WO2022194093A1 - Use of cell-free fat extract for treating non-hypertrophic scars - Google Patents

Abstract

Provided is the use of a cell-free fat extract for treating non-hypertrophic scars. Specifically, provided is the use of a cell-free fat extract in the preparation of a composition or a formulation for preventing and/or treating non-hypertrophic scars. The cell-free fat extract has an excellent therapeutic effect on non-hypertrophic scars.

Description

Cell-free adipose extract for the treatment of non-hypertrophic scars technical field

The present invention relates to the field of medicine, in particular to the use of acellular fat extracts for the treatment of non-hypertrophic scars.

Background technique

Non-hypertrophic scars are commonly seen in surgery, after trauma, and infections such as acne and smallpox. The reason for the formation is the defect of the dermis and subcutaneous tissue of the skin, which is related to the loss of collagen and elastin in the subsequent healing process. Usually non-hypertrophic scars do not affect the patient's life, but if the lesions exist on the head and face, it may seriously affect the patient's appearance.

Non-hypertrophic scars are characterized by changes in appearance such as hard texture, poor flatness, and hyperpigmentation. Laser, surgery, and injection filling are the main treatments for non-hypertrophic scars. However, the existing treatment methods for non-hypertrophic scars have many disadvantages, such as poor treatment effect, short treatment duration and high treatment cost. Therefore, the existing treatment methods cannot effectively treat non-hypertrophic scars.

Therefore, there is a need in the art to develop a drug that is effective in treating non-hypertrophic scars.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide the use of a cell-free fat extract in the prevention and/or treatment of non-hypertrophic scars.

The first aspect of the present invention provides the use of a cell-free fat extract for preparing a composition or preparation for preventing and/or treating non-hypertrophic scars.

In another preferred embodiment, the non-hypertrophic scar includes a depressed scar.

In another preferred embodiment, the non-hypertrophic scar includes a non-hypertrophic scar on the skin.

In another preferred embodiment, the skin includes facial skin.

In another preferred embodiment, the prevention and/or treatment of non-hypertrophic scars comprises one or more methods selected from the group consisting of:

(i) Improve the degree of scarring skin defects;

(ii) reducing the stiffness of the scar texture;

(iii) Improve scar skin smoothness;

(iv) Lighten skin pigmentation; and/or

(v) Improve the elasticity of scarred skin.

In another preferred embodiment, the cell-free fat extract is a cell-free fat extract prepared from human or non-human mammalian fat.

In another preferred embodiment, the non-human mammal is monkey, orangutan, cow, pig, dog, sheep, mouse or rabbit.

In another preferred example, the compositions or preparations include pharmaceutical compositions or preparations, food compositions or preparations, health product compositions or preparations, or dietary supplements.

In another preferred embodiment, the composition or preparation further includes a pharmaceutically, food, health product or dietary acceptable carrier.

In another preferred embodiment, the composition or preparation further includes other drugs for preventing and/or treating non-hypertrophic scars.

In another preferred embodiment, the other drugs for preventing and/or treating non-hypertrophic scars are selected from the group consisting of dermal fillers, anti-wrinkle drugs, ingredients for nourishing the skin, or a combination thereof.

In another preferred embodiment, the skin filling material is selected from the group consisting of hyaluronic acid, collagen, polymer degradable materials, or a combination thereof.

In another preferred embodiment, the skin filling material is hyaluronic acid or a pharmaceutically acceptable salt thereof, preferably sodium hyaluronate.

In another preferred embodiment, the weight ratio of the cell-free fat extract to the hyaluronic acid or a pharmaceutically acceptable salt thereof is 0.5-1.5:0.5-1.5, preferably 0.8-1.2:0.8 -1.2, preferably 1:1.

In another preferred embodiment, the anti-wrinkle drug includes botulinum toxin.

In another preferred example, the ingredients for nourishing the skin include vitamins.

In another preferred embodiment, the vitamin component is selected from the group consisting of vitamin B1, vitamin B2, vitamin B12, vitamin C, vitamin D, vitamin E, or a combination thereof.

In another preferred embodiment, the dosage form of the composition or preparation is an oral preparation, an external preparation or an injection preparation.

In another preferred embodiment, the injection preparation is an intravenous injection or an intramuscular injection.

In another preferred embodiment, the injection preparation is a subcutaneous injection preparation.

In another preferred embodiment, the injection preparation is a dermal injection preparation.

In another preferred embodiment, the injection preparation is a subcutaneous injection preparation at a non-hypertrophic scar.

In another preferred embodiment, the injection preparation is an intradermal injection preparation at a non-hypertrophic scar.

In another preferred embodiment, the dosage form of the composition or preparation is a solid dosage form, a semi-solid dosage form, or a liquid dosage form, such as solution, gel, cream, lotion, ointment, cream, paste, cake, powder, Patches etc.

In another preferred embodiment, the dosage form of the composition or preparation is powder, granule, capsule, injection, tincture, oral liquid, tablet or lozenge.

In another preferred embodiment, the composition or preparation is administered externally, topically, or by injection.

In another preferred embodiment, the cell-free fat extract does not contain cells and does not contain lipid droplets.

In another preferred embodiment, the lipid droplets are oil droplets released after fat cells are disrupted.

In another preferred embodiment, the "does not contain lipid droplets" means that in the cell-free fat extract, the volume of oil droplets accounts for less than 1% of the total liquid, preferably less than 0.5%, more preferably less than 0.1%.

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

In another preferred embodiment, the "cell-free" refers to the average number of cells in 1 ml of cell-free fat extract ≤ 1, preferably ≤ 0.5, more preferably ≤ 0.1, or 0.

In another preferred example, the cell-free fat extract is a naturally-obtained nano-fat extract without added components.

In another preferred example, the "no added ingredients" means that, except for the rinsing step, no solution, solvent, small molecule, chemical agent, and biological additive are added during the preparation of the fat extract.

In another preferred embodiment, the cell-free adipose extract is prepared by centrifuging adipose tissue after emulsification.

In another preferred embodiment, the cell-free fat extract is a cell-free fat extract.

In another preferred embodiment, the cell-free fat extract is prepared by the following method:

(1) providing an adipose tissue raw material, crushing the adipose tissue raw material, and rinsing (such as with physiological saline), thereby obtaining rinsed adipose tissue;

(2) centrifuging the rinsed adipose tissue to obtain a layered mixture;

(3) for the layered mixture, remove the upper oil layer and the lower water layer, and collect the middle layer (ie, the fat layer containing adipocytes);

(4) Emulsifying the intermediate layer to obtain an emulsified fat mixture (also referred to as nano-fat);

(5) subjecting the emulsified fat mixture to centrifugation to obtain an intermediate liquid layer, i.e., the initial fat extract; and

(6) Filtration and sterilization of the primary fat extract to obtain a cell-free fat extract.

A second aspect of the present invention provides a method for preparing a cell-free fat extract, the method comprising the steps of:

(1) providing an adipose tissue raw material, crushing the adipose tissue raw material, and rinsing (such as with physiological saline), thereby obtaining rinsed adipose tissue;

(2) centrifuging the rinsed adipose tissue to obtain a layered mixture;

(3) for the layered mixture, remove the upper oil layer and the lower water layer, and collect the middle layer (ie, the fat layer containing adipocytes);

(4) Emulsifying the intermediate layer to obtain an emulsified fat mixture (also referred to as nano-fat);

(5) subjecting the emulsified fat mixture to centrifugation to obtain an intermediate liquid layer, i.e., the initial fat extract; and

(6) Filtration and sterilization of the primary fat extract to obtain a cell-free fat extract.

In another preferred embodiment, the cell-free fat extract is as described in the first aspect of the present invention.

In another preferred example, in the step (2), the centrifugation is performed at 800-2500g, preferably 800-2000g, more preferably 1000-1500g, and most preferably 1100-1300g.

In another preferred example, in the step (2), the centrifugation time is 1-15 minutes, preferably 1-10 minutes, more preferably 1-8 minutes, and optimally 1-5 minutes.

In another preferred embodiment, the temperature of the centrifugation is 2-6°C.

In another preferred example, in the step (4), the emulsification is mechanical emulsification.

In another preferred embodiment, the mechanical emulsification is performed mechanically by repeated blowing through a syringe (eg 20-200 times, preferably 20-150 times, more preferably 20-100 times, more preferably 30-50 times). emulsification.

In another preferred example, the blowing method is to repeatedly push and beat at a constant speed with two 10ml injection syringes connected to a tee tube.

In another preferred embodiment, in the step (4), the emulsification is a method of crushing by a tissue homogenizer.

In another preferred example, in the step (5), after centrifugation, the emulsified fat mixture is layered into four layers, the first layer is an oil layer, the second layer is a residual adipose tissue layer, and the third layer is a liquid layer layer (ie, the middle liquid layer), and the fourth layer is the cell/tissue debris sedimentation layer.

In another preferred example, in the step (5), the centrifugation is performed at 1000-4000g, preferably 1000-3000g, more preferably 1500-2500g, and most preferably 1800-2200g.

In another preferred example, in the step (5), the centrifugation time is 1-15 minutes, preferably 1-10 minutes, more preferably 2-8 minutes, and optimally 3-7 minutes.

In another preferred embodiment, the temperature of the centrifugation is 2-6°C.

In another preferred example, in the step (5), the first layer, the second layer, the third layer and the fourth layer are arranged in order from top to bottom.

In another preferred embodiment, in the step (5), the intermediate liquid layer is a transparent or substantially transparent layer.

In another preferred example, in the step (6), the filter bag can remove the adipocytes in the primary fat extract.

In another preferred example, in the step (6), the filtration and sterilization are performed through a filter (eg, a 0.22 μm microporous membrane).

In another preferred embodiment, the filter is a microporous membrane filter.

In another preferred example, the pore size of the microporous filter membrane is 0.05-0.8 μm, preferably 0.1-0.5 μm, more preferably 0.1-0.4 μm, more preferably 0.15-0.3 μm, more preferably 0.2-0.25 μm, optimally 0.22 μm.

In another preferred embodiment, in the step (6), the filtration and sterilization are firstly passed through a first filter that can filter out cells, and then passed through a second filter that can filter out pathogens (such as bacteria). filter (eg, 0.22 μm filter).

In another preferred embodiment, the step (6) further includes sub-packaging the fat extract to form a sub-packaged product. (The subpackaged extract can be stored at -20℃ for later use; it can be used directly after thawing at low temperature (such as -4℃) or normal temperature, or it can be stored at low temperature (such as 4℃) for a period of time after thawing, and then used ).

The third aspect of the present invention provides a cell-free fat extract, which is prepared by the method described in the second aspect of the present invention.

The fourth aspect of the present invention provides a composition or formulation comprising (a) the cell-free fat extract according to the third aspect of the present invention; and (b) pharmaceutically, food, Nutraceutical or dietary acceptable carrier or excipient.

In another preferred example, the composition is a pharmaceutical composition, a food composition, a health product composition or a dietary supplement.

In another preferred embodiment, the dosage form of the composition or preparation is an oral preparation, an external preparation or an injection preparation.

In another preferred embodiment, the dosage form of the composition or preparation is powder, granule, capsule, injection, tincture, oral liquid, tablet or lozenge.

In another preferred embodiment, the injection is an intravenous injection or an intramuscular injection.

In another preferred embodiment, the injection is a subcutaneous injection.

In another preferred embodiment, the injection is a dermal injection preparation.

In another preferred embodiment, the injection is a subcutaneous injection at a non-hypertrophic scar.

In another preferred embodiment, the injection preparation is an intradermal injection preparation at a non-hypertrophic scar.

In another preferred embodiment, the dosage form of the composition or preparation is a solid dosage form, a semi-solid dosage form, or a liquid dosage form, such as solution, gel, cream, lotion, ointment, cream, paste, cake, powder, Patches etc.

In another preferred example, in the composition or preparation, the mass percentage of the cell-free fat extract is 5 wt %, preferably 1-20 wt %, based on the total weight of the composition or preparation.

The fifth aspect of the present invention provides a method for preparing the composition or preparation according to the fourth aspect of the present invention, the method comprising the steps of: mixing the cell-free fat extract according to the third aspect of the present invention with a pharmaceutical It is mixed with acceptable carriers or excipients on food, health product or diet to form a composition or preparation.

The sixth aspect of the present invention provides a method for preventing and/or treating non-hypertrophic scars by administering the cell-free adipose extract according to the third aspect of the present invention to a subject in need thereof.

In another preferred embodiment, the subject is a human or a non-human mammal.

In another preferred embodiment, the non-human mammals include rodents, such as rats and mice.

In another preferred embodiment, the administration mode is oral administration, topical administration or injection administration.

In another preferred embodiment, the injection is subcutaneous injection.

In another preferred embodiment, the injection is subcutaneous injection at a non-hypertrophic scar.

In another preferred embodiment, the injection is intradermal injection at a non-hypertrophic scar.

In another preferred example, the injection dose is 0.01-0.03 mL/1 cm ² .

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 specifically described in the following (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.

Description of drawings

Figure 1 shows the therapeutic effect of CEFFE on facial scars, where "before treatment" is before CEFFE treatment, and "after treatment" is the 3rd month after the last CEFFE treatment.

Figure 2 shows the therapeutic effect of CEFFE + sodium hyaluronate mixture on facial scars, where "before treatment" is before CEFFE + sodium hyaluronate mixture treatment, "after treatment" is after the last CEFFE + and sodium hyaluronate mixture treatment 3rd month.

Figure 3 shows the change of skin elasticity before and after CEFFE administration (n=6), where "Pre" is before CEFFE treatment, "3m" is the 3rd month after the last CEFFE injection treatment, "*" represents p<0.05, with Statistical significance.

Detailed ways

After extensive and in-depth research, the present inventors have developed for the first time that a cell-free fat extract has excellent therapeutic effects on non-hypertrophic scars. The present invention has been completed on this basis.

the term

Unless otherwise defined, 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.

As used herein, the terms "comprising," "including," and "containing" are used interchangeably to include not only open definitions, but also semi-closed, and closed definitions. In other words, the terms include "consisting of", "consisting essentially of".

Cell free fat extract (CEFFE) and preparation method thereof

As used herein, the terms "cell-free adipose extract of the present invention", "extract of the present invention", "fat extract of the present invention" and the like are used interchangeably to refer to the process during the preparation of the fat extract (other than the rinsing step) ) An adipose tissue-derived extract (or extract) prepared without the addition of any solutions, solvents, small molecules, chemicals, and biological additives. A typical method for preparing the extract of the present invention is as described above in the second aspect of the present invention. In addition, it should be understood that although the extract of the present invention does not have to add any additives (or added components) during the preparation process, some or small amounts of safe substances (such as small amounts) that do not negatively or adversely affect the activity of the extract of the present invention may also be added. water).

The cell-free fat extract of the present invention can be derived from human adipose tissue, which is purified from nano-fat by removing oil and cell/extracellular matrix fractions after centrifugation, and is a cell-free, easy-to-prepare, rich in various growth factor liquid.

In a preferred embodiment of the present invention, the cell-free fat extract is a cell-free fat extract.

Preferably, the cell-free fat extract of the present invention is prepared by the method as described in the second aspect of the present invention.

Typically, the cell-free fat extracts of the present invention are prepared by the following methods:

(1) providing an adipose tissue raw material, crushing the adipose tissue raw material, and rinsing (such as with physiological saline), thereby obtaining rinsed adipose tissue;

(2) centrifuging the rinsed adipose tissue to obtain a layered mixture;

(3) for the layered mixture, remove the upper oil layer and the lower water layer, and collect the middle layer (ie, the fat layer containing adipocytes);

(4) Emulsifying the intermediate layer to obtain an emulsified fat mixture (also referred to as nano-fat);

(5) subjecting the emulsified fat mixture to centrifugation to obtain an intermediate liquid layer, i.e., the initial fat extract; and

(6) Filtration and sterilization of the primary fat extract to obtain a cell-free fat extract.

In another preferred example, in the step (2), the centrifugation is performed at 800-2500g, preferably 800-2000g, more preferably 1000-1500g, and most preferably 1100-1300g.

In another preferred example, in the step (2), the centrifugation time is 1-15 minutes, preferably 1-10 minutes, more preferably 1-8 minutes, and optimally 1-5 minutes.

In another preferred example, in the step (4), the emulsification is mechanical emulsification.

In another preferred embodiment, the mechanical emulsification is performed mechanically by repeated blowing through a syringe (eg 20-200 times, preferably 20-150 times, more preferably 20-100 times, more preferably 30-50 times). emulsification.

In another preferred example, the blowing method is to repeatedly push and beat at a constant speed with two 10ml injection syringes connected to a tee tube.

In another preferred embodiment, in the step (4), the emulsification is a method of crushing by a tissue homogenizer.

In another preferred example, in the step (5), after centrifugation, the emulsified fat mixture is layered into four layers, the first layer is an oil layer, the second layer is a residual adipose tissue layer, and the third layer is a liquid layer layer (ie, the middle liquid layer), and the fourth layer is the cell/tissue debris sedimentation layer.

In another preferred example, in the step (5), the centrifugation is performed at 1000-4000g, preferably 1000-3000g, more preferably 1500-2500g, and most preferably 1800-2200g.

In another preferred example, in the step (5), the centrifugation time is 1-15 minutes, preferably 1-10 minutes, more preferably 2-8 minutes, and optimally 3-7 minutes.

In another preferred example, in the step (5), the first layer, the second layer, the third layer and the fourth layer are arranged in order from top to bottom.

In another preferred embodiment, in the step (5), the intermediate liquid layer is a transparent or substantially transparent layer.

In another preferred example, in the step (6), the filter bag can remove the adipocytes in the primary fat extract.

In another preferred example, in the step (6), the filtration and sterilization are performed through a filter (eg, a 0.22 μm microporous membrane).

In another preferred embodiment, the filter is a microporous membrane filter.

In another preferred example, the pore size of the microporous filter membrane is 0.05-0.8 μm, preferably 0.1-0.5 μm, more preferably 0.1-0.4 μm, more preferably 0.15-0.3 μm, more preferably 0.2-0.25 μm, optimally 0.22 μm.

In another preferred embodiment, in the step (6), the filtration and sterilization are firstly passed through a first filter that can filter out cells, and then passed through a second filter that can filter out pathogens (such as bacteria). filter (eg, 0.22 μm filter).

In another preferred embodiment, the step (6) further includes sub-packaging the fat extract to form a sub-packaged product. (The subpackaged extract can be stored at -20℃ for later use; it can be used directly after thawing at low temperature (such as -4℃) or normal temperature, or it can be stored at low temperature (such as 4℃) for a period of time after thawing, and then used ).

use

The present invention provides the use of a cell-free adipose extract for the preparation of a composition or formulation for the prevention and/or treatment of non-hypertrophic scars.

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. "Prevention" as used herein also includes delaying the onset of the disease and/or its attendant symptoms and reducing the risk of the disease in a subject.

"Treatment" as used in the present invention includes delaying and stopping the progression of the disease, or eliminating the disease, and does not require 100% inhibition, elimination and reversal. In some embodiments, the cell-free adipose extract of the present invention reduces, inhibits and/or reverses non-hyperplastic scarring, eg, by at least about 10%, compared to the absence of the cell-free adipose extract 28 of the present invention. At least about 30%, at least about 50%, or at least about 80%.

In the present invention, the skin site of the non-hypertrophic scar is not particularly limited. Preferably, the non-hypertrophic scar includes a facial non-hypertrophic scar.

In a preferred embodiment of the present invention, the non-hypertrophic scar includes a depressed scar.

In another preferred embodiment, the non-hypertrophic scar includes a non-hypertrophic scar on the skin.

In another preferred embodiment, the skin includes facial skin.

In a preferred embodiment of the present invention, the prevention and/or treatment of non-hypertrophic scars includes one or more methods selected from the group consisting of:

(i) Improve the degree of scarring skin defects;

(ii) reducing the stiffness of the scar texture;

(iii) Improve scar skin smoothness;

(iv) Lighten skin pigmentation; and/or

(v) Improve the elasticity of scarred skin.

The present invention also provides a method of preventing and/or treating non-hypertrophic scars by administering the acellular adipose extract according to the present invention to a subject in need thereof.

In another preferred embodiment, the subject is a human or a non-human mammal.

In another preferred embodiment, the non-human mammals include rodents, such as rats and mice.

Composition and Administration

The compositions described in the present invention include (but are not limited to): pharmaceutical compositions, food compositions, health care compositions, dietary supplements, and the like.

Typically, the cell-free fat extracts of the present invention can be prepared into pharmaceutical compositions such as tablets, capsules, powders, microparticles, solutions, lozenges, jellies, creams, elixirs, suspensions, Dosage forms such as tinctures, poultices, liniments, lotions, and aerosols. Pharmaceutical compositions can be prepared by generally known preparation techniques, and suitable pharmaceutical additives can be added to the medicament.

The composition of the present invention may also include a pharmaceutically, food, health product or dietary acceptable carrier. "Pharmaceutically, food, nutraceutical or dietary acceptable carrier" means: one or more compatible solid or liquid filler or gel substances, which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "Compatibility" as used herein means that the components of the composition can be admixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Pharmaceutical, food, health product or dietary acceptable carrier Examples of acceptable carrier moieties are 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, glycerin, mannitol, sorbitol) etc.), emulsifiers (such as Tween

), wetting agents (such as sodium lauryl sulfate), colorants, flavors, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

The mode of administration of the composition of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, parenteral (intravenous, intramuscular), topical, and preferred modes of administration are oral administration and injection. For example, the injection administration is subcutaneous or intradermal injection, preferably intradermal injection at a non-hypertrophic scar.

The dosage form of the composition or preparation of the present invention is an oral preparation, an external preparation or an injection preparation. Typically, 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 (a) fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, For example, glycerol; (d) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) Absorption accelerators such as quaternary amine compounds; (g) wetting agents such as cetyl alcohol and glyceryl monostearate; (h) adsorbents such as kaolin; and (i) lubricants such as talc, hard Calcium fatty acid, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain buffering agents.

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

Liquid dosage forms for oral administration or administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, 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.

Besides these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.

In addition to the active ingredient, suspensions may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances and the like.

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.

Dosage forms for topical administration or administration of the compounds of this invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required if necessary.

The cell-free adipose extracts of the present invention may be administered or administered alone or in combination with other agents for preventing and/or treating non-hypertrophic scars.

When the composition is administered, a safe and effective amount of the cell-free fat extract of the present invention is suitable for human or non-human animals (such as rats, mice, dogs, cats, cows, chickens, ducks, etc.) in need of treatment, wherein the administration The current dose is the effective dose that can be considered as acceptable in pharmacy, food or health care products. As used herein, the term "safe and effective amount" refers to an amount that produces 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 with 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, etc. different. For example, for a person weighing 60 kg, the daily dosage is usually 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 the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.

The main advantages of the present invention include:

1. The present invention finds for the first time that acellular fat extract has excellent therapeutic effect on non-hypertrophic scars.

2. The cell-free fat extract of the present invention is a cell-free component that can avoid cell-related problems in clinical applications, including, for example, genetic stability after cell processing, cell viability and viability after injection, Multiple administration and storage of cells, and immunogenicity of cells when using allogeneic fat, the cell-free fat extract of the present invention has the advantages of higher safety and lower side effects in the prevention and treatment of non-hypertrophic scars .

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In the following examples, the experimental methods without specific conditions are usually in accordance with conventional conditions, or in accordance with the conditions suggested by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Example 1

1. Preparation of Cell-Free Fat Extract (CEFFE)

This clinical trial was ethically approved by Shanghai Ninth People's Hospital, and the subjects signed the informed consent. The preparation method of cell-free adipose tissue extract is as follows:

(1) Adipose tissue was obtained from women with non-hypertrophic scars. After subcutaneous injection of tumescent solution in the abdomen for anesthesia, a 3mm liposuction cannula with a large lateral hole (2mm x 7mm) was used to connect a 20mL syringe, and radial suction was performed under artificial negative pressure. , the obtained fat was kept upright, and after removing the swelling fluid, rinsed 3 times with normal saline.

(2) Take the rinsed adipose tissue, place it in a centrifuge tube, put it in a centrifuge and centrifuge at 1200g for 3 minutes at 4°C to obtain a layered mixture.

(3) For the layered mixture, the upper oil layer and the lower water layer are removed, and the middle layer (ie, the fat layer containing adipocytes) is collected.

(4) For the middle layer, use two 10ml injection syringes to connect a tee tube to repeatedly push and beat 30 times at a constant speed to perform mechanical emulsification and obtain a mechanically emulsified fat mixture (also called nano fat).

(5) Centrifuge the mechanically emulsified fat mixture at 2000g at 4°C for 5 minutes to obtain a layered mixture. The layered mixture is divided into 4 layers, the first layer is the oil layer, and the second layer is the residual adipose tissue layer , the third layer is the liquid layer, the fourth layer is the cell/tissue debris precipitation layer, remove the oil layer and the residual adipose tissue layer, absorb the liquid layer, avoid the contamination of the cell/tissue debris precipitation layer during the absorption process, and obtain the fat primary extract.

(6) The obtained primary fat extract was sterilized by filtration through a 0.22 μm filter, thereby sterilizing and removing possible mixed living cells, thereby obtaining a cell-free fat extract (CEFFE).

2. Experimental method

2.1 Subjects of administration

This clinical trial was ethically approved by Shanghai Ninth People's Hospital, and the subjects signed the informed consent.

2.2 Method of administration

Six subjects with non-hypertrophic scars were injected with CEFFE prepared from self-liposuction. When injected, CEFFE and/or with sodium hyaluronate were slowly injected with a 30-gauge needle under the skin of the pitted scar, every 1 cm ² area of the scar Tissues were treated with 0.02ml CEFFE or 0.02ml CEFFE+sodium hyaluronate mixture (the weight ratio of CEFFE and sodium hyaluronate was 1:1) for a total of 3 treatments, and the administration interval was 2 weeks.

3. Detection and evaluation

3.1 General observation

Before the injection treatment and the 1st and 3rd month after the last treatment, the subjects used the instrument Visia to take pictures, including the degree of scar depression, whether it was accompanied by itching, hardness, etc., to evaluate the comprehensive changes of the scar after treatment.

3.2 Objective evaluation

Scar texture: All the enrolled patients should not be tested by skin elasticity tester CK-MPA580 before injection treatment and in the first and third months after the last treatment. The detection includes skin elasticity, evaluation of texture changes after scar treatment, and objective reflection of scar differences before and after treatment.

4. Statistical analysis

All data were analyzed by SPSS software, using paired t test, p < 0.05 means there is a statistical difference.

5. Experimental results

5.1 General observation

Three doctors who were not involved in this study participated in the subjective evaluation of the overall improvement. By comparing the photos of patients before and after CEFFE, CEFFE + sodium hyaluronate mixed solution injection treatment (as shown in Figure 1 and Figure 2), from Figure 1- 2 It can be seen that, compared with before CEFFE, CEFFE + sodium hyaluronate mixed solution treatment, after three times of CEFFE, CEFFE + sodium hyaluronate mixed solution injection treatment, the degree of scar skin defect in patients was significantly improved, the scar texture became softer, the flatness improved, and the pigmentation was improved. Sedimentation was significantly reduced, indicating that the combination of CEFFE, CEFFE and sodium hyaluronate has an excellent therapeutic effect on non-hypertrophic scars.

5.2 Objective evaluation

The changes of scar skin were detected by skin elasticity detector CK-MPA580, and the index of R2 (maximum elastic extension) was used as the elasticity change. The changes of skin elasticity before and after CEFFE treatment are shown in Figure 3. It can be seen from Figure 3 that, compared with before CEFFE treatment, the maximum elastic extension R2 of the scar at the third month after the last CEFFE injection treatment was significantly increased, and the skin extension ability was improved. It shows that CEFFE has excellent therapeutic effect on non-hypertrophic scars.

in conclusion

The results of the examples of the present invention show that CEFFE alone or combined with sodium hyaluronate are injected into non-hypertrophic scars, and it is found that the degree of scar skin defect is significantly improved, the texture of the scar is softened, the flatness is improved, and the pigmentation is reduced. The surrounding normal tissue is close to and improves the elasticity of the scarred skin, and the treatment effect is obvious. CEFFE alone or in combination with sodium hyaluronate is safe and effective in the treatment of non-hypertrophic scars with mild side effects. It can be used as a new treatment plan for non-hypertrophic scars.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content 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 the present application.

Claims (15)

1. Use of a cell-free fat extract, characterized in that it is used to prepare a composition or preparation for preventing and/or treating non-hypertrophic scars.
2. The use of claim 1, wherein the non-hypertrophic scar comprises a depressed scar.
3. The use according to claim 1, wherein the prevention and/or treatment of non-hypertrophic scars comprises one or more ways selected from the group consisting of:

   (i) Improve the degree of scarring skin defects;

   (ii) reducing the stiffness of the scar texture;

   (iii) Improve scar skin smoothness;

   (iv) Lighten skin pigmentation; and/or

   (v) Improve the elasticity of scarred skin.
4. The use according to claim 1, wherein the composition or preparation comprises a pharmaceutical composition or preparation, a food composition or preparation, a health product composition or preparation or a dietary supplement.
5. The use according to claim 1, wherein the dosage form of the composition or preparation is an oral preparation, an external preparation or an injection preparation.
6. The use according to claim 5, wherein the injection preparation is a subcutaneous injection preparation.
7. The use according to claim 1, wherein the dosage form of the composition or preparation is a subcutaneous injection preparation at a non-hypertrophic scar.
8. The use according to claim 1, wherein the composition or preparation further comprises other drugs for preventing and/or treating non-hypertrophic scars.
9. The use according to claim 8, wherein the other drugs for preventing and/or treating non-hypertrophic scars are selected from the group consisting of dermal fillers, anti-wrinkle drugs, ingredients for nourishing the skin, or a combination thereof.
10. The use according to claim 9, wherein the skin filling material is selected from the group consisting of hyaluronic acid, collagen, polymer degradable materials, or a combination thereof.
11. The use according to claim 9, wherein the skin filling material is hyaluronic acid or a pharmaceutically acceptable salt thereof.
12. The use according to claim 10, wherein the weight ratio of the cell-free fat extract to the hyaluronic acid or a pharmaceutically acceptable salt thereof is 0.5-1.5:0.5-1.5.
13. The use according to claim 10, wherein the weight ratio of the cell-free fat extract to the hyaluronic acid or a pharmaceutically acceptable salt thereof is 0.8-1.2:0.8-1.2.
14. The use according to claim 1, wherein the cell-free fat extract is prepared by the following method:

    (1) providing an adipose tissue raw material, crushing the adipose tissue raw material, and rinsing (such as with physiological saline), thereby obtaining rinsed adipose tissue;

    (2) centrifuging the rinsed adipose tissue to obtain a layered mixture;

    (3) for the layered mixture, remove the upper oil layer and the lower water layer, and collect the middle layer (ie, the fat layer containing adipocytes);

    (4) Emulsifying the intermediate layer to obtain an emulsified fat mixture (also referred to as nano-fat);

    (5) subjecting the emulsified fat mixture to centrifugation to obtain an intermediate liquid layer, i.e., the initial fat extract; and

    (6) Filtration and sterilization of the primary fat extract to obtain a cell-free fat extract.
15. A method of preventing and/or treating non-hypertrophic scars, characterized in that a cell-free adipose extract is administered to a subject in need thereof.

Images