Use of cell-free fat extract against alzheimer's disease

Abstract

Provided is use of a cell-free fat extract (CEFFE Pro 2.0) in the preparation of a pharmaceutical composition or a health product composition for preventing and / or treating Alzheimer's disease. Treatment of a Caenorhabditis elegans pathology model of Alzheimer's disease with the cell-free fat extract demonstrates that the cell-free fat extract can effectively prolong paralysis of Caenorhabditis elegans of Alzheimer's disease. In a cell experiment, the cell-free fat extract has the effects of promoting the proliferation of SH-SY5Y cells and improving the cell growth state, inhibiting cell agglomeration and shrinkage, and inhibiting cellular damage. The provided cell-free fat extract exhibits potential against Alzheimer's disease and can be used for preparing a pharmaceutical composition or a health product composition for preventing and / or treating Alzheimer's disease.

Description

Application of adipose decellularized active protein in the fight against Alzheimer's disease Technical Field

[0001] The present invention relates to the field of biomaterials, and in particular to application of adipose decellularized active protein in treating Alzheimer's disease. Background Art

[0002] Adipose tissue not only contains a variety of cells, such as adipocytes, adipose stem cells, and vascular endothelial cells, but also secretes a large number of growth factors related to tissue regeneration and repair. It also acts as a signaling molecule that regulates various physiological processes. Active proteins in fat have a wide range of applications in medicine, healthcare, cosmetics, and other fields. Adipose decellularized active protein (named CEFFE Pro 2.0) is an active protein extracted from decellularized human adipose tissue. The specific implementation plan is detailed in patent 202410066388.9.

[0003] Alzheimer's Disease (AD) is a progressive neurodegenerative disease. Clinically, it is characterized by comprehensive dementia manifestations such as memory dysfunction, aphasia, agnosia, visual-spatial skill impairment, executive dysfunction, and personality and behavioral changes, accounting for more than 75% of dementia patients. With the increasing aging of the population in my country, AD will become one of the major diseases affecting the health of the elderly, and the effective prevention and treatment of AD has become a major issue. The pathogenesis of AD is very complex, among which the "Aβ protein cascade hypothesis" has been the most studied. Aβ exerts neurotoxicity by inducing cell apoptosis, stimulating inflammatory cascade reactions, generating oxidative stress, acting on mitochondria, affecting energy metabolism, exacerbating Tau protein phosphorylation and NFT formation, and lowering the threshold of excitability.

[0004] AD has a complex etiology, a long course, and multiple pathogenesis stages, requiring long-term medication. Currently available anti-Alzheimer's drugs include acetylcholinesterase inhibitors (such as galantamine) and N-methyl-D-aspartate receptor (NMDA) receptor antagonists (such as memantine). However, these drugs are expensive and can easily cause side effects such as hallucinations, confusion, dizziness, headaches, and fatigue. Currently, clinically used anti-AD drugs can only alleviate patients' symptoms but cannot completely cure them.

[0005] Therefore, it is urgent to discover more effective anti-AD drugs. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for preventing and / or treating Alzheimer's disease by using adipose decellularized active protein.

[0007] In a first aspect of the present invention, there is provided a use of adipose decellularized active protein in preparing a composition for preventing and / or treating Alzheimer's disease and / or its symptoms.

[0008] In another preferred embodiment, the composition is a pharmaceutical composition and / or a health product composition.

[0009] In another preferred embodiment, in the composition, the weight fraction of the adipose decellularized active protein accounts for 1-99wt% of the total weight of the composition, for example, 5wt%, 10wt%, 20wt%, 50wt%, 70wt%, 90wt%.

[0010] In another preferred embodiment, the Alzheimer's disease is caused by factors selected from the group consisting of aging, family history, head trauma, low education level, thyroid disease, maternal childbearing age that is too high or too low, viral infection, gene mutation, or a combination thereof.

[0011] In another preferred embodiment, the Alzheimer's disease refers to Alzheimer's disease caused by Aβ deposition, Tau protein hyperphosphorylation, aging and oxidative stress, and / or neuroinflammation.

[0012] In another preferred embodiment, the symptoms of Alzheimer's disease are selected from the group consisting of: situational memory impairment, memory loss, decreased judgment, emotional indifference, time orientation disorder, limited vocabulary, aphasia, apraxia and agnosia, urinary incontinence, inability to take care of oneself in daily life, mild hemiparesis, or a combination thereof.

[0013] In another preferred embodiment, the prevention and / or treatment of Alzheimer's disease includes one or more features selected from the following group:

[0014] (i) Reduce the duration of paralysis;

[0015] (ii) promoting the proliferation of neurons and / or neural cells;

[0016] (iii) improve the activity of nerve cells;

[0017] (iv) inhibiting damage to neurons and / or nerve cells;

[0018] (v) Has neuroprotective effects.

[0019] In another preferred embodiment, the effective administration concentration of the adipose decellularization active protein is 10-500 μg / mL, preferably 20-300 μg / mL.

[0020] In another preferred embodiment, the adipose decellularized active protein does not contain cells and does not contain lipid droplets.

[0021] In another preferred embodiment, the lipid droplets are oil droplets released after the adipocytes are broken.

[0022] In another preferred embodiment, the “does not contain fat droplets” means that in the adipose decellularized biological material, the volume of oil droplets accounts for less than 1% of the total liquid, preferably less than 0.5%, and more preferably less than 0.1%.

[0023] In another preferred embodiment, the cells are selected from the group consisting of endothelial cells, adipose stem cells, macrophages, and stromal cells.

[0024] In another preferred embodiment, the “cell-free” refers to that the average number of cells in 1 ml of adipose decellularized biological material is ≤1, preferably ≤0.5, more preferably ≤0.1, or 0.

[0025] In another preferred embodiment, the adipose decellularized active protein is prepared by the following method, which comprises the following steps:

[0026] (1) Provide adipose tissue;

[0027] (2) pre-treating the adipose tissue to remove blood and swelling fluid from the adipose tissue;

[0028] (3) adding tissue lysis solution to the rinsed adipose tissue, mixing and lysing, then allowing to stand and separate, and taking the middle layer;

[0029] Optionally (4) repeat step (3) 1-3 times to combine the intermediate layers;

[0030] (5) filtering the middle layer to obtain a lysed adipose tissue fluid;

[0031] (6) performing virus inactivation on the lysed adipose tissue fluid to obtain a virus-inactivated tissue lysate; and

[0032] (7) Anion chromatography is used to separate and obtain adipose decellularized active protein.

[0033] In another preferred embodiment, in step (1), the adipose tissue is adipose tissue from which fascia has been removed.

[0034] In another preferred embodiment, in step (2), the pretreatment includes rinsing.

[0035] In another preferred embodiment, in step (2), the rinsing refers to rinsing with physiological saline or phosphate buffer.

[0036] In another preferred embodiment, the step (2) comprises: rinsing the adipose tissue with physiological saline or phosphate buffer to remove blood and swelling fluid.

[0037] In another preferred embodiment, in step (3), the tissue lysis solution system comprises: 100-200 mmol / L NaCl, 20-80 mmol / L Tris, 1-10 mmol / L EDTA-2Na, and 20-80 mmol / L NaH2PO4-Na2HPO4 buffer system.

[0038] In another preferred embodiment, in step (3), the tissue lysis solution system includes: 150 mmol / L NaCl, 50 mmol / L Tris, 5 mmol / L EDTA-2Na, and 50 mmol / L NaH2PO4-Na2HPO4 buffer system.

[0039] In another preferred embodiment, in step (3), the pH of the tissue lysis solution system is 8.5-11.5, preferably 9.5-11.0, for example 10.5.

[0040] In another preferred embodiment, in step (3), the mixing refers to homogenization using a tissue homogenizer.

[0041] In another preferred embodiment, in step (3), the homogenization is carried out at 2-10°C, preferably at 2-5°C in a water bath, for example at 4°C in a circulating water bath.

[0042] In another preferred embodiment, in step (3), the rotation speed of the homogenate is 8000 to 15000 rpm, for example, 10000 rpm.

[0043] In another preferred embodiment, in step (3), the homogenization time is 20-60 minutes, for example, 30 minutes.

[0044] In another preferred embodiment, in step (3), the static stratification refers to placing the homogenate in a chromatography refrigerator for static stratification.

[0045] In another preferred embodiment, in step (3), the intermediate layer is an intermediate solution layer, i.e., an aqueous protein solution.

[0046] In another preferred embodiment, the step (3) comprises: adding tissue lysis solution to the rinsed adipose tissue, then placing the tissue in a tissue homogenizer, homogenizing at a speed of 8000 to 15000 rpm in a circulating water bath at 2-10°C for 20-60 minutes, placing the homogenate in a chromatography refrigerator to stand and separate the layers, and taking the middle solution layer, i.e., the aqueous protein solution.

[0047] In another preferred embodiment, in step (5), the filtering refers to filtering with a filter, and preferably the pore size of the filter is 0.1-0.5 μm, such as 0.22 μm.

[0048] In another preferred embodiment, the step (7) further includes a pre-treatment step of replacing the buffer solution.

[0049] In another preferred embodiment, in step (7), the anion chromatography separation includes a primary purification by anion chromatography and a secondary purification by high-resolution anion chromatography.

[0050] In another preferred embodiment, the step (7) further includes post-processing steps of replacing the buffer solution and removing viruses by filtration.

[0051] In another preferred embodiment, the step (7) includes the following steps:

[0052] (7a) replacing the buffer of the virus-inactivated tissue lysate;

[0053] (7b) primary purification by anion chromatography;

[0054] (7c) performing ultrafiltration on the ultrafiltration membrane package to replace the buffer of the tissue lysate;

[0055] (7d) secondary purification by high-resolution anion chromatography;

[0056] (7e) performing a second ultrafiltration change using an ultrafiltration membrane package to replace the buffer of the tissue lysate;

[0057] (7f) Virus removal filtration.

[0058] In another preferred embodiment, in step (7b), the anion chromatography column filler used in the primary purification is Q sepharose.

[0059] In another preferred embodiment, in step (7b), the primary purification includes: regeneration of the chromatography column, equilibration of the chromatography column, sample loading, a first elution, a second elution, elution of the chromatography medium with an eluent, and sample collection.

[0060] In another preferred embodiment, in step (7b), the sample to be purified once is a tissue lysate after replacement of the buffer.

[0061] In another preferred embodiment, in step (7b), the flow rate of loading the sample for the primary purification is 100 to 300 cm / h.

[0062] In another preferred embodiment, in step (7b), the retention time of the sample subjected to the primary purification is more than 3 min, preferably more than 5 min.

[0063] In another preferred embodiment, in step (7b), the primary purification eluent comprises Tris and sodium chloride.

[0064] In another preferred embodiment, in step (7b), the eluent for the primary purification comprises 10-50 mmol / L Tris, 300-800 mmol / L sodium chloride, preferably 20 mmol / L Tris, 600 mmol / L sodium chloride.

[0065] In another preferred embodiment, in step (7b), the pH of the eluate of the primary purification is 8.0-11.0, preferably 9.0-10.5, for example 10.0.

[0066] In another preferred embodiment, in step (7b), the elution method of the primary purification is isocratic elution.

[0067] In another preferred embodiment, in step (7b), the sample collection for the primary purification refers to collecting the 50mAu-50mAu ultraviolet peak.

[0068] In another preferred embodiment, in step (7c), the sample obtained from the first purification is ultrafiltered once through an ultrafiltration membrane pack and the solution is replaced with 20-80 mmol / L Tris, for example, 50 mmol / L Tris.

[0069] In another preferred embodiment, in step (7d), the anion chromatography column filler used in the secondary purification is Q high performance.

[0070] In another preferred embodiment, in step (7d), the secondary purification includes: chromatography column regeneration, chromatography column equilibration, sample loading, a first elution, a second elution, elution of the chromatography medium with an eluent, and sample collection.

[0071] In another preferred embodiment, in step (7d), the secondarily purified sample is a tissue lysate after ultrafiltration through an ultrafiltration membrane pack and a one-time fluid exchange.

[0072] In another preferred embodiment, in step (7d), the flow rate of loading the secondary purified sample is 100 to 300 cm / h.

[0073] In another preferred embodiment, in step (7d), the retention time of the secondary purified sample is more than 3 minutes, preferably more than 5 minutes.

[0074] In another preferred embodiment, in step (7d), the secondary purification eluent comprises Tris and sodium chloride.

[0075] In another preferred embodiment, in step (7d), the secondary purification eluent comprises 10-50 mmol / L Tris, 300-800 mmol / L sodium chloride, preferably 20 mmol / L Tris, 600 mmol / L sodium chloride.

[0076] In another preferred embodiment, in step (7d), the pH of the secondary purification eluate is 8.0-11.0, preferably 9.0-10.5, for example 10.0.

[0077] In another preferred embodiment, in step (7d), the elution method of the secondary purification is isocratic elution.

[0078] In another preferred embodiment, in step (7d), the sample collection for secondary purification refers to collecting the 50mAu-50mAu ultraviolet peak.

[0079] In another preferred embodiment, in step (7), the ultrafiltration membrane packages used in the ultrafiltration primary liquid exchange and the ultrafiltration secondary liquid exchange are each independently Consieve UFC RC (3kDa).

[0080] In another preferred embodiment, in step (7), the ultrafiltration membrane package ultrafiltration first liquid exchange and the ultrafiltration membrane package ultrafiltration second liquid exchange each independently include the following steps: ultrafiltration membrane package endotoxin removal, ultrafiltration membrane package balancing, sample concentration, and sample liquid exchange.

[0081] In another preferred embodiment, in step (7e), the sample input by the ultrafiltration membrane package for secondary ultrafiltration and liquid exchange is an active protein obtained by secondary purification by high-resolution anion chromatography.

[0082] In another preferred embodiment, in step (7e), the ultrafiltration membrane package is ultrafiltered twice to replace the buffer solution with the formulation buffer solution.

[0083] In another preferred embodiment, in step (7), the inlet pressures of the ultrafiltration membrane package for the first ultrafiltration liquid exchange and the ultrafiltration membrane package for the second ultrafiltration liquid exchange are each independently less than 0.3 MPa, preferably 0.15 MPa.

[0084] In another preferred embodiment, in step (7e), the protein concentration of the ultrafiltration membrane bag after the second ultrafiltration liquid change is 5 mg / mL.

[0085] In another preferred embodiment, in step (7f), the virus removal filtration refers to using a virus removal nanofiltration filter to remove viruses.

[0086] In another preferred embodiment, in step (7f), the virus removal nanofiltration filter is ViruClear VF (20 nm pore size).

[0087] In another preferred embodiment, the step (7) further comprises: bottling the active protein solution obtained in step (7f) and placing it at 4°C for a long time.

[0088] In another preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient and / or carrier.

[0089] In another preferred embodiment, the health care product composition further comprises an excipient and / or carrier acceptable in health care science.

[0090] In another preferred embodiment, the dosage form of the composition is selected from the following group: solid preparation, liquid preparation or semisolid preparation.

[0091] In another preferred embodiment, the dosage form of the composition is selected from the following group: tablets, enteric-coated sustained-release preparations, injections, capsules, powders, ointments, powders, oral solutions, and suspensions.

[0092] In another preferred embodiment, the composition is administered to the subject by the following means: oral administration, transdermal injection, intravenous injection, intramuscular injection, intraperitoneal injection, or intracranial injection.

[0093] In a second aspect of the present invention, a method for preventing and / or treating Alzheimer's disease and / or its symptoms is provided, comprising the steps of administering a safe and effective amount of adipose decellularized active protein to a subject in need thereof.

[0094] In another preferred embodiment, the subject is a human or non-human mammal, such as a mouse, rat, rabbit, cat, dog, cow, sheep, monkey, etc.

[0095] In another preferred embodiment, the effective administration concentration of the adipose decellularization active protein is 10-500 μg / mL, preferably 20-300 μg / mL.

[0096] In another preferred embodiment, the adipose decellularized active protein is as described in the first aspect of the present invention.

[0097] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] Figure 1. The nematode CL4176 paralysis model confirms that CEFFE Pro 2.0 can prolong the duration of paralysis in nematodes, indicating that CEFFE Pro 2.0 has potential anti-Alzheimer's disease effects.

[0099] Figure 2. CEFFE Pro 2.0 can promote the proliferation of SH-SY5Y cells when the cell culture medium is maintained in MEM / F12.

[0100] Figure 3. The cell culture medium was changed from MEM / F12 to RPMI and incubated with CEFFE Pro 2.0. CEFFE Pro 2.0 can effectively inhibit the aggregation and shrinkage of SH-SY5Y cells, promote cell spreading growth, and improve cell survival.

[0101] Figure 4. The cell culture medium was changed from MEM / F12 to RPMI and incubated with CEFFE Pro 2.0. CEFFE Pro 2.0 can significantly promote cell proliferation.

[0102] Figure 5. CEFFE Pro 2.0 for Aβ 25-35 It has a protective effect on SH-SY5Y cell damage induced by TNF-α.

[0103] Figure 6. CEFFE Pro 2.0 has a protective effect on L-Glu-induced SH-SY5Y cell damage. DETAILED DESCRIPTION

[0104] After extensive and in-depth research, the inventors have discovered for the first time an active ingredient capable of treating Alzheimer's disease, adipose-derived activated protein. This active protein can be used to prepare a drug for the treatment of Alzheimer's disease. This adipose-derived activated protein is acellular, sterile, and virus-free, retaining a significant amount of active protein factors. Its acellular and fat-free nature makes it biosafe and non-immunogenic during allogeneic transplantation. Furthermore, fat is often directly disposed of as waste material from liposuction procedures, making it cost-effective.

[0105] In terms of treatment, this adipose decellularized active protein can effectively relieve muscle paralysis, promote the proliferation of nerve cells, improve the growth state of nerve cells, inhibit the damage of nerve cells, and has outstanding neuroprotective effects.

[0106] Therefore, the adipose decellularized active protein of the present invention has a very broad application prospect in treating Alzheimer's disease. On this basis, the present invention was completed.

[0107] the term

[0108] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0109] As used herein, the terms "comprise," "include," and "contain" are used interchangeably to include not only closed definitions but also semi-closed and open definitions. In other words, the terms include "consisting of," "consisting essentially of."

[0110] As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0111] In the present invention, the term "prevention" refers to a method of preventing the onset of a disease and / or its attendant symptoms or protecting a subject from acquiring a disease. As used herein, "prevention" also includes delaying the onset of a disease and / or its attendant symptoms and reducing the risk of a subject acquiring a disease.

[0112] The term "treatment" as used herein includes delaying and stopping the progression of the disease, or eliminating the disease, and does not require 100% inhibition, elimination, or reversal. In some embodiments, the composition or pharmaceutical composition of the present invention reduces, inhibits, and / or reverses Alzheimer's disease by, for example, at least about 10%, at least about 30%, at least about 50%, or at least about 80%, compared to the level observed in the absence of the composition or pharmaceutical composition active ingredient combination of the present invention.

[0113] Composition and administration

[0114] The compositions of the present invention include (but are not limited to): pharmaceutical compositions, health care product compositions, etc.

[0115] Typically, the adipose decellularized active protein of the present invention can be prepared into a pharmaceutical composition, such as a dosage form of tablets, capsules, powders, microgranules, solutions, lozenges, jellies, creams, spirits, suspensions, tinctures, poultices, liniments, lotions, and aerosols. The pharmaceutical composition can be prepared by commonly known preparation techniques, and suitable pharmaceutical additives can be added to the medicine.

[0116] The composition of the present invention may also include a pharmaceutically and health-care acceptable carrier. "Pharmaceutically and health-care acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compound of the present invention and with each other without significantly reducing the efficacy of the compound. Some examples of pharmaceutically and health-care acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0117] There is no particular limitation on the administration of the composition of the present invention. Representative administration methods include (but are not limited to): oral administration, parenteral administration (intravenous administration, intramuscular administration), intraperitoneal injection, local administration, and intracranial injection. The preferred administration methods are oral administration and injection administration.

[0118] The dosage form of the composition or preparation of the present invention is an oral preparation or an injection preparation. Representatively, solid dosage forms for oral administration or administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0119] Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other materials well known in the art. They may contain opacifying agents.

[0120] Liquid dosage forms for oral administration or administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0121] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and flavoring agents.

[0122] In addition to the active ingredients, suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0123] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0124] The adipose decellularized active protein of the present invention can be administered or administered alone, or in combination with other drugs for preventing and / or treating Alzheimer's disease and / or its symptoms.

[0125] When administering the composition, a safe and effective amount of the adipose decellularized active protein of the present invention is administered to a human or non-human animal (such as rats, mice, dogs, cats, cattle, chickens, ducks, etc.) in need of treatment, wherein the dosage during administration is an effective dosage that is acceptable in pharmaceuticals, foods, or health products. As used herein, the term "safe and effective amount" refers to an amount that produces a function or activity in humans and / or animals and is acceptable to humans and / or animals. One of ordinary skill in the art will appreciate that the "safe and effective amount" may vary depending on the form of the pharmaceutical composition, the route of administration, the excipients of the drug used, the severity of the disease, and the combination with other drugs. For example, for a person weighing 60 kg, the daily dosage is generally 0.1 to 1000 mg, preferably 1 to 600 mg, and more preferably 2 to 300 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician.

[0126] The main advantages of the present invention include:

[0127] (1) The active ingredient of the present invention, adipose decellularized active protein, can effectively treat Alzheimer's disease.

[0128] (2) The active ingredient of the present invention, the adipose decellularized active protein, is a cell-free component that can avoid cell-related problems in clinical applications, such as genetic stability after cell processing, cell activity and survival rate after injection, multiple-dose storage of cells, and immunogenicity of cells when allogeneic fat is used. The adipose decellularized active protein of the present invention has the advantages of higher safety and lower side effects in the prevention and treatment of Alzheimer's disease.

[0129] (3) The active ingredient of the present invention, adipose decellularized active protein, is extracted from fat, which is medical waste in liposuction surgery and is completely non-scarce. Moreover, since it is a cell-free component, it avoids immunogenicity and also has excellent therapeutic effects.

[0130] (4) The active ingredient of the present invention, the adipose decellularized active protein, is rich in multiple active factors.

[0131] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which no specific conditions are specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0132] It should be noted that CEFFE Pro 2.0 in the specific embodiment is the adipose decellularized active protein mentioned above, which is an active protein extracted from adipose tissue. The extraction method includes: collecting an adipose tissue sample, removing blood and swelling fluid in the adipose tissue, and then lysing the adipocytes in a suspended state. After filtering and inactivating viruses in the obtained lysed adipose tissue fluid, anion chromatography is used to separate and obtain the active protein in the adipose tissue.

[0133] Example 1 Extraction of active proteins from human adipose tissue

[0134] The present invention provides a method for preparing adipose decellularized active protein CEFFE Pro 2.0.

[0135] 1. Preparation of adipose tissue lysate: Obtain adipose tissue (from Shanghai Ninth People's Hospital, donated by patients undergoing liposuction with informed consent) and remove fascia. Rinse with saline or phosphate buffer to remove blood and edema. Add tissue lysis buffer (150 mmol / L NaCl, 50 mmol / L Tris, 5 mmol / L EDTA-2Na, 50 mmol / L NaH2PO4-Na2HPO4 buffer, pH 10.5) to the rinsed adipose tissue. Homogenize in a tissue homogenizer at 10,000 rpm in a circulating water bath (4°C) for 30 minutes. Place the homogenate in a chromatography freezer to allow separation. The intermediate solution layer (i.e., the aqueous protein solution) is collected and this step is repeated once. The intermediate homogenates are combined and filtered through a 0.22 μm filter to obtain adipose tissue lysate. Viruses are inactivated in the adipose tissue lysate to obtain a virus-inactivated tissue lysate, and the buffer is replaced.

[0136] 2. Purification of active protein by anion chromatography: The chromatography column filler in this step is Q sepharose, and the chromatography column is regenerated, equilibrated, sample loaded (the tissue lysate after buffer exchange is loaded onto the chromatography medium at a linear flow rate of 100-300 cm / h and a retention time ≥5 minutes), washed once, washed twice, and finally eluted with eluent (20 mmol / L Tris, 600 mmol / L sodium chloride, pH 10.0), collecting the 50 mAu-50 mAu UV peak.

[0137] 3. Further purification of the active protein by high-resolution anion chromatography: The chromatography column filler in this step is Q high performance, and the chromatography column regeneration, chromatography column balance, sample treatment and loading (the active protein eluted from the Q sepharose in the previous step is ultrafiltered through an ultrafiltration membrane and replaced with 50 mmol / L Tris, pH 10.0. The active protein solution after the buffer replacement is loaded on the chromatography medium with a linear flow rate of 100 to 300 cm / h and a retention time of ≥5 minutes), a single elution, a secondary elution, and finally, the chromatography medium is eluted with an eluent (20 mmol / L Tris, 600 mmol / L sodium chloride, pH 10.0), and the 50 mAu-50 mAu UV peak is collected.

[0138] 4. Ultrafiltration and Fluid Exchange for Preparation of Active Protein Preparation: The ultrafiltration membrane used in this step is Consieve UFC RC (3 kDa). Ultrafiltration membrane detoxification, ultrafiltration membrane balance, sample concentration, and fluid exchange are performed separately (the active protein eluted from the previous step is ultrafiltered using the Q High Performance membrane and replaced with the formulation buffer. The inlet pressure is maintained at less than 0.15 MPa until the pH and conductivity at the outflow end are consistent with those of the formulation buffer. The final active protein concentration is 5 mg / ml.

[0139] 5. Virus removal, filtration and filling: The virus removal nanofiltration filter used in this step is ViruClear VF (20 nm pore size). The active protein replaced into the formulation buffer in the previous step is filtered through the virus removal nanofiltration filter. After filtration, the active protein solution is filled into penicillin bottles, 1 ml / vial, and stored at 4°C for a long time.

[0140] Example 2: CEFFE Pro 2.0 can effectively delay muscle paralysis in AD Caenorhabditis elegans

[0141] 1. Materials and Reagents

[0142] 1.1 Caenorhabditis elegans: Caenorhabditis elegans strain CL4176 (Bristol strain) was purchased from the Caenorhabditis Genetics Center (CGC) at the University of Minnesota. The nematode has a well-defined and genetically tractable nervous system, which provides an effective model for exploring fundamental mechanistic pathways that may underlie complex human neurological diseases.

[0143] 1.2 Test sample: CEFFE Pro 2.0, stock solution concentration is 5 mg / ml, water-soluble.

[0144] 1.3 Nematode culture conditions: Nematodes were cultured on NGM (nematode growth medium) solid culture medium with OP50 at a temperature of 16°C and a humidity of 40-60%.

[0145] 2. Experimental methods

[0146] 2.1 Plate Preparation: Dilute CEFFE Pro 2.0 with ddH2O to the desired experimental concentration. Mix with 1 ml of E. coli OP50 pellet. Pipette 400 μL onto the surface of NGM solid medium (6 cm Petri dish) and allow to dry before use. Caffeine is used as a positive control at a concentration of 6.27 mM.

[0147] 2.2 Experimental process: After synchronizing the CL4176 strain on the control plate and the drug plate, the culture was cultured at 16°C for 48 hours, then induced at 23°C, and the cells were counted after 40 hours.

[0148] 2.3 Statistical Methods: The Log-rank (Mantel-Cox) test was used to compare differences in paralysis severity. Data were analyzed using GraphPad Prism v5.0. “*” represents p < 0.05, “**” represents p < 0.01, and “***” represents p < 0.001.

[0149] Example 3: CEFFE Pro 2.0 can effectively promote the proliferation of SH-SY5Y cells

[0150] 1. Cell Information: SH-SY5Y cells (human neuroblastoma cells) were purchased from Wuhan Punosai Life Science Co., Ltd. and cultured in MEM / F12 medium containing 15% fetal bovine serum and 1% double-antibody.

[0151] SH-SY5Y cells, derived from SK-N-SH cells, possess a functionally mature neuronal phenotype. Their morphology and physiological and biochemical functions are similar to those of normal neurons, making them suitable alternatives to primary neuronal cultures. This cell line was established in 1970 from a four-year-old girl with metastatic myeloid neuroblastoma. Since its initial discovery, the SH-SY5Y cell line has been widely used in in vitro neuroscience research, such as investigating the molecular mechanisms of AD, neurotoxicity, and neurodegeneration.

[0152] 2. Experimental methods

[0153] 2.1 Cell plating: After cell digestion, prepare a cell suspension, inoculate 5000 cells / well into a 96-well plate, and culture in a 37°C incubator for 24 hours.

[0154] 2.2 Drug Treatment: The next day, different concentrations of CEFFE Pro 2.0 working solution were prepared using MEM / F12 medium (the same medium as for routine culture). The original medium in the air was aspirated and replaced with freshly prepared culture medium containing CEFFE Pro 2.0. The concentrations of CEFFE Pro 2.0 were set as follows: 0 (blank group), 0.5, 1, 2.5, 5, 10, 25, 50, 100, and 200 μg / mL. Three replicates were set for each group, and culture was continued for 72 hours.

[0155] 2.3 CCK8 detection: After 72 hours of drug incubation, the 96-well plate was removed and each well was replaced with a working solution containing 10% CCK8 solution. The plates were incubated in a 37°C incubator for 1.5 hours and then detected using a microplate reader at dual wavelengths of 450 and 650 nm.

[0156] Example 4: CEFFE Pro 2.0 can alleviate the shrinkage and aggregation of SH-SY5Y cells caused by a sudden change of culture medium.

[0157] 1. Cell information: Same as Example 3

[0158] 2. Experimental methods

[0159] 2.1 Cell plating: After cell digestion, prepare a cell suspension, inoculate 5000 cells / well into a 96-well plate, and culture in a 37°C incubator for 24 hours.

[0160] 2.2 Drug Treatment: The next day, different concentrations of CEFFE Pro 2.0 working solution were prepared using RPMI medium (a medium different from conventional culture medium). The original MEM / F12 medium in the air was aspirated and replaced with the newly prepared culture medium containing CEFFE Pro 2.0. The concentrations of CEFFE Pro 2.0 were set as follows: 0 (blank group), 0.5, 1, 2.5, 5, 10, 25, 50, 100, and 200 μg / mL. Three replicates were set for each group, and the culture was continued for 72 hours.

[0161] 2.3 Observation and photography under a microscope: After 72 hours of drug incubation, remove the 96-well plate, observe the cell status under a microscope, and select typical fields of view for photography.

[0162] Example 5: CEFFE Pro 2.0 significantly promotes the proliferation of SH-SY5Y cells in RPMI medium

[0163] 1. Cell information: Same as Example 3

[0164] 2. Experimental methods

[0165] 2.1 Cell plating: After cell digestion, prepare a cell suspension, inoculate 5000 cells / well into a 96-well plate, and culture in a 37°C incubator for 24 hours.

[0166] 2.2 Drug Treatment: The next day, different concentrations of CEFFE Pro 2.0 working solution were prepared using RPMI medium (a medium different from that used for conventional culture). The original MEM / F12 medium in the wells was aspirated and replaced with the newly prepared culture medium containing CEFFE Pro 2.0. The concentrations of CEFFE Pro 2.0 were set as follows: 0 (blank group), 0.5, 1, 2.5, 5, 10, 25, 50, 100, and 200 μg / mL. Three replicates were set for each group, and the cells were cultured for 72 hours.

[0167] 2.3 CCK8 detection: After 72 hours of drug incubation, the 96-well plate was removed and each well was replaced with a working solution containing 10% CCK8 solution. The plates were placed in a 37°C incubator and incubated for 1.5 hours. The plates were then detected using a microplate reader at dual wavelengths of 450 and 650 nm.

[0168] Example 6: Effect of CEFFE Pro 2.0 on Aβ 25-35 Induced SH-SY5Y neuronal cell damage has a protective effect

[0169] 1. Cell information: Same as Example 3

[0170] 2. Experimental methods

[0171] 2.1 Aβ 25-35 Preparation and aging treatment: Aβ is decomposed from amyloid precursor protein (APP). 25-35 Fragments have certain neurotoxicity in cell culture. Therefore, in vitro experiments often use Aβ 25-35 Construct AD cell model. 1 mg of Aβ 25-35 The powder was dissolved in 4.717 ml of ddH2O to a concentration of 200 uM. The solution was sterilized by filtration using a 0.22 uM filter. The solution was sealed and placed in a 37°C incubator for aging. After one week, the solution was removed and aliquoted and stored at -20°C.

[0172] 2.2 Aβ 25-35 Establishment of SH-SY5Y cell injury model: Take cells in logarithmic growth phase, digest them and prepare cell suspension, inoculate 8000 cells / well in 96-well plates. After 48 hours, discard the culture medium and add Aβ at a final concentration of 80uM. 25-35 Solution, the control group was added with an equal amount of serum-free medium to establish Aβ 25- 35SH-SY5Y cell injury model.

[0173] 2.3 Treatment: Aβ 25-35 The SH-SY5Y cells were damaged and treated with drugs at the same time. The drug groups were added with CEFFE Pro 2.0 at final concentrations of 45, 90, and 180 μM, respectively. Each group had 3 replicates and was cultured for 48 hours.

[0174] 2.4 CCK8 assay: Each well was replaced with a working solution containing 10% CCK8 solution, incubated in a 37°C incubator for 1.5 hours, and then detected using a microplate reader at dual wavelengths of 450 and 650 nm.

[0175] Example 7: CEFFE Pro 2.0 protects SH-SY5Y cells from L-glutamate (L-Glu)-induced damage

[0176] 1. Cell information: Same as Example 3

[0177] 2. Experimental methods

[0178] 2.1 L-Glu Preparation: Excitotoxic damage caused by L-Glu accumulation in the extracellular fluid is the initiating factor of neuronal death. Using the glutamate injury model for neuroprotective drug screening and mechanism of action research has become an effective method for discovering potential pharmacological drugs. Weigh 20 mg of L-Glu powder and dissolve it in 6.8 ml of FBS-free culture medium. Sterilize by filtering through a 0.22 μm filter. Aliquot and store in a -20°C refrigerator.

[0179] 2.2 Establishment of an L-Glu-Induced SH-SY5Y Cell Injury Model: Logarithmically growing cells were obtained, digested, and prepared into a cell suspension. 8,000 cells were seeded per well in a 96-well plate. After 48 hours, the culture medium was discarded and an L-Glu solution with a final concentration of 10 mM was added. The control group received an equal volume of serum-free culture medium to establish an L-Glu-induced SH-SY5Y cell injury model.

[0180] 2.3 Drug treatment: Drug treatment was performed at the same time as the L-Glu-induced SH-SY5Y cell injury model was established. CEFFE Pro 2.0 was added to the drug treatment groups at final concentrations of 45, 90, and 180 μM, respectively. Each group had 3 replicates and was cultured for 48 hours.

[0181] 2.4 CCK8 assay: Each well was replaced with a working solution containing 10% CCK8 solution, incubated in a 37°C incubator for 1.5 hours, and then detected using a microplate reader at dual wavelengths of 450 and 650 nm.

[0182] Experiments were conducted according to the above scheme. The experimental results obtained in Example 2 showed that CEFFE Pro 2.0 can alleviate muscle paralysis in Caenorhabditis elegans (nematode strain CL4176), thereby prolonging the duration of paralysis. As shown in Figure 1, 20ug / ml of CEFFE Pro 2.0 had the strongest anti-paralysis effect, indicating that CEFFE Pro 2.0 has potential anti-Alzheimer's disease effects.

[0183] Example 3 further confirmed through cell experiments that 100ug / ml and 200ug / ml of CEFFE Pro 2.0 can effectively increase the proliferation activity of SH-SY5Y cells, indicating that CEFFE Pro 2.0 has potential neurotrophic activity and the effect of promoting the growth vitality of nerve cells.

[0184] Example 4: Sudden replacement of the culture medium (from MEM / F12 medium to RPMI medium) induced SH-SY5Y cell damage, leading to cell shrinkage, agglomeration, and slowed growth. This may be related to the inability of cells to adapt to changes in nutrients. Cells treated with CEFFE Pro 2.0 were able to effectively resist cell damage and deterioration caused by the culture medium replacement. As shown in Figure 3, images of each group of cells were taken under a microscope. CEFFE Pro 2.0 at a concentration of 2.5 μg / ml was able to effectively adjust the cell state, reduce cell agglomeration, and promote cell expansion from clumps to adherent growth. When the concentration reached 25 μg / ml, all cells had expanded and grown, cell agglomeration had completely disappeared, and cell growth was significantly improved. This effect was significantly enhanced as the concentration of CEFFE Pro 2.0 increased. CEFFE Pro 2.0 showed a concentration gradient-dependent regulation of the growth state of SH-SY5Y cells.

[0185] In Example 5, the cells photographed in Example 4 were subjected to a proliferation activity assay using a CCK8 assay. The results, as shown in Figure 4, show that CEFFE Pro 2.0 can significantly enhance the proliferation of SH-SY5Y cells starting at 10 μg / ml. Furthermore, as the concentration of CEFFE Pro 2.0 increases, the proliferative activity becomes stronger, showing a concentration gradient dependence.

[0186] In Example 6, Aβ was selected 25-35 As an inducing drug, CEFFE Pro 2.0 was used to act on SY5Y cells to construct an in vitro cell injury model to detect the effect of CEFFE Pro 2.0 on Aβ 25-35 The inhibitory effect of toxicity on CEFFE Pro 2.0 was further demonstrated, which further clarified the neuroprotective function of CEFFE Pro 2.0. The experimental data showed that, as shown in Figure 5, Aβ 25-35At a concentration of 40uM, it has a certain inhibitory effect on cell proliferation activity and can induce cell apoptosis. After adding CEFFE Pro 2.0 treatment, the cell viability is significantly improved. The higher the drug concentration, the better its resistance to Aβ 25- 35 Therefore, it is believed that CEFFE Pro 2.0 has an outstanding neuroprotective effect.

[0187] In Example 7, L-Glu was selected as an inducing drug. It has neurotoxicity and can cause neuronal damage by binding to its nerve cells, thereby exerting a neurotoxic effect. L-Glu was acted on SH-SY5Y cells to construct another in vitro cell damage model to detect the inhibitory effect of CEFFE Pro 2.0 on L-Glu toxicity, thereby illustrating the neuroprotective function of CEFFE Pro 2.0. Experimental data show that, as shown in Figure 6, L-Glu has obvious toxicity to cells at a concentration of 10mM, significantly reducing cell proliferation ability, and after adding CEFFE Pro 2.0 treatment, the viability of the cells is significantly improved, and the higher the drug concentration, the stronger its ability to resist the cell damage toxicity caused by L-Glu. Therefore, it is believed that CEFFE Pro2.0 has an outstanding neuroprotective effect.

[0188] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. Use of adipose decellularized active protein in the preparation of a composition for preventing and / or treating Alzheimer's disease and / or its symptoms.

2. The use according to claim 1, characterized in that The Alzheimer's disease is Alzheimer's disease caused by a factor selected from the group consisting of aging, family history, head trauma, low education level, thyroid disease, maternal childbearing age, viral infection, gene mutation, or a combination thereof.

3. The use according to claim 1, characterized in that The Alzheimer's disease refers to Alzheimer's disease caused by Aβ deposition, Tau protein hyperphosphorylation, aging and oxidative stress, and / or neuroinflammation.

4. The use according to claim 1, wherein The prevention and / or treatment of Alzheimer's disease comprises one or more features selected from the following group: (i) Reduce the duration of paralysis; (ii) promoting the proliferation of neurons and / or neural cells; (iii) improve the activity of nerve cells; (iv) inhibiting damage to neurons and / or nerve cells; (v) Has neuroprotective effects.

5. The use according to claim 1, characterized in that The adipose decellularized active protein does not contain cells and fat droplets.

6. The use according to claim 6, characterized in that The term "free of fat droplets" means that the volume of oil droplets in the adipose decellularized biological material accounts for less than 1% of the total liquid, preferably less than 0.5%, and more preferably less than 0.1%; The term “containing no cells” means that the average number of cells in 1 ml of adipose decellularized biological material is ≤1, preferably ≤0.5, more preferably ≤0.1, or 0.

7. The use according to claim 1, characterized in that The composition is a pharmaceutical composition or a health care product composition.

8. The use according to claim 1, characterized in that The dosage form of the composition is selected from the following group: tablets, enteric-coated sustained-release preparations, injections, capsules, powders, ointments, powders, oral solutions, and suspensions.

9. The use according to claim 1, characterized in that The adipose decellularized active protein is prepared by the following method, which comprises the following steps: (1) Provide adipose tissue; (2) pre-treating the adipose tissue to remove blood and swelling fluid from the adipose tissue; (3) adding tissue lysis solution to the rinsed adipose tissue, mixing and lysing, then allowing to stand and separate, and taking the middle layer; Optionally (4) repeat step (3) 1-3 times to combine the intermediate layers; (5) filtering the middle layer to obtain a lysed adipose tissue fluid; (6) performing virus inactivation on the lysed adipose tissue fluid to obtain a virus-inactivated tissue lysate; and (7) Anion chromatography is used to separate and obtain adipose decellularized active protein.

10. The use according to claim 9, characterized in that The anion chromatography separation includes a primary purification by anion chromatography and a secondary purification by high-resolution anion chromatography.

Images