KR20190008013A - A novel pharmaceutical composition for treating Alzheimer's disease - Google Patents

Abstract

The present invention relates to a novel pharmaceutical composite for treating an Alzheimer′s disease. The present invention provides a pharmaceutical composite for treating an Alzheimer′s disease, including a therapeutically effective amount of hyaluronic acid as an active ingredient. The hyaluronic acid is of a low or high molecular weight. The hyaluronic acid of the low molecular weight has 5-200 kDa of the molecular weight. The purpose of the present invention is to provide a novel and safe medicine for an Alzheimer′s disease, capable of removing Aβ and tau peptide, causing neurotoxicity in the brain of a patient with Alzheimer′s disease.

Description

[0001] The present invention relates to a novel pharmaceutical composition for treating Alzheimer's disease,

The present invention relates to a pharmaceutical composition, and more particularly, to a pharmaceutical composition for treating Alzheimer's disease and a method for treating Alzheimer's disease using the same.

Alzheimer's disease (AD), which accounts for 60-80% of the population suffering from dementia, is associated with memory loss and cognitive impairment due to the loss of neurons, . Familial Alzheimer's disease (familial AD) and familial Alzheimer's disease (sporadic AD), which are predominantly of elderly populations and whose exact cause can not be determined.

When a brain tissue of an AD patient is examined, a senile plaque and a neurofibrillary tangle are distinguished. These lesions are beta-amyloid (hereinafter abbreviated as "Aβ") and tau peptide are insoluble fibril and it made to form a (Glenner & Wong, Biochem Biophys Res Commun 120: 885-890, 1984; Grundke-Iqbal et al Proc Natl Acad Sci USA 83:........ 4913-4917, 1986).

Aβ and tau peptides are already cytotoxic in water-soluble oligomers. Accumulation of Aβ and tau peptides is a major cause of AD by inducing synaptic dysfunction or apoptosis. By activating the clearance of A [beta] and tau peptides, it is possible to prevent excessive accumulation of oligomeric peptides, thereby providing a basis for AD therapy.

Aβ is produced by proteolytic cleavage of the amyloid precursor protein (APP) of the cell membrane and secreted out of the cell or secreted by the endosome (Kinoshita et al ., J. Cell Sci . 116: 3339-3346, 2003), ER, trans-Golgi network, Regina et al ., Proc Natl Acad Sci USA 109: 2077-2082, 2012). Secreted Aβ may enter the cell through receptor-mediated endocytosis (Frank et al ., Nat. Rev. Neurosci. 8: 499-509, 2007). Aβ, which is present outside neurons, is an insulin-degrading enzyme (Zhao et al . Neurobiol. Aging 28 (6): 824-30, 2007), neprilysin, Iwata et al . Science 292: 1550 -1552, 2001), matrix metalloproteinase-9 (Miners et al ., Brain Pathol . 18: 240-252, 2008), glutamate carboxypeptidase II (Nalivaeva et al ( J. Neurochem. 120: 167-185, 2012)) or by glial phagocytosis. Meanwhile, in the intracellular Aβ is ubiquitin-proteasome path (ubiquitin-proteasome pathway, Perez et al, J. Alzheimers Dis 40:.. 1-17, 2014) or the self-extinguishing-lysosomal path (autophagic-lysosomal pathway, Cho et al ., Autophagy 10: 1761-1775, 2014). Oligomeric forms of Aβ, which are well known for their toxic form, are highly insoluble or highly molecular (Masafumi & Tamotsu, FEBS Journal 277: 1348-1358, 2010) and are likely to be removed by the lysosomal pathway rather than the proteasome pathway, In neurons, which are posterior cells, they are highly dependent on the self-digestion-lysosomal pathway (Jin et al ., Exp. Mol. Med. 44: 89-98, 2012). It is well known that tau peptides, which serve to stabilize microtubules within cells, are hyperphosphorylated in pathological states to form tau oligomers, which are removed via the autolytic-lysosomal pathway (Chesser et al. , Front Neurol. 4: 122, 2013). The self-digestion-lysosomal pathway refers to the process of removing substances that are present inside or outside the cell through lysosomes. Numerous studies have reported various problems in the autosynthetic-lysosomal pathway in AD. An increased endogenous abundance of soluble Aβ and an abnormally enlarged endosome have been found (Cataldo et al ., Am. J. Pathol . 173: 370-384, 2008), lysosomal hydrolase ) Was found around the senile plaque outside the cell. In addition, around the senile spots are surrounded by dystrophic neurites, which contain many lysosomal-like organs such as autophagosomes, amphisomes, and lysosomes (Cataldo et al ........ , J. Neuropathol Exp Neurol 55, 704-715, 1996), this increase in ER (vesicle) seems to be due to the accumulation of Aβ (Gowrishankar et al, Proc Natl Acad Sci USA 112: E3699-E3708, 2015). Abnormal accumulation of autophagic vesicles can cause problems with retrograde axonal transport, which is closely related to dysosomal dysfunction (Lee et al ., J. Neurosci . 31: 7817 -7830, 2011). In patients with AD, lysosomal enzymes are over-activated or inactivated (Lauren et al. , J. Neurochem . 140: 703-717, 2017) and their location is higher in organs other than lysosomes (Cataldo et al ., J. Neurosci. 17: 6142-6151, 1997). (Lauren et al. , J. Neurochem . 140: 703-717, 2017), lysosomal calcium storage / release (Coen et al ., J. Cell Biol . 198, 23 -35, 2012) and lysosomal acidification (Lee et al ., Cell 141: 1146-1158, 2010). These features are notable when attention is paid to early-stage patients with cognitive abilities that are normal but develop into AD (Cataldo et al. , Am. J. Pathol . 157, 277-286, 2000) , It is anticipated that treatment of the autosynthetic-lysosomal pathway at an early stage may be an effective and fundamental treatment.

On the other hand, hyaluronic acid (hereinafter abbreviated as 'HA') consists of a repetition of D-glucuronic acid and N-acetyl-D-glucosamine (Necas et al ., Veterinarni Medicina 53 (8): 397-411, 2008), which is a nonsulfated-glycosaminoglycan in the form of a chain and is widely distributed in connective tissues, epithelial tissues, ). HA is a major component of the extracellular matrix and is involved in various cellular responses such as cell proliferation, migration, and inflammation. Molecular weights vary widely from a few kDa to a few MDa and are known to function differently depending on their size (Necas et al ., Veterinarni Medicina 53 (8): 397-411, 2008).

However, the use of HA as a therapeutic agent for Alzheimer's disease has not been reported so far.

Donepezil has been reported as a choline esterase inhibitor (USE985864) as an FDA approved drug for Alzheimer's disease.

However, the choline esterase inhibitor, including donepezil, has side effects such as nausea, diarrhea and vomiting, plays a role in delaying the cognitive decline of the drug's action mechanism and Alzheimer's disease, It is not an ultimate therapeutic agent for inhibiting neuronal death due to neurotoxicity caused by A [beta] or tau peptide.

Therefore, it is required to develop a fundamental therapeutic agent capable of reversing the disease and reducing the side effects of the therapeutic agent for Alzheimer's disease by inhibiting neurotoxicity and neuronal cell death in Alzheimer's disease patients.

The object of the present invention is to provide a novel therapeutic agent for Alzheimer's disease that can remove Aβ and tau peptides which cause neurotoxicity in the brain of a patient suffering from Alzheimer's disease, in order to solve various problems including the above- do. However, the scope of the present invention is not limited thereto.

According to one aspect of the present invention, there is provided a pharmaceutical composition for treating Alzheimer's disease comprising a therapeutically effective amount of hyaluronic acid as an active ingredient.

According to another aspect of the present invention, there is provided a method for treating a patient suffering from Alzheimer's disease comprising administering a therapeutically effective amount of hyaluronic acid to a patient suffering from Alzheimer's disease.

Since the pharmaceutical composition for treating Alzheimer's disease of the present invention uses hyaluronic acid having proven stability as an active ingredient such as a filler for skin cosmetics as an effective ingredient, there is almost no side effect, and a nerve endings in the brain of a patient suffering from Alzheimer's disease Since it can efficiently remove toxic A [beta] and tau peptides, it enables fundamental treatment of Alzheimer's disease. Moreover, since hyaluronic acid can be easily separated from natural materials and can be easily synthesized, it is possible to create a great value added economically. However, the scope of the present invention is not limited by the above effects.

FIG. 1 is a graph showing changes in cell death rate when low molecular weight hyaluronic acid (LMW-HA) and high molecular weight hyaluronic acid (HMW-HA) were treated in HT22 cells treated with A? _1-42 oligomer Fig.
FIG. 2 is a photograph of a hippocampal cell cultured in a mouse primordium treated with A? _1-42 oligomer, LMW-HA or HMW-HA and observed with a fluorescence microscope.
FIG. 3 is a graph showing the results of observing the degree of apoptosis of cultured hippocampal cells treated with Aβ _1-42 oligomer of FIG. 2 and LMW-HA or HMW-HA.
Drug 4-Methylumbelliferone to 4 is the cell death caused by Aβ _1-42 to be expressed within the cell in HT22 cells inhibit the LMW-HA or a hyaluronic acid synthase (HAS) activity of the gene for synthesis of HA (4-MU) Of the present invention are counted through a fluorescence microscope.
FIG. 5 is a photograph showing the results of western blot analysis of the change of Aβ _1-42 oligomer accumulated in cells inside SH-SY5Y cells by HA treatment.
FIG. 6 is a photograph of a phenomenon in which the amount of EGFP-CINCCKVL (GFP-8) overexpressed in SH-SY5Y cells is changed by treatment with bupylymycin A1 (Baf-A1), which is a rysosomal inhibitor, through a fluorescence microscope.
FIG. 7 is a photograph showing the result of Western blot analysis of the change in the amount of GFP-8 caused by the lysosome function-lowering effect of Baf-A1.
8 is a photograph showing the results of Western blot analysis of the amount of GFP-8 that is changed by treatment with A? _1-42 oligomer and LMW-HA in SH-SY5Y cells.
Figure 9 shows the effect of tau peptide agglutination inhibitors, methylene blue (MB), LMW-HA, or HMW-HA, on the formation of tau peptide aggregates when overexpressing GFP-tau in SH-SY5Y cells under fluorescent microscopy And the tau spot is counted.
10 is a photograph showing the results of western blot analysis and filter-trap analysis of the total expression amount of tau peptide and the amount of aggregate in the cells observed in FIG.
11 shows the total amount of expression of tau peptides and the amount of agglutinates when 4-MU, LMW-HA, or HMW-HA were treated with SH-SY5Y stably overexpressing HA-tagged tau peptide, And filter-trap analysis.
FIG. 12 is a graph showing the results of a shrine water examination test showing the effect of inhibiting memory loss by Aβ caused by low molecular weight hyaluronic acid according to an embodiment of the present invention:
Saline: saline solution,
Aβ _1-42 : beta amyloid alone group,
HA: low molecular weight hyaluronic acid alone group,
Aβ _1-42 + HA: group receiving beta amyloid and low molecular weight hyaluronic acid.

Definition of Terms

As used herein, the term " autophagic-lysosomal pathway " (hereinafter abbreviated as " ALP ") refers to the formation of an abnormal ahotophagosome when an abnormally folded protein is generated Refers to a pathway in which the autolysome fuses with the lysosome to form an autolysosome thereby removing the abnormally folded protein. It is known that ALP causes the elimination of abnormally generated A [beta] and tau peptides, which are presumed to be a pathogen of Alzheimer's disease. Thus, it is argued that ALP defects are related to degenerative brain diseases such as Alzheimer's disease and Parkinson's disease .

In the process of inducing autophagy, Tor activity is inhibited, and yeast IF2α kinase, GCN2, and eIF2α-regulated transcription activator, GCN4, play an important role.

In addition, it has been reported that the combination of Atg13 and Atg1, a protein involved in self-digestion, plays an important role in inducing autogenesis. However, in the case of Tor kinase, Atg13 protein induces hyperphosphorylation and attenuates Atg1 binding to inhibit autogenous formation. The role of each constituent protein in the induction of self-digestion is not well known.

As used herein, the term " tau peptide " refers to a protein that exerts the ability to induce stabilization of microtubules. The protein can develop into a disease such as Alzheimer's disease due to the pathology of the nervous system when the tau peptide becomes imperfect to stabilize the microtubules in oligodendrocytes and astrocytes rich in neurons.

In addition, the tau peptide stabilizes the microtubules, as well as the protein that is mainly activated in the distal portion of the axon, which provides flexibility. It stabilizes microtubules with a bulbous protein called tubulin, It works to help assembly. Moreover, the tau peptide achieves control of microtubule stability through phosphorylation of homologous proteins, and hyperphosphorylation of the tau peptide can cause spiral and straight filament entanglement, and this entanglement is a pathology of Alzheimer ' s disease .

When the brain to be affected by the Alzheimer's disease is examined, all six homologous proteins of the tau peptide are hyperphosphorylated in a spiral filament often paired. Also, strange aggregates have been reported in any other degenerative brain disease.

DETAILED DESCRIPTION OF THE INVENTION

According to one aspect of the present invention, there is provided a pharmaceutical composition for treating Alzheimer's disease comprising a therapeutically effective amount of hyaluronic acid as an active ingredient.

In the above pharmaceutical composition, the hyaluronic acid may be a low molecular weight hyaluronic acid or a high molecular weight hyaluronic acid, preferably a low molecular weight hyaluronic acid, but may be used in a low molecular weight hyaluronic acid. The low molecular weight hyaluronic acid may have a molecular weight of 5 to 200 kDa, and the high molecular weight hyaluronic acid may have a molecular weight of 500 kDa to 10 MDa.

The pharmaceutical composition according to one embodiment of the present invention further comprises an inert ingredient, including a pharmaceutically acceptable carrier. As used herein, " pharmaceutically acceptable carrier " is a term referring to a composition, specifically a component other than the active ingredient of the pharmaceutical composition. Examples of pharmaceutically acceptable carriers include binders, disintegrants, diluents, fillers, lubricants, solubilizers or emulsifiers and salts. Such carriers may have a wide variety of forms depending upon the preparation desired for parenteral administration, including, for example, oral or intravenous administration. There can be other with reference to that in a pharmaceutical acceptable carrier are described in the following literature ^{(Remington's Pharmaceutical Sciences, 19 th} ed., Mack Publishing Company, Easton, PA, 1995).

In addition, the pharmaceutical composition according to one embodiment of the present invention may be administered at a dose of 0.1 mg / kg to 1 g / kg, more preferably at a dose of 0.1 mg / kg to 500 mg / kg . On the other hand, the dose can be appropriately adjusted according to the age, sex and condition of the patient.

The pharmaceutical composition according to one embodiment of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intracerebroventricular, intrathecal, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, intraocular, intravitreal, transdermal, subcutaneous Intraperitoneal, intranasal, enteral, topical, sublingual or rectal administration. For injectable forms, the pharmaceutical compositions of the present invention can be formulated into glycerol, liquid polyethylene glycols and mixtures thereof and dispersions in oils. Under normal storage and use conditions, these products may contain preservatives to prevent the growth of microorganisms.

In addition, the pharmaceutical composition according to an embodiment of the present invention may be administered through a method of administration such as intramuscular or intravenous injection rather than a direct delivery method to the brain such as an intravenous administration or intracerebral administration. In this case, The active ingredient, hyaluronic acid, must pass through the blood brain barrier (hereinafter abbreviated as BBB). To this end, the hyaluronic acid according to an embodiment of the present invention may be formulated and administered together with a carrier capable of binding to various known BBB-passing peptides by covalent or noncovalent bonding, or through other BBBs. Peptides disclosed in Korean Patent Laid-open Publication No. 2015-0083600, European Patent Publication No. EP1819723A, US Pat. No. 4801575, US8900551 and the like can be used as the peptide for BBB passage. Examples of the carrier capable of passing through the BBB include European Patents EP 2308514, Korean Patent No. 1081445, Korean Patent No. 228456, US Patent No. US6419949, and the like can be utilized.

The pharmaceutical composition according to one embodiment of the present invention may be formulated into oral or parenteral dosage forms according to the route of administration as described above.

In the case of a preparation for oral administration, the composition of the present invention may be formulated into a powder, a granule, a tablet, a pill, a sugar, a tablet, a liquid, a gel, a syrup, a slurry, . For example, an oral preparation can be obtained by combining the active ingredient with a solid excipient, then milling it, adding suitable auxiliaries, and then processing the mixture into a granular mixture. Examples of suitable excipients include, but are not limited to, sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, Cellulose such as methylcellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose and the like, fillers such as gelatin, polyvinylpyrrolidone and the like. In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may optionally be added as a disintegrant. Further, the pharmaceutical composition of the present invention may further comprise an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

For parenteral administration, they may be formulated in the form of injections, eye drops, ointment creams, lotions, oils, gels, aerosols and nasal inhalers by methods known in the art. These formulations are described in Remington's Pharmaceutical Science, 15th edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a commonly known formulary for all pharmaceutical chemistries.

The therapeutically effective amount may be a dose of 0.1 mg / kg to 1 g / kg, more preferably 0.1 mg / kg to 500 mg / kg.

The pharmaceutical composition may additionally comprise one or more other Alzheimer's therapeutic compounds as an active ingredient.

In the above pharmaceutical composition, the compound for treating Alzheimer's disease is selected from the group consisting of donepezil, ribvastigmine, galantamine, memantine, bapineuzumab, solanezumab, PF-04360365, MABT5102A, GSK933776A, gantenerumab, gammagard, octagam, CAD106, ACC001, UB311, V950 or AD01 / AD02.

According to another aspect of the present invention, there is also provided a method for treating a patient suffering from Alzheimer's disease comprising administering a therapeutically effective amount of hyaluronic acid to a patient suffering from Alzheimer's disease.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. It is to be understood that both the foregoing general description and the following detailed description of the present invention are exemplary and explanatory and are intended to provide further explanation of the invention as claimed to one skilled in the art. The scope of the present invention is not limited to the following examples. Rather, these embodiments are provided so that this disclosure will be more thorough and complete, and will fully convey the concept of the invention to those skilled in the art. In the drawings, the thickness and size of each layer are exaggerated for convenience and clarity of explanation.

Example 1: Preparation of hyaluronic acid

The present inventors purchased low molecular weight hyaluronic acid (LMW-HA, 8-15 kDa) and high molecular weight hyaluronic acid (HMA-HA, 1.75-2 MDa) from Sigma-Aldrich (USA)

Experimental Example 1: Inhibition of cytotoxicity by beta-amyloid oligomer

1-1: HT22 cells

The inventors of the present invention found that HT22 cells were treated with only 5 μM of oligomeric Aβ _1-42 (hereinafter abbreviated as "Aβ _1-42 oligomer") or only PBS as a control group, LMW-HA and HMW-HA Were treated with the concentrations of 20 and 200 μg / ml for 48 hours, and the degree of apoptosis was stained with propidium iodide, and the number of dead cells was counted under fluorescence microscope One). Statistical analysis was performed using a two-tailed unpaired t- test, and each group of experiments was repeated three times. * P < 0.05. As a result, as shown in Fig. 1, the degree of apoptosis of HT22 cells not treated with beta-amyloid oligomer was not changed by treatment with LMW-HA and HMW-HA. This demonstrates that LMW-HA and HMW-HA themselves are safe substances with little effect on cell survival. However, HT22 cells pretreated with beta - amyloid oligomers showed more than twice the degree of apoptosis compared with the control group. When treated with LMW-HA, the degree of apoptosis was reduced to less than half the degree of apoptosis, and it was confirmed that the amount of beta-amyloid oligomer was equivalent to that of the control group without pretreatment. In the case of HMW-HA, cell death was inhibited to a level corresponding to LMW-HA at a low concentration of 20 μg / ml, but cell death was not inhibited at a high concentration of 200 μg / ml. This suggests that LMW-HA inhibits apoptosis by beta-amyloid oligomers, and that HMW-HA has a low activity to inhibit apoptosis after conversion (degradation) to LMW-HA However, an excessive amount of HMW-HA may not be easily converted into LMW-HA. However, in long-term in vivo activity, the effect can be seen.

1-2: Mouse hippocampal neuron

Therefore, the present inventors analyzed whether or not Aβ-induced apoptosis can be suppressed in cultured hippocampal cultured cells, which are also involved in Aβ-induced apoptosis in Alzheimer's patients as well as established HT22 cells.

Specifically, similarly to Experimental Example 1-1, mouse hippocampus-cultured cells were seeded at a concentration of ⁵ × 10 ⁶ cells / ml and cultured for 6 days to obtain a cell-saturation degree of 70%. When the cells were saturated with 5 μM of a beta-amyloid oligomer (PBS), LMW-HA and HMW-HA were treated at a concentration of 100 μg / ml, respectively, and the degree of apoptosis was measured. The cells were treated with PBS (oligomeric Aβ1-42) After staining with propidium iodide, the cells were observed with a fluorescence microscope (Fig. 2), and the killed cells were counted (Fig. 3). * P < 0.05

As a result, as shown in FIGS. 2 and 3, LMW-HA efficiently inhibited cell death by beta-amyloid oligomer, and HMW-HA was found to be somewhat inferior in cell death inhibition.

Then, the present inventors investigated whether the HA inhibitory effect of HA is effective not only on the externally treated beta-amyloid oligomer but also on A [beta] expressed in the cell. In order to investigate whether the HA (pGFP-Ub) (PGFP-Ub-A [beta] _1-42 ) transfected with a beta-amyloid oligomer and inducing apoptosis by A [beta] in the cells after artificially overexpressing the beta-amyloid oligomer, 4-MU, 100 μg / ml LMW-HA and 100 μg / ml HMW-HA for 40 hours, respectively, and the degree of apoptosis was analyzed in the same manner as described above (FIG. * P <0.05, ** P < 0.01 As a result, as shown in Fig. 3, 4-MU, but is rather promote cell death, LMW-HA inhibited the apoptosis of HT22 cells overexpressing Aβ very remarkable. The above results demonstrate that hyaluronic acid of the present invention can inhibit not only extrinsic Aβ but also apoptosis caused by endogenous Aβ, which indirectly inhibits the death of nerve cells by promoting intracellular Aβ scavenging activity It shows.

Experimental Example 2: Confirmation of intracellular A? Removal

In order to confirm whether HA according to an embodiment of the present invention plays a role of eliminating Aβ oligomers accumulated in actual cells, the inventors of the present invention have found that the change of Aβ oligomer delivered into cells upon HA treatment of SH-SY5Y cells Were analyzed by Western blot analysis.

Specifically, SH-SY5Y cells were pretreated with LMW-HA at concentrations of 5, 20 and 50 μg / ml, respectively, and treated with 1 μM Aβ _1-42 oligomer for 12 hours after 12 hours. Cells were then lysed with a lysis buffer and electrophoresed with polyacrylamide gels. After electrophoresis, the cells were electrotransferred to nylon membrane, and the cells were treated with 4G8 / 6E10 antibody (Biolegend Company, # SIG39220, # SIG39320) Western blot analysis was performed using an anti-calnexin antibody (Santa Cruz, SC-11397) (Fig. 5). As a result, as shown in Fig. 5, A [beta] _1-42 oligomer had subcellular fractionation to track the quantitative change of A [beta] _{l -42} oligomer introduced into and transported through the endocytic pathway into the cell, To isolate microsome-fractions containing endosomes, lysosomes, and the like. By Western blot analysis using antibodies specific for the microsomal marker calnexin and antibodies specific for the A? _1-42 oligomer, 4G8 and 6E10, the Aβ _1-42 oligomer contained in the quantitatively identical amount of microsomes Was decreased as HA concentration increased. These results suggest that HA may contribute to the quantitative regulation of A? _1-42 oligomers.

Experimental Example 3: Confirmation of the mechanism of beta amyloid oligomer removal

The CINCCKVL sequence (SEQ ID NO: 1) targets the protein molecule bearing it to the lysosome and allows it to degrade in the lysosome. Thus, the present inventors prepared a plasmid DNA (pEGFP-CINCCKVL) expressing a fusion protein of EGFP protein and CINCCKVL in order to confirm the role of HA in the removal of target protein by lysosome. Then, in order to confirm whether the expression level of GFP-8 can represent the lysosomal activity, SH-SY5Y cells were transfected with the plasmid DNA to overexpress GFP-8, and a V-type ATPase 20 nM of bafilomycin A1 (hereinafter abbreviated as 'Baf-A1') which induces lysosome inhibition in a manner inhibiting the activity of V-type ATPase was treated for 4 hours (FIG. 6). As a result, it can be seen that the signal of GFP-8 was increased in the experimental group treated with Baf-A1, as shown in Fig.

In addition, the present inventors performed Western blot analysis using an antibody specific for LC3, which is a marker for determining whether the anti-GFP antibody and Baf-A1 are functioning. Specifically, SH-SY5Y cells were transfected with the plasmid DNA for 12 hours, after which 20 nM Baf-A1 was treated for 4 hours. The transformed cells were harvested and the cell lysate was electrophoresed and transferred to a nylon membrane. The anti-GFP antibody (Santa Cruz Biotechnology, Inc., SC8334) and anti-LC3 antibody (Novous Inc., NB100- 2220) (Fig. 7). As shown in FIG. 7, it was confirmed that the amount of GFP-8 was increased in the Western blot analysis. This suggests that there is a close relationship between the increase in the amount of GFP-8 and the decrease in the function of the lysosome.

Thus, the present inventors analyzed whether Aβ _1-42 oligomer inhibits the function of lysosomes and whether HA can restore such inhibition by using a lysosome activity assay system using GFP-8.

Specifically, SH-SY5Y cells were transfected with the plasmid DNA for 9 hours and treated with PBS or 100 μg LMW-HA for 26 hours in the presence or absence of 4 μM Aβ _1-42 oligomer. Then, as described above, the treated cells were harvested to prepare a cell lysate, followed by Western analysis using an anti-GFP antibody and anti-alpha-tubulin antibody as a control (FIG. 8). As a result, GFP-8 was increased by overexpressing GFP-8 in SH-SY5Y cells and then treating Aβ _1-42 oligomer. On the other hand, it was confirmed that GFP-8, which was increased by Aβ _1-42 oligomer when LMW-HA was further treated, decreased to the control level. These results indicate that LMW-HA may play an important role in restoring the degradation of lysosome by Aβ _1-42 oligomers. This suggests that the effect of HA on the accumulation of A? _1-42 oligomer, which is the result of the experiment of Experimental Example 1-1, is related to the activation of lysosome function.

Experimental Example 4: Confirmation of elimination of intracellular tau peptide aggregates

Overexpression of tau peptides in SH-SY5Y cells results in the aggregation of tau peptides within the cells, which can be accomplished by performing a filter-trap assay or observing tau dotting by fluorescence microscopy Can be confirmed. The increase in tau peptide aggregates in in vitro assays is an important evidence for tau-pathology. Therefore, in order to confirm the possibility that HA is involved in controlling the tau pathology that forms another major axis of Alzheimer's pathology, the HA according to an embodiment of the present invention removes the intracellular tau peptide aggregate GFP-tau spot analysis was performed in order to confirm whether or not it is possible. Specifically, a plasmid DNA GFP-tau designed to express GFP and tau peptide in the form of a fusion protein was first prepared. Then, SH-SY5Y cells were transfected with the GFP-tau for 6 hours, and 4 μM of methylene blue, 100 μg / mg of LMW-HA or 100 μg / mg of HMW-HA, which is a HA synthase inhibitor, , And GFP-tau spots were counted (Figure 9). As a result, as shown in FIG. 9, when GFP-tau was overexpressed in SH-SY5Y, it was confirmed that the treatment of methylene blue and LMW-HA as a tau peptide aggregation inhibitor decreased the GFP-tau spot compared to the control. On the other hand, HMW-HA was not significantly different from the control group.

Next, we harvested the cells used for GFP-tau spot analysis, and performed a Western analysis and a filter-trap assay to confirm the change in total tau peptide. Specifically, the cells were harvested to prepare a cell lysate, subjected to polyacrylamide gel electrophoresis and transfer to a nylon membrane, and subjected to western blotting using an anti-Ta antibody, HT7, to determine the total amount of tau peptide , And the degree of tau peptide aggregation was observed by filter-trap analysis using anti-GFP antibody (FIG. 10). As a result, as shown in FIG. 10, both LMW-HA and HMW-HA had the effect of reducing the total amount of tau peptide at a level similar to that of MB, which is known to inhibit the aggregation of tau peptides. The effect of inhibiting tau peptide aggregation was more pronounced in LMW-HA than in HMW-HA.

Further, in order to clearly clarify the relationship between HA and HA, the present inventors treated 4-MU with SH-SY5Y, in which HA-attached tau peptide is stably overexpressed, and then treated with 4-MU to remove the tau peptide The total amount and aggregation formation were increased. Specifically, a plasmid DNA HA-tau (a fusion protein in which a human ON4R tau peptide is labeled with HA) designed to express HA-attached tau peptide was stably transfected into SH-SY5Y cells, and SH-SY5Y / HA-tau cells. The cells were treated with 0.4 mM 4-MU, 100 μg / mg LMW-HA or 100 μg / mg HMW-HA for 24 hours, harvested and Western blot analysis and filter-trap analysis using HT17 (Fig. 11). As a result, as shown in FIG. 11, 4-MU increased total amount of tau peptide and aggregate formation, whereas HA according to one embodiment of the present invention suppressed total amount of tau peptide and aggregate formation, HA significantly reduced the formation of aggregates of tau peptides. This suggests that HA may play a major role in the quantitative regulation of tau peptides.

Experimental Example 5: Confirmation of efficacy by animal experiment

To confirm whether HA can improve cognitive function in Alzheimer model animals, we injected Aβ _1-42 peptide into normal mice through intracerebroventricular injection to induce memory decline, HA was inhibited by the inhibition of memory capacity.

Specifically, in normal C57BL / 6J mice (each group n = 10) the Aβ _1-42 peptide (0.1 mg / kg) and the dose or Aβ _1-42 peptide via intraventricular administration of PBS was dissolved in PBS to 8 trying After a 5-day course, dissolved LMW-HA (1.2 mg / kg) was intravenously administered intravenously and a novel object recognition test consisting of a schedule of 2 days of compliance, 1 day of training and 2 days of testing was performed . The mice were allowed to explore freely for 7 minutes at 24-hour intervals in a white plastic chamber (22 cm wide x 27 cm long x 30 cm high). In the training session, the mice were exposed to two objects placed in the chamber for 7 minutes. In the testing trial, one of the familiar things was replaced with a new thing. The preference index was defined as the ratio of the total time spent searching for the object to the time spent searching for new objects (Fig. 12).

HA is known to degrade very rapidly when it is injected into the body, such as in the case of tissue, which is left for about 2-3 days. The mouse model used above was a model for inducing acute memory decline temporarily by injecting a large amount of Aβ, which was suitable for showing sufficient effect even if a single injected HA remained short. As shown in Fig. 12, in the two-time trial, the group injected with A? _1-42 alone did not distinguish between the new object and the existing object, whereas the group injected with A? _1-42 and HA coincided with the control Level preference for new objects. These results suggest that HA may be effective in restoring memory by alleviating Aβ - related AD, and may further serve as an AD treatment agent.

As described above, HA according to an embodiment of the present invention not only significantly inhibits the formation of A [beta] oligomer and tau peptide aggregates causing cytotoxicity as a pathogenesis of Alzheimer's disease in neurons, Since the memory loss caused by Aβ is restored to normal levels, it can be used as an effective remedy for Alzheimer's disease.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. I will understand. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

Claims (6)

A pharmaceutical composition for the treatment of Alzheimer's disease comprising a therapeutically effective amount of hyaluronic acid as an active ingredient. The method according to claim 1,
Wherein the hyaluronic acid is a low molecular weight hyaluronic acid or a high molecular weight hyaluronic acid. 3. The method of claim 2,
Wherein said low molecular weight hyaluronic acid has a molecular weight of from 5 to 200 kDa. 3. The method of claim 2,
Wherein the high molecular weight hyaluronic acid has a molecular weight of 500 kDa to 10 MDa. The method according to claim 1,
&Lt; / RTI &gt; further comprising at least one other Alzheimer's treating compound. 6. The method of claim 5,
The compound for treating Alzheimer's disease is selected from the group consisting of donepezil, ribvastigmine, galantamine, memantine, bapineuzumab, solanezumab, PF-04360365, Wherein said composition is selected from the group consisting of MABT5102A, GSK933776A, gantenerumab, gammagard, octagam, CAD106, ACC001, UB311, V950 or AD01 / AD02.

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