Use of borate derivatives in the treatment of neurodegenerative diseases

Abstract

The present invention relates to the use of borate derivatives in the treatment of the neurodegenerative disease amyotrophic lateral sclerosis (ALS), and more particularly to the development and administration of pharmaceutical formulations comprising at least one borate derivative disclosed in the present invention.

Description

[Technical Field]

[0001] The present invention relates to the use of borate derivatives in the treatment of the neurodegenerative disease amyotrophic lateral sclerosis (ALS), and more particularly to the development and administration of pharmaceutical formulations comprising at least one borate derivative disclosed in the present invention. [Background technology]

[0002] Amyotrophic lateral sclerosis (ALS) is defined as a neurodegenerative disease characterized by progressive muscle paralysis reflecting degeneration of motor neurons in the primary motor cortex, brainstem, and spinal cord. Critical connections between the central nervous system and skeletal muscles are gradually lost, resulting in paralysis, respiratory failure, and death (1, 2).

[0003] At least 90% of ALS cases are sporadic, and the familial inheritance pathway is unknown (Non-Patent Document 3). The remaining 10% of cases may be associated with familial inheritance of various gene mutations. The identification of mutations in the superoxide dismutase 1 (SOD1) gene in 1993 sparked the first major wave of molecular research in ALS (Non-Patent Document 4). SOD1 is a protein expressed in almost all cell types and catalyzes the detoxification of superoxide. More than 150 mutations in SOD1 have been exclusively associated with ALS. Transgenic mice overexpressing mutant SOD1 exhibit ALS-like phenotypes and are considered the cornerstone of ALS research.

[0004] Boron compounds have found many applications in our lives. In particular, they are used in cleaning and bleaching agents, dyes, toothpastes, textiles, and textile dyes. Recent in vitro studies have demonstrated that different boron derivatives may be effective in many therapeutic models, such as the treatment of obesity and wound healing due to their antibacterial / anti-inflammatory properties (Non-Patent Documents 5, 6, 7, 8). Studies showing that boron derivatives inhibit the growth of cancer cells indicate that they may be promising agents for cancer therapy (Non-Patent Documents 9, 10, 11, 12). Research is ongoing into whether boron derivatives have therapeutic and / or preventive effects in neurodegenerative diseases.

[0005] Currently, two drugs are approved for the treatment of ALS. One is riluzole, which has been on the market for 25 years and has been proven to extend patients' lifespan by approximately several months (Non-Patent Document 13), and the other is edaravone, which was clinically approved in 2017 (Non-Patent Document 14). However, although these drugs have become part of routine treatment, unfortunately, they have not had a significant positive impact on the course of the disease and survival time.

[0006] Over the past few years, over 60 molecules have been investigated as potential treatments for ALS, and although some of these molecules have shown promise as drug candidates, unfortunately, the majority of them have not demonstrated sufficient clinical efficacy when phase 1 trials were conducted (Non-Patent Document 15). As mentioned above, the FDA-approved drugs currently used to treat ALS have failed to significantly improve the progression of ALS. Therefore, research aimed at developing novel drug candidates aimed at significantly improving the course of the disease has become increasingly important.

[0007] Like most neurodegenerative diseases, ALS is thought to be partly caused by neuroinflammation (Non-Patent Document 16). The present invention was developed based on the idea that boron compounds, which have been confirmed to have antimicrobial and anti-inflammatory properties in studies conducted in the healthcare field, may also be effective agents for the treatment of ALS disease.

[0008] Therefore, sodium pentaborate pentahydrate (NaB; NaBO·5HO) derivatives, whose antimicrobial, anti-inflammatory, and wound-healing properties have been demonstrated in previous studies (Non-Patent Documents 7, 17), and sodium perborate tetrahydrate (SPT; NaBO·4HO) derivatives, which are still used today as bleaching agents (Non-Patent Document 18), are used in the present invention.

[0009] Boron is a naturally occurring trace element with the symbol B (Non-Patent Document 19) and atomic number 5 (Non-Patent Document 19). It does not occur in nature in its elemental form; instead, it usually forms organoboron complexes with oxygen and sodium. Boron is also an essential nutrient for many organisms. In particular, organoboron complexes are crucial for the biological activity of organisms. In healthy tissues, boron exists in the form of boric acid or borate salts. The total amount in the body is approximately 3-20 mg, but the amount varies depending on the tissue structure (Non-Patent Documents 20, 21, 22).

[0010] Borates are odorless white crystals that dissolve readily in water. The simplest borates are boron oxide (B2O3) and boric acid (H2BO3).

[0011] The chemical name of sodium pentaborate pentahydrate (NaB; NaB5O8·5H2O) is a boron-based compound, sodium; tetrakis(oxoboranyloxy)boranide; pentahydrate (IUPAC).

[0012] The chemical name for sodium perborate tetrahydrate (SPT; NaBO3·4H2O) is a boron-based compound, sodium; 3-oxidedioxaborirane; tetrahydrate (IUPAC).

[0013] State-of-the-art patent literature (TR2019 / 08285, TR2018 / 03493) discloses the use of boron-based borate derivatives, alone and / or in combination, for the treatment of various diseases.

[0014] The state-of-the-art patent application EP 1 790 349 discloses the treatment of viral or bacterial infections with sodium pentaborate pentahydrate (NaB5O2·5H2O).

[0015] State-of-the-art patent application WO 2014168592 discloses an antiviral gel containing sodium pentaborate pentahydrate and used to treat infections associated with the herpes simplex virus.

[0016] In a report published by Fortune Business Insight on the estimated market size of drugs used in neurodegenerative diseases, 2019-2026 (Non-Patent Document 23), the neurodegenerative disease drug market is geographically classified into five major regions. These regions can be listed as North America, Asia Pacific, Latin America, the Middle East and Africa, and Europe, including Turkey. The United States, located in North America, has the largest market size and is expected to hold a market share of $62,786.2 million in 2026. [Prior art documents] [Non-patent literature]

[0017] [Non-Patent Document 1] Munsat, TL, Andres, PL, Finison, L., Conlon, T., & Thibodeau, L. (1988). The natural history of motoneuron loss in amyotrophic lateral sclerosis. Neurology, 38(3), 409-409. [Non-patent document 2] Swash, M., & Schwartz, MS (1995). Motor neuron disease: the clinical syndrome. In Motor Neuron Disease (pp. 1-17). Springer, London. [Non-patent document 3] Taylor, J.P., Brown, R.H., & Cleveland, D.W. (2016). Decoding ALS: from genes to mechanism. Nature, 539(7628), 197 - 206.

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[0018] The object of the present invention relates to the use of boron-based borate derivatives in the treatment of amyotrophic lateral sclerosis (ALS).

[0019] More specifically, the present invention relates to the use of the compounds sodium perborate tetrahydrate and sodium pentaborate pentahydrate in the treatment of ALS disease.

[0020] Another object of the present invention relates to realizing and preparing pharmaceutical formulations comprising at least one borate derivative as described above in the present invention.

[0021] A further object of the present invention relates to the development of pharmaceutical formulations comprising a therapeutically effective amount of at least one pharmaceutically acceptable boric acid derivative and at least one pharmaceutically acceptable excipient, and their use as drugs in the treatment of amyotrophic lateral sclerosis (ALS).

[0022] The use of borate derivatives in the treatment of neurodegenerative diseases realized to achieve the object of the present invention is illustrated in the attached figure: [Brief explanation of the drawings]

[0023] [Figure 1] The graph shows the motor performance of groups of mice before (t=0) and after borate administration. Data represent the longest dwell time of three test trials for each mouse (n=8 / group) ± SEM (**P≦0.01). [Figure 2] As a result of microarray analysis performed on spinal cord material from transgenic and wild-type experimental groups (n = 3 / group) that received NaB, SPT, and water only, gene expression in the other groups compared to the transgenic control group is presented in the form of a heat map (statistical differences between groups were determined by volcano plot analysis (two-way ANOVA; P value ≤ 0.05) and a fold change of ±2 or more was considered statistically significant). [Figure 3] As a result of the pathway analysis, some of the pathways for which SPT and NaB derivatives were found to be effective (PANTHER Gene Ontology (GO) Classification and Pathway System) and the number of genes located in these pathways (total number of genes: 2677, total number of hits: 443) are shown. [Figure 4] Normal SHSY5Y cells (left) and SHSY5U cells transformed into cholinergic neurons (right) are shown. [Figure 5] Gene expression levels in normal and differentiated SHST5Y cells are shown. [Figure 6] Non-toxic concentrations of amyloid beta and sodium pentaborate are shown. [Figure 7] The effect of sodium pentaborate treatment on amyloid-beta plaques is shown (A. negative control; B. amyloid-beta only; C. amyloid-beta combined with sodium pentaborate). [Figure 8] 1 shows the effect of sodium pentaborate on amyloid-β plaques. DETAILED DESCRIPTION OF THE INVENTION

[0024] The subject matter of the present invention relates to the use of boron-based borate derivatives as drugs for the treatment of neurodegenerative diseases, including at least one of amyotrophic lateral sclerosis (ALS), Parkinson's disease, Huntington's disease, and Alzheimer's disease.

[0025] As used herein, the term "pharmaceutically acceptable" refers to a molecule or compound that does not produce any adverse allergic effects or other untoward reactions when administered to an animal or human.

[0026] As used herein, the term "excipient" refers to any pharmaceutically acceptable auxiliary substance, such as a preservative, filler, lubricant, pH adjuster, disintegrant, surfactant, solvent, viscosity agent, emulsifier, dispersant, etc., that can be used in the study of pharmaceutical formulations.

[0027] As used herein, the term "treatment" refers to the prevention or alleviation or complete elimination of the onset or progression of the condition to which the term applies, or one or more symptoms of that condition.

[0028] As used herein, the term "therapeutically effective amount" refers to the dosage of a drug that, when administered to a patient in need of such treatment, produces a specific pharmacological response.

[0029] The boron-based borate derivatives for therapeutic use disclosed in this invention are the compounds sodium pentaborate pentahydrate (NaB; NaB5O8·5H2O) and sodium perborate tetrahydrate (SPT; NaBO3·4H2O), either alone or in combination.

[0030] The pharmaceutical formulations of the present invention can be prepared using standard techniques or state-of-the-art known manufacturing methods.

[0031] The pharmaceutical preparation of the present invention is formulated for use in the treatment of amyotrophic lateral sclerosis (ALS).

[0032] According to the present invention, the compounds sodium pentaborate pentahydrate (NaB) and sodium perborate tetrahydrate (SPT) can be administered orally, systemically, parenterally, nasally, or enterally. In a preferred embodiment of the present invention, the compound(s) can be administered orally or enterally in a suitable formulation and dosage form. More specifically, during enteral administration, the compound is used as a pharmaceutical preparation, preferably as a solution, together with at least one excipient. Herein, the preferred excipient is a solvent, which is essentially water.

[0033] The benefits of the compounds of the present invention (therapeutically effective amount received in a single administration; dosage) are adjusted to be effective for achieving the desired therapeutic response for a pharmaceutical form specific to the administration method. Thus, dosage and excipients can be adjusted depending on the desired therapeutic effect, the route of administration, the desired duration of treatment, and other criteria.

[0034] The specific dose level for any patient (human or mouse) can be adjusted on a treatment-specific basis depending on a variety of criteria, including body weight, general health, sex, diet, duration and route of administration, intestinal absorption and excretion characteristics, combination with other drugs, and severity.

[0035] In a preferred embodiment of the invention, the total daily dose of compound(s) for use in the treatment of ALS may be in an amount of 0.001 to 200 mg / kg body weight / day for SPT and 0.001 to 1000 mg / kg body weight / day for NaB, administered in single or divided doses.

[0036] The in vivo study used the SOD1 G93A transgenic mouse model, one of the most popular animal models for modeling ALS disease. In this study (n = 8), SPT and NaB were administered to the animals by oral gavage every other day. The motor functions of these transgenic animals receiving SPT and NaB throughout the experiment were compared with those of the transgenic control group (water). SPT was observed to improve rotarod performance by 88.4% in the SOD1 G93A model compared with the control group (P < 0.01). The group receiving NaB showed a 62.45% improvement in rotarod performance compared with the control group. The group receiving water showed a worsening of symptoms over time, with a decline in rotarod performance observed (Figure 1).

[0037] In a study conducted in connection with the present invention, a neuroscoring system (NPL 24) specifically developed for the SOD1 G93A transgenic mouse model was used to observe that in the group receiving SPT and NaB, animals that were still in stage 1 (NS1) at the end of the experiment showed milder classical symptoms of this stage, such as hindlimb contractions, tremors, and ataxia, which were significantly weaker than those in the control group (Table 3).

[0038] In addition to measuring motor function, to investigate whether the derivatives induce any gene-based changes in the central nervous system, we collected brain and spinal cord samples from wild-type and transgenic control (water) animals. RNA was isolated from these samples and subjected to microarray analysis in the NaB and SPT groups (n = 3). Gene expression profiles for each group were analyzed and displayed as heat maps. We observed that the expression of genes active in many metabolic pathways in the brain and spinal cord tissues of the transgenic control (water) group was altered compared to the wild-type control group. Interestingly, in the transgenic animals receiving SPT and NaB, these derivatives were effective in these pathways, reversing gene expression at a statistically significant rate, approximating gene expression levels in wild-type animals (Figure 2). Furthermore, pathway analysis (PANTHER Gene Ontology (GO) Classification and Pathway System) showed that these derivatives were effective in increasing the expression of genes related to many metabolic pathways, particularly in transgenic control animals (Figure 3), and caused positive changes in the expression of genes involved in pathways related to neurodegenerative diseases such as Parkinson's disease, Huntington's disease, and Alzheimer's disease.

[0039] The positive findings resulting from this application, over a very short period of approximately 21 days, indicate that long-term borate application can produce much more effective changes and may serve as a prelude to future clinical trials.

[0040] The following examples are intended to better illustrate the subject matter of the present invention without limiting it to these examples.

[0041] Example Example 1 Information on the experimental animals used In the experimental tests carried out to achieve the present invention, the mouse models to which the compounds were administered were as follows: (Table 1) Experimental animals JPEG2025531459000007.jpg44134

[0042] Example 2 Experimental Groups In the experimental tests carried out to achieve the present invention, experimental animals were divided into groups and modeled as follows depending on the compound or control (water) group to be administered: (Table 2) Experimental groups JPEG2025531459000008.jpg93131

[0043] Example 3: Administration of substances SPT or NaB derivatives were vortexed at high speed and dissolved in the mice's drinking water at 200 mg / kg per mouse. The borate solution prepared in drinking water was administered orally every other day for 3 weeks. The control group received only the same amount of drinking water by oral gavage.

[0044] Example 4 Motor Function Test: To understand whether the administered borate derivatives caused any difference in the motor coordination of the animals, especially the transgenic mice, the animals were subjected to several motor function tests before and after administration.

[0045] a) Rotarod test: Mice were allowed to acclimate to the rotarod apparatus for 1 day before motor function testing, and performance was recorded at the end of day 2. Motor performance was measured three times for a maximum of 180 seconds at a constant speed of 14 rpm (25), and the longest time spent on the apparatus was recorded.

[0046] b) Neuroscoring: To observe the early and late stages of the disease that developed during the experiment, wild-type and transgenic animals were subjected to neuroscoring as described in the study by Hatzipetros et al. (24). Thus, the aim was to observe whether the administration of the animals caused any differences in the onset, course, and severity of ALS symptoms. To this end, the disease course was graded using the following scoring system: 1. Neuroscore 0 (NS0, presymptomatic): When suspended by the tail, the mouse can fully extend its hind limbs outward from the lateral midline in a normal manner and remain in this position for at least 2 seconds. If the mouse is able to walk, normal gait is observed. 2. NS1 (first symptoms): When suspended by the tail, mice are unable to fully extend their hind limbs outwards from the lateral midline and are observed to either collapse partially backward or exhibit trembling of the hind limbs (tremors). Towards the end of this phase, impaired gait and slow gait (ataxia) begin. The first symptoms usually appear at day 90. 3. NS2 (Partial Paralysis, Paralysis): When suspended by the tail, the mouse has joint movement, even though the hind limbs are partially or completely collapsed or cannot be fully extended. If the mouse is ambulatory, it can use its hind limbs for forward movement, but its toes will bend backward at least twice during a 90 cm walk, or it will move with a limp. If placed on its left or right side, the mouse will be able to return to its original position within 10 seconds. 4. NS3 (Paralysis): When suspended by the tail, the mouse is observed to have rigid paralysis or minimal joint movement in the hind limbs. If the mouse is ambulatory, it is observed to have forward movement but is unable to use its hind limbs. When placed on its left or right side, the mouse is observed to be able to return to its original position within 10 seconds. 5. NS4 (painless endpoint): When suspended by the tail, the mouse is observed to have rigid paralysis in the hind limbs. If the mouse is ambulatory, no movement is observed, and when placed on its left or right side, the mouse is unable to return to its original position within 10 seconds, i.e., there is no righting reflex.

[0047] According to this scoring system, when symptoms observed in animals are divided into phases according to the order in which they occur, the period from 50 to 80 days after birth can be considered presymptomatic. Since the first symptoms begin to appear from day 90, this date can be considered the beginning of the early symptom phase and the beginning of Neuroscore 1. Although this period can vary, it can last from 20 to 38 days and encompasses the Neuroscore 2 phase. On average, at day 120, this process is referred to as late symptomatic. In females, this period lasts slightly longer, but on average it lasts from 5 to 10 days, and during this late symptomatic period, Neuroscore 3 and 4 phases are also observed (Non-Patent Document 24). Table 3. Detailed comparison of phenotypic characteristics of the NS1 phase in the experimental and control groups before and after administration of borate derivatives (wt: wild type, tg.: transgenic, s: symptoms, s1: hindlimb contraction, s2: tremor, s3: ataxia). JPEG2025531459000009.jpg79135

[0048] Statistical methods and data used: Methods: ANOVA and t-test α: 0.05 (type 1 error) σ: 0.11 (standard deviation between groups) δ: 0.8 (group difference) m: 1 (ratio of control group to other groups) n: 8

[0049] Evaluating the effectiveness of sodium pentaborate for Alzheimer's disease The inventors investigate the effectiveness of sodium pentaborate (NaB) as a boron derivative for another neurodegenerative disease, Alzheimer's disease. The test procedure is as follows:

[0050] Obtaining cell cultures SHSY5Y cells stocked in cryotubes at -80°C or liquid nitrogen were kept in a 37°C water bath until completely lysed. The lysed cells were transferred to a 15 ml Falcon tube containing 5 ml of cell culture medium and centrifuged at 300 x g for 5 minutes. After centrifugation, the medium above the cells was removed, and the cells were lysed with 1 ml of fresh medium using a pipette. Next, the cells were transferred to a cell flask containing 12 ml of DMEM / F12 medium and incubated in 5% CO. 2 The mixture was incubated in an incubator containing HCl at 37°C for 24±2 hours.

[0051] The next day, the medium on the adherent cells was replaced and incubation continued until the cells formed an 85-90% monolayer. The medium from the monolayer cells was removed and the cells were washed with 5 ml of PBS. Next, 3 ml of 0.25% trypsin / EDTA was added to the flask and incubated until the cells were completely removed. Five ml of medium was added to the cells removed from the surface of the flask, and all the cells were placed in a Falcon tube and centrifuged at 300 x g for 5 minutes. Finally, half of the resulting cells were seeded into a new flask and incubated in 5% CO. 2 The mixture was incubated at 37°C in an incubator containing HCl.

[0052] Differentiation of SHSY5Y cells into neuronal cells SHSY5Y cells were seeded in a 6-well plate with 250 × 10 cells per well and incubated in a medium containing DMEM / F12, 10% FBS, and 1% PSA at 5% CO . 2 The cells were incubated at 37°C for 24 hours. The medium on the cells was then removed, and each well was washed with 1 ml of PBS. 2 ml of differentiation medium (1% FBS, 10 μM / ml RA) was then added to each well, and the cells were incubated for 72 hours.

[0053] After the incubation period, the medium on the cells was removed and the cells were washed again with 1 ml of PBS. Then, 2 ml of differentiation medium (1% FBS, 10 μM / ml RA, 10 μg / ml BDNF) was added to the cells and incubated for 72 hours. The same procedure was repeated on day 7. By the end of day 10, the neurons were ready.

[0054] Neuronal cell characterization For characterization, we investigated the expression of acetylcholinesterase (AChE), choline acetyltransferase (ChAT), choline transporter 1 (CHT1), and vesicular acetylcholine transporter (VAChT) genes. To this end, RNA was isolated from differentiated cells. First, cells were detached from the surface with trypsin and washed three times with PBS. 400 μl of Trizol was added to the resulting cell pellet and incubated for 5 minutes. Next, 80 μl of cold chloroform was added and the mixture was centrifuged at 12,000 x g for 15 minutes. The clear supernatant formed after centrifugation was transferred to a new tube, and 400 μl of isopropanol was added and incubated for 10 minutes. The mixture was centrifuged at 12,000 x g for 10 minutes. The resulting RNA pellet was washed with 75% ethanol to remove excess salt. After drying, 50 μl of nucleic acid enzyme-free water was added to the pellet, and the RNA content was measured using a Nanodrop device.

[0055] After RNA isolation was completed, cDNA synthesis from the RNA was performed according to the kit's protocol (iScript cDNA Synthesis Kit, Biorad). The resulting cDNA was adjusted to 100 μl and used for RT-PCR. The reaction mixture was prepared by adding 2.5 μl of SYBR green, 1 μl of primers (F+R), 1 μl of cDNA, and 5.5 μl of nuclease-free water to a 96-well PCR plate with a total volume of 10 μl. The prepared plate was placed in an ABI StepOnePlus device, and RT-PCR was performed.

[0056] Cytotoxicity analysis The maximum non-toxic doses of NaB and amyloid beta (Aβ) in neuronal cells were determined by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)method (MTS), which relies on mitochondrial dehydrogenase enzyme activity. Within the experimental range, cells were treated with different concentrations of amyloid beta (6, 4, 2, 1, and 0.5 μg / ml) and NaB (100, 50, 20, 10, and 5 μg / ml).

[0057] After the incubation period (24 hours), the cell culture medium was removed and replaced with 10% MTS solution prepared in cell culture medium. The cells were incubated at 37°C, 5% CO 2 The cells were cultured in the medium for 2 hours. After incubation, absorbance measurements were performed at 490 nm using an ELISA plate reader. From the obtained absorbance values, cell viability was calculated according to the following formula: ((mean cell absorbance / median negative control absorbance)*100).

[0058] Obtaining an Alzheimer's disease model and evaluating the active ingredients The differentiated cells were treated with 2 ml of amyloid beta, the maximum non-toxic dose that had been determined in advance and prepared in neuronal culture medium, at 37°C and 5% CO. 2 After incubation, the cells were washed three times with 1 ml of PBS. Next, the maximum non-toxic amount of sodium pentaborate pentahydrate was prepared in 2 ml of neuronal medium and added to the cells. The cells were incubated at 37°C and 5% CO. 2 The mixture was cultured for 24 hours.

[0059] Immunocytochemical analysis The old medium from the incubated cells was removed, and the cells were washed three times with 1 ml of PBS. Cells were labeled according to the experimental group: NK (negative control), OAβ (amyloid beta only), ONaB (sodium pentaborate pentahydrate only), or Aβ + NaB (amyloid beta combined with sodium pentaborate).

[0060] First, cells were fixed with 1 ml of 2% paraformaldehyde for 30 minutes at room temperature, then washed three times with PBS. To permeabilize the cells, 750 μl of 0.1% Triton X-100 was added to the cells and incubated for 10 minutes at room temperature. Then, 5% BSA was added to the cells and incubated for 30 minutes at room temperature.

[0061] After the blocking step was completed, the liquid on the cells was removed, and an amyloid beta antibody (Cell Signaling, #12843) was added at a 1:200 ratio in 5% BSA, and the cells were incubated overnight at 4°C. After the incubation period, the cells were washed three times with PBS. Next, a secondary antibody (AlexaFluor 488, Thermo) was added at a 1:1000 ratio and incubated for 1 hour at room temperature in the dark.

[0062] After incubation, cells were washed three times with PBS. For nuclear staining, DAPI (Abcam, #ab285390) was added at a 1:1000 ratio and incubated for 5 minutes at room temperature in the dark. Finally, cells were washed three times with PBS and analyzed under a fluorescence microscope (Zeiss).

[0063] Data evaluation Data obtained from cytotoxicity assays were evaluated using MS Excel. Data obtained from immunocytochemistry studies were analyzed using Image J (National Institutes of Health) and MS Excel.

[0064] Results obtained

[0065] Differentiation of SHSY5Y cells into neuronal cells SHSY5Y cells have a stable karyotype consisting of 47 chromosomes and can be differentiated from a neuroblast-like state into mature human neurons through various mechanisms, including the application of specific brain-derived neurotrophic factors such as retinoic acid, phorbol esters, and BDNF. Previous studies have shown that SHSY5Y cells can differentiate into adrenergic, cholinergic, and dopaminergic neurons, depending on the method applied. Therefore, in this study, SHSY5Y cells were selected to obtain cholinergic neurons. Neurons obtained after the 10-day protocol are shown in Figure 4.

[0066] Neuronal cell characterization Cholinergic neurons exist in the central (brain and spinal cord) and peripheral (motor neurons and autonomic nervous system) nervous systems and use a neurotransmitter called acetylcholine for neurotransmission. Acetylcholine is active in regulating muscle contraction and other neurotransmissions. Cholinergic neurons play an important role in cognitive processes such as learning, memory, attention, and concentration, and acetylcholine is heavily involved in regulating these processes. Therefore, to characterize cholinergic neurons, the ChAT and VAChT genes, which encode enzymes and proteins essential for the synthesis and transport of acetylcholine, the CHT1 gene, which is involved in the reuptake of choline into neurons, and the AChE gene, which is responsible for the degradation of acetylcholine in the synaptic cleft, were identified and characterized. When gene expression levels were compared between cells differentiated with and without a differentiation protocol, we found a 5-fold increase in gene expression for the ChAT gene, a 16-fold increase in the VAChT gene, a 13-fold increase in the CHT1 gene, and a 28-fold increase in the AChE gene (Figure 5). This indicates that after the differentiation protocol applied to the cells, the cells acquired the characteristics of cholinergic neurons.

[0067] Cytotoxicity analysis Cytotoxicity assays are used to determine toxic and non-toxic concentrations of compounds. In this study, non-toxic concentrations of amyloid beta and sodium pentaborate were determined by MTS analysis.

[0068] This colorimetric assay is based on the reduction of the MTS tetrazolium compound by NAD(P)H-dependent dehydrogenase enzymes within metabolically active living cells to produce a colored, water-soluble formazan product.

[0069] According to the results of the analysis, the non-toxic concentration of amyloid beta was determined to be 4 μg / ml, and the non-toxic dose of sodium pentaborate was determined to be 5 μg / ml (shown in FIG. 6).

[0070] Evaluation of Alzheimer's disease models and testing of active ingredients To generate an Alzheimer's disease model, amyloid beta was administered to SHSY5Y cells differentiated into cholinergic neurons for 24 hours, resulting in the formation of amyloid beta plaques in the cells. The plaque formation was assessed by fluorescent staining. While no plaques were detected in the NK and ONaB groups, 75% of the cells in the OAβ group formed amyloid beta plaques. When 5 μg / ml of NaB alone was administered to cells with amyloid beta plaque formation for 24 hours, 92.42% of the plaques formed in the Aβ + NaB group were dissolved, compared with the OAβ group. These results indicate that sodium pentaborate has potential for the treatment of Alzheimer's disease through Aβ plaque disruption. These results are shown in the column graph in Figure 8.

Claims

1. Use of borate derivatives as drugs in the treatment of neurodegenerative diseases.

2. 2. The use according to claim 1, characterized in that the borate derivative is used as a drug in the treatment of amyotrophic lateral sclerosis (ALS).

3. The borate derivative is sodium pentaborate pentahydrate (NaB; NaB ５ O ８ ・5H ２ O) and sodium perborate tetrahydrate (SPT; NaBO ３ ・4H ２ 3. The use according to claim 2, characterized in that the compound is any one of the compounds of formula (I) and (II), or a mixture thereof.

4. 4. Use according to claim 3, characterized in that it comprises at least one excipient.

5. 5. The use according to claim 4, characterized in that it comprises one or more excipients selected from the group consisting of pharmaceutically acceptable preservatives, fillers, lubricants, pH adjusters, disintegrants, surfactants, solvents, viscosity agents, emulsifiers and dispersants.

6. 6. Use according to claim 4 or 5, characterized in that the excipient is a solvent.

7. Use according to any one of claims 1 to 6, characterized in that it is administered by any of the following routes: oral, systemic, parenteral, nasal and enteral.

8. Use according to any one of claims 1 to 7, characterized in that the administered daily dose of the borate derivative is between 0.001 and 200 mg / kg body weight / day for SPT and between 0.001 and 1000 mg / kg body weight / day for NaB.

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