WO2021077683A1 - Hyaluronic acid gel for hyaluronic acid injection and use method for hyaluronic acid gel - Google Patents
Hyaluronic acid gel for hyaluronic acid injection and use method for hyaluronic acid gel Download PDFInfo
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- WO2021077683A1 WO2021077683A1 PCT/CN2020/082589 CN2020082589W WO2021077683A1 WO 2021077683 A1 WO2021077683 A1 WO 2021077683A1 CN 2020082589 W CN2020082589 W CN 2020082589W WO 2021077683 A1 WO2021077683 A1 WO 2021077683A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
- A61K8/04—Dispersions; Emulsions
- A61K8/042—Gels
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
- A61K8/735—Mucopolysaccharides, e.g. hyaluronic acid; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/91—Injection
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- the present invention relates to the field of biomedicine, in particular to a hyaluronic acid gel for water-light injection, and also relates to a method of using the hyaluronic acid gel for water-light injection.
- Water and light injection is currently one of the most extensive medical beauty projects at home and abroad. It can effectively solve the problem of skin care products that are not easily absorbed through the epidermal barrier. It can quickly and effectively replenish the skin, thereby effectively improving skin quality and delaying senescence.
- Water-light injection originated from Mesotherapy proposed by French doctor Michal Pistor in 1952. The idea is to inject a small amount of therapeutic substance into the local superficial surface. After decades of hard work, the current water-light acupuncture technology was developed.
- Water-light injection uses negative pressure technology to accurately pierce several extremely thin needles into the skin about 1 to 2 mm, and directly transport the repairing substance through the epidermal layer into the epidermis and the dermis or the dermis layer, which solves the effective ingredients in skin care products.
- Hyaluronic acid also known as hyaluronic acid, is an acidic mucopolysaccharide inherent in the human body. It is widely used in various skin care products. It is a super moisturizing agent naturally present in human skin and can maintain moisture 1,000 times its own weight. The moisture level of the skin is closely related to the content and molecular weight of hyaluronic acid. High molecular weight hyaluronic acid has stronger water retention properties. With the increase of age and the destruction of daily active oxygen free radicals, the endogenous hyaluronic acid in the skin is continuously degraded and lost, and the water retention function is gradually weakened, which leads to various skin aging symptoms, such as dryness, roughness, and dullness. , Relaxation, wrinkles, etc. (Stern et al. Clinics in Dermatology 2008, 26: 106-122; Papakonstantinou et al., Dermatoendocrinol 2012, 4: 253-258).
- Supplementing exogenous hyaluronic acid is an important measure to improve skin texture, delay aging symptoms and restore youthfulness.
- the hyaluronic acid applied on the skin surface is difficult to be effectively absorbed, especially the high molecular weight hyaluronic acid with super moisturizing ability.
- Water-light injection of hyaluronic acid can directly penetrate the epidermal barrier, and can immediately replenish hyaluronic acid in the superficial layer of the skin to achieve the effect of rapid moisturizing and moisturizing and achieve the ultimate goal of improving skin quality.
- water-light injection is mainly superficial injection to supplement exogenous hyaluronic acid, and it also contains other restorative nutrients as appropriate.
- CN 106074213A discloses a hyaluronic acid gel for water and light injection, which consists of 10-30 parts of sodium hyaluronate, 0.5-3 parts of glycerin, 0.5-1.5 parts of mannitol, and 1-9 parts of mannitol. The pH adjuster and 25 to 45 parts of isotonic adjuster.
- CN 105233270A discloses a pharmaceutical composition for water-light therapy and its application.
- composition includes: 0.01-0.5wt% high molecular weight sodium hyaluronate, 0.01-0.3wt% medium molecular weight sodium hyaluronate, 0.01-1wt% % Low molecular weight sodium hyaluronate, 0.01-1wt% oligomeric sodium hyaluronate, 0.01-5wt% collagen, 0.05-1.8wt% adjuvant, recombinant human epidermal growth factor, the balance is water, reconstituted
- concentration of human epidermal growth factor is 0.5-10 ⁇ g/ml; the molecular weight of high molecular weight sodium hyaluronate is 1.3-2 million daltons, the molecular weight of medium molecular weight sodium hyaluronate is 500,000-1 million daltons, and the low molecular weight is transparent
- the molecular weight of sodium phosphate is 50,000 to 300,000 daltons, and the molecular weight of oligomeric sodium hyaluronate is
- hyaluronic acid is rapidly degraded into small molecular fragments and enters the body fluid circulation to be absorbed and metabolized, and loses the function of moisturizing and moisturizing in the skin; in the skin, the half-life of hyaluronic acid is less than 1 Tian (Laurent et al., Exp Physiol 1991, 76: 695-703). Therefore, although water injection can quickly replenish hyaluronic acid in the skin, it can only moisturize and moisturize in a short time, and it is difficult to achieve long-term effects such as improving skin texture, delaying aging and restoring youthfulness. For example, the research results of Amin et al.
- Cross-linking modification can delay the degradation and absorption of hyaluronic acid in the body, such as 1,4-butanediol diglycidyl ether (BDDE) cross-linked hyaluronic acid gel (such as Rui Lan Etc.)
- BDDE 1,4-butanediol diglycidyl ether
- the degradation and absorption time under the skin can be as long as 6 months, and it is widely used for wrinkle injection and filling.
- cross-linked hyaluronic acid gel products are very high (usually not less than 20mg/mL) (Edsman et al., Dermatol Surg 2012, 38:1170-1179), that is, the water content is usually not more than 98% (cross-linked).
- the water content of hyaluronic acid is about 49 times its own weight), and its moisturizing and moisturizing properties are poor; and these cross-linked hyaluronic acid gel products also have greater gel strength and need to be crushed into particles before they can be used.
- this application provides a disulfide bond cross-linked hyaluronic acid gel for water-light injection, which has longer degradation and absorption time and longer hydration and moisturizing maintenance than existing hyaluronic acid Time, impurity toxicity is small, and it is suitable for water-light injection, and does not produce the bulging phenomenon that current cross-linked hyaluronic acid gel products are prone to. Therefore, water-light injection can more effectively improve the skin texture.
- a hyaluronic acid gel for water-light injection wherein the hyaluronic acid is disulfide bond cross-linked hyaluronic acid, and the content of the hyaluronic acid is not more than 10 mg/mL.
- the content of disulfide crosslinked hyaluronic acid is expressed as the weight (mg/mL) of crosslinked hyaluronic acid contained in the unit volume of the disulfide crosslinked hyaluronic acid gel.
- the content of the cross-linked hyaluronic acid for water-light injection of the present invention is preferably between 4-8 mg/mL, particularly preferably between 5.5-7 mg/mL.
- the disulfide bond cross-linked hyaluronic acid gel used for water-light injection of the present invention has a low content, which is only current
- the cross-linked hyaluronic acid gel product is less than 50%; therefore, the disulfide bond cross-linked hyaluronic acid gel for water injection of the present invention has lower strength, better fluidity, and is easy to pass water
- the ultra-fine needle of the light needle is used for injection and achieves uniform distribution in the superficial subcutaneous layer.
- the water content of the disulfide cross-linked hyaluronic acid gel for water-light injection of the present invention is not less than 99% (99 times or more of the weight of the disulfide cross-linked hyaluronic acid), and can further It absorbs water and retains water to about 1000 times the weight of disulfide cross-linked hyaluronic acid, so it has a water retention capacity similar to that of non-cross-linked hyaluronic acid.
- a method similar to that disclosed in the applicant's prior art patent document CN 101721349A can be used to prepare the disulfide bond cross-linked hyaluronic acid gel for water light injection of the present invention.
- the preparation method realizes disulfide bond cross-linking by the hyaluronic acid sulfhydryl derivative aqueous solution under the oxidation of oxygen (oxygen in the air and/or dissolved oxygen in the aqueous solution), without the need to add a cross-linking agent, and the by-products are Water has many advantages such as simple production process and no impurities, which avoids the current complicated process of cross-linking hyaluronic acid gel products and the potential toxic and side effects caused by the residual cross-linking agent.
- the above-mentioned preparation method of the disulfide bond cross-linked hyaluronic acid gel for water injection does not require a purification process, and is particularly suitable for preparing crosslinked gels with low strength and certain fluidity.
- Liangel is suitable for water injection.
- the hyaluronic acid sulfhydryl derivative disclosed in the applicant’s prior art patent document CN 102399295A can be used to prepare the disulfide cross-linked hyaluronic acid gel for water injection of the present invention. glue.
- These hyaluronic acid sulfhydryl derivatives better maintain the initial structure, physiological functions and biocompatibility of hyaluronic acid, and can achieve effective disulfide bond cross-linking, delaying metabolic absorption in the body, and therefore have better properties than non-crosslinking Combined with hyaluronic acid for longer hydrating and moisturizing maintenance time.
- the hyaluronic acid of the present invention can be in the form of its salt (such as sodium salt, potassium salt, zinc salt, calcium salt, etc.) and/or its derivative form (such as carboxymethylated hyaluronic acid, acetylated hyaluronic acid, etc.) Acid, etc.).
- its salt such as sodium salt, potassium salt, zinc salt, calcium salt, etc.
- its derivative form such as carboxymethylated hyaluronic acid, acetylated hyaluronic acid, etc.) Acid, etc.
- the hyaluronic acid sulfhydryl derivatives refer to hyaluronic acid derivatives containing sulfhydryl groups, which can be prepared by sulfhydryl modification of hyaluronic acid, and also include various hyaluronic acid derivatives further through sulfhydryl groups.
- the sulfhydryl derivative prepared by chemical modification.
- the side chain carboxyl groups, side chain hydroxyl groups, and reducing end groups of hyaluronic acid or its salts or derivatives are usually reactive functional groups that can be modified by sulfhydrylization, as disclosed in previous documents such as WO 2009/006780A1.
- the preparation methods of hyaluronic acid sulfhydryl derivatives can be used to prepare the hyaluronic acid sulfhydryl derivatives of the present invention.
- the sulfhydryl derivatives of hyaluronic acid with various sulfhydryl content can be used to prepare the disulfide bond cross-linked hyaluronic acid gel for water-light injection of the present invention.
- the sulfhydryl content is expressed as the number of micromoles of sulfhydryl groups per gram of hyaluronic acid sulfhydryl derivative ( ⁇ mol/g).
- the preferred sulfhydryl content is between 10-50 ⁇ mol/g, and the particularly preferred sulfhydryl content is between 20-40 ⁇ mol/g. between g.
- Another object of the present invention is to provide a method for using the disulfide bond cross-linked hyaluronic acid gel for water injection.
- the water-light injection method well known to those skilled in the art can be used directly.
- Water injection such as Derma (Demasa) and other water-light injection instruments use five-needle or nine-needle ultra-fine negative pressure needles to perform water-light injection.
- the content of the disulfide cross-linked hyaluronic acid gel used for water-light injection of the present invention is high (for example, when 4-10 mg/mL)
- the pushing force for direct water-light injection is greater
- it can be appropriately diluted to a lower content (usually less than 4mg/mL) before the water light injection, and the water light injection can be carried out smoothly.
- the dilution process can be carried out in a syringe connected by a two-way or a three-way joint.
- One syringe is equipped with the disulfide cross-linked hyaluronic acid gel for water-light injection of the present invention, and the other syringe is equipped with Physical dilution and mixing can be achieved by reciprocating physiological saline, buffer solution that is isotonic with body fluids or nutrient active substance solution (such as solutions containing growth factors, amino acids, vitamins, etc.).
- physiological saline, buffer solution that is isotonic with body fluids or nutrient active substance solution such as solutions containing growth factors, amino acids, vitamins, etc.
- nutrient active substance solution such as solutions containing growth factors, amino acids, vitamins, etc.
- the disulfide cross-linked hyaluronic acid gel used for water-light injection of the present invention overcomes the shortcomings of current cross-linked hyaluronic acid gel products when used for water-light injection, and has a lower content and better Fluidity, easy to inject through the ultra-fine needle of the water-light needle, can achieve good uniform distribution in the superficial subcutaneous layer, without bulging; at the same time, it has a longer hydrating and moisturizing effect than uncrosslinked hyaluronic acid, which can be effective Improve skin texture.
- the disulfide bond cross-linked hyaluronic acid gel for water-light injection of the present invention can be conveniently prepared by hyaluronic acid sulfhydryl derivatives, and has the advantages of not requiring the addition of cross-linking agents, simple preparation process, and no impurities, etc. Many advantages can avoid the current complicated process of cross-linking hyaluronic acid gel products and the toxic and side effects caused by the residual cross-linking agent.
- disulfide cross-linked hyaluronic acid gel for water-light injection of the present invention can also be physically mixed with other nutrient active substances before use to achieve the synergistic effect of water-light treatment.
- Figure 2 The effect of disulfide cross-linked sodium hyaluronate gel on skin elasticity after water injection.
- Figure 3 The effect of disulfide cross-linked sodium hyaluronate gel on the depth of skin wrinkles after water injection.
- Hyaluronic acid sulfhydryl derivatives are prepared from sodium hyaluronate with a molecular weight of 180KDa as a raw material and prepared by the method reported by Shu et al. (Shu et al., Biomacromolecules 2002, 3:1304-1311). The sulfhydryl content is 24 ⁇ mol/g and 38 ⁇ mol, respectively. /g and 57 ⁇ mol/g.
- Example 2 Evaluation of the injectable properties of disulfide bond cross-linked hyaluronic acid gel
- Adopt Derma (Demasha) 2nd generation water-light injection instrument test the injectability of the gel prepared in Example 1 through conventional water-light injection needles (nine needles), the test results are as follows:
- the content of cross-linked hyaluronic acid is 3mg/mL and 4mg/mL, it usually has good injectability; and when the content is ⁇ 5.5mg/mL, it is difficult to inject through the water-light injection device.
- Example 3 Evaluation of the injectable properties of disulfide cross-linked hyaluronic acid gel after dilution
- the gel prepared in Example 1 (the content of cross-linked hyaluronic acid are: 5.5mg/mL, 6.5mg/mL, 7mg/mL, 8mg/mL and 10mg/mL; the sulfhydryl content of the sulfhydryl derivative is 38 ⁇ mol /g)
- the dilution process is carried out in a syringe connected by a two-way connector.
- One syringe contains the gel prepared in Example 1 (1mL), and the other syringe contains an appropriate volume of normal saline (1mL, 1.5mL, 3mL). Push back and forth 60 to 80 times for physical dilution and mixing.
- Adopt Derma (Demasa) 2nd generation water-light injection instrument test the injectability of the diluted gel through a conventional water-light injection needle (nine needles), the test results are as follows:
- Example 4 Evaluation of water absorption and water retention performance of disulfide bond cross-linked hyaluronic acid gel
- Example 3 Using the same dilution process as in Example 3, the gel prepared in Example 1 and physiological saline were physically mixed and diluted according to a volume ratio of 1:2, and then put into a glass bottle and left standing at room temperature for 1 hour. No obvious The delamination of the water phase and the gel phase indicates that the gel has good water absorption and water retention properties (up to about 1000 times the weight of cross-linked hyaluronic acid).
- Example 5 Evaluation of the injectable properties of disulfide cross-linked hyaluronic acid gel after dilution
- Example 3 Using the same mixing and dilution process as in Example 3, the gel prepared in Example 1 (content is 6.5mg/mL; the sulfhydryl content of the sulfhydryl derivative is 57 ⁇ mol/g) and 5 kinds of aqueous solutions (each containing 0.5mg/ml) mL vitamin C, vitamin B2, arginine, lysine and glutamine) are physically mixed and diluted according to the volume ratio of 1:1 and 1:2.
- Adopt Derma (Demasha) 2nd generation water-light injection instrument test the injectability of each diluted gel through a conventional water-light injection needle (nine needles); the test results show that each diluted gel has good injectability.
- Hyaluronic acid sulfhydryl derivatives are prepared using sodium hyaluronate with molecular weights of 300KDa and 1,500KDa as raw materials, prepared by the method reported by Shu et al. (Shu et al., Biomacromolecules 2002, 3:1304-1311), and their sulfhydryl content is 33 ⁇ mol/ g and 40 ⁇ mol/g.
- Example 7 Injectable properties of disulfide bond cross-linked hyaluronic acid gel
- Adopt Derma (Demasha) 2nd generation water-light injection device test the injectability of the gel prepared in Example 6 through conventional water-light injection needles (nine needles).
- the test results of this example show that when the content is 3 mg/mL and 4 mg/mL, it usually has good injectability; while the concentration is ⁇ 5.5 mg/mL, it is difficult to inject through the water-light injection device.
- Example 8 Evaluation of the injectable properties of disulfide cross-linked hyaluronic acid gel after dilution
- Example 6 The gel prepared in Example 6 (contents: 5.5 mg/mL, 6.5 mg/mL, 7 mg/mL, 8 mg/mL, and 10 mg/mL) and physiological saline were in a volume ratio of 1:1, 1:1.5, and 1. :2 Perform physical mixing and dilution. The dilution process is the same as in Example 3.
- Adopt Derma (Demasha) 2nd generation water-light injection instrument test the injectability of the diluted gel through a conventional water-light injection needle (nine needles). The test results of this embodiment show that when diluted to a content ⁇ 4 mg/mL, it has good injectability.
- Example 9 Evaluation of hydro-injection effect of disulfide bond cross-linked hyaluronic acid gel
- the samples used in the test group were: 2 mL of the gel prepared in implementation 1 (the content of cross-linked hyaluronic acid was 6.5 mg/mL; the sulfhydryl content of the thiolated derivative was 38 ⁇ mol/g), which was mixed and diluted with 3 mL of physiological saline before use.
- the mixing and dilution process is the same as in Example 3; the total sample volume is 5 mL, and the final content of cross-linked sodium hyaluronate is 2.6 mg/mL.
- the sample used in the control group was a non-crosslinked sodium hyaluronate aqueous solution (molecular weight 1.5MDa) with the same volume and the same concentration.
- the relative increase in skin moisture before and after injection (%) was tested with a skin moisture meter, the relative increase in skin elasticity (%) before and after injection was tested with a skin elasticity meter, and the relative decrease in average wrinkle depth before and after injection was tested with a fast three-dimensional imaging system Evaluation.
- test results of this embodiment are shown in Figures 1-3.
- the disulfide cross-linked hyaluronic acid gel has a better hydro-light treatment effect, and the maintenance time is longer; at the same time, all test subjects in the treatment group did not find bulging phenomenon, indicating two Sulfur-bonded cross-linked hyaluronic acid gel has good fluidity and can be evenly distributed on the superficial layer of water injection.
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Abstract
Disclosed in the present invention is a hyaluronic acid gel for hyaluronic acid injection. The hyaluronic acid gel is a disulfide bond crosslinked hyaluronic acid, and the content of hyaluronic acid in the gel is not more than 10 mg/mL. The crosslinked hyaluronic acid gel provided by the present invention has unique advantages when used for hyaluronic acid injection, is easily injected by means of an extremely fine needle of a hyaluronic acid syringe, and can realize good and uniform distribution in a subcutaneous layer without bulging; moreover, the hyaluronic acid gel has a moisturizing effect longer than that of uncrosslinked hyaluronic acid, and can effectively improve skin texture.
Description
本发明涉及生物医药领域，特别涉及一种用于水光注射的透明质酸凝胶，还涉及所述用于水光注射的透明质酸凝胶的使用方法。The present invention relates to the field of biomedicine, in particular to a hyaluronic acid gel for water-light injection, and also relates to a method of using the hyaluronic acid gel for water-light injection.
近年来国内外医疗美容行业发展迅速，研发了各类新兴美容技术。水光注射是当前国内外开展最为广泛的医疗美容项目之一，能有效解决体表使用护肤品不易透过表皮屏障吸收的问题，可快速有效给予肌肤补水补养，因而可有效改善肤质及延缓衰老。水光注射源于法国医生Michal Pistor于1952年提出的中胚层疗法(Mesotherapy)，其理念是在局部浅表注射微量治疗物质；之后经过几十年的努力开发出了当前的水光针技术。水光注射通过负压技术将数个极细的针头精确刺入皮下约1～2毫米处，透过表皮层直接将修复物质输送入表皮和真皮之间或真皮层，解决了护肤品中有效成分无法有效穿透表皮屏障的问题。In recent years, the domestic and foreign medical beauty industry has developed rapidly, and various emerging beauty technologies have been developed. Water and light injection is currently one of the most extensive medical beauty projects at home and abroad. It can effectively solve the problem of skin care products that are not easily absorbed through the epidermal barrier. It can quickly and effectively replenish the skin, thereby effectively improving skin quality and delaying senescence. Water-light injection originated from Mesotherapy proposed by French doctor Michal Pistor in 1952. The idea is to inject a small amount of therapeutic substance into the local superficial surface. After decades of hard work, the current water-light acupuncture technology was developed. Water-light injection uses negative pressure technology to accurately pierce several extremely thin needles into the skin about 1 to 2 mm, and directly transport the repairing substance through the epidermal layer into the epidermis and the dermis or the dermis layer, which solves the effective ingredients in skin care products. The problem of inability to effectively penetrate the epidermal barrier.
透明质酸又名玻尿酸，是一种人体内固有的酸性黏多糖，被普遍应用于各种护肤品，是人体肌肤中天然存在的超强保湿剂，可维持自身重量1000倍的水份。肌肤的湿润水平与透明质酸含量及分子量密切相关，高分子量的透明质酸具有更强的保水性能。随着年龄的增长以及日常活性氧自由基的破环作用，肌肤中内源性透明质酸不断被降解流失，保水功能逐渐弱化，因而导致了各种肌肤衰老症状，如干燥、粗糙、暗沉、松弛、皱纹等等(Stern等，Clinics in Dermatology 2008,26:106-122；Papakonstantinou等，Dermatoendocrinol 2012,4:253-258)。Hyaluronic acid, also known as hyaluronic acid, is an acidic mucopolysaccharide inherent in the human body. It is widely used in various skin care products. It is a super moisturizing agent naturally present in human skin and can maintain moisture 1,000 times its own weight. The moisture level of the skin is closely related to the content and molecular weight of hyaluronic acid. High molecular weight hyaluronic acid has stronger water retention properties. With the increase of age and the destruction of daily active oxygen free radicals, the endogenous hyaluronic acid in the skin is continuously degraded and lost, and the water retention function is gradually weakened, which leads to various skin aging symptoms, such as dryness, roughness, and dullness. , Relaxation, wrinkles, etc. (Stern et al. Clinics in Dermatology 2008, 26: 106-122; Papakonstantinou et al., Dermatoendocrinol 2012, 4: 253-258).
补充外源性透明质酸是改善肌肤肤质、延缓衰老症状及恢复年轻态的重要措施。然而由于表皮屏障的存在，涂抹在肌肤表面的透明质酸很难被有效吸收，特别是具有超强保湿能力的高分子量透明质酸。水光注射透明质酸可直接透过表皮屏障，可即刻在肌肤浅表层补充透明质酸，以实现快速补水保湿的功效及达到改善肤质的终极目的。当前水光注射以浅表注射补充外源性透明质酸为主，同时也适当含有其它修复营养物质。CN 106074213A公开了一种用于水光注射的透明质酸凝胶，其组成包括10～30份的透明质酸钠、0.5～3份的甘油、0.5～1.5份的甘露醇、1～9份的pH调节剂和25～45份的等渗调节剂。CN 105233270A公开了用于水光治疗的药物组合物及其应用，其组成包括：0.01～0.5wt％的高分子量透明质酸钠、0.01～0.3wt％的中分子量透明质酸钠、0.01～1wt％的低分子量透明质酸钠、0.01～1wt％的寡聚透明质酸钠、0.01～5wt％的胶原蛋白、0.05～1.8wt％的辅剂、重组人表皮生长因子，余量为水，重组人表皮 生长因子的浓度为0.5～10μg/ml；高分子量透明质酸钠的分子量为130～200万道尔顿，中分子量透明质酸钠的分子量为50～100万道尔顿，低分子量透明质酸钠的分子量为5～30万道尔顿，寡聚透明质酸钠的分子量为3000～8000道尔顿。Supplementing exogenous hyaluronic acid is an important measure to improve skin texture, delay aging symptoms and restore youthfulness. However, due to the existence of the epidermal barrier, the hyaluronic acid applied on the skin surface is difficult to be effectively absorbed, especially the high molecular weight hyaluronic acid with super moisturizing ability. Water-light injection of hyaluronic acid can directly penetrate the epidermal barrier, and can immediately replenish hyaluronic acid in the superficial layer of the skin to achieve the effect of rapid moisturizing and moisturizing and achieve the ultimate goal of improving skin quality. At present, water-light injection is mainly superficial injection to supplement exogenous hyaluronic acid, and it also contains other restorative nutrients as appropriate. CN 106074213A discloses a hyaluronic acid gel for water and light injection, which consists of 10-30 parts of sodium hyaluronate, 0.5-3 parts of glycerin, 0.5-1.5 parts of mannitol, and 1-9 parts of mannitol. The pH adjuster and 25 to 45 parts of isotonic adjuster. CN 105233270A discloses a pharmaceutical composition for water-light therapy and its application. Its composition includes: 0.01-0.5wt% high molecular weight sodium hyaluronate, 0.01-0.3wt% medium molecular weight sodium hyaluronate, 0.01-1wt% % Low molecular weight sodium hyaluronate, 0.01-1wt% oligomeric sodium hyaluronate, 0.01-5wt% collagen, 0.05-1.8wt% adjuvant, recombinant human epidermal growth factor, the balance is water, reconstituted The concentration of human epidermal growth factor is 0.5-10μg/ml; the molecular weight of high molecular weight sodium hyaluronate is 1.3-2 million daltons, the molecular weight of medium molecular weight sodium hyaluronate is 500,000-1 million daltons, and the low molecular weight is transparent The molecular weight of sodium phosphate is 50,000 to 300,000 daltons, and the molecular weight of oligomeric sodium hyaluronate is 3,000 to 8,000 daltons.
然而，透明质酸在体内透明质酸酶的作用下，被快速降解成小分子片段进入体液循环被吸收代谢，失去了在肌肤局部补水保湿的功能；在皮肤中，透明质酸的半衰期小于1天(Laurent等，Exp Physiol 1991,76:695-703)。因此，水光注射虽然可在肌肤局部快速补充透明质酸，但只能在短时间内起到补水保湿，难以实现改善肤质、延缓衰老及恢复年轻态等较长期效果。例如，Amin等(Dermatol Surg 2006,32:1467-1472)和El-Domyati等(International Journal of Dermatology 2012,51:913-919)的研究结果均表明透明质酸水光注射的疗效有待进一步提高。However, under the action of hyaluronidase in the body, hyaluronic acid is rapidly degraded into small molecular fragments and enters the body fluid circulation to be absorbed and metabolized, and loses the function of moisturizing and moisturizing in the skin; in the skin, the half-life of hyaluronic acid is less than 1 Tian (Laurent et al., Exp Physiol 1991, 76: 695-703). Therefore, although water injection can quickly replenish hyaluronic acid in the skin, it can only moisturize and moisturize in a short time, and it is difficult to achieve long-term effects such as improving skin texture, delaying aging and restoring youthfulness. For example, the research results of Amin et al. (Dermatol Surg 2006, 32: 1467-1472) and El-Domyati et al. (International Journal of Dermatology 2012, 51: 913-919) both show that the efficacy of hyaluronic acid water injection needs to be further improved.
交联改性可延缓透明质酸在体内的降解吸收，例如1,4-丁二醇二缩水甘油醚(BDDE)交联透明质酸凝胶(如瑞蓝
等)在皮下的降解吸收时间可长达6个月以上，被广泛用于皱纹注射填充。然而，当前交联透明质酸凝胶产品的含量很高(通常不小于20mg/mL)(Edsman等，Dermatol Surg 2012,38:1170-1179)，亦即含水量通常不大于98％(交联透明质酸自身重量约49倍的含水量)，补水保湿性能较差；并且，该等交联透明质酸凝胶产品还具有较大的凝胶强度，需粉碎为颗粒状后才能使用，且推挤力较大，很难通过水光针极细针头进行浅表层注射；此外，该等交联透明质酸凝胶产品的流动性差，无法在水光注射的浅表层均匀分布，且容易形成鼓包且在短期内无法降解消失。因此，当前交联透明质酸凝胶产品用于水光注射用途存在诸多局限性。
Cross-linking modification can delay the degradation and absorption of hyaluronic acid in the body, such as 1,4-butanediol diglycidyl ether (BDDE) cross-linked hyaluronic acid gel (such as Rui Lan Etc.) The degradation and absorption time under the skin can be as long as 6 months, and it is widely used for wrinkle injection and filling. However, the current content of cross-linked hyaluronic acid gel products is very high (usually not less than 20mg/mL) (Edsman et al., Dermatol Surg 2012, 38:1170-1179), that is, the water content is usually not more than 98% (cross-linked The water content of hyaluronic acid is about 49 times its own weight), and its moisturizing and moisturizing properties are poor; and these cross-linked hyaluronic acid gel products also have greater gel strength and need to be crushed into particles before they can be used. The pushing force is large, and it is difficult to perform superficial injection through the ultra-fine needle of the water-light needle; in addition, these cross-linked hyaluronic acid gel products have poor fluidity and cannot be evenly distributed in the superficial layer of the water-light injection, and are easy to form Bulging and cannot be degraded and disappeared in a short period of time. Therefore, current cross-linked hyaluronic acid gel products have many limitations for water-light injection applications.
发明内容Summary of the invention
为了解决现有技术中水光注射透明质酸在体内由于降解吸收导致补水保湿效果维持时间短的问题，同时也为了解决现有技术中交联透明质酸凝胶虽然降解吸收时间长但不适合于水光注射的问题，本申请提供了一种用于水光注射的二硫键交联透明质酸凝胶，具有比现有透明质酸更长的降解吸收时间和更长的补水保湿维持时间，杂质毒性小，同时适合于水光注射用途，且不会产生当前交联透明质酸凝胶产品易出现的鼓包现象，因而可通过水光注射更加有效地改善肌肤肤质。In order to solve the problem that the water-light injection hyaluronic acid in the body is degraded and absorbed in the body, the maintenance time of the moisturizing and moisturizing effect is short. At the same time, it is also to solve the problem that the cross-linked hyaluronic acid gel in the prior art has a long degradation and absorption time but is not suitable Regarding the problem of water-light injection, this application provides a disulfide bond cross-linked hyaluronic acid gel for water-light injection, which has longer degradation and absorption time and longer hydration and moisturizing maintenance than existing hyaluronic acid Time, impurity toxicity is small, and it is suitable for water-light injection, and does not produce the bulging phenomenon that current cross-linked hyaluronic acid gel products are prone to. Therefore, water-light injection can more effectively improve the skin texture.
本发明通过以下技术方案实现：The present invention is realized through the following technical solutions:
一种用于水光注射的透明质酸凝胶，其中所述透明质酸是二硫键交联透明质酸，其在透明质酸中的含量不大于10mg/mL。A hyaluronic acid gel for water-light injection, wherein the hyaluronic acid is disulfide bond cross-linked hyaluronic acid, and the content of the hyaluronic acid is not more than 10 mg/mL.
在本发明中，二硫键交联透明质酸的含量表示为二硫键交联透明质酸凝胶单位体积中含 有的交联透明质酸的重量(mg/mL)。本发明所述的用于水光注射的交联透明质酸的含量优选在4～8mg/mL之间，特别优选在5.5～7mg/mL之间。In the present invention, the content of disulfide crosslinked hyaluronic acid is expressed as the weight (mg/mL) of crosslinked hyaluronic acid contained in the unit volume of the disulfide crosslinked hyaluronic acid gel. The content of the cross-linked hyaluronic acid for water-light injection of the present invention is preferably between 4-8 mg/mL, particularly preferably between 5.5-7 mg/mL.
与当前交联透明质酸凝胶产品的含量(通常不小于20mg/mL)相比较，本发明所述用于水光注射的二硫键交联透明质酸凝胶的含量低，仅为当前交联透明质酸凝胶产品的50％以下；因此，本发明所述用于水光注射的二硫键交联透明质酸凝胶的强度更低，具有更好的流动性，易于通过水光针极细针头进行注射及实现在皮下浅表层的均匀分布。此外，本发明所述用于水光注射的二硫键交联透明质酸凝胶的含水量不小于99％(二硫键交联透明质酸自身重量的99倍及以上)，且可进一步吸水保水至二硫键交联透明质酸自身重量的约1000倍，因此具有与非交联透明质酸相近的保水能力。Compared with the current content of cross-linked hyaluronic acid gel products (usually not less than 20 mg/mL), the disulfide bond cross-linked hyaluronic acid gel used for water-light injection of the present invention has a low content, which is only current The cross-linked hyaluronic acid gel product is less than 50%; therefore, the disulfide bond cross-linked hyaluronic acid gel for water injection of the present invention has lower strength, better fluidity, and is easy to pass water The ultra-fine needle of the light needle is used for injection and achieves uniform distribution in the superficial subcutaneous layer. In addition, the water content of the disulfide cross-linked hyaluronic acid gel for water-light injection of the present invention is not less than 99% (99 times or more of the weight of the disulfide cross-linked hyaluronic acid), and can further It absorbs water and retains water to about 1000 times the weight of disulfide cross-linked hyaluronic acid, so it has a water retention capacity similar to that of non-cross-linked hyaluronic acid.
在本发明中，可采用类似于本发明申请人在先技术专利文献CN 101721349A所公开的方法来制备本发明所述的用于水光注射的二硫键交联透明质酸凝胶。该制备方法通过透明质酸巯基化衍生物水溶液在氧气(空气中的氧气和/或水溶液中的溶解氧)的氧化作用下实现二硫键交联，具有不需要添加交联剂，副产物为水，生产工艺简单以及不含杂质等许多优点，避免了当前交联透明质酸凝胶产品的复杂工艺及交联剂残余引起的潜在毒副作用。In the present invention, a method similar to that disclosed in the applicant's prior art patent document CN 101721349A can be used to prepare the disulfide bond cross-linked hyaluronic acid gel for water light injection of the present invention. The preparation method realizes disulfide bond cross-linking by the hyaluronic acid sulfhydryl derivative aqueous solution under the oxidation of oxygen (oxygen in the air and/or dissolved oxygen in the aqueous solution), without the need to add a cross-linking agent, and the by-products are Water has many advantages such as simple production process and no impurities, which avoids the current complicated process of cross-linking hyaluronic acid gel products and the potential toxic and side effects caused by the residual cross-linking agent.
如本领域技术人员所熟知，在当前的交联透明质酸凝胶产品的制备工艺中，常规采用浸泡透析方式进行纯化，亦即通过将交联反应后的凝胶浸泡于大量的水或缓冲盐溶液中，以透析除去交联凝胶中残余的交联剂等杂质；该纯化过程要求交联凝胶具有较高的强度，因此当前交联透明质酸凝胶产品均具有较高的强度，不适合于水光注射用途。与此相反，在本发明中，所述用于水光注射的二硫键交联透明质酸凝胶的上述制备方法不需要纯化过程，特别合适于制备强度较低、具有一定流动性的交联凝胶，适合于水光注射用途。As is well known to those skilled in the art, in the current preparation process of cross-linked hyaluronic acid gel products, immersion dialysis is conventionally used for purification, that is, by immersing the gel after the cross-linking reaction in a large amount of water or buffer In the salt solution, the residual cross-linking agent and other impurities in the cross-linked gel are removed by dialysis; this purification process requires the cross-linked gel to have high strength, so current cross-linked hyaluronic acid gel products have high strength , Not suitable for water injection purposes. In contrast, in the present invention, the above-mentioned preparation method of the disulfide bond cross-linked hyaluronic acid gel for water injection does not require a purification process, and is particularly suitable for preparing crosslinked gels with low strength and certain fluidity. Liangel is suitable for water injection.
在本发明中，可采用本发明申请人在先技术专利文献CN 102399295A所公开的透明质酸巯基化衍生物来制备本发明所述的用于水光注射的二硫键交联透明质酸凝胶。该等透明质酸巯基化衍生物较好保持了透明质酸的初始结构、生理功能和生物相容性，且可实现有效的二硫键交联，延缓体内的代谢吸收，因此具有比非交联透明质酸更长的补水保湿维持时间。In the present invention, the hyaluronic acid sulfhydryl derivative disclosed in the applicant’s prior art patent document CN 102399295A can be used to prepare the disulfide cross-linked hyaluronic acid gel for water injection of the present invention. glue. These hyaluronic acid sulfhydryl derivatives better maintain the initial structure, physiological functions and biocompatibility of hyaluronic acid, and can achieve effective disulfide bond cross-linking, delaying metabolic absorption in the body, and therefore have better properties than non-crosslinking Combined with hyaluronic acid for longer hydrating and moisturizing maintenance time.
本发明所述的透明质酸可以是其盐的形式(如钠盐、钾盐、锌盐、钙盐等)和/或其衍生物的形式(如羧甲基化透明质酸、乙酰化透明质酸等)。The hyaluronic acid of the present invention can be in the form of its salt (such as sodium salt, potassium salt, zinc salt, calcium salt, etc.) and/or its derivative form (such as carboxymethylated hyaluronic acid, acetylated hyaluronic acid, etc.) Acid, etc.).
在本发明中，所述的透明质酸巯基化衍生物是指含有巯基的透明质酸衍生物，可通过透明质酸的巯基化改性制备，也包括各种透明质酸衍生物进一步通过巯基化改性所制备的巯基化衍生物。透明质酸或其盐或其衍生物的侧链羧基、侧链羟基、以及还原性端基等通常是可进行巯基化改性的活性官能团，WO 2009/006780A1等在先文件所公开的多种透明质酸巯基化 衍生物制备方法均可用于制备本发明所述的透明质酸巯基化衍生物。In the present invention, the hyaluronic acid sulfhydryl derivatives refer to hyaluronic acid derivatives containing sulfhydryl groups, which can be prepared by sulfhydryl modification of hyaluronic acid, and also include various hyaluronic acid derivatives further through sulfhydryl groups. The sulfhydryl derivative prepared by chemical modification. The side chain carboxyl groups, side chain hydroxyl groups, and reducing end groups of hyaluronic acid or its salts or derivatives are usually reactive functional groups that can be modified by sulfhydrylization, as disclosed in previous documents such as WO 2009/006780A1. The preparation methods of hyaluronic acid sulfhydryl derivatives can be used to prepare the hyaluronic acid sulfhydryl derivatives of the present invention.
在本发明中，各种巯基含量的透明质酸巯基化衍生物均可用于制备本发明所述的用于水光注射的二硫键交联透明质酸凝胶。巯基含量表示为每克透明质酸巯基化衍生物中含有的巯基的微摩尔数(μmol/g)，优选的巯基含量在10～50μmol/g之间，特别优选的巯基含量在20～40μmol/g之间。In the present invention, the sulfhydryl derivatives of hyaluronic acid with various sulfhydryl content can be used to prepare the disulfide bond cross-linked hyaluronic acid gel for water-light injection of the present invention. The sulfhydryl content is expressed as the number of micromoles of sulfhydryl groups per gram of hyaluronic acid sulfhydryl derivative (μmol/g). The preferred sulfhydryl content is between 10-50μmol/g, and the particularly preferred sulfhydryl content is between 20-40μmol/g. between g.
本发明的再一个目的是提供所述的用于水光注射的二硫键交联透明质酸凝胶的使用方法。Another object of the present invention is to provide a method for using the disulfide bond cross-linked hyaluronic acid gel for water injection.
当本发明所述的用于水光注射的二硫键交联透明质酸凝胶的含量较低时(通常小于4mg/mL时)，可直接采用本领域技术人员所熟知水光注射方式进行水光注射，如采用Derma
(德玛莎)等水光注射仪通过五针或九针等极细负压针头进行水光注射。然而，当本发明所述的用于水光注射的二硫键交联透明质酸凝胶的含量较高时(如4～10mg/mL时)，直接进行水光注射的推挤力较大，此时可在水光注射前适当稀释至较低含量(通常小于4mg/mL时)，即可顺利进行水光注射。稀释过程可在两通或三通接头连接的注射器中进行，其中一支注射器装有本发明所述的用于水光注射的二硫键交联透明质酸凝胶，另一只注射器装有生理盐水、与体液等渗的缓冲液或营养活性物质溶液(如含有生长因子、氨基酸、维生素等的溶液)，两支注射器往复推挤即可实现物理稀释混合。当前水光注射除了补充透明质酸外，通常也希望同时适当补充其它修复营养物质；该需求可方便通过上述物理混合过程实现，以实现协同的水光注射补水保湿等效果。
When the content of the disulfide cross-linked hyaluronic acid gel for water-light injection of the present invention is low (usually less than 4 mg/mL), the water-light injection method well known to those skilled in the art can be used directly. Water injection, such as Derma (Demasa) and other water-light injection instruments use five-needle or nine-needle ultra-fine negative pressure needles to perform water-light injection. However, when the content of the disulfide cross-linked hyaluronic acid gel used for water-light injection of the present invention is high (for example, when 4-10 mg/mL), the pushing force for direct water-light injection is greater At this time, it can be appropriately diluted to a lower content (usually less than 4mg/mL) before the water light injection, and the water light injection can be carried out smoothly. The dilution process can be carried out in a syringe connected by a two-way or a three-way joint. One syringe is equipped with the disulfide cross-linked hyaluronic acid gel for water-light injection of the present invention, and the other syringe is equipped with Physical dilution and mixing can be achieved by reciprocating physiological saline, buffer solution that is isotonic with body fluids or nutrient active substance solution (such as solutions containing growth factors, amino acids, vitamins, etc.). In addition to supplementing hyaluronic acid, current water-light injections usually also wish to appropriately supplement other restorative nutrients at the same time; this requirement can be easily achieved through the above-mentioned physical mixing process to achieve synergistic water-light injection moisturizing and moisturizing effects.
本发明的有益效果是：The beneficial effects of the present invention are:
本发明所述的用于水光注射的二硫键交联透明质酸凝胶克服了当前交联透明质酸凝胶产品用于水光注射时的缺点，具有更低的含量和更好的流动性，易于通过水光针极细针头进行注射，可在皮下浅表层实现良好的均匀分布，不会出现鼓包现象；同时具有比未交联透明质酸更长时间的补水保湿效果，可有效改善肌肤肤质。The disulfide cross-linked hyaluronic acid gel used for water-light injection of the present invention overcomes the shortcomings of current cross-linked hyaluronic acid gel products when used for water-light injection, and has a lower content and better Fluidity, easy to inject through the ultra-fine needle of the water-light needle, can achieve good uniform distribution in the superficial subcutaneous layer, without bulging; at the same time, it has a longer hydrating and moisturizing effect than uncrosslinked hyaluronic acid, which can be effective Improve skin texture.
本发明所述的用于水光注射的二硫键交联透明质酸凝胶可方便地通过透明质酸巯基化衍生物制备，具有不需要添加交联剂、制备工艺简单、不含杂质等许多优点，可避免了当前交联透明质酸凝胶产品的复杂工艺及交联剂残余引起的毒副作用。The disulfide bond cross-linked hyaluronic acid gel for water-light injection of the present invention can be conveniently prepared by hyaluronic acid sulfhydryl derivatives, and has the advantages of not requiring the addition of cross-linking agents, simple preparation process, and no impurities, etc. Many advantages can avoid the current complicated process of cross-linking hyaluronic acid gel products and the toxic and side effects caused by the residual cross-linking agent.
此外，本发明所述的用于水光注射的二硫键交联透明质酸凝胶还可以方便地在使用前与其它营养活性物质进行物理混合，以实现水光治疗的协同效果。In addition, the disulfide cross-linked hyaluronic acid gel for water-light injection of the present invention can also be physically mixed with other nutrient active substances before use to achieve the synergistic effect of water-light treatment.
图1.二硫键交联透明质酸钠凝胶水光注射后对皮肤含水量的影响Figure 1. The effect of disulfide bond cross-linked sodium hyaluronate gel on skin moisture content after water injection
图2.二硫键交联透明质酸钠凝胶水光注射后对皮肤弹性的影响。Figure 2. The effect of disulfide cross-linked sodium hyaluronate gel on skin elasticity after water injection.
图3.二硫键交联透明质酸钠凝胶水光注射后对皮肤皱纹深度的影响。Figure 3. The effect of disulfide cross-linked sodium hyaluronate gel on the depth of skin wrinkles after water injection.
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。The embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.
实施例1：二硫键交联透明质酸凝胶的制备Example 1: Preparation of disulfide bond cross-linked hyaluronic acid gel
透明质酸巯基化衍生物以180KDa分子量的透明质酸钠为原料，采用Shu等所报道的方法制备(Shu等，Biomacromolecules 2002,3:1304-1311)，其巯基含量分别为24μmol/g、38μmol/g和57μmol/g。Hyaluronic acid sulfhydryl derivatives are prepared from sodium hyaluronate with a molecular weight of 180KDa as a raw material and prepared by the method reported by Shu et al. (Shu et al., Biomacromolecules 2002, 3:1304-1311). The sulfhydryl content is 24μmol/g and 38μmol, respectively. /g and 57μmol/g.
将上述透明质酸巯基化衍生物溶解得到10ml水溶液，含量分别为3、4、5.5、6.5、7、8和10mg/mL；调节溶液pH值为7.4，除菌过滤后转移入25ml无菌玻璃容器；室温密闭静置保存4周，溶液失去流动性并形成二硫键交联水凝胶。Dissolve the above-mentioned hyaluronic acid sulfhydryl derivatives to obtain 10ml of aqueous solution with the contents of 3, 4, 5.5, 6.5, 7, 8 and 10mg/mL respectively; adjust the pH of the solution to 7.4, and transfer to 25ml of sterile glass after sterilization and filtration Container: sealed and stored at room temperature for 4 weeks, the solution loses fluidity and forms a disulfide bond cross-linked hydrogel.
实施例2：二硫键交联透明质酸凝胶的可注射性能评价Example 2: Evaluation of the injectable properties of disulfide bond cross-linked hyaluronic acid gel
采用Derma
(德玛莎)2代水光注射仪，测试实施例1所制备凝胶的通过常规水光注射针头(九针)的可注射性，试验结果如下：
Adopt Derma (Demasha) 2nd generation water-light injection instrument, test the injectability of the gel prepared in Example 1 through conventional water-light injection needles (nine needles), the test results are as follows:
当交联透明质酸的含量为3mg/mL和4mg/mL时，通常具有良好的可注射性；而含量≥5.5mg/mL，则很难通过水光注射仪进行注射。When the content of cross-linked hyaluronic acid is 3mg/mL and 4mg/mL, it usually has good injectability; and when the content is ≥5.5mg/mL, it is difficult to inject through the water-light injection device.
实施例3：二硫键交联透明质酸凝胶稀释后的可注射性能评价Example 3: Evaluation of the injectable properties of disulfide cross-linked hyaluronic acid gel after dilution
实施例1所制备的凝胶(交联透明质酸的含量分别为：5.5mg/mL、6.5mg/mL、7mg/mL、8mg/mL和10mg/mL；巯基化衍生物的巯基含量为38μmol/g)与生理盐水按照体积比1:1、1:1.5 和1:2进行物理混合稀释。稀释过程在两通接头连接的注射器中进行，其中一支注射器装有实施例1所制备的凝胶(1mL)，另一只注射器装有适当体积的生理盐水(1mL、1.5mL、3mL)，往返推挤60～80次进行物理稀释混合。The gel prepared in Example 1 (the content of cross-linked hyaluronic acid are: 5.5mg/mL, 6.5mg/mL, 7mg/mL, 8mg/mL and 10mg/mL; the sulfhydryl content of the sulfhydryl derivative is 38μmol /g) Physically mix and dilute with physiological saline in a volume ratio of 1:1, 1:1.5, and 1:2. The dilution process is carried out in a syringe connected by a two-way connector. One syringe contains the gel prepared in Example 1 (1mL), and the other syringe contains an appropriate volume of normal saline (1mL, 1.5mL, 3mL). Push back and forth 60 to 80 times for physical dilution and mixing.
采用Derma
(德玛莎)2代水光注射仪，测试稀释后凝胶通过常规水光注射针头(九针)的可注射性，试验结果如下：
Adopt Derma (Demasa) 2nd generation water-light injection instrument, test the injectability of the diluted gel through a conventional water-light injection needle (nine needles), the test results are as follows:
当稀释至含量≤4mg/mL时，即可实现通过水光注射仪进行注射；稀释后含量越低，可注射性越好。而当前交联透明质酸凝胶产品(如瑞蓝
等)的强度大且粒径较大，在同等条件下即使按照更高倍数的体积比进行稀释，仍极易堵塞注射针头。
When it is diluted to a content of ≤4mg/mL, injection can be achieved through a water-light injection instrument; the lower the content after dilution, the better the injectability. The current cross-linked hyaluronic acid gel products (such as Rui Lan Etc.) has high strength and large particle size. Even if it is diluted according to a higher volume ratio under the same conditions, it is still very easy to block the injection needle.
实施例4：二硫键交联透明质酸凝胶的吸水保水性能评价Example 4: Evaluation of water absorption and water retention performance of disulfide bond cross-linked hyaluronic acid gel
采用与实施例3相同的稀释过程，将实施例1所制备的凝胶与生理盐水按照体积比1:2进行物理混合稀释，然后置入玻璃瓶，室温静置1小时，均未观察到明显的水相与凝胶相的分层现象，表明凝胶具有良好的吸水保水性能(可高达交联透明质酸自身重量的约1000倍)。Using the same dilution process as in Example 3, the gel prepared in Example 1 and physiological saline were physically mixed and diluted according to a volume ratio of 1:2, and then put into a glass bottle and left standing at room temperature for 1 hour. No obvious The delamination of the water phase and the gel phase indicates that the gel has good water absorption and water retention properties (up to about 1000 times the weight of cross-linked hyaluronic acid).
实施例5：二硫键交联透明质酸凝胶稀释后的可注射性能评价Example 5: Evaluation of the injectable properties of disulfide cross-linked hyaluronic acid gel after dilution
采用与实施例3相同的混合稀释过程，实施例1所制备的凝胶(含量为6.5mg/mL；巯基化衍生物的巯基含量为57μmol/g)分别与5种水溶液(分别含有0.5mg/mL维生素C、维生素B2、精氨酸、赖氨酸和谷氨酰胺)按照体积比1:1及1:2进行物理混合稀释。采用Derma
(德玛莎)2代水光注射仪，测试各稀释后凝胶通过常规水光注射针头(九针)的可注射性；试验结果表明各稀释后凝胶均具有良好的可注射性能。
Using the same mixing and dilution process as in Example 3, the gel prepared in Example 1 (content is 6.5mg/mL; the sulfhydryl content of the sulfhydryl derivative is 57μmol/g) and 5 kinds of aqueous solutions (each containing 0.5mg/ml) mL vitamin C, vitamin B2, arginine, lysine and glutamine) are physically mixed and diluted according to the volume ratio of 1:1 and 1:2. Adopt Derma (Demasha) 2nd generation water-light injection instrument, test the injectability of each diluted gel through a conventional water-light injection needle (nine needles); the test results show that each diluted gel has good injectability.
实施例6：二硫键交联透明质酸凝胶的制备Example 6: Preparation of disulfide bond cross-linked hyaluronic acid gel
透明质酸巯基化衍生物以300KDa和1,500KDa分子量的透明质酸钠为原料，采用Shu等所报道的方法制备(Shu等，Biomacromolecules 2002,3:1304-1311)，其巯基含量分别为33μmol/g 和40μmol/g。Hyaluronic acid sulfhydryl derivatives are prepared using sodium hyaluronate with molecular weights of 300KDa and 1,500KDa as raw materials, prepared by the method reported by Shu et al. (Shu et al., Biomacromolecules 2002, 3:1304-1311), and their sulfhydryl content is 33μmol/ g and 40μmol/g.
将上述透明质酸巯基化衍生物溶解得到10ml水溶液，含量分别为3、4、5.5、6.5、7、8和10mg/mL；调节溶液pH值为7.4，除菌过滤后转移入25ml无菌玻璃瓶；室温密闭静置保存4周，溶液失去流动性并形成二硫键交联水凝胶。Dissolve the above-mentioned hyaluronic acid sulfhydryl derivatives to obtain 10ml of aqueous solution with the contents of 3, 4, 5.5, 6.5, 7, 8 and 10mg/mL respectively; adjust the pH of the solution to 7.4, and transfer to 25ml of sterile glass after sterilization and filtration Bottle: sealed and stored at room temperature for 4 weeks, the solution loses fluidity and forms a disulfide bond cross-linked hydrogel.
实施例7：二硫键交联透明质酸凝胶的可注射性能Example 7: Injectable properties of disulfide bond cross-linked hyaluronic acid gel
采用Derma
(德玛莎)2代水光注射仪，测试实施例6所制备凝胶的通过常规水光注射针头(九针)的可注射性。本实施例的试验结果表明：当含量为3mg/mL和4mg/mL时，通常具有较好的可注射性；而浓度≥5.5mg/mL，则很难通过水光注射仪进行注射。
Adopt Derma (Demasha) 2nd generation water-light injection device, test the injectability of the gel prepared in Example 6 through conventional water-light injection needles (nine needles). The test results of this example show that when the content is 3 mg/mL and 4 mg/mL, it usually has good injectability; while the concentration is ≥ 5.5 mg/mL, it is difficult to inject through the water-light injection device.
实施例8：二硫键交联透明质酸凝胶稀释后的可注射性能评价Example 8: Evaluation of the injectable properties of disulfide cross-linked hyaluronic acid gel after dilution
实施例6所制备的凝胶(含量分别为：5.5mg/mL、6.5mg/mL、7mg/mL、8mg/mL和10mg/mL)与生理盐水按照体积比1:1、1:1.5和1:2进行物理混合稀释。稀释过程与实施例3相同。The gel prepared in Example 6 (contents: 5.5 mg/mL, 6.5 mg/mL, 7 mg/mL, 8 mg/mL, and 10 mg/mL) and physiological saline were in a volume ratio of 1:1, 1:1.5, and 1. :2 Perform physical mixing and dilution. The dilution process is the same as in Example 3.
采用Derma
(德玛莎)2代水光注射仪，测试稀释后凝胶通过常规水光注射针头(九针)的可注射性。本实施例的试验结果表明当稀释至含量≤4mg/mL时，具有良好的可注射性。
Adopt Derma (Demasha) 2nd generation water-light injection instrument, test the injectability of the diluted gel through a conventional water-light injection needle (nine needles). The test results of this embodiment show that when diluted to a content ≤ 4 mg/mL, it has good injectability.
实施例9：二硫键交联透明质酸凝胶的水光注射效果评价Example 9: Evaluation of hydro-injection effect of disulfide bond cross-linked hyaluronic acid gel
选取健康志愿者8名(试验组4名，对照组4名)分别进行面部水光注射(Derma
2代水光注射仪，常规九针水光注射针头)。
Eight healthy volunteers (4 in the test group and 4 in the control group) were selected for facial water and light injection (Derma 2nd generation water-light injection instrument, conventional nine-needle water-light injection needle).
试验组使用的样品为：2mL实施1制备的凝胶(交联透明质酸的含量为6.5mg/mL；巯基化衍生物的巯基含量为38μmol/g)，使用前与3mL生理盐水混合稀释，混合稀释过程与实施例3相同；样品总体积为5mL，交联透明质酸钠的最终含量为2.6mg/mL。对照组使用的样品为相同体积相同浓度的非交联透明质酸钠水溶液(分子量1.5MDa)。The samples used in the test group were: 2 mL of the gel prepared in implementation 1 (the content of cross-linked hyaluronic acid was 6.5 mg/mL; the sulfhydryl content of the thiolated derivative was 38 μmol/g), which was mixed and diluted with 3 mL of physiological saline before use. The mixing and dilution process is the same as in Example 3; the total sample volume is 5 mL, and the final content of cross-linked sodium hyaluronate is 2.6 mg/mL. The sample used in the control group was a non-crosslinked sodium hyaluronate aqueous solution (molecular weight 1.5MDa) with the same volume and the same concentration.
在面部水光注射后的不同时间点，分别在相同部位进行皮肤含水量测试以及皱纹和皮肤弹性的改善测试。At different time points after the facial water injection, the skin moisture content test and the improvement test of wrinkles and skin elasticity were performed on the same part.
注射前后皮肤水分相对增加量(％)采用皮肤水分测定仪进行测试，注射前后皮肤弹性相对增加量(％)采用皮肤弹性测定仪进行测试，注射前后皱纹平均深度相对减少值采用快速三维成像系统进行评价。The relative increase in skin moisture before and after injection (%) was tested with a skin moisture meter, the relative increase in skin elasticity (%) before and after injection was tested with a skin elasticity meter, and the relative decrease in average wrinkle depth before and after injection was tested with a fast three-dimensional imaging system Evaluation.
本实施例的试验结果参见图1-3。与非交联透明质酸对照相比较，二硫键交联透明质酸凝胶具有更好水光治疗效果，且维持时间更长；同时治疗组的所有试验对象均未发现鼓包现象， 表明二硫键交联透明质酸凝胶具有良好的流动性，可在水光注射的浅表层均匀分布。The test results of this embodiment are shown in Figures 1-3. Compared with the non-cross-linked hyaluronic acid control photograph, the disulfide cross-linked hyaluronic acid gel has a better hydro-light treatment effect, and the maintenance time is longer; at the same time, all test subjects in the treatment group did not find bulging phenomenon, indicating two Sulfur-bonded cross-linked hyaluronic acid gel has good fluidity and can be evenly distributed on the superficial layer of water injection.
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受实施例的限制，其它任何未背离本发明的精神实质与原理下所做的改变、修饰、组合、替代、简化均应为等效替换方式，都包含在本发明的保护范围之内。The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the embodiments, and any other changes, modifications, combinations, substitutions, and simplifications made without departing from the spirit and principle of the present invention All should be equivalent replacement methods, and they are all included in the protection scope of the present invention.
Claims (10)
- 一种用于水光注射的透明质酸凝胶，其特征在于，所述透明质酸是二硫键交联的透明质酸，且其中二硫键交联透明质酸的含量不大于10mg/mL。A hyaluronic acid gel for water-light injection, characterized in that the hyaluronic acid is disulfide bond cross-linked hyaluronic acid, and the content of disulfide bond cross-linked hyaluronic acid is not more than 10 mg/ mL.
- 根据权利要求1所述的用于水光注射的透明质酸凝胶，其特征在于，所述的二硫键交联透明质酸的含量在4～8mg/mL之间。The hyaluronic acid gel for water-light injection according to claim 1, wherein the content of the disulfide bond cross-linked hyaluronic acid is between 4 and 8 mg/mL.
- 根据权利要求2所述的用于水光注射的透明质酸凝胶，其特征在于，所述的二硫键交联透明质酸的含量在5.5～7mg/mL之间。The hyaluronic acid gel for water-light injection according to claim 2, wherein the content of the disulfide bond cross-linked hyaluronic acid is between 5.5 and 7 mg/mL.
- 根据权利要求1所述的用于水光注射的透明质酸凝胶，其特征在于，所述用于水光注射的交联透明质酸凝胶通过透明质酸巯基化衍生物制备。The hyaluronic acid gel for water light injection according to claim 1, wherein the cross-linked hyaluronic acid gel for water light injection is prepared by hyaluronic acid sulfhydryl derivatives.
- 根据权利要求4所述的用于水光注射的透明质酸凝胶，其特征在于，所述透明质酸巯基化衍生物的巯基含量在10～50μmol/g之间。The hyaluronic acid gel for water and light injection according to claim 4, wherein the sulfhydryl content of the hyaluronic acid sulfhydryl derivative is between 10-50 μmol/g.
- 根据权利要求5所述的用于水光注射的透明质酸凝胶，其特征在于，所述透明质酸巯基化衍生物的巯基含量在20～40μmol/g之间。The hyaluronic acid gel for water and light injection according to claim 5, wherein the sulfhydryl content of the hyaluronic acid sulfhydryl derivative is between 20-40 μmol/g.
- 根据权利要求1-6之一所述的用于水光注射的透明质酸凝胶的使用方法，其特征在于，在使用前将所述二硫键交联透明质酸凝胶与稀释液进行物理混合稀释至二硫键交联透明质酸的含量不大于4mg/mL。The method of using hyaluronic acid gel for water-light injection according to any one of claims 1-6, wherein the disulfide cross-linked hyaluronic acid gel is combined with a diluent before use. Physically mix and dilute until the content of disulfide cross-linked hyaluronic acid is not more than 4mg/mL.
- 根据权利要求7所述的用于水光注射的透明质酸凝胶的使用方法，其特征在于，所述的稀释液是生理盐水或与体液等渗的缓冲盐溶液。The method for using hyaluronic acid gel for water-light injection according to claim 7, wherein the diluent is physiological saline or a buffered salt solution that is isotonic with body fluids.
- 根据权利要求7所述的用于水光注射的透明质酸凝胶的使用方法，其特征在于，所述的稀释液含有营养活性物质。The method for using hyaluronic acid gel for hydro-light injection according to claim 7, wherein the diluent contains nutrient active substances.
- 根据权利要求9所述的用于水光注射的透明质酸凝胶的使用方法，其特征在于，所述营养活性物质是氨基酸、维生素或生长因子。The method of using hyaluronic acid gel for water-light injection according to claim 9, wherein the nutrient active substance is an amino acid, a vitamin or a growth factor.
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| CN110812270B (en) * | 2019-10-23 | 2021-09-17 | 常州百瑞吉生物医药有限公司 | Hyaluronic acid gel composition for water light injection and preparation method thereof |
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| CN115671030A (en) * | 2021-07-21 | 2023-02-03 | 杭州协合医疗用品有限公司 | Melanocyte-containing sodium hyaluronate hydrogel capable of being injected by water light needle, preparation method and application |
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