KR20240058735A - Composition for preventing or treating diseases mediated by microglia inflammasome - Google Patents

Abstract

The present invention relates to a composition for preventing or treating inflammatory body-mediated diseases in microglial cells, and more specifically, by inhibiting the phosphorylation of Syk, AMPK, and Akt in microglial cells activated by amyloid-beta. The present invention relates to a composition for preventing or treating inflammatory-mediated diseases in microglial cells, comprising as an active ingredient a compound or a pharmaceutically acceptable salt thereof that has the effect of inhibiting inflammatory-some activation.
The akhak composition provided by the present invention inhibits the phosphorylation of Syk, AMPK, and Akt, which are the mechanisms of inflammatory body activation induced by amyloid beta in microglial cells, and inhibits the production of ASC polymer, interleukin-1β, and caspase-1. By doing so, it can exhibit an excellent preventive or therapeutic effect on inflammatory body-mediated diseases in microglial cells.

Description

Composition for preventing or treating diseases mediated by microglia inflammasome}

The present invention relates to a composition for preventing or treating inflammatory body-mediated diseases in microglial cells, and more specifically, by inhibiting the phosphorylation of Syk, AMPK, and Akt in microglial cells activated by amyloid-beta. The present invention relates to a composition for preventing or treating inflammatory-mediated diseases in microglial cells, comprising as an active ingredient a compound or a pharmaceutically acceptable salt thereof that has the effect of inhibiting inflammatory-some activation.

Alzheimer's disease (AD) is the most common degenerative brain disease that causes dementia, and symptoms of the progressive neurodegenerative disease include neuron loss and cognitive impairment. Alzheimer's disease exhibits neuropathological hallmarks of extracellular amyloid-β:Aβ plaques and intracellular neurofibrillary tangles. Amyloid-beta is a sequential cleavage of amyloid-β precursor protein (APP) mediated by Beta-secretase 1 (BACE1) and presenilin/γ-secretase. is created by The production and aggregation of amyloid-beta plays an important role in triggering complex pathologies that lead to neurodegeneration, tangles of nerve fibers, inflammation, and neuron loss.

Activation of microglial cells, indicative of neuroinflammation, is another pathological characteristic in the brains of patients with Alzheimer's disease. Additionally, microglia are involved in many CNS (central nervous system) diseases, including chronic neurodegenerative diseases such as AD, PD (Parkinson's disease), HIV (human immunodeficiency virus) dementia, and multiple sclerosis. system) and acts as a potential pathogen in diseases (Block et al., Nat Rev Neurosci, 8:57-69, 2007). Activated glial cells and leukocytes produce a variety of inflammatory mediators, including anaphylatoxin complement, cytokines, chemokines, and prostaglandins (Gelderblom et al., Stroke 40(5):1849-57, 2009; Iadecola et al., Nature Neuroscience 14(11):1363-1368, 2011).

Studies on the inflammasome complex in peripheral tissues are related to the production and secretion of inflammatory cytokines and apototic and pyroptotic cell death (Lamkanfi et al., Annu Rev Cell Dev Biol. 28:137-61). NLRP1 (NACHT, LRR and PYD domains-containing protein 1) and NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasomes are NLRP1/3 receptor, ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) , a cytoplasmic polymer composed of procaspase-1, procaspase-11 (homologous to human precursor caspase-4 or 5), and X-linked inhibitor of apoptosis (XIAP). It is a complex. By activation and homo-oligomerization of NLRP1 and NLRP3 receptors, precursor caspase-1 is automatically activated and converted to cleaved caspase-1, forming NLRP1 and NLRP3 inflammasomes. Chronic accumulation of Aβ promotes microglial activation in Alzheimer's disease. Increased IL-1β levels are associated with the accumulation of Aβ. IL-1β is produced in an inactive pro-form for activation and secretion of caspase-1, and caspase-1 activity is regulated by inflammasomes. The NLRP3 inflasome senses inflammatory crystals and aggregated proteins such as Aβ and is associated with chronic inflammatory diseases. Inflammatory substances induced by activation of NLRP inflammasomes play a role in mediating synaptic dysfunction, cognitive impairment, and restriction of microglial function. The progression of Alzheimer's disease can be effectively prevented through treatment that inhibits the activation of NLRP inflammasomes and inflammasome-derived cytokines, which play a key role in Aβ-mediated inflammatory responses.

Accordingly, the present inventors conducted repeated studies to find a substance that exhibits a preventive or therapeutic effect for neuroinflammatory diseases such as Alzheimer's by inhibiting the activation of NLRP3 inflammatory bodies in microglial cells. As a result, a compound exhibiting the structure of Formula 1 or a pharmaceutical thereof was found. It was confirmed that an acceptable salt had this effect, and the present invention was completed.

Therefore, the object of the present invention is to provide a pharmaceutical for preventing or treating inflammasome-mediated diseases in microglia, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient: Provided is a composition:

[Formula 1]

Another object of the present invention is to provide a reagent composition for inhibiting inflammatory bodies in microglial cells, comprising 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 method of inhibiting inflammatory bodies in microglial cells, comprising treating animals other than humans with a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof: will be:

[Formula 1]

In order to achieve the object of the present invention described above, the present invention provides an inflammasome-mediated inflammasome in microglia containing a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient. Provided is a pharmaceutical composition for preventing or treating diseases:

[Formula 1]

In order to achieve another object of the present invention, the present invention provides a reagent composition for inhibiting inflammatory bodies in microglial cells, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient. Provides:

[Formula 1]

In order to achieve another object of the present invention, the present invention provides a method for treating animals other than humans with a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof, in microglial cells. Methods for inhibiting inflammasomes are provided:

[Formula 1]

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating inflammasome-mediated diseases in microglia, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient: :

[Formula 1]

The inflammasome is a large multiprotein complex that plays an important role in innate immunity, involved in the production of proinflammatory cytokines interleukin-1β (IL-1β) and IL-18. These related cytokines cause a variety of biological effects related to infection, inflammation, and autoimmune processes. They are produced as inactive precursors, pro-IL-1β and pro-IL-18, and share a common maturation mechanism requiring caspase-1. Caspase-1 itself is synthesized as the enzyme precursor (zymogen) of pro-caspase-1 and, through an autocatalytic process, generates two subunits that form active caspase-1. . Activation of caspase-1 occurs within the inflammasome after its assembly.

The microglial cells are immune cells of the central nervous system and play important roles such as maintaining homeostasis and controlling signal transmission of nerve cells as well as immune function. Microglial cells in the aging brain produce harmful signaling substances such as various inflammatory cytokines and reactive oxygen species, increasing the likelihood of causing disease. It is known that microglia play an important role in several neurological diseases, such as Alzheimer's disease, Parkinson's disease, or neuropathic pain.

In one embodiment of the present invention, the pharmaceutical composition according to the present invention may inhibit the activity of inflammatory bodies.

In the present invention, for the purpose of the present invention, inhibition of the activity of the inflammasome includes inhibition of the production of the inflammasome, inhibition of cytokine production due to the inflammasome, or ASC (adaptor protein apoptosis-associated spec-like protein containing a caspase-recruitment domain) polymer (oligomer). ) may inhibit formation. Preferably, the pharmaceutical composition according to the present invention can inhibit the activity of inflammatory bodies by inhibiting the formation of ASC oligomers, which corresponds to the stage before the formation of inflammatory bodies, but is not limited thereto.

In the present invention, the inflammasomes are caspase-1-activating protein complexes, including 1) NLRP3 (NOD-like receptor family, pyrin domain-containing 3), a sensor protein, 2) linking protein ( It consists of ASC (adaptor protein apoptosis-associated spec-like protein containing a caspase-recruitment domain), which is an adapter protein, and 3) procaspase-1, which is an effector protein. The above-mentioned inflammasome components are assembled when microbial infection or tissue injury occurs. The inflammasomes activated by assembly in the cytosol activate caspase-1 to produce interleukin (IL)-1β or interleukin-18. It is secreted and performs the host's innate immune defense function. Preferably, the inflammasome may be an NLRP3 inflammasome, but is not limited thereto.

In the present invention, when the NLRP3 inflammasome is excessively activated, it can induce and/or promote the onset of inflammasome-mediated inflammatory disease. In particular, excessive activity of NLRP3 inflammasomes in microglial cells can cause neuroinflammatory diseases such as Alzheimer's.

Therefore, in the present invention, the inflammatory body-mediated disease in microglial cells refers to an abnormal disease that occurs when inflammatory bodies are abnormally and excessively activated, preferably Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, multiple sclerosis, It may be selected from the group consisting of amyotrophic lateral sclerosis, and inflammatory spinal cord injury, but is not limited thereto.

Meanwhile, in the present invention, “prevention” may include without limitation any act of blocking, suppressing or delaying the symptoms of an inflammatory mediated disease in microglial cells using the pharmaceutical composition according to the present invention. there is.

Additionally, in the present invention, “treatment” may include, without limitation, any action that improves or benefits the symptoms of an inflammatory medium-mediated disease in microglial cells using the pharmaceutical composition according to the present invention.

In the present invention, the pharmaceutical composition may be in the form of a capsule, tablet, granule, injection, ointment, powder, or beverage, and the pharmaceutical composition may be intended for human subjects.

The pharmaceutical composition of the present invention is not limited to these, but can be formulated and used in the form of oral dosage forms such as powders, granules, capsules, tablets, and aqueous suspensions, external preparations, suppositories, and sterile injection solutions according to conventional methods. You can. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavorings, etc. for oral administration, and buffers, preservatives, and analgesics for injections. Topics, solubilizers, isotonic agents, stabilizers, etc. can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives, etc. can be used. The dosage form of the pharmaceutical composition according to the present invention can be prepared in various ways by mixing it with a pharmaceutically acceptable carrier as described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be manufactured in the form of unit dose ampoules or multiple doses. there is. In addition, it can be formulated into solutions, suspensions, tablets, capsules, sustained-release preparations, etc.

Meanwhile, examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil may be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. may be additionally included.

The route of administration of the pharmaceutical composition according to the present invention is not limited to these, but is oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, and topical. , sublingual or rectal. Oral or parenteral administration is preferred. As used herein, the term “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention can also be administered in the form of a suppository for rectal administration.

The pharmaceutical composition of the present invention may vary depending on several factors, including the activity of the specific compound used, age, body weight, general health, gender, diet, time of administration, route of administration, excretion rate, drug formulation, and severity of the specific disease to be prevented or treated. It may vary in various ways, and the dosage of the pharmaceutical composition may vary depending on the patient's condition, body weight, degree of disease, drug form, route and period of administration, but may be appropriately selected by a person skilled in the art, and may range from 0.0001 to 50 mg/day. It can be administered in kg or 0.001 to 50 mg/kg. Administration may be administered once a day, or may be administered several times. The above dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention can be formulated into pills, dragees, capsules, solutions, gels, syrups, slurries, and suspensions.

As used in the present invention, the term "pharmaceutically acceptable salt" refers to a salt of a compound to be administered prepared from a non-toxic acid including a pharmaceutically acceptable inorganic acid, organic acid, solvate, hydrate or clathrate thereof. indicates. Examples of these inorganic acids include hydrochloric acid, hydrobromic acid, iodic acid, nitric acid, sulfuric acid, phosphoric acid, acetic acid, hexafluorophosphoric acid, citric acid, gluconic acid, benzoic acid, propionic acid, butyric acid, sulfosalicylic acid, maleic acid, lauric acid, malic acid, and fumaric acid. , succinic acid, tartaric acid, amsonic acid, palm acid, p-toluenesulfonic acid and mesylic acid. Suitable organic acids may be selected, for example, from the aliphatic, aromatic, carboxyl and sulfonic classes of organic acids, such as formic acid, acetic acid, propionic acid, succinic acid, camphorsulfonic acid, citric acid, fumaric acid, gluconic acid, isethionic acid, Lactic acid, malic acid, mucilic acid, tartaric acid, para-toluenesulfonic acid, glycolic acid, glucuronic acid, maleic acid, furoic acid, glutamic acid, benzoic acid, atranilic acid, salicylic acid, phenylacetic acid, mandelic acid, emponic acid (palm acid), These include methanesulfonic acid, ethanesulfonic acid, pantothenic acid, benzenesulfonic acid (besylate), stearic acid, sulfanilic acid, alginic acid, and galacturonic acid. Additionally, pharmaceutically acceptable salts include, but are not limited to, alkaline earth metal salts (e.g., calcium or magnesium), alkali metal salts (e.g., sodium-dependent or potassium) and ammonium salts. Includes.

As used herein, the term “composition” or “pharmaceutical composition” refers to a mixture of at least one compound useful within the invention and a pharmaceutically acceptable carrier. Pharmaceutical compositions facilitate administration of the compounds to a patient or subject. A variety of techniques exist in the art for administering compounds, including but not limited to intravenous, oral, aerosol, parenteral, ocular, pulmonary, and topical administration.

The present invention also provides a reagent composition for inhibiting inflammatory bodies in microglial cells, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:

[Formula 1]

The present invention also provides a method of inhibiting inflammatory bodies in microglial cells, comprising treating animals other than humans with a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:

[Formula 1]

The types of animals are not limited and may include rats, mice, rabbits, goats, cows, horses, dogs, monkeys, etc.

The akhak composition provided by the present invention inhibits the phosphorylation of Syk, AMPK, and Akt, which are the mechanisms of inflammatory body activation induced by amyloid beta in microglial cells, and inhibits the production of ASC polymer, interleukin-1β, and caspase-1. By doing so, it can exhibit an excellent preventive or therapeutic effect on inflammatory body-mediated diseases in microglial cells.

Figure 1 shows the results of evaluating the amounts of IL-1β, 1L-18, and ASC secreted into the medium to evaluate the activation of NLRP3 inflammasomes in microglial cells stimulated with LPS and Aβ.
Figure 2 shows the results of observing through staining the aggregation of ASC protein in microglial cells stimulated with LPS and Aβ.
Figure 3 shows the results of evaluating the phosphorylation of Syk, AMPK, and Akt to confirm the mechanism by which the compound of Formula 1 (FA) inhibits Aβ-induced inflammatory body activation in microglial cells.
Figure 4 shows the results of confirming the cognitive function recovery of the compound of formula 1 (FA) in the ADLP Alzheimer's animal model through a Y-maze experiment.
Figures 5 to 7 show the degree of activation of microglial cells (Figure 5), the amount of accumulation of Aβ (Figure 6), and the amount of accumulation of phosphorylated tau protein (Figure 5) to evaluate the efficacy of the compound (FA) of Formula 1 in the ADLP Alzheimer's animal model. This is the result of confirming 7).
Figures 8 to 10 show that when activation of inflammatory bodies by LPS is induced in microglial cells, the compound of Chemical Formula 1 (FA) activates cleaved caspase-1 and IL-18, which are activation markers of inflammatory bodies (Figure 8), pro This result confirms that the increase in IL-1β (Figure 9) is suppressed and that this occurs through AMPK-AKT (Figure 10).
Figure 11 is a result confirming that when LPS is administered to a normal animal model, activation of inflammatory bodies in microglial cells appears, and that the compound of Formula 1 (FA) can inhibit this.
Figure 12 shows the results confirming that the compound of Formula 1 (FA) exhibits a microglial cell-dependent drug effect.

Hereinafter, the present invention will be explained in detail by the following examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited thereto.

1. In vitro inflammatory cell inhibitory activity

In cells, lippopolysaccaride (LPS) and Aβ are used to mimic the mechanism by which inflammatory bodies are stimulated in Alzheimer's disease. Microglial cells were treated with 10 ng/ml of LPS for 3 hours as a primary stimulus, and treated with 5 μM of Aβ for 24 hours as a secondary stimulus before use in the experiment. The degree of activation of NLRP3 inflammasomes can be confirmed by the secretion of ASC protein, which forms a corpuscular structure with the NLRP3 protein, and the amount of inflammatory cytokines secreted into the medium by caspase1 activated by the inflammasomes.

When the proteins in the medium in which the cells were cultured were confirmed by western blot, secreted IL-1β, 1L-18 cytokines, and ASC increased (Figure 1).

In addition, it was confirmed through staining that ASC protein was aggregated within the cells (Figure 2).

At this time, when cells were treated with 100 μM or 200 μM compound (FA) of Formula 1 for 1 hour before treating Aβ as a secondary stimulus, the expression and secretion of cytokines and aggregation of ASC protein were all significantly reduced (Figure 1 and Figure 2).

It was confirmed that the efficacy of the compound of Formula 1 is because the drug restores the phosphorylation of Syk, AMPK, and Akt, which are the mechanisms of Aβ-induced inflammatory body activation in microglial cells (Figure 3).

2. In vivo efficacy evaluation

The effect of the compound of Formula 1 was confirmed in an animal model using ADLP mice, an Alzheimer's disease model mouse that overexpresses Aβ and tau proteins. For 6 months to 8 weeks, when amyloid aggregates and phosphorylated tau protein were accumulating in the brains of the mice, 5 mg/kg of the compound of Formula 1 was diluted in phosphatate buffered saline (PBS) and orally administered daily.

Afterwards, when the degree of memory was measured through a behavioral experiment using the Y-maze, the rats administered the drug showed a significantly higher level of memory compared to the ADLP rats injected only with PBS (Figure 4).

In addition, when the degree of accumulation of pathological proteins was confirmed by staining the rat brain, the degree of microglial activation (Figure 5), accumulation of Aβ (Figure 6), and accumulation of phosphorylated tau protein (Figure 7) were all There was significant recovery.

3. Inflammasome activation by LPS stimulation rather than Aβ and control using the compound of Formula 1

The compound of Formula 1 restores the activation of inflammatory bodies caused by LPS in microglial cells through regulating the activity of AMPK. When the activation of inflammatory bodies by LPS was induced by treating primary microglial cells with LPS for 24 hours, cleaved caspase-1 and IL-18, which are markers of inflammatory bodies seen in western blot (Figure 8), pro It can be seen that IL-1β (Figure 9) increases. At this time, when ASA, ibuprofen, and the compound of Formula 1, which are NSAID drugs, are treated respectively, it can be seen that only the compound of Formula 1 shows a significant recovery effect. It was confirmed at the protein level that this phenomenon occurs through AMPK-AKT (FIG. 10).

4. Confirmation of the drug action mechanism by confirming the effect of the compound of formula 1 in an animal model that caused an inflammatory response by in vivo administration of LPS and the effect in TREM2 KO mice.

After administering the compound of formula 1 to B6 animals without pathology for 2 months, I.p. LPS was administered by injection to induce an inflammatory response. When confirmed by staining, it was confirmed that ASC speck significantly increased in microglial cells within the brain due to LPS. On the other hand, no increase in ASC was observed in the group administered the compound of Formula 1. Through this, it was confirmed that the compound of Formula 1 can effectively prevent the activation of inflammatory bodies in microglial cells caused by an inflammatory response even if it is not Alzheimer's disease. Meanwhile, syk kinase, which is affected by the compound of Formula 1, operates downstream of various receptors, and it is well known that TREM2, a well-known inflammation-related receptor for microglial cells, also has syk as a signaling system beneath it. Therefore, through the TREM2 KO model, we sought to confirm that the activation of inflammasomes is regulated by syk-ampk signals. In fact, the activity of inflammatory bodies in TREM2 KO mice was significantly lower than that in normal mice even when administered LPS, and the level was similar to that in mice administered the compound of Formula 1 (FIG. 11).

5. Confirmation of microglial cell-dependent drug responsiveness of the compound of Formula 1

We sought to confirm that the compound of Formula 1 has the most important effect on microglial cells when administered in an animal model, and that the drug effect is microglial-dependent.

To eliminate microglial cells, PLX3397 was mixed with feed and administered to an Alzheimer's disease animal model (ADLP) for 3 months.

As a result, iba1, a marker of microglial cells, was significantly decreased when confirmed by staining (Figures 12a, b). When the compound of formula 1 was administered to mice fed PLX feed, the effects of the compound of formula 1 on various Alzheimer's pathologies were not confirmed, unlike what was confirmed in general animal models. Memory ability (Figure 12c), accumulation of amyloid plaques (Figure 12d, e), and accumulation of phosphorylated tau protein (Figure 12f, g) all showed no significant changes. Through this, it was confirmed that the compound of Formula 1 was effective only in the presence of microglial cells.

The akhak composition provided by the present invention inhibits the phosphorylation of Syk, AMPK, and Akt, which are the mechanisms of inflammatory body activation induced by amyloid beta in microglial cells, and inhibits the production of ASC polymer, interleukin-1β, and caspase-1. By doing so, it can exhibit excellent preventive or therapeutic effects on inflammatory body-mediated diseases in microglial cells, and thus has very high industrial applicability.

Claims (7)

A pharmaceutical composition for preventing or treating inflammasome-mediated diseases in microglia, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

The pharmaceutical composition according to claim 1, wherein the inflammasome is an NLRP3 inflammasome.
The method of claim 1, wherein the compound or a pharmaceutically acceptable salt thereof is used in microglial cells as ASC (adaptor protein apoptosis-associated spec-like protein containing a caspase-recruitment domain) polymer, interleukin-1β (IL-1β), or A pharmaceutical composition characterized by inhibiting the production of caspase-1.
The pharmaceutical method according to claim 1, wherein the inflammatory body-mediated disease in microglial cells is a disease mediated by activation of inflammatory bodies in microglial cells by amyloid-beta or lipopolysaccharide (LPS). Composition.
The method of claim 1, wherein the inflammasome-mediated disease in microglial cells is a group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, multiple sclerosis, amyotrophic lateral sclerosis, and inflammatory spinal cord injury. A pharmaceutical composition selected from .
A reagent composition for inhibiting inflammatory bodies in microglial cells, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

A method of inhibiting inflammatory bodies in microglial cells, comprising treating an animal other than a human with a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
[Formula 1]