3 apos; application of sialic acid lactose in preparation of medicine for treating Alzheimer disease

Abstract

The invention discloses an application of 3 '-sialic acid lactose in preparation of a medicine for treating Alzheimer's disease. The invention further discloses application of the 3 '-sialic acid lactose or the medicinal salt thereof in preparation of a medicine for inhibiting Tau protein aggregation, and a medicinal composition containing the 3'-sialic acid lactose. The specific sialic acid derivative can effectively block pathological aggregation of tau, and the treatment effect of the medicine on AD is remarkably improved. The 3, 3 '-sialic acid lactose is low in acute toxicity, has no genetic toxicity and reproductive toxicity after being taken for a long time at a high dosage, and has relatively good medicinal safety and relatively good medicinal prospects.

Description

Technical Field

[0001] The present invention relates to the application of a sialic acid derivative, in particular to the application of 3'-sialyllactose in the preparation of a drug for treating Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD), commonly known as senile dementia, is a common neurodegenerative disease in the elderly, characterized by learning and memory impairment, aphasia, apraxia, agnosia, personality and behavioral changes, and other comprehensive dementia. AD is the most common cause of dementia in the elderly, but there is currently a lack of effective therapeutic drugs. AD includes two main neuropathological characteristics: (1) amyloid plaques (amyloid plaques) accumulated outside cells by β-amyloid protein (β-amyloid, Aβ); (2) neurofibrillary tangles (NFTs) formed in cells mainly by abnormal hyperphosphorylated tau protein. The number of NFTs (rather than the number of amyloid plaques) is significantly positively correlated with the degree of dementia in AD patients (Chu and Liu 2019). Existing drugs have limited effect on reversing the course of AD, and a large number of studies have turned to therapeutic strategies that target and inhibit tau pathology.

[0003] Current treatments for AD primarily include the following: cholinesterase inhibitors (such as donepezil, galantamine, rivastine, and huperzine A), noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists (such as memantine), Aβ monoclonal antibodies (such as Aduhelm and Leqembi), and low-molecular-weight oligosaccharide compounds (such as sodium mannitol). These drugs have limited efficacy in treating AD (Iqbal, Liu et al. 2018) and cannot block the pathological aggregation of tau, making them insufficient for clinical use.

[0004] Sialic acid is a type of acidic monosaccharide with a 9-carbon backbone. It is negatively charged and highly hydrophilic. It is widely found at the termini of glycoproteins and glycolipids and is involved in various biological processes. In humans, sialic acid exists in tissues and body fluids such as saliva, breast milk, and cerebrospinal fluid in the form of N-acetylneuraminic acid (Neu5Ac). In breast milk, Neu5Ac forms specific oligosaccharides: 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL). Other mammals, including pigs, cattle, and sheep, also contain Neu5Gc (N-glycolylneuraminic acid), but humans cannot synthesize Neu5Gc.

[0005] Early studies have shown that long-term use of drugs that enhance cholinergic function can promote neuronal sialylation and significantly improve learning ability in animals. Stimulating brain polysialic acid synthesis has a protective effect on brain damage and repair (Liu, Simpson et al. 2023). Furthermore, sialic acid-binding immunoglobulin-like lectins 3 (CD33) inhibit the uptake and clearance of Aβ by glial cells by recognizing and binding to sialic acid on glycoproteins or glycolipids (the specific ligand is still unknown) (Griciuc, Serrano-Pozo et al. 2013; Griciuc, Patel et al. 2019). Competitive binding of free sialic acid analogs to CD33 can promote Aβ uptake and clearance (Miles, Hermans et al. 2019). However, the effects of sialic acid and its derivatives on tau protein aggregation have not been reported, and existing technologies do not demonstrate the therapeutic effects of sialic acid and its derivatives on AD. Summary of the Invention

[0006] Purpose of the invention: The purpose of the present invention is to provide the use of 3'-sialyllactose or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating Alzheimer's disease, so as to solve the problem of how to prepare a drug for treating Alzheimer's disease. The second purpose of the present invention is to propose the use of 3'-sialyllactose or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting Tau protein aggregation, so as to solve the problem of how to prepare a drug for inhibiting Tau protein aggregation. The third purpose of the present invention is to propose a pharmaceutical composition for treating Alzheimer's disease, so as to solve the problem that the efficacy of existing drugs for AD is limited. The fourth purpose of the present invention is to propose a pharmaceutical composition for inhibiting Tau protein aggregation, so as to solve the problem that existing drugs cannot block the pathological aggregation process of tau.

[0007] Technical solution: The present invention discloses the use of 3'-sialyllactose or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating Alzheimer's disease.

[0008] The pharmaceutically acceptable salt forms of 3'-sialyllactose in the present invention include various modified forms such as 3'-sialyllactose sodium salt. Through screening, the present invention discovered that the application of 3'-sialyllactose can effectively inhibit the insoluble aggregation of tau protein, thereby treating neurodegenerative diseases such as Alzheimer's disease. The molecular structure of 3'-sialyllactose is as follows:

[0009]

[0010] The second aspect of the present invention discloses the use of 3'-sialyllactose or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting Tau protein aggregation.

[0011] Preferably, the aggregation of the Tau protein is insoluble aggregation.

[0012] Preferably, the concentration of 3'-sialyllactose or a pharmaceutically acceptable salt thereof in the above-mentioned drug for treating Alzheimer's disease or drug for inhibiting Tau protein aggregation is 2-20 μM.

[0013] The third aspect of the present invention discloses a pharmaceutical composition for treating Alzheimer's disease, comprising an effective amount of 3'-sialyllactose or a pharmaceutically acceptable salt thereof.

[0014] The fourth aspect of the present invention discloses a pharmaceutical composition for inhibiting Tau protein aggregation, comprising an effective amount of 3'-sialyllactose or a pharmaceutically acceptable salt thereof.

[0015] Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients, such as water, stabilizers, antioxidants, osmotic pressure regulators, etc.

[0016] Preferably, the pharmaceutical composition is in the form of at least one of an injection, a powder injection, a nanoparticle formulation, a sustained-release gel, and a tablet. Injections include intravenous / intrathecal injections, nanoparticle formulations may use liposomes or recombinant proteins as carriers, and powder injections may be lyophilized powder injections.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0018] The present invention effectively blocks the pathological aggregation of tau through the use of specific sialic acid derivatives, significantly enhancing the therapeutic efficacy of the drug for AD. 3'-sialyllactose has low acute toxicity and exhibits no genetic or reproductive toxicity with long-term, high-dose administration, demonstrating good pharmaceutical safety and promising pharmaceutical prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a diagram showing the screening results of sialic acid and its derivatives for inhibiting AD P-tau-induced tau aggregation;

[0020] Figure 2 Screening results for sialic acid and its derivatives to reduce P-tau-induced tau aggregation in HEK293T cells;

[0021] Figure 3 These are the results of in vivo efficacy experiments on sialic acid and its derivatives. DETAILED DESCRIPTION

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0023] Example 1: In vitro screening of sialic acid and its derivatives capable of inhibiting tau aggregation, the method is as follows:

[0024]

[0025] Pathological tau protein (AD P-tau) extracted from the brain of AD patients can capture normal tau protein in cells and induce pathological aggregation of tau. In order to explore the effects of Neu5Ac, Neu5Gc, 3'-SL and 6'-SL on tau aggregation induced by AD P-tau, according to the experimental method in the reference (Gu, J., W.Xu, et al. (2020). "Truncation of tau selectively facilitates its pathological activities." J Biol Chem.), HeLa cells were first transfected with pCI / HA-tau. 151-391 Human tau expressed in a highly aggregated form with a hemagglutinin (HA) tag 151-391 Truncated HA-tau 151-391 ), AD P-tau was added 6 hours after transfection to induce HA-tau 151-391 Aggregation, in the experimental group, AD P-tau was added with Neu5Ac, Neu5Gc, 3'-SL or 6'-SL at a final concentration of 6.5μM, and in the control group, AD P-tau was added with normal saline. Cells were collected 48 hours after transfection and immunofluorescence was performed with HA antibody to detect HA-tau. 151-391 The proportion of aggregated cells.

[0026] The results are as follows Figure 1 As shown, Figure 1 Figure A in the middle is the immunofluorescence result. The arrows indicate cells containing tau aggregates. Figure 1 Figure B is a statistical graph showing the proportion of cells containing tau aggregates to HA-positive cells in each group. Figure 1 Con is the control group. Figure 1 It can be seen that after the addition of AD P-tau, about 15% of HA-positive cells showed tau. 151-391 3'-SL significantly reduced the proportion of cells containing tau aggregates to 12%, while Neu5Gc and Neu5Ac had no similar effect, and 6'-SL even increased the proportion of cells containing tau aggregates (e.g. Figure 1 (As shown in Figure B). Therefore Figure 1 The results showed that 3'-SL can specifically inhibit tau aggregation induced by AD P-tau, while other types of sialic acid and its derivatives not only have no inhibitory effect, but may also promote tau aggregation induced by AD P-tau.

[0027] Example 2: To further verify the effect of various types of sialic acid in inhibiting tau aggregation in cells, human HA-tau was expressed in HEK293T cells using the same method as in Example 1. 151-391 Six hours after transfection, AD P-tau was added to HEK293T cells to induce insoluble aggregation of tau. The experimental group was added with AD P-tau and different final concentrations (2, 6.5, 20, 60 μM) of 3'-SL. The non-induced group was not added with AD P-tau and only with normal saline. The control group (Con) was added with ADP-tau and normal saline. 48 hours after transfection, the cells were lysed with RIPA lysis buffer and insoluble HA-tau was separated by ultracentrifugation. 151-391 and soluble HA-tau 151-391 Part, the content of insoluble and soluble tau was analyzed by Western blot detection using antibodies against HA.

[0028] The results are as follows Figure 2 As shown, Figure 2 Figure A is a WB graph showing that 3'-SL at different concentrations inhibits tau aggregation. Figure B is a statistical graph showing the ratio of insoluble tau to soluble tau in Figure A. Figures C and E are WB graphs showing that Neu5Ac, Neu5Gc, and 6'-SL at a final concentration of 6.5 μM inhibit tau aggregation. Figures D and F are statistical graphs showing the ratio of insoluble tau to soluble tau in Figures C and E, respectively. Figure 2 As shown in Figures A and B, the AD P-tau-treated group showed a significant increase in insoluble tau compared to the saline-treated group (NoS), indicating that AD P-tau induces the formation of insoluble tau. Compared to the AD P-tau-treated group without 3'-SL (0 μM 3'-SL), only the 6.5 μM 3'-SL group significantly inhibited AD P-tau-induced tau aggregation in cells. Other 3'-SL concentrations had no inhibitory effect. Notably, the 20 μM 3'-SL group showed a strong tendency to promote tau aggregation. These results demonstrate that 3'-SL significantly inhibits tau aggregation, with an effective concentration range of 2 to 20 μM. The corresponding intravenous dose in humans is 15.5 to 155 mg / kg, and the oral dose is 77.5 to 772.5 mg / kg.

[0029] On the contrary, by Figure 2 As shown in Figures C and D, 6.5 μM Neu5Gc or 6'-SL significantly increased the insoluble aggregation of tau; Figure 2 As shown in Figures E and F, 6.5 μM Neu5Ac had no significant effect on tau aggregation.

[0030] Example 3: In vivo efficacy experiment of sialic acid and its derivatives, the method is as follows:

[0031] Human tau transgenic mice expressing the P301L mutation were used as experimental animals. Tau transgenic mice aged 7-9 months were randomly divided into 8 groups. Homologous wild-type C57BL / 6J mice were used as blank control group. The model group was injected with normal saline by tail vein. The experimental groups were injected with normal saline solution of Neu5Ac, Neu5Gc, 3'-SL and 6'-SL by tail vein respectively. The single dose was 50 mg / kg. The drug was administered once every two days. After 4 weeks of continuous administration, the water maze test was performed to measure the latency of each group of mice to find the platform. Figure 3 The results showed that the latency of the Neu5Ac, 6'-SL, and Neu5Gc groups to find the plateau was no different from that of the model group, indicating that the three have no therapeutic effect on AD. Only the latency of the 3'-SL group was significantly lower than that of the model group and close to that of the blank control group, indicating that 3'-SL can effectively alleviate the cognitive impairment of AD and has a good therapeutic effect on AD.

Claims

1. Use of 3'-sialyllactose or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating Alzheimer's disease.

2. Use of 3'-sialyllactose or a pharmaceutically acceptable salt thereof in the preparation of drugs for inhibiting Tau protein aggregation.

3. The use according to claim 2, characterized in that The aggregates are insoluble aggregates.

4. The use according to any one of claims 1 to 3, characterized in that The concentration of 3'-sialyllactose or a pharmaceutically acceptable salt thereof in the drug for treating Alzheimer's disease or the drug for inhibiting Tau protein aggregation is 2-20 μM.

5. A pharmaceutical composition for treating Alzheimer's disease, characterized in that: Contains an effective amount of 3'-sialyllactose or a pharmaceutically acceptable salt thereof.

6. A pharmaceutical composition for inhibiting Tau protein aggregation, characterized in that: Contains an effective amount of 3'-sialyllactose or a pharmaceutically acceptable salt thereof.

7. The pharmaceutical composition according to claim 5 or 6, characterized in that Pharmaceutically acceptable excipients are also included.

8. The pharmaceutical composition according to claim 7, characterized in that The dosage form of the pharmaceutical composition includes at least one of injection, powder injection, nanoparticle preparation, sustained-release gel, and tablet.

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