KR20240024033A - Effective administration methods for Compounds inhibiting mTOR signaling and composition for preventing or treating neurological diseases comprising the same as an active ingredient - Google Patents

Abstract

The present invention relates to a method of administering a pharmaceutical composition containing a novel compound that inhibits the mTOR signaling pathway as an active ingredient. Methods of administering via oral administration and transdermal patch instead of intraperitoneal injection were presented and compared. The compound of the present invention was found to inhibit mTORC1, activate autophagy, and alleviate a series of conditions associated with Alzheimer's-type dementia in dementia model animals, resulting in neurological diseases including Alzheimer's-type dementia, degenerative brain disease, and mTOR disease. It is a pharmaceutical composition that can prevent or treat, and it can be usefully used as an administration method for patients with degenerative brain disease or mental development disorders such as autism, who need to take it continuously after onset by using oral administration or transdermal patches instead of injections. there is.

Description

Effective administration methods for compounds inhibiting mTOR signaling and composition for preventing or treating neurological diseases comprising the same as an active ingredient}

The present invention relates to an efficient administration route for a composition for preventing or treating neurological diseases containing a compound that inhibits the mTOR signaling pathway as an active ingredient.

The main cause of degenerative brain diseases, including dementia, is neuronal death due to abnormal protein homeostasis (abnormal proteostasis) of causative proteins, and controlling this can be a causative treatment method. Alzheimer's dementia (AD), a representative degenerative brain disease, causes gradual impairment of short-term memory and cognitive function, and the accumulation of senile plaques with Aβ in brain tissue and intracellular neurofibrillary tangles (NFTs) created by Tau hyperphosphorylation. This is observed as the main lesion.

The Aβ is produced when β-secretase and γ-secretase cleave APP due to mutations in Amyloid Precusor Protein (APP), presenillin-I (PS-I), or Presenillin-II (PS-II), and its abnormal structure ( β-sheet), it accumulates rather than being removed, and mutations such as APP, PS-I, PS-II, and tau become a genetic factor in the development of Alzheimer's disease (FAD). Both the senile plaques and NFTs are generated and accumulated due to abnormal protein homeostasis and induce death of surrounding nerve cells, and Aβ oligomer and hyperphosphorylated tau oligomer are transmitted to surrounding nerve cells through synapses, gradually causing more cells to die. do.

Until now, no treatment for Alzheimer's type dementia has been developed that treats the cause of dementia other than drugs that relieve symptoms. More than 100 candidate drugs for removing senile plaques, including β-secretase inhibitors, γ-secretase inhibitors, and monoantibodies that recognize Aβ and help dismantle senile plaques, have entered clinical trials. However, even if the candidate drugs remove senile plaques, there is no Clinical results showed that cognitive function was not recovering, and almost all of them were declared failures and discontinued throughout 2018 and 2019. The U.S. Food and Drug Administration (FDA) granted accelerated approval in 2021 for Aducanumab (Biogen), a monoclonal antibody that binds to Aβ aggregates and induces the disassembly of senile plaques, as a treatment for early-stage Alzheimer's-type dementia, but controversy continues over its efficacy. there is. The main dementia treatment currently in use is not a drug that treats the cause of the disease, but an acetylcholine enzyme inhibitor (Ache esterase inhibitor) that delays the decline in mental function.

The mTOR (mammalian target of rapamycin) complex regulates intracellular protein synthesis and autophagy and plays a role in causing cell growth. Specifically, mTOR is a serine/threonine kinase that combines internal and external signals and acts as the center of a signaling hub that regulates cell metabolism, growth, proliferation and survival. Therefore, abnormalities in mTOR signaling are found in many diseases, and excessive mTOR signaling is particularly problematic in neurological diseases and cancer (Lipton & Sahin (2014) Neuron 84: 275-291).

In the brain tissue of patients with Alzheimer's dementia, Aβ and NFT accumulation and pathological accumulation of hyperphosphorylated tau are associated with increased phosphorylation of S6K (S2481), a downstream signal transducer, due to increased mTOR signaling or activation of mTORC1 (mammalian target of rapamycin complex 1). Proportionality has been shown (Li et al. (2005) FEBS J. 272: 4211-4220). In addition, AKT, which activates mTORC1, is activated by mTORC2 and is increased in the temporal lobe brain of Alzheimer's dementia patients, and inhibition of mTOR signaling shows physiological and behavioral relief in animal models of Aβ accumulation. A decrease in the amount of Aβ due to drugs or genetic manipulation is accompanied by a decrease in the activity of the mTOR pathway, and inhibition of the mTOR pathway also causes the disease in Parkinson's disease (PD) and Huntington's disease (HD), which are types of degenerative brain diseases. It has been found to alleviate (Lipton & Sahin (2014) Neuron 84: 275-291).

Meanwhile, the mTOR complex consists of mTORC1, which has mTOR, mLST8, DEPTOR, Tti1/Tel2, RAPTOR, and PRAS40 as component proteins, and mTORC2 (mammalian It exists as a target of rapamycin complex 2), and both are suggested to be related to brain diseases and autism, but the two complexes have different functions (Saxton & Sabatini (2017) Cell 168: 960-976). The mTORC2 induces cell survival and proliferation in response to external signals and functions to interact at the appropriate time and place. For example, mTORC2 is activated in response to PI3K stimulation by growth factors such as insulin, and phosphorylates AGC kinases such as AKT, SGK, and PKC to redistribute the actin cytoskeleton and promote cell survival and proliferation. Additionally, neuroplasticity, such as metabotropic glutamate receptor-mediated long-term depression (mGluR-LTD), is also regulated by mTORC2.

In the process of neurogenesis, mTOR signaling in neural progenitor cells is a key regulator of maintaining stemness and differentiation of neurons and glial cells. Increased mTOR signaling due to mutations in Phosphatase and tensin homolog (PTEN) or Tuberous sclerosis 1/2 (TSC1/2), which are upstream genes of the mTORC1 pathway, has a significant impact on neural structure and differentiation (Lipton & Sahin (2014) Neuron 84: 275-291). Neurons with TSC1/2 deletion have an increased number of axons, and hippocampal neurons have a larger soma and larger dendritic arbors, but a reduced number of dendritic spines (Tavazoie et al. (Tavazoie et al. 2005) Nat. Neurosci. 8: 1727-1734). Even in animal models in which Tsc1 is deleted, large neurons and neurons and glial cells with little plasticity are found (Uhlmann et al. (2002) Ann. Neurol. 52: 285-296).

The above-mentioned functional distinction between mTORC1 and mTORC2 was reported in developing raptor and rictor knockout (KO) mice. Brain-specific raptor KO transgenic mice showed microcephaly, reduced neuron size, cell death, and high mortality rate in early life (Cloetta et al. (2013) J. Neurosci. 33: 7799-7810). Additionally, in oligodendrocytes of the spinal cord, mTORC1 promoted the initiation and thickness of myelination.

mTORC2 rictor KO mice show microencephaly, a decrease in the soma size of neurons and a decrease in dendrite length (Thomanetz et al. (2013) J. Cell Biol. 201: 293-308), and S6K1 and 4EBP1 phosphorylation. Since there is no change in , this appears to be a phenomenon unrelated to mTORC2. mTORC2 organizes the cytoskeleton by activating Rho and Rac and acts on synapse formation and function. However, if it is too activated, the number of dendritic spines increases but does not mature.

When rapamycin, a representative inhibitor of mTOR, is administered long-term to dementia model animals, the loss of memory and cognitive function associated with dementia is recovered, Aβ42 is reduced, and autophagy is increased, delaying or delaying the progression of dementia disease. suppress (Spilman et al. (2010) PLoS One 4: e9979). However, rapamycin has an immunosuppressive function and has severe side effects, so it cannot be used as a treatment for dementia that requires long-term administration. Rapamycin inhibits mTORC1, but long-term administration also inhibits mTORC2 (Sarbassov et al. (2006) Mol. Cell 22: 159-168). Accordingly, it is unclear whether the dementia treatment effect of rapamycin is due to mTORC1 inhibition or mTORC2 inhibition.

Arc (activity-regulated cytoskeleton-associated protein) is one of the immediate early genes whose expression is rapidly induced by synaptic activation. Arc mRNA is rapidly transported and located in a recently activated dendritic spine in a translation-inhibited state. Afterwards, when the synapse where Arc mRNA is located is activated at a relatively low frequency (<10Hz) due to synaptic activation, the activation signal of mGluR1/5 is received, Arc mRNA is translated, and the generated Arc protein is AMPA (α-Amino- It promotes endocytosis of 3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors, causing long term depression (LTD), which reduces synaptic activity (Wilkerson et al. (2018) Semin. Cell Dev. Biol. 77: 51-62). The mGluR-dependent LTD that occurs in the hippocampus contributes to synaptic plasticity and plays a key role in new memory formation and cognitive function.

The nonamyloidogenic pathway, in which Aβ is not produced from APP, occurs in the cell membrane, while the amyloidogenic pathway in which Aβ is produced occurs in the endosome, not the cell membrane (Rajendran & Annaert (2012) Traffic 13: 759-770). Arc protein binds γ-secretase to the endosome where APP and β-secretase are located, thereby promoting Aβ production through the amyloid production process of APP (Wu et al. (2011) Cell 147: 615-628). Therefore, Arc protein is a key regulator that determines activity-dependent Aβ production, which increases as neurons become activated. Recently, it was revealed that mTORC2 is required for LTD to occur due to mGluR signaling, which leads to the synthesis of Arc protein in the hippocampus (Zhu et al. (2018) Nat. Neurosci. 21: 799-802). Aβ oligomer also activates the mTOR signaling pathway and increases Arc protein production (Caccamo et al. (2011) J. Biol. Chem. 286: 8924-8932; Lancor et al. (2004) J. Neurosci. 24: 10191- 10200), signaling by Aβ oligomer is mediated by PrPc and mGluR5 (Um et al. (2013) Neuron 79: 887-902). Considering that mGluR-dependent LTD formation is mediated by Arc protein-mediated internalization of AMPA receptor-containing endosomes, the activity-dependent generation process of Aβ is also expected to require mTORC2. Therefore, mTORC2 becomes a target protein for developing a treatment for Alzheimer's type dementia that inhibits Aβ production.

Rapamycin and its rapalog, a rapamycin derivative, inhibit mTORC1 activity and have been expected to promote autophagy. However, they do not specifically inhibit phosphorylation of ULK1 Ser758 and TFEB Ser142, which are mTORC1 substrates involved in autophagy (Choo) et al. (2008) Proc. Natl. Acad. Sci. USA 105: 17414-17419). Therefore, in order to use mTORC1 inhibitors to promote autophagy, there is a need to develop new mTORC1 inhibitors with an inhibition mechanism different from that of rapamycin.

mTORopathy is a genetic disease in which nervous system abnormalities occur due to somatic mutations of genes in the mTOR signaling pathway in germ cells or nerve cells (Karalis & Bateup (2021) Dev. Neurosci. 43: 143 -158). Epilepsy, autism spectrum disorder, tuberous sclerosis (TSC), and Fragile Mutations in the phosphatase and tensin homolog (PTEN) gene, an inhibitor of PI3K/mTOR signaling, are a typical example of mTORopathy, and mTOR excessive signaling causes macrocephaly, autism, epilepsy, and mental retardation (Nguyen et al. (2015) Epilepsia 56: 636-646).

The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating mTORopathy, which contains a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

Another object of the present invention is to provide a patch for preventing or treating mTORopathy containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating neurological diseases induced by nerve damage, comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a patch for preventing or treating neurological diseases induced by nerve damage, containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating degenerative brain diseases containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a patch for preventing or treating degenerative brain diseases containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating mTORopathy, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In addition, the present invention provides a patch for preventing or treating mTORopathy containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

Additionally, the present invention provides a pharmaceutical composition for preventing or treating neurological diseases induced by nerve damage, comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

Additionally, the present invention provides a patch for preventing or treating neurological diseases induced by nerve damage, comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating degenerative brain diseases, comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a patch for preventing or treating degenerative brain diseases containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

A pharmaceutical composition for preventing or treating degenerative brain diseases, such as Alzheimer's type dementia, a pharmaceutical composition for preventing or treating mTOR disease, and/or comprising the novel compound of the present invention or a pharmaceutically acceptable salt thereof. It is a pharmaceutical composition for promoting nerve regeneration and can be used by administration methods such as intraperitoneal injection, oral administration, and transdermal patch.

Figure 1 is a graph showing the blood concentration trend of HN1703 according to the route of administration.
Figure 2 is a table of pharmacokinetic parameters comparing oral administration and intraperitoneal injection of the candidate drug.
Figure 3 is a graph of the concentration trend of the candidate drug in the blood and brain according to the route of administration.
Figure 4 is a comparison table with new drugs or drugs under development for similar indications as the candidate substance.
Figure 5 shows a comparison of efficacy tests after daily intraperitoneal injection or oral administration of the candidate substance to 5xFAD mice, an animal model of dementia, for 2 weeks. After performing the Y-shaped maze test, the recovered efficiency was also compared with rapamycin and donepezil.
Figure 6 shows a water maze test performed after daily intraperitoneal or oral administration of the candidate material to 5xFAD mice, an animal model of dementia, for 2 weeks to compare their efficacy.
Figure 7 shows a comparison of efficacy by performing a passive avoidance test after intraperitoneally or orally administering the candidate substance to 5xFAD mice, an animal model of dementia, daily for 2 weeks.
Figure 8 is a diagram showing the inhibition of amyloid plaque formation and activation of autophagy following oral administration of the candidate substance to 5xFAD mice, an animal model of dementia, confirmed by immunohistochemical staining.
Figure 9 is a diagram confirming the brain inflammatory response and nerve regeneration effect following oral administration of the candidate substance to 5xFAD mice, an animal model of dementia, using immunohistochemical staining.

Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. In the following description, detailed descriptions of techniques well known to those skilled in the art may be omitted. Additionally, when describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of the field to which the present invention belongs.

Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part is said to “include” a certain element, this means that it may further include other elements rather than excluding other elements, unless specifically stated to the contrary.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating mTORopathy, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

One aspect of the present invention provides an O-alkylated norathyriol derivative compound represented by the following formula (A) or a pharmaceutically acceptable salt thereof:

[Formula A]

(In the above equation,

R1 and R2 are each a substituent selected from the group consisting of hydrogen, hydroxy, halogen, C1 to C10 straight or branched alkyl, and C1 to C10 straight or branched alkoxy,

R3 and R4 are each a substituent selected from the group consisting of hydrogen, halogen, and straight or branched chain alkyl of C1 to C10.)

In one embodiment of the present invention, R1 and R2 are each a substituent selected from the group consisting of hydrogen, hydroxy, and straight or branched chain alkyl of C1 to C5, and R3 and R4 are each hydrogen, and C1 to C5 straight or branched alkyl. It may be one substituent selected from the group consisting of C5 straight or branched alkyl.

In one embodiment of the present invention, R1 and R2 are each a substituent selected from the group consisting of hydrogen and C1 to C3 straight or branched alkyl, and R3 and R4 are each hydrogen and C1 to C3 straight or branched alkyl. It may be one substituent selected from the group consisting of branched alkyl.

In one embodiment of the present invention, the O-alkylated norathyriol derivative compound represented by Formula A is norathyriol (1,3,6,7-tetrahydroxy-9H-xanthen-) represented by Formula 2 below. 9-one) can be synthesized through sequential alkylation reactions.

[Formula 2]

In one embodiment of the present invention, the O-alkylated norathyriol derivative compound represented by Formula A may be one selected from the group of compounds represented by Formula 1, Formula 3, and Formula 4, Preferably, it is a compound represented by Formula 1, but is not limited thereto.

[Formula 3]

[Formula 4]

One aspect of the present invention involves producing a compound represented by Formula A2 by reacting norathiriol represented by Formula 2 below with a compound represented by R1R2X1X2 in Scheme 1 below in the presence of an organic solvent and a base catalyst. Provided is a method for producing an O-alkylated norathyriol derivative compound comprising:

[Scheme 1]

(In the above reaction equation,

X1 and X2 are each one of chloro, bromo, iodo, tosylate and mesylate,

R1 and R2 are each a substituent selected from the group consisting of hydrogen, hydroxy, halogen, C1 to C10 straight or branched alkyl, and C1 to C10 straight or branched alkoxy.)

In one embodiment of the present invention, 7,9-dihydroxy-10H-[1,3]dioxolo[4,5-b], which is an O-alkylated norathyriol derivative compound represented by the following formula (3) ]Xanthen-10-one (7,9-dihydroxy-10H-[1,3]dioxolo[4,5-b]xanthen-10-one) can be synthesized through the mechanism of Scheme 1 above:

[Formula 3]

In one embodiment of the present invention, the organic solvent is dimethyl sulfoxide, dimethylformamide, acetone, tetrahydrofuran, benzene, toluene, ether, methanol, hexane, cyclohexane, pyridine, acetic acid, carbon tetrachloride, chloroform, and dichloro. It may be one or more selected from the group consisting of methane and water, for example, dimethyl formamide (DMF).

In one embodiment of the present invention, the base catalyst may be any one or more selected from the group consisting of KOH, NaOH, K2CO3, Na2CO3, KHCO3, NaHCO3 and NaH, for example, NaHCO3.

In one embodiment of the present invention, the step of producing the compound represented by Formula A2 is performed at 60°C to 100°C, for example, 75°C to 85°C, for example, at 70°C for 2 to 5 hours, for example. This can be done by stirring for 4 hours.

Another aspect of the present invention is to prepare an O-alkylated norathyriol derivative compound represented by Formula A2 below in the presence of an organic solvent and a base catalyst to a compound represented by R3-X3 in Scheme 2 below or Scheme 3 below. Provides a method for producing an O-alkylated norathyriol derivative compound comprising the step of reacting with a compound represented by R-4-X4 to produce a compound represented by the following formula A3 or A4:

[Scheme 2]

[Scheme 3]

(In the above reaction equation,

X3 and X4 are each one of chloro, bromo, iodo, tosylate and mesylate,

R1 and R2 are each a substituent selected from the group consisting of hydrogen, hydroxy, halogen, C1 to C10 straight or branched alkyl, and C1 to C10 straight or branched alkoxy,

R3 and R4 are each a substituent selected from the group consisting of hydrogen, halogen, and straight or branched chain alkyl of C1 to C10.)

In one embodiment of the present invention, 9-hydroxy-7-methoxy-10H-[1,3]dioxolo[4,5], which is an O-alkylated norathyriol derivative compound represented by the following formula (4) -b]xanthen-10-one (9-hydroxy-7-methoxy-10H-[1,3]dioxolo[4,5-b]xanthen-10-one) can be synthesized through the mechanism of Scheme 2 above. :

[Formula 4]

In one embodiment of the present invention, 7,9-dimethoxy-10H-[1,3]dioxolo[4,5-b], which is an O-alkylated norathyriol derivative compound represented by the following formula (1) ]Xanthen-10-one (7,9-dimethoxy-10H-[1,3]dioxolo[4,5-b]xanthen-10-one) can be synthesized through the mechanism of Scheme 3 above:

[Formula 1]

In one embodiment of the present invention, the organic solvent is dimethyl sulfoxide, dimethylformamide, acetone, tetrahydrofuran, benzene, toluene, ether, methanol, hexane, cyclohexane, pyridine, acetic acid, carbon tetrachloride, chloroform, and dichloro. It may be one or more selected from the group consisting of methane and water, for example, acetone or dimethylformamide (DMF).

In one embodiment of the present invention, the base catalyst may be any one or more selected from the group consisting of KOH, NaOH, K2CO3, Na2CO3, KHCO3, NaHCO3 and NaH, for example, K2CO3 or NaH.

In one embodiment of the present invention, the step of producing the compound represented by Formula A3 or A4 may be performed by stirring at room temperature for 10 minutes to 2 hours, for example, 30 minutes to 1 hour.

The active substance of the present invention can be used in the form of a pharmaceutically acceptable salt, and an acid addition salt formed by a pharmaceutically acceptable free acid is useful as the salt. The term “pharmaceutically acceptable salt” refers to a concentration that has an effective effect that is relatively non-toxic and harmless to patients, and is defined as any organic or It means inorganic addition salt. For these salts, inorganic acids and organic acids can be used as free acids. Hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, perchloric acid, and phosphoric acid can be used as inorganic acids, and citric acid, acetic acid, lactic acid, maleic acid, and fumarine can be used as organic acids. Acids, gluconic acid, methanesulfonic acid, glyconic acid, succinic acid, tartaric acid, galacturonic acid, embonic acid, glutamic acid, aspartic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethane sulfuric acid Fonic acid, 4-toluenesulfonic acid, salicylic acid, citric acid, benzoic acid, or malonic acid can be used. Additionally, these salts include alkali metal salts (sodium salts, potassium salts, etc.) and alkaline earth metal salts (calcium salts, magnesium salts, etc.). For example, acid addition salts include acetate, aspartate, benzate, besylate, bicarbonate/carbonate, bisulfate/sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, Gluceptate, gluconate, glucuronate, hexafluorophosphate, hybenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, mally. ate, malonate, mesylate, methyl sulfate, naphthylate, 2-naphsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, saccharide Latex, stearate, succinate, tartrate, tosylate, trifluoroacetate, aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, Potassium, sodium, tromethamine, zinc salt, etc. may be included, and among these, hydrochloride or trifluoroacetate is preferable.

The acid addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the active substance in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile, etc., adding an organic acid or an inorganic acid, filtering and drying the resulting precipitate. It can be manufactured by distilling the solvent and excess acid under reduced pressure, drying it, or crystallizing it in an organic solvent.

Additionally, a pharmaceutically acceptable metal salt can be prepared using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically appropriate to prepare sodium, potassium, or calcium salts as metal salts. Additionally, the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate).

Furthermore, the present invention includes not only the active substance and its pharmaceutically acceptable salt, but also all possible solvates, hydrates, isomers, optical isomers, etc. that can be prepared therefrom.

The pharmaceutical composition of the present invention may further include adjuvants in addition to the active ingredients. The auxiliary agent may be any one known in the art without limitation, but the effect may be increased by further including, for example, Freund's complete auxiliary agent or incomplete auxiliary agent.

The pharmaceutical composition according to the present invention can be prepared by incorporating the active ingredient into a pharmaceutically acceptable carrier. Here, pharmaceutically acceptable carriers include carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, Examples include calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions according to conventional methods. .

When formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations contain the active ingredient plus at least one excipient, such as starch, calcium carbonate, sucrose, lactose, and gelatin. It can be prepared by mixing etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used diluents such as water and liquid paraffin, they contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. You can. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.

The pharmaceutical composition according to the present invention can be administered to an individual through various routes. All modes of administration are contemplated, for example, by oral, intravenous, intramuscular, subcutaneous, or intraperitoneal injection.

The dosage of the pharmaceutical composition according to the present invention is selected taking into account the age, weight, gender, physical condition, etc. of the individual. It is obvious that the concentration of the active ingredient included in the pharmaceutical composition can be selected in various ways depending on the target, and is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg/ml. If the concentration is less than 0.01 ㎍/ml, pharmaceutical activity may not appear, and if it exceeds 5,000 ㎍/ml, it may be toxic to the human body.

The “mTOR disease” of the present invention is preferably a neurological disease, and the “neurological disease” refers to a neurological disease caused by hyperactivation of the mTOR signaling pathway or abnormal proteostasis of proteins.

In the present invention, the “neurological disease” includes Alzheimer's disease, the main etiology of which is senile plaques and NFTs formed by hyperactivation of the mTOR signaling pathway and abnormal protein homeostasis.

In the present invention, the “neurological disease” is a concept that includes mTOR disease, a genetic disease in which neurological abnormalities are caused by overactivation of the mTOR signaling pathway due to germline or somatic mutations in nerve cells, and epilepsy (Moloney) et al. (2021) Brain Comm. 3: 1-21), autism spectrum disorder (ASD) (Winden et al. (2018) Ann. Rev. Neurosci. 41: 1-23), macrocephaly (Butler et al., (2005) J. Med. Genet. 42: 318-321), tuberous sclerosis complex (TSC) (Crino (2015) Cold Spring Harb. Perspect. Med. 5: a022442), Seizure (Harvey et al. (2008) Epilepsia 49: 146-155), Fragile X syndrome (Sharma et al. (2010) J. Neurosci. 30: 694-702), or intellectual disability (Dentel et al. (2019) Neuron 104: 1032-1033).

In the present invention, the "neurological disease" is a concept that includes degenerative brain disease for which promoting protein homeostasis can be a causal treatment method, and includes dementia (Sancesario & Bernardini (2018) Ann. Transl. Med . 6: 340), Parkinson's disease (Hague et al. (2005) J. Neural Neurosurg. Psychiatry 76: 1058-1063), Alzheimer's type dementia (Wenk (2003) J. Clinic. Psychiatry 64: 7-10), peak It may be any one selected from the group consisting of Dickson (1998) Brain Pathol. 8: 339-354), or Huntington's disease (Hague et al. (2005) J. Neural Neurosurg. Psychiatry 76: 1058-1063).

Since memory loss, traumatic central nervous system disease, spinal cord injury disease, peripheral nerve damage, amyotrophic axonal sclerosis, or peripheral nerve disease are also accompanied by degeneration and death of nerve cells, a therapeutic effect can be expected if new nerve regeneration is promoted. , and thus may be included in the above “neurological diseases”.

According to one embodiment of the present invention, the composition may inhibit the formation of amyloid plaques, and the inhibition of amyloid plaque formation may be induced by activation of autophagy.

According to one embodiment of the present invention, the composition may promote nerve regeneration (neurogenesis), and the nerve regeneration may promote division, differentiation, migration, or survival of neural stem cells or neural progenitor cells.

According to one embodiment of the present invention, the composition may increase cognitive function, and the cognitive function may be short-term or long-term memory.

According to one embodiment of the present invention, the mTOR disease includes epilepsy, autism spectrum disorder (ASD), macrocephaly, tuberous sclerosis complex (TSC), seizures, and vulnerability. It may be a condition selected from the group consisting of Fragile X syndrome and intellectual disability.

According to one embodiment of the present invention, the composition may be oral or injectable, and the injectable may be administered by a method selected from the group consisting of intradermal injection, subcutaneous injection, intravenous injection, intramuscular injection, and intraperitoneal injection. there is.

According to one embodiment of the present invention, the injection drug may have an increased maximum concentration in the blood compared to the reference value for oral administration, and may have an increased half-life in the blood compared to the reference value for oral administration.

The term “injection” used in the present invention refers to a method of administering medication by injecting a drug solution into a part of the body using a syringe and needle for the purpose of acting locally or systemically, and is more effective than conventional oral administration, suppository administration, or topical administration. It is a method of administration that allows the effect of the drug to be obtained accurately and quickly, and can effectively deliver the drug to the body even in cases where the drug cannot be administered orally or in situations where the drug is denatured by digestive juices and absorption is difficult. Injection is largely intradermal. There are injections, subcutaneous injections, intramuscular injections, intravenous injections, and intraarterial injections. Intradermal injection is characterized by slow absorption and visible reactions. It is an injection method that can be used to diagnose or prevent diseases or to check antibiotic side effects before surgery. Subcutaneous injection is a method of administering medicine to the connective tissue under the skin and is absorbed quickly, while intramuscular injection is a method of injecting into the muscle tissue of the buttocks or upper arm so that the medicine is absorbed quickly due to the high vascular distribution of the muscle. . Intravenous injection is a method of administering medication directly into a vein. It is an injection method that allows the medication to act quickly and administer an accurate amount. It is a method of administration used when the medication cannot be injected into the muscle or subcutaneously due to its irritating properties. , large amounts of medication can be administered. Arterial injection is a method of injecting a drug into an artery that flows into the affected area, and a direct effect of the drug on the affected area can be expected.

Injectables for use in the above injections refer to aqueous, water-soluble, oily, suspended, emulsified, solid (administration after dissolution) formulations, and are formulated for injection purposes to administer drugs so that they act directly in the body without passing through the digestive tract. It means a standardized product. The conditions for injections are 1) free of excipients, 2) completely sterile, 3) not containing pyrogenic substances, 4) having an osmotic pressure similar to serum osmolality, 5) having a pH similar to serum pH, and 6) If it is a preparation that does not chemically react with components of body tissue, it can be used as an injection.

In addition, the above injection may contain solvents, solubilizers, buffers, isotonic agents, stabilizers, sulfating agents, analgesics, and suspending agents as additives according to the Ministry of Food and Drug Safety drug additive guidelines. “Additives” that can be mixed into injections are substances other than the active ingredients contained in the preparation and are used for the purposes of increasing the usefulness of the medicine, facilitating formulation, stabilizing the preparation, and improving the appearance. Additives include excipients, stabilizers, preservatives, buffers, coagulants, suspending agents, emulsifiers, fragrances, solubilizers, colorants, thickeners, etc., as needed. However, the additives used do not have direct pharmacological effects at the dosage of the preparation, are safe, and do not change the therapeutic effect of the preparation or interfere with the test, and can be summarized as follows.

1) Improved stability, bioavailability, etc.

2) Maintaining the quality of the formulation during preservation or use

3) Improving pharmacoeconomics by controlling the physical properties of medicines

“Solvents” that can be mixed with the above additives include distilled water for injection, 0.9% sodium chloride injection, IV solution, dextrose injection, dextrose + sodium chloride injection, PEG, lactated IV solution, ethanol, and propylene glycol. , non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, benzene benzoate, etc. may be mixed. “Solubilizers” may include sodium benzoate, sodium salicylate, sodium acetate, urethane monoethyl acetamide, butazolidine, propylene glycol, Tween, nicotinic acid amide, hexamine, and dimethyl acetamide. . “Buffers” may include weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, gums, etc. Sodium chloride may be mixed as an “isotonizing” agent, and “stabilizers” include sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2 S2 O3), sodium sulfite (Na2 SO3), nitrogen gas (N2), and ethylenediamine. Tetraacetic acid, etc. may be mixed. “Sulfurizing agents” may include sodium bisulfide 0.1%, sodium formaldehyde, sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, and acetone sodium bisulfite. “Analgesics” may include benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, calcium gluconate, etc., and “suspending agents” may include sodium CMC, sodium alginate, Tween 80, aluminum monostearate, etc. there is.

In addition, the present invention provides a patch for preventing or treating mTORopathy containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

According to one embodiment of the present invention, the patch may be administered intradermally or subcutaneously.

“Intradermal or subcutaneous administration” of the present invention is also referred to as “transdermal delivery”, and the term “transdermal delivery” refers to delivery of the active agent through the stratum corneum such that upon penetration it contacts the intradermal space without significant pain. Since the stratum corneum has no nerves, it can be penetrated without nerve stimulation. The terms “transdermal” and “intradermal” are used interchangeably herein. The stratum corneum consists mainly of several layers of dead skin cells and has no developed blood vessels. Therefore, the stratum corneum often poses a strong barrier to the transdermal delivery of active agents, especially charged macromolecules such as peptides. Unlike active agents delivered via subcutaneous injection, which mostly provide complete entry into the bloodstream, many factors (and barriers) can affect the pharmacokinetics of drugs delivered via the transdermal route. For example, the site of application, the thickness, integrity, and hydration state of the skin, the thickness and density of fatty tissue beneath the skin at the application site, the size of the drug molecule, and the permeability and pH state of the membrane of the transdermal device, all of which affect transdermally delivered drugs. may affect the bioavailability of In certain embodiments, transdermal delivery is achieved at a depth of less than or equal to about 700 μm, or less than or equal to about 600 μm, or less than or equal to about 500 μm, or less than or equal to about 400 μm, or less than or equal to about 300 μm, or less than or equal to about 250 μm, or less than or equal to about 150 μm. It involves skin penetration through the stratum corneum and into the dermis. In certain embodiments, the average needle depth of penetration is approximately 800 μm, or about 700 μm, or about 600 μm, or about 500 μm, or about 400 μm, or about 300 μm, or about 250 μm, or about 150 μm. .

Additionally, “intradermal or subcutaneous administration” of the present invention may be delivered transdermally in the form of a “microneedle patch.” Injections using conventional needles have disadvantages such as risk of infection, large doses, difficulty in dose control, skin flaking, needle phobia, and pain. To address these shortcomings, the “microneedle patch” is attracting attention as a means to replace conventional injections. Microneedle patches are hypodermic syringes with millimeter-sized needles that have been actively developed over the past few decades to replace other types of small needles. In addition, microneedles have better transdermal permeability than existing needles, are painless, and can continuously absorb the loaded drug. A patch with these microneedles attached is called a microneedle patch, and by using a flexible base, it can be used to suit the individual's skin shape and attachment area.

Additionally, the present invention provides a pharmaceutical composition for preventing or treating neurological diseases induced by nerve damage, comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

According to one embodiment of the present invention, the neurological disease may be selected from the group consisting of traumatic central nervous system disease, spinal cord injury disease, peripheral nerve damage, amyotrophic axonal sclerosis, and peripheral nerve disease, and the neurological disease is cognitive dysfunction. It may include, and the cognitive dysfunction may be selected from the group consisting of learning disability, memory loss, mild cognitive impairment, depression, agnosia, amnesia, aphasia, apraxia, and Binswanger's disease.

Additionally, the present invention provides a patch for preventing or treating neurological diseases induced by nerve damage, comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating degenerative brain diseases, comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

According to one embodiment of the present invention, the degenerative brain disease may be selected from the group consisting of dementia, Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, epilepsy, stroke, stroke, ischemic brain disease, and memory loss, and is preferably It is usually Alzheimer's type dementia, but is not limited to this.

According to one embodiment of the present invention, the degenerative brain disease may include cognitive dysfunction, and the cognitive dysfunction includes learning disability, memory loss, mild cognitive impairment, depression, agnosia, amnesia, aphasia, apraxia, and It may be selected from the group consisting of Binswanger's disease.

In addition, the present invention provides a patch for preventing or treating degenerative brain diseases containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

Hereinafter, the present invention will be described in more detail by examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

<Experimental example>

Evaluation of blood and brain concentrations of HN-1703 according to the route of administration

- Purpose of experiment

In this experiment, blood and brain concentrations over time after oral and intraperitoneal administration were investigated to evaluate the pharmacokinetics of HN-1703 according to the route of administration.

- method

Intraperitoneal administration of HN-1703 to C57BL/6 mice

Mice were raised in a breeding chamber with light and dark alternating cycles every 12 hours, had free access to food and water, and were raised in compliance with animal experiment ethics regulations. C57BL/6 mice (Samtaco) were intraperitoneally injected with HN-1703 at a dose of 20 mg/kg into 8-week-old male animals. The drug was dissolved in 20% DMSO: 10% tween 80: 70% saline (administration vehicle) and then injected. To investigate blood concentration, 0.1 mL of blood was collected from the orbit through a heparin-coated microtubule at 0, 10, 15, 30 minutes, 1, 2, 4, 8, and 24 hours, divided into 2 groups and 4 points each (Group A) : 5, 30 min, 2, 8 hr, n=6/ Group B: 15 min, 1, 4, 24 hr, n=6) Blood was collected. Whole blood is centrifuged at 13000 rpm for 1 minute to collect only the plasma, divided into 30 μL each and stored in a deep freezer until analysis. After the final blood collection, brain tissue is extracted after lethal exsanguination by opening the diaphragm and cutting the abdominal aorta. For the extracted tissue, the blood on the surface of the tissue is removed using Kimwipes, and then the total weight of the tissue is weighed and recorded using a 2 mL EP Tube with the weight recorded in advance. To each tissue, add 4 times the total weight of physiological saline and make 20% homogenates using a tissue homogenizer. Samples are divided into 30 μL portions and stored in a deep freezer until analysis. The concentration of HN-1703 and its three bioactive metabolites (HN-1701, HN-1702, HN-2103) among the pretreated samples was quantified using LC-MS/MS.

Oral administration of HN-1703 to C57BL/6 mice

C57BL/6 mice (Samtaco) were orally administered HN-1703 at a dose of 20 mg/kg using a sonde to 8-week-old male animals. The drug was dissolved (or suspended) in 20% DMSO: 10% tween 80: 70% saline (administration vehicle) and then administered orally as a single dose using an oral sonde at a dose of 50 mg/kg. At 5, 15, 30 min, 1, 2, 4, 8, and 24 hr, 0.1 mL of blood was injected from the orbit through heparin-coated microtubules into 2 groups at 4 points each (Group A: 5, 30 min, 2, 8 hr, n=6/ Group B: 15 min, 1, 4, 24 hr, n=6) Blood was collected. Whole blood is centrifuged at 13000 rpm for 1 minute to collect only the plasma, divided into 30 μL each and stored in a deep freezer until analysis. After the final blood collection, brain tissue is extracted after lethal exsanguination by opening the diaphragm and cutting the abdominal aorta. For the extracted tissue, the blood on the surface of the tissue is removed using Kimwipes, and then the total weight of the tissue is weighed and recorded using a 2 mL EP Tube with the weight recorded in advance. To each tissue, add 4 times the total weight of physiological saline and make 20% homogenates using a tissue homogenizer. Samples are divided into 30 μL portions and stored in a deep freezer until analysis. Among the pretreated samples, the concentration of HN-1703 and its three bioactive metabolites (HN-1701, HN-1702, HN-2103) was quantified using LC-MS/MS.

LC-MS/MS analysis

Establish LC-MS/MS-based analysis conditions for HN-1703 and three bioactive metabolites (HN-1701, HN-1702, HN-2103). Mass spectrometer conditions are determined by evaluating the conditions of precursor ion scan, product ion scan, fragmentor, and collision energy using the Agilent 6430 triple quadrupole system. HPLC conditions are established by examining various analytical column and mobile phase conditions.

The biological sample pretreatment method for the analysis of HN-1703 and three types of bioactive metabolites (HN-1701, HN-1702, HN-2103) in mouse plasma and brain tissue was determined by comparing the liquid-liquid extraction method and the protein precipitation method to determine the limit of quantification. Established by taking into account such factors. By applying an established analysis method, HN-1703 and three bioactive metabolites (HN-1701, HN-1702, HN-2103) were analyzed in mouse plasma and brain tissue.

Analysis of research results

For drug concentration results in plasma, pharmacokinetic parameters such as Cmax, Tmax, AUC, T1/2, Vd, and CL were calculated using non-compartmental analysis.

- When administered intraperitoneally, bioavailability is calculated using the formula (dose normalized AUCIP)/(dose normalized AUCIV)×100 (%).

- When administered orally, bioavailability is calculated using the formula (dose normalized AUCPO)/(dose normalized AUCIV)×100 (%).

In vivo efficacy evaluation of HN-1703 on 5xFAD transgenic mice, an animal model of dementia

- Purpose of experiment

This experiment was conducted to: 1) evaluate the effect of HN-1703 on Alzheimer's-type dementia-related diseases, specifically, its efficacy in improving memory and cognitive function depending on the route of administration;

- method

Intraperitoneal administration of HN-1703 to 5xFAD mice

5xFAD animals were 6-month-old (all born within 3 days) male heterozygotes that were administered HN-1703 in three doses for 14 days. Each group consisted of 15 wild type littermates, 8 mice administered with vehicle (DMSO administered) on 5xFAD, 9 mice administered with 1 mg/kg administered on 5xFAD, 9 mice administered with 5 mg/kg, 20 mg/kg administered group, and control group. Rapamycin 1mg/kg in 5xFAD was injected intraperitoneally to 9 animals once a day at the same time in a volume of 100 μl. The behavior of mice was measured during the dark cycle in which mice are active, with light and dark alternating in a 12-hour period in a breeding chamber. Food and water were freely accessible, and they were bred in compliance with Kyung Hee University's animal experiment ethics regulations. To get used to the behavior measurer's hand and smell, it was placed on the palm of the hand for 5 days and habituation was performed for 10 minutes each day until it did not escape the palm for 2 minutes, and then cognitive function was measured. The behavioral measurement room maintained humidity (40-60%) and temperature (22-26℃), and reflected light was maintained to prevent sound and light effects. Behavioral measures were measured sequentially for 18 days using the Y-maze, Morris Water Maze, and Passive Avoidance test.

Oral administration of HN-1703 to 5xFAD mice

5xFAD animals were 6-month-old (all born within 3 days) male heterozygotes that were administered HN-1703 in three doses for 14 days. Each group included 12 wild type littermates, 9 mice in the 5xFAD with vehicle (administered with DMSO), 8 mice in the 1 mg/kg dose group, 9 mice in the 5 mg/kg dose group, 9 mice in the 20 mg/kg group, and As a control group, Donepezil 5mg/kg in 5xFAD was orally administered to 5 animals once a day at the same time using a sonde. The behavior of mice was measured during the dark cycle in which mice are active, with light and dark alternating in a 12-hour period in a breeding chamber. Food and water were freely accessible, and they were bred in compliance with Kyung Hee University's animal experiment ethics regulations. To get used to the behavior measurer's hand and smell, it was placed on the palm of the hand for 5 days and habituation was performed for 10 minutes each day until it did not escape the palm for 2 minutes, and then cognitive function was measured. The behavioral measurement room maintained humidity (40-60%) and temperature (22-26℃), and reflected light was maintained to prevent sound and light effects. Behavioral measures were measured sequentially for 18 days using the Y-maze, Morris Water Maze, and Passive Avoidance test.

Y-maze test

A rat was placed in a Y-shaped maze, and when the rat explored the maze, it was measured whether it alternated between each maze in the center and chose a new maze, or forgot the maze it had passed through and selected the same maze again, and calculated the number of alterations as a percentage. For detailed measurement methods, refer to Heo H et al. (J Ethnopharmacol, 2009), and the spatial memory and short-term memory of mice were tested. In the Y-shaped maze, the mouse was filmed entering each arm for 8 minutes, 10 minutes, and 20 minutes, and the formula for formulating the probability of success was obtained by dividing the number of times it entered each of the three arms by the total number of times it entered each arm - 2 (%). did. All results were statistically processed using the ANOVA test.

passive avoidance test

The passive avoidance test was performed in the order following the Y-maze test as described by Heo et al. (J Ethnopharmacol, 2009). Due to the avoidance effect caused by strong light, the mouse is trained to avoid from a bright room to a dark room 3 times a day for 4 days to become familiar with the environment, and when it avoids within 30 seconds (Acquisition time), it is placed in an electric foot in a dark room at the same time the next day. After applying shock (0.5 mA, 30 g standard) for 3 seconds, the mouse was placed in a bright room again exactly 24 hours later, and the avoidance response due to light, that is, the retention time required to move from a bright room to a dark room, was measured. The latency time was measured for 720 seconds (confinement experiment) and data were collected for each group.

water maze test

A water maze test designed by Richard Morris to test spatial memory. It was performed in a round, rust-free pool with a white interior surface (diameter 120 cm; height 60 cm). The pool was 50 cm deep in water (maintained at 25.0 ± 1.0 °C). The tank containing this was divided into four equal parts, and a platform (about 12 cm in diameter) was placed in the middle of one of the sections at a depth of 3 mm from the water surface. On the first day, the rats were allowed to practice free swimming for 1 minute without the platform footrest, and from the second day, they mixed paint in the water so that the foothold was not visible, and trained the rats for 1 minute each to find the platform using spatial clues on the tank, with more than 6 mice in each group. After sufficient rest, they were placed in each quadrant and measured with a video camera. A training test was conducted to find the footrest in the aquarium four times a day for four days, and in the final experiment on the 6th day, the foothold in the aquarium was removed and the time the animal stayed in the area where the foothold was was measured (probe trial). Captured video pictures were analyzed with a video tracking system (Ethovision water maze program, Noldus Information Technology, Wogeningen, Netherlands). The information analyzed included swimming time in the target quadrant and the number of times the swimmer crossed the virtual platform to find the removed platform.

Immunohistochemical staining of 5xFAD mouse brain tissue

The brain section immunostaining method was modified from that described by Heo H et al. (J Ethnopharmacol, 2009). Rats were perfused transcardially with 4% PFA. After fixation by soaking in 4% PFA for 4 hours, brain tissue was non-frozen in 30% sucrose, frozen in optimal cutting temperature (OCT) mixture, and stored at -80 °C. Brain tissue pieces were sectioned in the coronal direction at a thickness of 35 μm. Brain tissue sections were immersed in storage solution (30% glycerol, 30% ethylene glycol, PBS) and stored at 4°C. Stored brain slices were permeabilized for 20 min in 0.5% Triton Afterwards, the cells were blocked in a free-floating state for 2 hours in 15% standard serum, 3% bovine serum albumin (BSA, bio-WORLD, Dublin, OH, USA) and 0.1% Triton The sections were shaken with primary antibody at 4°C for 16 hours. Afterwards, a secondary antibody complementary to the primary antibody was attached at room temperature (22±1°C) for 45 minutes. Next, the nuclei were washed with physiological saline (PBS) containing 1 μg/ml PI (propidiumiodide, Sigma) or DAPI (1:4000), placed on a glass slide, and encapsulated using an aqueous mount. Observations were made using a focal scanning microscope (LSM 810 Carl Zeiss, Oberkochen, Germany).

Primary antibodies include LC3-II (MBL, PM036), 6E10 (biolegend, 803001) [LC3/6E10; Antibodies such as ICC double staining], Iba-1 (Wako, 019-19741) [ThioS/Iba1/PI triple staining], NeuN (Millipore, MAB377B), and GFAP (Chemicon) were used as secondary antibodies. used Alexa 488, Cy3, Alexa 488 (Invitrogen, A21202), Cy3 (Jackson lab, 715-165-151), and Alexa 488 (Jackson lab, 711-546-152).

<Example 1> Measurement of blood and brain concentrations of compositions by intraperitoneal injection and oral administration

Figure 1 shows 0, 10, 15, 30 min, 1, 2, 4, 8 after intraperitoneal injection of the composition at 20 mg/kg or oral administration at 50 mg/kg to C57BL/6 mice (male, 8 weeks). Blood was collected after 24 h (sparse sampling) to measure the blood concentration of the composition.

Figure 2 is a table of pharmacokinetic parameters comparing oral administration and intraperitoneal injection of the candidate drug. The highest concentration of HN1703 in the blood is 20 mg/kg, which is 267.8 ng/ml 15 minutes after intraperitoneal injection, and the half-life is 23.7 hours. When administered orally at 50 mg/kg, the concentration is 65.5 ng/ml after 4 hours and the half-life is 12.7 hours. The bioavailability (BA) when administered orally is approximately 30.5% compared to when administered intraperitoneally. Therefore, it is reasonable to administer an oral dose of about three times that of intraperitoneal administration (60 mg/kg), but considering the solubility of HN1703, it was judged that there is a possibility that HN1703 may precipitate in the gastrointestinal tract after oral administration, so 50 mg/kg was administered.

Figure 3 is a graph of the concentration trend of the candidate drug in the blood and brain according to the route of administration. 24 hours after administration of the candidate HN1703, the AUC in plasma (h*ng/ml) was 406.3 h*ng/ml after intraperitoneal injection, 1023.2 h*ng/ml after oral administration, and the AUC in brain tissue (h*ng) /ml) is 220 h*ng/ml after intraperitoneal injection, 351.7 h*ng/ml after oral administration, and the Brain/plasma ratio of HN1703 is 50 mg/kg, 0.54 24 hours after intraperitoneal injection, and 0.54 after oral administration. It is 0.34.

Figure 4 is a comparison table with new drugs or drugs under development for similar indications as the candidate substance. The brain/plasma ratio of HN1703 is similar or superior compared to new drugs or drugs under development for similar indications.

<Example 2> Evaluation of memory and cognitive function improvement in 5xFAD rats

Figures 5-7 compare efficacy tests after intraperitoneal or oral administration of the candidate material to 5xFAD mice, an animal model of dementia, once a day for two weeks. The restored efficiency of memory and cognitive ability was compared with rapamycin and donepezil. These are the analysis results of the maze, passive avoidance test, and water maze test.

Figure 5 compares the efficiency recovered after performing the Y-shaped maze test. This is the result of a Y-maze test analysis showing that HN-1703 has the effect of restoring deteriorated cognitive function when administered to 5xFAD transgenic mice, an animal model for dementia. In the intraperitoneal injection experiment, 5xFAD mice (5xFAD-DMSO) administered only Vehicle DMSO showed approximately 50.22% to 55.73% alteration (P<0.001 compared to the normal group) at 8, 10, and 12 minutes, but HN in 5xFAD In the groups administered 1, 5, and 20 mg/kg of -1703, the recovery rate was 63.46% to 73.95%, close to 78% of normal animals, and in the case of oral administration of 12.5, 25, and 50 mg/kg of HN-1703 to 5xFAD, the The alteration recovery rate was up to 73.5%, which was higher than the normal group's 67.75 - 71.24% (significance P<0.001-0.01), and therefore oral administration (50 mg/kg) improved memory better than intraperitoneal injection (20 mg/kg). It seemed. On the other hand, when rapamycin (normal dosage 1mg/kg) was administered intraperitoneally, there was little recovery compared to DMSO administration, and when Donepezil (normal dosage 5mg/kg) was administered orally, there was no recovery in memory ability.

Figure 6 shows a water maze test performed after daily intraperitoneal or oral administration of the candidate material to 5xFAD mice, an animal model of dementia, for 2 weeks to compare their efficacy. In the intraperitoneally injected water maze test, the 5xFAD-DMSO group had a greater latency to find a foothold by 14 seconds compared to normal rats on the last day of the training period, and HN-1703 was administered at 1 mg/kg, 5 mg/kg, and 20 mg/kg. The learning speed of the group administered mg/kg rapidly improved, and after 4 days of training, the learning speed decreased similar to that of the normal group in the groups administered 5 mg/kg and 20 mg/kg. The swimming distance was similar in all experimental groups, but the swimming time in the target quadrant (quadrant 1) was statistically significantly reduced to 7 seconds in the 5xFAD-DMSO group compared to approximately 17.14 seconds in the normal group (P<0.001), and in the HN-1703 group. recovered spatial awareness in a dose-dependent manner from about 12.48 seconds to about 19.31 seconds (P<0.01), and the 20 mg/kg group swam for more time than the normal group. The number of times swimming past the target platform was about 0.43 times (P<0.001) in the 5xFAD-DMSO group, which was 15% of the 2.7 times in the normal group, and increased to 1.14 times in the rapamycin group, but was not statistically significant. In the HN-1703 administration group, the dose increased 4.6 to 5.6 times in a concentration-dependent manner, from 2 to 2.43 times (P<0.01).

In the water maze test oral administration group, compared to the 5xFAD-DMSO group, the latency time required to find a foothold during the training period was significantly decreased in the groups administered 12.5 mg/kg and 25 mg/kg of HN-1703 along with the normal group. The swimming distance was similar in all experimental groups, but the swimming time in the target quadrant (quadrant 1) was statistically significantly reduced to approximately 5.15 seconds in the 5xFAD-DMSO group compared to approximately 18.02 seconds in the normal group (P<0.001), and in the Donepezil administered group ( The recovery of about 10.08 seconds) was not statistically significant, but the HN-1703 administration group recovered spatial perception in a dose-dependent manner from about 13.88 seconds to about 16.05 seconds, and statistically (P< 0.001) swam significantly more time. The number of times the 5xFAD-DMSO group swam past the place where the target platform was was about 0.125 times, which was very less than the normal group's 1.8 times. In the Donepezil administered group, it increased significantly but was not statistically significant, and in the HN-1703 administered group it was about 1.63 to about 2.13. The circuit recovered significantly (P<0.001 - 0.01) in a dose-dependent manner up to 13 to 17 times, and spatial perception improved more in the HN-1703 25 mg/kg administered group than the normal group. Therefore, it was confirmed that the spatial perception, which was reduced in 5xFAD mice compared to the normal group, recovered to the level of the normal group or higher, similar to the intraperitoneal injection of 20 mg/kg of HN-1703 in the 25 mg/kg group.

Figure 7 shows a comparison of the efficacy of the candidate substance by performing a passive avoidance test (24 hours after electrical stimulation) after daily intraperitoneal or oral administration of the candidate substance to 5xFAD mice, an animal model of dementia, for 2 weeks. In the passive avoidance test of intraperitoneal injection experimental animals, the latency time of the 5xFAD-DMSO group (192.25 seconds, P<0.001) was significantly reduced compared to the normal group (about 593.53 seconds), but HN-1703 was administered at 1 mg/kg and 5 mg/kg. kg and 20 mg/kg administered mice showed a significant recovery in latency time, ranging from 534.33 seconds, 561.11 seconds to 626.5 seconds (P<0.001 - 0.01), and the rapamycin administered group also significantly increased, but the significance was low (461 seconds, P<0.05).

In the passive avoidance test of orally administered experimental animals, the latency time of the 5xFAD-DMSO group (about 174.7 seconds, P<0.001) was significantly reduced compared to the normal group (about 542.87 seconds), but HN-1703 12.5 mg/kg (586.3 seconds, P<0.001), 25 mg/kg (642.38 seconds, P<0.001), and 50 mg/kg (579.4 seconds, P<0.001) administration groups all showed avoidance times that were more than three times longer than those in the normal group. . In the group administered Donepezil, recovery was lower than normal but greater (512 seconds, P<0.05). Therefore, it was confirmed that the memory, which had been reduced in 5xFAD mice compared to the normal group, recovered to above the normal group in the HN-1703 12.5 - 50 mg/kg administered group, similar to the 20 mg/kg intraperitoneal injection.

<Example 3> Removal of amyloid plaques in 5xFAD mice, alleviation of brain inflammatory response, and confirmation of increased nerve regeneration

Figures 8 and 9 show the brains of each group of 5xFAD mice whose memory was measured in the above examples, stained for amyloid plaques, and GFAP and Iba-1 antibodies showing activated brain inflammatory response; This diagram confirmed the removal of amyloid plaques, alleviation of brain inflammatory response, and increased nerve regeneration by oral administration of the candidate material through immunostaining with Ki67 antibody, which stains proliferating cells.

Figure 8 shows the results of immunostaining with Thiflavin-S staining or β-amyloid antibody 6E10 on the brain tissue of dementia animals administered orally with the candidate substance. As a result, amyloid was significantly reduced in the group administered the candidate substance and significantly decreased compared to the group administered Donepezil. To investigate whether amyloid plaque removal was due to autophagy activation, immunostaining with LC3 antibody, an autophagy activation marker, showed that autophagy significantly increased in a dose-dependent manner and increased more than the Donepezil administered group.

Figure 9 shows the results of staining with a GFAP antibody, which stains activated astrocytes, and an Iba-antibody, which stains microglia, to investigate brain inflammatory response and nerve regeneration. As a result, activated astrocytes and microglia were observed in a dementia animal model. It increased significantly, and significantly decreased in a dose-dependent manner after oral administration of HN1703, and decreased significantly compared to the Donepezil administered group.

In addition, immunostaining of the subgranular zone of the dentate gyrus, where neural stem cells proliferating in the hippocampus exist, with Ki67 antibody showed an increase in the 50 mg/kg dose group. This is interpreted as an increase in neural stem cells in the subgranular zone, which is opposite to the increase in GFAP immunostained cells.

Preparation example of medicine

The active substance according to the present invention can be formulated in various forms depending on the purpose. The following is an example of several formulation methods containing the effective substance according to the present invention as an active ingredient, but the present invention is not limited thereto.

<Drug Preparation Example 1> Preparation of powder

Active substance 2g

lactose 1 g

After mixing the above ingredients, they were filled into an airtight bubble to prepare a powder.

<Drug Preparation Example 2> Preparation of tablets

Active substance 100 mg

corn starch 100 mg

lactose 100 mg

Magnesium stearate 2mg

After mixing the above ingredients, tablets were manufactured by tableting according to a conventional tablet manufacturing method.

<Drug Preparation Example 3> Preparation of capsules

Active substance 100 mg

corn starch 100 mg

lactose 100 mg

Magnesium stearate 2mg

After mixing the above ingredients, a capsule was prepared by filling a gelatin capsule according to a typical capsule manufacturing method.

<Pharmaceutical Preparation Example 4> Preparation of injections

Active substance 10 ㎍/㎖

Dilute hydrochloric acid BP until pH reaches 3.5

Sodium Chloride BP for Injection Up to 1 ml

The active substance according to the present invention was dissolved in an appropriate volume of sodium chloride BP for injection, the pH of the resulting solution was adjusted to pH 3.5 using diluted hydrochloric acid BP, and the volume was adjusted using sodium chloride BP for injection and thoroughly mixed. . The solution was filled into a 5 ml Type I ampoule made of transparent glass, sealed under an upper grid of air by dissolving the glass, and sterilized by autoclaving at 120°C for 15 minutes or more to prepare an injection solution.

<Drug Preparation Example 5> Preparation of nasal absorbent (Nasal spray)

Active substance 1.0 g

Sodium Acetate 0.3g

Methylparaben 0.1g

Propylparaben 0.02g

sodium chloride Appropriate amount

HCl or NaOH pH adjustment appropriate amount

Purified water Appropriate amount

According to the usual manufacturing method of a nasal absorbent, it is prepared to contain 3 mg of the active substance per 1 mL of saline water (0.9% NaCl, w/v, the solvent is purified water), filled into an opaque spray container, and sterilized to prepare a nasal absorbent. Manufactured.

<Drug Preparation Example 6> Preparation of liquid preparation

Active substance 100mg

Iseonghwadang 10 g

mannitol 5 g

Purified water Appropriate amount

According to the usual liquid preparation method, add and dissolve each ingredient in purified water, add lemon zest, mix the above ingredients, add purified water to adjust the total to 100 mL, fill in a brown bottle, and sterilize to prepare the liquid. did.

So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is set forth in particular in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

Claims (29)

A pharmaceutical composition for preventing or treating mTORopathy comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]
. According to clause 1,
The composition inhibits the formation of amyloid plaques. According to clause 2,
A composition that inhibits the formation of amyloid plaques by activating autophagy. According to clause 1,
The composition promotes nerve regeneration (neurogenesis). According to clause 4,
A composition wherein the nerve regeneration promotes division, differentiation, migration, or survival of neural stem cells or neural progenitor cells. According to clause 1,
The composition is a composition that increases cognitive function. According to clause 6,
A composition wherein the cognitive function is short-term or long-term memory. According to clause 1,
The mTOR disease includes epilepsy, autism spectrum disorder (ASD), macrocephaly, tuberous sclerosis complex (TSC), seizures, Fragile A composition, wherein the composition is a condition selected from the group consisting of disorders. According to clause 1,
The composition is an oral or injectable composition. According to clause 9,
A composition wherein the injectable agent is administered by a method selected from the group consisting of intradermal injection, subcutaneous injection, intravenous injection, intramuscular injection, and intraperitoneal injection. According to clause 9,
A composition in which the injection drug has an increased maximum concentration in the blood compared to the baseline value of oral administration. According to clause 9,
A composition in which the injection has an increased half-life in the blood compared to the baseline value of oral administration. A patch for the prevention or treatment of mTORopathy containing a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]
. According to clause 13,
The patch is administered intradermally or subcutaneously. A pharmaceutical composition for the prevention or treatment of neurological diseases induced by nerve damage, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]
. According to clause 15,
The composition inhibits the formation of amyloid plaques. According to clause 16,
A composition that inhibits the formation of amyloid plaques by activating autophagy. According to clause 15,
The composition promotes nerve regeneration (neurogenesis). According to clause 18,
A composition wherein the nerve regeneration promotes division, differentiation, migration, or survival of neural stem cells or neural progenitor cells. According to clause 15,
The composition, wherein the neurological disease is selected from the group consisting of traumatic central nervous system disease, spinal cord injury disease, peripheral nerve injury, amyotrophic axonal sclerosis, and peripheral nerve disease. According to clause 15,
A composition wherein the neurological disease includes cognitive dysfunction. According to clause 21,
The composition, wherein the cognitive dysfunction is selected from the group consisting of learning disability, memory loss, mild cognitive impairment, depression, agnosia, amnesia, aphasia, apraxia, and Binswanger's disease. A patch for the prevention or treatment of neurological diseases induced by nerve damage containing a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]
. A pharmaceutical composition for the prevention or treatment of degenerative brain disease comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]
. According to clause 24,
The composition, wherein the degenerative brain disease is selected from the group consisting of dementia, Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, epilepsy, stroke, stroke, ischemic brain disease, and memory loss. According to clause 25,
A composition wherein the degenerative brain disease is Alzheimer's disease. According to clause 24,
A composition wherein the degenerative brain disease includes cognitive dysfunction. According to clause 27,
The composition, wherein the cognitive dysfunction is selected from the group consisting of learning disability, memory loss, mild cognitive impairment, depression, agnosia, amnesia, aphasia, apraxia, and Binswanger's disease. A patch for the prevention or treatment of degenerative brain disease containing a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]
.