KR20240147092A - Pharmaceutical composition for preventing or treating brain damage due to neurological disease - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating brain damage caused by a neurological disease. The present invention provides a pharmaceutical composition for preventing or treating brain damage containing amlexanox.

Description

{PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING BRAIN DAMAGE DUE TO NEUROLOGICAL DISEASE}

The present invention relates to a pharmaceutical composition for preventing or treating brain damage caused by a neurological disease.

Zinc in the brain has various physiological functions, including synaptic function, intracellular signaling, regulation of various genes (zinc finger transcription factors), autophagy, lysosomal function, and neurogenesis, and therefore zinc homeostasis is essential for maintaining normal brain function.

Some of the zinc in the brain is stored in synaptic vesicles by the zinc transporter 3 (ZnT3), and is released from excitatory synaptic terminals during synaptic activity. Zinc is present in the terminal portions of glutamatergic axon terminals throughout the mammalian central nervous system, including the hippocampus, cerebral cortex, and spinal cord (Danscher et al., 1985; Frederickson, 1989).

It is known that secretion and accumulation of zinc contribute to neuronal cell death in several disease states such as epilepsy (Frederickson et al., 1988; Suh et al., 2001), cerebral ischemia (Tonder et al., 1990; Koh et al., 1996), traumatic brain injury (Suh et al., 2000b), and hypoglycemia (Suh et al., 2004; Suh et al., 2007; Suh et al., 2008). In particular, zinc is known to contribute to the development of dementia by binding to amyloid beta peptide (Bush et al., 1994; Suh et al., 2000a; Cherny et al., 2001). This phenomenon is known to induce intracellular zinc deficiency and worsen dementia symptoms.

Moreover, zinc deficiency is frequent in the elderly population, which can cause autophagy and lysosomal dysfunction, leading to waste accumulation and brain aging. Therefore, normalizing zinc homeostasis is expected to help delay aging through normalizing lysosomal function.

The fundamental cause of vascular dementia is also accompanied by aging of vascular endothelial cells and surrounding cells, decreased autophagy, accumulation of lipid waste products, inflammatory response resulting from these, and breakdown of the BBB, so it is thought that maintaining zinc homeostasis in these cells will also be helpful.

In the case of Alzheimer's dementia, the accumulation of Abeta and subsequent accumulation of p-tau are related to the decline in lysosomal function, so a method that induces an increase in zinc in lysosomes may be helpful as a treatment to reduce toxic proteins.

In addition, it is possible that synaptic zinc deficiency indirectly contributes to cognitive decline in dementia by impairing BDNF/Trk signaling between neurons, and therefore, a method to improve cognitive function through normalization of synaptic zinc may be helpful.

Most central nervous system cells are cells that have completed cell division, and therefore, in the case of humans, they must survive and function for at least several decades. In cells that can divide, such as blood cells, accumulated toxic waste can be processed through cell division and apoptosis, but nerve cells that last a long time are inevitably vulnerable to the accumulation of toxic waste. In other words, if the function of lysosomes, the most important organelle for removing waste, declines, the central nervous system is the most affected.

In the central nervous system, there are not only neurons but also various types of “supportive cells” such as astrocytes, microglia, and oligodendrocytes. These cells are not only affected by toxic wastes but also play a role in absorbing and removing toxic wastes released from neurons. However, when overexposed to toxic wastes, they can contribute to damage neurons by being involved in inflammatory responses. For example, astrocytes have recently been known to change into type 1 astrocytes that damage neurons, and their role needs to be elucidated. Microglia also have phagocytic functions and contribute to the removal of wastes, but when activated, they can cause inflammation and secrete cytokines, which can also damage neurons. In addition, vascular cells (endocytes, pericytes), CSF metabolism-related cells (ependymal cells, arachnoid granulation cells) are thought to also experience functional decline and indirectly contribute to neuronal damage.

There are various mechanisms by which toxic wastes are generated in cells. For example, toxic proteins or wastes are generated through various pathways such as when genetic damage causes the generation of mutated proteins, when protein folding is not performed properly due to ER stress, decreased ubiquitin/proteasome function, abnormal metal metabolism, and oxidative damage. However, when lysosome function is normal, it is possible to process them. The problem is that in long-lived cells such as neurons, the effects of wastes accumulate over the long term and lead to decreased lysosome function. In particular, most toxic proteins alkalize the pH of lysosomes, creating a vicious cycle. Therefore, if a method is developed to enhance lysosome function, especially a method that is effective in a state where the v-ATPAse function that acidifies lysosomes is reduced, it will be possible to reduce the accumulation of toxic wastes and develop a fundamental treatment method for brain damage caused by neurological diseases. Furthermore, p-tau accumulation also occurs in vascular dementia, and there is a possibility that enhancing lysosome function to remove it may be effective in treating vascular dementia.

In order to maintain the health of the central nervous system throughout the increasingly longer human lifespan, it is very important to effectively and continuously remove waste products from nerve cells. However, there is no drug or method that effectively promotes this, and in particular, there is no drug that effectively enhances the function of lysosomes, the main organelle for removing waste products.

The present inventors discovered that zinc in lysosomes enhances the function of lysosomes in neurons and astrocytes. Therefore, it is possible that appropriate regulation of zinc metabolism in the nervous system can promote the removal of toxic waste products in the nervous system through the enhancement of lysosomal function. It is expected that this strategy can be used to prevent and treat Alzheimer's disease and vascular dementia accompanied by a decline in lysosomal function (Fig. 1).

To date, no treatment for neurological diseases has been developed that targets increased waste removal within neurons. Furthermore, no strategy has been attempted to achieve this by regulating zinc metabolism in the nervous system. There is a need to develop drugs that can suppress brain damage through mechanisms that enhance lysosomal function by regulating intracellular zinc concentration and inhibiting PDE.

Publication of Patent Publication No. 10-2022-0018552

The present invention aims to provide a pharmaceutical composition for preventing or treating brain damage containing amlexanox.

The present invention also aims to provide a food composition for preventing or improving brain damage containing amlexanox.

The present invention also aims to provide a health functional food for preventing or improving brain damage, including amlexanox.

The present invention also aims to provide a composition for inducing an increase in the concentration of zinc in lysosomes, comprising amlexanox.

The purposes of the present disclosure are not limited to the purposes mentioned above, and other purposes and advantages of the present disclosure which are not mentioned can be understood by the following description, and will be more clearly understood by the embodiments of the present disclosure. In addition, it will be easily understood that the purposes and advantages of the present disclosure can be realized by the means and combinations thereof indicated in the claims.

The present invention provides a pharmaceutical composition for preventing or treating brain damage comprising amlexanox.

The chemical formula of the above Amlexanox is 2-amino-7-isopropyl-5-oxo-5H-[1]benzopyrano[2,3-b] pyridine-3-carboxylic acid, the molecular formula is C ₁₆ H ₁₄ N ₂ O ₄ , and the molecular weight is 298.30. Amlexanox is an odorless, yellowish-white crystalline powder, and its structural formula is as follows.

[Structural formula 1]

The above amlexanox inhibits phosphodiesterase.

The above amlexanox increases the amount of zinc in lysosomes.

The above amlexanox inhibits cell death caused by reactive oxygen species.

The above amlexanox inhibits the activity of stellate cells.

The above amlexanox is included in a concentration of 0.01 μM or more and less than 10.0 μM with respect to the total amount of the composition.

The above brain damage is caused by a neurological disease.

The present invention also provides a food composition for preventing or improving brain damage, comprising amlexanox.

The above amlexanox inhibits phosphodiesterase.

The present invention also provides a health functional food for preventing or improving brain damage, including amlexanox.

The present invention also provides a composition for inducing an increase in the concentration of zinc in lysosomes, comprising amlexanox.

The above amlexanox inhibits phosphodiesterase.

The above amlexanox induces acidification of lysosomes.

The present invention can provide a pharmaceutical composition for preventing or treating brain damage comprising amlexanox.

The present invention can also provide a food composition for preventing or improving brain damage containing amlexanox.

The present invention can also provide a health functional food for preventing or improving brain damage, including amlexanox.

The present invention can also provide a composition for inducing an increase in the concentration of zinc in lysosomes, including amlexanox.

Figure 1 shows the physiological mechanism of lysosome dysfunction and zinc in neurodegenerative brain diseases.
Figure 2 shows the results of a phosphodiesterase inhibition experiment of Amlexanox.
Figure 3 shows the results of measuring changes in intracellular zinc concentration due to Amlexanox treatment.
Figure 4 shows the changes in pH and cathepsin activity of lysosomes due to treatment with amlexanox.
Figure 5 shows the cell death inhibition effect by Amlexanox treatment.
Figure 6 shows the results of confirming the effect of Amlexanox on neurological disorders and nerve damage caused by traumatic brain injury.
Figure 7 shows the results of confirming whether motor nerve recovery in a mouse model was achieved by treatment with Amlexanox.
Figure 8 shows the results of observing whether microglial cell activity was suppressed after brain damage caused by Amlexanox treatment.

The term "treatment" as used herein refers to an approach for obtaining beneficial or desirable clinical results, whether or not detectable beneficial or desirable clinical results for the purposes of the present invention, and also includes alleviation of symptoms, reduction in the extent of a disease, stabilization (i.e., not worsening) of a disease, delaying or slowing down the progression of a disease, ameliorating or temporarily alleviating and alleviating a disease state, whether partial or total. The term "prevention" as used herein includes, but is not limited to, all acts of using the pharmaceutical composition to suppress or delay a disease. Accordingly, the present invention refers to both therapeutic treatments and preventive measures.

The pharmaceutical composition of the present invention can be administered in a therapeutically effective amount, which is an amount of the active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue, animal or human, as thought by a researcher, veterinarian, physician or other clinician, i.e. an amount that induces alleviation of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dosage and frequency of administration of the pharmaceutical composition of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be readily determined by those skilled in the art, and can be adjusted according to various factors including the type of disease, the severity of the disease, the content of the active ingredient and other ingredients contained in the composition, the type of formulation, and the age, weight, general health, sex and diet of the patient, the time of administration, the route of administration and the secretion rate of the composition, the treatment period, and drugs used concomitantly.

In the present invention, the term "food composition" means a food that, regardless of the nutrients provided to a subject consuming it, acts beneficially on one or more functions of the organism to provide a better state of health. As a result, the food composition can be used for the prevention, improvement, or treatment of a disease or a disease-causing factor.

The food composition of the present invention can be preferably formulated as a food composition by additionally including one or more food-wise acceptable or pharmaceutically acceptable carriers in addition to the effective ingredients described above.

In the present invention, "health functional food" includes food manufactured and processed using raw materials or ingredients having functionality useful to the human body according to Act No. 6727 on Health Functional Foods, and means a food with high medical and healthcare effects processed so that, in addition to providing nutrition, it efficiently exhibits bio-regulatory functions such as prevention of brain damage, bio-defense, immunity, and recovery for the purpose of the present invention.

The amount of Amlexanox mixed can be appropriately changed depending on the intended use (prevention, health, or therapeutic treatment), and it is preferable that Amlexanox be included in an amount of 0.01 to 95 wt% based on the total weight of the food composition, and more preferably, it is included in an amount of 1 to 80 wt%. If the content is less than 0.01 wt%, the efficacy of administration may be reduced, and if it exceeds 95 wt%, there may be difficulties in formulation.

There is no particular limitation on the type of the above food. Examples of foods to which the oyster mushroom extract of the present invention can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, etc., and include all health foods in the conventional sense.

When the amlexanox composition of the present invention is used as a health functional food, it may additionally contain an additional ingredient that does not have a detrimental effect on the brain damage improvement or prevention effect of amlexanox.

Hereinafter, the present invention will be described in more detail through examples, but it is obvious that the present invention is not limited by the following examples.

Exam example

1. Confirmation of the phosphodiesterase inhibition effect of Amlexanox

The relative activity (% activity) according to the concentration of Amlexanox (10 uM) for various phosphodiesterase isomers was confirmed, and the phosphodiesterase inhibition experiment of Amlexanox was performed, and the results are shown in Fig. 2. As shown in Fig. 2, it was confirmed that Amlexanox has an excellent inhibitory effect on all isoforms including PDE3. In addition, in order to directly confirm the phenomenon of increased intracellular cAMP due to PDE inhibition, astrocytes from 3-day-old mice were cultured, and Amlexanox was treated to cells at about 2 weeks. As a result of treating astrocytes with AMX and measuring the change in intracellular cAMP, it was confirmed that the cAMP level was significantly increased. In addition, after injecting intracellular soluble amyloid beta and over-injecting Tau and SOD1 toxic proteins to construct AD and ALS in vitro systems, we confirmed the change in toxic proteins due to Amlexanox, and confirmed that the toxic proteins were reduced in all cell disease models.

2. Confirmation of increased intracellular zinc concentration by Amlexanox treatment

The results of measuring the change in intracellular zinc concentration after treating with amlexanox are shown in Fig. 3. After culturing 3-day-old mouse astrocytes on PLL-coated glass for 2 weeks, they were stained with zinc staining samples, FluoZin-3AM and Zinpyr-1, and lysosome staining sample, Lysotracter, for 30 minutes, and treated with amlexanox. The change in zinc within the cells, especially within lysosomes, was confirmed using confocal microscopy. As a result, it was observed that the increase in zinc was located in lysosomes.

3. Confirmation of pH change in lysosomes by treatment with amlexanox

After culturing mouse astrocytes on PLL-coated glass for 2 weeks on 3-day-old plates, the cells were stained with Lysosensor DND189, which can detect changes in the pH of lysosomes, and Cathepsin L, a protein known as lysosomes. The changes were then confirmed by treating with AMX, and the results are shown in Fig. 4. When Bafilomycin A1 is treated, the pH of lysosomes increases and the activity of lysosome-specific proteins decreases, but when AMX is treated, the pH becomes acidic and the activity of lysosome-specific proteins increases.

4. Confirmation of cell death inhibition effect by Amlexanox treatment

After culturing mouse astrocytes on the third day of life for two weeks, apoptosis was induced by treating _them with _H2O2 , and the inhibitory effect of Amlexanox was observed through an LDH assay, and the results are shown in Fig. 5. It was confirmed that Amlexanox inhibits apoptosis caused by reactive oxygen species.

5. Confirmation of improvement in neurological disorders caused by traumatic brain injury by Amlexanox treatment

As shown in Figs. 6 and 7, in order to investigate the effect of Amlexanox on neurological disorders and nerve damage caused by traumatic brain injury, which is one of the animal models of vascular dementia, Amlexanox (5 mg/kg) was administered once a day for a week after inducing the injury (Fig. 6B). To examine the neurological disorders occurring after the injury, the neurological severity score (NSS) test was performed and observed. The NSS measures neurological damage with an 18-point score by a researcher who was not involved in the treatment, and evaluates reflexes, flexibility, movement, and coordination. 1 point means that a specific task was not performed, and a higher score means more severe damage, while a score of 0 indicates better health. As a result, it was confirmed that the NSS score was lower in the group administered Amlexanox than in the group administered vehicle after traumatic brain injury (Fig. 6C and D). In addition, one week after traumatic brain injury, the activity of astrocytes and the number of neurons were significantly reduced in both the CA1 of the hippocampus and the GCL and Hilus of the DG, and it was found that this astrocyte activity and neuron reduction were suppressed in the experimental group administered the novel PDE inhibitor (Fig. 6E). In addition, it was confirmed that motor neurons were recovered in the mouse model administered with Amlexanox (Fig. 7). This indicates that the administration of Amlexanox improves neurological disorders that occur after traumatic brain injury and also suppresses the activity of astrocytes and neuronal death.

6. Confirmation of inhibition of microglial cell activity after brain damage by Amlexanox treatment

We observed whether microglia activity was inhibited after brain damage by Amlexanox treatment, and the results are shown in Fig. 8. As shown in Fig. 8, it was observed that microglia activity occurring after traumatic brain damage was inhibited by Amlexanox treatment, and the activity of M1 microglia, an inflammatory phenotype, was significantly inhibited.

From the above experimental results, it is judged that Amlexanox has the activity control and neuroprotective effects of glial cells, and furthermore, has the potential to overcome neuronal damage and cognitive dysfunction caused by vascular dementia.

Claims (13)

A pharmaceutical composition for preventing or treating brain damage comprising amlexanox. In the first paragraph,
The above amlexanox is a pharmaceutical composition that inhibits phosphodiesterase. In the first paragraph,
The above amlexanox is a pharmaceutical composition that increases the amount of zinc in lysosomes. In the first paragraph,
The above amlexanox is a pharmaceutical composition that inhibits cell death caused by reactive oxygen species. In the first paragraph,
The above amlexanox is a pharmaceutical composition that inhibits the activity of stellate cells. In the first paragraph,
A pharmaceutical composition, wherein the above amlexanox is included in a concentration of 0.01 μM or more and less than 10.0 μM with respect to the total amount of the composition. In the first paragraph,
A pharmaceutical composition wherein the above brain damage is caused by a neurological disease. A food composition for preventing or improving brain damage comprising amlexanox. In Article 8,
A food composition wherein the above amlexanox inhibits phosphodiesterase. A health functional food containing amlexanox for preventing or improving brain damage. A composition for inducing an increase in the concentration of zinc in lysosomes, comprising amlexanox. In Article 11,
The above amlexanox is a composition for inducing an increase in the concentration of zinc in lysosomes, which inhibits phosphodiesterase. In Article 12,
The above amlexanox is a composition for inducing an increase in the concentration of zinc in lysosomes, which induces acidification of lysosomes.