WO2025056010A1 - New skin repair preparation - Google Patents

Abstract

The use of human serum albumin in the preparation of a medicament for improving the skin condition of a subject. A skin repair preparation, comprising human serum albumin and an optional pharmaceutically acceptable carrier. The young and non-invasive recombinant human serum albumin is used for injection into the mesoderm of a subject, achieving a good skin improvement effect.

Description

A new skin repair preparation

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Chinese patent application number CN202311205659.6 and filing date September 15, 2023, the entire contents of which are hereby incorporated by reference.

Technical Field

The present invention relates to the field of medical cosmetology, and more specifically, to the application of human serum albumin in improving the skin condition of a subject.

Background Art

The mesoderm of the skin is located between the epidermis and the dermis, and plays a key role in the skin's absorption of the active ingredients of topical beauty products and the storage of collagen and elastin in the inner layer of the skin. Mesotherapy was first proposed by French doctor C. Pistoire in 1952. It uses ultra-micro-penetration technology to directly deliver a variety of high-concentration skin nutrients through the epidermis to the subcutaneous tissue through positioning, stratification, and quantitative methods to achieve the purpose of improving skin condition and anti-aging. At present, this method of directly providing "nutrients" to the deep skin through special injections has become an important means of skin anti-aging.

Depending on the different mechanisms of action of different nutrients on the skin, existing mesotherapy has different focuses, mainly including locking in moisture, hemostasis and coagulation, repair and anti-inflammation, and promoting tissue regeneration. Commonly used nutrients include hyaluronic acid, platelet-rich plasma, polydeoxynucleotides, glutathione, vitamin C, etc.

Human albumin is synthesized in the liver and transported to various tissues and organs of the body through the circulatory system. The intravascular albumin content accounts for 30%-40% of the total body storage, and 60% of the extravascular albumin is distributed in tissues such as the skin, muscles, and intestinal mucosa. Albumin is extremely hydrophilic and can freely pass through the capillary wall, dynamically exchange between the circulating blood and the extracellular fluid, and maintain the balance of the colloidal osmotic pressure of the blood and the extracellular fluid. In addition, albumin also has the functions and effects of scavenging free radicals, regulating cell apoptosis, maintaining acid-base balance and anti-coagulation. It is an important component for maintaining hemodynamic stability and an important component for transporting oxygen.

Summary of the invention

The present invention provides the use of human serum albumin in the manufacture of a medicament for improving the skin condition of a subject.

The present invention also provides a skin repair preparation, comprising: human serum albumin, and optionally a pharmaceutically acceptable carrier.

The human serum albumin in the present invention can be human serum albumin prepared from human blood or recombinantly produced. A particularly preferred human serum albumin is young and undamaged human serum albumin.

In some embodiments, the drug may further include sodium hyaluronate to improve delivery efficiency and usage experience.

In some embodiments, the drug may further include additional functional components, such as nicotinamide, α-lipoic acid, carnosine, resveratrol, and the like.

Preferably, the medicament is for mesodermal injection into a subject.

Those skilled in the art will appreciate that the medicine provided by the present invention can be used for patients suffering from skin diseases, and can also be used for healthy subjects who need to improve their skin condition.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1, before and after treatment comparison, the front and right side photos of subject 001 before and after treatment, observing the overall skin condition and improvement of brown spots. Under Visia, the brown spot percentile improved from 25% to 33%. A Front, left side before treatment, right side after treatment. B Right side, left side before treatment, right side after treatment.

Figure 2, before and after treatment comparison, the front and right side photos of subject 002 before and after treatment, observing the overall skin condition and improvement of brown spots. Under Visia, the brown spot percentile improved from 27% to 92%. A front, left side before treatment, right side after treatment. B right side, left side before treatment, right side after treatment.

Figure 3, before and after treatment comparison, the front and right side photos of subject 003 before and after treatment, observing the overall skin condition and improvement of brown spots. Under Visia, there was no significant improvement in the percentile of brown spots. A Front, left side before treatment, right side after treatment. B Right side, left side before treatment, right side after treatment.

Figure 4, before and after treatment comparison, the front and right side photos of subject 004 before and after treatment, observing the overall skin condition and improvement of brown spots. Under Visia, the brown spot percentile improved from 54% to 69%. A front, left side before treatment, right side after treatment. B right side, left side before treatment, right side after treatment.

Figure 5, before and after treatment comparison, the front and right side photos of subject 005 before and after treatment, observing the overall skin condition and improvement of brown spots. Under Visia, the brown spot percentile improved from 16% to 19%. A front, left side before treatment, right side after treatment. B right side, left side before treatment, right side after treatment.

Figure 6, before and after treatment comparison, the front and right side photos of subject 006 before and after treatment, observing the overall skin condition and improvement of brown spots. Under Visia, the brown spot percentile improved from 44% to 48%. A front, left side before treatment, right side after treatment. B right side, left side before treatment, right side after treatment.

Figure 7, before and after treatment comparison, the front and right side photos of subject 007 before and after treatment, observing the overall skin condition and improvement of brown spots. Under Visia, the brown spot percentile improved from 29% to 30%. A Front, left side before treatment, right side after treatment. B Right side, left side before treatment, right side after treatment.

Figure 8, before and after treatment comparison, the front and right side photos of subject 008 before and after treatment, observing the overall skin condition and improvement of brown spots. Under Visia, there was no significant improvement in the percentile of brown spots. A Front, left side before treatment, right side after treatment. B Right side, left side before treatment, right side after treatment.

Figure 9, before and after treatment comparison, the front and right side photos of subject 009 before and after treatment, observing the overall skin condition and improvement of brown spots. Under Visia, the brown spot percentile improved from 3% to 11%. A front, left side before treatment, right side after treatment. B right side, left side before treatment, right side after treatment.

Figure 10, before and after treatment comparison, the front and right side photos of subject 010 before and after treatment, observing the overall skin condition and improvement of brown spots. Under Visia, the brown spot percentile improved from 22% to 33%. A Front, left side before treatment, right side after treatment. B Right side, left side before treatment, right side after treatment.

Figure 11, before and after treatment comparison, the front and right side photos of subject 011 before and after treatment, observing the overall skin condition and improvement of brown spots. Under Visia, the brown spot percentile improved from 39% to 50%. A Front, left side before treatment, right side after treatment. B Right side, left side before treatment, right side after treatment.

Figure 12, before and after treatment comparison, the front and right side photos of subject 012 before and after treatment, observing the overall skin condition and improvement of brown spots. Under Visia, there was no significant improvement in the percentile of brown spots. A Front, left side before treatment, right side after treatment. B Right side, left side before treatment, right side after treatment.

Figure 13: Viscosity curves of sodium hyaluronate with different molecular weights.

Figure 14: Viscosity curves of complex preparations of sodium hyaluronate with different molecular weights and recombinant albumin with different concentrations.

Figure 15: Affinity detection graph of recombinant human serum albumin and various ligands.

Figure 16: Comparison before and after treatment, the right side and frontal photos of subject 013 before and after treatment, observing the overall skin condition and the improvement of pores and red areas. Under Visia, the pores and red areas were effectively improved.

Figure 17: Comparison before and after treatment, the right side of subject 029 was photographed before and after treatment to observe the overall skin condition and the improvement of the red area. Under Visia, the red area was effectively improved.

Figure 18: Comparison before and after treatment, the right side of subject 018 was photographed before and after treatment, observing the overall skin condition and the improvement of red areas, brown spots and texture. Under Visia, the red areas, brown spots and texture were effectively improved.

DETAILED DESCRIPTION

The term "human serum albumin" (HSA) used in this article has the well-known meaning in the art, with CAS NO of 70024-90-7 and molecular weight of 66kD.

The term "human serum albumin" or "HSA" includes wild-type human serum albumin (see, for example, Gene name: ALB; NCBI ID: 213; UniProtKB-P02768), and also includes functional mutants or modified forms thereof. It will be appreciated by those skilled in the art that certain modifications may be made to wild-type human serum albumin, such as the addition, deletion or substitution of one or more amino acid residues, without substantially affecting its biological function. Therefore, the term "human serum albumin" used herein also encompasses such modified mutants or modified forms of human serum albumin.

Human serum albumin can be prepared by a variety of methods. Common commercially available HSA is often produced from human blood, and it can also be produced by recombinant methods. HSA products prepared by different processes have certain differences in performance, including the degree of oxidation, degree of saccharification, and biological activity.

Herein, "human serum albumin" may also be referred to as "human albumin", "human albumin" and the like.

Young healthy human serum albumin

The term "young undamaged human serum albumin" or "young undamaged HSA" as used herein refers to HSA in a fresh state without significant damage. "Damage" herein includes oxidation, glycation, carbonylation, and the like.

The skilled person will understand that the term "young, undamaged" HSA does not strictly require that the HSA has absolutely no damage. It is very difficult to prepare HSA without any damage. For the purposes of the present invention, HSA containing limited damage is still acceptable and can still be referred to as young, undamaged HSA.

"Young, intact" HSA typically exhibits at least one of the following four properties, such as two, three, and preferably four: (1) a higher proportion of free sulfhydryl groups at the Cys-34 residue; (2) lower levels of advanced glycation end products (AGEs); (3) lower levels of carbonylation; and (4) lower levels of homocysteine. Young, intact HSA typically has a higher proportion of free sulfhydryl groups and lower levels of AGEs, carbonyls, and homocysteine.

In some embodiments, "young and intact" HSA exhibits at least one of the following four properties, such as two, three, and preferably four: (1) the proportion of free thiol groups of Cys-34 residues determined by the Ellman method is greater than 50%, such as 70%, especially 80%, preferably 90%, and more preferably greater than 95%; (2) the level of advanced glycation end products (AGE) determined by ELISA (CLOUD-CLONE Co., CEB353Ge) is less than 60 μg/g protein, preferably 40 μg/g protein, and more preferably less than 30 μg/g protein; (3) the carbonyl level determined by the Protein Carbonyl Content Assay Kit is less than 1.7 nmol/mg protein, preferably less than 1.5 nmol/mg protein (4) the level of homocysteine measured by ELISA (Jianglai, JL10022) is lower than 5 nmol/g protein, preferably 3.5 nmol/g protein, and more preferably lower than 2 nmol/g protein.

In some embodiments, the present invention provides a preparation of young, undamaged human serum albumin (HSA) that exhibits at least one, e.g., two, three, and preferably four, of the following four properties when compared to an endogenous HSA preparation prepared from the plasma of a young individual: (1) a higher proportion of free sulfhydryl groups at Cys-34 residues; (2) lower levels of advanced glycation end products (AGEs); (3) lower levels of carbonylation; and (4) lower levels of homocystinylation.

The term "young individual" generally refers to a person under 30 years old, preferably under 18 years old, more preferably under 3 years old, or even under 1 year old.

The term "preparation" refers to a material prepared by a certain process. For example, an HSA preparation can be an HSA sample prepared from a specific individual or from multiple individuals, which contains a population of human serum albumin molecules. A "preparation" can be a packaged commercial product or a crude product prepared in the form of a solution or lyophilized powder.

Free sulfhydryl group: There is only one free sulfhydryl group in wild-type HSA, which is located at the Cys-34 residue. It is easy for a person skilled in the art to understand that a functional mutant of HSA may contain an amino acid sequence slightly different from that of wild-type HSA, and the Cys-34 residue may be located at a slightly different position from position 34. This can be easily determined by homology alignment and the like.

In some embodiments, the proportion of free thiol groups on the Cys-34 residue is determined by the Ellman method. Preferably, the proportion of free thiol groups is greater than 70%, particularly 80%, preferably 90%, more preferably greater than 95%.

AGE: Serum albumin is a plasma protein that is highly sensitive to glycation. This process, also known as the Maillard reaction, is a slow, non-enzymatic reaction that initially involves the attachment of glucose or derivatives to the free amine groups of albumin to form a reversible Schiff base product, resulting in the formation of a stable fructosamine residue (ketoamine) after the Amadori rearrangement. The Amadori product can then cyclize to form pyranose or furanose adducts. Further modifications of these early glycation products, such as rearrangement, oxidation, polymerization, and cleavage, produce irreversible conjugates called advanced glycation end products (AGE). The mass concentration of AGE was measured using an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions (CLOUD-CLONE Co., CEB353Ge). The results were normalized by the HSA concentration of each reaction and expressed as micrograms of AGE per gram of protein.

In some embodiments, the AGE level is determined by ELISA (CLOUD-CLONE Co., CEB353Ge).Preferably, the AGE level is lower than 60 μg/g protein, preferably lower than 40 μg/g protein, more preferably lower than 30 μg/g protein.

Carbonyl: Carbonylation here refers to the formation of carbonyl groups on the amino acid residues of HSA. Protein Carbonyl Content Assay Kit (ab126287) was used to quantify carbonyl groups in HSA. The method is based on the reaction of DNPH with protein carbonyls, which form DNP hydrazones and can be quantified using a microplate reader at an absorbance of 375 nm. Carbonyl molar concentrations were quantified using Protein Carbonyl Content Assay Kit (Abcam, ab126287) according to the manual. The results were normalized by the HSA concentration of each reaction and expressed as nanomoles carbonyl per milligram of protein.

In some embodiments, the level of carbonyl is determined by Protein Carbonyl Content Assay Kit. Preferably, the level of carbonyl is less than 1.7 nmol/mg protein, preferably less than 1.5 nmol/mg protein.

Homocysteine (HCY): Homocysteine (HCY) is metabolized from the essential amino acid Met, which has been found to be involved in protein post-translational modifications (PTMs). HCY can be linked to proteins through an isopeptide bond (N-Hcy-protein) with lysine (Lys) residues or through a disulfide bond (S-Hcy-protein) with Cys-34 residues. N-homocystinylation is an important post-translational modification that affects the structure and function of proteins and causes protein damage. N-homocystinylation can be measured by Hcy-thiolactone produced only by Hcy. The molar concentration of Hcy can be quantitatively determined by ELISA and HPLC. In our study, the molar concentration of Hcy was measured by the ELISA method according to the manufacturer's instructions (Jianglai, JL10022). The results were normalized by the HSA concentration of each reaction and expressed as nmol Hcy per gram of protein.

In some embodiments, the level of homocysteine is determined by ELISA (Jianglai, JL10022). Preferably, the level of homocysteine is lower than 5 nmol/g protein, preferably lower than 3.5 nmol/g protein, more preferably lower than 2 nmol/g protein.

Those skilled in the art will appreciate that the above four parameters can be measured by other generally recognized methods, and the numerical results of the parameters measured by different methods may be different. Those skilled in the art can easily compare the results measured from different methods through limited experiments.

In a preferred embodiment, the proportion of free sulfhydryl groups on the Cys-34 residue is greater than 80%, the AGE level is less than 30 μg/g protein, the carbonyl level is less than 1.5 nmol/mg protein, and the homocysteine level is less than 2 nmol/g protein.

Young, intact HSA preparations can be recombinantly produced or purified from human plasma. Preferably, the HSA preparation is recombinantly produced.

The rHSA used in this study was from Progen and was produced by recombinant means.

Skin repair preparations

Another aspect of the present invention provides a skin repair preparation, comprising: human serum albumin, and optionally a pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable carrier" as used herein refers to solid or liquid diluents, fillers, antioxidants, stabilizers and other substances that can be safely administered. Depending on the route of administration, various carriers well known in the art can be used, including, but not limited to, sugars, starch, cellulose and its derivatives, maltose, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffer, emulsifiers, isotonic saline, and/or pyrogen-free water, etc.

The term "optional" means that it may or may not be present. For example, "optional pharmaceutically acceptable carrier" means that the preparation may or may not contain a pharmaceutically acceptable carrier, which can be determined by those skilled in the art according to specific circumstances.

The term "therapeutically effective amount" as used herein refers to the amount of active substance sufficient to cause the biological or medical response desired by the clinician in the subject. The "therapeutically effective amount" can be determined by those skilled in the art based on factors such as the route of administration, the subject's weight, age, and condition. For example, a typical daily dose range can be 0.01 mg to 100 mg of active ingredient per kg body weight.

The drug provided by the present invention can be prepared into clinically acceptable dosage forms such as injections. The drug of the present invention can be administered to a subject by any appropriate route, for example, by mesodermal injection and the like.

Medical and aesthetic applications of human serum albumin

The present invention is based on the understanding of the basic physical and chemical properties and functions of human serum albumin, and uses human serum albumin to promote skin regeneration and repair and anti-aging. In some embodiments, mesodermal injection is used as a treatment method to observe the effect of albumin in improving the overall condition of the skin and alleviating skin problems such as spots, wrinkles, texture, pores, brown spots, and red areas (inflammation).

Those skilled in the art will appreciate that the human serum albumin in the present invention may be human serum albumin obtained from human blood or may be recombinantly produced.

The particularly preferred human serum albumin is young and undamaged human serum albumin. The molecular structure of young and undamaged recombinant human serum albumin prepared by genetic engineering technology is completely consistent with the natural newly synthesized albumin of the human body. In addition to the basic physiological functions of albumin, the natural conformation and physicochemical properties of the protein are maintained to the greatest extent during the preparation process. It has the young and undamaged characteristics of strong reducing power, low glycation modification, low carbonylation and homocysteine modification (see PCT international patent application PCT/CN2021/112093), thus having stronger hydrophilicity, antioxidant, anti-glycation, Anti-inflammatory and other functions. The above characteristics are the basic elements required for ideal skin anti-aging and repair products. Therefore, young and undamaged recombinant human serum albumin is expected to become a new star in mesotherapy besides traditional commonly used nutrients.

The young, undamaged recombinant human serum albumin used in the embodiments of the present invention is produced by Progi Inc. and is obtained by genetic engineering means using Pichia yeast fermentation and purification. The protein conformation is consistent with natural human serum albumin, and the purity exceeds 99.999999%. The proportion of Cys34 amino acid residues that provide reducing power in the reduced state exceeds 99%, and the overall protein post-modification levels such as glycation, carbonylation and homocysteineization are lower than those of commercially available blood-derived human serum albumin (see PCT/CN2021/112093).

In some embodiments of the present invention, 3 consecutive young non-damaged recombinant human serum albumin mesoderm injection treatments were completed in 12 volunteer subjects, and the treatment was evaluated by taking photos with the VISIA system before and one month after each treatment. In order to eliminate factors that interfere with the efficacy evaluation to the greatest extent, the subjects did not receive other medical beauty treatments except daily skin care from one month before treatment to one month after the last treatment, and fixed equipment, environment, relatively fixed operators, etc. were used to reduce possible interference factors and systematic errors in the process of injection, photography, and data evaluation.

In other embodiments of the present invention, after 4 months of follow-up observation among 30 volunteer subjects, it was proved that the use of recombinant albumin dermal mesotherapy injection for mesodermal injection can effectively improve the overall condition of the skin, especially pigmentation, pores, texture and sensitivity problems, and achieved statistically significant improvements.

Young, non-damaged recombinant human serum albumin has good safety and tolerance for mesodermal injection. The treatment effect analysis uses a combination of the GAIS method and the VISIA system score. The GAIS method is for two professional doctors to score the overall skin improvement based on the photographs and calculate the average value. The VISIA system score is a percentile quantitative value derived from the system's own data. It was found that the vast majority of subjects have achieved varying degrees of improvement in the overall skin condition, spots, wrinkles, texture, pores, brown spots, and red areas (inflammation). The VISIA system can also derive absolute value scores, which can be verified with the results of the GAIS method and VISIA percentile scores.

Due to the low viscosity of young and non-damaged recombinant albumin solutions, slight leakage of the syringe is prone to occur during mesoderm injection, as well as leakage caused by skin tissue extrusion when the pinhole is closed, which reduces the drug delivery efficiency. At the same time, young and non-damaged recombinant albumin solutions are very easy to be absorbed and metabolized through the blood-interstitial fluid material exchange in the dermis, and the effect is maintained for about 1 week, and it is impossible to continue to stay in the dermis to play a role. Therefore, the present invention further explores the composite formula of young and non-damaged recombinant albumin and sodium hyaluronate, and adjusts the overall viscosity of the composite preparation by adjusting the ratio of high, medium and low molecular weight sodium hyaluronate, thereby optimizing the product delivery efficiency and bioavailability, so that the effect is maintained for more than 6 weeks, and the use experience of doctors and patients is also improved.

Since sodium hyaluronate in different molecular weight ranges has different viscosity, filling support and moisturizing properties, the present invention attempts to combine recombinant human serum albumin with high molecular weight (2050kD-2950kD), medium molecular weight (800kD- The viscosity and stability of the composite preparations at different compounding ratios were tested, and the results shown in Table 5 and Figure 13 were obtained.

The present invention also tried to adjust the albumin concentration in the process of adjusting the composite formulation of albumin and sodium hyaluronate, and found that the albumin concentration had little effect on the overall viscosity and stability of the composite formulation. The specific data are shown in Table 6, Table 7 and Figure 14.

Taking advantage of the fact that albumin can bind to other proteins, lipids and other biomacromolecules, as well as various small molecule compounds, and can be a natural drug carrier for slow and sustained release, the present invention further attempted to add nicotinamide, α-lipoic acid, carnosine, resveratrol and other components with skin repair and anti-aging functions to young and undamaged recombinant human serum albumin to form a composite formula, and preliminarily observed the affinity of the above components to young and undamaged human serum albumin. The results are shown in Table 8 and Figure 15.

Example

Example 1: Mesotherapy trial using young intact recombinant human serum albumin

In this example, there were 12 subjects (female, aged 27-41 years old), each of whom received young and non-damaged human serum albumin mesoderm injection treatment once a month for 3 consecutive times. To avoid interference with the efficacy evaluation, all subjects did not receive any other medical beauty treatments from one month before the first treatment to one month after the last treatment.

The subjects collected data before the first treatment, one month after the first treatment, one month after the second treatment, and one month after the third treatment. The data before the first treatment was used as the initial baseline value for comparison and evaluation of the subsequent treatment effect. The treatment effect was evaluated by taking photos using a VISIA skin analyzer (produced by Canfield, USA, model 7.1.0). The image data of the front, left (45°) and right (45°) positions were collected under standard white light, 365nm ultraviolet light and cross-polarized light (Figures 1-12), and two professional doctors were asked to give GAIS scores for the overall improvement of the facial skin based on the image data, and then the mean was calculated (Table 1). In addition, the system's own data export and analysis functions were used for qualitative and quantitative evaluation (Tables 2-4).

The specific operation steps are as follows: the subject first cleans the face, takes a photo with the VISIA system after the skin dries naturally, and then applies anesthetic ointment (Tsinghua Unisplendour) for 30 minutes. After removing the anesthetic ointment, a young and non-damaged recombinant human serum albumin (see PCT/CN2021/112093) mesodermal injection is performed. The albumin concentration is 200mg/mL. The injection instrument uses a DermaShine electronic syringe, Panace-DS-30 model, and the injection needle uses a DermaShine brand disposable sterile injection needle. The injection depth is 0.8mm-1.2mm, the single injection volume is 0.0167ml-0.0250ml, and a total of 5ml is injected. After the full face injection is completed, apply a medical moisturizing dressing to calm the skin for 20 minutes, and then follow the routine precautions after mesodermal treatment for care.

Example 2: Young and undamaged recombinant human serum albumin mesodermal therapy can improve the facial skin condition of subjects

The fourth evaluation results collected in the experiment described in Example 1 (ie, the VISIA photography results one month after the third treatment) were directly compared with the initial baseline status collected before the first treatment, and the results described in Figures 1 to 12 were obtained.

The overall skin condition of most subjects was improved to varying degrees. The improvements mainly included skin translucency, reduction of acne marks and pigmentation, delicate skin texture, smaller pores, improved acne inflammation, reduced redness, and reduced fine lines under the eyes.

The overall improvement effect before and after treatment was evaluated by two professional doctors according to the VISIA system data and the GAIS scoring method, and the average value was obtained. The scoring is shown in Table 1, where 0 points are no improvement, 1 point is mild improvement, 2 points are moderate improvement, and 3 points are significant improvement. Among the 12 subjects, except for 2 subjects (05 and 07) with 0 points and no improvement, 10 subjects achieved different degrees of overall improvement, and 8 subjects achieved mild, mild-moderate, and moderate improvements.

Table 1. GAIS overall improvement score table before and after treatment

Example 3: Young and undamaged recombinant human serum albumin mesodermal therapy can alleviate facial skin problems in subjects

The fourth evaluation result collected in the experiment described in Example 1 (i.e., the VISIA photography result one month after the third treatment) was directly compared with the initial baseline state collected before the first treatment, and the VISIA system was used to automatically The image information was quantified and exported using software, and the quantitative results shown in Tables 2 to 4 were obtained for spots, wrinkles, textures, pores, brown spots, and red areas (inflammation).

The evaluation in this embodiment is expressed in percentile form, that is, the percentile of the state of each VISIA photo record within the comparable range of the system is given. The larger the value, the greater the probability of exceeding the comparable object, that is, the better the skin condition.

Table 2 shows the scores of various indicators before and after treatment derived from the analysis of VISIA images taken from the front side (front view), among which 41.7% of the subjects had improved spots, 41.7% of the subjects had improved wrinkles, 41.7% of the subjects had improved texture, 41.7% of the subjects had improved pores, 75% of the subjects had improved brown spots, and 25% of the subjects had improved red areas (inflammation).

Table 2. Percentile score table of frontal image information before and after treatment

Table 3 shows the scores of various indicators before and after treatment derived from the analysis of the VISIA images taken on the right, among which 50% of the subjects had improved spots, 41.7% of the subjects had improved wrinkles, 41.7% of the subjects had improved texture, 41.7% of the subjects had improved pores, 75% of the subjects had improved brown spots, and 25% of the subjects had improved red areas (inflammation).

Table 3. Percentile score table of right image information before and after treatment

Table 4 shows the scores of various indicators before and after treatment derived from the analysis of the VISIA images taken on the left, among which 50% of the subjects had improved spots, 33.3% of the subjects had improved wrinkles, 8.3% of the subjects had improved texture, 33.3% of the subjects had improved pores, 66.7% of the subjects had improved brown spots, and 50% of the subjects had improved red areas (inflammation).

Table 4. Percentile score table of left image information before and after treatment

Example 4: Exploration of Sodium Hyaluronate Concentration and Ratio

Young and undamaged recombinant human serum albumin has low viscosity. After mesoderm injection, some protein will leak out, affecting the product's effect and user experience. Therefore, we consider adding sodium hyaluronate components to the product formula. While increasing the product viscosity and reducing leakage, it can also give full play to the super moisturizing and water-locking properties of sodium hyaluronate. Referring to the viscosity of existing products on the market, we screened the types and concentrations of sodium hyaluronate, tested the viscosity of low, medium and high molecular weight sodium hyaluronate at different concentrations, and drew viscosity curves.

The experimental process is as follows: prepare sodium hyaluronate solutions of different concentrations and molecular weights, use a viscosity tester to test the viscosity, and draw a viscosity curve. The test conditions are: 3# rotor, 30rpm speed. Sodium hyaluronate was purchased from Shandong Zhongshan Biotechnology Co., Ltd., and the viscosity tester was purchased from Shanghai Hengping Instrument Factory, model: SNB-1.

Table 5: Viscosity analysis of sodium hyaluronate with different molecular weights and concentrations

Note: - indicates the viscosity exceeds the upper limit of instrument detection.

Example 5: Exploration of viscosity of composite preparations of recombinant human serum albumin and sodium hyaluronate at different concentrations

Combined with the viscosity curves of sodium hyaluronate with different molecular weights, different concentrations of sodium hyaluronate and different concentrations of recombinant human serum albumin were selected to prepare mixed solutions, and the viscosity of the mixed solutions was tested.

The experimental process is as follows: select sodium hyaluronate of high, medium and low molecular weight, prepare 0.5% and 1% solutions respectively, and then mix with recombinant human serum albumin to ensure that the final concentration of recombinant human serum albumin is 0, 25, 50, 75, 100 mg/ml, use viscosity testing instrument to test viscosity, draw viscosity curve, test conditions: 3# rotor, 30 rpm speed. Sodium hyaluronate was purchased from Shandong Zhongshan Biotechnology Co., Ltd., and the viscosity testing instrument was purchased from Shanghai Hengping Instrument Factory, model: SNB-1.

Table 6: Viscosity analysis of sodium hyaluronate and recombinant human serum albumin with different concentrations

Example 6: Determination of compound formulation formula

Based on the viscosity curve observed in Example 5, and making full use of the respective characteristics of high, medium and low molecular weight sodium hyaluronate, it is proposed to compound the above-mentioned sodium hyaluronates with different molecular weights in different proportions and study the viscosity and stability of the compounded solution.

The experimental process is as follows: select sodium hyaluronate of high, medium and low molecular weight, prepare solutions with concentrations of 0.5% and 1% according to Table 7, and then mix with recombinant human serum albumin to ensure that the final concentration of recombinant human serum albumin is 50 and 100 mg/ml, and use a viscosity tester to test the viscosity and draw a viscosity curve. The test conditions are: 3# rotor, 30 rpm speed. Sodium hyaluronate was purchased from Shandong Zhongshan Biotechnology Co., Ltd., and the viscosity tester was purchased from Shanghai Hengping Instrument Factory, model: SNB-1.

After comprehensive comparison of viscosity and stability research data, Scheme 1 was determined as the final formulation scheme, and a preliminary stability study was conducted. The 25°C accelerated test has been carried out for more than 9 months, and the compound preparation is still stable.

Table 7: Analysis of the formula of sodium hyaluronate and recombinant human serum albumin compound preparation

Example 7: Study on the affinity between functional small molecule compounds and recombinant albumin

Based on the formula of Scheme 1 determined in Example 6, we further studied the binding ability of functional small molecule compounds with recombinant albumin. The small molecule compounds include nicotinamide (NAD), L-carnosine, and α-lipoic acid. The research method is optical surface plasmon resonance, and the main equipment is molecular interaction analyzer, model: cytiva/Biacore 8K.

Experimental process: Prepare HAS-CM5 chip by coupling Series S Sensor Chip CM5 chip with protein sample; accurately weigh small molecule ligand, dissolve in appropriate solvent and dilute to working concentration; detect the affinity between receptor and ligand; the detection data is automatically processed by BIAcoreTM Insight Evaluation Software according to the Single cycle kinetics/Steady state affinity model to calculate the binding rate constant ka, dissociation rate constant kd, and dissociation constant KD:kd/ka.

The affinity of recombinant human albumin and ligands were tested respectively, and the affinity constants KD were Naproxen: 260μM, nicotinamide: 127μM, L-carnosine: 165μM and α-lipoic acid greater than 2000μM. Referring to the affinity results of positive control Naproxen and negative control Quinine small molecules and the affinity constant KD range of protein and small molecule binding ( ^10-3-10-6 ), it can be seen that ^nicotinamide and L-carnosine can have specific interaction with recombinant human albumin and have strong affinity; α-lipoic acid has no specific interaction with recombinant human albumin.

Table 8: Affinity analysis results of recombinant albumin and small molecules

Example 8: Evaluation of the efficacy and safety of albumin in improving skin quality

Clinical Data

Volunteer selection criteria:

(1) The subjects were aged 18 years or older and in good health;

(2) Clinical manifestations include dull facial complexion, flushing sensitivity, dry lines and fine wrinkles;

(3) had not received other facial cosmetic treatments in the 3 months before the trial and during the entire observation period;

(4) Agree to the project information and sign the project informed consent form;

(5) Willing to comply with the trial requirements and complete the entire trial, and able to complete follow-up as required.

Volunteer exclusion criteria:

(1) Pregnant or breastfeeding women;

(2) Those who have taken oral or topical drugs prohibited by the test (glucocorticoids, immunosuppressants and antiallergic drugs) within 14 days before the test, or have participated in similar tests or received facial treatments in the past three months;

(3) Skin diseases (e.g. psoriasis, eczema, skin cancer, etc.), or obvious erythema, wounds, abrasions, tattoos, etc. on or near the test area;

(4) those with coagulation disorders or taking prescription or over-the-counter blood thinning drugs (anticoagulants);

(5) severe complications (such as heart disease), autoimmune diseases, systemic blood diseases, exacerbation of chronic physical diseases, etc.;

(6) Immunosuppressive diseases (such as HIV positive) and use of immunosuppressive drugs;

(7) History of diseases that are prone to irritation, such as recurrence of herpes in the treatment area;

(8) Those with severe scarring constitution;

(9) Experts or professionals believe that there are other iatrogenic reasons that may affect the test results;

(10) Researchers participating in other clinical trials;

(11) Those who are not voluntary participants or are unable to complete the required content according to the trial requirements.

Experimental Materials and Methods

Test materials

(1) Recombinant albumin dermal mesotherapy injection (manufacturer: Shenzhen Progi Pharmaceutical Technology Co., Ltd.);

(2)VISIA skin image analyzer (produced by Canfield, USA, and represented by Guangzhou Hanma);

(3) Dermalab multifunctional skin tester;

(4) DermaShine electronic injector.

Preparation before treatment

Before treatment, the patient was informed of the treatment method, postoperative reactions and precautions and signed a preoperative informed consent form.

After cleansing, take 5 photos of the patient's face from the front, 45 degrees to the left and right, and 90 degrees to the left and right in the same environment, with the same camera, at the same angle and the same brightness.

The VISIA skin detector conducts a comprehensive monitoring of the skin's spots, wrinkles, texture, pores, UV spots, brown spots, red areas and porphyria from the left, frontal and right sides respectively, and then the multifunctional skin detector detects the skin's erythema and melanin index.

Treatment

Recombinant albumin dermal mesotherapy samples were used for mesodermal injection, where the albumin concentration was 100mg/mL, the injection instrument used was a DermaShine electronic syringe, Panace-DS-30 model, the injection needle used was a DermaShine brand disposable sterile injection needle, the injection depth was 0.8mm-1.2mm, the single injection volume was 0.0167ml-0.0250ml, and a total of 5ml was injected into the whole face. After the injection, a medical moisturizing dressing was applied to calm the skin for 20 minutes, and the subsequent care was carried out according to the conventional precautions after mesodermal treatment.

Once every four weeks, three times in total.

Treatment time points: week 0, week 4, week 8 (W0, W4, W8);

Testing time points: Week 0, Week 4, Week 8, Week 12 (W0, W4, W8, W12). Participants were revisited at W0 (before the first treatment), W4 (before the second treatment), W8 (before the third treatment), and W12 (4 weeks after treatment), and photos were taken and skin tests were performed with the help of instruments.

All subjects will return to the research center for efficacy and safety assessments at 4, 8, and 12 weeks after the first treatment.

Clinical effect evaluation

At week 0 (W0) before treatment and week 12 (W12) after the first treatment, the investigators and subjects evaluated:

(1) Dermlab facial test for erythema index and melanin index.

(2) Analysis of VISIA photo data.

(3) Evaluation of facial improvement effect: -1 point = worsening, 0 point = no improvement, 1 point = partial improvement, 2 points = significant improvement, 3 points = very significant improvement.

(4) Two doctors who were not involved in the treatment performed GPS scoring on facial fine lines, pigmentation, and telangiectasia based on the clinical data of the subjects. Wrinkles: Mild wrinkles are fine lines that appear only under certain facial expressions, such as the shallow wrinkles around the eyes when smiling. These wrinkles are usually short and shallow, with a small number and a relatively limited distribution = 1-2 points; moderate wrinkles are shallow wrinkles that can be clearly seen even when no expression is made, with moderate length, and may be distributed on the forehead, corners of the eyes, corners of the mouth, etc. = 3-4 points; severe wrinkles are deep and long wrinkles, with a large number and widely distributed in various areas of the face. They may be accompanied by skin sag and sagging = 5-6 points. Pigmentation: A small number of light pigmentation, with only a few lighter pigmentation spots on the face, such as freckles or mild sunburn, with a small diameter, the number may be between a few and a dozen, and the distribution is relatively scattered = 1-2 points; moderate pigmentation, with a certain number of pigmentation spots, slightly darker in color, slightly larger in diameter, and may be concentrated in certain specific areas, such as the cheeks or the bridge of the nose = 3-4 points; a large number of dark pigmentation spots, with a large number of dark pigmentation spots on the face, including chloasma, with a large diameter, a large number of spots, and a wide distribution = 5-6 points. Telangiectasia: A small number of mild telangiectasias, with only a small number of fine red blood streaks on the face, which are difficult to detect without careful observation, and the distribution is relatively limited = 1 point; moderate telangiectasias, with relatively obvious red blood streaks, the number is relatively large, and they are distributed in the cheeks and other parts, which are easier to detect = 2-3 points; severe telangiectasias, with a large number of obvious telangiectasias on the face, and the red blood streaks are obvious and widely distributed, which may affect the uniformity of facial skin color = 4 points.

Safety Assessment

During the follow-up visit, the subjects were evaluated for adverse reactions and asked whether the original symptoms of the target area worsened or new skin lesions appeared, such as itching, dryness, desquamation, rash, etc., and whether it interfered with their daily life.

Adverse reactions are graded as follows: severe - adverse reactions interfere with daily activities, the subjects feel that the symptoms are obvious, cannot be tolerated, and need to stop treatment; moderate - adverse reactions interfere with daily life. The subjects feel that the symptoms are obvious, but can be tolerated. No need to stop treatment; mild - adverse reactions are tolerable and the subjects can occasionally feel them. If the responsible doctor believes that the subject is not suitable for continuing the trial, or the subject requests to stop, the trial of the subject will be stopped, and the reason and time of the stop will be recorded.

Statistical analysis

SPSS software was used for data processing. The measurement data were expressed as mean ± standard deviation (x ± s). The t test was used for statistical analysis. The difference was considered statistically significant when p < 0.05.

Study Results

Demographics

A total of 30 subjects were included in this study, all of whom were female, aged 27-53 years old, with varying degrees of facial symptoms such as dull skin tone, spots, flushing, sensitive skin, dry lines and fine lines.

Clinical effect evaluation

Skin tester evaluation

The subjects were measured for erythema and melanin index using a Dermlab skin detector. The left or right side was selected by a random number table method for measuring the erythema index (EI), and the remaining side was measured for the melanin index (MI). Five points were measured, including 2 cm above the eyebrow, the outer side of the outer canthus, the highest point of the zygomatic bone, 2 cm beside the corner of the mouth, and the midpoint of the line between the corner of the mouth and the mandible. The five measured values were averaged and a paired sample t-test was performed. The data all obeyed the normal distribution, and the measured data before and after treatment were significantly correlated. Melanin index (Melanin) is an indicator for evaluating skin pigmentation, which is used to evaluate the level of pigmentation on the skin. The higher the index, the more pigment on the skin. Erythema index (Erythema) is an indicator for evaluating the degree of skin redness, which is used to evaluate the level of redness (hemoglobin) on the skin. The higher the index, the higher the degree of skin redness. The statistical analysis results are as follows:

Statistics of paired samples of melanin index

P＜0.05

Statistics of paired samples of erythema index

P＜0.05

The analysis results showed that the melanin index (MI) and erythema index (EI) decreased after treatment compared with the mean values before treatment, and the decrease was statistically significant.

VISIA Assessment

After the subjects took VISIA photos, the photos were analyzed by VISIA software to obtain the scores of various indicators. After taking the average of the three scores of the same volunteer in the same shot (front view, left 45°, right 45°), a paired sample t-test was performed. The analysis data conformed to the normal distribution, and the score data before and after treatment were significantly correlated. The statistical analysis results are as follows:

Visia texture score paired sample statistics

P＜0.05

Pore score paired sample statistics

P＜0.05

Brown spot paired sample statistics

P＜0.05

Red Zone Paired Sample Statistics

P＜0.05

From the above statistics, we can see that the characteristic scores of texture, pores, brown spots, and red areas in the VISIA scores before and after treatment were all reduced, and the p values were all less than 0.05, indicating that the improvements in the above observation indicators were statistically significant.

After analyzing the spots and wrinkles, although the overall scores were slightly reduced, the p value was greater than 0.05, and the reduction result was not statistically significant.

GPS Rating

Professional doctors who were not involved in the treatment performed GPS scores on pigmentation, telangiectasia, and wrinkles before and after treatment, and performed paired sample t-tests on the scores. The score data conformed to the normal distribution, and there was a significant correlation between the score data before and after treatment.

The statistical analysis results are as follows:

Statistics of paired samples of color spot GPS scores

P＜0.05

Telangiectasia GPS score paired sample statistics

P＜0.05

Statistics of paired samples of wrinkle GPS scores

P＜0.05

The results showed that the subjects' pigmentation, telangiectasia and wrinkle scores were improved after treatment, and the improvement was statistically significant. Figures 16-18 respectively selected representative cases of improvement effects, which were significantly improved in pores, red areas, brown spots and texture.

Subjective rating

The subjects scored the facial improvement effect according to the GAIS scale. The scoring results are as follows:

GAIS score

Among them, 7 cases were very significantly improved, 12 cases were significantly improved, and 10 cases were partially improved, with an improvement rate of 96.7%.

Safety Assessment

During the 4-month study, no serious adverse reactions were reported and the subjects tolerated the drug well.

in conclusion

This study evaluated the safety and efficacy of recombinant albumin dermal mesotherapy injection in improving problematic skin.

After 4 months of follow-up observation, the Dermlab test showed that the skin erythema-melanin index decreased after treatment compared with before treatment, and the decrease was statistically significant. VISIA photo data analysis showed that the skin texture, pores, brown spots, and red area scores all decreased, and the decrease was statistically significant. Professional doctors objectively evaluated the GPS scoring results and showed that the subjects' spots, capillary dilation, and wrinkle scores all decreased, and the decrease was statistically significant. The subjective feelings of the subjects also proved its effectiveness, with an improvement rate of 96.7%.

During the clinical study, no serious adverse reactions were reported and the subjects tolerated the drug well.

In summary, mesodermal injection therapy using recombinant albumin dermal mesotherapy can effectively improve skin quality, pigmentation and sensitivity problems.

The implementation methods of the present disclosure are not limited to the above-mentioned embodiments. Without departing from the spirit and scope of the present disclosure, ordinary technicians in this field can make various changes and improvements to the present disclosure in form and details, and these are considered to fall within the protection scope of the present disclosure.

Claims (26)

Use of human serum albumin in the manufacture of a medicament for improving the skin condition of a subject. The use of claim 1, wherein the human serum albumin is human serum albumin prepared from human blood. The use of claim 1, wherein the human serum albumin is young and undamaged human serum albumin. The use of claim 3, wherein the young, undamaged human serum albumin exhibits at least one, for example two, three, and preferably four, of the following four properties when compared to an endogenous human serum albumin preparation prepared from plasma of young individuals: (1) a higher proportion of free sulfhydryl groups on the Cys-34 residue; (2) a lower level of advanced glycation end products (AGE); (3) a lower level of carbonylation; and (4) a lower level of homocysteine. The use of claim 3, wherein the young undamaged human serum albumin exhibits at least one of the following four properties, for example, two, three, and preferably four: (1) the proportion of free thiol groups on the Cys-34 residue determined by the Ellman method is greater than 50%, for example, 70%, in particular 80%, preferably 90%, and more preferably greater than 95%; (2) the level of advanced glycation end products (AGE) determined by ELISA (CLOUD-CLONE Co., CEB353Ge) is lower than 60 μg/g protein, preferably lower than 40 μg/g protein, and more preferably lower than 30 μg/g protein; (3) the carbonyl level determined by the Protein Carbonyl Content Assay Kit is lower than 1.7 nmol/mg protein, and preferably lower than 1.5 nmol/mg protein; (4) the level of homocysteine determined by ELISA (Jianglai, JL10022) is lower than 5 nmol/g protein, preferably lower than 3.5 nmol/g protein, and more preferably lower than 2 nmol/g protein. The use of claim 3, wherein the proportion of free thiol groups on the Cys-34 residue is greater than 80%, the AGE level is lower than 30 μg/g protein, the carbonyl level is lower than 1.5 nmol/mg protein, and the homocysteine level is lower than 2 nmol/g protein. The use according to any one of claims 1 to 6, wherein the drug further comprises sodium hyaluronate. The use of claim 7, wherein the concentration of human serum albumin in the drug is 100 mg/ml and the concentration of sodium hyaluronate is 1.00%. The use according to any one of claims 1 to 8, wherein the medicine further comprises another functional component. The use according to any one of claims 1 to 9, wherein the functional component is selected from the group consisting of the following members: nicotinamide, α-lipoic acid, carnosine, and resveratrol. The use according to any one of claims 1 to 10, wherein the drug is used for mesodermal injection into a subject. The use according to any one of claims 1 to 11, wherein the drug is in the form of an injection. The use according to any one of claims 1 to 12, wherein the subject is a healthy subject in need of improving skin condition. A skin repair preparation comprising: human serum albumin, and optionally a pharmaceutically acceptable carrier. The skin repair preparation according to claim 14, wherein the human serum albumin is human serum albumin prepared from human blood. The skin repair preparation of claim 14, wherein the human serum albumin is young and undamaged human serum albumin. The skin repair preparation of claim 16, wherein the young, undamaged human serum albumin exhibits at least one, e.g., two, three, and preferably four, of the following four properties when compared to an endogenous human serum albumin preparation prepared from plasma of young individuals: (1) a higher proportion of free sulfhydryl groups at Cys-34 residues; (2) lower levels of advanced glycation end products (AGEs); (3) lower levels of carbonylation; and (4) lower levels of homocystinylation. The skin repair preparation of claim 16, wherein the young, undamaged human serum albumin exhibits at least one of the following four properties, for example, two, three, and preferably four: (1) the proportion of free thiol groups on the Cys-34 residue determined by the Ellman method is greater than 50%, for example, 70%, in particular 80%, preferably 90%, and more preferably greater than 95%; (2) the level of advanced glycation end products (AGE) determined by ELISA (CLOUD-CLONE Co., CEB353Ge) is lower than 60 μg/g protein, preferably lower than 40 μg/g protein, and more preferably lower than 30 μg/g protein; (3) the carbonyl level determined by the Protein Carbonyl Content Assay Kit is lower than 1.7 nmol/mg protein, and preferably lower than 1.5 nmol/mg protein; (4) the level of homocysteine determined by ELISA (Jianglai, JL10022) is lower than 5 nmol/g protein, preferably lower than 3.5 nmol/g protein, and more preferably lower than 2 nmol/g protein. The skin repair preparation of claim 16, wherein the proportion of free sulfhydryl groups on the Cys-34 residue is greater than 80%, the AGE level is lower than 30 μg/g protein, the carbonyl level is lower than 1.5 nmol/mg protein, and the homocysteine level is lower than 2 nmol/g protein. The skin repair preparation according to any one of claims 14 to 19, wherein the preparation further comprises sodium hyaluronate. The skin repair preparation according to any one of claims 14 to 20, wherein the concentration of human serum albumin in the preparation is 100 mg/ml and the concentration of sodium hyaluronate is 1.00%. The skin repair preparation according to any one of claims 14 to 21, wherein the preparation further comprises an additional functional component. The skin repair preparation according to any one of claims 14 to 22, wherein the functional component is selected from the group consisting of niacinamide, α-lipoic acid, carnosine, and resveratrol. The skin repair preparation according to any one of claims 14 to 23, wherein the preparation is for mesodermal injection into a subject. The skin repair preparation according to any one of claims 14 to 24, wherein the preparation is in the form of an injectable dosage form. The skin repair preparation according to any one of claims 14 to 25, wherein the preparation is for use in healthy subjects in need of improving skin condition.