WO2017026923A1 - Stable cosmetic composition based on immobilized hyaluronidase and preparation method thereof - Google Patents

Abstract

The invention relates to the field of cosmetology and pertains to agents based on a hyaluronidase enzyme. A hyaluronidase- based cosmetic cream, containing: immobilized hyaluronidase as an enzyme, which is covalently bonded to a polymer carrier; a cosmetic base consisting of Lipoderm 4/1 and water; and sodium benzoate as a preservative.

Description

 Stable cosmetic composition based on

 immobilized hyaluronidase and method thereof

The invention relates to the field of cosmetology and relates to

cosmetics based on the enzyme hyaluronidase.

The enzyme hyaluronidase is able to break down acidic

mucopolysaccharides, including hyaluronic acid. Process

depolymerization of the latter to free hexoses increases tissue and vascular permeability, which can be used for disorders and cosmetic defects accompanied by growth of connective tissue. Various sources are known regarding the use of hyaluronidase to eliminate such defects.

Patent RU 2286139, 10.27.2006, refers to face and body skin care products, and aimed at eliminating pustular rashes and cicatricial changes in the skin. The anti-acne cream contains a cosmetic base, eftederm, malavit, diclofenac, tincture of calendula, juniper oil, eucalyptus oil, cedar oil, tea tree oil, riboflavin (vitamin B2), lidase.

U.S. Patent Application 20140328826 discloses a method for treating keloid scars, hypertrophic scars or scars by administering a composition comprising: low molecular weight hyaluronic acid, hyaluronidase, and optionally a pharmaceutically acceptable carrier and / or excipient. However, a serious obstacle to the penetration of high molecular weight compounds such as enzymes into the cell is the external

cell membrane consisting of protein and lipid layers.

The creation of immobilized enzymes helps to solve these problems.

The main problem in creating effective enzyme-based creams is to increase the stability of the protein molecule while maintaining its specific activity. The recognized most effective way to stabilize protein molecules and create prolonged proteins is immobilization (covalent conjugation) on high molecular weight carriers.

 The proposed cosmetic cream contains immobilized hyaluronidase, a substance belonging to the modern class of enzymatic preparations of prolonged action. The technological novelty of the cream lies in the use of immobilized hyaluronidase in which a polymer carrier, a derivative of polyethylene piperazine, belonging to a new class of water-soluble derivatives of heteroaliphatic aliphatic polyamines is used as a carrier and stabilizer of the enzyme.

 A polymer carrier is a polymer matrix that protects the protein molecule of the enzyme (active substance) from numerous body effects that lower specific activity.

 Typically, immobilized enzymes are artificially produced complexes or conjugates of an enzyme with a water-insoluble carrier. Immobilized hyaluronidase, the substance has good solubility in water and in water-salt solutions, contribute to the retention of water in the composition, thereby improving the moisturizing properties of the cream.

- Known cosmetic compositions containing immobilized enzymes (lipolytic lipases, proteases and proteolytic enzymes for the breakdown of glycoproteins in the skin and release oligosaccharides) for local use (US4556554 (A) - 1985-12-03). There is no concrete mention of hyaluronidase and its purpose.

RU 2539390 01/20/2015 C1 discloses cosmetic compositions based on chondroitin sulfate, hyaluronidase, liposomes, beta-cyclodextrins, harpagophytum extract, ginger extract. In the description of the source, it is noted that the introduction of a polymer carrier into the composition helps immobilize hyaluronidase and increase its activity. However, there is no evidence of an anti-scar effect. These compositions are designed to be applied to the joint area.

As the closest analogue, the source RU 2471867 C2, which describes cosmetic compositions based on hyaluronidase, is described. Mentioned the possibility of creating funds against scars.

Object of the invention: the development of a new storage stable effective cosmetic product based on immobilized hyaluronidase.

The problem is solved by a cosmetic cream containing

immobilized hyaluronidase covalently bound to an active polymer carrier, a cosmetic base consisting of 4/1 lipoderm and water, a sodium benzoate preservative in the following ratios

components, mass. %: lipoderm - 10-20 immobilized hyaluronidase - 0.2-0.6 sodium benzoate - 0.05 - 0.1 flavoring - up to 0.3 s distilled water - the rest.

Any hyaluronidase covalently linked to the polymer carrier can be used. As an immobilizing polymer,

for example, various polymers suitable for application to the skin are used, such as polyethylene glycol, hydroxyethyl starch,

polyvinylpyrrolidone, polysaccharides, silicones, etc. (e.g.

obtained by the methods described in WO2010021570, US 7521434, RU 2409669, http: //www.ncbi.nlm.nih.gov/pmc/articles/PMC3563746/) ·

Preferably, immobilized hyaluronidase is used in which the enzyme is covalently chemically bonded to the polymer support A-CO-NH-B, where A-CO is a fragment of the carboxyl function of the Ν-oxide derivative of poly-1,4-ethylene piperazine and B-ΝΗ is the amino group of hyaluronidase and has hyaluronidase activity.

As the carrier, a carboxyl-containing Ν-oxide derivative of poly-1,4-ethylene piperazine with a molecular weight of 20,000 to 40,000 Da can be used.

- Immobilization of hyaluronidase with a carboxyl-containing derivative of Ν-oxide poly-1,4-ethylene piperazine is carried out by carbodiimide or hydrazide methods in aqueous solutions.

The hydrazide method involves the azidation reaction of a hydrazide derivative of a carboxyalkyl substituted--oxide of poly-1,4-ethylene piperazine (PEP) in the presence of sodium nitrite, followed by a conjugation reaction of the activated PEP derivative with hyaluronidase. The immobilized hyaluronidase is purified by ultrafiltration on a membrane cassette with a lower cut-off limit of 5 kDa, followed by freeze drying of the purified solution of the preparation. The carbodiimide method involves the conjugation of hyaluronidase with a carboxyalkyl-substituted--oxide of poly-1,4-ethylene piperazine in the presence of a condensing agent of water-soluble carbodiimide, followed by purification by ultrafiltration on a membrane cassette with a lower cut-off limit of 5 kDa particles and freeze drying.

The source of hyaluronidase can be bovine or sheep testes.

As a basis, it is proposed to use lipoderm 4/1. LILO DERM 4/1 (LIPODERM 4/1) is a self-emulsifying cream base with pronounced emollient and water-retaining properties, intended for the manufacture of cosmetic and pharmaceutical creams and oil-in-water emulsions of various consistencies. Composition Lipoderm 4/1: glycerol monostearate, stearic alcohol, cetyl alcohol, diethylene glycol stearate, PEG-400 stearate, Polysorbate 20, PEG-40 hydrogenated castor oil, dipropylene glycol. The selected foundation determines the effective softening of the skin, increasing its elasticity, activating the processes of cell renewal, smoothing small wrinkles. It has been unexpectedly discovered that this base significantly increases the effectiveness of hyaluronidase.

As a flavoring, any flavoring acceptable in cosmetic products can be used to give a pleasant smell. It can be either a substance that gives both a monoaromat (for example, apricot, orange, lemon, banana), and a bouquet of aromas (for example, “chocolate surprise”, “Iris cream”).

The proposed cream based on immobilized hyaluronidase has the following preferred applications: - Prevention of scar formation after cosmetic procedures, tattoo removal, laser resurfacing of the skin, dermabrasion, mid and deep exfoliations, hair removal;

- The consequences of acne;

- Defects in the nail plate caused by diseases of non-fungal etiology;

- Scars after photodynamic procedures, burns, including sunburn.

- Fresh and mature scars: post-traumatic, post-burn and postoperative scars.

Effect: high efficiency, elimination or a significant decrease in the color intensity of cicatricial lesions compared with surrounding tissues.

Production technology:

1. Heat the aqueous and oily phase (lipoderm 4/1) separately to 80-85 C

2. Stir for 2-5 minutes at T = 80-85 ° C, then cool to T = 60-65 ° C and mix until smooth

3. With stirring, bring the emulsion to T = 32-35 ° C and introduce the active substance previously dissolved in water (immobilized

hyaluronidase) and a preservative dissolved in water (sodium benzoate)

4. Continue mixing until the emulsion reaches room temperature.

The possibility of carrying out the invention may be

demonstrated by the following examples.

b Example 1 lipoderm - 20 immobilized hyaluronidase - 0.4 sodium benzoate 0.05 Apricot flavoring - 0.1 distilled water - up to 100%. Example 2 lipoderm - 10 immobilized hyaluronidase - 0.6 sodium benzoate - 0.1 - distilled water - up to 100%.

Example 3 A study of the effectiveness of a cream to prevent scarring after cosmetic procedures.

Three groups of patients were formed after tattoo removal, laser resurfacing of the skin, deep peeling, and also after hair removal. Group 1-cream according to example 2 without immobilization of hyaluronidase. Group 2 - cream according to example 2 with immobilized hyaluronidase and a base made of a polyethylene oxide gel (should I give an example with a gel. Group 3 - cream according to example 2.

Patients applied the cream twice a day, compliance (following the instructions of the cosmetologist) in the groups ranged from about 85 to 88% of patients. The cream was easy to apply and stayed on the skin for many hours. In the study, there were practically no side effects associated with the use of the cream.

Scarring was observed in 94% of patients in group 1, 87% in group 2. Moreover, scars in the cream group were observed only in 26% of cases and were much less than in the control group. A final evaluation at the end of the third month showed that the difference between the two control groups and the third group was statistically significant.

It was noted that the cream according to the invention restores the normal color of the skin and normalizes the hydration of scar tissue. At

using other substrates instead of lipoderm, the color in the affected area was different.

The proposed cream can prevent the formation of scars and scars, make them almost invisible, more elastic, and also relieve itching and pain in the first days of applying the cream.

Claims

Claim 1. Cosmetic cream based on hyaluronidase containing a cosmetic base, characterized in that immobilized hyaluronidase covalently bound to a polymeric carrier is used as an enzyme, while lipoderm 4/1 and water are used as a cosmetic base, sodium benzoate is a preservative in the following ratios of components, mass. %: lipoderm - 10-20 immobilized hyaluronidase - 0.2-0.6 sodium benzoate - 0.05 - 0.1 flavoring - up to 0.3 distilled water - the rest. 2. The cream according to claim 1, characterized in that the immobilized hyaluronidase is used, in which the enzyme is linked by a covalent chemical bond to the polymer carrier A-CO-NH-B, where A-CO is a fragment of the carboxyl function of the poly-1 окс-oxide derivative, 4-ethylene piperazine, and the B-ΝΗ-amino group of hyaluronidase and has hyaluronidase activity. 3. Cream according to claim 1 or claim 2, characterized in that the source Hyaluronidase is a bovine or sheep testis. 4. The cream according to claim 2, characterized in that a carboxyl-containing derivative of Ν-oxide of poly-1,4-ethylene piperazine with a molecular weight of 20,000 to 40,000 Da is used as a carrier. 5. The method of obtaining a cosmetic cream based on hyaluronidase according to any one of p. 1-4, characterized in that the water and lipoderm 4/1 are heated separately to 80-85 ° C, stirred for 2-5 minutes at 80-85 ° C, then cooled to 60-65 ° C and stirred until homogeneous, continue stirring until the temperature of the resulting emulsion reaches 32-35 ° C and introduced previously dissolved in water immobilized hyaluronidase and sodium benzoate dissolved in water continue stirring until room temperature is reached. Yu