An anti-Alzheimer's composition containing human albumin, its preparation method and application

Abstract

The present invention discloses an anti-Alzheimer's disease composition containing human albumin, its preparation method and application, which relates to the field of medicine. The anti-Alzheimer's disease composition of the present invention comprises 5 to 40 parts by mass of human albumin; 1 to 20 parts of polyunsaturated fatty acid or its ester, and the balance is deionized water. The composition and preparation method of the anti-Alzheimer's disease composition of the present invention are simple, and the anti-Alzheimer's disease effect is good, having a wide application prospect in the field of medicine.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to an anti-Alzheimer's disease composition containing human albumin, its preparation method and application. Background Art

[0002] Alzheimer's disease (AD) is a brain disease with insidious onset and no typical symptoms during the onset process, so it is very easy to be ignored. After cardiovascular and cerebrovascular diseases and malignant tumors, it has become the third major disease causing disability and death in the elderly. Summary of the Invention

[0003] In view of the problems and deficiencies of the prior art, the present invention provides an anti-Alzheimer's disease composition containing human albumin, its preparation method and application. The composition of the present invention includes human albumin and polyunsaturated fatty acids.

[0004] Human serum albumin (HSA) is a monomeric protein produced by the liver. Mature human serum albumin contains 585 amino acid residues and has a molecular weight of 66KD. Blood-derived human serum albumin products are therapeutic drugs extracted from human plasma and are currently widely used in the treatment of liver ascites, burns and other related diseases.

[0005] Polyunsaturated fatty acids (PUFAs) are mainly derived from marine organisms or deep-sea fish, etc. The content of PUFAs in red blood cells can improve central nervous system inflammation and oxidative damage, reduce the release of pro-inflammatory cytokines, increase the synthesis of neurotrophic factors, inhibit apoptosis, promote nerve development and regeneration, and thus improve memory ability.

[0006] The solution of the present invention is as follows:

[0007] In the first aspect of the present invention, there is provided an anti-Alzheimer's disease composition containing human albumin, characterized in that the composition includes the following components in parts by mass: the mass fraction of human albumin is 5 to 40 parts; the mass fraction of polyunsaturated fatty acids (PUFAs) is 1 to 20 parts. Preferably, the balance of the composition is deionized water.

[0008] Furthermore, the human albumin is selected from at least one of recombinant human albumin and blood-derived human serum albumin.

[0009] Further, the polyunsaturated fatty acids are selected from two of docosahexaenoic acid (22:6; DHA) or its esters, docosapentaenoic acid (22:5; DPA) or its esters, and eicosapentaenoic acid (20:5; EPA) or its esters. Preferably, the esters are selected from at least one of methyl polyunsaturated fatty acid esters and ethyl polyunsaturated fatty acid esters. More preferably, the methyl polyunsaturated fatty acid esters include at least one of methyl docosapentaenoate, methyl docosahexaenoate, and methyl eicosapentaenoate; and the ethyl polyunsaturated fatty acid esters include at least one of ethyl docosapentaenoate, ethyl docosahexaenoate, and ethyl eicosapentaenoate.

[0010] Further, the mass ratio of two of the polyunsaturated fatty acids or their esters, namely docosahexaenoic acid (22:6; DHA) or its esters, docosapentaenoic acid (22:5; DPA) or its esters, and eicosapentaenoic acid (20:5; EPA) or its esters, is 1:(0.05 - 20). For example, it can be 1:0.05, 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, and any range therebetween.

[0011] Preferably, the mass ratio of two of the polyunsaturated fatty acids or their esters, namely docosahexaenoic acid (22:6; DHA) or its esters, docosapentaenoic acid (22:5; DPA) or its esters, and eicosapentaenoic acid (20:5; EPA) or its esters, is 1:(0.1 - 10).

[0012] Further, the composition further includes additives acceptable in the pharmaceutical field.

[0013] Preferably, the additives include at least one of polysorbates, polyoxyethylene fatty acid esters, parabens, gum arabic, tragacanth gum, and stearates.

[0014] Further, the composition can also be used in combination with other drugs for treating Alzheimer's disease. Preferably, the drugs for treating Alzheimer's disease include at least one of galantamine, donepezil, and huperzine A.

[0015] In the second aspect of the present invention, there is provided the use of the composition as described above in the preparation of anti - Alzheimer's disease drugs.

[0016] Further, the drug can be administered orally and by injection.

[0017] Furthermore, the pharmaceutical dosage form includes at least one of oral liquid, soft capsule, emulsion, and injection.

[0018] Furthermore, the injection administration includes at least one of intramuscular injection and intravenous injection.

[0019] Furthermore, when the drug is administered orally, it needs to be through a sustained-release route, including at least one of capsules, nanoparticles, microcapsules, and microsphere drugs.

[0020] It should be noted that the present invention does not specifically limit the type of the product. Those skilled in the art can understand that for other products, as long as the composition described in the present application is added and plays the same role as the composition in the present invention, they should also be within the protection scope of the present invention.

[0021] In the third aspect of the present invention, a method for preparing the composition is provided. The method includes the following steps:

[0022] S1: Mix different kinds of polyunsaturated fatty acid solutions with the specified parts by mass evenly.

[0023] S2: Add the polyunsaturated fatty acid solution obtained in step S1 to an aqueous solution of human albumin or a human albumin salt buffer with the specified parts by mass, and make up the balance with deionized water to obtain a mixed solution of the composition. Preferably, the human albumin salt buffer includes at least one of human albumin phosphate buffer, human albumin citrate buffer, and human albumin acetate buffer. Preferably, the human albumin buffer salt solution is human albumin PBS buffer.

[0024] Furthermore, the conditions for even mixing in step S1 are: 40 - 60 °C, 50 - 100 rpm, and stir for 10 - 30 min.

[0025] Furthermore, step S2 also includes a high-pressure homogenization step. Preferably, the conditions for high-pressure homogenization are to use a high-pressure homogenizer at 400 - 1200 N and perform high-pressure homogenization for 10 - 30 min.

[0026] Furthermore, the method also includes a step of aseptically filtering the mixed solution of the composition prepared in step S2, and the filtration diameter is 0.1 μm.

[0027] The beneficial effects of the present invention include but are not limited to:

[0028] 1. The present invention provides an anti-Alzheimer's disease composition containing human albumin. Using human albumin as the main raw material, through the synergistic effect of human albumin and two polyunsaturated fatty acids, it has a good anti-Alzheimer's disease effect, with simple composition and better biocompatibility.

[0029] 2. The present invention also provides a method for preparing an anti-Alzheimer's disease composition containing human albumin, which has a simple manufacturing process and high application prospects. Detailed Embodiments

[0030] The present invention will be described in detail below in conjunction with embodiments. However, the present invention is not limited to these embodiments. Unless otherwise specified, the raw materials and catalysts in the embodiments of the present invention are purchased through commercial channels.

[0031] Definition: Recombinant human albumin refers to human albumin prepared by recombinant DNA technology for treatment and prevention.

[0032] Blood-derived human blood albumin refers to human albumin isolated and purified from the blood, plasma or specific immune plasma of healthy humans.

[0033] Source of materials: Recombinant human albumin: Tonghua Anruite Biopharmaceutical Co., Ltd., batch number: ART101S;

[0034] Docosahexaenoic acid (DHA) (batch number: D100925-100mg, Shanghai Aladdin Biochemical Technology Co., Ltd.);

[0035] Eicosapentaenoic acid (EPA) (batch number: C472120-5mg, Shanghai Aladdin Biochemical Technology Co., Ltd.);

[0036] Docosapentaenoic acid (DPA) (batch number: CCFD200505-25MG, Sinopharm Chemical Reagent Co., Ltd.);

[0037] Amyloid-β 1-42 ) Mw: 4514.04, (batch number: A494432-2.5mg, Shanghai Aladdin Biochemical Technology Co., Ltd.);

[0038] Dimethyl sulfoxide (DMSO) (batch number: D103277-250ml, Shanghai Aladdin Biochemical Technology Co., Ltd.);

[0039] Hexafluoroisopropanol (batch number: 213-059-4, Shanghai Macklin Biochemical Co., Ltd.);

[0040] Physiological sodium chloride solution (batch number: 20241019, Sichuan Kelun Pharmaceutical Co., Ltd.);

[0041] Isoflurane for animals (batch number: R510-22-16, Shenzhen Reword Biotechnology Co., Ltd.);

[0042] MTT assay kit (batch number: C0009S, Shanghai Beyotime Biotechnology Co., Ltd.);

[0043] SPF-grade mice (batch number: CS-008, Liaoning Changsheng Biotechnology Co., Ltd.);

[0044] SH-SY5Y (human neuroblastoma cells) (batch number: SNL-092, Wuhan Shang'en Biotechnology Co., Ltd.);

[0045] Equipment:

[0046] Water bath (HH.S21-8, Shanghai Boxun Medical Biological Instrument Co., Ltd.);

[0047] Multifunctional fluorescence microplate reader (SPMax 3500FL type, Shanghai Flash Spectrum Biotechnology Co., Ltd.);

[0048] Analytical balance (BSA224S type, Sartorius Scientific Instruments Beijing Co., Ltd.);

[0049] Brain stereotaxic apparatus (model DB053, Beijing Zhishu Duobao Biotechnology Co., Ltd.);

[0050] Morris water maze video analysis system (model DB001X, Beijing Zhishu Duobao Biotechnology Co., Ltd.);

[0051] Y maze stimulator (model DB003, Beijing Zhishu Duobao Biotechnology Co., Ltd.);

[0052] Small animal activity recorder (model DB-YLS-1C, Beijing Zhishu Duobao Biotechnology Co., Ltd.);

[0053] Digital display disperser (model IKAT-25, IKA (Guangzhou) Instrument Equipment Co., Ltd.);

[0054] Digital display electrothermal constant temperature water bath (model HH-4, Shanghai Yiheng Instruments Co., Ltd.);

[0055] Other reagents and instruments used are all commercially available, unless otherwise specified.

[0056] Example 1

[0057] Prepare a recombinant human albumin anti-Alzheimer's disease composition containing the following components in parts by mass: The active ingredients are shown in Table 1.

[0058] Table 1 Component table of various recombinant human albumin anti-Alzheimer's disease compositions

[0059]

[0060] Prepare an anti-Alzheimer's disease composition according to the component information in Table 1. The specific method is as follows:

[0061] (1) Take the mass fraction of polyunsaturated fatty acids, stir at 40 - 60 °C and 50 - 100 rpm for 10 - 30 min to mix evenly;

[0062] (2) Cool the temperature of the unsaturated fatty acid solution after mixing evenly to 30 - 35 °C, add it to the mass fraction of human albumin, and use a high-pressure homogenizer to homogenize at 400 - 1200 N for 10 - 30 min to obtain a mixed solution of the anti-Alzheimer's disease composition.

[0063] (3) Sterile filter the mixed solution of the anti-Alzheimer's disease composition through a 0.1 μm filter to obtain an anti-Alzheimer's disease composition containing human albumin.

[0064] Test Example 1: Cell Activity Experiment

[0065] 1 μg / μl Aβ 1-42 Solution Preparation:

[0066] Precisely weigh 1 mg of Aβ 1-42 powder and dissolve it in 20 μl of anhydrous DMSO. After complete dissolution, add 980 μl of normal saline, mix evenly, and refrigerate in a 4 °C refrigerator to obtain 1 μg / μl Aβ 1-42 solution.

[0067] Inoculate SH-SY5Y cells in the logarithmic growth phase at a density of 5×10 3 cells / 100 μl into a 96-well plate and culture at 37 °C for 24 hours. Then perform the following grouping treatments, with each group treated for 24 hours: Blank control group: only SH-SY5Y cells (group 38); Positive control group (treated with Aβ 1-42 , group 39): SH-SY5Y cells + 1 μg / μl Aβ 1-42 ; Sample group: SH-SY5Y cells + 1 μg / μl Aβ 1-42 + 10 μg / μl sample (experimental group numbers: 1 - 37). After treatment, incubate the SH-SY5Y cells in each group with MTT (final concentration in the incubation system is 0.5 mg / mL) for 4 hours, and then incubate with 10% SDS solution at 37 °C for another 15 minutes. Measure the absorbance at 560 nm using an enzyme-linked immunosorbent assay reader. Calculate the cell viability, where the OD 560nm value of the experimental group is denoted as As, and the OD 560nm values of the control group and the blank group are denoted as Ac and Ab, and all samples are measured three times. Calculate the cell activity according to the following formula.

[0068] The experimental data are shown in Table 2.

[0069] Cell viability (%) = [(As - Ab) / (Ac - Ab)] × 100%

[0070] As: Absorbance of the experimental group (including cells, culture medium, MTT solution, and anti - Alzheimer's disease composition);

[0071] Ac: Absorbance of the control group (including cells, culture medium, MTT solution, without anti - Alzheimer's disease composition);

[0072] Ab: Absorbance of the blank group (including culture medium, MTT solution, without cells and anti - Alzheimer's disease composition).

[0073] Table 2 Experimental results of cell activity test

[0074]

[0075] Note: * represents the difference between the recombinant human protein group and the blood - derived human albumin group, P ≤ 0.05, # represents the difference between the positive control group and the blank group, P ≤ 0.05.

[0076] As can be seen from Table 2, the effects of a recombinant human albumin anti - Alzheimer's disease composition on the viability of SH - SY5Y cells: Compared with the data results of the bovine serum albumin group (37), the results of both the blood - derived human albumin group (7 - 12) and the recombinant human albumin group (1 - 6) are much higher than those of the bovine serum albumin group, indicating that human albumin is more conducive to the growth of SH - SY5Y cells; the effect of the recombinant human albumin group (1 - 6) is better than that of the blood - derived human albumin group (7 - 12), and the cell survival rate is above 129%. The combination of adding DPA, EPA, and albumin (including blood - derived albumin) at the same ratio (experimental groups 3 and 9) is better than the combination of adding DPA, DHA, and albumin (including blood - derived albumin) at the same ratio (experimental groups 27 and 29) and the combination of EPA, DHA, and albumin (including blood - derived albumin) (experimental groups 28 and 30). The experimental results of adding DHA, EPA, and DPA alone show that in terms of cell survival rate, DPA and EPA are more significant than DHA in cell proliferation ability. The addition of a three - fatty - acid composition (35 - 36) does not improve the survival rate of SH - SY5Y cells compared with the addition of a two - fatty - acid composition (1 - 12). Therefore, the combination of two unsaturated fatty acids, DPA and EPA, is preferred. The cell survival rate of the blank control group (only containing cells, culture medium, and MTT solution, group 38) is 100%, and the cell survival rate of the positive control group is only 70%, indicating that the anti - Alzheimer's disease composition prepared by this method has a significant difference compared with the blank control and the positive control, suggesting that the recombinant human albumin anti - Alzheimer's disease composition has a role in promoting cell proliferation of SH - SY5Y.

[0077] Meanwhile, in the experimental groups (groups 13 - 17) where only single components of recombinant human albumin, DHA, EPA, and DPA were added, the experimental data showed the lowest cell survival rate compared to the experimental groups (groups 1 - 12, groups 18 - 36), indicating that the single component had the weakest effect on cell proliferation activity. At the same time, when only two of the three functional components were added, the data results of the experimental groups (groups 18 - 26) were 82% - 108%, which were much lower than the data of 119% in the experimental groups (groups 1 - 12). This shows that when the three active components of this anti - Alzheimer's disease composition co - exist, it can maintain a high cell activity. For the optimal formulation ratio, the experimental groups (groups 1 - 6) were much higher than other groups (groups 7 - 30), with a cell activity value > 129%, indicating that an anti - Alzheimer's disease composition containing recombinant human albumin prepared by this method has strong vitality for the growth of human neuroblastoma cells.

[0078] Test Example 2: Animal behavioral experiments for Alzheimer's disease

[0079] There were 190 SPF - grade mice, with an equal number of males and females, and a body weight of 25 - 30 g. After purchase, the environment was kept clean, and they were fed with special feed, with unrestricted diet and water. All animal surgeries were carried out in accordance with the "Guide for the Care and Use of Laboratory Animals". Based on the experimental results of cell activity tests, experimental groups (groups 1 - 12) and groups (27 - 30) were selected as the sample groups to conduct animal experiments.

[0080] After 7 days of adaptive feeding for 190 mice, they were randomly divided into a blank group, a sham - operation group, a model group, and a sample group (groups: 1 - 19), a total of 19 groups, with 10 mice in each group. The mice were anesthetized with isoflurane for animals and fixed on a stereotaxic apparatus for mice. Except for the blank group and the sham - operation group, the other groups of mice were injected with 3 μl of 1 μg / μl Aβ 1-42 solution at a constant speed for 2 min and the needle was left in place for 5 min to ensure sufficient diffusion of the solution. Penicillin sodium powder was sprinkled on the surgical site to prevent infection, and the scalp was sutured. After modeling, 0.1 ml of 40,000 U penicillin sodium solution was intramuscularly injected for 3 consecutive days. The mice in the sham - operation group were injected with 3 μl of normal saline into the lateral ventricle at the same position, and the postoperative treatment method was the same as above.

[0081] The blank group, the sham - operation group, and the model group of mice were intragastrically administered with 0.5% CMA - Na solution, and the mice in the sample group (groups: 1 - 16) were intragastrically administered with 80 mg / kg of the corresponding composition for 14 consecutive days. The experimental mice were respectively subjected to Y - maze and Morris water - maze experiments to verify the changes in their cognitive abilities.

[0082] (1)Y-maze experiment: Referring to the method of Kim DH et al. (Neurobiol Learn Mem, 2007, 87(4): 536-546), the Y-maze experiment was conducted. The Y-maze consists of arms A, B, and C at 120°. It is made of iron plates with black paint on both the inner and outer surfaces. The arm length is 47 cm, the height is 16 cm, and the width is 3 cm. The intersection of the three arms at one end is an equilateral triangle. During the experiment, a layer of fresh bedding was laid at the bottom and replaced after each mouse's experiment. 70% ethanol was used to eliminate the smell left by the previous mouse. Before the experiment started, the mouse was placed at one end facing arm A and allowed to freely explore the maze for 10 min. The total number of times entering the arms (when the whole body except the tail enters the arm is counted as one entry) and the order of entering the arms were recorded, and the spontaneous alternation response rate was calculated. The formula is: spontaneous alternation response rate = [number of correct alternation responses / (total number - 2)] × 100%. The data results are shown in Table 3.

[0083] Table 3 Results of Y-maze test experiment

[0084]

[0085] Note: # represents the difference between the model group and the blank group, P ≤ 0.05, * represents the difference between the recombinant human protein-administered group and the blood-derived human albumin-administered group, P ≤ 0.05.

[0086] According to the data in Table 3, compared with the blank group (81.5%), the spontaneous alternation response rate of the model group mice decreased (52.2%), indicating that intracerebroventricular injection of Aβ 1-42 caused damage to the short-term memory of mice; compared with the model group, the spontaneous alternation response rate of the mice in groups (1-16) increased, indicating that albumin could relieve the short-term memory damage caused by intracerebroventricular injection of Aβ 1-42 in mice, and the data of groups (1-6) were better than those of groups (7-12), indicating that recombinant albumin was superior to blood-derived human albumin in relieving the short-term memory damage caused by injection of Aβ 1-42 caused.

[0087] (2)Morris water maze experiment: The Morris water maze experiment was carried out with reference to the method of Mutlu O (Drug Target Insights, 2015, 9(1): 1-8). The Morris water maze consists of a circular pool with a diameter of 120 cm and a height of 40 cm, and an automatic video recording and analysis system. After adding water, black ink was added to prevent interference, and the water temperature was maintained at about 25 °C. The pool was surrounded by black curtains, and different ornaments were hung on the curtains as references. The water maze was divided into four quadrants: southeast, northeast, southwest, and northwest. A black circular platform 1 cm below the water surface was placed in the center of a certain quadrant. During the test, the mice were placed into the water facing the pool wall of a certain quadrant, and the time required for the mice to find and climb onto the platform, that is, the escape latency, was recorded according to the water maze video tracking system. Each mouse was trained 2 times a day for 5 consecutive days for the above-mentioned spatial navigation experiment. If it exceeded 60 s in the previous few trainings, the mouse was guided to stay on the platform for 10 s. The maximum escape latency in this experiment was set at 120 s, that is, if the mouse did not find the platform within 120 s, the escape latency was recorded as 120 s. The data results are shown in Table 4.

[0088] Table 4 Results of Morris water maze test

[0089]

[0090] Note: # represents the difference between the model group and the blank group after the fifth day of administration and the first day of administration, P≤0.05, * represents the difference between the recombinant human protein group and the blood-derived human albumin group after the fifth day of administration and the first day of administration, P≤0.05.

[0091] Compared with the blank group, the escape latency of the mice in the model group was prolonged from the second day, indicating that intracerebroventricular injection of Aβ 1-42 caused memory impairment in spatial navigation of mice. The escape latency of the mice in the sham operation group (group 18) was not significantly different from that of the blank group, indicating that the operation did not cause damage to the spatial navigation cognitive function of the mice; compared with the model group, the escape latency of the mice intragastrically administered with albumin composition (groups: 1-16) was significantly shortened from the fourth day; the escape latency of the mice intragastrically administered with recombinant albumin composition (groups: 1-6) was significantly shortened from the fifth day and was lower than that of the mice intragastrically administered with blood-derived human albumin composition (groups: 7-12), indicating that the recombinant human albumin group (1-6) had a better effect than the blood-derived human albumin group (7-12).

[0092] As described above, these are only embodiments of the present invention. The protection scope of the present invention is not limited by these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, various modifications and variations can be made to the present invention. Any modification, equivalent replacement, improvement, etc. made within the technical idea and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of a composition containing human albumin in the preparation of an anti-Alzheimer's disease drug, characterized in that: The composition is composed of any of the following: (1) Recombinant human albumin: 5 parts, DPA: 0.5 parts, EPA: 0.5 parts; (2) Recombinant human albumin: 10 parts, DPA: 1.5 parts, EPA: 3 parts; (3) Recombinant human albumin: 20 parts, DPA: 3 parts, EPA: 5 parts; (4) Recombinant human albumin: 25 parts, DPA: 5 parts, EPA: 8 parts; (5) Recombinant human albumin: 30 parts, DPA: 8 parts, EPA: 10 parts; (6) Recombinant human albumin: 40 parts, DPA: 10 parts, EPA: 10 parts; (7) Hematogenous human albumin: 5 parts, DPA: 0.5 parts, EPA: 0.5 parts; (8) Hematogenous human albumin: 10 parts, DPA: 1.5 parts, EPA: 3 parts; (9) Hematogenous human albumin: 20 parts, DPA: 3 parts, EPA: 5 parts; (10) Hematogenous human albumin: 25 parts, DPA: 5 parts, EPA: 8 parts; (11) Hematogenous human albumin: 30 parts, DPA: 8 parts, EPA: 10 parts; (12) Hematogenous human albumin: 40 parts, DPA: 10 parts, EPA: 10 parts.

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