KR20070122315A - Soft tissue filler composition comprising autologous dermal cell and hyaluronic acid - Google Patents

Abstract

A soft tissue filler composition comprising an autologous dermal cell and hyaluronic acid is provided to alleviate and remove skin wrinkles and scars naturally without side effects, and improve rapidness and durability of therapeutic effects. A soft tissue filler composition for alleviating or treating skin defects caused by deletion of a dermal layer comprises (a) 1x10^7 to 8X10^7 cell/ml of an autologous dermal cell which is isolated and cultured from a patient himself, and a cell extract obtained from the cell cultured medium containing collagen protein and (b) 2.0-20 mg/ml of hyaluronic acid, and is formulated as injection, wherein the autologous dermal cell comprises a dermal fibroblast stem cell, a dermal fibroblast transit-amplifying cell and a dermal fibroblast.

Description

Injectable human soft tissue filler composition containing autologous dermal cells and hyaluronic acid {SOFT TISSUE FILLER COMPOSITION COMPRISING AUTOLOGOUS DERMAL CELL AND HYALURONIC ACID}

The present invention relates to a human soft tissue filler composition for injection containing autologous dermal cells and hyaluronic acid as an active ingredient useful for the relief and removal of wrinkles and scars.

Filler of human soft tissue dates back to the 19th century. In the 1960s, siloxane filling was used, but side effects are severe and are no longer used. About 20 years ago, wrinkles and scars were treated through injections of bovine collagen protein. However, not only allergic reactions but also tissue absorption was fast, resulting in only a few months of therapeutic effects. In order to solve this problem, a method of preparing and injecting collagen protein using the patient's own skin has been developed, but a large amount of skin is required to produce an effective amount of collagen protein, and collagen protein still remains in the tissue within a short time after injection. There was a problem of being absorbed.

In addition, different sizes of medium collagen proteins have been used as soft tissue fillers in the past few years, but the therapeutic effect has been short, about 6 months, and induced allergic reactions. In order to solve the immune response of the heavy collagen protein, autologen extracted from human placenta (Autologen) was used, but the effect was maintained for only a few months. Fibrel, a collagen product extracted from pigs, has been used for fine wrinkles after FDA approval in 1990, but it is difficult to expect continuous treatment effects and caused side effects of kernel formation at the injection site. In addition, Artecol, which is a mixture of bovine collagen and microspore, can achieve its long-term therapeutic effect by producing collagen itself through local dermal stimulation to prevent collagen from being absorbed after the procedure. Although there is a number, there is a problem that the particles always protrude to the treatment site.

On the other hand, biodegradable, non-animal stabilized hyaluronic acid gels of Restyland ^™ (Q-Med Scandivavia, USA) mainly remove wrinkles and make the lips bulky. It was approved by the US FDA on December 12, 2003 as an injectable injection. Hyaluronic acid, taken from the placenta as gluconate aldehyde, acts to provide nutrients to tissues and cells, provides a liquid environment for tissue metabolism, and makes the skin elastic and smooth. The advantage of using hyaluronic acid for soft tissue filling is that it is a natural ingredient, safe and free of side effects, while its duration is as short as six months.

Existing self-derived skin cell injection products took 3 to 9 months to take effect per dosage unit, and no immediate effect was achieved. In addition, when hyaluronic acid is used to remove wrinkles or perform a procedure that enlarges the lips, an immediate effect appears, but there is a problem that the therapeutic effect is maintained for about 6 months.

Therefore, the present inventors have intensively researched to develop a long-lasting human soft tissue filler that immediately exerts a therapeutic effect in alleviating and removing wrinkles and scars. The present invention was completed by finding that a soft tissue filler composition containing hyaluronic acid as an active ingredient has a natural and excellent therapeutic effect and is effective in removing wrinkles and scars.

Accordingly, it is an object of the present invention to provide an immediate, excellent and lasting human soft tissue filler that is effective in treating skin defects caused by dermal defects.

In order to achieve the above object, the present invention provides a human soft tissue filler composition for injection containing autologous dermal cells and hyaluronic acid obtained by isolating, culturing and propagating the patient's own skin as an active ingredient.

Preferably, the autologous dermal cells in the composition of the present invention are 1 × 10 ⁷ to 8X10 ⁷ per ml of injection, and the hyaluronic acid is 2.0 to 20 mg / ml.

In the present invention, autologous dermal cells refer to dermal fibroblast stem cells, dermal fibroblast transit amplifying cells and / or dermal fibroblasts and isolated from the patient's own cells in vitro. Obtained by propagation and incubation at.

The dermal cells isolated from the patient's own cells according to the present invention and cultured and grown in vitro are composed of three cells: the dermal stem cells, dermal metastasis-amplified cells and dermal fibroblasts. In addition, collagen proteins obtained from the patient's own cells, proliferated and cultured in vitro and obtained with autologous dermal cells may be included in the cell culture medium of the present invention.

In a preferred embodiment of the soft tissue filler of the invention, the dermal stem cells, dermal metastasis-amplified cells, dermal fibroblasts and collagen protein in the filler are derived from the patient's own small amount of skin (1-30 mm 2). The dermal tissue collected from the patient is separated into single dermal cells by digestion and then in vitro cultured and propagated in a serum-free medium containing growth factor and active factor in the cell culture chamber. A composite filler comprising cells, dermal fibroblasts and collagen protein is obtained.

According to a preferred embodiment of the present invention, the culture medium used for in vitro serum-free culture and propagation in the production method of the present invention does not include animal-derived serum but instead contains growth factors and active factors. The growth factor usable in the serum-free culture of the present invention may be selected from the group consisting of epidermal growth factor, dermal growth factor, basic fibroblast growth factor, and mixtures thereof. The active factor may be selected from the group consisting of cortical hormone, Bovine Pituitary Extract (BPE), Insulin, hydrocortisone, and mixtures thereof. Serum-free culture medium, growth factors and active factors usable in the present invention can be used commercially available products (Cascad, Sigma, Hyclne, USA). The content of growth factor in the serum-free culture of the present invention is 1 to 10 ng / ml, preferably 2 to 5 ng / ml, the content of the active factor is 0.1 to 1%, preferably 0.2 to 0.5%.

In a preferred embodiment of the production method according to the invention, in vitro culture and propagation using a serum-free medium containing growth factors and active factors as described above is preferably 6 to 10 weeks.

Hyaluronic acid usable in the present invention may be natural hyaluronic acid isolated from skin or animal sources and modified hyaluronic acid resulting from fermentation of genetically modified microorganisms, for example, Shandong Boxer Dal You can use the products shared by the company. It is also preferable to use hyaluronic acid having a molecular weight of at least 1,000,000 daltons.

According to the present invention, the soft tissue filler composition for removing wrinkles and scars is isolated from the patient's own dermal tissue, expanded and cultured in vitro to obtain dermal stem cells, dermal metastasis-amplified cells and / or dermal fibroblasts. The cells are prepared by adding 2.0 to 20 mg / ml of 1 to 4 ml of hyaluronic acid such that the final density of the cells is 1 × 10 ⁷ to 8X10 ⁷ / ml.

Since hyaluronic acid is a natural ingredient, it can be used as a dermal supplement because it is safe and has no side effects, but it has a disadvantage of short duration because of a 6-month maintenance period, and wrinkle removal using only autologous cells can achieve a lasting cosmetic effect. Satisfaction is high because the wrinkles or concave scars of the site are very safely improved, but there is a disadvantage in that the expression of such a therapeutic effect is slow. That is, the present invention is characterized by achieving the object of expressing and sustaining a rapid therapeutic effect by complementing the problems of using them alone by using a mixture of autologous cells and hyaluronic acid.

The compositions of the present invention can be formulated primarily into products for injection into human skin, which are performed according to conventional methods known to those skilled in the art. In one example, the compositions of the present invention may further comprise suitable excipients in the form of solutions, thick solutions, suspensions or gels and adapted for injection into the skin. Here, suitable excipients should provide concentrations that are well tolerated, stable, easy and enable good use. Examples of such excipients include phosphate buffers, bacteriostatic saline, propylene glycol, starch, sucrose and sorbitol.

In addition, the compositions of the present invention and the injections derived therefrom further comprise additives such as inert, pharmaceutically acceptable carriers or diluents, thickeners, emulsifiers, preservatives and mixtures thereof. Suitable examples of such additives include preparations commonly used in pharmaceutical and skin care preparations. More preferably, examples of inert pharmaceutically acceptable carriers or diluents include saline and purified water. Suitable thickeners include acrylamide copolymers, carbomers, hydroxyethylcellulose, polyacrylic acid, polymethacrylic acid and polyvinyl alcohol.

Suitable emulsifiers include capryl / capric triglyceride, stearate-7, cetyl alcohol, cetylphosphate, isostearate-11 and sodium isostearate. Preservatives confer resistance to the attack and toxicity of microorganisms to the compositions of the present invention. Suitable examples include alcohols, optional parabens, diazolidinyl urea, DMDM hydantoin, phenoxyethanol and iodoprovinyl butylcarbamate. Examples of additives other than those listed above may also be used in the present invention as is commonly known to those skilled in the art.

Since the injection of the present invention as described above uses dermal cells derived from the patient itself, it can maintain a continuous effect for many years without causing rejection or side effects. In addition, the injection of the present invention can expect an immediate therapeutic effect by hyaluronic acid in the dosage unit, while the problem that hyaluronic acid is short-acting is solved by autologous skin cells that are effective only 3 to 9 months after administration. The effect can be sustained over the years. Therefore, the injection of the present invention has the advantage that it is possible to maintain the therapeutic effect continuously and to be safe as well as to immediately show a clear filling and restoring effect after being applied to the affected area.

In addition, autologous dermal cells used in the injection of the present invention have an enhanced function of collagen protein production by these cells because the environment in which they are grown by hyaluronic acid is maintained. In other words, the injection according to the present invention produces more collagen protein at the injected site, thus making the skin firm and exerting wrinkles and scars. In addition, because it improves the local skin environment, when used on the face, for example, the facial expression is natural after treatment, and the beauty effect of looking young is excellent.

Injectables according to the present invention have the advantage of superior therapeutic effect, natural and long-lasting, compared to bovine collagen protein injections, collagen preparations from its own skin, Botox injections and the like.

Therefore, the scope of the present invention includes a method for alleviating or treating such wrinkles or scars by injecting the injection into a skin defect area such as a wrinkle or scar area caused by a dermal defect. The method is useful for alleviating or treating facial and neck wrinkles, pregnancy patterns, or wrist wrinkles, and can be used effectively to make lips full.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1 Preparation of Human Soft Tissue Filler Composition for Injection

end. Extraction of healthy skin of its own

After disinfecting the patient's ear or inside the arm with 70% alcohol, the local surface was anesthetized with 10% lidocaine and 1: 100,000 adrenaline. 4 mm 2 dermal tissue was collected from the anesthesia site using a skin extractor, and then immersed in tissue preservation solution. At this time, DMEM (Dulbeco's Modified Eagle medium, Gibco BRL Co., Ltd.) to which 10% fetal bovine serum (FBS) was added was used as a tissue preservation solution.

I. Preparation of autologous dermal cell culture

The skin tissue was placed in a 35 mm culture vessel, and 2 ml of 0.05% pancreatin / EDTA solution was added thereto, followed by reaction for 10 minutes in a CO ₂ incubator at 37 ° C., and 5 ml of DMEM solution containing 10% FBS was added. To terminate the digestion reaction.

After digestion, the skin tissue was cut and broken into suction tubes to separate cells. The crushed cells were centrifuged at 25 ° C. at 1000 rpm / min for 5 minutes to remove the supernatant and only cell precipitate was recovered.

1 ml of dermal cell culture solution (Cascade, Mut. 106, its main component is basal culture solution, growth factor EGF concentration is 1 ng / ml, activity factor BPE concentration is 0.2%) in the cell precipitate isolated as described above. Addition was carried out in a CO ₂ incubator at 37 ° C., 5% CO ₂ conditions.

Once every two to three days the cell culture was replaced with fresh new ones and subcultured until 50-70% of the cells were cultured. The cell culture was digested with pancretin and centrifuged to remove the supernatant and incubated with a 1: 3 proliferation fold.

The cell precipitate obtained by centrifuging the culture broth cultured in vitro for 4-6 weeks contains an active ingredient of 1X10 ⁷ to 8X10 ⁷ cells per milliliter, so that 5% glucose injection solution or 2-20 mg / ml of hyaluronic acid 1 to Injection was prepared by adding 4 ml.

All. Viral and immune response testing for product validation

1) Virus scan

1 ml of the cell suspension for injection containing dermal cells cultured in (1-1) of Example 1 was tested for HIV and hepatitis virus in a Beijing infectious disease hospital (according to the HIV and hepatitis test criteria of KFDA). ), It was confirmed that the dermal cells isolated and cultured according to the present invention are cells free of viruses.

2) Immune response test

In addition, subcutaneous experiments were performed on the skin of the patient with 0.1 ml of the cell suspension at 4 weeks of culture to confirm that there was no immune response. At this time, the subcutaneous experiment was carried out in the same manner as the penicillin subcutaneous experiment method (projection of 0.05ml white penicillin white suspension in the arm epidermis of the inner side of the cuff, and observed for 30 minutes, if there is no red swelling phenomenon of a large area).

3) Finished product inspection

In addition, aseptic tests were carried out based on the Chinese Biological Product Regulations (2000 Edition) Rule <Bioproduct Sterility Testing Regulations> Clause A / B to confirm that the injection of the present invention meets aseptic requirements.

Thus prepared injection of the present invention is a pale white cell suspension, its transport and storage was carried out in a dry, clean and free of corrosive gas, avoiding direct sunlight under 2 to 10 ℃ temperature conditions in a medium pressure environment.

Example 2 Clinical Trial

In order to investigate the therapeutic effect and stability of the soft tissue filler composition injection according to the present invention was carried out as follows.

end. Subject Selection Criteria

Patients between the ages of 18 and 60 years old were those who had applied for the treatment due to wrinkles on the face, such as forehead wrinkles, brow wrinkles (folds between the eyebrows), and nasolabial folds around the mouth. Patients with infectious diseases, acute and chronic infectious diseases, patients with wrinkles, pregnant women, severe heart, liver, kidney disease patients, and those with sensitive constitutions were excluded. Eleven patients were selected for blood and urine, electrocardiogram, hepatic and renal function, HIV and HBs Ag test.

I. Injection manufacturing

Injections prepared according to Example 1 were used in clinical trials.

All. cure

After the wrinkles of the subjects were disinfected with 70% alcohol, the epidermis was anesthetized by injection of 1% lidocaine or by applying 2.5% lidocaine and 2.5% prilocaine ointment. 1.5 ml of the injection prepared in (1-1) of Example 1 was placed in a 3 ml syringe, and several points were taken with a 4.5 cm needle having a length of 2.2 cm at an angle (at this time, the angle between the injection needle and the skin was 20 °). To 45 °), and an injection was injected between the upper and middle layers of the dermis. 15-20 points were taken per injection site, and 0.05-0.1 ml of the cell mixture was injected per point. At the time of injection, the skin was pulled hard until the skin became white and space was left at the injection site. After injection into all wrinkles or scars, the injection site was massaged for 2 hours with an ice bag.

la. Observation after surgery

Local injections within 24 hours after the procedure may cause mild swelling or numbness, but no other local or systemic reactions. Thus, once every two weeks, a total of three injections were performed at the same dose, followed by a 12-month follow up.

hemp. Treatment effect analysis

Wrinkle improvement effect was evaluated according to the following criteria, the results are shown in Table 1:

:: 해당 부위의 주름이 제거되고 피부가 완전히 회복됨.

:: Wrinkles removed and skin fully restored.

: 해당 부위의 주름이 옅어지고, 시술 전보다 선명히 개선됨.

: Wrinkles in the area become thinner and clearer than before procedure.

x: Almost no change before and after the procedure in wrinkle improvement.

part Therapeutic effect

Success rate (

and

) Forehead wrinkles 1/3 (33.3) 1/3 (33.3) 1/3 (33.3) 66.6% Fine wrinkles 2/4 (50) 2/4 (50) 100% Circumference 2/4 (50) 2/4 (50) 100% Sum 5/11 (45.5) 5/11 (45.5) 1/11 (9.1) 91%

As shown in Table 1, in subjects injected with a mixture of autologous dermal cells and hyaluronic acid according to the present invention, hyaluronic acid is absorbed over time and autologous dermal metastasis-amplified cells are constantly matured to dermal fiber. By transforming into blasts to form autologous collagen, the thickness and density of the dermal layer was increased, wrinkles and concave scars were improved, the elasticity and gloss of the skin were recovered, and the cosmetic effect was continuously maintained.

During the course of treatment, one subject had a slight discomfort due to red acne at the injection site during the third injection, which improved after 2 days without any relevant measures, and other adverse reactions during the course of treatment. It was not triggered. Therefore, the composition of the present invention was confirmed that the stability is good.

As described above, the human soft tissue filler composition of the present invention can maintain the therapeutic effect for a long time without causing an immune response and side effects by using autologous dermal cells, and can quickly exhibit the therapeutic effect by containing hyaluronic acid. Therefore, it can be useful for alleviating and treating wrinkles and scars as well as improving the skin's shine and elasticity.

Claims (12)

(a) a cell extract obtained from a cell culture comprising autologous dermal cells and collagen protein isolated and cultured from the patient's own skin; And (b) containing hyaluronic acid as an active ingredient, Compositions for alleviating or treating skin defects caused by dermal defects. The method of claim 1, wherein the self-dermal cell composition is characterized in that a concentration of 1X10 ⁷ to 8X10 ⁷ gae / ㎖. The composition of claim 2, wherein the autologous dermal cells are contained at a concentration of 1 × ^{10 7} to 5 × ^{10 7} cells / ml. The composition of claim 1, wherein the autologous dermal cells comprise dermal fibroblas tstem cells, dermal fibroblast tansit-amplifying cells and dermal fibroblasts. The composition of claim 1, wherein the hyaluronic acid is contained at a concentration of 2.0 to 20 mg / ml. The composition of claim 1 wherein the hyaluronic acid is a natural hyaluronic acid isolated from an animal source or a modified hyaluronic acid resulting from fermentation of a genetically modified microorganism. The composition of claim 1 wherein the hyaluronic acid has a molecular weight of at least 1,000,000 daltons. An injection for the alleviation or treatment of skin defects caused by dermal defects, formulated with the composition of claim 1. The injection of claim 8 comprising a pharmaceutically acceptable excipient and / or excipient. 10. The injection of claim 9 wherein the additive is selected from the group consisting of carriers, diluents, thickeners, emulsifiers, preservatives and mixtures thereof. The injection according to claim 8, wherein the alleviation or treatment of skin defects caused by dermal defects is for improving wrinkles, removing scars or making the lips bulky. The injection according to claim 11, wherein the improvement of wrinkles is improvement of wrinkles of the face and neck, improvement of pregnancy patterns, or improvement of wrinkles on the back of the hand.