Application of Fatty Acellular Active Protein in Anti-Alzheimer's Disease

Abstract

The present invention discloses the use of a cell-free adipose-derived active protein (CEFFE Pro 2.0) in the preparation of a pharmaceutical composition for preventing and / or treating Alzheimer's disease. The present invention uses the cell-free adipose-derived active protein to treat a pathological model of Alzheimer's disease in Caenorhabditis elegans, and the results show that the cell-free adipose-derived active protein can effectively prolong the paralysis time of Caenorhabditis elegans with Alzheimer's disease. In cell experiments, the cell-free adipose-derived active protein has the effects of promoting the proliferation of SH-SY5Y cells, improving the cell growth state, inhibiting cell aggregation and shrinkage, and inhibiting cell damage. The cell-free adipose-derived active protein provided by the present invention has the potential for anti-Alzheimer's disease and can be used in the preparation of a pharmaceutical composition for preventing and / or treating Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to the field of biological materials, and particularly to the application of adipose-derived acellular active protein in the treatment of Alzheimer's disease. Background Art

[0002] Adipose tissue not only contains various cells, such as adipocytes, adipose stem cells, vascular endothelial cells, etc., but also secretes a large number of growth factors related to tissue regeneration and repair. At the same time, it can also participate in the regulation of various physiological processes as signal molecules. The active proteins in adipose tissue have wide application values in the fields of medicine, healthcare, cosmetics, etc. Adipose-derived acellular active protein (named CEFFE Pro 2.0) is an active protein extracted from human adipose tissue after decellularization. The specific implementation is described in detail in Patent 202410066388.9.

[0003] Alzheimer's disease (AD) is a progressive neurodegenerative disease. Clinically, it is mainly 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 aggravation of the aging degree of the population in our country, AD will become one of the main diseases affecting the health of the elderly. Effective prevention and treatment of AD have become a major issue. The pathogenesis of AD is very complex, and the "Aβ protein cascade hypothesis" has been studied the most. Aβ exerts neurotoxicity through inducing apoptosis, triggering inflammatory cascades, generating oxidative stress, acting on mitochondria, affecting energy metabolism, exacerbating the phosphorylation of Tau protein and the formation of NFT, and reducing the excitatory threshold and other pathways.

[0004] AD has complex etiologies, a long course of disease, and many pathogenesis links, and requires long-term medication. Currently, the anti-Alzheimer's drugs on the market are acetylcholinesterase inhibitors (such as galantamine) and N-methyl-D-aspartic acid receptor (NMDA receptor) antagonists (such as memantine). However, the above drugs are expensive and prone to side effects such as hallucinations, confusion, dizziness, headache, and fatigue after taking the medicine. Currently, the anti-AD drugs used clinically can only relieve the patient's condition and cannot be completely cured.

[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 the use of adipose-derived acellular active protein for the prevention and / or treatment of Alzheimer's disease.

[0007] In a first aspect of the present invention, there is provided the use of a decellularized adipose-derived active protein in the preparation of 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.

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

[0010] In another preferred embodiment, the Alzheimer's disease is Alzheimer's disease caused by factors selected from the following group: aging, family history, head trauma, low educational level, thyroid disease, too high or too low maternal age at childbearing, 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, hyperphosphorylation of Tau protein, aging and oxidative stress, and / or neuroinflammation.

[0012] In another preferred embodiment, the symptoms of the Alzheimer's disease are selected from the following group: episodic memory impairment, memory decline, decreased judgment ability, apathy, time disorientation, few speech words, aphasia, apraxia and agnosia, urinary incontinence, inability to take care of daily life, hemiparesis, or a combination thereof.

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

[0014] (i) Slowing down the time of paralysis;

[0015] (ii) Promoting the proliferation of neurons and / or nerve cells;

[0016] (iii) Improving the viability of nerve cells;

[0017] (iv) Inhibiting the damage of neurons and / or nerve cells;

[0018] (v) Having a neuroprotective effect.

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

[0020] In another preferred embodiment, the decellularized adipose-derived 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 fragmentation of adipocytes.

[0022] In another preferred example, the statement of "not containing lipid droplets" means that in the fat acellular biomaterial, the percentage of the volume of oil droplets in the total liquid is less than 1%, preferably less than 0.5%, and more preferably less than 0.1%.

[0023] In another preferred example, the cells are selected from the group consisting of: endothelial cells, adipose stem cells, macrophage blood cells, and stromal cells.

[0024] In another preferred example, the statement of "not containing cells" means that the average number of cells in 1 ml of the fat acellular biomaterial is ≤1, preferably ≤0.5, more preferably ≤0.1, or 0.

[0025] In another preferred example, the fat acellular active protein is prepared by the following method, which includes the following steps:

[0026] (1) Provide adipose tissue;

[0027] (2) Pretreat the adipose tissue to remove the blood and swelling fluid in the adipose tissue;

[0028] (3) Add tissue lysate to the rinsed adipose tissue and mix for lysis, then let it stand for stratification and take the middle layer;

[0029] Optionally (4) Repeat step (3) 1 - 3 times and combine the middle layers;

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

[0031] (6) Inactivate the virus in the lysed adipose tissue fluid to obtain virus-inactivated tissue lysate; and

[0032] (7) Use anion chromatography for separation to obtain the fat acellular active protein.

[0033] In another preferred example, in step (1), the adipose tissue is adipose tissue with fascia removed.

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

[0035] In another preferred example, in step (2), the rinsing means rinsing with physiological saline or phosphate buffer solution.

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

[0037] In another preferred example, in step (3), the tissue lysate system comprises: 100 - 200 mmol / L NaCl, 20 - 80 mmol / L Tris, 1 - 10 mmol / L EDTA-2Na, 20 - 80 mmol / L NaH 2 PO 4 -Na 2 HPO 4 buffer system.

[0038] In another preferred example, in step (3), the tissue lysate system comprises: 150 mmol / L NaCl, 50 mmol / L Tris, 5 mmol / L EDTA-2Na, 50 mmol / L NaH 2 PO 4 -Na 2 HPO 4 buffer system.

[0039] In another preferred example, in step (3), the pH of the tissue lysate system is 8.5 - 11.5, preferably 9.5 - 11.0, such as 10.5.

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

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

[0042] In another preferred example, in step (3), the rotation speed of the homogenization is 8000 - 15000 revolutions per minute, such as 10000 revolutions per minute.

[0043] In another preferred example, in step (3), the time of the homogenization is 20 - 60 minutes, such as 30 min.

[0044] In another preferred example, in step (3), the static layering refers to placing the homogenized homogenate in a chromatography cold cabinet for static layering.

[0045] In another preferred example, in step (3), the middle layer is the middle solution layer, that is, the aqueous protein solution.

[0046] In another preferred example, step (3) includes: adding a tissue lysate to the rinsed adipose tissue, then placing it in a tissue homogenizer, homogenizing at a rotation speed of 8000 - 15000 revolutions per minute for 20 - 60 minutes under a 2 - 10 °C circulating water bath, placing the homogenate in a chromatography cold cabinet for static layering, and taking the middle solution layer, that is, the aqueous protein solution.

[0047] In another preferred example, in step (5), the filtration refers to filtration using a filter. Preferably, the pore size of the filter is 0.1 - 0.5 μm, such as 0.22 μm.

[0048] In another preferred example, step (7) further includes a pretreatment step for replacing the buffer solution.

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

[0050] In another preferred example, step (7) further includes: post - treatment steps of replacing the buffer solution and virus - removal filtration.

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

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

[0053] (7b) One - time purification by anion chromatography;

[0054] (7c) Ultrafiltration membrane package for one - time buffer replacement by ultrafiltration to replace the buffer solution of the tissue lysate;

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

[0056] (7e) Ultrafiltration membrane package for second - time buffer replacement by ultrafiltration to replace the buffer solution of the tissue lysate;

[0057] (7f) Virus - removal filtration.

[0058] In another preferred example, in step (7b), the packing material of the anion chromatography column used for the one - time purification is Qsepharose.

[0059] In another preferred example, in step (7b), the one - time purification includes the steps of: column regeneration, column equilibration, sample loading, first elution, second elution, eluting the chromatography medium with the eluent, and collecting the sample.

[0060] In another preferred example, in step (7b), the sample for the one - time purification is the tissue lysate after replacing the buffer solution.

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

[0062] In another preferred example, in step (7b), the retention time of sample loading for the one - time purification exceeds 3 min, preferably exceeds 5 min.

[0063] In another preferred example, in step (7b), the eluate of the primary purification includes Tris and sodium chloride.

[0064] In another preferred example, in step (7b), the eluate of the primary purification includes 10 - 50 mmol / L Tris and 300 - 800 mmol / L sodium chloride, preferably 20 mmol / L Tris and 600 mmol / L sodium chloride.

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

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

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

[0068] In another preferred example, in step (7c), the sample obtained from the primary purification is ultrafiltered through an ultrafiltration membrane cassette for one buffer exchange and then replaced with 20 - 80 mmol / L Tris, such as 50 mmol / L Tris.

[0069] In another preferred example, in step (7d), the packing material of the anion chromatography column used for the secondary purification is Q high performance.

[0070] In another preferred example, in step (7d), the secondary purification includes the steps of column regeneration, column equilibration, sample loading, primary washing, secondary washing, eluting the chromatography medium with eluate, and collecting the sample.

[0071] In another preferred example, in step (7d), the sample for the secondary purification is the tissue lysate after one buffer exchange by ultrafiltration through an ultrafiltration membrane cassette.

[0072] In another preferred example, in step (7d), the flow rate of sample loading for the secondary purification is 100 - 300 cm / h.

[0073] In another preferred example, in step (7d), the retention time of sample loading for the secondary purification exceeds 3 min, preferably exceeds 5 min.

[0074] In another preferred example, in step (7d), the eluate of the secondary purification includes Tris and sodium chloride.

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

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

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

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

[0079] In another preferred example, in step (7), the ultrafiltration membrane cartridges used in the first ultrafiltration membrane cartridge liquid change and the second ultrafiltration membrane cartridge liquid change are each independently Consieve UFC RC (3 kDa).

[0080] In another preferred example, in step (7), the first ultrafiltration membrane cartridge liquid change and the second ultrafiltration membrane cartridge liquid change each independently include the following steps: removing endotoxin from the ultrafiltration membrane cartridge, balancing the ultrafiltration membrane cartridge, concentrating the sample, and changing the sample liquid.

[0081] In another preferred example, in step (7e), the sample input for the second ultrafiltration membrane cartridge liquid change is the active protein obtained by secondary purification of high-resolution anion chromatography.

[0082] In another preferred example, in step (7e), the second ultrafiltration membrane cartridge liquid change replaces the buffer with the formulation buffer.

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

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

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

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

[0087] In another preferred embodiment, step (7) further comprises: filling the active protein solution obtained in step (7f) and storing 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 dosage form of the composition is selected from the group consisting of: solid preparations, liquid preparations or semi-solid preparations.

[0090] In another preferred embodiment, the dosage form of the composition is selected from the group consisting of: tablets, enteric-coated sustained-release preparations, injections, capsules, powders, ointments, powders, oral liquids, suspensions.

[0091] In another preferred embodiment, the composition is administered to the subject by the following means: oral administration, percutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intracranial injection.

[0092] In a second aspect of the present invention, there is provided a method for preventing and / or treating Alzheimer's disease and / or its symptoms, comprising the step of: administering a safe and effective amount of fat acellular active protein to a subject in need thereof.

[0093] 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.

[0094] In another preferred embodiment, the effective administration concentration of the fat acellular active protein is 10 - 500 μg / mL, preferably 20 - 300 μg / mL.

[0095] In another preferred embodiment, the fat acellular active protein is as described in the first aspect of the present invention.

[0096] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Figure 1 The nematode CL4176 paralysis model confirmed that CEFFE Pro 2.0 can prolong the paralysis time of nematodes, indicating that CEFFE Pro 2.0 has a potential effect against Alzheimer's disease.

[0098] Figure 2 By keeping the cell culture medium as MEM / F12, CEFFE Pro 2.0 can promote the proliferation of SH-SY5Y cells.

[0099] Figure 3, The cell culture medium was changed from MEM / F12 to RPMI for culturing, and CEFFE Pro 2.0 was added for incubation. CEFFE Pro 2.0 can effectively inhibit the aggregation and shrinkage of SH-SY5Y cells, promote cell spreading growth, and improve the cell survival state.

[0100] Figure 4 , The cell culture medium was changed from MEM / F12 to RPMI for culturing, and CEFFE Pro 2.0 was added for incubation. CEFFE Pro 2.0 can significantly promote the proliferation ability of cells.

[0101] Figure 5 , CEFFE Pro 2.0 has a protective effect on Aβ 25-35 -induced SH-SY5Y cell damage.

[0102] Figure 6 , CEFFE Pro 2.0 has a protective effect on L-Glu-induced SH-SY5Y cell damage. Detailed implementation mode

[0103] After extensive and in-depth research, the present inventor has first discovered an active ingredient capable of treating Alzheimer's disease, named adipose acellular active protein, which can be used to prepare drugs for treating Alzheimer's disease. The adipose acellular active protein itself is cell-free, sterile, and virus-free, and retains a large number of active protein factors. However, due to its advantages of being cell-free and fat-free, its biological safety is excellent, and it has no immunogenicity during allogeneic transplantation. Moreover, fat is often directly treated as waste from liposuction surgery, so the cost is low.

[0104] In terms of treatment, the adipose acellular active protein can effectively relieve muscle paralysis, promote the proliferation of nerve cells, improve the growth state of nerve cells, and inhibit nerve cell damage, showing prominent neuroprotective effects.

[0105] Therefore, the adipose acellular active protein of the present invention has very broad application prospects for treating Alzheimer's disease. Based on this, the present invention has been completed.

[0106] Terms

[0107] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0108] As used herein, the terms "comprising", "including", and "containing" can be used interchangeably, including not only closed definitions but also semi-closed and open definitions. In other words, the said terms include "consisting of" and "consisting essentially of".

[0109] As used herein, when referring to a specifically recited numerical value, the term "about" means that the value can 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.).

[0110] In the present invention, the term "prevent" refers to a method of preventing the onset of a disease and / or its attendant symptoms or protecting a subject from acquiring a disease. "Prevent" as used herein also includes delaying the onset of a disease and / or its attendant symptoms and reducing the risk of a subject contracting the disease.

[0111] "Treatment" as described in the present invention includes retarding and terminating the progression of a disease, or eliminating the disease, and does not require 100% inhibition, eradication, and reversal. In some embodiments, the compositions or pharmaceutical compositions described in the present invention alleviate, inhibit, and / or reverse 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 levels observed in the absence of the active ingredient of the compositions or pharmaceutical compositions described in the present invention.

[0112] Compositions and Administration

[0113] The compositions described in the present invention include (but are not limited to): pharmaceutical compositions, etc.

[0114] Typically, the adipose-derived acellular active protein of the present invention can be formulated into pharmaceutical compositions, such as dosage forms like tablets, capsules, powders, microparticles, solutions, lozenges, gels, cream formulations, spirits, suspensions, tinctures, poultices, liniments, lotions, and aerosols. The pharmaceutical compositions can be prepared by commonly known preparation techniques, and suitable pharmaceutical additives can be added to the drugs.

[0115] The compositions of the present invention may also include a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatible" herein means that the components in the composition can be admixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers are cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, 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 ), wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0116] There is no particular limitation on the mode of administration of the composition of the present invention. Representative modes of administration include (but are not limited to): oral, parenteral (intravenous, intramuscular), intraperitoneal injection, topical administration, intracranial injection. Preferred modes of administration are oral administration and injection administration.

[0117] 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 drug delivery 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 dibasic calcium phosphate, or is mixed with the following components: (a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, such as hydroxypropylmethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants, such as glycerol; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin wax; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also contain buffering agents.

[0118] Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and casings, such as enteric coatings and other materials well known in the art. They may contain opacifying agents.

[0119] Liquid dosage forms for oral administration or drug delivery include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, the liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers, and emulsifying agents, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3 - butanediol, dimethylformamide, and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0120] In addition to these inert diluents, the composition may also contain adjuvants, such as wetting agents, emulsifying agents, and suspending agents, sweetening agents, flavoring agents, and fragrances.

[0121] In addition to the active ingredient, the suspension may contain suspending agents, such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, and agar, or mixtures of these substances.

[0122] Compositions for parenteral injection may comprise a physiologically acceptable sterile aqueous or non-aqueous solution, dispersion, suspension or emulsion, and a sterile powder for reconstitution into a sterile injectable solution or dispersion. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0123] The acellular adipose-derived active protein of the present invention can be administered alone or in combination with other drugs for preventing and / or treating Alzheimer's disease and / or its symptoms.

[0124] When administering the composition, a safe and effective amount of the acellular adipose-derived active protein of the present invention is administered to a human or non-human animal in need of treatment (such as rats, mice, dogs, cats, cows, chickens, ducks, etc.), and the dosage during administration is an effective dosage considered pharmaceutically acceptable. 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. Those of ordinary skill in the art should understand that the "safe and effective amount" may vary depending on factors such as the form of the pharmaceutical composition, the route of administration, the excipients used in the drug, the severity of the disease, and the combination with other drugs. For example, for a person weighing 60 kg, the daily dosage is usually 0.1 - 1000 mg, preferably 1 - 600 mg, more preferably 2 - 300 mg. Of course, the specific dosage should also consider factors such as the route of administration and the health status of the patient, which are within the scope of the skills of a skilled physician.

[0125] The main advantages of the present invention include:

[0126] (1) The active ingredient, acellular adipose-derived active protein of the present invention can effectively treat Alzheimer's disease.

[0127] (2) The active ingredient, acellular adipose-derived active protein of the present invention is a cell-free component, which can avoid cell-related problems in clinical applications, such as genetic stability after cell processing, cell viability and survival rate after injection, storage of cells for multiple administrations, and immunogenicity of cells when using allogeneic adipose tissue. The acellular adipose-derived active protein described in the present invention has the advantages of high safety and low side effects in the prevention and treatment of Alzheimer's disease.

[0128] (3) The active ingredient, acellular adipose-derived active protein of the present invention is extracted from adipose tissue, which is a 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.

[0129] (4) The active ingredient, acellular adipose-derived active protein of the present invention is rich in a variety of active factors.

[0130] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0131] It should be particularly noted that CEFFE Pro 2.0 in the specific embodiment is the adipose acellular active protein mentioned above, which is an active protein extracted from adipose tissue. The extraction method includes: collecting adipose tissue samples, removing blood and swelling fluid from the adipose tissue, and then lysing adipocytes in a suspended state. The obtained lysed adipose tissue fluid is filtered and inactivated for viruses, and then the active protein in the adipose tissue is obtained by anion chromatography separation.

[0132] Example 1 Extraction of Active Protein from Human Adipose Tissue

[0133] The present invention provides a preparation method of adipose acellular active protein CEFFE Pro 2.0.

[0134] 1. Preparation of adipose tissue lysate: Take adipose (from Shanghai Ninth People's Hospital, donated by liposuction patients with patient informed consent) and remove fascia, and rinse with physiological saline or phosphate buffer to remove blood and swelling fluid; add tissue lysate (150 mmol / L NaCl, 50 mmol / L Tris, 5 mmol / L EDTA-2Na, 50 mmol / L NaH2PO4-Na2HPO4 buffer system, pH 10.5) to the rinsed adipose tissue, then place it in a tissue homogenizer and homogenize at a speed of 10,000 revolutions per minute in a circulating water bath (4 °C) for 30 minutes. The homogenate is placed in a chromatography cold cabinet to stand and separate layers, and the middle solution layer, that is, the aqueous phase protein solution, is taken. Repeat this step once, combine the middle layer homogenates, and filter with a 0.22 μm filter to obtain adipose tissue lysate. Inactivate the viruses in the adipose tissue lysate to obtain virus-inactivated tissue lysate and replace the buffer.

[0135] 2. Anion chromatography purification of active protein: The packing material of the chromatography column in this step is Q sepharose. The chromatography column is regenerated, equilibrated, the sample is loaded (the tissue lysate after replacing the buffer is loaded onto the chromatography medium, linear flow rate 100 - 300 cm / h, retention time ≥ 5 minutes), washed once, washed a second time, 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 ultraviolet peak is collected.

[0136] 3. Further purification of the active protein by high-resolution anion chromatography: The packing material of the chromatography column in this step is Q high performance. The chromatography column is regenerated, equilibrated, the sample is processed and loaded (the active protein eluted by Q sepharose in the previous step is ultrafiltered and buffer exchanged using an ultrafiltration membrane cassette, replaced with 50 mmol / L Tris, pH 10.0, and the active protein solution after replacing the buffer is loaded onto the chromatography medium, with a linear flow rate of 100 - 300 cm / h and a retention time of ≥5 minutes), washed once, washed twice, 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.

[0137] 4. Preparation of the active protein preparation by ultrafiltration and buffer exchange: The ultrafiltration membrane cassette in this step is Consieve UFC RC (3 kDa). The ultrafiltration membrane cassette is endotoxin-removed, equilibrated, the sample is concentrated and buffer exchanged (the active protein eluted by Q High Performance in the previous step is ultrafiltered and buffer exchanged using an ultrafiltration membrane cassette, replaced with the formulation buffer, maintaining the inlet pressure less than 0.15 MPa until the pH and conductivity at the outlet are the same as those of the formulation buffer, and the final concentration of the active protein is 5 mg / ml.

[0138] 5. Virus removal filtration and filling: The virus removal nanofiltration filter in this step is ViruClear VF (20 nm pore size). The active protein replaced with the formulation buffer in the previous step is filtered through the virus removal nanofiltration filter, and after filtration, the active protein solution is filled into vials, 1 ml / vial, and stored at 4°C for a long time.

[0139] Example 2. CEFFE Pro 2.0 can effectively delay the time of muscle paralysis and paralysis in AD Caenorhabditis elegans

[0140] 1. Materials and reagents

[0141] 1.1 Caenorhabditis elegans: The Caenorhabditis elegans strain CL4176 (Bristol strain) was purchased from the Caenorhabditis Genetics Center / CGC at the University of Minnesota, USA. The nematodes have a very clear and genetically easy-to-control nervous system, which provides an effective model for exploring the basic mechanistic pathways that may underlie complex human neurological diseases.

[0142] 1.2 Test article: CEFFE Pro 2.0, with a stock solution concentration of 5 mg / ml and is water-soluble.

[0143] 1.3 Nematode culture conditions: Nematodes were cultured on NGM (nematode growth medium) solid medium with OP50 growth, at a culture temperature of 16 °C and a humidity of 40 - 60%.

[0144] 2. Experimental methods

[0145] 2.1 Plate preparation: Dilute CEFFE Pro 2.0 to a specific experimental concentration with ddH 2 O, mix it evenly with 1 ml of Escherichia coli OP50 precipitate, pipette 400 μL and spread it on the surface of NGM solid medium (6 cm petri dish), and use it for experiments after drying. Caffeine is the Positive control with a concentration of 6.27 mM.

[0146] 2.2 Experimental process: After synchronizing the CL4176 strain on the control plate and the drug plate, culture it at 16 °C for 48 hours, then transfer it to 23 °C for induction, and start counting after 40 hours.

[0147] 2.3 Statistical method: The statistical method used for comparing the differences in paralysis degree is the Log-rank (Mantel-Cox) Test. Data analysis was performed using GraphPad.Prism.v5.0. "*" represents p < 0.05, "**" represents p < 0.01, and "***" represents p < 0.001.

[0148] Example 3. CEFFE Pro 2.0 can effectively promote the proliferation of SH-SY5Y cells

[0149] 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.

[0150] The SH-SY5Y cell line is derived from the SK-N-SH cell line and has a functional mature neuron phenotype. Its cell morphology and physiological and biochemical functions are similar to those of normal nerve cells, and it can be used to replace primary neuron cultures. This cell line was established from the metastatic bone marrow neuroblastoma of a four-year-old girl in 1970. Since the first discovery of the SH-SY5Y cell line, it has been widely used in in vitro research in the field of neuroscience, such as studying the molecular mechanisms of AD, neurotoxicity, and neurodegeneration.

[0151] 2. Experimental methods

[0152] 2.1 Cell seeding: After digesting the cells to make a cell suspension, seed the cells in a 96-well plate at a cell density of 5000 cells / well and culture them in an incubator at 37 °C for 24 hours.

[0153] 2.2 Drug treatment: On the next day, prepare working solutions of CEFFE Pro 2.0 at different concentrations using MEM / F12 medium (the same medium as used in routine culture). Aspirate the original medium in the wells, and replace it with the newly prepared culture medium containing CEFFE Pro 2.0. The concentrations of CEFFE Pro 2.0 are set as: 0 (blank group), 0.5, 1, 2.5, 5, 10, 25, 50, 100, 200 μg / mL. Each group has 3 replicate wells, and continue to culture for 72 hours.

[0154] 2.3 CCK8 assay: After 72 hours of drug incubation, take out the 96-well plate, and replace the medium in each well with the working solution containing 10% CCK8 solution. Incubate it in a 37°C incubator for 1.5 hours, and then detect it using a microplate reader at dual wavelengths of 450 and 650 nm.

[0155] Example 4: CEFFE Pro 2.0 can relieve the agglomeration and shrinkage state of SH-SY5Y cells caused by sudden replacement of the culture medium

[0156] 1. Cell information: The same as in Example 3

[0157] 2. Experimental method

[0158] 2.1 Cell seeding: After cell digestion, prepare a cell suspension and seed it in a 96-well plate at a cell density of 5000 cells / well. Incubate it in a 37°C incubator for 24 hours.

[0159] 2.2 Drug treatment: On the next day, prepare working solutions of CEFFE Pro 2.0 at different concentrations using RPMI medium (a medium different from that used in routine culture). Aspirate the original MEM / F12 medium in the wells, and replace it with the newly prepared culture medium containing CEFFE Pro 2.0. The concentrations of CEFFE Pro 2.0 are set as: 0 (blank group), 0.5, 1, 2.5, 5, 10, 25, 50, 100, 200 μg / mL. Each group has 3 replicate wells, and continue to culture for 72 hours.

[0160] 2.3 Microscopic observation and photography: After 72 hours of drug incubation, take out the 96-well plate, observe the cell state under a microscope, and select typical fields of view for photography.

[0161] Example 5: CEFFE Pro 2.0 significantly promotes the proliferation ability of SH-SY5Y cells in RPMI medium

[0162] 1. Cell information: The same as in Example 3

[0163] 2. Experimental method

[0164] 2.1 Cell seeding: After the cells were digested, they were made into a cell suspension and seeded in a 96-well plate at a cell density of 5000 cells / well, and then cultured in an incubator at 37°C for 24 hours.

[0165] 2.2 Drug treatment: The next day, working solutions of different concentrations of CEFFE Pro 2.0 were prepared using RPMI medium (a medium different from the conventional culture medium). 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, 200 μg / mL, with 3 replicates in each group, and the cells were cultured for another 72 hours.

[0166] 2.3 CCK8 assay: After 72 hours of drug incubation, the 96-well plate was taken out, and the working solution containing 10% CCK8 solution was replaced in each well. Then it was incubated in an incubator at 37°C for 1.5 hours, and then detected using a microplate reader at dual wavelengths of 450 and 650 nm.

[0167] Example 6. CEFFE Pro 2.0 has a protective effect on Aβ 25-35 induced SH-SY5Y neuronal cell injury

[0168] 1. Cell information: The same as in Example 3

[0169] 2. Experimental method

[0170] 2.1 Preparation and aging treatment of Aβ 25-35 Aβ is decomposed from amyloid precursor protein (APP). Aβ 25-35 fragments have certain neurotoxicity in cell culture. Therefore, in vitro experiments often use Aβ 25-35 to construct an AD cell model. 1 mg of Aβ 25-35 powder was dissolved in 4.717 ml of ddH 2 O to obtain a concentration of 200 μM. It was filtered and sterilized using a 0.22 μM filter, sealed, and placed in an incubator at 37°C for aging treatment. After one week, it was taken out, aliquoted, and stored at -20°C.

[0171] 2.2 Establishment of an Aβ 25-35 induced SH-SY5Y cell injury model: Cells in the logarithmic growth phase were taken. After the cells were digested, they were made into a cell suspension and seeded in a 96-well plate at a cell density of 8000 cells / well. After 48 hours, the medium was discarded, and a solution of Aβ 25-35 with a final concentration of 80 μM was added. The control group was added with an equal volume of serum-free medium to establish an Aβ 25-35 induced SH-SY5Y cell injury model.

[0172] 2.3 Administration treatment: Aβ 25-35 While establishing the model of Aβ-induced SH-SY5Y cell injury, administration treatment was carried out. The administration groups were respectively added with CEFFE Pro 2.0 with final concentrations of 45, 90, and 180 uM. There were 3 replicate wells in each group, and the cells were cultured for another 48 hours.

[0173] 2.4 CCK8 assay: The working solution containing 10% CCK8 solution was replaced in each well, and the plate was incubated in an incubator at 37°C for 1.5 hours. Subsequently, detection was carried out using a microplate reader at dual wavelengths of 450 and 650 nm.

[0174] Example 7: CEFFE Pro 2.0 has a protective effect on L-glutamate (L-Glu)-induced SH-SY5Y cell injury

[0175] 1. Cell information: The same as in Example 3

[0176] 2. Experimental method

[0177] 2.1 Preparation of L-Glu: The accumulation of L-Glu in the extracellular fluid causing excitotoxic injury is the initiating factor for neuronal death. Using the glutamate injury model for screening neuroprotective drugs and studying the mechanism of drug action has become one of the effective methods to discover the potential pharmacological value of drugs. Weigh 20 mg of L-Glu powder, dissolve it in 6.8 ml of serum-free medium, filter and sterilize it with a 0.22-μm filter, and dispense and store it in a -20°C refrigerator.

[0178] 2.2 Establishment of the L-Glu-induced SH-SY5Y cell injury model: Cells in the logarithmic growth phase were taken. After digestion, the cells were made into a cell suspension and inoculated into 96-well plates at a cell density of 8000 cells / well. After 48 hours, the culture medium was discarded, and a L-Glu solution with a final concentration of 10 mM was added. The control group was added with an equal volume of serum-free medium to establish the L-Glu-induced SH-SY5Y cell injury model.

[0179] 2.3 Administration treatment: While establishing the model of L-Glu-induced SH-SY5Y cell injury, administration treatment was carried out. The administration groups were respectively added with CEFFE Pro 2.0 with final concentrations of 45, 90, and 180 uM. There were 3 replicate wells in each group, and the cells were cultured for another 48 hours.

[0180] 2.4 CCK8 assay: The working solution containing 10% CCK8 solution was replaced in each well, and the plate was incubated in an incubator at 37°C for 1.5 hours. Subsequently, detection was carried out using a microplate reader at dual wavelengths of 450 and 650 nm.

[0181] According to the above-mentioned protocol, the experimental results obtained in Example 2 showed that CEFFE Pro 2.0 could relieve the muscle paralysis of Caenorhabditis elegans (nematode strain CL4176), thus prolonging the paralysis time. As Figure 1 shown, 20 μg / ml of CEFFE Pro 2.0 had the strongest anti-paralysis effect, indicating that CEFFE Pro 2.0 had the potential effect of anti-Alzheimer's disease.

[0182] Example 3 further confirmed through cell experiments that 100 μg / ml and 200 μg / ml of CEFFE Pro 2.0 could effectively improve the proliferation activity of SH-SY5Y cells, indicating that CEFFE Pro 2.0 had the potential neurotrophic activity and the effect of promoting the growth vitality of nerve cells.

[0183] In Example 4, SH-SY5Y cell damage was induced by suddenly changing the culture medium (changing from MEM / F12 medium to RPMI medium), resulting in cell shrinkage and aggregation and slow growth. This might be related to the change in nutrient components that made the cells unable to adapt. The cells treated with CEFFE Pro 2.0 could effectively resist the cell damage and deteriorated state caused by the change of the culture medium. As Figure 3 shown, cell pictures of each group were obtained by taking pictures under the microscope. CEFFE Pro 2.0 at a concentration of 2.5 μg / ml could effectively adjust the cell state, reduce cell aggregation, and promote the cells to spread out from the clumps and adhere to grow. When the concentration reached 25 μg / ml, all the cells had spread out and grown, and the cell aggregation phenomenon had completely disappeared. The cell growth state was significantly improved. With the increase of the concentration of CEFFE Pro 2.0, this effect was significantly enhanced. The adjustment of the growth state of SH-SY5Y cells by CEFFE Pro 2.0 showed a concentration gradient dependence.

[0184] In Example 5, the proliferative viability of the cells that had been photographed in Example 4 was detected using the CCK8 assay. The results were as Figure 4 shown. CEFFE Pro 2.0 could significantly improve the proliferative ability of SH-SY5Y cells. This effect could be exerted starting from 10 μg / ml, and with the increase of the concentration of CEFFE Pro 2.0, the pro-proliferative activity was stronger, showing a concentration gradient dependence.

[0185] In Example 6, Aβ 25-35 was selected as the inducing drug to act on SY5Y cells to construct an in vitro cell damage model to detect the inhibitory effect of CEFFE Pro 2.0 on the toxicity of Aβ 25-35 and further clarify the neuroprotective function of CEFFE Pro 2.0. The experimental data showed that, as Figure 5 shown, Aβ25-35 It has a certain inhibitory effect on the proliferation activity of cells at a concentration of 40 μM, can induce apoptosis in cells, and after treatment with CEFFE Pro 2.0, the cell viability is significantly improved. The higher the drug concentration, the stronger its ability to resist the cytotoxicity caused by Aβ 25-35 induced cell toxicity. Therefore, it is considered that CEFFE Pro 2.0 has prominent neuroprotective effects.

[0186] In Example 7, L-Glu was selected as the inducing drug, which has neurotoxicity. It can bind to nerve cells and cause neuron damage, thereby exerting neurotoxic effects. L-Glu was applied to SH-SY5Y cells to construct another in vitro cell injury model to detect the inhibitory effect of CEFFE Pro 2.0 on the toxicity of L-Glu, and further clarify the neuroprotective function of CEFFE Pro 2.0. The experimental data show that, as Figure 6 shown, L-Glu has obvious toxicity to cells at a concentration of 10 mM, significantly reducing the cell proliferation ability. After treatment with CEFFE Pro 2.0, the cell viability is significantly improved. The higher the drug concentration, the stronger its ability to resist the cell injury toxicity caused by L-Glu. Therefore, it is considered that CEFFE Pro 2.0 has prominent neuroprotective effects.

[0187] All documents mentioned in the present invention are cited in this application as references, just as if each document was cited separately as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. Use of adipose decellularized active protein in preparing a composition for treating Alzheimer's disease, in, The Alzheimer's disease is Alzheimer's disease caused by Aβ deposition or nerve damage; The adipose decellularized active protein is prepared by a method comprising 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 fat tissue, mixing and lysing, then standing and stratifying, and taking the middle layer; Optionally (4) repeat step (3) 1-3 times to merge the middle layers; (5) filtering the middle layer to obtain a lysed fat tissue fluid; (6) inactivating viruses in the lysed fat tissue fluid to obtain a virus-inactivated tissue lysate; and (7) Using anion chromatography to separate, obtain adipose decellularized active protein, Wherein, in step (3), the tissue lysis solution system is: 100-200 mmol / L NaCl, 20-80 mmol / LTris, 1-10 mmol / L EDTA-2Na, 20-80 mmol / L NaH2PO4-Na2HPO4 buffer system; The step (7) comprises the following steps: (7a) replacing the buffer of the virus-inactivated tissue lysate; (7b) primary purification by anion chromatography; (7c) ultrafiltration through an ultrafiltration membrane package to replace the buffer of the tissue lysate; (7d) secondary purification by high-resolution anion chromatography; (7e) performing a second ultrafiltration change using an ultrafiltration membrane package to replace the buffer of the tissue lysate; (7f) Virus removal filtration, In step (7b), the eluent for the primary purification is 10-50 mmol / L Tris, 300-800 mmol / L sodium chloride; In step (7b), the anion chromatography column filler used in the primary purification is Q sepharose; In step (7b), the sample collection of the first purification refers to collecting the 50mAu-50mAu UV peak; In step (7d), the eluent for the secondary purification is 10-50 mmol / L Tris, 300-800 mmol / L sodium chloride; In step (7d), the anion chromatography column filler used in the secondary purification is Q high performance; In step (7d), the sample collection for secondary purification refers to collecting the 50mAu-50mAu ultraviolet peak.

2. The use according to claim 1, characterized in that The method for treating 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.

3. The use according to claim 1, characterized in that The adipose decellularized active protein contains no cells and no lipid droplets; Wherein, 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; The term “containing no cells” means that the average number of cells in 1 ml of adipose decellularized biological material is ≤ 1.

4. The use according to claim 3, characterized in that The lipid droplets are oil droplets released after the fat cells are broken; The cells are selected from the group consisting of endothelial cells, adipose stem cells, macrophages, and stromal cells.

5. The use according to claim 3, 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 0.5% of the total liquid; The term “containing no cells” means that the average number of cells in 1 ml of adipose decellularized biological material is ≤ 0.

5.

6. The use according to claim 3, 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 0.1% of the total liquid; The term “containing no cells” means that the average number of cells in 1 ml of adipose decellularized biological material is ≤ 0.

1.

7. The use according to claim 3, characterized in that The term “containing no cells” means that the average number of cells in 1 ml of adipose decellularized biological material is 0.

8. The use according to claim 1, characterized in that The composition is a pharmaceutical composition.

9. The use according to claim 1, characterized in that The dosage form of the composition is selected from the following group: tablets, injections, capsules, ointments, powders, and oral solutions.

10. The use according to claim 1, characterized in that The dosage form of the composition is selected from the following group: enteric-coated sustained-release preparations, powders, and suspensions.

11. The use according to claim 1, characterized in that The composition is administered to the subject by the following methods: oral administration, percutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, and intracranial injection.

12. The use according to claim 1, characterized in that In step (3), the tissue lysis solution system is: 150 mmol / L NaCl, 50 mmol / L Tris, 5 mmol / L EDTA-2Na, 50 mmol / L NaH2PO4-Na2HPO4 buffer system.

13. The use according to claim 1, characterized in that In step (7b), the eluent for the primary purification is 20 mmol / L Tris, 600 mmol / L sodium chloride.

14. The use according to claim 1, characterized in that In step (7d), the eluent for the secondary purification is 20 mmol / L Tris, 600 mmol / L sodium chloride.

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