Method of administering spine-forming compounds

Abstract

Provided herein are methods for generating functional spines on a neuron, the method comprising transiently contacting the neuron with an effective amount of a spine-forming compound, wherein the transient contact initiates spine-forming activity resulting in the formation of new functional spines, and the effective amount of transient contact terminates after initiation and before the functional spines mature. Disclosed herein are methods for treating subjects suffering from neurodegenerative diseases or conditions that would benefit from spine formation. The disease or condition may be characterized by a loss of spine density in the diseased neurons, which in turn results in the loss or reduction of one or more functional properties in the subject.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit under § 119(e) of U.S. Provisional Application No. 63 / 456,685, filed on 3 April 2023, which is incorporated herein by reference in its entirety.

[0002] Provided herein are methods and medications for initiating spine formation in subjects that require it. [Background technology]

[0003] Human neurodegenerative diseases are among the most common age-related conditions. Many of these diseases result from a reduction in the ability of aging or diseased neurons to produce functional nerve spines and synapses in sufficient quantities to communicate with one another. A key underlying marker for many of these neurodegenerative diseases is the loss of adequate density of functional dendritic spines. The resulting pathology leads to one or more functional declines in memory, mobility, and other important human characteristics.

[0004] Current treatments for most or all human neurodegenerative diseases primarily work to slow further functional decline in patients. That is, these treatments restore previously lost functions. To slow the progression of the disease or its associated symptoms without causing significant recovery.

[0005] Furthermore, these treatments require the substantially continuous maintenance of therapeutic drug concentrations in the brain for optimal effectiveness. For example, acetylcholine is a neurotransmitter that, in addition to other functions, plays a role in muscle movement, short-term memory, and learning. Neurodegenerative diseases involving the loss of one or more of these functional attributes are often treated with cholinesterase inhibitors. Such inhibitors reduce the breakdown of acetylcholine in the brain and require the substantially continuous presence of an effective amount of the inhibitor. Nevertheless, the pathology of the disease persists without decline.

[0006] Many elderly patients with neurodegenerative diseases who are receiving treatment also have one or more additional conditions or illnesses that require drug therapy. This imposes further constraints on the attending physician, limiting the selection of medications used to avoid harmful drug interactions.

[0007] Given the increasing incidence of neurodegenerative diseases, at least partially attributable to the aging population, methods for treating such diseases are needed. Ideally, such methods would confer restorative properties, minimize harmful drug interactions, reduce severity, or eliminate side effects. Such methods would represent a paradigm shift in the treatment of these diseases. [Overview of the Initiative] [Means for solving the problem]

[0008] Disclosed herein are methods for treating subjects suffering from neurodegenerative diseases or conditions that would benefit from spine formation. The diseases or conditions may be characterized by a loss of spine density in the affected neurons, which in turn results in the loss or reduction of one or more functional properties in the subject.

[0009] The methods described herein initiate spine formation activity in mammalian neurons in a very short time (e.g., within one hour of administration). The formation of functional dendritic spines, which can form functional synapses with axons from other neurons, occurs over a longer period of several hours or more (e.g., about four hours). The resulting synapses restore functional communication between these neurons, resulting in the repair of some or more of the normal functional properties controlled by the neurons.

[0010] The compounds used in the methods described below have very short half-lives in mice, approximately 30 minutes or less, and in one case, the half-life is 24 minutes. Furthermore, the concentrations of these compounds in brain tissue and serum are extremely low, on the nanomolar level. Such properties result in a transient presence in the brain before being eliminated from the body. For example, in mice, approximately 96% of the drug is eliminated from the body within the first two hours after administration. Without being limited to any theory, we assume that the compounds used in the methods herein simply initiate or signal spine formation, and that the neurons independently continue the process through maturation.

[0011] In one embodiment, a method is provided for increasing spine density in aged or diseased neurons by initiating spine formation with such neurons through transient contact with the compounds described herein or a composition containing such compounds. Such transient contact initiates the repair of neural communication by increasing the number of functional dendritic spines (synapse-forming spines) on the neurons. Nevertheless, our in vivo study using the compound once daily for four days demonstrated a significant difference between treated diseased mice and untreated diseased mice. This is surprising and unexpected, as the in vivo concentration of the administered compound decreased by approximately 96% within the first two hours, demonstrating its effectiveness at the nanomolar level.

[0012] Again, without limiting to any theory, the compounds described herein stimulate both spine formation and dendritic spine formation, which are capable of forming functional synapses with axons from other neurons, following a transient contact ("guiding signal") after more than 96% of the compound is no longer present, resulting in a substantial portion of the biological transformation.

[0013] This combination of continued biological transformation after the elimination of most of the compounds, coupled with a short half-life, is intended to enable a reduction in drug side effects and / or prevention of potential drug interactions in subjects currently being treated with one or more additional pharmaceutical agents.

[0014] While not limited to any particular theory, the data suggest that the compounds described herein initiate spine-forming activity in neurons, resulting in the formation of spines capable of generating functional synapses. Although the half-lives of these compounds are relatively short in mammals, they are still capable of successfully initiating and maturing spine formation even after several half-lives have expired. This suggests that these compounds activate or turn on spine-forming activity, including maturation, which has been reduced or lost due to neurological conditions. The fact that such spine-forming activity, including maturation, occurs successfully after the in vivo concentration of the compound approaches zero suggests that initiating spine-forming activity is all that is required. Based on the above, the neurological conditions underlying the loss of spine density in affected neurons may be primarily due to the condition impairing the activation of spine-forming activity. If so, the ability of the compounds described herein to activate spine-forming activity addresses a major component of disease progression in which the loss of spine density in affected neurons leads to loss of function (e.g., loss of short-term memory, loss of muscle function, etc.).

[0015] In any case, the data herein demonstrate that the compounds described herein initiate spine-forming activity. Furthermore, this activity results in spine maturation and synapse formation with adjacent axons, despite rapid in vivo clearance of the activating compounds. Combined, these data provide important insights into the pathology of such neurological conditions, as well as methods and dosages for offsetting the spine density loss associated with that condition. Accordingly, the methods described herein provide a therapeutic protocol in which a unit dose of the compound is administered once or possibly twice daily to achieve spine-forming activity linked to synapse formation.

[0016] Given that the amount of compound required to initiate and produce functional spine-forming activity resulting in functional synapses is very limited, the methods and unit doses described herein significantly target the amount of compound required to induce mammalian spine formation, despite rapid in vivo clearance of the compound from mammals, and limited administration of as little as one or two doses per day. In other words, it is considered that only the mimicry of spine-forming activity is required to bring about the desired formation of functional synapses.

[0017] In one embodiment, a method for generating a functional spine on a neuron, wherein the neuron contains an effective amount of a compound of formula I: [ka] or transient contact with a pharmaceutically acceptable salt, isotope-enriched analog, tautomer, prodrug, stereoisomer, or mixture of stereoisomers thereof, In the formula, R is hydrogen, C1-C4 alkyl, halo, hydroxyl, amino, cyano, or nitro. q is between 2 and 8. This transient contact initiates spine-forming activity, which leads to the formation of new functional spines. A method is provided wherein an effective amount of said transient contact ends after initiation and before complete maturation of said functional spine.

[0018] In one embodiment, a compound, or a salt, stereoisomer, mixture of stereoisomers, or metabolite thereof, of formula I is provided, wherein the maximum concentration in brain tissue is about 30 nM or less.

[0019] In one embodiment, the compound is

Chemical formula

[0020] In one embodiment, a method for repairing neurons having a disease in a mammal suffering from a neurodegenerative disease, the method comprising administering to the mammal a once-daily dose of an effective amount of a compound of formula I:

Chemical formula

[0021] In one embodiment, an effective amount of the compound of formula I provides a concentration of up to 30 nM in brain tissue.

[0022] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof, wherein R is hydrogen, q is 4 or 6, mammal is human, and the effective dose is about 0.1 to 0.4 mg / kg, preferably about 0.1 to 0.4 mg / kg / day.

[0023] In one embodiment, the mammal is a human, and the compound of formula I is administered at a dose of about 0.25 mg / kg, preferably about 0.25 mg / kg / day.

[0024] In one embodiment, the neuronal disease or disorder is a neurodegenerative disease. In one embodiment, the neurodegenerative disease is selected from Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson's disease, frontotemporal dementia, and Huntington's disease.

[0025] In one embodiment, the neurodegenerative disease is Alzheimer's disease.

[0026] In one embodiment, neurodegenerative disease is recognized as being in the early or middle stage, but not the late stage, as these terms are recognized in the art.

[0027] In one embodiment, the administered dose is therapeutically effective in treating neuronal diseases or disorders.

[0028] In one embodiment, a method is provided for repairing aged or diseased neurons in mammals that require repair by initiating the formation of dendritic spines according to a method provided herein.

[0029] In one embodiment, in mammals suffering from reduced spine density and therefore impaired functional synapses, a unit dose for initiating the formation of stable functional synapses, wherein the unit dose is approximately 0.4 mg / kg / day or less of a compound of formula IA: [ka] or comprising a pharmaceutically acceptable salt thereof, isotope-enriched analogue, tautomer, prodrug, stereoisomer, or mixture of stereoisomers, q is between 2 and 8. A unit dose is provided in which the compound has a serum half-life of less than 3 hours, and furthermore, the unit dose provides stable functional synapse formation in the mammal.

[0030] In one embodiment, a method is provided for generating a functional spine on a neuron, wherein the neuron is subjected to an effective amount of a compound of formula I: [ka] or transient contact with a pharmaceutically acceptable salt, isotope-enriched analog, tautomer, prodrug, stereoisomer, or mixture of stereoisomers thereof, In the formula, R is hydrogen, C1-C4 alkyl, halo, hydroxyl, amino, cyano, or nitro. q is between 2 and 8. This transient contact initiates spine-forming activity, which leads to the formation of new functional spines. The method involves an effective amount of transient contact being terminated after initiation and before the functional spine is fully mature.

[0031] In one embodiment, a method is provided for initiating spine-forming activity in a mammal suffering from a neurological condition, characterized in that the neuron has impaired spine-forming activity, characterized in that it has reduced ability to initiate spine-forming activation resulting in the formation of new functional spines, wherein the neuron is activated by an effective amount of a compound of formula I: [ka] or a pharmaceutically acceptable salt thereof, isotope-enriched analogue, tautomer, prodrug, stereoisomer, or mixture of stereoisomers, In the formula, R is hydrogen, C1-C4 alkyl, halo, hydroxyl, amino, cyano, or nitro. q is between 2 and 8. This transient contact initiates spine-forming activity, which leads to the formation of new functional spines. The method involves an effective amount of transient contact being terminated after initiation and before the functional spine is fully mature. [Brief explanation of the drawing]

[0032] [Figure 1] This shows the escape latency (seconds, y-axis) versus the time in days (x-axis) in one embodiment according to Example 1. [Figure 2A] The frequency, duration in the zone (seconds), distance traveled (cm), and velocity (cm / s) (y-axis) of wild-type (WT) mice and 3xTg-AD mice treated with the vehicle and with compound 1 at 3 and 30 mg / kg, respectively, are shown in one embodiment according to Example 1. [Figure 2B] The frequency, duration in the zone (seconds), distance traveled (cm), and velocity (cm / s) (y-axis) of wild-type (WT) mice and 3xTg-AD mice treated with the vehicle and with compound 1 at 3 and 30 mg / kg, respectively, are shown in one embodiment according to Example 1. [Figure 2C] The frequency, duration in the zone (seconds), distance traveled (cm), and velocity (cm / s) (y-axis) of wild-type (WT) mice and 3xTg-AD mice treated with the vehicle and with compound 1 at 3 and 30 mg / kg, respectively, are shown in one embodiment according to Example 1. [Figure 2D] The frequency, duration in the zone (seconds), distance traveled (cm), and velocity (cm / s) (y-axis) of wild-type (WT) mice and 3xTg-AD mice treated with the vehicle and with compound 1 at 3 and 30 mg / kg, respectively, are shown in one embodiment according to Example 1. [Figure 3]The cumulative duration of inactivity (multiple values ​​may be shown) of WT and 3xTg-AD mice treated with the vehicle and with compound 1 at 3 and 30 mg / kg in one embodiment according to Example 1. [Figure 4] The PSD95 (Panels A1a-A1d), co-localization (Panels A2a-A2d), and SYN (Panels A3a-A3d) of WT and 3xTg-AD mice treated with the vehicle and with Compound 1 at 3 and 30 mg / kg in one embodiment according to Example 1 are shown. [Figure 5] The following shows the PSD95 (Figure 5A), SYN (Figure 5B), and colocalized PSD95 / SYN (Figure 5C) spots in WT and 3xTg-AD mice treated with vehicle and with compound 1 at 3 and 30 mg / kg in one embodiment according to Example 1: Lane A - WT vehicle; B - 3xTg-AD vehicle; C - 3xTg-AD compound 1, 3 mg / kg; D - 3xTg-AD compound 1, 30 mg / kg. [Figure 6] An image of a dendritic spine in one embodiment according to Example 1 is shown. [Figure 7] This chart shows the total number of mushrooms, stubby cells, and shin spines per 100 μm in WT and 3xTg-AD mice treated with vehicle and compound 1 at 3 and 30 mg / kg, according to one embodiment of Example 1. [Figure 8] This chart shows the number of spines per 20 μm in WT, untreated (control), vehicle-treated, and 3xTg-AD mice treated with compound 1 at 0.1, 0.3, 1, 3, and 10 μM concentrations, according to one embodiment of Example 1. [Figure 9]This figure shows various gel bands of WT and 3xTg-AD mice treated with a vehicle and with compound 1 at 3 and 30 mg / kg in one embodiment according to Example 1. Lane A - WT vehicle; B - 3xTg-AD vehicle; C - 3xTg-AD compound 1, 3 mg / kg; D - 3xTg-AD compound 1, 30 mg / kg; E - WT vehicle; F - 3xTg-AD vehicle; G - 3xTg-AD compound 1, 3 mg / kg; H - 3xTg-AD compound 1, 30 mg / kg. [Figure 10] This figure shows the levels of various proteins targeting Alzheimer's disease in WT and 3xTg-AD mice treated with a vehicle and with compound 1 at 3 and 30 mg / kg, according to one embodiment of Example 1. [Figure 11] The immunohistochemistry of a specific protein in 3xTg-AD mice treated with a vehicle and with compound 1 at 3 and 30 mg / kg is shown in one embodiment according to Example 1. [Figure 12] This bar graph shows the levels of amyloid beta, tau, and phosphorylated tau in 3xTg-AD mice treated with the vehicle and with compound 1 at 3 and 30 mg / kg in one embodiment according to Example 1. [Figure 13A] In one embodiment, to provide a total diameter of approximately 4-5 μm for positioning presynaptic and postsynaptic structures, selected neurite segments of at least 20 μm are provided, with a width of approximately 0.3 μm and a boundary of approximately 2 μm around the segment. [Figure 13B] This is a schematic diagram showing the anatomical structure of the neuronal junction and the locations of the presynaptic and postsynaptic structures. [Figure 14] A bar graph shows DIV14 rat hippocampal neurons treated with the compounds described herein for 4 hours in one embodiment according to Example 1. The Y-axis represents dendritic spines per 20 μm, and the x-axis represents the concentration of the test compound in μM. Group 1: co-localized synaptobrevin / PSD95; Group 2: synaptobrevin alone; Group 3: PSD95 alone. [Figure 15] The graph shows the walking score in TDP-43 mice treated with compound 1 and vehicle, in one embodiment according to Example 2, where daily treatment was initiated on day 14. [Figure 16] The graph shows the kyphosis score in compound 1 and vehicle-treated TDP-43 mice in one embodiment according to Example 2, where daily treatment was initiated on day 14. [Figure 17] The graph shows the tremor scores in TDP-43 mice treated with compound 1 and vehicle, in one embodiment according to Example 2, where daily treatment was initiated on day 14. [Figure 18] The graph shows the survival time of TDP-43 ALS mice treated with compound 1 in one embodiment according to Example 2. [Figure 19] This shows the basal and apical spine density of layer 5 of the motor cortex in WT and Ubqln2 mice treated with the vehicle, and in WT and Ubqln2 mice treated with compound 1 (10 mg / kg). [Figure 20] This image shows dendritic spines in layer 5 of the motor cortex. [Figure 21] This shows the basal and apical spine density of the medial prefrontal cortex in WT and Ubqln2 mice treated with the vehicle, as well as in WT and Ubqln2 mice treated with compound 1 (10 mg / kg). [Figure 22] Images of dendritic spines in the medial prefrontal cortex are shown. [Figure 23] The basal and apical spine densities of the hippocampal CA1 region in WT and Ubqln2 mice treated with the vehicle, and in WT and Ubqln2 mice treated with compound 1 (10 mg / kg). [Figure 24] This image shows dendritic spines in the CA1 region of the hippocampus. [Modes for carrying out the invention]

[0033] Generally, the compounds and methods described herein provide the administration of compounds at spine-forming initiation concentrations useful for the treatment, prevention, or reversal of neuronal diseases or disorders. In some embodiments, the compositions and methods provide spine-forming initiation concentrations that are useful for the treatment of neuronal diseases or disorders, reduce drug interactions, minimize side effects, and are low enough to expand a suitable patient population.

[0034] definition The following description illustrates exemplary embodiments of the Technology. However, such description should be understood not as limiting the scope of the Disclosure, but rather as being provided solely as an example of the embodiments.

[0035] When used herein, the following words, phrases, and symbols are intended to have the meanings set forth below in general, except to the extent that the context in which they are used indicates otherwise.

[0036] Terms such as "spine formation" refer, in their usual, conventional sense, to the formation (e.g., initiation, growth, and / or maturation) of dendritic spines on neurons. In some embodiments, spine formation includes an increase in spine density. In some embodiments, the compounds provided herein promote spine formation without affecting the normal distribution of spine morphology. In further embodiments, spine formation may result in a change in the distribution of spine morphology. Promotion is measured in comparison to a control without administration of the compound.

[0037] As used herein, the term “dendrite” refers to a branched extension of a neuron cell. Dendrites are typically responsible for receiving electrochemical signals transmitted from the axon of an adjacent neuron. The terms “dendritic spine” or “dendrite spine” or “spine” refer to a protoplasmic projection on a neuron cell (e.g., a dendrite). In some embodiments, a dendritic spine may be described as having a membranous neck that can terminate at a microcephaly (e.g., head). Depending on their shape, dendritic spines are classified, for example, as headless, thin, stubby, mushroom, or branch. Dendritic spine density refers to the total number of dendritic spines per unit length of a neuron cell. For example, dendritic spine density may be given as the number of dendritic spines per micron or per 20 microns.

[0038] Terms such as “dendritic spine initiation” refer, in their usual conventional sense, to the process that results in an increase in the number of dendritic spines or an increase in the development of dendritic spines. Terms such as “dendritic spine morphology” refer, in their usual conventional sense, to the physical characteristics (e.g., shape and structure) of dendritic spines. Improvement of dendritic spine morphology is a change in morphology (e.g., an increase in length or width) that results in an increase in functionality (e.g., an increase in the contact area between neurons). Exemplary methods for evaluating such features, as known in the art and disclosed herein, include measuring the dimensions (i.e., length and width) of dendritic spines. Thus, the term “improving dendritic spine morphology” generally refers to an increase in the length, width, or both of the length and width of dendritic spines.

[0039] "Binding" refers to the formation of a complex between at least two different species (e.g., chemical compounds including biomolecules, or cells) that are close enough to react or interact. For example, binding of two different species (e.g., proteins and compounds described herein) may result in the formation of a complex in which the species interact via non-covalent or covalent bonds. In some embodiments, the resulting complex is formed by the interaction of two different species (e.g., proteins and compounds described herein) via non-covalent bonds (e.g., electrostatic, van der Waals, or hydrophobic).

[0040] Where defined herein, terms such as “activation,” “to activate,” and “being activated” in relation to protein-activator (e.g., agonist) interactions mean that the activator (e.g., a compound described herein) has a positive effect (e.g., an improvement) on the activity or function of the protein compared to the activity or function of the protein in the absence of the activator (e.g., a compound described herein).

[0041] Where defined herein, terms such as “inhibit,” “inhibit,” and “inhibiting” should be given the meanings commonly understood by those skilled in the art. In reference to protein-inhibitor (e.g., antagonist) interactions, the terms “inhibit,” “inhibit,” and “inhibiting” mean a negative effect (e.g., reduction) on the functional activity of a protein compared to the functional activity of the protein in the absence of the inhibitor (e.g., a compound described herein).

[0042] References to values ​​or parameters "about" in this specification include (and describe) embodiments relating to the value or parameter itself. In certain embodiments, the term "about" includes ±10% of the indicated amount. In other embodiments, the term "about" includes ±5% of the indicated amount. In certain other embodiments, the term "about" includes ±1% of the indicated amount. Also, the term "about X" includes the description "X". Furthermore, the singular "a" and "the" include plural referents unless the context explicitly indicates otherwise. Thus, for example, a reference to "the compound" includes multiple such compounds, and a reference to "the assay" includes one or more assays and their equivalents known to those skilled in the art.

[0043] "Halogen" or "halo" includes fluoro, chloro, bromo, and iodine.

[0044] The terms “optional” or “optionally” mean that the event or situation described thereafter may or may not occur, and the description includes both the cases in which the event or situation occurs and the cases in which it does not occur. The term “optionally substituted” means that any one or more hydrogen atoms on a given atom or group may or may not be replaced by a non-hydrogen part. When referring to a group, the term “optionally substituted” is intended to be interpreted as describing a group substituted by non-substituents and by the indicated or defined substituent(s).

[0045] Some compounds exist as tautomers. For example, amide-containing compounds may exist in equilibrium with imido acid tautomers. Regardless of which tautomer is shown, and regardless of the nature of the equilibrium between the tautomers, a compound is understood by those skilled in the art to contain all tautomers.

[0046] Any formula or structure provided herein is also intended to represent both the isotopically labeled and unlabeled forms of a compound. An isotopically labeled compound has a structure as shown by the formula provided herein, except that one or more atoms are replaced by an isotope having the indicated atomic weight or mass number. Examples of isotopes that can be incorporated into the compounds of the present disclosure, or counterions therefor, include, but are not limited to, 2 H (deuterium, D), 3 H (tritium), 11 C, 13 C, 14 C, 15 N, 18 F, 31 P, 32 P, 35 S, 36 Cl and 125 isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine such as I. Various isotopically labeled compounds under the present disclosure can, for example, 3 H, and 14 C, etc., radioactive isotopes incorporated therein are possible. Such isotopically labeled compounds can be useful in metabolic studies, kinetics studies, detection or imaging techniques such as positron emission tomography (PET) or single photon emission computed tomography (SPECT), including drug or substrate tissue distribution assays, or in the radioactive treatment of patients.

[0047] The present disclosure also includes "deuterated analogs" of a compound, and counterions therefor, in which 1 to n hydrogens are replaced by deuterium, where n is the number of hydrogens in the molecule. Such compounds can be synthesized by well-known means in the art, for example, by using starting materials in which one or more hydrogens are replaced by deuterium.

[0048] The isotopically labeled compounds and prodrugs thereof of the present disclosure can generally be prepared by replacing readily available isotopically labeled reagents with non-isotopically labeled reagents and carrying out the procedures disclosed in the schemes or in the examples and preparations described below.

[0049] The concentration of such heavier isotopes, specifically deuterium, can be defined by the isotopic enrichment factor. In the compounds of this disclosure, any atom not specifically designated as a particular isotope means that it represents any stable isotope of that atom. Unless otherwise specified, where a position is specifically designated as "H" or "hydrogen," that position is understood to have hydrogen in its naturally occurring isotopic composition. Thus, in the compounds of this disclosure, any atom specifically designated as deuterium (D) means that it is rich in deuterium at the designated position at a level exceeding that which occurs naturally.

[0050] The compounds described herein may exist as salts, such as pharmaceutically acceptable salts. The compounds can form salts, such as acid salts and / or base salts. Pharmaceutically acceptable salts, hydrates, solvates, tautomers, polymorphs, and prodrugs of the compounds described herein are also provided. “Pharmaceutically acceptable” or “physiologically acceptable” refers to compounds, salts, compositions, dosage forms, and other materials useful for preparing pharmaceutical compositions suitable for veterinary or human pharmaceutical use. Salts of the compounds described herein can be prepared according to the procedures described herein and as known in the art.

[0051] The term "pharmaceutically acceptable salt" of a given compound refers to a salt that retains the biological efficacy and properties of the given compound and is biologically desirable or otherwise desirable. Examples of "pharmaceutically acceptable salts" or "physiologically acceptable salts" include salts with inorganic acids and salts with organic acids. In addition, if the compounds described herein are obtained as acid addition salts, the free base can be obtained by basicizing a solution of the acidic salt. Conversely, if the product is a free base, the addition salt, in particular a pharmaceutically acceptable addition salt, can be produced by dissolving the free base in a suitable solvent and treating the solution with an acid, following conventional procedures for preparing acid addition salts from base compounds. Those skilled in the art will recognize various synthetic methodologies that can be used to prepare non-toxic, pharmaceutically acceptable addition salts. pharmaceutically acceptable acid addition salts can be prepared from inorganic and organic acids. Examples of salts derived from inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. Examples of salts derived from organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, isobutyric acid, suberic acid, and lactic acid. Similarly, pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. Examples of salts derived from inorganic bases include salts of sodium, potassium, lithium, ammonium, calcium, and magnesium.Salts derived from organic bases include, for example, alkylamines (i.e., NH2(alkyl)), dialkylamines (i.e., HN(alkyl)2), trialkylamines (i.e., N(alkyl)3), substituted alkylamines (i.e., NH2(substituted alkyl)), di(substituted alkyl)amines (i.e., HN(substituted alkyl)2), tri(substituted alkyl)amines (i.e., N(substituted alkyl)3), alkenylamines (i.e., NH2(alkenyl)), dialkenylamines (i.e., HN(alkenyl)2), trialkenylamines (i.e., N(alkenyl)3), substituted alkenylamines (i.e., NH2(substituted alkenyl)), di(substituted alkenyl)amines (i.e., HN(substituted alkenyl)2), tri(substituted alkenyl)amines (i.e., N(substituted alkenyl)3), mono-, di-, and Examples of preferred amines include, but are not limited to, primary, secondary, and tertiary amines such as tri-cycloalkylamines (i.e., NH2(cycloalkyl), HN(cycloalkyl)2, N(cycloalkyl)3), mono-, di-, or tri-arylamines (i.e., NH2(aryl), HN(aryl)2, N(aryl)3), or mixed amines. Specific examples of preferred amines include, but are not limited to, isopropylamine, trimethylamine, diethylamine, tri(isopropyl)amine, tri(n-propyl)amine, ethanolamine, 2-dimethylaminoethanol, piperazine, piperidine, morpholine, and N-ethylpiperidine. Methods for preparing salts also involve mixing compounds by redox reactions with active metals or ion exchange, for example, due to differences in the solubility of the salts.

[0052] A "solvate" is the solid form of a compound in which a solvent molecule is incorporated. Solvates are formed by the interaction between a solvent and a compound. A hydrate is a solvate in which the solvent is water. Solvates of salts of the compounds described herein are also provided.

[0053] As used herein, “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes all kinds of solvents, dispersion media, fillers, coatings, antibacterial and antifungal agents, isotonic agents and absorption retarders, etc. The use of such media and agents as pharmaceutically active substances is well known in the art. Their use in therapeutic compositions is intended unless conventional media or agents are incompatible with the active ingredient. Auxiliary active ingredients may also be incorporated into the composition.

[0054] "Treatment" or "treating" provides a beneficial or desirable outcome, such as improvement of one or more clinical indicators of a disease or disability. Beneficial or desirable outcomes may include: reducing or improving one or more symptoms of a disability, reducing the degree of the disability (e.g., stabilizing the disability, preventing or delaying its worsening or progression), providing partial or complete remission of the disability, enhancing the effectiveness of another medicine, improving quality of life, and / or extending the survival of the patient population.

[0055] "Prevention" or "prevention" means blocking the onset of a disease or disorder, or its symptoms. In some embodiments, the compound may be administered to subjects (including humans) at risk of disease or condition, for example, those with a family history of the disease or condition. Prevention may include delaying the attainment of a predetermined disease milestone, or reducing the appearance or progression of indicators of disease or disorder.

[0056] "Dose" refers to a single, separate administration. "Dose cycle" refers to two or more doses, where the doses are separated for a certain period of time.

[0057] "Subject" refers to a mammal, such as a human, or other animal, that may benefit from the administration of the compounds described herein. The methods described herein may be useful in the therapeutic and / or veterinary use of humans. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. When the subject is human, the subject may be referred to as a "patient."

[0058] The term "therapeutic dose" of a compound as used herein means an amount sufficient to induce treatment of the disease or disorder described herein when administered to a subject. The therapeutic dose may vary depending on the subject, the disease or disorder being treated, the subject's weight and age, the severity of the disease or disorder, any complications of the disease or disorder, and the mode of administration, and is generally determined by a physician.

[0059] The term "refractory" means that a subject with a disease or disorder has previously resisted treatment for that disease or disorder. For example, one or more symptoms of the disease or disorder persist following treatment.

[0060] The term "marker" refers to a characteristic of an object that indicates a risk of developing a disease or disorder. For example, a marker may be a genetic indicator associated with a disease or disorder, a personal history of a disease or disorder, a family history, such as a genetic relative or multiple relatives who have had the disease or disorder, or a related disease or disorder, or a test result or symptom.

[0061] The term "transient" in relation to the presence of a drug in vivo means that an effective amount of the drug is present only for a short period, while the biological process resulting in the biological endpoint (in this case, functional dendritic spines) continues favorably after the drug has fallen below the effective amount or disappeared (less than 5% of the effective amount of drug remains in the serum). For a drug with a half-life of 24 minutes, the drug is considered absent less than approximately 2 hours after administration. Those skilled in the art will understand that the half-life of a compound in one mammal may differ from that of another. Therefore, the half-lives of the compounds described herein may differ from those measured in rodents when administered to humans.

[0062] The term "repair" refers to the generation of new functional synapses.

[0063] The term "functional" refers to the ability of spines or synapses to communicate with other neurons.

[0064] The term “neurodegenerative disease” refers to one of a subset of neuronal diseases or disorders that are incurable. These diseases are debilitating, involve progressive degeneration and / or death of nerve cells, and ultimately lead to the death of the individual. These diseases are classified into at least two components: those affecting movement (called ataxia) or those affecting mental function (called dementia). Such neurodegenerative diseases include, but are not limited to, dementia (including, just a few examples, Alzheimer's disease (AD) and frontotemporal dementia), amyotrophic lateral sclerosis (ALS), Parkinson's disease, and Huntington's disease. Examples of neuronal diseases that are not neurodegenerative include Fragility X syndrome, autism, chronic stress, and attention deficit / hyperactivity disorder (ADHD).

[0065] The terms “early stage,” “mid-stage,” and “late stage” for each neurodegenerative disease are well-known, and many define terms familiar to the attending physician. In Alzheimer’s disease (AD), these stages are defined in their entirety at https: / / www.alz.org / alzheimers-dementia / stages, which are incorporated herein by reference. Thus, patients in the early stage of AD may experience forgetfulness, including difficulty recalling the correct word or name immediately, remembering names when introduced to new people, difficulty performing tasks in social or work settings, forgetting materials they have just read, losing or misplacing valuables, and experiencing increased trouble with planning or organizing. Patients with mid-stage AD may experience difficulty expressing thoughts and performing routine tasks without assistance, including: forgetting events or personal history; experiencing mood swings or withdrawal, especially in socially or emotionally challenging situations; being unable to recall personal information such as addresses or phone numbers, and the high school or college they attended; experiencing confusion about where they are or what day of the week it is; needing assistance to choose appropriate clothing for the season or situation; having trouble controlling their bladder and bowels; experiencing changes in sleep patterns, such as sleeping during the day and becoming restless at night; and showing an increased tendency to wander and get lost. Patients with late-stage AD may experience a lack of ability to respond to their environment and require assistance and personal care, including: loss of awareness of recent experiences and their surroundings; experiencing changes in physical abilities, including walking, sitting, and eventually swallowing; having difficulty communicating; and being more susceptible to infections, particularly pneumonia.

[0066] In ALS, these stages are defined in their entirety, as incorporated herein by reference: https: / / alsnewstoday.com / stages-of-als / and https: / / www.mda.org / disease / amyotrophic-lateral-sclerosis / signs-and-symptoms / stages-of-als. Thus, a person in the early stages of ALS may exhibit symptoms including: muscles may be weak and limp, or stiff, tense, and spasming; muscle spasms and contractions that result in loss of muscle mass (atrophy) (fasciculations); symptoms may be limited to a single area of ​​the body, or mild symptoms may affect two or more areas; the person may experience fatigue, poor balance, slurred speech, weak grip strength, stumbling while walking, or other mild symptoms. Patients with ALS in the mid-stage may exhibit the following: paralysis of some muscles while others are weakened or unaffected; persistent fasciculations; contractures of unused muscles, where the joints become stiff, painful, and sometimes deformed; in the event of a fall, the person may be unable to stand up on their own; weakness of the swallowing muscles may cause choking and significant difficulty in managing food and saliva; weakness of the respiratory muscles may cause respiratory failure, especially when lying down; some people experience uncontrollable and inappropriate laughter or crying fits (effects of emotional dysregulation). Patients with ALS in the late stage may exhibit the following: paralysis of most spontaneous muscles; severe impairment of the diaphragm; very limited mobility requiring assistance for most personal needs; respiratory problems may cause fatigue, confused thinking, headaches, and susceptibility to pneumonia; speech or eating and drinking by mouth may be impossible.

[0067] In Parkinson's disease (PD), there are five distinct components of the disease, as described in detail at https: / / www.parkinson.org / Understanding-Parkinsons / What-is-Parkinsons / Stages-of-Parkinsons, which is incorporated herein by reference in its entirety. These stages are further classified as the early stage (components 1 and 2 of the five components), the middle stage (components 2 and 3 of the five components), and the late stage (components 4 and 5 of the five components). Other neurodegenerative diseases also have clearly defined stages of the disease. Stage 1: During this early stage, the person generally has mild symptoms that do not interfere with daily activities, tremors and other movement symptoms occur only on one side of the body, and there are changes in posture, gait, and facial expression. Stage 2: Symptoms begin to worsen, tremors, rigidity, and other movement symptoms affect both sides of the body, and there may be gait problems and poor posture, and the person is still able to live independently, but daily tasks become more difficult and time-consuming. Stage 3: Considered the middle stage, characterized by loss of balance and slow movement, with falls being more common. The person is still completely independent, but symptoms significantly impair activities such as dressing and eating. Stage 4: Symptoms are severe and limited; the person can stand without assistance, but movement may require a walker, and the person needs assistance with daily living activities and cannot live independently. Stage 5: Symptoms are intractable, and leg stiffness may make it impossible to stand or walk. The person may require a wheelchair or be bedridden, requiring 24-hour care for all activities, and may experience hallucinations and delusions.

[0068] The stages of Huntington's disease (HD) are described in their entirety at https: / / hopes.stanford.edu / stages-of-huntingtons-disease / , which are incorporated herein by reference. Early stage symptoms include motor symptoms of the limbs, including involuntary contractions of the fingers, toes, and face; mild loss of coordination; difficulty performing complex movements; cognitive symptoms include difficulty thinking through complex tasks; and behavioral symptoms include depression, irritability, and disinhibition. Middle stage Huntington's disease symptoms may result in difficulty working or driving and inability to perform household chores; motor symptoms include chorea, difficulty with voluntary motor tasks such as walking, balance problems, and dysphagia; cognitive symptoms include difficulty organizing information and thinking clearly, as well as inability to solve problems; and behavioral symptoms include an increase in the severity of those in the early stage, plus apathy and decreased sexual desire. Late symptoms include difficulties in all aspects of life, including speech, and patients may remain bedridden. Motor symptoms include severe difficulty with spontaneous movement, rigidity, dystonia, and bradykinesia. Cognitive symptoms are impaired in daily life, and behavioral symptoms include apathy (which may adequately counteract depression) and psychosis.

[0069] Treatment methods and use Provided herein are methods for preventing, repairing, and / or treating neuronal diseases or disorders by administering a sufficient amount of a compound to a subject to provide in the brain a concentration that promotes spine formation in one or more neurons, as described herein. Also provided are the compounds described herein for use in the prevention, treatment, or repair of neuronal diseases or disorders, wherein the compound is administered at a spine formation-initiating dose.

[0070] Unit dose In one embodiment, in mammals suffering from reduced spine density and therefore impaired functional synapses, a unit dose for initiating the formation of stable functional synapses, wherein the unit dose is approximately 0.4 mg / kg / day or less of a compound of formula IA: [ka] or comprising a pharmaceutically acceptable salt thereof, isotope-enriched analogue, tautomer, prodrug, stereoisomer, or mixture of stereoisomers, q is between 2 and 8. The compound has a serum half-life of less than 3 hours, and the unit dose is such that it provides stable, functional synapse formation in the mammal.

[0071] In one embodiment, the unit dose contains approximately 0.1 mg / kg / day to approximately 0.4 mg / kg / day. In one embodiment, the decrease in functional spine density is a result of a neurological condition. In one embodiment, the unit dose is prescribed for once-daily administration.

[0072] Medication regimen In further embodiments, compositions and methods are provided for preventing, repairing, and / or treating neuronal diseases or disorders / neurodegenerative diseases or disorders by inducing spine formation, wherein the amount of drug administered is effective in providing a concentration in the brain that induces spine formation and increases spines. In some embodiments, spine formation is induced within less than 5 minutes, less than 10 minutes, less than 30 minutes, or less than 60 minutes of administration.

[0073] Surprisingly, as described herein, very low concentrations of spine-forming compounds with half-lives of less than 30 minutes (when measured in vivo in rodents) are sufficient to induce spine formation. Such low concentrations have been found to provide a guiding signal to neurons to induce spine formation. Equally surprising is the fact that, despite the absence of drugs from mammals, the induced spines are maintained for extended periods following administration of low doses of the compounds described herein. This combination of a very short half-life in the brain and a very low dose is considered novel for producing long-lasting effects on neurons.

[0074] The dose-response curves of the compounds described herein may be nonlinear. The compounds described herein initially provide a spine-forming initiation concentration in cerebral fluid or brain tissue, and then cross the blood-brain barrier at a rate sufficient to be subsequently eliminated.

[0075] In some embodiments, following a first dose, the compound, its salt, stereoisomer, mixture of stereoisomers, or metabolite is administered to brain fluid (e.g., serum) or brain tissue. max It reaches C max The effect reaches the brain within 5–10 minutes and then decreases rather rapidly due to its short half-life. However, once initiated, spine development continues even after the concentration of the compound in the extracellular fluid falls below the spine-forming initiation concentration. Therefore, a single dose can induce dendritic spines, and the induction can be maintained for approximately 4 hours or more following administration to the subject, for example, approximately 24 hours, 48 ​​hours, 72 hours, 96 hours, or up to a week.

[0076] Spine-initiating concentrations of the compounds described herein, as provided herein, increase synaptic density or improve synaptic morphology in brain regions. For example, the increase in synaptic density or improvement in synaptic morphology may occur in the hippocampus. The increase in synaptic density or improvement in synaptic morphology may occur in the prefrontal cortex. The increase in synaptic density or improvement in synaptic morphology may occur in the motor cortex. Spine-initiating concentrations of the compounds described herein, as provided herein, may increase the density of mushroom and / or stubby spines in brain regions, e.g., the hippocampus, prefrontal cortex, or motor cortex, or another brain region.

[0077] To achieve a desired in vivo concentration, the spine-initiating dose of the compounds described herein may be based on the body weight of the subject. The effective dose to achieve the spine-initiating concentration may be determined by allometric scaling. In some embodiments, the mammal is human, and the effective dose is about 0.1–0.4 mg / kg. The effective dose in humans may be less than about 0.5 mg / kg, or about 0.2, 0.25, 0.3, 0.35, or 0.4 mg / kg / day, preferably about 0.4 mg / kg / day. The dose in human subjects may be about 0.2–0.4 mg / kg. The dose may be effective for the treatment, prevention, or repair of neuronal diseases or disorders, such as the neurodegenerative diseases described herein.

[0078] Furthermore, administration is preferably continued on a daily schedule or a five-day-a-week schedule.

[0079] Neuronal diseases or disorders include Alexander disease, Alpers disease, Alzheimer's disease, depression, perinatal asphyxia, Parkinson's disease dementia ("PD dementia"), amyotrophic lateral sclerosis, telangiectatic ataxia, Batten disease (also known as Spielmeier-Voigt-Sjögren-Batten disease), cavernous encephalopathy (e.g., bovine spongiform encephalopathy (mad cow disease), kuru disease, Creutzfeldt-Jakob disease, fatal familial insomnia, Canavan disease, Cockayne syndrome, corticobasal degeneration, fragility X syndrome, and pre- Cranotemporal dementia, Gerstmann-Streusler-Scheinker syndrome, Huntington's disease, HIV-related dementia, Kennedy disease, Krabbe disease, Lewy body dementia, Machado-Joseph disease (spinocerebellar ataxia type 3), multiple sclerosis, multiple system atrophy, narcolepsy, neuroborreliosis, Parkinson's disease, Pelizaeus-Merzbacher disease, Pick's disease, primary lateral sclerosis, prion disease, Refsum disease, Sandhoff disease, Schilder's disease, subacute combined degeneration of the spinal cord secondary to pernicious anemia, schizophrenia, spinocerebellar ataxia ( (Multiple types with various characteristics), spinal muscular atrophy, Steele-Richardson-Olsewski disease, spinal fistula, drug-induced parkinsonism, progressive supranuclear palsy, corticobasal degeneration, progressive bulbar palsy (PBP), pseudobulbar palsy, progressive muscular atrophy, spinal muscular atrophy, Kennedy disease, multiple system atrophy, idiopathic parkinson's disease, autosomal dominant parkinson's disease, familial, type 1 (PARK1), Parkinson's disease 3, autosomal dominant Lewy body (PARK3), Parkinson's disease 4, autosomal dominant Lewy body (PARK 4) It may be Parkinson's disease 5 (PARK5), Parkinson's disease 6, autosomal recessive early-onset (PARK6), Parkinson's disease 2, autosomal recessive juvenile (PARK2), Parkinson's disease 7, autosomal recessive early-onset (PARK7), Parkinson's disease 8 (PARK8), Parkinson's disease 9 (PARK9), Parkinson's disease 10 (PARK10), Parkinson's disease 11 (PARK11), Parkinson's disease 12 (PARK12), Parkinson's disease 13 (PARK13), or mitochondrial Parkinson's disease.In some embodiments, neuronal diseases include Alzheimer's disease, Parkinson's disease, Parkinsonian dementia, autism, stroke, post-traumatic stress disorder (PTSD), traumatic brain injury (TBD), chronic traumatic encephalopathy (CTE), schizophrenia, dementia (e.g., common dementia), attention deficit / hyperactivity disorder (ADHD), amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD) (e.g., FTLD-tau, FTLD-TDP, or FTLD-FUS), memory loss (e.g., age-related memory loss), hypertensive encephalopathy, or chronic stress.

[0080] In one preferred embodiment, the neuronal disease or disorder is a neurodegenerative disease selected from Alzheimer's disease, Parkinson's disease, ALS, frontotemporal dementia, or traumatic brain injury.

[0081] In one embodiment, any one of the above diseases is excluded from the scope of the present invention.

[0082] In some embodiments, the neuronal disease is Alzheimer's disease (AD). Alzheimer's disease is characterized by symptoms of memory loss in the early stages of the disease. Apoε4 carriers are at increased risk of developing AD. Experiencing traumatic brain injury (TBI) is another risk factor for developing AD, and studies have shown that people who have experienced TBI have a significantly increased risk of AD. Cognitive decline is correlated with progressive synaptic loss. As the disease progresses, symptoms include confusion, loss of long-term memory, paraphasia, vocabulary loss, aggression, irritability, and / or mood swings. In more advanced stages of the disease, there is loss of physical function. People with Alzheimer's disease (AD) exhibit many characteristic neurological disorders, including increased oxidative stress, mitochondrial dysfunction, synaptic dysfunction, disruption of calcium homeostasis, deposition of senile plaques and neurofibrillary tangles, and brain atrophy. AD-related disorders include senile dementia of the AD type (SDAT), frontotemporal dementia (FTD), vascular dementia, mild cognitive impairment (MCI), and age-related memory impairment (AAMI). In some embodiments, a method is provided for treating or preventing Alzheimer's disease, comprising administering a spine-forming initiation dose of one of the compounds described herein to a patient in need of a method for treating or preventing Alzheimer's disease. In some embodiments, the neuronal disease or disorder is selected from Alzheimer's disease, Parkinson's disease, Parkinsonian dementia, autism, fragility X syndrome, stroke, and traumatic brain injury. Surprisingly, treatment with the compounds described herein can treat AD or its symptoms without altering the pathology of Aβ or Tau.

[0083] Neuronal diseases or disorders may be associated with spinal cord injury or complications of spinal cord injury. Spinal cord injury (SCI) may be accompanied by one or more of several acute complications. In some embodiments, complications of spinal cord injury may include respiratory complications, cardiovascular complications, hypotension, bradycardia, neurogenic shock, autonomic hyperreflexia, secondary immunodeficiency, syringomyelia, neuropathic arthropathy, Charcot arthropathy, loss of motor control, loss of sensory function, quadriplegia, paraplegia, loss of bladder control, convulsions, loss of sexual function, numbness, loss of balance, autonomic hyperreflexia, deep vein thrombosis, spasticity, pain, neuropathic pain, syringomyelia, neurogenic heterotopic ossification, shock, bradyarrhythmia, hypotension, heterotopic pulsation, abnormal thermoregulation, altered sweat secretion, vasodilation, autonomic hyperreflexia, thromboembolism, pressure ulcers, or heterotopic ossification, or a combination thereof.

[0084] Neuronal diseases or disorders may be associated with traumatic brain injury or stroke. Neuronal diseases or disorders may be mental disorders such as depression or schizophrenia. In either case, the disease or disorder may be treatable or ameliorative by increasing dendritic spine density in mammals. In some embodiments, compared to when treatment was initiated, the mean dendritic spine density following administration of a spine-forming initiation dose of the compound described herein increases by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%, or by any range between any two numbers including the endpoints. The increase may be maintained for about 4 hours, about 24 hours or longer after dose administration.

[0085] This method offers significant benefits in stages of neurodegenerative disease or disorder where many neurons remain largely intact, as new spines are thought to be able to form functional synapses with surrounding neurons. For example, the method provided herein may be beneficial in patients with 0%–20% functional loss (early stage disease) or 20%–60% functional loss (mid-stage disease). For example, the neuronal / neurological disease or disorder may be Alzheimer's disease, and subjects may present with mild cognitive impairment, mild dementia, or moderate dementia.

[0086] Amyotrophic lateral sclerosis (ALS) can be staged according to the patient's symptoms. One staging system used is the Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R). The ALSFRS provides a physician-generated estimate of the degree of functional impairment in a patient that can be assessed to evaluate disease progression. The ALSFRS-R includes 12 questions asked of the physician to assess the patient's impression of the level of functional impairment when performing 12 common tasks, including speaking, salivation, swallowing, writing, cutting food, grooming and hygiene, turning in bed, walking, climbing stairs, dyspnea, orthopnea, and respiratory failure. Each task is rated on a 5-point scale from 0 = unable to do to 4 = normal ability. The scores for the individual items are summed up to generate a score report between 1 (lowest functioning) and 48 (normal functioning). (A score of zero would indicate an inconsistent loss of ability.) In some embodiments, the neuronal disease or disorder is amyotrophic lateral sclerosis (ALSFRS-R), and the subject has an ALSFRS-R score of 38–47. In some embodiments, the neuronal disease or disorder is amyotrophic lateral sclerosis (ALSFRS-R), and the subject has an ALSFRS-R score of 20–38.

[0087] The clinical stages of ALS are defined by the involvement of central nervous system (CNS) regions with debilitation, wasting, seizures, dysphagia, or dysarthria, and regions defined in the same manner as the El Escorial criteria. Involvement of a single CNS region (onset of symptoms) defines Stage 1, a second CNS region defines Stage 2, and three CNS regions define Stage 3. Respiratory involvement sufficient to require assisted ventilation or dysphagia sufficient to require a gastrostomy defines Stage 4. Stage 5 is death. In some embodiments, the neuronal disease or disorder is amyotrophic lateral sclerosis (ALS), and the subject is clinically stage 1 or stage 2.

[0088] Traumatic brain injury (TBI) is classified as mild, moderate, and severe. A mild form of TBI is a concussion. These can be caused by a blow, impact, or vibration to the head or body that causes the brain to move rapidly inside the skull. Even without loss of consciousness, mild TBI is diagnosed if the person has memory loss about events immediately before or after the injury. Symptoms of mild TBI include headache, dizziness, insomnia, decreased concentration and attention, depression, anxiety, mood swings, balance problems, slower information processing speed, and reduced ability to learn and recall new things. In moderate TBI, there is usually an abnormal CT, PET, or MRI finding after the injury. Loss of consciousness may last from minutes to hours. People experiencing moderate TBI usually have confusion that lasts from days to weeks. However, physical, cognitive, and / or behavioral impairments may persist for several months or may be permanent. Severe traumatic brain injury (TBI) is usually accompanied by prolonged unconsciousness or coma lasting days, weeks, or months. Symptoms of severe TBI may include loss of consciousness, headache, nausea, vomiting, lack of coordination, dizziness, balance problems, dilated pupils (one or more), slurred speech, behavioral or mood swings, loss of coordination, restlessness, and agitation. Patients with severe TBI can improve significantly, including returning to a life very similar to their pre-injury life. However, some are left with permanent physical, cognitive, or behavioral impairments. Rehabilitation is almost always necessary in severe traumatic brain injury. In some embodiments, a neuronal disorder or injury is a traumatic brain injury, and the patient suffers from a mild or moderate traumatic brain injury but not a severe one.

[0089] In traumatic brain injury, the recovery process has three stages. In the first stage, the patient is unconscious. In the second stage, consciousness is fully restored, which is indicated by the end of the post-traumatic memory loss period, and evidence of rapid improvement in basic physical and mental functions continues. The rate of recovery is, in most cases, within six months of the injury, which represents the end of the second stage. In the third stage, which may last for several months, both the patient and their relatives adapt to the patient's remaining disability. See Bond, The stages of recovery from severe head injury with special reference to late outcome, Int. Rehabil. Med. (1979) Vol.1(4):155-9. Therefore, in some embodiments, the neuronal disease or disorder is traumatic brain injury, and the patient is in the second or third stage of recovery.

[0090] In some embodiments, the dendritic spine density following administration of a spine-forming initiation dose of the compound described herein increases by about 20% to about 100% compared to the time at which treatment with the compound described herein is initiated.

[0091] In some embodiments, the time required to achieve the spine density or spine morphology changes described herein, e.g., average dendritic spine density, average spine density, or average number of spines per neuron, is as short as 5 minutes, e.g., 30 minutes, 45 minutes, 60 minutes, or 4 hours. The change in spine density is maintained for at least about 4 hours, about 24 hours, about 48 hours, or about 72 hours following administration.

[0092] compound In some embodiments, the compound is the compound described in International Patent Publication No. 2019 / 028164. In some embodiments, the compound is the compound of formula I. [ka] or a pharmaceutically acceptable salt thereof, isotope-enriched analog, tautomer, prodrug, stereoisomer, or mixture of stereoisomers, In the formula, R is hydrogen, C1-C4 alkyl, halo, hydroxyl, amino, cyano, or nitro. q is between 2 and 8.

[0093] In some embodiments, the provided compound is selected from the following, or a pharmaceutically acceptable salt or solvate thereof: [ka]

[0094] The efficacy of compound 1, which promotes spine formation in mice with spinal cord injury, has also been demonstrated in a recent study by Fogarty et al., “Novel regenerative drug, SPG302 promotes functional recovery of diaphragm muscle activity after cervical spinal cord injury”, The Journal of Physiology 0.0 (2023) pp 1-20.

[0095] Combination therapy In one embodiment, the doses of the compounds described herein may be used in combination with one or more additional therapeutic agents used and / or under development to treat neuronal diseases or disorders.

[0096] When used for the treatment or prevention of the diseases and disorders described herein, the doses of the compounds or pharmaceutical compositions described herein may be administered together with one or more additional therapeutic agents, for example, additional therapeutic agents approved for use in the treatment or prevention of a particular disease or disorder, more specifically, agents considered to form the current standard of care. Where combination therapy is envisioned, the activators may be administered simultaneously, separately, or sequentially in one or more pharmaceutical compositions.

[0097] Recent strategies for treating AD, which may be combined with the methods provided herein, involve controlling the production or aggregation state of specific isoforms of Aβ peptides. Additional strategies include the prevention, reduction, or elimination of toxic forms of phosphorylated tau. Other strategies include small molecule targeting of enzymes that play a role in Aβ peptide production through the processing of amyloid precursor proteins to reduce the abundance of Aβ peptides in the brain. In addition, there is accumulating information on the role of non-amyloid neuropathy, such as tauopathy or the sporadic inheritance of specific mutations in the apolipoprotein E gene, which is prompting the development of additional strategies to combat neurodegeneration.

[0098] One or more additional medications include: Tacrine, Donepezil, Galantamine, Rivastigmine, Memantine, Levodopa, Carbidopa, Rislid, Rasagiline, Tolcapone, Entacapone, Clozapine, Desipramine, Citalopram, Nortriptyline, Paroxetine, Atomoxetine, Venlafaxine, Amantadine, Donepezil, Rivastigmine, Bromocriptine, Cabergoline, Pergolide, Pramipexole, Ropinirole, Rotigotine, Apomorphine, Benserazide, Selegiline, Omigapil, CEP-1347, Isradipine, DOPA, Lithium, Riluzole, Levetiracetam, Ezogabine, Pregabalin, Rufinamide, Ferbamate, Carbamazepi It may be valproic acid, sodium valproate, lamotrigine, phenytoin, oxcarbazepine, ethosuximide, gabapentin, thiagabin, topiramate, vigabatrin, phenobarbital, primidone, clonazepam, interferon beta-la, interferon beta-lb, mitoxantrone, natalizumab, fingolimod, natalizumab, teriflunomide, dimethyl fumarate, glatiramer, ATOH1 gene therapy, ozanezumab, arimochromol, tiracemtiveb, dexpramipexole, pridopidine, or galantamine, or phosphoglycerate kinase (PGK) as described in US2018 / 0147263. In some embodiments, one or more additional therapeutic agents may be acetylcholinesterase inhibitors (AChEIs), such as acotiamide, alphapinene, ambenonium, demepotassium, DFP (diisopropyl fluorophosphate), donepezil, edrophonium, galantamine, huperzine A, lactucopicrin, radostigyl, neostigmine, physostigmine, pyridostigmine, diflozin, echothiopart, rivastigmine, rosmarinic acid, tacrine, ungeremin, zanapezil, ganstigmine, fencerin, phenethylnorcymserine (PENC), cymserine, thiacymserine, SPH1371 (galantamine plus), ER127528, RS1259, or F3796.In some embodiments, one or more additional therapeutic agents may be amyloid-depleting antibodies, such as bapineozumab, solanezumab, gantenerumab, crenezumab, ponezumab, BAN2401, or aducanumab.

[0099] One or more additional medications include chloral hydrate, estazolam, flurazepam hydrochloride, pentobarbital, pentobarbital sodium, phenobarbital sodium, secobarbital sodium, temazepam, triazolam, zaleplon, or hypnotic sedatives such as zolpidem tartrate, acetazolamide sodium, carbamazepine, clonazepam, dipotassium clorazepate, diazepam, divalproex sodium, ethosuximide, fosphenytoin sodium, gabapentin, lamotrigine, magnesium sulfate, and phenobarbital. Anticonvulsants such as phenobarbital sodium, phenytoin, phenytoin sodium, primidone, thiagabine hydrochloride, topiramate, sodium valproate, or valproic acid, amitriptyline hydrochloride, amitriptyline pamoate, amoxapine, bupropion hydrochloride, citalopram hydrobromide, clomipramine hydrochloride, desipramine hydrochloride, doxepin hydrochloride, fluoxetine hydrochloride, imipramine hydrochloride, imipramine pamoate, mirtazapine, nefazodone hydrochloride, nortriptyline hydrochloride, paroxetine hydrochloride, and phenelzine sulfate. Antidepressants such as sertraline hydrochloride, tranylcypromine sulfate, trimipramine maleate, or venlafaxine hydrochloride; anxiolytics such as alprazolam, buspirone hydrochloride, chlordiazepoxide, chlordiazepoxide hydrochloride, dipotassium clorazepate, diazepam, doxepin hydrochloride, hydroxyzine emponate, hydroxyzine hydrochloride, hydroxyzine pamoate, lorazepam, meprobamate, midazolam hydrochloride, or oxazepam; chlorpromazine hydrochloride, clozapine, fluphenazine decanoate, fluphenazine Antipsychotic drugs such as enanthate esters, fluphenazine hydrochloride, haloperidol, haloperidol decanoate, haloperidol lactate, roxapine hydrochloride, roxapine succinate, mesolidazine besylate, morindone hydrochloride, olanzapine, perphenazine, pimozide, prochlorperazine, quetiapine fumarate, risperidone, thioridazine hydrochloride, thiothixen, thiothixen hydrochloride, or trifloperazine hydrochloride, amphetamine sulfate, caffeine, dextroamphetamine sulfate, dosapram hydrochloride, methamphetamine hydrochloride,Central nervous system stimulants such as methylphenidate hydrochloride, modafinil, pemoline, or phentermine hydrochloride; antiparkinson's disease drugs such as amantadine hydrochloride, benztropine mesylate, biperiden hydrochloride, biperiden lactate, bromocriptine mesylate, carbidopa-levodopa, entacapone, levodopa, pergolide mesylate, pramipexole dihydrochloride, ropinirole hydrochloride, selegiline hydrochloride, tolcapone, or trihexyphenidyl hydrochloride; bupropion hydrochloride, donepezil hydrochloride, droperidol, and fluvoxamine maleate. Central nervous system drugs such as salts, lithium carbonate, lithium citrate, naratriptan hydrochloride, nicotine polarilex, nicotine transdermal system, propofol, rizatriptan benzoate, sibutramine hydrochloride monohydrate, sumatriptan succinate, tacrine hydrochloride, or zolmitriptan; cholinergic agents (e.g., parasympathetic stimulants) such as betanethyl chloride, edrophonium chloride, neostigmine bromide, neostigmine methylsulfate, physostigmine salicylate, or pyridostigmine bromide; astropine sulfate; dicyclochromium Anticholinergics such as scopolamine hydrochloride, glycopyrrolate, hyoscyamine, hyoscyamine sulfate, propantheline bromide, scopolamine, butylscopolamine bromide, or scopolamine hydrobromide; adrenergic agonists (sympathomimetic agents) such as dobutamine hydrochloride, dopamine hydrochloride, metalaminol bitartrate, norepinephrine bitartrate, phenylephrine hydrochloride, pseudoephedrine hydrochloride, or pseudoephedrine sulfate; dihydroergotamine mesylate, ergotamine tartrate, methyserzide maleate, or propranolol Adrenergic blockers (sympathetic nerve blockers) such as hydrochloride, skeletal muscle relaxants such as baclofen, carisoprodol, chlorzoxazone, cyclobenzaprine hydrochloride, dantrolene sodium, methocarbamol, or tizanidine hydrochloride, neuromuscular blockers such as atracurium besylate, cisatracurium besylate, doxacrium chloride, mibacrium chloride, pancuronium bromide, pipecuronium bromide, rapacronium bromide, rocuronium bromide, succinylcholine chloride, tubocurarine chloride, or vecuronium bromide, or betamethasone,It may be an adrenocortical steroid such as betamethasone acetate or betamethasone sodium phosphate, betamethasone sodium phosphate, cortisone acetate, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, fludrocortisone acetate, hydrocortisone, hydrocortisone acetate, hydrocortisone cypionate, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, prednisolone, prednisolone acetate, prednisolone sodium phosphate, prednisolone tebutate, prednisone, triamcinolone, triamcinolone acetonide, or triamcinolone acetate.

[0100] kit Also provided herein is a kit comprising a spine-forming initiation dose of the compound or its pharmaceutical composition thereof, optionally a second activator, and a suitable package. In one embodiment, the kit further includes instructions for use. In one embodiment, the kit includes a spine-forming initiation dose of the compound or its pharmaceutical composition thereof, and a label and / or instructions for use relating to the use of the pharmaceutical composition in the treatment of neuronal diseases or conditions as described herein.

[0101] Furthermore, provided herein are manufactured articles comprising a spine-forming initiation dose of the compound described herein, or its pharmaceutical composition, in a suitable container. The container may be a vial, jar, ampoule, pre-filled syringe, nebulizer, aerosol dispenser, dropper, or bag for intravenous injection.

[0102] Pharmaceutical composition and administration mode The compounds described herein in spine-forming initiation doses may be administered in the form of pharmaceutical compositions. Therefore, also provided herein are pharmaceutical compositions comprising the compounds described herein in spine-forming initiation doses and one or more pharmaceutically acceptable excipients. Suitable pharmaceutically acceptable excipients include, for example, inert solid diluents and fillers, diluents including sterile aqueous solutions and various organic solvents, permeability enhancers, solubilizers, and adjuvants. Such compositions are prepared in manner well known in the pharmaceutical field. See, for example, Remington's Pharmaceutical Sciences, Mace Publishing Co., Philadelphia, Pa. 17th Ed. (1985) and Modern Pharmaceutics, Marcel Dekker, Inc. 3rd Ed. (GS Banker & C.T. Rhodes, Eds.).

[0103] Pharmaceutical compositions may be administered by various methods, including, for example, nasal, rectal, intrachuccal, intranasal, and transdermal routes. In certain embodiments, pharmaceutical compositions may be administered by intra-arterial injection, intravenous, intraperitoneal ("ip"), parenteral, intramuscular, subcutaneous, oral, topical, or as inhalants.

[0104] One mode of administration is, for example, parenteral administration by injection. Forms into which the pharmaceutical compositions described herein can be incorporated for administration by injection include, for example, aqueous or oily suspensions or emulsions containing sesame oil, corn oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose, or sterile aqueous solutions, and similar pharmaceutical vehicles.

[0105] Oral administration may be another route for administering the compositions described herein. Administration may be carried out, for example, via capsules or enteric-coated tablets. When preparing a pharmaceutical composition comprising at least one of the compounds described herein, or a pharmaceutically acceptable salt or solvate thereof, the active ingredient is usually diluted with an excipient and / or encapsulated in such a carrier, which may be in the form of a capsule, sachet, paper, or other container. Where the excipient acts as a diluent, it functions as a vehicle, carrier, or medium for the active ingredient and may be in the form of a solid, semi-solid, or liquid material. Thus, the composition may be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solid or liquid media), ointments, soft and hard gelatin capsules, sterile injections, and sterile packaged powders, for example, containing up to 10% by weight of the active compound.

[0106] Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The formulation may further contain lubricants, wetting agents, emulsifiers and suspending agents such as talc, magnesium stearate, and mineral oil, as well as preservatives, sweeteners, and flavoring agents such as methyl and propyl hydroxybenzoic acid.

[0107] The pharmaceutical composition and any containers into which it is dispensed may be sterilized. The pharmaceutical composition may also contain adjuvants such as preservatives, stabilizers, emulsifiers, or suspending agents, wetting agents, salts for altering osmotic pressure, viscosity modifiers, or buffers.

[0108] At least one of the compounds described herein, such as the compounds described herein or their pharmaceutically acceptable salts or solvates, can be formulated to provide rapid, sustained, or delayed release of the active ingredient after administration to a subject using procedures known in the art. Controlled-release drug delivery systems for oral administration include osmotic pump systems and dissolution systems containing polymer-coated reservoirs or drug-polymer matrix formulations. Examples of controlled-release systems are described in U.S. Patents 3,845,770, 4,326,525, 4,902,514, and 5,616,345. Another formulation for use in the manner disclosed herein is a transdermal delivery device ("patch"). Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds described herein in controlled amounts. The construction and use of transdermal patches for drug delivery are well known in the art. See, for example, U.S. Patent Nos. 5,023,252, 4,992,445, and 5,001,139. ​​Such patches may be constructed for continuous, pulsating, or on-demand delivery of the drug.

[0109] To prepare solid compositions such as tablets, the main active ingredient may be mixed with a pharmaceutically acceptable excipient to form a solid pre-formulation composition containing a homogeneous mixture of the compounds described herein, or mixtures of their pharmaceutically acceptable salts or solvates. When these pre-formulation compositions are referred to as homogeneous, the active ingredient is uniformly dispersed throughout the composition, and therefore the composition can be easily subdivided into equally effective unit dosage forms such as tablets, pills, and capsules.

[0110] Tablets or pills of the compounds described herein may be coated or otherwise formulated to provide a dosage form that offers the benefit of long-lasting action or to protect from the acidic conditions of the stomach. For example, a tablet or pill may contain an inner dose component and an outer dose component, the latter in the form of an envelope covering the former. The two components may be separated by an enteric coating that helps to resist disintegration in the stomach and allow the inner component to pass through the duodenum intact or to delay its release. A variety of materials may be used for such enteric coatings or coatings, and such materials include several polymer acids and mixtures of polymer acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0111] Pharmaceutical compositions may be formulated for nasal administration. Such pharmaceutical compositions may contain one or more active ingredients, such as compounds described herein or pharmaceutically acceptable salts or solvates thereof, in various physical states. For example, the active ingredients may be dissolved or suspended in a liquid carrier. The active ingredients may be in a dry form. The dry form may be a powder. The active ingredients in the powder may be amorphous or crystalline. For example, compounds described herein or pharmaceutically acceptable salts or solvates thereof may be amorphous or crystalline. Crystalline active materials may be hydrates or solvates.

[0112] Solid compounds, or their salts or crystals, may be present in the formulation at selected average particle sizes. The particles may have average particle sizes (at their longest dimension) ranging from 10 nm, 100 nm, 300 nm, 500 nm, 1 μm, 10 μm, 50 μm, 100 μm, 300 μm, or 500 μm, or between any two of these values.

[0113] Administration may be by inhalation or inhalation. Compositions for inhalation or inhalation may include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, as well as powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described herein. In some embodiments, compositions are administered orally or via nasal respiratory routes. The effects may be topical or systemic. In certain embodiments, the effects are topical to the cranial tissue. In other embodiments, compositions in pharmaceutically acceptable solvents may be sprayed using an inert gas. The sprayed solution may be inhaled directly from the spraying device, or the spraying device may be attached to a face mask tent or an intermittent positive pressure respirator. Solutions, suspensions, or powder compositions may be administered preferably orally or nasally from a device for delivering formulations in an appropriate manner. Pharmaceutical compositions for inhalation or inhalation may be aerosols.

[0114] The pharmaceutical composition may comprise a liquid suspension or solution containing an active ingredient in an amount of approximately 0.05%, 0.1%, 0.3%, 0.5%, 0.7%, 1%, 2%, 3%, 4%, or 5% w / w. The liquid may comprise water and / or alcohol. The liquid may comprise a pH adjuster so that the pH is in the range of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in between.

[0115] The pharmaceutical composition may contain pharmaceutically acceptable preservatives. Suitable preservatives for use herein include, but are not limited to, phenylethyl alcohol, benzalkonium chloride, benzoic acid, or benzoates such as sodium benzoate, which protect the solution from contamination by pathogenic particles. In certain embodiments, the pharmaceutical composition contains about 0.01% to about 1.0% w / w of benzalkonium chloride, or about 0.01% to about 1% v / w of phenylethyl alcohol. The preservative may also be present in an amount of about 0.01% to about 1%, preferably about 0.002% to about 0.02%, relative to the total weight or total volume of the composition.

[0116] The pharmaceutical composition may also contain one or more emulsifiers, wetting agents, or suspending agents in amounts of approximately 0.01% to approximately 90%, or approximately 0.01% to approximately 50%, or approximately 0.01% to approximately 25%, or approximately 0.01% to approximately 10%, or approximately 0.01% to approximately 1% w / w.Such agents for use herein include, but are not limited to, polyethylene sorbitan monooleate (polysorbate 80), polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 65 (polyoxyethylene (20) sorbitan tristearate), polyoxyethylene (20) sorbitan monooleate, polyoxyethylene (20) sorbitan monopalmitate, polyoxyethylene (20) sorbitan monostearate; lecithin; alginic acid; sodium alginate; al Potassium ginate; ammonium alginate; calcium alginate; propane-1,2-diol alginate; agar; carrageenan; locust bean gum; guar gum; tragacanth; acacia; xanthan gum; karaya gum; pectin; amidated pectin; ammonium phosphatide; microcrystalline cellulose; methylcellulose; hydroxypropylcellulose; hydroxypropyl methylcellulose; ethyl methylcellulose; carboxymethylcellulose; sodium, potassium, and calcium salts of fatty acids; mono- and Diglycerides; Acetates of mono- and di-glycerides of fatty acids; Lactates of mono- and di-glycerides of fatty acids; Citrates of mono- and di-glycerides of fatty acids; Tartaric acid esters of mono- and di-glycerides of fatty acids; Mono- and di-acetyl tartaric acid esters of mono- and di-glycerides of fatty acids; Mixed acetates and tartaric acid esters of mono- and di-glycerides of fatty acids; Sucrose esters of fatty acids; Sucrose glycerides; Polyglycerol esters of fatty acids; Polyglycerol esters of castor oil condensation polymers of fatty acids Examples of polyoxyethylene sorbitan fatty acid esters or polysorbates include, but are not limited to, polyglycerol esters; propane-1,2-diol esters of fatty acids; sodium stearoyl-2-lactate; calcium stearoyl-2-lactate; stearoyl tartrate; sorbitan monostearate; sorbitan tristearate; sorbitan monolaurate; sorbitan monooleate; sorbitan monopalmitate; quillaja extract; polyglycerol esters of dimerized fatty acids of soybean oil; oxidatively polymerized soybean oil; and pectin extract.

[0117] In further embodiments, the pharmaceutical composition for nasal administration may be provided in powder form. For example, the powdered nasal composition can be used directly as a unit dosage form. If desired, the powder can be filled into capsules, such as hard gelatin capsules. The contents of the capsule or single-dose device can be administered, for example, using an inhaler.

[0118] Therefore, a method for treating neuronal disease or disorder may include the step of administering a pharmaceutical composition comprising the compound described herein, or a salt thereof, intranasally to a subject requiring a method for treating neuronal disease or disorder. [Examples]

[0119] Example 1 Transgenic mice and treatment protocols The study used 6-month-old 3xTg-AD and wild-type (WT) female homozygous mice. Characterization of 3xTg-AD mice was previously described. Additional steps are found in Oddo, S., et al. (2003). Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic dysfunction, Neuron 39, 409-21. Trujillo-Estrada, et al. (2019).

[0120] Two different doses of compound 1, 3 mg / kg and 30 mg / kg, were used. 3xTg-AD and WT mice were randomly divided into groups (n=8-11): WT vehicle (n=11), 3xTG-AD vehicle (n=10), 3xTG-AD compound 1, 3 mg / kg (n=10), 3xTG-AD compound 1, 10 mg / kg (n=11), and 30 mg / kg (n=10). Compound 1 (dissolved in 5% dimethyl sulfoxide (DMSO) and diluted to 5% DMSO with phosphate-buffered saline (PBS)) was administered intraperitoneally once daily for 4 weeks. Animals treated with the vehicle (5% DMSO in PBS) were used as a negative control group. WT mice were used as a comparator to confirm neurodegeneration in 3xTG-AD mice. After all behavioral experiments were completed, the mice were euthanized.

[0121] After euthanasia, the animals were perfused transcardiacally with 0.1 M phosphate-buffered saline pH 7.4. Next, one hemisphere of the brain was stained with Golgi solution using the superGolgi Kit (Bioenno Tech LLC, Santa Ana, CA), and synaptosomes were prepared. The other hemisphere was then used for immunohistochemistry as described below.

[0122] Golgi staining of dendritic spines Brain cells were incubated in the impregnation solution for 11 days, followed by incubation in the post-impregnation solution for 2 days. Once neuronal impregnation was complete, thick (150 μm) free-floating sections were obtained using an HA752 vibratome (Campden Instruments Ltd, Lafayette, IN) and collected sequentially in mounting buffer. Each section mounted on a coated slide was stained and post-stained for 20 minutes, dehydrated in serial ethanol, cleared with xylene, and mounted on a coverslip with DPX (VWR, Visalia, CA, USA) mounting medium.

[0123] Dendritic spine analysis Using Neurolucida software from Microbrightfield Bioscience (MBF Bioscience, Williston, VT, USA), stereometric analysis and quantification were performed to determine the number of spines in the radial laminae (sr) of the hippocampal CA1 region and the prefrontal cortex. Briefly, all second sections throughout the entire anterior-posterior range of each region under consideration were used. Spines were counted using a 100× / 1.4 objective lens (5-6 sections per animal, n=5 per group).

[0124] Synaptosome extract In short, hippocampi from non-Golgi-treated hemispheres were homogenized (using a Dounce homogenizer) in DEPC-treated water (Ambion) supplemented with 0.32 M sucrose, 20 mM Tris-HCl, 0.5 M EDTA, and 0.5 M EGTA (pH 7.4) containing a complete protease (Sigma) and phosphatase inhibitor cocktail (Sigma). After homogenization, the crude synaptosome fraction (synaptosomes + mitochondria) was isolated by two consecutive centrifugations (1,500 × g, 10 min, followed by 12,500 × g, 20 min; 4°C). The protein content of the synaptosome fraction was determined using the Bradford assay. For Western blotting (WB) experiments, the synaptosome preparations were transiently stored at -80°C.

[0125] Immunoblot Equal amounts of protein (20 μg) were separated on a 10% Bis-Tris gel (Invitrogen, Carlsbad, CA) and transferred to a nitrocellulose membrane. The membrane was blocked for 1 hour in a 5% (w / v) suspension of bovine serum albumin (BSA; Gemini Bio-Products, West Sacramento, CA, USA) in 0.2% Tween® 20 Tris buffered saline (pH 7.5). After blocking, the membrane was incubated overnight at 4°C with one of the following primary antibodies: anti-drebrin (1:1000; Enzo Life Sciences), anti-GluA1 (1:1000; Cell Signaling), anti-p-GluA1 (Ser845; 1:1000; Cell Signaling), anti-postsynaptic density protein 95 (PSD95; 1:1000; Cell Signaling), anti-synaptic vesicle glycoprotein 2 (SV2A; 1:1000; Abcam), anti-fascin (1:1000; Abcam), anti-p-fascin (1:1000; Abcam), anti-synaptophysin (1:2000; Abcam), and anti-β-tubulin (1:5000; Cell Signaling). The membrane was washed in tween®-TBS for 20 minutes and incubated with a specific secondary antibody diluted 1:10000 (Pierce Biotechnology) at 20°C for 60 minutes. The blot was developed using Super Signal (ThermoFisher Scientific, Rockford, IL, USA).

[0126] immunohistochemistry Floating sections (40 μm thick) without coronal planes were pre-treated with 3% H2O2 / 3% methanol in Tris-buffered saline (TBS) for 30 minutes to block endogenous peroxidase activity. After washing with TBS, the sections were incubated for 15 minutes in TBS containing 0.1% TritionX-100 (TBST), and then for 30 minutes in TBST containing 2% bovine serum albumin (BSA, Sigma-Aldrich). The sections were incubated overnight at room temperature with TBS + 5% normal horse serum containing 6E10 (1:1000; BioLegend, San Diego, CA, USA), anti-HT7 (1:500; Thermo Scientific), and anti-AT180 (1:500; Thermo Scientific). Next, the sections were incubated at 20°C for 1 hour in a 1:500 mixture of biotinylated anti-mouse, TBS + 2% BSA + 5% normal serum, followed by incubation with Vector ABC Kit and DAB reagent (Vector Laboratories, Burlingame, CA, USA) to visualize the staining.

[0127] For double fluorescence staining, sections were incubated overnight at 4°C with anti-PSD95 (1:250; Invitrogen) and anti-synaptophysin (1:700; Sigma). Next, sections were incubated for 1 hour with secondary goat anti-mouse alexa-fluor555 in synaptophysin and with goat anti-rabbit alexa-fluor488 in PSD95 antibody (Invitrogen). The sections were then mounted and covered with a coverslip using Fluoromount-G (Southern Biothech).

[0128] quantitative analysis Biochemical data were quantitatively analyzed using ImageJ1.36b software. For synaptophysin / PSD95 quantification, fluorescence sections were imaged using a Leica DM2500 laser scanning confocal with the same laser and detection settings. Using a 63x oil immersion objective lens (zoom=4), Z-stacks (0.34 μm spacing, within a depth of 3 μm) were collected for each section (6 sections per animal, n=5) for each target region (CA1, radial layer). The number of synaptic points and the number of co-localizations between PSD95 and synaptophysin points (number of co-localization spots at distances of 200 nm or less) were quantified using Bitplane Imaris software (spot function).

[0129] Aβ and tau ELISA Aβ and tau levels were measured using the V-Plex Aβ Peptide Panel 1 kit and the Phospho-Thr231 / Total Tau kit, respectively. For the Aβ ELISA, 150 μl / well of Diluent35 was added to the plate and incubated at room temperature for 1 hour. After washing, 25 μl of detection antibody solution and 25 μl of sample (soluble fraction, S1), calibrator or control were added to each well and incubated at 4°C for 24 hours. The samples were then washed, 150 μl of Read buffer was added to each well, and the plate was read using a MESO QuickPlex SQ120 instrument. For the tau ELISA, 150 μl / well of Blocker A solution was first added and incubated at room temperature for 1 hour. The plate was then washed, and 25 μl of calibrator or sample was added to each well. The plate was then washed again and incubated in detection antibody solution (25 μl) at room temperature for 1 hour. Finally, the samples were washed, 150 μl of Read buffer was added, and the data was read using a MESO QuickPlex SQ120 instrument. The obtained data were normalized by the protein concentration of each sample.

[0130] In vitro assay In short, neurons from the hippocampus of three neonatal rats were cultured, placed in 24-well plates, and cultured for 14 days. On day 1, the culture medium was replaced with plain medium. On day 4, mitosis of glial cells was stopped, and 5 nM araC was added to avoid background staining, multidimensional focal plains, and excessive nutrient consumption. On day 14, compound 1 was added to the culture wells at several concentrations (0.1, 0.3, 1, 3, and 10 μM) using 0.1% DMSO as the solvent. On day 15, cells were fixed onto coverslips using methanol and double-stained with an antibody against PSD95 for synaptic counting of spines on basal dendrites and with an antibody against synaptobrevin II for presynaptic imaging. DAPI staining was used for nuclear counting. The number of spines per 20 μm long dendrite was compared to the untreated control group. The vehicle group was treated with 0.1% DMSO.

[0131] For immunocytochemistry, coverslips containing primary rat hippocampal neurons from day 15 were fixed with 100% frozen methanol and permeabilized with 0.02% Triton® X-100. The neurons were then incubated in BSA at room temperature for 1 hour, followed by incubation with their respective antibodies: mouse anti-PSD95 (Thermofisher) and rabbit anti-synaptobrevin II (SYSY). Subsequently, they were incubated with secondary antibodies: Alexa Fluor488 conjugate anti-mouse antibody and Alexa Fluor594 conjugate anti-rabbit antibody. Coverslips were mounted on slides using a fluorescent mounting medium containing DAPI (Gbi). Approximately 10 images were taken from different regions of a total of three coverslips per treatment group. Imaging was performed using a BX43 Olympus microscope driven by standard Olympus "CellSens" software. Images were taken using a DP74 camera (Olympus) under a 60x water immersion objective lens. Dendritic spines were automatically detected using ImageJ software. To perform analysis on PSD95 content puncta, the exclusion threshold was set 5 points above the background. Irrelevant material such as cell bodies, glia, and synapses from the focal plane were dimmed. The ImageJ plugin "NeuronJ" was used to estimate neurite lengths.

[0132] statistical analysis All experiments were conducted blinded, and data were analyzed using one-way or two-way analysis of variance (ANOVA), followed by Tukey comparisons using Graphpad Prism8® software (Graphpad Prism Inc., San Diego, CA, USA). Significance was set at a 95% confidence level. All values ​​are presented as mean ± SEM.

[0133] result In vivo experiments demonstrate that treatment with compound 1 mitigates behavioral impairment in 3xTg-AD mice. To evaluate the cognitive effects of compound 1, 6-month-old WT mice and 3xTg-AD mice were administered either vehicle or compound 1 at 3 or 30 mg / kg (ip) daily for 4 weeks, behavioral assessments were performed during the 4 weeks, and the mice were subsequently sacrificed for histological and biochemical assays in the brains of the same animals. To assess whether compound 1 rescues cognitive function in 3xTg-AD mice, we evaluated spatial memory in vehicle and compound 1-treated WT and 3xTg-AD mice using the Morris water maze test (Figure 1). Vehicle-treated 3xTg-AD mice showed severe impairment in learning during acquisition of spatial tasks compared to vehicle-treated WT mice. These deficits were reversed by compound 1 treatment at both 3 and 30 mg / kg doses (Figure 1). In addition, to assess memory retention, mice were tested 24 hours after the last training test. Vehicle-treated 3xTg-AD mice showed significant impairment in memory retention, as measured by the frequency of visits to target areas and the time spent there (Figures 2A-2B). Treatment of 3xTg-AD mice with compound 1 at 3 and 30 mg / kg significantly improved memory retention in these assays. Furthermore, the cognitive impairment observed in 3xTg-AD vehicle mice was not due to motor defects, as no significant changes were observed in travel distance or speed in any of the treatment groups (including WT and 3xTg-AD mice) (Figures 2C-2D).

[0134] To further characterize the memory-enhancing effects of compound 1 in 3xTg-AD mice, we used another hippocampus-dependent task, contextual fear conditioning (CFC) paradigm (Figure 3). Vehicle-treated 3xTg-AD mice showed a significantly reduced freeze response to the conditioned context compared to wild-type vehicle mice. The duration of the freeze response significantly increased after compound 1 treatment in 3xTg-AD mice, and was similar to that of wild-type mice. Overall, these data suggest that compound 1 reverses hippocampal learning and memory deficits in 3xTg-AD mice.

[0135] Experiments demonstrate that compound 1 restores the number of postsynaptic points and their co-localization with presynaptic factors in 3xTg-AD mice. Next, we used immunofluorescence microscopy to assess whether the improvement in compound 1-induced 3xTg-AD mice on a hippocampus-dependent memory task was associated with changes in presynaptic and postsynaptic protein points in the radial langue of hippocampal CA1. Analysis of immunolabeled sections of the postsynaptic scaffold protein PSD95 revealed a significant defect in the number of postsynaptic points (Figure 4 panels A1a-A1d and Figure 5A) in vehicle-treated 3xTg-AD mice (Figure 4 panel A1b) compared to WT vehicle mice (Figure 4 panel A1a). Postsynaptic points were restored by compound 1 treatment at both 3 and 30 mg / kg doses (Figure 4 panels A1c and A1d). In 3xTg-AD mice, a decrease in the number of presynaptic factors containing synaptophysins was also observed, but this was not rescued by treatment with compound 1 at 3 or 30 mg / kg (Figure 4 panels A2a-A2d and Figure 5B). However, treatment with compound 1 (30 mg / kg) rescued the deficiency of colocalization of PSD95- and synaptophysin-labeled factors (Figure 4 panels A3a-A3d) in 3xTg-AD mice at 30 mg / kg (Figure 5C), which was considered an immunohistochemical measure of functional synapses.

[0136] Experiments show that compound 1 reverses a significant decrease in dendritic spine density in 3xTg-AD mice and improves spine formation in vitro. Therefore, we attempted to verify that compound 1 increases dendritic spine density in a 3xTg-AD model, as expected and as suggested by both behavioral immunohistochemical data. We performed Golgi staining and stereochemical quantification of dendritic spines in the radial layer (sr) of hippocampal CA1 (Figure 6 panels A1 and A2). Stereochemical quantification showed that vehicle-treated 3xTg-AD mice showed a significant reduction in dendritic spine density (Figure 6 panels A1 and A2), particularly in mushroom and stubby spine profiles (Figure 7), compared to vehicle-treated WT mice. These significant defects in dendritic spines (approximately 35–50%) in 3xTg-AD mice were reversed by treatment with compound 1 at 3 and 30 mg / kg (Figure 6 panels A1, A3, and A4), increasing spine density in 3xTg-AD mice to levels not statistically different from those seen in vehicle-treated WT mice. Surprisingly, treatment with compound 1 overcame the level of spine loss that occurred before treatment was initiated at 6 months, as well as all spine loss that occurred during 4 weeks of treatment.

[0137] We also evaluated the in vitro effect of compound 1 on dendritic spine density on primary hippocampal neurons from rats (Figure 8; see also Figure 14). Treatment with compound 1 at concentrations of 0.1, 0.3, 1, 3, and 10 μM resulted in a statistically significant increase in mean spine density (spines / 20 μm) compared to the vehicle group (37.41±5.88, 33.72±2.75, 35.65±3.68, 45.97±3.89, and 24.71±2.33, respectively, compared to 12.54±1.81 in the vehicle control). Treatment with the vehicle (0.1% DMSO) did not increase mean spine density compared to the untreated control group (14.76±1.02 vs. 12.54±1.81, respectively).

[0138] To further understand the nature of compound 1's synaptic rescue effect, we measured the levels of several key synaptic proteins, including drebrin, GluA1, p-GluA1, PSD95, synaptic vesicle glycoprotein A (SV2A), phascin, and synaptophysin, by Western blotting of hippocampal synaptosomes (Figures 9 and 10) prepared from a subset of animals tested in behavioral assays. Changes in the levels and activity of these synaptic markers are associated with memory impairment in AD. Consistent with the observed spine-deficient 3xTg-AD mice, WB analysis revealed a significant decrease in steady-state levels of drebrin, PSD95, and p-GluA1 / GluA1 ratio in 3xTg-AD vehicle mice compared to WT vehicle mice. The levels of these proteins were significantly increased with compound 1 treatment (Figures 9 and 10). These data suggest that the structural effect of compound 1 on dendritic spine density is accompanied by an increase in key scaffolds, actin regulation, and glutamatergic signaling proteins involved in the formation and plasticity of glutamatergic synapses.

[0139] The experiment demonstrates that compound 1 does not alter Aβ or Tau levels in 3xTg-AD mice. Since the accumulation of Aβ and hyperphosphorylated tau is a characteristic feature of AD molecular pathology, any drug affecting AD progression could alter one or both of these features, even if it does not directly target these pathways. However, as measured by immunohistochemistry (Figure 11) and ELISA (Figure 12A), Aβ levels were not altered by compound 1 treatment in 3xTg-AD mice. Furthermore, immunohistochemistry and ELISA analysis revealed that neither steady-state Tau (HT7) nor phospho-Tau species recognized by AT180 (Thr231) were altered by compound 1 treatment (Figures 11 and 12B). The data suggest that the effect of compound 1 on the restoration of synaptic and cognitive deficits in 3xTg-AD is not associated with any changes in Aβ and / or tau levels.

[0140] Example 2 Homozygous transgenic TDP-43 mice typically begin to die by day 23. This study utilized a mouse model of rapidly progressing ALS. Developed by the Kumar-Sing [Wils, 2010] lab, this model overexpresses human TDP-43 under the control of the Thy-1 promoter. In the majority of ALS cases, TDP-43, primarily a nuclear mRNA-binding protein, is mislocalized to the cytoplasm and subsequently modified and cleaved post-translation. This misregulation of TDP-43 mapping to brain regions affected by ALS (and FTD) appears to be a major driving factor in the pathogenesis. Therefore, mutations in TDP-43 that promote the formation of TDP-43 cytoplasmic inclusions are responsible for ALS in humans. Homozygous mice with the human TDP-43 transgene (indicated as TAR4 / 4) develop motor symptoms, tremors, and postural phenotypes within the first two weeks of life, which rapidly progress to death, typically between 22 and 25 days postnatally (PND), starting at the beginning of the fourth week of life. This model is used to screen for candidate therapeutic agents that can significantly impact disease progression [Becker, 2017].

[0141] In our ongoing study at USC, homozygous TDP-43 mice were treated daily with compound 1 (15 mg / kg, intraperitoneal [IP]) from PND14, the point at which the ALS-related phenotype is already present, until euthanasia was indicated. Significant improvements in motor function were observed in gait (Figure 15), posture (Figure 16), and tremor (Figure 17). On the scale used, wild-type mice scored 0 (no disability).

[0142] All vehicle-treated mice died by day 24, but compound 1-treated mice survived until PND33,34 (Figure 18). Thus, the treatment regimen regenerated lost synapses and produced significant improvements in both motor function and survival, even when intervention was not initiated until after the onset of symptoms in this highly aggressive model of ALS-like pathogenesis.

[0143] Example 3 Neurons from the hippocampus of neonatal rats were cultured, placed in 24-well plates, and cultured for 14 days. On day 1, the culture medium was replaced with plain medium. On day 4, 5 nM araC was added to halt glial cell mitosis and to avoid background staining, multidimensional focal plains, and excessive nutrient consumption. On day 14, the test compound was added to the culture wells at several concentrations (1, 10, 50, and 100 nM) using DMSO as the solvent. The medium was replaced with the test compound, and the cells were washed to remove the test compound at various time points (2 min, 5 min, 10 min, 30 min, 45 min, 1 hour, and 2 hours). Cells were fixed onto coverslips using methanol and double-stained with antibody against PSD95 for synaptic counting of spines on basal dendrites and antibody against synaptobrevin II for presynaptic imaging. DAPI staining was used for cell nuclear counting. The number of spines per 20 μm-long dendrite will be compared to that of the untreated control group. The vehicle group will be treated with 0.1% DMSO.

[0144] Example 4 This study used Ubqln2 P497S mice and wild-type (WT) mice. The characterization of Ubqln2 P497S mice was previously described in Le, N et al, "Motor neuron disease, TDP-43 pathology, and memory deficits in mice expressing ALS-FTD-linked UBQLN2 mutations." Proceedings of the National Academy of Sciences 113.47(2016):E7580-E7589.

[0145] Ubqln2 P497S mice and wild-type (WT) mice were divided into three groups (n=9): WT vehicle, Ubqln2 vehicle, and Ubqln2 compound 1 (10 mg / kg). Compound 1 (dissolved in 5% dimethyl sulfoxide (DMSO) and diluted to 5% DMSO with phosphate-buffered saline (PBS)) was administered once daily by intraperitoneal injection for 4 weeks. Animals treated with the vehicle (5% DMSO in PBS) were used as a negative control group. WT mice were used as the control group. After completion of treatment, the mice were euthanized.

[0146] Golgi staining and dendritic spine density analysis were performed using the same procedure as described in Example 1.

[0147] result Figure 19 shows the spine density of the motor cortex layer 5 in the basal and apical dendrites of the WT vehicle, Ubqln2 vehicle, and Ubqln2 compound 1 (10 mg / kg) groups. Figure 20 shows the postmortem Golgi staining of the dendritic spines. Figures 21 and 22 show the spine density and Golgi staining of the medial prefrontal cortex of each group, respectively. Figures 23 and 24 show the spine density and Golgi staining of the hippocampal CA1 area of ​​each group, respectively.

[0148] In vivo experiments and imaging of dendritic spines show that treatment of Ubqln2 P497S mice with compound 1 at 10 mg / kg increases dendritic spine density compared to a vehicle-treated Ubqln2 control group. This study complements the findings of the 3xTg-AD mouse study described in Example 1 and demonstrates the efficacy of compound 1 in a Ubqln2 mouse model for ALS / FTD.

[0149] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as they are generally understood by those skilled in the art to which this disclosure belongs.

[0150] The disclosures described herein as illustrative can be preferably implemented without any elements(s) or limitations(s) not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” and “containing” should be read broadly and non-restrictively. Additionally, the terms and expressions used herein are for illustrative purposes only, not limitation, and no intention is made to exclude such terms and any equivalents of the exhibited and described features or parts thereof, although it is acknowledged that various modifications are possible.

[0151] Therefore, although this disclosure has been specifically disclosed through preferred embodiments, it should be understood that any optional features, modifications, improvements, and variations of the disclosure embodied herein can be reused by those skilled in the art, and such modifications, improvements, and variations are deemed to be within the scope of this disclosure. The materials, methods, and examples provided herein represent preferred embodiments and are illustrative; they are not intended to be limitations on the scope of this disclosure.

[0152] All publications, patent applications, patents, and other references referred to herein are expressly incorporated by reference to the same extent that each is incorporated by reference individually. In case of any conflict, this specification, including definitions, shall prevail.

Claims

1. A method for repairing diseased neurons in mammals suffering from neurodegenerative diseases, wherein the method is An effective dose of the compound of formula I administered once daily to the aforementioned mammal: 【Chemistry 11】 or administering a pharmaceutically acceptable salt thereof, isotope-enriched analogue, tautomer, prodrug, stereoisomer, or mixture of stereoisomers, In the formula, R is hydrogen, C １ -C ４ Alkyl, halo, hydroxyl, amino, cyano, or nitro, q is between 2 and 8. The compound comes into contact with the neuron, initiating the repair of the neuron by the subsequent formation of new dendritic spines, the spines of which form functional synapses with axons on other neurons, and the repair continues in the absence of the effective amount of the compound. A method for characterizing the aforementioned neurodegenerative disease as being in an early or intermediate stage.

2. The method according to claim 1, wherein the neurodegenerative disease includes ataxia.

3. The method according to claim 1, wherein the neurodegenerative disease includes cognitive loss.

4. The method according to claim 1, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson's disease, frontotemporal dementia, and Huntington's disease.

5. The method according to claim 1, wherein the neurodegenerative disease includes dementia.

6. The method according to claim 5, wherein the dementia is caused by AD or frontotemporal dementia.

7. The method according to claim 1, wherein the mammal is a human and the effective dose is 0.4 mg / kg / day or less.

8. The method according to claim 7, wherein the effective amount is about 0.1 to about 0.4 mg / kg / day.

9. The method according to claim 1, wherein the neurodegenerative disease is Alzheimer's disease.

10. The method according to claim 9, wherein the Alzheimer's disease is in an early stage.

11. The method according to claim 9, wherein the Alzheimer's disease is in the intermediate stage.

12. The method according to claim 1, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).

13. The method according to claim 12, wherein the ALS is in an early stage.

14. The method according to claim 12, wherein the ALS is in the intermediate stage.

15. The method according to claim 1, wherein the compound has a serum half-life of less than 30 minutes in rodents.

16. A unit dose of a compound of formula IA for initiating the formation of stable functional synapses in mammals suffering from reduced spine density and consequently impaired functional synapses, wherein the unit dose is approximately 0.4 mg / kg / day or less: 【Chemistry 12】 or comprising a pharmaceutically acceptable salt thereof, isotope-enriched analogue, tautomer, prodrug, stereoisomer, or mixture of stereoisomers, q is between 2 and 8. A unit dose of the compound having a serum half-life of less than 3 hours, and further, the unit dose provides stable functional synapse formation in the mammal.

17. The unit dose according to claim 1, wherein the unit dose comprises approximately 0.1 mg / kg / day to approximately 0.4 mg / kg / day.

18. The unit dose according to claim 1, wherein the decrease in functional spine density is a result of a neurological condition.

19. The unit dose according to claim 1, wherein the unit dose is prescribed for administration once daily.

20. A method for generating a functional spine on a neuron, wherein the method comprises a compound of formula I in an effective amount on the neuron: 【Chemistry 13】 or transient contact with a pharmaceutically acceptable salt, isotope-enriched analog, tautomer, prodrug, stereoisomer, or mixture of stereoisomers thereof, In the formula, R is hydrogen, C １ -C ４ Alkyl, halo, hydroxyl, amino, cyano, or nitro, q is between 2 and 8. The aforementioned transient contact initiates spine-forming activity, which leads to the formation of new functional spines. A method wherein an effective amount of the transient contact is terminated after initiation and before the functional spine is fully mature.

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