FIGS. 8A to 8C).

Acid-induced damage was allowed to proceed on 24 wells in either pH 7.4 or 6.0 ECF containing MK801, CNQX, and nimodipine (see FIG. 8). Cell damage was determined by measurement of lactate dehydrogenase (LDH) release at various time points (FIGS. 8A and 8B) (Koh and Choi, J. Neurosci., 1987, 20: 83-90), as well as live / dead. The assay was performed by fluorescent staining of the cells. Compared to neuronal treatment at pH 7.4, 1 hour acid incubation (pH 6.0) induced a time-dependent increase in LDH release (FIG. 8A). After 24 hours, a maximum LDH release of 45.7% + 5.4% was induced (n = 25 wells). Larger cell damage was induced by continuous treatment at pH 6.0 (FIG. 8B, n = 20). Consistent with the LDH assay, live / dead cell staining with fluorescein diacetate and propidium iodide showed a similar increase in cell death upon 1 hour acid treatment (data not shown). 1 hour incubation with pH 6.5 ECF also induced significantly less LDH release compared to pH 6.0 ECF (n = 8 wells, data not shown).

The effect of amiloride and PcTX venom on acid-induced LDH release was tested to determine whether ASIC activation is involved in acid-induced glutamate receptor-independent nerve injury. LDH release was significantly reduced by adding either 100 μΜ amiloride or 100 ng / mL PcTX venom 10 minutes before and 1 hour before acid incubation (FIG. 8C). At 24 hours, LDH release decreased from 45.3% + 3.8% to 31.1% + 2.5% with amiloride and 27.9% + 2.6% with PcTX poison ( n = 20-27, p <0.01). Addition of amiloride or PcTX venom for 1 hour in ECF at pH 7.4 did not affect baseline LDH release, but prolonged incubation with amiloride alone (eg, 5 hours) Release was increased (n = 8, data not shown).

(E) Activation of homomeric ASIC1a is responsible for acidosis-induced damage FIG. 9 shows a series of data representing typical data suggesting that ASIC1a may be involved in acid-induced damage in vitro It is a graph. FIG. 9A shows typical data representing inhibition of acid-induced LDH release by decreasing [Ca ²⁺ ] _e (n = 11-12, ^** p <0.01 pH 6.0, 1.3). Compare with Ca ²⁺ ). FIG. 9B shows typical data showing that acid incubation increased LDH release in COS-7 cells transfected with ASIC1a but not in untransfected COS-7 cells (n = 8-20). Amiloride (100 μM) inhibited acid-induced LDH release in cells transfected with ASIC1a. ^* P <0.05 for 7.4 vs 6.0 and for 6.0 vs 6.0 + amiloride. FIG. 9C shows typical data representing lack of inductive damage in ASIC ^{− / −} neurons and protection by amiloride and PcTX toxin (each group n = 8, p> 0.05). FIG. 9D shows typical data representing an acid-induced increase in LDH release in cultured cortical neurons under OGD conditions (n = 5). LDH release induced by the 1 hour OGD / acidosis combination was not inhibited by Trolox and L-NAME (n = 8-11). OGD did not increase acid-induced LDH release in ASIC1 ^{− / −} neurons. ^** p <0.01 for pH 7.4 vs. pH 6.0 and ^* p <0.05 for pH 6.0 vs. 6.0 + PcTX poison. MK801, CNQX, and nimodipine were included in the ECF in all experiments (FIGS. 9A-D).

Neurons were treated with ECF pH 6.0 in the presence of normal or reduced [Ca ²⁺ ] _e to see if Ca ²⁺ invasion plays a role in acid induction (see FIG. 9). ). Reduction of Ca ²⁺ from 1.3 to 0.2 mM inhibited acid-induced LDH release (n = 11-12, p <0.01; FIG. 9A), similar to ASIC1a inhibition by PcTX toxin (40 0.0% + 4.1% to 21.9% + 2.5%). Ca ²⁺ -free solutions were not tested because they can activate large inward currents through Ca ²⁺ sensitive cation channels. Otherwise, it can complicate data interpretation. Inhibition of acid damage by both non-specific and specific ASIC1a inhibitors amiloride and PcTX, and by reducing [Ca ²⁺ ] _e , activation of Ca ²⁺ permeable ASIC1a is acid-induced. Suggests that it may be involved in nerve damage.

Acid damage in untransfected COS-7 cells and COS-7 cells transfected with ASIC1a was tested to provide further evidence that ASIC1a activation was involved in acid damage. COS-7 is a cell line commonly used for the expression of ASICs because it lacks endogenous channels. After confluence (36-48 hours after plating), cells were treated with ECF at either pH 7.4 or 6.0 for 1 hour. LDH release was measured 24 hours after acid incubation. Treatment of untransfected COS-7 cells with pH 6.0 ECF did not induce an increase in LDH release when compared to pH 7.4 treated cells (10. vs. pH 7.4). 3% + 0.8% and 9.4% + 0.7% for pH 6.0, N = 19 and 20 wells; p> 0.05, FIG. 9B). However, in COS-7 cells stably transfected with ASIC1a, 1 hour incubation at pH 6.0 increased from 15.5% + 2.4% to 24.0% + 2.9%. LDH release was significantly increased (n = 8 wells, p <0.05). Addition of amiloride (100 μM) inhibited acid-induced LDH release in these cells (FIG. 9B).

Acid damage in CHO cells transiently transfected with GFP alone or cDNA encoding GFP + ASIC1a was also studied. After transfection (24-36 hours), cells were incubated for 1 hour at acidity (pH 6.0) and assayed for cell damage after 24 hours of acid incubation. One hour acid incubation greatly reduced the number of surviving GFP positive cells in the GFP / ASIC1a group, but not in the group transfected with GFP alone (data not shown).

Cytotoxicity experiments in cortical neurons cultured from ASIC ^{+ / +} and ASIC1 ^{− / −} mice were performed to further demonstrate the involvement of ASIC1a in acidosis-induced nerve injury. Furthermore, 1 hour acid incubation of ASIC ^{+ / +} neurons at pH 6.0 induced substantial LDH release caused by amiloride and PcTX toxin (n = 8-12). However, 1 hour acid treatment of ASIC1 ^{− / −} neurons did not induce a significant increase in LDH release at the 24 hour time point (13.8% + 0.9% versus pH 7.4, and , 14.2% + 1.3%, pH 6.0, N = 8, p> 0.05). This suggests resistance of these neurons to acid damage (FIG. 9C). Furthermore, knockout of the ASIC1 gene also excluded the effect of acid-induced LDH release of amiloride and PcTX venom (FIG. 9C, n = 8 respectively). This further indicated that inhibition of acid-induced damage of cortical neurons by amiloride and PcTX toxin was due to inhibition of the ASIC1 subunit (FIG. 8C). In contrast to acid incubation, 1 hour treatment of ASIC1 ^{− / −} neurons with 1 mM NMDA + 10 μM glycine (in Mg ²⁺ -free [pH 7.4] ECF) produced a maximum LDH release at 24 hours. Of 84.8% + 1.4% (n = 4, FIG. 9C). This showed a normal response to other cell damage processes.

(F) Modeled ischemia increases glutamate-independent neuronal cell damage induced by acidosis via ASIC. The magnitude of ASIC current depends on cellular components and neurochemistry of swollen cerebral ischemic cells It can be increased by chemical ingredients, arachidonic acid, and lactic acid. Furthermore, more importantly, the suppression of ASIC current sensitivity can be dramatically reduced by modeled ischemia (FIGS. 5A and 5C). Activation of ASIC in an ischemic state is expected to result in greater neuronal damage. To test this hypothesis, neurons were subjected to 1 hour acid treatment under oxygen and glucose depletion (OGD) conditions. MK801, CNQX, and nimodipine were included in all solutions to inhibit glutamate receptor-mediated cell damage in cells associated with voltage-dependent Ca ²⁺ channels and OGD. One hour incubation with pH 7.4 ECF under OGD conditions induced only 27.1% + 3.5% of maximal LDH release at 24 hours (n = 5, FIG. 9D). This finding is consistent with previous reports that one hour of OGD does not induce substantial cell damage due to inhibition of glutamate receptors and voltage-dependent Ca ²⁺ channels (Aarts et al., 2003). However, when combined with acidosis (pH 6.0), 1 hour OGD induces 73.9% + 4.3% of maximal LDH release (n = 5, FIG. 9D, p <0.01). This is significantly greater than acid-induced LDH release when not under OGD conditions (see FIG. 8A, p <0.05). Addition of the ASIC1a inhibitor PcTX toxin (100 ng / mL) significantly induced acid / OGD-induced LDH release to 44.3% + 5.3% (n = 5, p <0.05). , FIG. 9D).

The same experiment was performed using cultured neurons derived from ASICl7 ^{− / −} mice. However, unlike the case of neurons containing ASICI, in ASIC1 ^{− / −} neurons, treatment with OGD and acid for 1 hour only slightly increased LDH release (26.1% + 2.7% to 30.4% + 3.5%, N = 10-12, FIG. 9D). This finding suggests that the increase in acid-induced damage by OGD can be a major cause of the increase in OGD-mediated toxicity by OGD.

It has been shown previously that activation of Ca ²⁺ permeable cation conductivity activated by reactive oxygen / nitrogen species can cause glutamate receptor-dependent nerve damage (Aarts et al, Cell, 2003). , 1 15: 863-877). Cell damage induced by long-term OGD can be dramatically reduced by either agents that directly recover free radicals (eg, Trolox) or agents that reduce free radical production (eg, L-NAME) . To investigate whether the combination of short-term OGD and acidosis that caused nerve damage involved a similar mechanism, we examined the effect of Trolox and L-NAME on OGD / acid-induced LDH release. As shown in FIG. 9D, neither Trolox (500 μM) or L-NAME (300 μM) had a significant effect on nerve damage induced by the 1 hour OGD / acidosis combination (n = 8-11). Additional experiments also showed that the ASIC inhibitors amiloride and PcTX toxin had no effect on the conductivity of the TRPM7 channel (Aarts et al, supra). Taken together, these findings strongly suggest that neuronal damage induced by the 1 hour OGD / acidosis combination, but not the TRPM7 channel, may play a major role in our study. To do.
(G) Activation of ASIC1a in ischemic brain injury in vivo FIG. 10 shows data representing ASICI inhibition and neuroprotection by ASICI gene knockout in cerebral ischemia in vivo. FIG. 10A shows a graph of typical data obtained from TTC-stained brain sections, which shows aCSF (n = 7), amiloride (n = 11), or PcTX toxin (n = 5). The stained volume in the brain from the injected rat (“infarct volume”) is shown. ^* P <0.05 and ^** p <0.01 compared to the group injected with aCSF. FIG. 10B shows a graph of exemplary data representing a reduction in infarct volume in brains from ASIC1 ^{− / −} mice (n = 6 for each group). ^* Compare with groups with p <0.05 and ^** p <0.01 + / +. FIG. 10C shows a decrease in infarct volume in brains derived from mice administered intraperitoneally with 10 mg / kg memantine (Mem) or with intraventricular administration of PcTX toxin (500 ng / mL). A typical data graph is shown. ^** p <0.01 Compared to aCSF injection and compared to memantine and memantine plus PcTX toxin (n = 5 for each group). FIG. 10D shows a graph of typical data representing a reduction in infarct volume in brains derived from either ASIC1 ^{+ / +} (wild type) or ASIC1 ^{− / −} mice administered memantine intraperitoneally (in each group). n = 5). ^* P <0.05, and ^** p <0.01.

Protective effects of amiloride and PcTX venom in a rat model of transient focal ischemia (Longa et al., Stroke, 1989, 20: 84-91), ASIC1a activation in vivo ischemic brain injury Tested to find out if involved in. Ischemia (100 minutes) was induced by transient middle cerebral artery occlusion (MCAO). A total of 6 μl artificial CSF (aCSF) alone, aCSF-containing amiloride (1 mM), or PcTX toxin (500 ng / mL) was injected into the ventricle 30 minutes before and 30 minutes after ischemia. The volume of intraventricular fluid and spinal fluid in 4 week old rats is estimated to be approximately 60 μl. Since the injected amiloride and PcTX appear to be uniformly distributed in the CSF, a concentration of −100 μM for amiloride and −50 ng / mL for PcTX is expected and this concentration is useful in cell culture experiments. This is the concentration that has been found to be. Infarct volume was measured by TTC staining at 24 hours after ischemia (Bederson et al., Stroke, 1986, 17: 1304-1308). Ischemia (100 min) resulted in an infarct volume of 329.5 + 25.6 mm ³ in rats infused with aCSF (n = 7), but rats infused with amiloride (n = 11, p <0.05). infarct volume) rats were injected with only 229.7 + 41.1 mm ³ and PcTX venom in (n = 5, p in <0.01) 130.4 + 55.0mm ³ (about 60% decrease) (FIG. 10A).

ASIC1 ^{− / −} mice were used to further demonstrate the involvement of SIC1a in ischemic brain injury in vivo. Male ASIC1 ^{+ / +} , ASIC1 ^+/− , and ASIC1 ^{− / −} mice (approximately 25 g, with a genetically similar C57B 16 background) were prepared as previously described (Stenzel-Poore et al, Lancet, 2003, 362: 1028-1037) and subjected to MCAO for 60 minutes. Consistent with protection by pharmacological inhibition of ASIC1a (above), − / − mice compared to + / + mice (84.6 + 10.6 mm ³ , N = 6, p <0.01). Showed a significantly smaller (about 61% decrease) infarct volume (32.9 + 4.7 mm ³ , N = 6). +/− also showed a reduced infarct volume (56.9 + 6.7 mm ³ , N = 6, p <0.05) (FIG. 10B).

To investigate whether inhibition of the ASIC1a channel or knockout of the ASIC1 gene could provide further protection in vivo, memantine (10 mg / kg) immediately after 60 minutes of MCAO in the presence of established glutamate receptor inhibition Was injected intraperitoneally (ip) in C57B 16 mice and brains of 0.4 μl total volume of aCSF alone or aCSF (500 ng / mL) containing PcTX toxin 15 min before and 15 min after ischemia. With room injection (i.c.v.). In control mice that received saline intraperitoneally and aCSF administered intraventricularly, MCAO for 60 minutes induced an infarct volume of 123.6 + 5.3 mm ³ (n = 5, FIG. 10C). In mice that received memantine intraperitoneally and aCSF intraventricularly, ischemia during the same period induced an infarct volume of 73.8 + 6.9 mm ³ (n = 5, p <0.01). However, mice injected with memantine and PcTX venom induced only 47.0 + 1.1 mm ³ infarct volume (n = 5, p <0.01 compared to both control and memantine groups, FIG. 10C). ). These data suggest that homomeric ASIC1a may provide additional protection in in vivo ischemia in the presence of established NMDA receptor inhibition. Further protection was also observed in ASIC1 ^{− / −} mice treated with pharmacological NMDA inhibition (FIG. 10D). In ASIC ^{+ / +} mice injected intraperitoneally with saline or 10 mg memantine per kg body weight, MCAO for 60 minutes is 101.4 + 9.4 mm ³ or 61.6 + 12.7 mm ³ , respectively. Infarct volume was induced (n = 5 in each group, FIG. 10D). However, in ASIC1 ^{− / −} mice injected with memantine, an infarct volume of 27.7 + 1.6 mm ³ (n = 5) was induced in the same ischemic period, and infarcts in ASIC1 ^{+ / +} mice injected with memantine It was significantly smaller than the volume (p <0.05).

Taken together, these data indicate that activation of Ca ²⁺ permeable ASIC1a is a novel glutamate-dependent biological mechanism underlying ischemic brain injury.

Example 2: Time window of PcTX neuroprotection This example describes a typical experiment that measures the neuroprotective effect of PcTX venom at different times after the onset of stroke in rodents (see Figure 11). Briefly, in rodents, cerebral ischemia (stroke) was induced by middle cerebral artery occlusion (MCAO). At the indicated time after induction, artificial cerebrospinal fluid (aCSF), PcTX venom (0.5 μL, 500 ng / mL total protein), or inactivated (boiled) venom is added to each rodent side. Injection into the ventricle. As shown in FIG. 11, administration of PcTX toxin resulted in a 60% reduction in stroke volume both at 1 and 3 hours after the onset of stroke. Furthermore, substantial stroke volume reduction can still be maintained if treatment is withdrawn for 5 hours after the onset of MCAO. Thus, neuroprotection by ASIC inhibition can have a long therapeutic time window after the onset of stroke, whereby stroke subjects can benefit from treatment performed hours after the stroke begins. This effect of ASIC inhibition on neuroprotection against stroke is much stronger than the effect of calcium channel inhibition of the NMDA receptor (a major target for experimental stroke treatment) using glutamate antagonists. To date, there have been no glutamate antagonists that exhibit such a selective profile as demonstrated by ASIC1a selective inhibition.

Example 3 Exemplary Cystine Knot Peptides This example describes exemplary cystine knot peptides such as full-length PcTx1 and deletion derivatives of PcTx, which are screened in cultured cells and are used in ischemic animals (eg, Rodents such as mice or rats) and / or administered to ischemic human subjects.

FIG. 12 shows the primary amino acid sequence (SEQ ID NO: 1) of a typical cystine knot peptide, PcTx1, designated as 50, represented by the one-letter code, and this peptide is represented in various ways compared to amino acid positions 1-40. It has typical peptide characteristics. Peptide 50 may include six cysteine residues that form cystine bonds 52, 54, 56 that give rise to a cystine knot motif 58. The peptide can also include one or more β-sheet regions 60 and positively charged regions 62. N-terminal region 64 and C-terminal region 66 may be adjacent to the cystine knot motif.

FIG. 13 shows a comparison of the PcTx1 peptide 50 of FIG. 12 aligned with various typical deletion derivatives of this peptide. These derivatives include N-terminal deletion 70 (SEQ ID NO: 2), partial C-terminal deletion 72 (SEQ ID NO: 3), complete C-terminal deletion 74 (SEQ ID NO: 4), and / or N / C-terminal deletion 76 (SEQ ID NO: 5) may be included. Other derivatives of PcTx1, for example, make any deletion, insertion, or substitution of one or more amino acids while maintaining at least about 25% or about 50% sequence similarity or identity of the original PcTx1 sequence. May be included.

Each PcTx1 derivative may be tested for its ability to selectively inhibit ASIC proteins and / or for effects on ischemia, if any. Any suitable test system can be used to perform this test, examples of which include any cell-based assay system and / or an animal model system described elsewhere in the present teachings. . PcTx1 derivatives can also or alternatively be tested in ischemic human subjects.

Example 4: Selectivity of PcTX toxin against ASIC1a This example demonstrates that PcTX toxin (and thus PcTx1 toxin) against ASIC1a alone compared to other ASIC proteins or combinations of ASIC proteins expressed in cultured cells. An experiment for measuring the selectivity of is described. COS-7 cells expressing the designated ASIC protein were treated with PcTX toxin (25 ng / mL in ASIC1a expressing cells and 500 ng / mL in ASIC2a, ASIC3 or ASIC1a + 2a expressing cells). Channel current was measured at a pH of half the maximum channel activation state (pH 0.5). As shown in FIG. 14, PcTX toxin inhibits most of the current mediated by ASIC1a homomeric channels at a protein concentration of 25 ng / mL and at 500 ng / mL, homomeric ASIC2a, ASIC3, or heteromers. There was no effect on the current mediated by the sex ASIC1a / ASIC2a (n = 3-6). At 500 ng / mL, PcTX venom is also mediated by other ligand-dependent channels (eg, NMDA and GABA consumer-dependent channels) and voltage-dependent channels (eg, Na ⁺ , Ca ²⁺ , and K ⁺ channels s) There was no effect on the current to be generated (n = 4-5). These experiments indicate that the PcTX toxin, ie, the PcTx1 peptide, is a specific inhibitor for homomeric ASIC1a. Using this cell-based assay system, the potency and selectivity of ASIC inhibition can be measured for various synthetic peptides or other candidate inhibitors (see, eg, Example 3).

Example 5: Nasal administration of PcTX toxin that is neuroprotective This example illustrates the effectiveness of nasally administering PcTX toxin to reduce ischemia-induced damage in an animal model system of stroke Enter the data. Intracerebral ischemia was induced by middle cerebral artery occlusion in male mice. One hour after initiating occlusion, animals were treated as controls or treated with PcTX venom (50 μL of 5 ng / mL PcTx venom (total protein) introduced nasally). As shown in FIG. 15, nasal administration of PcTX toxin resulted in a 55% reduction in ischemia-induced damage (ischemic damage), as defined by infarct volume, compared to control treatment. It was done. Nasal administration may be via a spray deposited as a substance in the nasal cavity rather than being inhaled into the lung and / or via an aerosol that is at least partially inhaled into the lung. In some examples, nasal administration may have many advantages over other routes of administration, such as more effective delivery to the brain and / or suitability for self-administration by ischemic subjects.

Example 6 Inhibition of ASIC1a Channels by Amiloride and Amiloride Analogs As shown in FIG. 16, amiloride and amiloride analogs benzamyl, phenamyl and EIPA inhibit ASIC1a current in a dose-dependent manner. Similarly, amiloride and the amiloride analogs benzamyl and EIPA inhibit the ASIC2a current in a dose-dependent manner (FIG. 17). Table 1 summarizes the inhibition of the ASIC1a channel by amiloride and amiloride analogs. Amiloride was an effective inhibitor of this channel with an IC ₅₀ of 7.7 μM.

Example 7 Reduction of Infarct Volume in Mice by Intraventricular Injection of Amiloride and Amiloride Analogue Mice were subjected to the middle cerebral artery occlusion (MCAO) described above for 60 minutes. Amiloride or amiloride analog benzamyl, bepridil, EIPA or KB-R7943 was administered by intracerebroventricular injection 1 hour after MCAO. Animals were evaluated one day after ischemia induction. As shown in FIG. 18, intracerebroventricular infusion of amiloride or the amiloride analog benzamyl, bepridil, EIPA or KB-R7943 effectively reduces infarct volume.

Example 8: Reduction of infarct volume in mice by intravenous infusion of amiloride Mice were subjected to the middle cerebral artery occlusion (MCAO) described above for 60 minutes. Amiloride was administered by intravenous infusion one, three or five hours after MCAO. Animals were evaluated one day after ischemia induction. As shown in FIG. 19, intravenous infusion of amiloride effectively reduces infarct volume. The effective penetration of amiloride into the CNS can be explained by the fact that the blood brain barrier is prone to subsequent cerebral ischemia / reperfusion. FIG. 20 shows that intravenous infusion of amiloride has a long therapeutic time window of 5 hours.

Example 9 Structure-Activity Relationship of Hydrophobic Amiloride Analogues on Various Channels As shown in Table 1, substitution of the C5 amino group of amiloride with an alkyl group resulted in an increase in the potency of the ASIC1a channel. The same substitution increased the effect of the ASIC3 channel (Kuduk et al, Bioorg. Med. Chem. Lett., 2009, 19: 2514-2518). The opposite result was obtained when a hydrophobic group was substituted on the guanidino portion of the structure. Indeed, benzamyl, a benzyl substituted guanidino analog, was the most potent ASIC1a inhibitor compound tested (IC50 = 4.9 μΜ). Taken together, these results indicated that amiloride is an effective inhibitor of ASUC1a with an IC ₅₀ of 7.7 μM. They also provide structure-activity relationships for designing amiloride analogs that can inhibit the ASIC1a channel (see FIG. 21). Thus, in some embodiments, amiloride analogs are generated by introducing changes to the guanidine portion of the amiloride structure. Since amiloride is only a very weak inhibitor of the Na ⁺ / Ca ²⁺ ion exchanger (IC50 = 1.1 mM), the amiloride analog is likewise a very weak inhibitor of the Na ⁺ / Ca ²⁺ ion exchanger. There is a possibility. In some embodiments, the amiloride analog is designed to have a high selectivity for ASIC1a compared to the ASIC3 channel. In other embodiments, a ring structure, such as a cyclic guanidine group, is introduced into the amiloride structure to increase the ASIC1a current inhibitory activity. It is also possible for one or more NH groups of amiloride to form hydrogen bonds internally with the amino group or ion channel at the 3-position.

In vivo results in mice showed that efficacy could be achieved at plasma concentrations of 32.5 μΜ (50 μl × 1 mM iv dose) and 12.5 μΜ total brain concentration (1 μl × 500 μΜ icv dose). That is, it is estimated that only a 10-fold increase in concentration is required to obtain an effective concentration suitable for acute treatment for human stroke. Therefore, novel analogs are screened for increased ASIC1a IC ₅₀ concentrations from values of 4-8 μM for amiloride and benzamyl to values lower than 1 μM.

In some embodiments, the amiloride analog comprises a methylated analog of benzamyl (Formulas 1-5 of FIG. 21) and an amidino analog of Benzamyl (Formula 6 of FIG. 21). In other embodiments, the amiloride analog comprises a single ring formed on the guanidine group. In other embodiments, the amiloride analog comprises an acyl guanidino group for increased inhibitory potency against the ASIC1a current.

Amiloride is dissolved in water at 1 mM and is effective in treating ischemia in a mouse model at a dose of 50 μl per injection. An equivalent dose in mg / kg based on a 65 kg human would be close to 40 mg and require an infusion dose of more than 160 ml. Similarly, benzamyl has a reported solubility of 0.4 mg / ml (1.7 mM) in 0.9% saline so that only 5 mg of benzamyl dihydrochloride in a 10 ml injection. It can only be administered. Therefore, amiloride analogs with high water solubility are desirable. In some embodiments, the amiloride analog includes a water-soluble group in the guanidino group, such as an N, N-dimethylamino group or a sugar, to improve water solubility. In some embodiments, the amiloride analog has a water solubility of 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM or more. In other embodiments, the amiloride analog is administered at a dose of 10 mg, 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, or 500 mg intravenously in a human in a single 10 ml injection. It has the solubility that makes it possible. In still other embodiments, the amiloride analog is administered at a dose of 10 mg, 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, or 500 mg into the human ventricle in a single 2 ml injection. It has a solubility that makes it possible to

The above disclosure can encompass one or more separate inventions, as well as independent applications. Each of these inventions will be described in its preferred form. These preferred forms include specific embodiments thereof as disclosed and shown herein, but are intended to be construed in a limited sense, since numerous variations are possible. is not. The subject matter of the present invention includes all novel and non-obvious combinations and subcombinations of the various elements, features, functions and / or properties disclosed herein. The following claims particularly point out certain combinations and subcombinations regarded as novel and non-obvious. Inventions embodied in other combinations and subcombinations of features, functions, elements and / or properties may be claimed in an application claiming priority from this or a related application. Such claims may relate to different inventions, to the same invention, or to the original claims, whether they are broad, narrow, the same or different. It is considered to be included within the subject matter of the disclosed invention.

Claims (6)

A pharmaceutical composition for reducing nerve damage in the central nervous system of an adult human subject comprising an amiloride analog or a pharmaceutically acceptable salt thereof, wherein the amiloride analog comprises phenamyl, bepridil, 5- ( N-methyl-N-isobutyl) amylolide, 5- (N, N-hexamethylene) amylolide and 5- (N, N-dimethyl) amylolide hydrochloride,
Said pharmaceutical composition, wherein the pharmaceutical composition is administered intravenously within 5 hours of the onset of an ischemic event and the nerve injury is acidosis-induced nerve injury. 2. The pharmaceutical composition of claim 1, wherein the amiloride analog or a pharmaceutically acceptable salt thereof is given in a dosage range of 0.1 mg to 10 mg per kg body weight. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is administered within 1 hour of the onset of an ischemic event. The said pharmaceutical composition of Claim 1 administered within 1 to 5 hours of onset of an ischemic event. The pharmaceutical composition according to claim 1, wherein the nerve damage is brain damage. 2. The pharmaceutical composition of claim 1, wherein the amiloride analog or pharmaceutically acceptable salt thereof is given in a dosage range of 0.1 mg to 30 mg per kg body weight.