JP2020079289A - Composition for reducing nervous system injury and methods of making and using the same - Google Patents

Abstract

To provide a composition comprising an amiloride and/or an amiloride analog which can be used for reducing nerve injury or nervous system injury in a subject.SOLUTION: A pharmaceutical composition for reducing nerve injury in the central nervous system of an adult human subject comprises an amiloride analog, or a pharmaceutically acceptable salt thereof. The amiloride analog is selected from the group consisting of phenamil, bepridil, 5-(N-methyl-N-isobutyl)amiloride, 5-(N,N-hexamethylene)amiloride and 5-(N,N-dimethyl)amiloride hydrochloride. The composition is administered intravenously within 5 hours of the onset of an ischemic event. The nerve injury is acidosis-induced nerve injury.SELECTED DRAWING: Figure 20

Description

The present application relates to the field of neurology. In particular, the present 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 conditions, including chemical and physical damage to the nervous system. Many types of nerve damage result in altered ion influx into nerve cells, thereby causing nerve cell death. Therefore, various ion channels may be candidates to mediate this change in ion flux, resulting in a reduced degree of nerve damage.

One aspect of the present application relates to a method for reducing nerve damage in a subject. The method comprises 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 intracerebroventricularly.

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 a related embodiment, the amiloride analog is benzamil, phenmil, 5-(N-ethyl-N-isobutyl)-amiloride (EIPA), bepridil, KB-R7943, 5-(N-methyl-N-. It is selected from the group consisting of isobutyl) amiloride, 5-(N,N-hexamethylene)amiloride and 5-(N,N-dimethyl)amiloride hydrochloride. In another related embodiment, the amiloride analog is benzamil. In another related embodiment, the amiloride analog is a methylated analog of benzamil. In another related embodiment, the amiloride analog comprises one ring formed on the guanidine group. In another related embodiment, the amiloride analog comprises an acylguanidino group. In another related embodiment, the amiloride analog comprises a water-soluble group formed on a guanidine group, which water-soluble group is a Ν,Ν-dimethylamino group or a sugar group.

In some embodiments, amiloride, an amiloride analog, or a pharmaceutically acceptable salt thereof is provided in a dose range of 0.1 mg to 10 mg/Kg body weight.

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

In some embodiments, the amiloride analog is benzamil, phenmil, EIPA bepridil, KB-R7943, 5-(N-methyl-N-isobutyl) amiloride, 5-(N,N-hexamethylene)amiloride. And 5-(N,N-dimethyl)amiloride hydrochloride. In a related embodiment, the amiloride analog is benzamil. In other embodiments, the amiloride analog is a methylated analog of benzamil, an amiloride analog containing one ring formed on a guanidine group, an amiloride analog containing an acylguanidino group, and a guanidine group. A water-soluble group which 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 altered ion influx into nerve cells. The method comprises 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, intracerebroventricularly or intramuscularly.

In other embodiments, the amiloride analog is benzamil, phenmil, EIPA bepridil, KB-R7943, 5-(N-methyl-N-isobutyl) amiloride, 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 benzamil. In other embodiments, the amiloride analog is a methylated analog of benzamil, an amiloride analog containing one ring formed on a guanidine group, an amiloride analog containing an acylguanidino group, and a guanidine group. A water-soluble group which 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 comprises administering a therapeutically effective amount of a pharmaceutical composition comprising amiloride, an amiloride analog, or a pharmaceutically acceptable salt thereof to a subject in need of such treatment.

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

In other embodiments, the amiloride analog is benzamil, phenmil, EIPA bepridil, KB-R7943, 5-(N-methyl-N-isobutyl) amiloride, 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 benzamil. In other embodiments, the amiloride analog is a methylated analog of benzamil, an amiloride analog containing one ring formed on a guanidine group, an amiloride analog containing an acylguanidino group, and a guanidine group. A water-soluble group which 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 done.

In some embodiments, the pharmaceutical composition comprises an amiloride analog or a pharmaceutically acceptable salt thereof, wherein the amiloride analog is benzamil, phenmil, 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 benzamil, one of which is formed on a guanidine group. A ring-containing amiloride analog, an acylguanidino group-containing amiloride analog, and a water-soluble group formed on a guanidine group, which is a Ν,Ν-dimethylamino group or a sugar group. Selected from the group consisting of the body.

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 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, intravenously, intrathecally, Formulated for intracerebroventricular or intramuscular injection.

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

FIG. 1 is a flow chart diagram illustrating an exemplary method for reducing nerve damage in a subject under ischemia. FIG. 2 is a flow chart 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 on the electrophysiology and pharmacology of acid-sensing ion channel (ASIC) proteins in cultured mouse cortical neurons. FIG. 4 is a further series of graphs representing representative data on electrophysiology and pharmacology of ASIC proteins in cultured mouse cortical neurons. FIG. 5 is a combination of graphs and traces representing representative data showing that an ischemia model can promote the activity of ASIC proteins 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 depicting typical data showing that ASIC1a may be involved in acid-induced damage in vitro. FIG. 10 is a series of graphs with data showing 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 time and type of treatment. FIG. 12 is a diagram of the primary amino acid sequence of an exemplary cystine-knot peptide, PcTx1, along with various exemplary peptides characterized. FIG. 13 is a comparative diagram aligning the cystine-knot peptide of FIG. 12 with various exemplary deletion derivatives of this peptide. FIG. 14 is an exemplary graph plotting the magnitude of calcium current measured in cells as a function of ASIC family members expressed in cells. FIG. 15 is a graph depicting typical data regarding the efficacy of nasal administration of PcTx venom in reducing ischemic damage in an animal model system. FIG. 16: Representative ASIC1a current traces in CHO cells treated with benzamil (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 by (panel C). FIG. 17: Representative ASIC2 current traces in CHO cells treated with benzamil (panel A) or 5-amiloride (panel B) and expressed in CHO cells by amiloride and amiloride analogs (panel C). FIG. 6 is a composite diagram showing dose-dependent inhibition of ASIC2 current. FIG. 18 is a graph showing reduction of infarct volume in mice by intracerebroventricular injection of amiloride or an amiloride analog. FIG. 19 is a composite diagram showing a reduction in infarct volume in mouse cortical tissue by intravenous infusion of saline or amiloride 60 minutes after MCAO. FIG. 20 is a composite diagram showing reduction of infarct volume in cortical tissues of mice by intravenous infusion of saline or amiloride 3 hours 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 movements, the sympathetic nervous system and the parasympathetic nervous system, and the autonomic nervous system that controls involuntary functions, and , All the 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, neurons, glial cells, astrocytes, cerebrospinal fluid (CSF) cells, interstitial cells, spinal protective coat cells, epidural cells. Cells (ie, cells outside the dura), cells in non-neural tissue that are adjacent to, in contact with, or distributed to neural tissue, epineurium, perineurium, endometrium, chordae, Contains cells such as bundles.

In some embodiments, the nerve injury is nervous system injury. In other embodiments, the nerve injury is brain injury. In some embodiments, the nerve damage is nervous system damage caused by altered ion influx into nerve cells. For example, stroke/cerebral ischemia is a major cause of morbidity and mortality. Post-synaptic glutamate receptor hyperactivation and subsequent Ca2+ toxicity play an important role in ischemic brain injury. The present application shows that activation of Ca2+ permeable acid-sensing ion channels (ASIC) is involved in acidosis-induced, glutamate receptor-independent ischemic brain injury and targets ASIC family members (ASIC). To provide a new direction for neuroprotection. The present application further provides novel inhibitors of ASICs 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 comprises 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 intracerebroventricularly.

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 a related embodiment, the amiloride analog is benzamil, phenmil, 5-(N-ethyl-N-isobutyl)-amiloride (EIPA), bepridil, KB-R7943, 5-(N-methyl-N-. It is selected from the group consisting of isobutyl) amiloride, 5-(N,N-hexamethylene)amiloride and 5-(N,N-dimethyl)amiloride hydrochloride. In another related embodiment, the amiloride analog is benzamil. In another related embodiment, the amiloride analog is a methylated analog of benzamil. In another related embodiment, the amiloride analog comprises one ring formed on the guanidine group. In another related embodiment, the amiloride analog comprises an acylguanidino group. In another related embodiment, the amiloride analog comprises a water-soluble group formed on a guanidine group, which water-soluble group is a Ν,Ν-dimethylamino group or a sugar group.

In some embodiments, amiloride, an amiloride analog, or a pharmaceutically acceptable salt thereof is provided in a dose range of 0.1 mg to 10 mg/Kg body weight. In other embodiments, the pharmaceutical composition is administered within 1 hour of the onset of the ischemic event, within 5 hours of the onset of the ischemic event, or within 1-5 hours of the onset of the ischemic event.

Another aspect of the present application relates to a method for treating brain injury in a subject. The method comprises 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 intracerebroventricularly.

In some embodiments, the amiloride analog is benzamil, phenmil, EIPA bepridil, KB-R7943, 5-(N-methyl-N-isobutyl) amiloride, 5-(N,N-hexamethylene)amiloride. And 5-(N,N-dimethyl)amiloride hydrochloride. In a related embodiment, the amiloride analog is benzamil. In other embodiments, the amiloride analog is a methylated analog of benzamil, an amiloride analog containing a single ring formed on a guanidine group, an amiloride analog containing an acylguanidino group, and a water-soluble group is Ν. , Ν-dimethylamino group or sugar group, selected from the group consisting of amiloride analogs containing one water-soluble group formed on the guanidine group.

Another aspect of the present application relates to a method for reducing nervous system damage caused by altered ion influx into nerve cells. The method comprises 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 comprises the step of administering a therapeutically effective amount of an active ingredient selected from the group consisting of amiloride, amiloride analogs, and salts thereof, intravenously or intrathecally to a subject in need of such treatment. The methods of the present application may provide one or more advantages over other methods of treating ischemia. These benefits include, among other things, (1) less ischemia-induced injury, (2) less therapeutic side effects (eg, by selecting a more specific therapeutic target), and/or (3) efficacy. Longer window of treatment may be included.

FIG. 1 shows a flowchart 20 with exemplary steps 22, 24 that may be performed in a method of reducing nerve damage in a subject in 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). The ASIC selective inhibitor may then be administered to the ischemic subject (shown in step 24). Administration of an inhibitor to an ischemic subject can be performed in a amount that is therapeutically effective for the subject to reduce ischemia-induced damage, eg, an amount that reduces the amount of brain damage resulting from stroke.

A possible explanation for the efficacy of the ischemia treatment of FIG. 1 may be provided by the data of the teachings herein (see, eg, Example 1). In particular, the damaging effects of ischemia may not be comparable to acidosis, ie, tissue/cell acidification due to ischemia may not be sufficient to cause ischemia-induced damage. Rather, ischemia-induced damage can often be caused by calcium influx into cells mediated by ASIC family members, particularly ASIC1a. Thus, selective inhibition of ASIC1a channel activity may reduce this deleterious calcium influx and thereby reduce ischemia-induced damage.

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

Nerve Damage The present application relates to pharmaceutical compositions and methods for reducing nerve damage in a subject. As used herein, the term "nerve damage" refers to physical interactions or trauma, bruising or compression or surgical trauma, pharmacological damage to blood vessels such as hemorrhagic or ischemic damage, or nerve damage. By degenerative or any other neurological disease, or any other factor that causes damage or adverse symptoms of nervous system tissues or cells is meant acute or chronic damage or adverse conditions to nervous system tissues or cells. In some embodiments, the nerve damage is caused by a cognitive disorder, a psychiatric disorder, a neurotransmitter-mediated disorder or a neuropathy. 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, such as neuronal death, or is implicated. Disease or condition associated with or related to the disease. In this case, the central feature of the disease may be cognitive (eg memory, attention, cognitive and/or thinking) disorders. These diseases include cognitive disorders caused by pathogens, such as HIV-related cognitive disorders or Lyme disease-related cognitive disorders. In some embodiments, the cognitive impairment is degenerative cognitive impairment. 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). , Aging memory impairment (AAMI) and epilepsy.

As used herein, the term "mental disorder" refers to and is intended to those mental disorders that are believed to cause or cause abnormal thoughts and cognition. Mental disorders are characterized by loss of reality, delusions, hallucinations (they have the perception of reality in the sense that they are clear, substantive, and located in an external, objective space) Consciousness and awareness in the absence of stimuli), changes in personality and/or disorganized thinking. Other common symptoms include abnormal or bizarre behaviour, as well as difficulties in social interaction and impairment of activities of daily living. Typical mental disorders are schizophrenia, bipolar disorder, psychotic 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, and norepinephrine, or of aminergic G protein-coupled receptors. Disorders are referred to or intended to be diseases or conditions that are believed to be associated with or related to, or related to or related to. Typical neurotransmitter-mediated disorders include spinal cord injury, diabetic neuropathy, allergic disease, and aging protection such as senile hair loss (alopecia), senile weight loss and senile visual impairment (cataract). Diseases associated with the geroprotective activity are mentioned. Abnormal neurotransmitter levels are associated with a variety of 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, psychotic disorders, depression and various allergic disorders.

As used herein, the term "neuropathy" is believed to be associated with, or associated with, nerve cell death and/or impairment of nerve cell function or reduction of nerve cell function, or a disease associated with or associated with it. Or, it refers to symptoms and intends them. Typical neurological indications include Alzheimer's disease, Huntington's chorea, amyotrophic lateral sclerosis (ALS), Parkinson's disease, canine cognitive impairment syndrome (CCDS), Lewy body disease, Menkes disease, Wilson disease, Crotzfeldt-Jakob disease, neurodegenerative diseases and conditions such as Farre's disease, acute or chronic diseases related to cerebral circulation such as ischemic or hemorrhagic stroke, or damage due to cerebral hemorrhage, aging memory loss (AAMI), Mild cognitive impairment (MCI), traumatic mildness (MCI), post-concussion syndrome, traumatic stress syndrome, adjuvant chemotherapy, traumatic brain injury (TBI), neuronal cell death-mediated eye disease, macular degeneration, old age Macular degeneration, autism (including autism spectrum syndrome), Asperger's syndrome, and Rett's syndrome, avulsion injury, spinal cord injury, myasthenia gravis, Guillain-Barre syndrome, multiple sclerosis, diabetes Sexual neuropathy, fibromyalgia, spinal cord injury-related neuropathy, schizophrenia, bipolar disorder, psychotic disorders, anxiety or depression, and chronic pain.

In some embodiments, nerve damage or nervous system damage is caused by altered ion influx into nerve cells or tissue. As used herein, the term "nervous tissue" refers to animal tissue including nerve cells, neural networks, glial cells, neuroinflammatory cells, and endothelial cells in contact with "nervous tissue". .. A “nerve cell” can be any type of nerve cell known to those of skill in the art, including, but not limited to, a neuron. As used herein, the term "neuron" refers to cells of ectodermal embryonic origin derived from any part of the animal's nervous system. Neurons express well-characterized neuron-specific markers such as the nerve fiber proteins NeuN (neuronal nuclear marker), MAP2, and class III tubulin. Included as nerve cells 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. Is a cell. "Glial cells" useful in the present invention include, but are not limited to, astrocytes, Schwann cells, and oligo dendritic cells. "Neuroinflammatory cells" useful in the present invention include cells of bone marrow origin such as, but not limited to, 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 conditions. Ischemia, as used herein, is a condition in which blood flow to organs and/or tissues is reduced. Decreased blood flow is due to many mechanisms such as partial or complete blockage (occlusion), stenosis (compression), and/or leakage/rupture of one or more blood vessels that provide blood flow to organs and/or tissues. Can be triggered. Ischemia can be caused by thrombosis, embolism, atherosclerosis, hypertension, bleeding, aneurysm, surgery, trauma, drugs and the like. Thus, diminished blood flow can be chronic, transient, acute or sporadic.

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

Ischemia-related symptoms can be the result of any of the ischemia. This result may be substantially coincident with the onset of ischemia (eg, a direct effect of ischemia) and/or may occur substantially after the onset of ischemia, and/or Alternatively, it may occur after ischemia has ended (eg, indirect 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 in addition, the symptoms may include local and/or systemic acidosis (lower pH), hypoxia (lower oxygen), free radical production, and the like.

In some embodiments, the ischemia-related condition is stroke. Stroke, as used herein, is cerebral ischemia caused by a decrease in blood supply to part (or all) of the brain. Symptoms caused by stroke can be sudden (eg, loss of consciousness) or have gradual onset over hours or days. Further, the stroke may be, in particular, a major ischemic stroke (complete stroke) or a lesser major transient ischemic stroke. Symptoms caused by a stroke include, for example, hemiplegia, hemiplegia, numbness on one side, weakness on one side, paralysis on one side, temporary weakness in the limbs, pain in the limbs, confusion, difficulty speaking, Difficulty understanding speech, difficulty seeing with one or both eyes, blurred vision, loss of vision, dizziness, dizziness, falling tendency, loss of physical coordination, sudden severe headache, wheezing, and/or Loss of consciousness. Alternatively, or additionally, the symptom may be a test and/or device, such as an ischemic blood test (eg, testing for mutated albumin, specific protein isoforms, damaged proteins, etc.), electrocardiogram, electroencephalogram, exercise It can be detected more easily or only by stress tests, brain CT or MRI scans and the like.

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

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

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 having a blood flow, including rodents (mouse, rat, etc.), dogs, cats, birds, sheep, goats, non-human primates, etc. .. The animal may be treated for its own purpose, eg, for veterinary purposes (eg, as treatment of pets). Alternatively, animals may use animal models of nerve injury, such as ischemia, to facilitate testing of drug candidates for human use, such as to study potency of drug candidates, time windows of efficacy, side effects, etc. Can be provided.

Ischemic subjects for treatment may be selected according to any suitable criteria. Typical criteria are any detectable ischemic symptom, history of ischemia, events that increase (or induce ischemia) ischemia (eg, surgery, 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 is ischemia that occurred at least about 1, 2, or 3 hours prior to initiation of treatment, or more than about 1, 12, or 6 hours prior to initiation of treatment. Multiple previous ischemic episodes (eg, transient ischemic attacks) may have occurred.

ASIC inhibitors, amiloride and amiloride analogs Inhibitors of ASIC family members, as used herein, refer to one or more of the ASIC family, that is, in particular ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, and ASIC4. A substance that reduces (partially, substantially, or completely) the activity of a member. In some examples, the inhibitor is one such as the ability of the cell membrane to pass an ion (eg, sodium, calcium, and/or potassium, for example) across the cell membrane (inside and/or outside the cell). It may reduce the channel activity of one or more members. The substance may be a compound (small molecule less than about 10 kDa, peptide, nucleic acid, lipid, etc.), a complex of two or more compounds, and/or a mixture. Furthermore, the substance may inhibit ASIC family members by any suitable mechanism, in particular 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 described in Waldmann, R.; , Et al. 1997, Nature 386, pp. 173-177, which is incorporated herein by reference.

The expression “ASIC1a inhibitor” refers to a product that is potentially or practically pharmaceutically useful as ASIC1a within the scope of proper pharmacological judgment, and includes pharmaceutically active species, Includes references to substances described, promoted, 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 after each exposure to the same (submaximal) concentration of inhibitor (eg, in cultured cells). It is substantially stronger inhibition in ASIC1a than in the case. The inhibitor is an ASIC1a relative to at least one other ASIC family member (such as ASIC1b, ASIC2a, ASIC2b, ASIC3, ASIC4, etc.) and/or relative to all other ASIC family members. Can be selectively inhibited. The strength of inhibition of a selective inhibitor is determined by the Ki value (inhibition constant) when compared to the inhibitor concentration at which inhibition occurs (eg, IC ₅₀ (inhibitor concentration that produces 50% of maximal inhibition)) or different ASIC family members. Or the dissociation constant). An ASIC1a selective inhibitor is at least about 2-fold, 4-fold, or 10-fold lower than the concentration for inhibition of at least one other ASIC family member or all other ASIC family members (1/2, 4 ASIC1a activity can be inhibited at concentrations of 1-fold, or 10-fold or less). Thus, an ASIC1a selective inhibitor is at least about 2-fold, 4-fold, or 10-fold lower than the concentration for inhibition of at least one other ASIC family member and/or all other ASIC family members (2 min. 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 can also be specific for ASIC1a. ASIC1a-specific inhibition, as used herein, is an inhibition that is substantially exclusive to ASIC1a as 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-fold lower (5% concentration or less) compared to inhibition of all other ASIC family members. Thus, an ASIC1a-specific inhibitor is, for example, at least about 20-fold lower (5% or less), such that inhibition of other ASIC family members is at least substantially (or completely) undetectable, It may have an IC ₅₀ and/or a K _i for ASIC1a inhibition compared to all other members of the ASIC family. It may inhibit ASIC1a at inhibitor concentrations. In some embodiments, the ASIC1a selective inhibitor has high potency against homomeric ASIC1a channels and is a commercially available amiloride-related ASIC1a inhibitor, eg, amiloride benzamil, phenamil, 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- It has high water solubility compared to 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 ASIC1a selective inhibitor and an inhibitor for a non-ASIC channel protein such as a non-ASIC calcium channel. it can. In some examples, the subject can be treated with an inhibitor of the NMDA receptor, such as an ASIC1a selective inhibitor and a glutamate antagonist.

The inhibitor may be a peptide or may include a peptide. The peptide will typically have any suitable number of amino acid subunits of at least about 10 and 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. Peptides having these cysteines form a “knot part” containing (1) a ring formed by two disulfide bonds and their linking skeleton, and (2) a third disulfide bond passing through the ring. You can. In some examples, the peptide can be a conotoxin from arachnids and/or conch snails. For example, the peptide may be the venom PcTx1 (psalmotoxin 1) from the tarantula (Psalmopoeus cambridgeii (Pc)).

In some instances, 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, peptides 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 aspects of peptides that may be suitable as inhibitors are described in Example 3 below.

Amino acid sequence alignment methods for comparing and creating amino acid sequence identity and similarity scores are well known in the art. Typical alignment methods that may be suitable include Smith and Waterman's “Best Fit”, Needleman and Wunsch's homology alignment algorithm “GAP”, Pearson and Lipman's similarity method “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 sequences 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 may be other with similar chemical properties (eg, charged or hydrophobic). It is expected to be substituted with an amino acid residue, resulting in less (or no) effect on the functional properties of the molecule. Where the sequences differ by conservative substitutions, the percent sequence identity may be adjusted upwards to provide "similarity" in the sequences, which modifies the conservative characteristics of the substitutions. For example, each conservative substitution may be scored as partial rather than as complete mismatch, thereby modifying 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 carried out in any suitable method, such as the program PC/GENE (Intelligenetics, Mountain View, Calif, USA). As described by Meyers and Miller, Computer Application. Biol Sci. , 4:11-17 (1988).

Amiloride is a pyrazine derivative containing a guanidine group, which has been used for the treatment of mild hypertension, and few side effects have been reported. Amiloride acts by directly inhibiting the epithelial sodium channel (ENaC), thereby inhibiting sodium reabsorption in the distal tubule end of the kidney, the connecting duct, and the collecting duct. It also promotes the loss of sodium and water from the body, but not potassium. As used herein, the term "amiloride" refers to both amiloride and salts of amiloride, such as amiloride hydrochloride.

Amiloride analog, as used herein, refers to a compound that has a slightly altered chemical structure but similar biological activity to amiloride. Examples of amiloride analogs include, but are not limited to, benzamil, 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)amiloride hydrochloride. As another example, 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 benzamil, formed on a guanidine group. A single ring-containing amiloride analog, an acylguanidino group-containing amiloride analog, and a water-soluble group formed on a guanidine group, the water-soluble group being an N,N-dimethylamino group or a sugar group. Is an amiloride analog. In some embodiments, the amiloride analog does not inhibit the 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 maintain 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 the ASIC2a and/or ASIC3 channels. In one embodiment, the amiloride analog exhibits high selectivity for ASIC1a compared to ASIC3 and/or ASIC2 channels.

As used herein, the term "amiloride analog" refers to both amiloride analogs and salts of amiloride analogs, such as 5-(N,N-dimethyl)amiloride hydrochloride.

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

Administration of Inhibitors Administration (or administration), as used herein, includes any suitable condition, and any route of exposure of the subject to the inhibitor at any suitable number of times. Administration can be self-administration or by someone else, such as a medical practitioner (eg, doctor, nurse, etc.). Administration includes infusion (especially, eg, intravenous, intramuscular, subcutaneous, intracerebral, intracerebroventricular, and/or intrathecal), ingestion (eg, using capsules, troches, liquid compositions, etc.), inhalation (eg, Aerosols that are inhaled through the nose and/or mouth (mean droplet diameters less than about 10 microns), absorption through the skin (eg, by skin patches), and/or transmucosal (especially, eg, buccal, nasal) And/or pulmonary mucosa) and the like. Mucosal administration can be accomplished, for example, by spraying (eg, nasal spray), aerosol for inhalation and the like. The spray may be a surface spray (droplets with an average diameter greater than about 50 microns) and/or a spatial spray (droplets with an average diameter of about 10-50 microns). .. In some examples, ischemia results in changes in the blood-brain barrier of an ischemic subject, such that an inhibitor introduced into the bloodstream of the subject (eg, by infusion and/or absorption) reaches the brain. Efficiency can be increased. Administration can be performed once or as treatment. Thus, administration may occur before ischemia is detected (eg, prophylactically), after a minor ischemic episode, during chronic ischemia, after a full stroke, and the like. In some embodiments, the amiloride or amiloride analog is administered intravenously. In other embodiments, the amiloride or amiloride analog is administered intracerebrally. 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, the amiloride or amiloride analog is administered intrathecally.

A therapeutically effective amount (or simply "effective amount") of 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, is the extent, incidence, and/or extent 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 (generally compared to a control subject group). The therapeutically effective amount of the inhibitor or inhibitors may be given in a single dose or in multiple doses of 0.1 to 50 ml per dose.

In some embodiments, the inhibitor is amiloride, an amiloride analog or a salt thereof, and 0.01 to 30 mg/Kg body weight, 0.01 to 10 mg/Kg body weight, 0.01 to 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 1 kg body weight, 0.03 to 3 mg per 1 kg body weight, 0.03 to 1 mg per 1 kg body weight, 0.03 to 0.3 mg per 1 kg body weight, 0.03 to 0.1 mg per 1 kg body weight, 0.1 to 30 mg per kg body weight, 0.1 to 10 mg per kg body weight, 0.1 to 3 mg per kg body weight, 0.1 to 1 mg per kg body weight, 0.1 to 0.3 mg per kg body weight, 1 kg per kg body weight 0.3-30 mg, 0.3-10 mg/kg body weight, 0.3-3 mg/kg body weight, 0.3-1 mg/kg body weight, 1-30 mg/kg body weight, 1-10 mg/kg body weight, 1 kg/kg body weight It is given as a daily dose (as a single dose or multiple doses) in the range of 1 to 3 mg, 3 to 30 mg per kg body weight, 3 to 10 mg per kg body weight or 10 to 30 mg per kg body weight. In one embodiment, the amiloride analog is benzamil, phenamil, EIPA, bepridil, KB-R7943, 5-(N-methyl-N-isobutyl)-amiloride, 5-(N,N-hexamethylene)-amiloride 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, the amiloride analog is formed on a methylated analog of benzamil, an amiloride analog containing one ring formed on a guanidine group, an amiloride analog containing an acylguanidino group, and a guanidine group. Selected from the group consisting of amiloride analogs, which are N,N-dimethylamino groups or sugar groups.

In other embodiments, the inhibitor is amiloride, an amiloride analog or a salt thereof, and is 0.1-1000 mg/dose, 0.1-300 mg/dose, 0.1-100 mg/dose, 0.1-1000 mg/dose. 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- 30 mg/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/ It is administered as a pharmaceutical composition formulated as a single dose in the range of 100-1000 mg/dose, 100-300 mg/dose, or 300-1000 mg/dose. In one embodiment, the amiloride analog is benzamil, phenamil, EIPA, bepridil, KB-R7943, 5-(N-methyl-N-isobutyl)-amiloride, 5-(N,N-hexamethylene)-amiloride 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, the amiloride analog is formed on a methylated analog of benzamil, an amiloride analog containing one ring formed on a guanidine group, an amiloride analog containing an acylguanidino group, and a guanidine group. Selected from the group consisting of amiloride analogs, which are N,N-dimethylamino groups or sugar groups. In some embodiments, the pharmaceutical composition is formulated for intravenous, intracerebral, intracerebroventricular, intrathecal 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), preservatives, one or more excipients, colorants, flavors, salts, defoamers and the like. The inhibitor may be present in the vehicle in a concentration that, when administered to an ischemic subject, provides a therapeutically effective amount of the inhibitor for the treatment of ischemia.

In some embodiments, amiloride analogs having greater water or fat solubility are produced. In certain embodiments, the amiloride analog comprises a water-soluble group in the guanidino group, such as a Ν,Ν-dimethylamino group or a sugar, to improve water solubility (Formulas 13-16, Figure 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 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 single 10 ml human dose. It has the solubility that makes it possible. In still other embodiments, the amiloride analog is intraventricularly 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 in a single 2 ml human dose. It has the solubility that enables

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

The screen may include any suitable assay system that measures the interaction between the ASIC protein and a set of candidate inhibitors. Typical assay systems include assays that are performed biochemically (eg, binding assays), particularly with cells grown in culture (“cultured cells”) and/or with organisms. May be included.

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

One or more ASIC inhibitors may be administered to a subject having nerve injury, eg, a subject in ischemia, to test the efficacy of the inhibitor for the treatment of nerve injury. The subject having an ischemic state may be a human or an animal. In some examples, an ischemic subject may provide an animal model system in ischemia and/or stroke.
Typical animal model systems include rodents (particularly mice and/or rats) with experimentally induced ischemic conditions. The ischemic state may, in particular, be induced mechanically (eg surgically) and/or by administration of a drug. In some instances, the ischemic condition may be induced by occlusion of a blood vessel, eg, stenosis 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, an amiloride analog, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, and the pharmaceutical composition is 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 benzamil, phenmil, EIPA bepridil, KB-7943, 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 comprises a methylated analog of benzamil, a single ring formed on a guanidine group. Comprising an amiloride analogue comprising, an amylolide analogue comprising an acylguanidino group, and an amiloride analogue comprising a water-soluble group formed on a guanidine group, the water-soluble group being 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 comprises 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 amiloride analog or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

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

In one embodiment, the amiloride analog is a methylated analog of benzamil. In another embodiment, the amiloride analog comprises one ring formed on the guanidine group. In another embodiment, the amiloride analog comprises an acylguanidino group. In yet another embodiment, the amiloride analog comprises a water-soluble group formed on the guanidine group, which is a N,N-dimethylamino group or 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 comprises PcTx1 or a PcTx1 derivative.

As used herein, "pharmaceutically acceptable carrier" includes 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 absorbing agents that may be necessary to prepare a particular therapeutic composition. It can be prepared from various substances. The use of such media and agents in combination with pharmaceutically active substances is well known in the art. Its use in therapeutic compositions is intended unless any conventional medium or agent is incompatible with the active ingredient. Optionally, the amiloride and/or amiloride analog may be mixed together in a pharmaceutical composition comprising a co-active ingredient that is not contraindicated with the amiloride and/or amiloride analogs described above.

In some embodiments, the pharmaceutical composition is formulated for intravenous infusion. In other embodiments, the pharmaceutical composition comprises amiloride and/or amiloride analog formulated for intravenous infusion. In other embodiments, the pharmaceutical composition comprises amiloride and/or amiloride analog formulated for intracerebroventricular injection. In other embodiments, the pharmaceutical composition comprises amiloride and/or amiloride analog formulated for intrathecal injection. In other embodiments, the pharmaceutical composition comprises amiloride and/or amiloride analog formulated for intramuscular injection. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include saline, bacteriostatic water, Cremophor EL ^™ (BASF, Parsippany, NJ) or liquids 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 (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the particle size required in the dispersed state and by the use of surfactants. Prevention of microbial activity can be achieved by various antibacterial and antifungal agents such as 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 injectable compositions is brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the amiloride and/or amiloride analog in the required amount in a suitable solvent, followed by sterile filtration. 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 lyophilisation, which gives the active ingredient and any additional ingredients from that solution which have been previously sterile filtered. Be done.

In some embodiments, the pharmaceutical composition is provided in dry form and is formulated in 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 manufactured from any pharmaceutically acceptable substance such as gelatin or a cellulose derivative.

In certain embodiments, the pharmaceutical composition is formulated for immediate release, sustained release, delayed release or combinations thereof. Sustained release, also known as extended release, timed or period release, modified release (CR), modified release (MR), continuous release (CR or Contin), slowly dissolves and releases the active ingredient over a period of time. This is the mechanism used in tablets or capsules, which is a pharmaceutical product. 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 that they maintain more stable drug levels in the bloodstream, which results in longer 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 fixed rate by maintaining a constant drug level for a specified period of time. This can be accomplished with a variety of formulations, including, but not limited to, liposomes, drug-polymer conjugates such as 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 sustained release of the rest of the active ingredient. In one embodiment, the pharmaceutical composition is formulated as a powder so that the immediate release active ingredient can be taken. In another embodiment, the pharmaceutical composition is formulated in liquid, gel, suspension or emulsion form. Such liquids, gels, suspensions or emulsions may be ingested by the subject in their neat form or encapsulated in capsules.

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

The following examples describe selected aspects and embodiments of the teachings of the present application, and 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 purposes of illustration and should not be construed as limiting the scope of the teachings herein.

Example 1: Neuroprotection in ischemia by inhibiting calcium permeable acid-sensing ion channels This example demonstrates the role of ASIC1a in mediating ischemic injury and the ability of ASIC1a inhibitors to reduce ischemic injury. The experiment showing is described (see FIGS. 2 to 10). Ca2+ toxicity may play a central role in ischemic brain injury. The mechanism by virulent Ca2+ loading in cells occurring in the ischemic brain has failed to show effective neuroprotection in stroke in multiple human trials of glutamate antagonists, so It became unclear. 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 channels (ASIC), which can induce 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 infusion of the ASIC1a inhibitor or knockout of the ASIC1a gene may result in an antagonist effect.

The normal brain requires the complete oxidation of glucose to meet its energy requirements. During ischemia, depletion of oxygen transforms the brain into anaerobic glycolysis. Accumulation of lactic acid as a by-product 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 system. To date, six 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 Neuronal cell culture After anesthesia with halothane, cerebral cortex was excised from E16 Swiss mice or PI ASIC1 ^+/+ and ASIC1 ^−/− mice and incubated with 0.05% trypsin-EDTA for 10 minutes at 37°C. Incubate. The tissue was then minced with a fire-polished glass pipette and placed in poly-L-ornithine coated 24-well plates or 25x25 mm glass coverslips at a density of 2.5x10 ⁵ cells per well or 10 ⁶ cells per coverslip. I plated. Neurons were cultured with MEM supplemented with 10% horse serum (for E16 culture) or Neurobasal medium supplemented with B27 (for PI culture) and used 12 days later for electrophysiology and toxicity studies. The proliferation of glial cells was suppressed 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) was (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 more reliable pH buffer effect. The patch electrode contained (in 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 damage assay-LDH measurement Cells were washed 3 times with ECF and randomly divided into treatment groups. MK801 (10 μΜ), CNQX (20 μΜ), and nimodipine (5 μΜ) were added to all groups to eliminate potential secondary activation of glutamate receptors and voltage-gated Ca ²⁺ channels. After the acid incubation, neurons were washed and incubated at 37°C in Neurobasal medium. LDH release was measured in culture medium using the LDH assay kit (Roche Molecualar Biochemicals). Medium (100 μL) was transferred from culture wells to 96-well plates and mixed with 100 μL of 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 incubation 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 μM Fura-2-acetoxymethyl ester for 40 minutes at 22° C., washed 3 times, and usually For 30 minutes in ECF. The coverslips were transferred to the perfusion chamber on an inverted microscope (Nikon TE300). The cells were illuminated with a xenon lamp and observed using a 40× UV fluorescent oil immersion objective, and video images were acquired using a cooled CCD camera (Sensys KAF 1401, Photometries). The digitized images were acquired on a PC controlled by Axon Imaging Workbench software (Axon Instruments) and analyzed. The shutter and filter wheel (Lambda 10-2) were controlled by software that allowed the cells to be illuminated with the excitation wavelength of 340 or 380 nm for the appropriate time. Fura-2 fluorescence was detected at an emission wavelength of 510 nm. Ratio images (340/380) were analyzed by averaging the pixel ratio values in the localized area of cells in the visual field. Values were extrapolated to SigmaPlot for further analysis.

Fluorescein-diacetate stain and propidium iodide uptake Cells were incubated for 30 minutes in ECF containing fluorocein-diacetate (FDA) (5 μΜ) and propidium iodide (PI) (2 μΜ) then dye free. It was 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% confluence, cells were co-transfected with FuGENE6 transfection reagent (Roche Molecular Biochemicals) with cDNA for ASIC and GFP in pc ^DNA3 vector. 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 in the presence of G418 for more than 5 generations. The cells were then examined for ASIC1a expression 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 glucose-free ECF of 6.0. 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, a slight decrease in pH from 7.38 to 7.28 (n=3) and 6.0 to 5.96 (n=4) with 1 hr OGD did.

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

Animals were killed 24 hours after ischemia with chloral hydrate. The brain was rapidly removed, coronally excised at 1 mm (mouse) or 2 mm (rat) intervals, and immersed in vital pigment (2%) 2,3,5-triphenyltetrazolium hydrochloride (TTC). To dye. The infarct area was calculated by subtracting the normal area stained with TTC in the ischemic hemisphere area from the non-ischemic hemisphere area. The infarct volume was calculated by summing the infarct areas of all parts and multiplying by the thickness of the section. Injection into the rat ventricle is stereotactic using a microsyringe pump with a cannula inserted stereotactically at the 0.8 mm posterior part of the cross suture, the 1.5 mm lateral part of the midline, and the 3.8 mm abdomen of the dura. Made by fixed technique. All manipulations and analyzes were performed by individuals blind to treatment groups.

Results (a) Acidosis activates ASICs in mouse cortical neurons Figures 3 and 4 show typical data on the electrophysiology and pharmacology of ASICs in cultured mouse cortical neurons. 3A and 3B are graphs showing the pH dependence of the ASIC current activated by the pH falling from 7.4 to the pH value shown. Dose-response curves were fitted to Hill's formula using a mean pH _0.5 of 6.18 + 0.06 (n=10). 3C and 3D are graphs showing current-voltage relationships for ASICs (n=5). ASIC current magnitudes at various voltages were normalized to the magnitude recorded at -60 mV. 4A and 4B are graphs showing dose-dependent inhibition of ASIC currents by amiloride. IC50 = 16.4 + 4.1 μΜ, N = 8. 4C and 4D are graphs showing inhibition of ASIC currents by PcTX venom. ^** P<0.01.

ASIC currents in cultured mouse cortical neurons were recorded (see Figure 3). At a maintenance potential of -60 mV, a rapid decrease in extracellular pH (pHe) to below 7.0 causes a large transient inward current with a small steady-state component in many neurons. (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 linear IV relationship and a reversal approximating the Na ⁺ equilibrium potential were obtained (n=6, FIGS. 3C and 3D). These data indicate that lowering 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 Figure 4). As shown in FIG. 4, amiloride dose-dependently inhibited ASIC currents 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 currents in cortical neurons is shown in Figures 4C and 4D. At 100 ng/mL, PcTX venom reversibly inhibited the peak size of the ASIC current to 47% + 7% (n=15, FIGS. 4C and 4D), which resulted in acid activation of the homomeric ASIC1a. A large 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 co-present in these neurons.

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

Since acidosis may 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 features of these channels ( (See FIG. 5). ASIC currents in neurons were recorded 1 hour after oxygen glucose deprivation (OGD). Briefly, a set of cultured cells was washed three times with glucose-free cell fluid (ECF) and subjected to OGD, whereas control cultured cells were washed with glucose-containing ECF. And subjected to incubation in a normal cell culture incubator. OGD was terminated after 1 hour by exchanging the glucose-free ECF for Neurobasal medium and incubating the culture in a normal incubator. ASIC currents were then recorded 1 hour after OGD if no neuronal morphology changes occurred. OGD treatment induced a modest increase in the magnitude of ASIC currents (1520 + 138 pA in control group, N=44; 1886 + 185 pA in nerve cells 1 h after ODA, N=40, p<0.05, figure). 5A and 5B). More importantly, OGD induced a dramatic reduction in ASIC-sensitive inhibition shown by an increase in the time constant of current decrease (814.7 + 58.9 ms in control neurons, N=6; after OGD. 1928.9 + 315.7 ms, N = 6, p <0.01, in Figures 5A and 5C). In cortical neurons cultured from ASIC1 ^−/− mice, the decrease in pH from 7.4 to 6.0 was similar to that in previous studies in hippocampal neurons (Wemmie et al., 2002). The current in the direction also did not activate (n=52). In these neurons, 1 hour OGD did not activate or enhance the acid-induced response (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 may be FIG. 6A shows a typical trace obtained with 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 approximately -17 mV (n=5) after correction of the liquid field potential. FIG. 6B shows representative traces and summary data showing inhibition of Ca ²⁺ mediated currents by amiloride and PcTX venom. The Ca ²⁺ mediated current magnitude peak was reduced to 26% + 2% of control value by 100 μM amiloride (n=6, p<0.01) and 22% by 100 ng/mL PcTX venom. + 0.9% (n=5, p<0.01). FIG. 7A shows a typical 340/380 nm ratio as a function of pH, showing the increase in [Ca ²⁺ ] _i with decreasing pH to 6.0. Neurons contain 1.3 mM CaCl ₂ with voltage-dependent Ca ²⁺ channel inhibitors (5 μΜ nimodipine and 1 μΜ ω-conotoxin MVIIC) and glutamate receptors (10 μΜ MK801 and 20 μΜ CNQX). Dipped in normal ECF. The inset of FIG. 7A shows a typical inhibition of acid-induced increase in [Ca ²⁺ ] _i by 100 μM amiloride. FIG. 7B shows typical summary data showing inhibition of acid-induced increase in [Ca ²⁺ ] _i by amiloride and PcTX venom. N=6-8, ^** p<0.01 Compared to 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, showing a lack of acid-induced [Ca ²⁺ ] _i increase in ASIC1 ^−/− neurons, which It shows that a normal reaction was shown to NMDA (n=8).

The Ca ²⁺ permeability of ASICs in cortical neurons was determined by standard ion substitution protocols (Jia et al, Neuron, 1996, 17:945-595) and Fura-2 fluorescent Ca ²⁺ imaging techniques (Chu et al, 2002, J. 87:2555-2561). Using a dipping solution containing 10 mM Ca ²⁺ (no Na ⁺ and K ⁺ ) as the sole charge carrier, at a holding potential of −60 mV, 15 out of 18 neurons, an inward direction greater than 50 pA The current was recorded. This shows significant Ca ²⁺ permeability of ASICs in most cortical neurons (FIG. 6A). Consistent with activation of the homomeric ASIC1a channel, the current evoked by 10 mM Ca ²⁺ was greatly inhibited by both the non-specific ASIC inhibitor amiloride and the ASIC1a-specific inhibitor PcTX venom (FIG. 6B). .. The Ca ²⁺ mediated current magnitude peak was reduced to 26% + 2% of control value by 100 μM amiloride (n=6, p<0.01) and 22% by 100 ng/mL PcTX venom. + 0.9% (n=5, p<0.01). Presence of inhibitors of other major Ca ²⁺ entry pathways (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 out of 20 neurons responded to pH decrease with a detectable increase in intracellular Ca ²⁺ ([Ca ²⁺ ] _i ) concentration (FIG. 7A). Generally, [Ca ²⁺ ] _i remains elevated during long perfusion of low pH solutions. In some cells, the [Ca ²⁺ ] _i increase lasted much longer than the period of acid perfusion (FIG. 7A). A long-lasting Ca ²⁺ response may be due to the fact that the ASIC response in intact neurons may be less sensitive than the response in whole cell recordings, or as a result of ASIC-mediated Ca ²⁺ entry It suggests that Ca ²⁺ is released from storage. Preincubation of neurons with 1 μΜ thapsigargin partially inhibited the persistent component of Ca ²⁺ increase. This indicates that release of Ca ²⁺ from intracellular stores may also contribute to acid-induced intracellular Ca ²⁺ accumulation (n=6, data not shown). Similar to the current ^mediated by Ca ²⁺ ions (FIG. 6B), both [Ca ²⁺ ] _i peak and sustained increase were associated with homomeric ASIC1a involvement in acid-induced [Ca ²⁺ ] _i increase. Consistently, it is largely inhibited by amiloride and PcTX venom (Figures 7A and 7B, n=6-8). Knockout of the ASICI gene eliminated the acid-induced increase in [Ca ²⁺ ] _i 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, lowering the pH to 5.0 or 4.0 activated a detectable current 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-gated and NMDA, GABA receptor-gated channels (data not shown).

(D) ASIC Inhibition Protects Acidosis-Induced Glutamate-Independent Nerve Damage Figure 8 shows that acid incubation induces glutamate receptor-independent nerve damage protected by ASIC inhibition. Typical data suggestive of behavior are shown. 8A and 8B show the time induced by 1 hour (FIG. 8A) or 24 hour (FIG. 8B) incubation of cortical neurons in ECF at pH 7.4 (black bars) or 6.0 (white bars). 3 shows a graph representing typical data for dependent LDH release. N=20-25 wells, ^* p<0.05, and ^** p<0.01, compared to the group at the same time point, pH 7.4 (acid-induced nerves with fluorescein diacetate (FDA)) Injury was also analyzed by staining the cell bodies of living neurons, as well as propidium iodide (PI) staining of the nuclei of dead neurons, Figure 8C with 100 μΜ amiloride or 100 ng/mL PcTX venom. , N=20-27, ^* p<0.05, and ^** p<0.01) showing inhibition of acid-induced LDH release.MK801, CNQX, and nimodipine showed ECF in all experiments. Contained therein (FIGS. 8A-C).

Acid-induced damage was allowed to proceed on 24 wells in ECF containing MK801, CNQX, and nimodipine at either pH 7.4 or 6.0 (see Figure 8). Cell damage was determined by measuring 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 cells/death. Assays were performed by fluorescent staining of cells. Incubation with acid for 1 h (pH 6.0) induced a time-dependent increase in LDH release compared to treatment of neurons with pH 7.4 (Fig. 8A). After 24 hours, a maximal LDH release of 45.7% + 5.4% was induced (n=25 wells). Sequential treatment with pH 6.0 induced greater cell damage (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 with 1 h of acid treatment (data not shown). Incubation with ECF at pH 6.5 for 1 hour also induced a significant but lesser amount of LDH release compared to that with ECF at pH 6.0 (n=8 wells, data not shown).

The effects of amiloride and PcTX venom on acid-induced LDH release were tested to determine if ASIC activation was involved in acid-induced glutamate receptor-independent nerve injury. LDH release was significantly reduced by the addition of either 100 μΜ amiloride or 100 ng/mL PcTX venom 10 min before and during 1 h of acid incubation (Figure 8C). At 24 hours, LDH release was reduced from 45.3% + 3.8% to 31.1% + 2.5% with amiloride and 27.9% + 2.6% with PcTX venom ( 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, whereas long-term incubation with amiloride alone (eg, 5 hours) did not produce LDH. Increased release (n=8, data not shown).

(E) Homomeric ASIC1a activation is responsible for acidosis-induced damage. Figure 9 presents a series of representative 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 reduction of [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 μΜ) inhibited acid-induced LDH release in ASIC1a-transfected cells. ^* P<0.05 for 7.4 vs 6.0 and for 6.0 vs 6.0 + amiloride. FIG. 9C shows typical data demonstrating the lack of induced damage in ASIC ^−/− neurons and protection by amiloride and PcTX venom (n=8 in each group, 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 venom. MK801, CNQX, and nimodipine were included in the ECF in all experiments (Figure 9A-D).

Neurons were treated with ECF at pH 6.0 in the presence of normal or diminished [Ca ²⁺ ] _e to see if Ca ²⁺ entry 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, similar to ASIC1a inhibition by PcTX venom (n=11-12, p<0.01; FIG. 9A) (40). 0.0% + 4.1% to 21.9% + 2.5%). Ca ²⁺ free solutions were not tested as they can activate large inward currents via 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 , is that activation of Ca ²⁺ permeable ASIC1a is acid-induced. It suggests that it may be involved in nerve damage.

Acid damage of untransfected COS-7 cells and COS-7 cells transfected with ASIC1a was tested to provide further evidence that activation of ASIC1a is involved in acid damage. COS-7 is a commonly used cell line for expression of ASICs due to its lack of 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 after 24 hours of acid incubation. Treatment of untransfected COS-7 cells with ECF at pH 6.0 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, Figure 9B). However, in COS-7 cells stably transfected with ASIC1a, a 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).

The acid damage of CHO cells transiently transfected with cDNA encoding GFP alone or GFP+ASIC1a was also studied. After transfection (24-36 hours), cells were incubated with acidity (pH 6.0) for 1 hour and assayed for cell damage damage 24 hours after acid incubation. A 1 hour acid incubation significantly reduced viable GFP-positive cells in the GFP/ASIC1a group, but not in the GFP alone-transfected group (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, a 1 hour acid incubation of ASIC ^+/+ neurons at pH 6.0 induced substantial LDH release mediated by amiloride and PcTX venom (n=8-12). However, acid treatment of ASIC1 ^−/− neurons for 1 hour did not induce a significant increase in LDH release at 24 hours (13.8% + 0.9% vs. pH 7.4, and , 14.2% + 1.3% vs. 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 abrogated the effects of acid-induced LDH release of amiloride and PcTX venom (FIG. 9C, n=8 each). Furthermore, this showed that inhibition of acid-induced damage of cortical neurons by amiloride and PcTX venom was due to inhibition of 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) resulted in maximum LDH release at 24 hours. Induced 84.8% + 1.4% (n=4, FIG. 9C). This indicated a normal response to other cell injury processes.

(F) Modeled ischemia increases glutamate-independent neuronal injury induced by acidosis via ASIC The magnitude of ASIC current depends on the cellular components and neurochemistry of swollen cerebral ischemic cells. Can be augmented by the active ingredient, arachidonic acid, and lactic acid. Moreover, and more importantly, the desensitization of ASIC currents can be dramatically reduced by modeled ischemia (FIGS. 5A and 5C). Activation of ASICs in ischemic conditions is expected to result in greater neuronal damage. To test this hypothesis, neurons were subjected to acid treatment for 1 hour under oxygen and glucose deprivation (OGD) conditions. MK801, CNQX, and nimodipine were included in all solutions to inhibit glutamate receptor-mediated cell damage in cells associated with voltage-gated Ca ²⁺ channels and OGD. Incubation with ECF at pH 7.4 for 1 hour 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 1 hour OGD does not induce substantial cell damage by inhibition of glutamate receptors and voltage-gated Ca ²⁺ channels (Aarts et al., 2003). However, when combined with acidosis (pH 6.0), 1 hour OGD induced a maximal LDH release of 73.9% + 4.3% (n=5, FIG. 9D, p<0.01). , Which is significantly greater than the acid-induced LDH release without OGD conditions (FIG. 8A, see p<0.05). Addition of the ASIC1a inhibitor PcTX venom (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 with cultured neurons derived from ASICl7 ^−/− mice. However, unlike the case of neurons containing ASICI, in ASIC1 ^−/− neurons, 1 hour treatment with combined OGD and acid only slightly increased LDH release (26.1% + From 2.7% to 30.4% + 3.5%, N = 10-12, Figure 9D). This finding suggests that increased acid-induced damage by OGD may be a major cause of OGD-induced increase in ASCICI-mediated toxicity.

It has previously been shown that activation of Ca ²⁺ permeable cation conductivity activated by reactive oxygen/nitrogen species can result in glutamate receptor-dependent nerve damage (Aarts et al, Cell, 2003). , 1 15:863-877). Long-term OGD-induced cell damage can be dramatically reduced by either agents that directly scavenge 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 injury involved a similar mechanism, the effects of trolox and L-NAME on OGD/acid-induced LDH release were tested. As shown in FIG. 9D, neither trolox (500 μM) nor L-NAME (300 μM) showed 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 venom had no effect on the conductivity of the TRPM7 channel (Aarts et al, supra). Taken together, these findings strongly suggest that activation of ASIC, but not TRPM7 channels, may play a major role in our studies in nerve damage induced by the 1 hour OGD/acidosis combination. To do.
(G) Activation of ASIC1a in In Vivo Ischemic Brain Injury FIG. 10 shows data representing neuroprotection by ASICI inhibition and ASICI gene knockout in cerebral ischemia in vivo. FIG. 10A shows a graph of typical data obtained from TTC-stained brain sections showing that aCSF (n=7), amiloride (n=11), or PcTX venom (n=5). Shown is the stained volume ("infarct volume") in the brain from the injected rat. ^* P<0.05 and ^** p<0.01 Compared with aCSF infused group. FIG. 10B shows a graph of typical data representing a reduction in infarct volume in the brain from ASIC1 ^−/− mice (n=6 for each group). Compared to groups with ^* p<0.05 and ^** p<0.01 +/+. FIG. 10C shows reduction of infarct volume in the brain from mice intraperitoneally administered with 10 mg/kg memantine (Mem) or with intraventricular administration of PcTX venom (500 ng/mL). A graph of typical data to be presented is shown. ^** Compared to p<0.01 aCSF infusion and compared to memantine and memantine plus PcTX venom (n=5 for each group). FIG. 10D shows a graph of typical data showing a reduction in infarct volume in the brain from either ASIC1 ^+/+ (wild type) or ASIC1 ^−/− mice treated with ip memantine (in each group). n=5). ^* P<0.05, and ^** p<0.01.

The protective effect of amiloride and PcTX venom in a rat model of transient focal ischemia (Longa et al., Stroke, 1989, 20:84-91) was demonstrated by activation of ASIC1a in ischemic brain injury in vivo. Was tested to see if they were 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 venom (500 ng/mL) was infused into the ventricles 30 and 30 minutes before ischemia. The volume of intraventricular fluid and spinal fluid in 4-week-old rats is estimated to be approximately 60 μl. Since the infused amiloride and PcTX appear to be evenly distributed in CSF, a concentration of -100 μΜ for amiloride and -50 ng/mL for PcTX is expected, which is effective in cell culture experiments. Is the concentration found to be The 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 aCSF infused rats (n=7), but amiloride infused rats (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) Was generated (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 (about 25 g, with a congenic 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 the mice ^{(84.6 + 10.6mm 3, N =} 6, p <0.01) , A significantly smaller (about 61% reduction) 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 additional protection in vivo, memantine (10 mg/kg) was immediately followed by 60 minutes MCAO in the presence of glutamate receptor inhibition. Was intraperitoneally (ip) injected into C57B 16 mice and brains of 0.4 μl total volume of aCSF alone or aCSF (500 ng/mL) containing PcTX venom 15 min before and 15 min after ischemia. With room injection (icv). MCAO for 60 minutes induced an infarct volume of 123.6 + 5.3 mm ³ in control mice in which saline was administered intraperitoneally and aCSF was administered intracerebroventricularly (n=5, FIG. 10C). In mice treated with 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, only 47.0 + 1.1 mm ³ infarct volume was induced in mice injected with memantine and PcTX venom (n=5, p<0.01 compared to both control and memantine groups, FIG. 10C). ). These data suggest that the homomeric ASIC1a may provide additional protection in in vivo ischemia in the presence of established NMDA receptor inhibition. Additional protection was also observed in ASIC1 ^−/− mice treated with pharmacological NMDA inhibition (FIG. 10D). In ASIC ^+/+ mice intraperitoneally infused with saline or 10 mg memantine per kg body weight, MCAO for 60 min was 101.4 + 9.4 mm ³ or 61.6 + 12.7 mm ³ , respectively. Infarct volume was induced (n=5 in each group, Figure 10D). However, in the same ischemic period, the infarct volume of 27.7 + 1.6 mm ³ (n=5) was induced in memantine-injected ASIC1 ^−/− mice, and the infarct of memantine-injected ASIC1 ^+/+ mice was induced. 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 measuring the neuroprotective effect of PcTX venom at different times after the onset of stroke in rodents (see Figure 11). Briefly, cerebral ischemia (stroke) was induced by middle cerebral artery occlusion (MCAO) in rodents. At the indicated times after induction, artificial cerebrospinal fluid (aCSF), PcTX venom (0.5 μL, 500 ng/mL total protein), or inactivated (boiled) venom was applied to the side of each rodent. Injected into the ventricles. As shown in FIG. 11, administration of PcTX venom resulted in a 60% reduction in stroke volume both 1 and 3 hours after stroke onset. Furthermore, if treatment is withheld for 5 hours after the onset of MCAO, a substantial reduction in stroke volume may still be maintained. Thus, neuroprotection by ASIC inhibition can have a long therapeutic time window after stroke onset, which allows stroke subjects to benefit from treatments that occur 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, with glutamate antagonists. To date, no glutamate antagonist has shown 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 were screened in cultured cells and tested 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 by the single letter code 50, for various peptides shown relative to amino acid positions 1-40. Has typical peptide characteristics. Peptide 50 may include 6 cysteine residues forming cystine bonds 52, 54, 56 which result in a cystine knot motif 58. The peptide may also include one or more β-sheet regions 60 and positively charged regions 62. N-terminal region 64 and C-terminal region 66 may flank the cystine knot motif.

Figure 13 shows a comparison of the PcTx1 peptide 50 of Figure 12 aligned with various exemplary deletion derivatives of this peptide. These derivatives are 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-terminal. /C-terminal deletion 76 (SEQ ID NO:5). Other derivatives of PcTx1 include, for example, any deletion, insertion, or substitution of one or more amino acids while maintaining at least about 25% or about 50% sequence similarity or identity to the original PcTx1 sequence. May be included.

Each PcTx1 derivative may be tested for its ability to selectively inhibit the ASIC protein and/or for its effect on ischemia, if any. Any suitable test system can be used to perform this test, including any cell-based assay system and/or animal model system described elsewhere in the teachings of this application. .. The PcTx1 derivative may also, or alternatively, be tested in an ischemic human subject.

Example 4: Selectivity of PcTX venom for ASIC1a This example demonstrates that PcTX venom for ASIC1a alone (and thus, PcTx1 toxin) compared to other ASIC proteins or combinations of ASIC proteins expressed in cultured cells. Described below is an experiment for measuring the selectivity of. COS-7 cells expressing the designated ASIC protein were treated with PcTX venom (25 ng/mL in ASIC1a expressing cells and 500 ng/mL in ASIC2a, ASIC3 or ASIC1a+2a expressing cells). Channel currents were measured at pH at half maximal channel activation (pH 0.5). As shown in FIG. 14, PcTX venom inhibited most of the current mediated by the homomeric channel of ASIC1a 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 sexual ASIC1a/ASIC2a (n=3-6). At 500 ng/mL, PcTX venom also mediated through other ligand-gated channels (eg, NMDA and GABA demander-gated channels) and voltage-gated channels (eg, Na ⁺ , Ca ²⁺ , and K ⁺ channels s). There was no effect on the applied current (n=4-5). These experiments show that PcTX venom, the PcTx1 peptide, is a specific inhibitor of the 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 Neuroprotective PcTX Toxin This example illustrates the efficacy of nasal administration of PcTX toxin to reduce ischemia-induced injury in an animal model system of stroke. Enter the data. Intracerebral ischemia was induced in male mice by occlusion of the middle cerebral artery. One hour after the onset of occlusion, animals were treated as controls or treated with PcTX venom (50 μL of 5 ng/mL (total protein) PcTx venom is introduced intranasally). As shown in Figure 15, nasal administration of PcTX venom results in a 55% reduction in ischemia-induced injury (ischemic injury), as defined by infarct volume, as compared to control treatment. Was done. Nasal administration may be via a spray that deposits as a substance in the nasal cavity rather than by inhalation into the lungs and/or via an aerosol that is at least partially inhaled into the lungs. In some cases, nasal administration can have a number of advantages over other routes of administration, such as more effective delivery to the brain and/or suitability for self-administration by an ischemic subject.

Example 6: Inhibition of ASIC1a Channels by Amiloride and Amiloride Analogs As shown in FIG. 16, amiloride and the amiloride analogs benzamyl, phenamil and EIPA inhibit ASIC1a currents in a dose dependent manner. Similarly, amiloride and the amiloride analogs benzamil and EIPA inhibit ASIC2a currents in a dose-dependent manner (Figure 17). Table 1 summarizes the inhibition of ASIC1a channels 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 Intracerebroventricular Infusion of Amiloride and Amiloride Analogs Mice were subjected to the above-described middle cerebral artery occlusion (MCAO) for 60 minutes. Amiloride or the amiloride analog benzamil, bepridel, EIPA or KB-R7943 was administered by intracerebroventricular infusion 1 hour after MCAO. Animals were evaluated 1 day after induction of ischemia. As shown in Figure 18, intracerebroventricular infusion of amiloride or the amiloride analog benzamil, bepridel, 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 above-described middle cerebral artery occlusion (MCAO) for 60 minutes. Amiloride was administered by intravenous infusion 1, 3, or 5 hours after MCAO. Animals were evaluated 1 day after induction of ischemia. As shown in FIG. 19, intravenous infusion of amiloride effectively reduces infarct volume. Effective penetration of amiloride into the CNS may be explained by the fact that the blood-brain barrier is prone to cerebral ischemia/reperfusion. FIG. 20 shows that intravenous infusion of amiloride has a long therapeutic time window of 5 hours.

Example 9: Structure-Activity Relationships of Hydrophobic Amiloride Analogs for Various Channels As shown in Table 1, replacing the C5 amino group of amiloride with an alkyl group resulted in increased 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. In fact, the benzyl-substituted guanidino analog, benzamil, was the most potent ASIC1a inhibitor compound tested (IC50=4.9 μΜ). Taken together, these results indicate 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 ASIC1a channels (see Figure 21). Thus, in some embodiments, amiloride analogs are produced by introducing changes in the guanidine moiety 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 analogue likewise has a very weak inhibitor of the Na ⁺ /Ca ²⁺ ion exchanger. Could be In some embodiments, amiloride analogs are designed to have high selectivity for ASIC1a over ASIC3 channels. In other embodiments, ring structures, eg, cyclic guanidine groups, are introduced into the amiloride structure to increase the inhibitory activity of ASIC1a currents. It is also possible for one or more NH groups of amiloride to internally form a hydrogen bond with the amino group or the ion channel at the 3-position.

In vivo results in mice showed that efficacy could be achieved at a plasma concentration of 32.5 μM (50 μl×1 mM iv dose) and a total brain concentration of 12.5 μM (1 μl×500 μM 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, new analogs are screened for increased IC ₅₀ concentration of ASIC1a from values of 4-8 μM for amiloride and benzamil to values below 1 μM.

In some embodiments, the amiloride analogs include methylated analogs of benzamil (Equations 1-5 in Figure 21) and amidino analogs of benzamil (Equation 6 in Figure 21). In other embodiments, the amiloride analog comprises one ring formed on a guanidine group. In other embodiments, the amiloride analog comprises an acylguanidino group for increased inhibitory potency against ASIC1a currents.

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, requiring an infusion dose of more than 160 ml. Similarly, benzamil has a reported solubility of 0.4 mg/ml (1.7 mM) in 0.9% saline, which results in only 5 mg of benzamil dihydrochloride in a 10 ml infusion. 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 at the guanidino group, such as an N,N-dimethylamino group or 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 intravenously to a human in a single 10 ml infusion 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. It has the solubility that makes it possible. In still other embodiments, the amiloride analog is administered to human ventricle in a single 2 ml infusion 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. It has the solubility that enables

The above disclosure may encompass one or more separate inventions, as well as independent applications. Each of these inventions is described in its preferred form. These preferred forms, including the particular embodiments thereof as disclosed and illustrated herein, are intended to be construed in a limited sense, as numerous variations are possible. is not. The subject matter of the inventions 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 sub-combinations that are considered novel and non-obvious. Inventions embodied in other combinations and subcombinations of features, functions, elements and/or properties may be claimed in applications claiming priority from this or a related application. Whether such claims are broad, narrow, the same, or different from the original claims, whether they relate to different inventions, the same invention, or the original claims, 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-( Selected from the group consisting of N-methyl-N-isobutyl) amiloride, 5-(N,N-hexamethylene) amiloride and 5-(N,N-dimethyl) amiloride hydrochloride,
The above-mentioned pharmaceutical composition, which is administered intravenously within 5 hours of the onset of an ischemic event, and the nerve damage is acidosis-induced nerve damage. The pharmaceutical composition according to claim 1, wherein the amiloride analog or a pharmaceutically acceptable salt thereof is given in a dose range of 0.1 mg to 10 mg per kg body weight. The pharmaceutical composition according to claim 1, which is administered within 1 hour of the onset of an ischemic event. The pharmaceutical composition according to claim 1, which is administered within 1 to 5 hours after the onset of an ischemic event. The pharmaceutical composition according to claim 1, wherein the nerve damage is brain damage. The pharmaceutical composition according to claim 1, wherein the amiloride analog or a pharmaceutically acceptable salt thereof is provided in a dose range of 0.1 mg to 30 mg per kg body weight.