Large molecule transdermal microemulsion system and its uses

Abstract

In a large molecule transdermal microemulsion system capable of effectively performing a planned action through the dermis and for the use thereof, the large molecule transdermal microemulsion system comprises an effective amount of a large molecule active substance capable of performing a planned action, wherein, based on the mass percentage of the total weight of the large molecule transdermal microemulsion system, the composition of the large molecule transdermal microemulsion system has an oil phase share of 7 to 36%, a water phase share of 57% to 88%, and an emulsifier share of 8% to 20%.

Description

Technology Field

[0001] The present invention belongs to the field of biotechnology and relates to a large molecule transdermal microemulsion system with a high volume and its uses. Background Technology

[0002] The transdermal system is a method by which drugs are absorbed through the skin, and it is a new route of administration that enables the treatment or prevention of diseases by allowing drugs to be absorbed into the human blood circulation through the skin and reach an effective blood concentration.

[0003] Transdermal drug delivery systems have several advantages; they effectively relieve patient pain during injection administration, effectively avoid irritation and side effects caused by oral drugs, improve the sustained-release effect of drugs, and increase the safety of drug use and treatment. Transdermal drug administration has several advantages, such as avoiding the hepatic first-pass effect, maintaining blood drug concentrations more stably, and high safety.

[0004] However, human skin primarily acts as a barrier, and this structure is advantageous for 'maintaining internal homeostasis and defending against the outside.' It protects the body from external stimuli while preventing the intrusion of foreign substances; for foreign substances to penetrate the skin, they must first pass through the epidermal layer and exert their function from within.

[0005] The skin consists of three parts: the epidermis, dermis, and subcutaneous tissue. Of these, the epidermis is the thinnest layer of the skin and is divided into the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum basale; the stratum basale is connected to the dermis through the basement membrane. The stratum corneum is the outermost layer of the epidermis and consists of 5 to 15 layers of keratinocytes and intercellular lipids; this structure is likened to a 'brick structure.' The thickness of the stratum corneum is 15 to 50 μm, and due to its very low water content (5% to 20%) and inactive metabolism, it acts as a major barrier against the transdermal absorption of chemical substances. Intercellular lipids are mainly composed of 45% to 50% ceramide, 25% cholesterol, 15% long-chain free fatty acids, and 5% other lipids. The dermis is located beneath the epidermis to support it and is composed primarily of connective tissue containing collagen fibers, elastic fibers, and the matrix. The dermis also contains other tissues such as nerves, blood vessels, lymphatic vessels, muscles, hair follicles, sebaceous glands, and large and small sweat glands.

[0006] Due to the dense stratum corneum structure of the skin, most drugs are not absorbed and do not reach systemic circulation; furthermore, bioavailability fails to meet clinical needs, and it is particularly difficult for large-molecule biologically active drugs to penetrate the skin and exert their effects. Therefore, improving drug transdermal penetration is a key challenge and a major obstacle in the research and development of transdermal formulations.

[0007] Commonly used physical transdermal penetration enhancement technologies include microneedles, iontophoresis, electroporation, ultrasound introduction, and needleless injection systems, while commonly used chemical penetration enhancers include transdermal penetration enhancers and ion pairs.

[0008] Transdermal penetration enhancers are substances that facilitate the transdermal absorption of drugs and are the most preferred method. Currently, transdermal administration systems have made rapid progress in drug development, and new products, such as drugs like estradiol and testosterone, are continuously being released. In the field of cosmetic medicine, emulsion-type transdermal absorption products are also being developed among high-end cosmetics to maximize effectiveness.

[0009] However, there is currently a shortage of products suitable for the transdermal absorption of large molecules. The problem to be solved

[0010] The present invention provides a large molecule transdermal microemulsion system capable of effectively performing a planned action through the skin and uses thereof. means of solving the problem

[0011] To this end, the present invention provides the following technical solution.

[0012] The present invention provides a large molecule transdermal microemulsion system comprising an effective amount of a large molecule active substance capable of performing a planned action, wherein, based on the mass percentage of the total weight of the transdermal microemulsion system, the composition of the large molecule transdermal microemulsion system has an oil phase share of 7 to 36%, an aqueous phase share of 57% to 88%, and an emulsifier share of 8% to 20%.

[0013] The large molecule transdermal microemulsion system provided by the present invention is also characterized in that the oil phase share is 20% to 30%, the aqueous phase share is 60% to 70%, and the emulsifier share is 8% to 12%; and / or the composition of the transdermal microemulsion system in a mass ratio of oil phase:aqueous phase:emulsifier is 2 to 2.5:6 to 7:1.

[0014] The large molecule transdermal microemulsion system provided by the present invention also has the large molecule active substance having a mass of 300 kDa or less or 150 kD or less, and / or, based on the mass percentage of the total weight of the transdermal microemulsion system, the effective amount of the large molecule active substance in the transdermal microemulsion system has a mass of 10% or less, or 5% or less, or 2% or less, or 1% or less, and preferably the large molecule active substance is a biomolecule capable of providing treatment, cosmetic and skin care; and also preferably, the large molecule active substance has the characteristic of being water-soluble.

[0015] The large molecule transdermal microemulsion system provided by the present invention further comprises, wherein the included large molecule active substance is selected from at least one or a plurality of Clostridium neurotoxin, BSA, soluble collagen, and elastin, and / or provided by a yeast extract included in the system, preferably, the Clostridium neurotoxin is a type A, B, C, D, E, F, or G botulinum toxin, and / or the light chain of the Clostridium neurotoxin comprises an amino acid sequence that matches at least 35%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 99%, or 100% of any one of SEQ ID NO: 1 to 7; and / or the heavy chain of the Clostridium neurotoxin has the characteristic of comprising an amino acid sequence that matches at least 35%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 99%, or 100% of any one of SEQ ID NO: 8 to 14.

[0016] The large molecule transdermal microemulsion system provided by the present invention also has the characteristic that, based on the mass percentage of the total weight of the system, the share of the effective amount of Clostridium neurotoxin in the system is 0.02% or less, preferably 0.00001% to 0.015%, more preferably 0.0005% to 0.015% or 0.0008% to 0.0015%, the share of the effective amount of soluble collagen, BSA, and elastin is all 5% or less, or 2% or less, or 1% or less, and the share of yeast extract is 1% to 10%.

[0017] The large molecule transdermal microemulsion system provided by the present invention is also characterized in that, based on the mass percentage occupied by the total weight of the transdermal microemulsion system, the composition of the system comprises 5% to 20% or 10% to 20% or 13% to 18% glycerin, 5% to 20% or 5% to 15% or 8% to 12% emulsifier, 1% to 10% or 3% to 8% butylene glycol or propylene glycol, 0.5% to 5% or 1% to 3% laurocapram, 0.3% to 3% or 1% to 2% carboxymethyl chitosan or chitosan derivative, 55% to 80% or 60% to 70% water, and the effective amount of the large molecule active substance.

[0018] The large molecule transdermal microemulsion system provided by the present invention also has the characteristic that the emulsifier is selected from any one or a plurality of polysorbate 80, polysorbate 60, polysorbate 20, and sorbitan oleate.

[0019] The large molecule transdermal microemulsion system provided by the present invention also contains a preservative, preferably containing a preservative having a mass percentage of 0.1 to 1% based on the mass percentage of the total weight of the transdermal microemulsion system, and furthermore, the preservative is characterized by being selected from any one or a plurality of phenoxyethanol, methylparaben, ethylparaben, propylparaben, benzyl alcohol, and sorbic acid.

[0020] The large molecule transdermal microemulsion system provided by the present invention also has the characteristic that the average particle size of the transdermal microemulsion system is 10 to 15 nm.

[0021] The present invention also provides a use for a large molecule transdermal microemulsion system to achieve therapeutic, cosmetic, and skin care effects.

[0022] The aforementioned use provided by the present invention is characterized by being used transdermally through one or more of the following methods: application, transdermal device, attachment, and spraying.

[0023] The present invention also provides the use of a large molecule transdermal microemulsion system in manufacturing drugs or products that provide therapeutic, cosmetic, and skin care effects. Effects of the invention

[0024] The large molecule transdermal penetrating microemulsion system and its use provided by the present invention enable the large molecule active substance to be effectively transdermally absorbed and to achieve a intended action by forming an oil-in-water microemulsion with an average particle size in the nanometer range, through appropriate proportions of the oil phase, water phase, and emulsifier in the composition of the large molecule transdermal penetrating microemulsion system. Brief explanation of the drawing

[0025] Figure 1 is the 4-parameter logistic standard curve of the BSA standard in Example 4. Figure 2 is a composite phenotype drawing of the toes of the mouse experiment in Example 5. Specific details for implementing the invention

[0026] Specific embodiments of the present invention are described below with reference to the drawings. Regarding the specific methods or materials used in the embodiments, those skilled in the art may make conventional alternative choices based on existing technology in accordance with the technical concept of the present invention, and such choices are not limited to those specified in the embodiments of the present invention.

[0027] Unless otherwise specified, the methods used in the examples are all conventional methods, and the materials, reagents, etc. used can be obtained through commercial channels unless otherwise specified.

[0028] An emulsion refers to a heterogeneous dispersed liquid formulation formed by two immiscible liquid phases, in which one phase is dispersed into another liquid phase in the form of small droplets. The phase forming the droplets is called the dispersed phase, internal phase, or discontinuous phase, and the other liquid phase is called the dispersion medium, external phase, or continuous phase.

[0029] An emulsion consists of an oil phase (indicated by O), an emulsifier, and a water phase (indicated by W). Depending on the type and properties of the emulsifier and the phase volume ratio (φ), an oil-in-water (O / W) or water-in-oil (W / O) type emulsion or a complex emulsion may be formed.

[0030] Aqueous: Water or aqueous solution. An aqueous solution refers to a substance in which a raw material that dissolves well in water is dissolved in water.

[0031] Oil phase: Refers to a state where raw materials that do not dissolve well in water are dissolved in an oily substance.

[0032] Emulsions are classified into ordinary emulsions, submicron emulsions, and nanoemulsions depending on the size of the emulsion particles, that is, the size of the deposits.

[0033] 1. Ordinary emulsion: The droplet size of an ordinary emulsion is generally 1 μm to 100 μm, and it is a milky white, opaque liquid.

[0034] 2. Submicron emulsions: The droplet size is generally 0.1 μm to 1.0 μm, and submicron emulsions are primarily used as carriers for gastrointestinal drug delivery. Intravenous emulsions must be submicron emulsions, and the particle size is generally in the range of 0.25 μm to 0.4 μm.

[0035] 3. Nanoemulsions: The size of the droplets is <100 nm, generally falling within the range of 10 to 100 nm, and nanoemulsions are also called microemulsions.

[0036] The large molecule transdermal microemulsion system provided by the present invention comprises an effective amount of a large molecule active substance capable of performing a predetermined action, and based on the mass percentage of the total weight of the transdermal microemulsion system, the composition of the large molecule transdermal microemulsion system has an oil phase share of 16 to 36% or 20 to 30%; an aqueous phase share of 55% to 88% or 60 to 70%; and an emulsifier share of 8% to 20% or 10% to 25%.

[0037] Macro-active substances refer to substances with relatively large molecular weights, and primarily refer to biomolecules.

[0038] The general name for Da is Dalton, which is the common unit of molecular weight and is the algebraic sum of the atomic weights of all atoms within a molecule. Numerically, the Dalton (Da) is equivalent to a relative molecular weight.

[0039] In biochemistry, molecular biology, and proteomics, kDa (kilodalton) is often used to denote large biological molecules such as proteins. 1 kDa refers to a molecule with a relative molecular weight of 1,000, and large molecules refer to biological substances with a relative molecular weight of 5,000 or more, or even exceeding one million, such as polypeptides, proteins, nucleic acids, polysaccharides, and antibodies.

[0040] The composition of the macromolecular transdermal microemulsion system provided by the present invention is as shown in Table 1:

[0041] Table 1. Based on mass percentage of the total weight of macromolecular transdermal microemulsion systems paid 7 to 36% premier 57% to 88% Emulsifier 8 to 20%

[0042] In Table 1, the composition of the large molecule transdermal microemulsion system of the present invention refers to three types: oil phase, water phase, and emulsifier, among which "an effective amount of large molecule active substance capable of performing the intended action" may be oil-based (dissolved in oil) or water-based (dissolved in water).

[0043] In one example, the macromolecular active material of the present invention is a biomolecule of about 300 kDa or less or about 150 kDa or less, preferably, the biomolecule is water-soluble and exists in the aqueous phase after addition.

[0044] In one example, the planned action can implement one or more of the therapeutic, cosmetic, and skin care effects.

[0045] In one example, the limitation of the macromolecular transdermal microemulsion system of the present invention satisfies one or two of the conditions listed in Table 2:

[0046] Table 2 First: Based on mass percentage of the total weight of the macromolecular transdermal delivery system: Oil phase: 20 to 30%; Water phase: 60 to 70%; Emulsifier 8 to 12% Second: Based on mass ratio, the composition of the above transdermal microemulsion system is as follows: Oil phase: Water phase: Emulsifier = 2 to 2.5:6 to 7:1

[0047] It is sufficient if the macromolecular active substance can exert an effect through the system, and based on the mass percentage of the total weight of the transdermal microemulsion system, the effective amount of the macromolecular active substance in the transdermal microemulsion system is such that the share of the macromolecular active substance is 10% or less, or 5% or less, or 2% or less, or 1% or less. Adjustments are made according to the specific macromolecular active substance within the said range, and after the final adjustment, the oil phase, water phase, and emulsifier of the entire microemulsion system must satisfy the aforementioned ratio or share requirements.

[0048] In one example, the included macromolecular active substance is selected from at least one or a plurality of Clostridium neurotoxin, soluble collagen and elastin, and / or is provided by yeast extract included in the system, that is, the included macromolecular active substance is one of several cases shown in Table 3.

[0049] Table 3 Types or origins of included macromolecular active substances first Select only one or a combination of Clostridium neurotoxin, soluble collagen, and elastin second Except for the first case, the transdermal microemulsion system contains yeast extract, and the macromolecular active substance is derived from the components of the yeast extract. third Different choices in the first case and combinations in the second case

[0050] For example, for any one of the cases shown in Table 3, the content is as follows, based on the mass percentage of the total weight of the transdermal microemulsion system:

[0051] The share of the effective amount of the above Clostridium neurotoxin is 0.02% or less, preferably 0.00001% to 0.015%, more preferably 0.0005% to 0.015% or 0.0008% to 0.0015%, and the content of the Clostridium neurotoxin is 10 ng / ml to 10 μg / ml;

[0052] The share of the effective amount of soluble collagen and the elastin is both 5% or less, or 2% or less, or 1% or less;

[0053] An effective amount is provided in a yeast extract having a content of 1 to 10% in the emulsion system, for example, if the yeast extract is 10% and the contained macromolecule is 5%, the effective amount is 5%;

[0054] Clostridium neurotoxins can be used for therapeutic and cosmetic purposes; for example, botulinum toxin exerts therapeutic effects in muscle relaxation and spasm relief by specifically binding to presynaptic membrane receptors on surrounding cholinergic nerve endings, cleaving the synapse-associated protein SNAP-25 to hinder the exocrine release of presynaptic membrane vesicles, and inhibiting the release of acetylcholine (Ach) from nerve endings to induce chemical denervation of muscles. The muscle chemical denervation effect of botulinum toxin forms the theoretical basis for controlling hypertonia, movement disorders, and dynamic wrinkles. In addition, botulinum toxin is also used for body shaping because the muscle volume decreases as it atrophies due to the muscle being unused when injected into the target muscle multiple times. Currently, the use of botulinum toxin in the field of disease treatment includes facial muscle spasms, idiopathic blepharospasm, spastic torticollis, spastic cerebral palsy, limb spasticity after stroke, tremors, and various other dystonia disorders. The use of botulinum toxin in the field of micro-invasive cosmetic procedures includes the removal of various dynamic wrinkles such as glabellar lines, forehead wrinkles, and crow's feet; facial contouring such as eyebrow height adjustment, jaw muscle relaxation, mandibular line lifting, and neck band injections; and body contouring such as masseter muscle reduction, gastrocnemius muscle reduction, and trapezius muscle reduction.

[0055] The term Clostridium neurotoxin in this specification refers to natural Clostridium neurotoxin and toxins having a structure and function similar to natural Clostridium neurotoxin.

[0056] Although there are differences in the amino acid sequences and immunogenicity of each type of toxin, they all exhibit similar molecular structures. Clostridium neurotoxin is produced by toxin-producing Clostridium as a non-toxic single-strand polypeptide, with a molecular weight of approximately 150 kD. It becomes active only after being cleaved into a double-stranded form by cellular or ex vivo proteases, and consists of a light chain (L strand, toxin amino terminus, 50 kD) and a heavy chain (H strand, toxin carboxy terminus, 100 kD) connected by disulfide bonds. The heavy chain is further composed of two domains: Hn (amino terminus, 50 kD) and Hc (carboxy terminus, 50 kD).

[0057] Furthermore, the Clostridium neurotoxin is a botulinum toxin of type A, B, C, D, E, F, or G, and / or the light chain of the Clostridium neurotoxin comprises an amino acid sequence that matches at least 35%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 99%, or 100% of any one of SEQ ID NO: 1 to 7; and / or the heavy chain of the Clostridium neurotoxin comprises an amino acid sequence that matches at least 35%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 99%, or 100% of any one of SEQ ID NO: 8 to 14. The sequences are as shown in Table 4.

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064] Botulinum toxin-related products have broad application prospects in fields such as clinical spasticity, glandular hypersecretion, the treatment of neuropathic pain, and cosmetic wrinkle removal. However, because it is a macromolecule, all current of these products are administered via injection, making it difficult for them to penetrate the skin directly to reach the site of action and exert their effects.

[0065] Currently, only type A and type B botulinum toxins are available as drugs, but most used in the medical field is type A botulinum toxin. Type A botulinum toxins currently used in clinical practice include Botox produced by Allergan in the United States, Dysport produced by Speywood in the United Kingdom, and Hengli (BTX-A) produced by the Lanzhou Institute of Biological Products in China. Botulinum toxin-related products have broad application prospects in fields such as clinical spasticity, glandular hypersecretion, treatment of neuropain, and cosmetic wrinkle removal. However, currently, all of these products are administered via injection. The large molecule transdermal microemulsion system has verified through efficacy experiments in mice that biomolecules such as botulinum toxin can exert their intended effects through the dermis.

[0066] Yeast extract (also known as yeast flavor, with the English name Yeast extract, abbreviated as YE) is a pure natural product in the form of a brown soluble paste or a light yellow powder, obtained by purifying proteins and nucleic acids within yeast cells using advanced modern biological technologies such as autolysis, enzymatic hydrolysis, separation, and concentration, based on protein-rich edible yeast as a raw material in accordance with the regulations of the Chinese Pharmacopoeia. The main components of yeast extract include monosaccharides, polysaccharides, minerals, and vitamins, in addition to amino acids and nucleotides.

[0067] The primary effects of yeast extract are skin regeneration, anti-aging, and moisturization. Yeast extract possesses excellent skin regenerative effects, achieving these benefits primarily through three mechanisms of action: (1) reducing the migration of pre-existing melanin to surface cells; (2) accelerating exfoliation by promoting the metabolism of keratinocytes containing melanin; and (3) regulating skin quality by promoting epidermal protein synthesis. Yeast extract has excellent free radical scavenging and anti-aging effects; it regulates skin texture, fine lines, and wrinkles, and increases skin elasticity to make the skin smooth and delicate. Furthermore, it can alleviate and prevent dull skin tone issues that occur during the premature aging process. Yeast extract also has powerful moisturizing effects: rich in natural moisturizing factors, it helps the stratum corneum retain moisture more effectively, provides a deep radiance to the skin, and prevents dryness; it strengthens skin barrier function and reduces transepidermal water loss by increasing the ceramide content of the stratum corneum; and it repairs damaged lipid barriers in the stratum corneum, thereby increasing skin resistance and regulating skin condition.

[0068] Soluble collagen is a natural protein that performs important functions in the skin. While collagen helps maintain skin elasticity and firmness, as we age, the collagen in the skin gradually decreases, leading to sagging and wrinkles. However, soluble collagen is a form that can penetrate deeper into the skin, allowing it to replenish collagen and improve skin condition.

[0069] Soluble collagen stimulates and promotes collagen synthesis to repair already damaged collagen and promote the production of new collagen, thereby improving skin elasticity and firmness and alleviating wrinkles and fine lines.

[0070] Elastin is an important component of the skin, and this elastic protein is the foundation of skin youth. Elastin peptides have benefits such as repairing skin damage, regenerating blood vessels, and promoting fibroblast proliferation. They also improve skin elasticity, making the skin smooth and firm.

[0071] As an example, the composition of the macromolecular transdermal microemulsion system of the present invention is as shown in Table 5:

[0072] Table 5 Composition name classification Based on the mass percentage of the total weight of the transdermal composition glycerin paid 5% to 20% or 10 to 20% or 13 to 18% Butylene glycol or propylene glycol 1% to 10% or 3 to 8% Azone 0.5 to 5% or 1 to 3% Emulsifier (e.g., any one or a plurality of polysorbate 80, polysorbate 60, polysorbate 20, and sorbitan oleate) Emulsifier 5% to 20% or 5 to 15% or 8 to 12% Carboxymethyl chitosan (chitosan) or chitosan derivatives premier 0.3% to 3% or 1 to 2% water 55 to 80% or 60 to 70% macromolecular active substances Type of ingredient and effective amount of the optional description above

[0073] Preferably, the example in Table 5 above is the composition of Table 6:

[0074] Table 6 Composition name classification Based on the mass percentage of the total weight of the transdermal composition glycerin paid 10 to 20% or 13 to 18% Butylene glycol or propylene glycol 3 to 8% Azone 1 to 3% Emulsifier (e.g., any one or a plurality of polysorbate 80, polysorbate 60, polysorbate 20, and sorbitan oleate) Emulsifier 5 to 15% or 8 to 12% Carboxymethyl chitosan (chitosan) or chitosan derivatives premier 1 to 2% water 55 to 80% or 60 to 70% macromolecular active substances It is water-soluble, and the content is 5% or less; or 2% or less; or 1% or less; or 0.05% or less; or 0.02% or less, preferably 0.00001% to 0.015%, and more preferably 0.0005% to 0.015% or 0.0008% to 0.0015%.

[0075] Chitosans are natural molecular alkaline polysaccharides that possess film-forming, antibacterial, anti-inflammatory, hemostatic, analgesic, and pruritic effects, as well as scar removal, wound healing promotion, and antioxidant properties. Chitosan has excellent inhibitory effects against microorganisms such as bacteria, yeast, and fungi, and Staphylococcus aureus (found on the skin) present on the general human epidermis Staphylococcus epidermidis ), E. coli( Escherichia coli ), Candida tropicalis( Candida tropicalis It exhibits a significant inhibitory effect against infections such as Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus pyogenes, which are commonly found in patients with skin conditions and burns. Chitosan possesses powerful moisturizing properties; as its moisturizing ability surpasses that of sodium hyaluronate, it is referred to as a "super moisturizer." Chitosan can adsorb heavy metals and residues found in cosmetics. Chitosan has regenerative capabilities that repair the skin barrier, making it useful for sensitive and acne-prone skin.

[0076] In one example, the macromolecular transdermal microemulsion system further comprises a preservative, preferably a preservative having a mass percentage of 0.1 to 1% based on the mass percentage of the total weight of the transdermal microemulsion system. Furthermore, the preservative is selected from any one or a plurality of phenoxyethanol, methylparaben, ethylparaben, propylparaben, benzyl alcohol, and sorbic acid.

[0077] The particle size of the particles of the macromolecular transdermal microemulsion system of the present invention is, on average, nanometer-sized, and preferably, on average 10 to 15 nm.

[0078] Table 5 is one specific example of the configuration of a large molecule transdermal microemulsion system provided by the present invention.

[0079] Table 5 Composition name classification Based on the mass percentage of the total weight of the transdermal composition glycerin paid 5% to 20% or 10 to 20% or 13 to 18% Butylene glycol or propylene glycol 1% to 10% or 3 to 8% Azone 0.5 to 5% or 1 to 3% Emulsifier (e.g., any one or a plurality of polysorbate 80, polysorbate 60, polysorbate 20, and sorbitan oleate) Emulsifier 5% to 20% or 5 to 15% or 8 to 12% Carboxymethyl chitosan (chitosan) or chitosan derivatives premier 0.3% to 3% or 1 to 2% water 55 to 80% or 60 to 70% Botulinum toxin type A 10 ng / ml to 10 μg / ml

[0080] The large molecule transdermal microemulsion system of the present invention is used to achieve therapeutic, cosmetic, and skin care effects, and when used, transdermal application is performed through one or more of the following methods: application, transdermal device, attachment, and spraying.

[0081] The large molecule transdermal microemulsion system of the present invention may also be used to manufacture drugs or products that provide therapeutic, cosmetic, and skin care effects.

[0082] In addition, in the present invention, water is primarily one or a combination of distilled water, drinking water, sterilized water, ultrapure water, and deionized water.

[0083] The description of some components related to the examples below is as follows:

[0084] The applications of superoxide dismutase in cosmetics mainly include the following: (1) as a cosmetic additive to prevent skin aging and provide moisturizing effects; and (2) used for the prevention and treatment of related skin diseases, which is widely utilized overseas. Currently, SOD is added to many high-end cosmetics both domestically and internationally, and is manufactured into mask packs, milk lotions, and creams.

[0085] Vitamins are essential organic substances for the human body. Vitamin A promotes the proliferation of epidermal cells and increases the formation of dermal collagen and elastin, providing anti-aging effects. Vitamin E is a natural antioxidant that boosts metabolism and improves skin elasticity. Vitamin C has strong antioxidant properties that promote collagen synthesis and inhibit its breakdown, while Vitamins C and E exhibit a synergistic effect in eliminating free radicals together.

[0086] In addition to essential vitamins for the human body, various plant-derived natural active ingredients have been proven to have anti-wrinkle and firming functions, such as Centella asiatica extract, Okamura sea salt extract, and calendula extract. These ingredients are currently widely used in cosmetics for anti-wrinkle and firming purposes.

[0087] Example 1

[0088] The formulation of the large molecule transdermal microemulsion system provided in this embodiment is as shown in Table 6:

[0089] Table 6 furtherance Usage (based on mass percentage) 1 glycerin 15% 2 Polysorbate-80 10% 3 Propylene glycol 5% 4 Laurocapram 2% 5 Carboxymethyl chitosan 1.5% 6 phenoxyethanol 0.5% 7 Botulinum toxin A 0.01% 8 purified water Remaining amount

[0090] Manufacturing method:

[0091] (1) Using an electronic balance, purified water, carboxymethyl chitosan, and botulinum toxin A were placed in 3L beaker 1 in order according to the mixing amounts and dissolved by stirring at room temperature (rotation speed: 400 to 500 rpm).

[0092] (2) Polysorbate-80, laurocapram, phenoxyethanol, propylene glycol, and glycerin were sequentially weighed into 2L beaker 2 according to the mixing amounts using an electronic balance and stirred uniformly at room temperature (rotation speed: 250 to 300 rpm).

[0093] (3) The solution from beaker 2 was added to beaker 1 and stirred uniformly at room temperature (rotation speed: 400 to 500 rpm).

[0094] (4) Purified water was added to the solution prepared in step (3) to make 1000g, and the mixture was stirred at room temperature to be uniformly mixed.

[0095] Example 2

[0096] The formulation of the large molecule transdermal microemulsion system provided in this embodiment is as shown in Table 7.

[0097] Table 7 furtherance Usage (based on mass percentage) 1 glycerin 15% 2 Polysorbate-80 10% 3 Butylene glycol 5% 4 Carboxymethyl chitosan 1.5% 5 Yeast extract 1% 6 Tocopherol (Vitamin E) 1% 7 Sodium ascorbic acid phosphate ester 1% 8 Superoxide dismutase (SOD) 0.5% 9 phenoxyethanol 0.5% 10 Retinyl palmitate 0.3% 11 (Everyday) Fragrance 0.2% 12 purified water Remaining amount

[0098] Manufacturing method:

[0099] (1) Using an electronic balance, purified water, carboxymethyl chitosan, yeast extract, superoxide dismutase (SOD), and sodium ascorbic acid phosphate ester were added in order to a 3L beaker 1 according to the mixing amounts, and stirred to dissolve at room temperature (rotation speed: 400 to 500 rpm).

[0100] (2) Polysorbate-80, tocopherol (vitamin E), retinyl palmitate, phenoxyethanol, (daily use) fragrance, butylene glycol, and glycerin were placed in order into a 2L beaker 2 using an electronic balance according to the mixing amounts, and were stirred uniformly at room temperature (rotation speed: 250 to 300 rpm).

[0101] (3) The solution from beaker 2 was added to beaker 1 and stirred uniformly at room temperature (rotation speed: 400 to 500 rpm).

[0102] (4) Purified water was added to the solution prepared in step (3) and stirred uniformly at room temperature.

[0103] Example 3

[0104] The formulation of the large molecule transdermal microemulsion system provided in this embodiment is as shown in Table 8.

[0105] Table 8 number furtherance Usage (based on mass percentage) 1 glycerin 15% 2 Polysorbate-80 10% 3 Butylene glycol 5% 4 Carboxymethyl chitosan 1.5% 5 Yeast extract 1% 6 Tocopherol (Vitamin E) 1% 7 Sodium ascorbic acid phosphate ester 1% 8 Superoxide dismutase (SOD) 0.5% 9 phenoxyethanol 0.5% 10 Retinyl palmitate 0.3% 11 (Everyday) Fragrance 0.2% 12 Soluble collagen 0.01% 13 Elastin 0.01% 14 Hydroxypropyltetrahydropyratriol 0.01% 15 Carnosine 0.01% 16 sodium polyglutamate 0.01% 17 Okamura seaweed extract 0.02% 18 Calendula extract 0.02% 19 Centella asiatica extract 0.02% 20 aloe extract 0.02% 21 biotin 0.01% 22 purified water Remaining amount

[0106] Manufacturing method:

[0107] (1) Using an electronic balance, purified water, hydroxypropyltetrahydropyranoltriol, carnosine, sodium polyglutamate, biotin, Okamura seaweed extract, calendula extract, Centella asiatica extract, aloe extract, carboxymethyl chitosan, sodium ascorbic acid phosphate ester, yeast extract, superoxide dismutase (SOD), soluble collagen, and elastin were weighed in order into a 3L beaker 1 according to the mixing amounts, and then dissolved by stirring at room temperature (rotation speed: 400 to 500 rpm).

[0108] (2) Polysorbate-80, tocopherol (vitamin E), retinyl palmitate, phenoxyethanol, (daily use) fragrance, butylene glycol, and glycerin were placed in order into a 2L beaker 2 using an electronic balance according to the mixing amounts, and were stirred uniformly at room temperature (rotation speed: 250 to 300 rpm).

[0109] (3) The solution from beaker 2 was added to beaker 1 and stirred uniformly at room temperature (rotation speed: 400 to 500 rpm).

[0110] (4) After adding purified water to the solution prepared in step (3) to make it 1000g, the solution was stirred at room temperature to be uniformly mixed.

[0111] The formulations prepared in Examples 1 to 3 have excellent emulsifying effects, high transparency, and no layer separation, and as a result measured with a nano particle size potentiometer, the average particle size is 10 to 15 nm, and in particular, the average particle size of the formulation prepared in Example 3 was 12.99 nm.

[0112] Comparative Example 1

[0113] The formulation of this comparative example is as shown in Table 9.

[0114] Table 9 furtherance Usage (based on mass percentage) oleic acid 10.283% Oleic alcohol 12.754% Propylene glycol 11.592% menthol 1.000% Laurocapram 2.000% Polysorbate 80 35.574% Sorbitan oleate 13.118% phenoxyethanol 0.500% Botulinum toxin type A 0.001% purified water 13.178%

[0115] Manufacturing method:

[0116] (1) The acidic ester was placed in a 2L beaker and stirred for 5 minutes; oleyl alcohol and oleic acid were weighed according to the mixture amount using an electronic balance and placed in a beaker and stirred for 5 minutes; laurocapram and phenoxyethanol were weighed according to the mixture amount using an electronic balance and placed in a beaker and stirred uniformly at room temperature (rotation speed: 200 to 250 rpm, 5 minutes).

[0117] (2) Weigh the propylene glycol and menthol according to the mixture amount using an electronic scale and place them in blue cap bottles in that order.

[0118] After heating to 60℃ (about 10 minutes) to completely dissolve, it was added to the solution obtained in step (1) and stirred uniformly at room temperature (rotation speed: 200 to 250 rpm, 5 minutes).

[0119] (3) 10 mg of botulinum toxin A was added to the solution prepared in (2) and mixed by stirring at room temperature (rotation speed: 200 to 250 rpm, 5 min).

[0120] (4) Add purified water to the solution prepared in step (3) to make it 1000g, and then at room temperature

[0121] It was stirred uniformly.

[0122] Comparative Examples 2 to 10

[0123] The formulations of Comparative Examples 2 to 10 are as shown in Table 10, and are as follows based on mass percentage:

[0124] Table 10 designation Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Comparative Example 6 Comparative Example 7 Comparative Example 8 Comparative Example 9 Comparative Example 10 glycerin 20.000% 15.000% 15.000% 15.000% 15.000% 15.000% 15.000% 15.000% 15.000% Polysorbate 80 2.000% 5.000% 5.000% 5.000% 5.000% 5.000% 10.000% 10.000% 10.000% Polysorbate-20 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 5.000% 10.000% Sorbitan oleate 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 3.750% 0.000% 0.000% Propylene glycol 2.000% 5.000% 5.000% 5.000% 5.000% 5.000% 5.000% 5.000% 5.000% Laurocapram (Azone) 2.000% 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% phenoxyethanol 0.500% 0.500% 0.500% 0.500% 0.500% 0.500% 0.500% 0.500% 0.500% Carboxymethyl chitosan (chitosan) 1.000% 1.000% 1.500% 1.500% 1.500% 1.500% 1.500% 1.500% 1.500% Yeast extract 1.000% 1.000% 1.000% 1.000% 1.000% 1.000% 1.000% 1.000% 1.000% Tocopherol (Vitamin E) 0.000% 0.000% 1.000% 1.000% 0.000% 0.300% 0.000% 0.550% 0.450% Sodium ascorbic acid phosphate ester 0.000% 0.000% 0.500% 0.500% 0.500% 0.500% 0.500% 0.500% 0.500% Retinyl palmitate 0.000% 0.000% 0.900% 0.000% 0.500% 0.300% 0.000% 0.550% 0.400% (Everyday) Fragrance 0.100% 0.050% 0.200% 0.200% 0.200% 0.200% 0.200% 0.200% 0.200% Soluble collagen 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% Witch hazel (Hamamelis mollis) extract 0.000% 0.000% 0.050% 0.050% 0.050% 0.050% 0.050% 0.050% 0.050% Centella asiatica extract 0.000% 0.000% 0.050% 0.050% 0.050% 0.050% 0.050% 0.050% 0.050% water 71.400% 72.450% 69.300% 70.200% 70.700% 70.600% 62.450% 60.100% 55.350%

[0125] According to each formulation of Comparative Examples 2 to 10, the oil phase was sufficiently mixed and dissolved, the aqueous phase was sufficiently mixed and dissolved, and then the two phases were mixed to produce the product. The specific manufacturing method can be selected using general techniques and can be referred to in the preceding examples, so it will not be explained further.

[0126] The appearance effects of Comparative Examples 2 to 10 manufactured are all inferior to those of Examples 1 to 3, and specific details are as shown in Table 11:

[0127] Table 11 Comparative example Appearance effect Comparative Example 2 Poor emulsion effect, poor transparency, layer separation Comparative Example 3 Poor emulsion effect, poor transparency, layer separation Comparative Example 4 Poor emulsion effect, poor transparency, layer separation Comparative Example 5 Poor emulsion effect, poor transparency, layer separation Comparative Example 6 Poor emulsion effect, poor transparency, layer separation Comparative Example 7 Poor emulsion effect, poor transparency, layer separation Comparative Example 8 Poor emulsion effect, poor transparency, layer separation Comparative Example 9 It is too sticky Comparative Example 10 It is too sticky

[0128] Example 4

[0129] In this experiment, transdermal efficiency was measured.

[0130] Botulinum toxin A added in Example 1 and Comparative Example 1 was replaced with BSA protein, the concentration of the BSA protein emulsion prepared in Example 1 was 1.0 mg / ml, and the concentration of the BSA protein emulsion prepared in Comparative Example 1 was 0.62 mg / ml, and a transdermal test was performed using the Franz diffusion cell method.

[0131] Experimental Equipment and Materials

[0132] 1. Reagents

[0133] Phosphate buffer, protein standard, sample, protein detection kit (ultra-high sensitivity);

[0134] 2. Consumables

[0135] Isolated porcine skin for transdermal evaluation, centrifuge tube, EP tube, pipette tip, pre-coated microwell plate;

[0136] 3. Device

[0137] TP-6 transdermal diffuser (Tianjin Jingtuo Instrument Technology Co., Ltd.), multifunction microplate reader (Thermo Scientific, Varioskan Lux), medical refrigerator (Qingdao Haier Biomedical Co., Ltd.), biochemical incubator (Shanghai Yiheng Scientific Instrument Co., Ltd.), pipette (Eppendorf AG).

[0138] 1) Preparation before the experiment:

[0139] (1) The pig skin required for the experiment was taken out of a refrigerator at -20℃, thawed at 4℃, and then washed with physiological saline solution.

[0140] (2) Before the experiment, the supply room, diffusion room (receiving room, receiving liquid from the supply room), and stirrer were cleaned.

[0141] (3) Add pure water to the water tank, and it is appropriate to match the height with the diffusion cell stand, and set the temperature and speed to 32℃.

[0142] (4) After the temperature of the TP-6 ​​transdermal diffusion device was raised to the set temperature, pig skin was placed between the supply chamber and the diffusion chamber. The solution was stirred at a constant speed to maintain an isothermal state and to ensure a uniform concentration of exudate.

[0143] 2. Experimental Method:

[0144] (1) Three experimental groups and one blank group were set up.

[0145] (2) Add BSA protein test solution: 150 μl (1 mg / ml) of the BSA protein emulsion test solution prepared in Example 1, 150 μl (0.62 mg / ml) of the BSA protein emulsion test solution prepared in Comparative Example 1, and 150 μl (1 mg / ml) of the BSA protein test solution dissolved in PBS were added to each of the three supply chambers above, and 150 μl of PBS was added to the blank group.

[0146] (3) After filling the chambers with PBS, four 1.5 ml EP tubes were prepared. Every hour, 400 μl of the liquid from each chamber was collected from the collection port to perform subsequent OD measurements. The sampling time and experimental group number were marked, and sampling was performed for a total of 6 hours. After sampling, an additional 400 μl of PBS was added to each chamber to maintain a constant volume of the chambers.

[0147] (4) All collected samples were prepared by storing them in a 4°C refrigerator.

[0148] 3. ELISA Experiment Detection:

[0149] 1) Reagent preparation:

[0150] a. Before use, all reagents were equilibrated at room temperature (18 to 25 °C). The detection wavelength was set to 450 nm according to the microplate reader instructions, and the device was preheated for 15 minutes before reading.

[0151] b. Washing buffer: 600 ml of washing buffer was prepared by diluting 20 ml of concentrated washing buffer in 580 ml of deionized water.

[0152] c. Standard Working Solution: First, the standard was centrifuged at 1000g for 1 minute, and 2ml of the standard sample dilution was added and mixed uniformly. 300μl of the 20000ng / ml standard dilution was aspirated into the first tube and mixed again to obtain a 6666.7ng / ml working solution. Then, 300μl of the solution was transferred from the previous tube to the next tube. Each tube was thoroughly mixed before the next transfer. The standard was diluted to 6 points to set the values ​​to 20000ng / ml, 6666.7ng / ml, 2222.2ng / ml, 740.7ng / ml, 246.9ng / ml, and 0ng / ml, respectively.

[0153] d. Working solution of detection reagent A: The required amount (100 μl / well) was calculated prior to the experiment. During the preparation, 100 to 200 μl more than the calculated amount was prepared. Before use, the stock solution tube was slightly centrifuged, and 100× concentrated detection reagent A was diluted to 1× working solution A using detection diluent A (e.g., 10 μl detection reagent A + 990 μl detection reagent A diluent).

[0154] e. Working solution of detection reagent B: The required amount (100 μl / well) was calculated before the experiment. During the preparation, 100 to 200 μl more than the calculated amount was prepared. Before use, the stock tube was gently centrifuged, and the 100× concentrated detection reagent B was diluted to 1× working solution B using detection diluent B (e.g., 10 μl detection reagent B + 990 μl detection reagent B diluent).

[0155] 2) Experimental Method

[0156] (1) Standard wells, sample wells, and blank wells were set up. Six different concentrations were added sequentially. A standard (including 0 wells, 50 μL / well) was added, and 50 μL of the sample to be measured (a sample collected from the sample collection port in the receiving room) was added to the other wells. Then, 50 μL / well of working solution of detection reagent A was immediately added, mixed by shaking gently, covered with the plate sealing film included in the kit, and incubated at 37°C for 60 minutes.

[0157] (2) Discard the liquid from all wells, add 350 μL of washing buffer to each well, soak for 60 seconds, then discard the liquid from each well again and dry by patting on clean absorbent paper. This washing step was repeated a total of 3 times.

[0158] (3) 100 μL of working solution of detection reagent B was added to each well, and the plate was covered with a sealing film and incubated at 37°C for 30 minutes.

[0159] (4) Discard the liquid from each well and repeat the 2-step washing process 5 times.

[0160] (5) 90 μL of TMB reagent was added to each well, covered with a new sealing film, and incubated at 37°C in the dark for 10 to 20 minutes.

[0161] (6) 50 μL of the termination solution was added to each well in the same order as the color solution was added, and the mixture was mixed by shaking gently.

[0162] (7) After checking that there were no bubbles or water vapor at the bottom of the microplate wells, the absorbance OD value of each well was measured at 450 nm and the microplate reader value was recorded.

[0163] 4. Inspection Data and Conclusion

[0164] (1) The 4-parameter logistic standard curve for BSA standard is as shown in Figure 1.

[0165] Creating a standard curve:

[0166] Processing detected standard protein OD value data:

[0167] The data derives a standard curve through 4-parameter fitting, and the 4-parameter fitting formula is as follows:

[0168]

[0169] Refer to Figure 1 for a specific standard curve.

[0170] (2) Sample detection result

[0171] Table 12 shows the concentration of BSA that passed through the skin over time in different samples, converted according to the standard curve.

[0172] Table 12 Time (h) BSA emulsion (ng / ml) prepared in Example 1 BSA emulsion (ng / ml) prepared in Comparative Example 1 PBS-dissolved BSA (ng / ml) 0 0 0 0 1.5 105.8 95.2 68.0 3 151.8 147.1 108.7 6 148.6 129.1 110.0 12 125.8 142.7 125.4 24 239.6 134.1 114.7

[0173] Transdermal calculations were performed based on the results:

[0174] (1) When the BSA emulsion protein test solution prepared in Example 1 was measured after 24 hours, the concentration in the receiving chamber was 239.6 ng / ml and the volume was 15 ml, the BSA concentration in the supply chamber was 1 mg / ml and the volume added to the supply chamber was 150 µl, and the transdermal efficiency of the BSA emulsion prepared in Example 1 was calculated to be 2.40%.

[0175] (2) When the BSA emulsion protein test solution prepared in Comparative Example 2 was measured after 24 hours, the concentration in the receiving chamber was 147.1 ng / ml and the volume was 15 ml, the BSA concentration in the supply chamber was 0.62 mg / ml and the volume added to the supply chamber was 150 µl, and through this, the transdermal efficiency of the BSA emulsion prepared in Example 5 was calculated to be 2.37%.

[0176] (3) When the BSA protein test solution dissolved in PBS was measured after 24 hours, the concentration in the receiving chamber was 125.4 ng / ml and the volume was 15 ml, the concentration in the supply chamber was 1 mg / ml and the volume added to the supply chamber was 150 µl, and the transdermal efficiency of PBS was calculated to be 1.25%.

[0177] Therefore, Example 1 and Comparative Example 2 showed excellent transdermal absorption effects compared to the control group, and the transdermal absorption effect of Example 1 was better than that of Comparative Example 2.

[0178] Example 5

[0179] An efficacy verification experiment was performed in mice using the botulinum toxin A microemulsion with a concentration of 10 μg / ml prepared in Example 1 and Comparative Example 1.

[0180] To compare the difference in transdermal penetration efficiency for the macromolecular active substances of Example 1 and Comparative Example 1, 17 to 19 g SPF grade CD-1 (ICR) mice were used. After anesthetizing with tribromoethanol, the skin on the right hind leg was depilated. Before each application, the application site was cleaned with a medical cotton swab soaked in 10% alcohol, dried, and then the drug was applied. 200 μl of botulinum toxin A microemulsion was applied once daily to the skin of the right hind leg of the mouse, and the treatment was continued for 7 consecutive days. Botulinum toxin A was not applied to the left hind leg of the mouse.

[0181] As a result of the experiment, 2 to 3 days after applying botulinum toxin A to mice, a toe fusion phenotype (fusion of toes 2 to 5) of the right hind leg appeared, and the experimental results of Example 1 and Comparative Example 1 are shown in Table 13, and the emulsion prepared with the formulation of Example 1 already showed toe fusion at D2; that is, 33% of the experimental mice had two toes fused and 33% had five toes fused; D3 toe fusion: 33% of the experimental mice had two toes fused and 33% had five toes fused; D4 toe fusion: 66% of the experimental mice had three toes fused and 33% had five toes fused; D5 toe fusion: 100% of the experimental mice had five toes fused.

[0182] The emulsion prepared with the composition of Comparative Example 1 did not show toe fusion in D2; D3 toe fusion: 33% of the experimental mice had two toes fused; D4 toe fusion: 66% of the experimental mice had two toes fused, and 33% had three toes fused; D5 toe fusion: 33% of the experimental mice had three toes fused, and 66% had four toes fused.

[0183] Table 13. Results of in vivo efficacy tests in mice of botulinum toxin A microemulsions prepared in Example 1 and Comparative Example 1, both with a concentration of 10 μg / ml. furtherance Example 1 Experimental Group Comparative Example 1 Experimental Group 1 D0 Lamination State 100% anynonymy 100% anynonymy 2 D1 Laminated State 100% anynonymy 100% anynonymy 3 D2 Laminated State 33% two-toe fusion, 33% five-toe fusion 100% anynonymy 4 D3 Laminated State 33% two-toe fusion, 33% five-toe fusion 33% two-toe fusion 5 D4 laminated state 66% three-toe fusion, 33% five-toe fusion 66% two-toe fusion, 33% three-toe fusion 6 D5 Laminated State 100% Five-Toe Fusion 33% three-toe fusion, 66% four-toe fusion

[0184] The results of Table 13 showed that the botulinum toxin A microemulsion prepared in Example 1 exhibited significantly superior efficacy results in mice compared to Comparative Example 1, and the microemulsion of Example 1 not only showed rapid onset of effect but also had superior efficacy compared to the microemulsion of Comparative Example 1. The toe fusion phenotypes in Example 1 and Comparative Example 1 are shown in Figure 2.

[0185] According to the literature, a toe fusion phenotype appeared after injecting botulinum toxin A into the gastrocnemius muscle of mice.

[0186] Accordingly, when botulinum toxin A was prepared as a microemulsion using the developed emulsion delivery system and applied to the skin of mice, a toe fusion phenotype identical to that after injecting botulinum toxin A was confirmed, thereby proving that the developed emulsion system works effectively in the body of mice and exhibits superior efficacy.

[0187] Example 7

[0188] Human efficacy test of the anti-wrinkle, firming cosmetic microemulsion prepared in Example 3

[0189] 1. Under normal circumstances, adult subjects used the product continuously for 28 days as instructed, and then evaluated whether the product had moisturizing, restorative, firming, and anti-wrinkle effects, and whether it was suitable for sensitive skin, mild, and non-irritating.

[0190] 2. Subjects: A total of 31 valid subjects completed the evaluation. They were healthy Chinese women with sensitive skin (selected via the SGS (SGS-CSTC Standards Technical Services Co., Ltd.) internal skin sensitivity questionnaire), aged from 31 to 60 years with an average age of 52.65 ± 6.08 years, and met the subject volunteer inclusion and exclusion criteria. The evaluation site was the face.

[0191] Subject Selection and Exclusion Criteria:

[0192] The subjects for this evaluation were selected from SGS’s CPCH efficacy laboratory subject information database, and healthy subjects who met the selection criteria below and did not meet the exclusion criteria were selected.

[0193] (1) Selection criteria

[0194] Healthy female, age: 28 to 60 years;

[0195] Race: Asian (Chinese);

[0196] The subject's face has sensitive skin (screened via the SGS internal skin sensitivity questionnaire);

[0197] Subjects themselves perceive discomfort, such as (non-persistent) itching or stinging on the face, as 4 to 7 points (0 to 9-point scale, self-assessment questionnaire);

[0198] Subjects themselves perceived their facial skin as dull, sagging, and lacking elasticity;

[0199] Visual evaluation of forehead wrinkles: Grade 3 to 6 (based on SGS internal atlas standards).

[0200] Random unilateral lateral canthal fold visual evaluation score grade 3 to 6 (SGS internal schematic standard)

[0201] Unilateral nasolabial fold visual evaluation score Grade 3 to 6 (based on SGS internal schematic)

[0202] Transepidermal water loss (TEWL) on one cheek > 15 g / h / m²

[0203] Measurement of moisture content in the stratum corneum of one cheek < 60 au

[0204] No distinct skin lesions, scars, hair, etc. on the facial skin

[0205] Adhere closely to the requirements of the evaluation project and be able to maintain regularity in daily life during the research period.

[0206] (2) Exclusion criteria

[0207] If any of the following conditions apply, the study will be excluded:

[0208] Persons who do not agree to sign the consent form;

[0209] Those who do not intend to comply with project requirements;

[0210] Persons simultaneously participating in other clinical studies;

[0211] Persons who used cosmetics or / and medicines on the day of evaluation;

[0212] Persons who have voluntarily reported being pregnant or breastfeeding;

[0213] Persons receiving drug treatment during the study period;

[0214] Subjects suffering from infectious skin diseases or atopic dermatitis;

[0215] Persons with skin abnormalities such as moles or telangiectasia in the evaluation area;

[0216] Subjects who received exfoliation or skin treatment within 3 months prior to participation in the evaluation;

[0217] Subjects who received immunosuppressant treatment within 3 months prior to participation in the evaluation;

[0218] Subjects who received systemic steroid treatment or phototherapy within one month prior to participation in the evaluation;

[0219] Persons who have used topical medications and / or cosmetics with special efficacy (claiming moisturizing, repair, firming, or anti-wrinkle effects) on the relevant area within 2 weeks prior to participating in the evaluation;

[0220] When measurement is difficult due to lesions, distinct marks, or other abnormalities in the evaluation site;

[0221] Subjects with severe reactions or allergies to cosmetics, drugs, or general light irradiation;

[0222] In addition to the above, if deemed unsuitable for evaluation at the discretion of the project manager.

[0223] 3. Method of Use: After cleansing, completely remove oil and keep the skin dry. Take two drops of an appropriate amount of essence (micro-emulsion) using a dropper and apply it in dots to wrinkled areas such as around the eyes, forehead, nasolabial folds, and chin, then massage until the essence (micro-emulsion) is absorbed. Daily use for 7 days was recommended. A usage cycle of once every 4 weeks was recommended (the product was stored at a constant temperature of 2 to 8°C in a refrigerator during periods of non-use).

[0224] 4. Evaluation period: Before product use (D0), 14 days after product use (D14), 28 days after product use (D28).

[0225] 5. Evaluation Parameters:

[0226] (1) Image collection

[0227] Primos CR was used for skin wrinkle and photo analysis through facial image collection. A smaller analysis value indicated that skin wrinkles had improved.

[0228] (2) Skin elasticity

[0229] The Cutometer® MPA580 skin elasticity meter was used for the skin elasticity test. As the R2, R5, and R7 measurement values ​​increased, it indicated that skin elasticity had improved.

[0230] (3) Skin tautness

[0231] The skin firmness maintenance effect was tested using the Cutometer® MPA580 skin elasticity meter. A decrease in the F4 measurement value indicated that the skin firmness maintenance effect had improved.

[0232] (4) Moisture content of the stratum corneum of the skin

[0233] The moisture content of the stratum corneum of the skin was measured using the Corneometer® CM825 skin moisture content meter. An increase in the measured value indicated an increase in the moisture content of the stratum corneum of the skin.

[0234] (5) Skin transepidermal water loss rate

[0235] The Tewameter®™ Hex skin moisture loss meter was used to measure the transepidermal water loss rate. A decrease in the measurement value indicated that the skin barrier had improved.

[0236] (6) TC value

[0237] The TC value is a secondary parameter in non-invasive testing, representing the amount of transepidermal water loss per unit time of water content within a unit area, and a decrease in the analysis value indicates that skin barrier function has improved.

[0238] (7) Skin moisture distribution

[0239] The MoistureMap MM 200 skin moisture distribution meter is a unique instrument that observes hydration distribution and texture characteristics based on capacitive imaging. MGL represents the average grayscale value of skin moisture distribution, and a smaller value indicates a higher moisture content.

[0240] (8) Skin glossiness

[0241] The Glossymeter® GL200 was used to measure skin glossiness. An increase in the measurement value indicated that skin glossiness had increased.

[0242] (9) Self-assessment by the subject

[0243] The subjects conducted self-assessments based on their usage situations.

[0244] 6. Design of Evaluation Plan:

[0245] (1) Before using the product (D0):

[0246] After the subject arrived at SGS, the face was cleansed using a facial cleanser and the skin was dried with a lint-free dry tissue. The subject then rested for 30 minutes in a laboratory at a temperature of 21±1℃ and a relative humidity of 50±10%, and a dermatologist performed a visual evaluation. If the inclusion criteria were met, the subject proceeded to the next evaluation step.

[0247] A laboratory technician measured the moisture content (Corneometer) and transepidermal water loss rate (Tewameter) of the facial skin's stratum corneum, and if the inclusion criteria were met, the process moved on to the next evaluation step.

[0248] Laboratory technicians collected Primos CR images, Moisture Map MM200, Cutometer, and Glossymeter measurements from eligible subjects.

[0249] (2) The subject completed the questionnaire.

[0250] The laboratory technician explained the product usage to the subject and distributed the product, and the subject used the sample on-site under the supervision of the laboratory technician and immediately completed a usage log in the event of an adverse reaction.

[0251] (3) 14 days after product use (D14)

[0252] After the subject arrived at SGS, the subject cleaned their face using a facial cleansing product and dried their skin with a lint-free dry tissue. The subject then sat quietly for 30 minutes in a laboratory at a temperature of 21±1℃ and a relative humidity of 50±5%, and a laboratory technician collected Primos CR images of the subject's face and measurements from the skin instruments Cutometer, Moisture Map MM200, Tewameter, Corneometer, and Glossymeter.

[0253] The laboratory technician weighed the product and checked the product usage log.

[0254] The subject left SGS.

[0255] (4) 28 days after product use (D28)

[0256] After the subject arrived at SGS, the subject cleaned their face using a facial cleansing product and dried their skin with a lint-free dry tissue. The subject then sat quietly for 30 minutes in a laboratory at a temperature of 21±1℃ and a relative humidity of 50±5%, and a laboratory technician collected Primos CR images of the subject's face and measurements from the skin instruments Cutometer, Moisture Map MM200, Tewameter, Corneometer, and Glossymeter.

[0257] The subject completed the usage questionnaire.

[0258] A laboratory technician weighed, retrieved, and inspected the product, and checked and retrieved the product usage log.

[0259] The subject left SGS.

[0260] (5) Data Statistics: The data was statistically processed using SPSS 28.0. A normality test was performed on the evaluation data. If the evaluation data followed a normal distribution, statistical analysis was performed using the T-test method, and if it followed a non-normal distribution, statistical analysis was performed using the rank sum test method. The grade data was statistically analyzed using the rank sum test. The significance level of the statistical method was set to P<0.05.

[0261] As a result of using the product continuously for 28 days with 31 healthy Chinese female subjects with sensitive skin, the evaluation results showed that under the evaluation conditions, the product demonstrated moisturizing, restorative, firming, and anti-wrinkle effects starting from the 14th day, and was suitable for sensitive skin, non-irritating, and gentle. The results are as follows.

[0262] (1) Device evaluation results (Table 14)

[0263]

[0264]

[0265]

[0266]

[0267]

[0268] (2) Subject self-assessment (satisfaction)

[0269] After 14 days of using the product, 94% of the subjects felt a deep moisturizing effect, 94% felt their skin was moist and firm, 94% felt the moisturizing effect was good, 90% felt their skin had become firmer, 87% felt their skin elasticity had improved, 87% felt their wrinkles had improved, 87% felt their fine lines had improved, 87% felt their overall skin condition had improved, 90% felt the product was suitable for sensitive skin, and 100% felt the product was mild and non-irritating.

[0270] After 28 days of using the product, 100% of the subjects felt that it had a deep moisturizing effect, 100% of the subjects felt that their skin was moist and firm, 100% felt that the moisturizing effect was good, 100% of the subjects felt that their skin had become more elastic, 97% of the subjects felt that their skin had become more elastic, 94% of the subjects felt that wrinkles had improved, 97% of the subjects felt that fine lines had improved, 90% of the subjects felt that the overall condition of their skin had improved, 100% of the subjects felt that the product was suitable for sensitive skin, 100% of the subjects felt that the product was mild and non-irritating, 100% of the subjects were satisfied with the overall effect / efficacy of the product, and 100% of the subjects stated that they intended to continue using the product.

[0271] Evaluation Conclusion:

[0272] As a result of using the product for 28 consecutive days by 31 healthy Chinese female subjects with sensitive skin, the lotion formulation of Example 3 was evaluated as having moisturizing, repairing, firming, and anti-wrinkle effects under the evaluation conditions, and was found to be suitable for sensitive skin, mild, and non-irritating.

[0273] To summarize the above:

[0274] It was confirmed through BSA transdermal efficiency experiments that the macromolecular substance transdermal microemulsion system provided by the present invention can effectively achieve transdermal absorption of macromolecular substances;

[0275] In addition, animal experiments using botulinum toxin demonstrated that the macromolecular substance transdermal microemulsion system provided by the present invention exhibits a very excellent transdermal absorption effect for macromolecular substances.

[0276] Furthermore, human skin application experiments containing macromolecular active ingredients also demonstrated that the macromolecular transdermal microemulsion system provided by the present invention possesses a very excellent transdermal absorption effect for macromolecular substances.

[0277] It should be clarified that the above embodiments are merely for illustrative purposes, and the scope of protection of the present invention is not limited to the specific scope of the embodiments.

Claims

Claim 1 A large molecule transdermal microemulsion system comprising an effective amount of a large molecule active substance capable of performing a planned action; wherein, based on the mass percentage occupied by the total weight of the transdermal microemulsion system, the composition of the large molecule transdermal microemulsion system is characterized in that the oil phase share is 7 to 36%, the water phase share is 57% to 88%, and the emulsifier share is 8% to 20%. Claim 2 A large molecule transdermal microemulsion system according to claim 1, wherein the oil phase share is 20% to 30%, the water phase share is 60% to 70%, and the emulsion share is 8% to 12%; and / or, based on mass ratio, the composition of the transdermal microemulsion system is: oil phase: water phase: emulsifier = 2 to 2.5:6 to 7:

1. Claim 3 A large molecule transdermal microemulsion system according to claim 1 or 2, wherein the large molecule active substance is 300 kDa or less or 150 kDa or less, and / or, based on the mass percentage occupied in the total weight of the transdermal microemulsion system, the effective amount of the large molecule active substance in the transdermal microemulsion system is such that the share of the large molecule active substance is 10% or less, or 5% or less, or 2% or less, or 1% or less, preferably, the large molecule active substance is a biomolecule capable of providing treatment, cosmetic and skin care, and also preferably, the large molecule active substance is water-soluble. Claim 4 In any one of claims 1 to 3, the contained macromolecular active substance is at least one or a plurality of Clostridium neurotoxins, BSA, soluble collagen, and elastin, and / or is provided in a yeast extract included in the system, preferably, the Clostridium neurotoxin is a type A, B, C, D, E, F, or G botulinum toxin, and / or the light chain of the Clostridium neurotoxin comprises an amino acid sequence having at least 35%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 99%, or 100% correspondence with any one of SEQ ID NO: 1 to 7; A large molecule transdermal microemulsion system characterized in that the heavy chain of the Clostridium neurotoxin comprises an amino acid sequence having at least 35%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 99%, or 100% correspondence with any one of SEQ ID NO: 8 to 14. Claim 5 A large molecule transdermal microemulsion system according to claim 4, wherein, based on the mass percentage of the total weight of the transdermal microemulsion system, the share of the effective amount of Clostridium neurotoxin in the system is 0.02% or less, preferably 0.00001% to 0.015%, more preferably 0.0005% to 0.015% or 0.0008% to 0.0015%; the share of the effective amount of soluble collagen, BSA, and elastin is all 5% or less, or 2% or less, or 1% or less; and the share of yeast extract is 1% to 10%. Claim 6 A large molecule transdermal microemulsion system according to any one of claims 1 to 5, wherein, based on the mass percentage occupying the total weight of the transdermal microemulsion system, the composition of the system comprises: 5% to 20% or 10% to 20% or 13% to 18% glycerin; 5% to 20% or 5% to 15% or 8% to 12% emulsifier; 1% to 10% or 3% to 8% butylene glycol or propylene glycol; 0.5% to 5% or 1% to 3% laurocapram; 0.3% to 3% or 1% to 2% carboxymethyl chitosan or chitosan derivative; 55% to 80% or 60% to 70% water; and the effective amount of the large molecule active substance. Claim 7 A large molecule transdermal microemulsion system according to claim 6, characterized in that the emulsifier is selected from any one or a plurality of polysorbate 80, polysorbate 60, polysorbate 20, and sorbitan oleate. Claim 8 A macromolecular transdermal microemulsion system according to any one of claims 1 to 7, further comprising a preservative, preferably comprising a preservative having a mass percentage of 0.1 to 1% based on the mass percentage of the total weight of the transdermal microemulsion system, and further wherein the preservative is selected from one or more of phenoxyethanol, methylparaben, ethylparaben, propylparaben, benzyl alcohol, and sorbic acid. Claim 9 A large molecule transdermal microemulsion system according to any one of claims 1 to 8, characterized in that the average particle size of the particles of the transdermal microemulsion system is 10 to 15 nm. Claim 10 Use of a large molecule transdermal microemulsion system according to any one of claims 1 to 9 for realizing therapeutic, cosmetic, and skin care effects. Claim 11 In item 10, the use is characterized by performing transdermal use through one or more of the following methods at use: application, transdermal device, attachment, and spraying. Claim 12 Use of a large molecule transdermal microemulsion system according to any one of claims 1 to 9 in the manufacture of drugs or products that provide therapeutic, cosmetic, and skin care effects.

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