WO2023083322A1 - 一种经皮给药的制剂材料、实现核酸皮肤递送的药物制剂及其制备方法和应用 - Google Patents

一种经皮给药的制剂材料、实现核酸皮肤递送的药物制剂及其制备方法和应用 Download PDF

Info

Publication number
WO2023083322A1
WO2023083322A1 PCT/CN2022/131486 CN2022131486W WO2023083322A1 WO 2023083322 A1 WO2023083322 A1 WO 2023083322A1 CN 2022131486 W CN2022131486 W CN 2022131486W WO 2023083322 A1 WO2023083322 A1 WO 2023083322A1
Authority
WO
WIPO (PCT)
Prior art keywords
choline bicarbonate
preparation
eutectic mixture
acid eutectic
choline
Prior art date
Application number
PCT/CN2022/131486
Other languages
English (en)
French (fr)
Inventor
胡勇
Original Assignee
武汉瑞佶生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 武汉瑞佶生物科技有限公司 filed Critical 武汉瑞佶生物科技有限公司
Publication of WO2023083322A1 publication Critical patent/WO2023083322A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics

Definitions

  • the invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a preparation material for transdermal administration, a pharmaceutical preparation for realizing nucleic acid skin delivery, a preparation method and application thereof.
  • nucleic acid drug-mediated gene therapy may be one of the effective solutions.
  • the technology of delivering nucleic acid drugs through local skin is still immature, because nucleic acid has a high molecular weight and is easy to degrade, making it difficult to achieve stable and effective delivery.
  • RNAs Several reports have demonstrated the local delivery of small RNAs using techniques such as spherical nucleic acids and self-assembling framework nucleic acids (El-Sagheer A H, Brown T. Click Nucleic Acid Ligation: Applications in Biology and Nanotechnology [J].
  • Microneedles have also been used to locally deliver nucleic acid drugs. Electroporation and peptide carrier methods are also being explored (Xt B, Fei J B, Ping W A, et al. Nucleic acid-based drug delivery strategies-ScienceDirect[J].Journal of Controlled Release,2020,323:240- 252).
  • Psoriasis commonly known as psoriasis
  • psoriasis is a chronic inflammatory skin disease with a long course of disease and a tendency to relapse, and some cases are almost unhealed for life, which has a great impact on the physical health and mental status of patients.
  • a large number of studies have proved that psoriasis is an immune-mediated inflammatory skin disease, and its pathogenesis is related to inflammatory cell infiltration and inflammatory factors.
  • the current treatment method is oral administration of vitamin D3 analogues, which can easily cause symptoms of hypercalcemia; another treatment method is external application of glucocorticoids, but the effect of glucocorticoids on skin lesions is temporary and prone to repeated attacks.
  • an object of the present invention is to provide a preparation material for transdermal administration.
  • Another object of the present invention is to provide a preparation method for the preparation material for transdermal administration.
  • Another object of the present invention is to provide the application of the preparation material for transdermal administration.
  • Another object of the present invention is to provide a pharmaceutical preparation for skin delivery of nucleic acids.
  • Another object of the present invention is to provide a preparation method of the pharmaceutical preparation.
  • Another object of the present invention is to provide the application of the pharmaceutical preparation.
  • Another object of the present invention is to provide a method for treating skin diseases.
  • the pharmaceutical preparation provided by the invention can stably and effectively deliver the nucleic acid drug through the skin to the subcutaneous tissue.
  • the present invention provides a preparation material for transdermal administration, comprising choline bicarbonate-geranic acid eutectic mixture and/or choline bicarbonate-phenylpropionic acid eutectic mixture;
  • the preparation material comprises bicarbonate choline-geranic acid eutectic mixture and bicarbonate choline-phenylpropionic acid eutectic mixture
  • the volume ratio of the eutectic mixture of phenylpropionic acid is (0.8 ⁇ 1.2):(0.8 ⁇ 1.2).
  • the volume ratio of the choline bicarbonate-geranic acid eutectic mixture to the choline bicarbonate-phenylpropionic acid eutectic mixture is (0.9-1.1):(0.9-1.1).
  • the invention provides the preparation method of the preparation material, comprising the following steps: preparing the choline bicarbonate-geranic acid eutectic mixture or the choline bicarbonate-phenylpropionic acid eutectic mixture, and obtaining it by dialysis;
  • the preparation material comprises choline bicarbonate-geranic acid eutectic mixture and choline bicarbonate-phenylpropionic acid eutectic mixture
  • the choline bicarbonate-geranic acid eutectic mixture is formed by mixing and reacting geranic acid ethanol solution and choline bicarbonate aqueous solution; the mass percentage of geranic acid in the geranic acid ethanol solution is 65% ⁇ 75%, the mass percentage of choline bicarbonate in the choline bicarbonate aqueous solution is 75% ⁇ 85%.
  • the choline bicarbonate-phenylpropionic acid eutectic mixture is formed by mixing and reacting phenylpropionic acid ethanol solution and choline bicarbonate aqueous solution; the mass percentage of phenylpropionic acid in the phenylpropionic acid ethanol solution is 65 % to 75%, the mass percentage of choline bicarbonate in the choline bicarbonate aqueous solution is 75% to 85%.
  • the temperature of the mixed reaction is 20-25°C
  • the mixed reaction is accompanied by stirring, and the mixed reaction ends when carbon dioxide gas is no longer generated, and the mixed reaction also includes standing in sequence after the mixed reaction , solvent removal and drying steps.
  • the dialysis material used in the dialysis has a molecular weight cut-off of 8000-10000 D, and the dialysis time is 65-80 hours.
  • the invention provides a pharmaceutical preparation for skin delivery of nucleic acid, which includes the preparation material and nucleic acid molecule; the ratio of the preparation material to the nucleic acid molecule is (0.8-1.2) mL: (0.8-1.2) mg.
  • the nucleic acid molecule is mRNA.
  • the present invention also provides the application of the preparation material and the pharmaceutical preparation in the preparation of medicines for treating skin diseases, and the skin diseases include psoriasis and skin damage.
  • the preparation material provided by the present invention is a kind of preparation material for general transdermal administration, which is used for local transdermal administration and can significantly enhance the penetration of active pharmaceutical ingredients, especially nucleic acids, to the skin;
  • the composition raw materials of the preparation materials only include choline bicarbonate, geranic acid and phenylpropionic acid, and have many potential advantages such as adjustability, wide applicability and excellent safety.
  • the preparation material provided by the present invention does not cause an increase in the level of immune factors, has good safety, and can significantly enhance the penetration of mRNA to the skin; external application of the EGF mRNA preparation provided in the examples of the present invention can significantly promote Skin wounds heal.
  • Fig. 1 is a schematic structural diagram of a pharmaceutical preparation for nucleic acid skin delivery provided by the present invention.
  • Fig. 2 is the result of quantitative PCR experiment testing the delivery efficiency of the preparation material CCC provided by the present invention to mRNA.
  • Fig. 3 is the luc mRNA-CCC preparation applied in embodiment 4, the fluorescence imaging result after 24h.
  • Fig. 4 is smear luc mRNA-CCC preparation in embodiment 5, the detection result of inflammatory factor TNFa content after 48h.
  • Fig. 5 is to smear EGF mRNA-CCC preparation in embodiment 6, observe skin damage repair situation after 10 days.
  • the invention provides a preparation material for transdermal administration, which comprises choline bicarbonate-geranic acid eutectic mixture and/or choline bicarbonate-phenylpropionic acid eutectic mixture.
  • the choline bicarbonate-geranic acid eutectic mixture and/or the choline bicarbonate-phenylpropionic acid eutectic mixture can promote the penetration of the active ingredients of the drug to the skin, especially the ability of mRNA to Penetration of the skin.
  • the preparation material includes choline bicarbonate-geranic acid eutectic mixture and choline bicarbonate-phenylpropionic acid eutectic mixture
  • the choline bicarbonate-geranic acid eutectic mixture The volume ratio of choline bicarbonate-phenylpropionic acid eutectic mixture is (0.8-1.2):(0.8-1.2), preferably (0.9-1.1):(0.9-1.1), more preferably 1:1.
  • the invention provides the preparation method of the preparation material, which comprises the following steps: preparing the choline bicarbonate-geranic acid eutectic mixture or the choline bicarbonate-phenylpropionic acid eutectic mixture and performing dialysis to obtain the preparation.
  • the choline bicarbonate-geranic acid eutectic mixture is formed by mixing and reacting geranic acid ethanol solution and choline bicarbonate aqueous solution.
  • the mass percent content of geranic acid in the ethanol solution of geranic acid is preferably 65% to 75%, more preferably 68% to 72%, most preferably 70%;
  • the mass percent content of choline bicarbonate is preferably 75% to 85%, more preferably 78% to 82%, most preferably 80%.
  • the geranic acid ethanol solution is prepared by dissolving the geranic acid in absolute ethanol, and the choline bicarbonate is dissolved in water to prepare an aqueous choline bicarbonate solution; then the prepared geranic acid ethanol solution and choline bicarbonate aqueous solution mixed reaction.
  • the molar ratio of geranic acid to choline bicarbonate is preferably (1.5-2.5):1, more preferably (1.6-2):1.
  • the volume ratio during mixing is calculated according to the determined molar ratio, and the mass percentages of geranic acid and choline bicarbonate in the geranic acid ethanol solution and the choline bicarbonate aqueous solution.
  • the temperature of the mixing reaction is preferably 20-25° C., and the mixing reaction is accompanied by stirring. In the present invention, there is no special limitation on the time of the mixing reaction, and it is preferred to end the mixing reaction when carbon dioxide gas is no longer generated.
  • the choline bicarbonate-geranic acid eutectic mixture of the present invention is obtained after the mixing reaction.
  • the standing temperature is preferably 35-45°C, more preferably 38-42°C, most preferably 40°C
  • the standing time is preferably 20-30h, more preferably 22-26h , most preferably 24h; the effect of standing still is to make the reactants fully react and settle.
  • the step of desolventization is preferably carried out.
  • the desolventization is preferably carried out by rotary evaporation, and the temperature of the rotary evaporation is preferably 55-65°C, more preferably 58-62°C.
  • the rotary evaporation time is preferably 1.5-2.5 h, more preferably 2 h.
  • drying is preferably carried out after the solvent removal step is completed; the drying temperature is preferably 55 to 65° C., more preferably 58 to 63° C., most preferably 60° C., and the drying time is preferably 45° C. ⁇ 55h, more preferably 48h.
  • the choline bicarbonate-geranic acid eutectic mixture is viscous after drying.
  • the choline bicarbonate-phenylpropionic acid eutectic mixture is formed by mixing and reacting phenylpropionic acid ethanol solution and choline bicarbonate aqueous solution; in the present invention, in the phenylpropionic acid ethanol solution
  • the mass percentage of phenylpropionic acid is preferably 65% to 75%, more preferably 68% to 72%, most preferably 70%
  • the mass percentage of choline bicarbonate in the aqueous choline bicarbonate solution is preferably 75% to 85%, more preferably 78% to 82%, most preferably 80%.
  • the phenylpropionic acid is dissolved in absolute ethanol to prepare an ethanol solution of phenylpropionic acid
  • choline bicarbonate is dissolved in water to prepare an aqueous solution of choline bicarbonate; then the prepared benzene Propionic acid ethanol solution and choline bicarbonate aqueous solution are mixed and reacted.
  • the molar ratio of phenylpropionic acid to choline bicarbonate is preferably (1.5-2.5):1, more preferably (1.6-2):1, and more preferably 1.77:1.
  • the volume ratio when mixing is calculated according to the mass percentage of phenylpropionic acid in ethanol solution of phenylpropionic acid and choline bicarbonate solution and choline bicarbonate solution determined according to the molar ratio.
  • the temperature of the mixing reaction is preferably 20-25°C.
  • the process of the mixing reaction is accompanied by stirring, and the method and rotational speed of the stirring are not particularly limited in the present invention.
  • there is no special limitation on the time of the mixing reaction and it is preferred to end the mixing reaction when carbon dioxide gas is no longer generated.
  • the choline bicarbonate-phenylpropionic acid eutectic mixture of the present invention is obtained.
  • the present invention preferably further includes the steps of standing, removing solvent and drying in sequence.
  • the standing temperature is preferably 35-45°C, more preferably 38-42°C, most preferably 40°C
  • the standing time is preferably 20-30h, more preferably 22-26h , most preferably 24h; the effect of standing still is to make the reactants fully react and settle.
  • the step of desolventization is preferably carried out.
  • the desolventization is preferably carried out by rotary evaporation, and the temperature of the rotary evaporation is preferably 55-65°C, more preferably 58-62°C.
  • the rotary evaporation time is preferably 1.5-2.5 h, more preferably 2 h.
  • drying is preferably carried out after the solvent removal step is completed; the drying temperature is preferably 55 to 65° C., more preferably 58 to 63° C., most preferably 60° C., and the drying time is preferably 45° C. ⁇ 55h, more preferably 48h.
  • the choline bicarbonate-phenylpropionic acid eutectic mixture is viscous after drying.
  • the preparation material includes choline bicarbonate-geranic acid eutectic mixture and choline bicarbonate-phenylpropionic acid eutectic mixture
  • choline bicarbonate-phenylpropionic acid eutectic mixture obtained by mixing the prepared bicarbonate choline-geranic acid eutectic mixture with bicarbonate choline-phenylpropionic acid eutectic mixture and dialyzing.
  • the molecular weight cut-off of the dialysis material used in the dialysis is preferably 8000-10000D
  • the dialysis material is preferably a dialysis bag
  • the dialysis time is preferably 65-80h, more preferably 70-75h, most preferably It is 72h.
  • the dialysate is preferably replaced once every 24 hours.
  • the dialysate outside the dialysis bag is preferably PBS, and the solubility of the PBS is preferably 80-120 mM, more preferably 90-110 mM, and most preferably 100 mM.
  • the function of the dialysis is to remove small molecule impurities such as ethanol.
  • the present invention also provides a pharmaceutical preparation for nucleic acid skin delivery, including the preparation material and nucleic acid molecule; the volume ratio of the preparation material and nucleic acid molecule is (0.8-1.2): (0.8-1.2), preferably (0.9-1.1): (0.9-1.1), most preferably 1:1.
  • the nucleic acid molecule is preferably mRNA.
  • the specific sequence of the mRNA is not particularly limited in the present invention, and any sequence of mRNA can be used. In the specific implementation process of the present invention, epidermal growth factor DGF, glyceraldehyde- 3-phosphate dehydrogenase GAPDH and luciferase LUC will be described as examples.
  • the nucleic acid molecule is preferably mixed with the preparation material in a liquid form; the present invention has no special limitation on the concentration of the nucleic acid molecule, any concentration is acceptable; the specific mRNA concentration is based on the effect of mRNA and the efficacy of the drug. determined by the needs.
  • the preparation method of the pharmaceutical preparation includes the following steps: mixing preparation materials and nucleic acid molecules, and dialysis. In the specific implementation process of the present invention, the preparation material and the nucleic acid molecule are mixed, shaken and dissolved, and then dialyzed.
  • the dialysis material used in the dialysis is preferably a dialysis bag, the molecular weight cut-off of the dialysis bag is preferably 8000-10000D, and the dialysis time is preferably 65-80 hours, more preferably 70-75 hours, most preferably 72 hours.
  • the dialysis solution is preferably replaced once every 24 hours.
  • the dialysate outside the dialysis bag is preferably PBS, and the solubility of the PBS is preferably 80-120 mM, more preferably 90-110 mM, and most preferably 100 mM.
  • the invention also provides the application of the preparation material and the pharmaceutical preparation in the preparation of medicines for treating skin diseases.
  • the skin diseases include but not limited to psoriasis and/or skin lesions.
  • the technical solutions provided by the present invention will be described in detail below in conjunction with the examples, but they should not be interpreted as limiting the protection scope of the present invention.
  • the present invention generally and/or specifically describes the materials and experimental methods used in the experiments. While many of the materials and methods of manipulation which are employed for the purposes of the invention are well known in the art, the invention has been described here in as much detail as possible. For methods not specified in detail, perform the conventional operations in the art or the operations suggested by the manufacturer's instructions.
  • choline bicarbonate solution was dissolved in 1.52mL water to prepare 80wt% choline bicarbonate solution;
  • Fig. 1 is a schematic structural diagram of the pharmaceutical preparation mRNA-CCC for skin delivery of nucleic acids provided by the present invention.
  • CAGE, CAPA, CAGE+mRNA (GAPDH), CAPA+mRNA (GAPDH), and CAGE+CAPA+mRNA (GAPDH) were prepared according to the methods described in Example 1 and Example 2, respectively.
  • mRNA is specific GAPDH mRNA.
  • balb/c mice aged 6-8 weeks were raised in ventilated cages under SPF conditions and a 12-h light and 12-h dark cycle.
  • Use depilatory cream to remove back hair apply 200 ⁇ l GAPDH mRNA-CCC preparation in the range of 1cm ⁇ 1cm, take mouse skin tissue after 24h, put it into liquid nitrogen for quick freezing, crush the quick frozen skin tissue into powder, homogenate and lyse in QIAzol (Add 1ml lysing agent per 1g tissue powder) to prepare tissue lysate for qPCR.
  • Quantitative PCR experiments were performed to detect the delivery efficiency of CCC to mRNA, and ⁇ -actin was used as an internal reference.
  • 1Standard gradient preparation of ⁇ -actin positive template the concentration of the positive template is 10 11 copies/ml, take 3 ⁇ l before the reaction and dilute 10 times (add 27 ⁇ l of water and mix well) to 10 10 , then dilute to 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 copies/ml, for future use.
  • the reaction conditions are: 93°C for 2min, then 93°C for 1min, 55°C for 2min, a total of 40 cycles.
  • GADPH upstream primer F AGGTCGGTGTGAACGGATTTG (SEQ ID No.1); GADPH downstream primer R: TGTAGACCATGTAGTTGAGGTCA (SEQ ID No.2)
  • ⁇ -actin upstream primer GGAGATTACTGCCCTGGCTCCTA (SEQ ID No.3)
  • ⁇ -actin downstream primer GACTCATCGTACTCCTGCTTGCTG (SEQ ID No.4)
  • LUC mRNA-CCC preparation Preparation of LUC mRNA-CCC preparation: Mix 2.5mL CAPA and 2.5mL CAGE, add 5mL LUC mRNA solution (the concentration of LUC mRNA is 1 ⁇ g/ ⁇ l), shake gently to dissolve, and then place it in a dialysis bag (molecular weight cut-off: 8000 ⁇ 10000D), placed in 10mM PBS and dialyzed for 72h to obtain the LUC mRNA-CCC preparation.
  • balb/c mice aged 6-8 weeks were raised in ventilated cages under SPF conditions and a 12-h light and 12-h dark cycle.
  • Use depilatory cream to remove back hair apply 200 ⁇ l LUC mRNA-CCC preparation in the range of 1cm ⁇ 1cm (LUC mRNA-CCC preparation is CAGE+CAPA+mRNA, set up control at the same time, apply CAGE+mRNA, CAPA+mRNA, mRNA respectively and PBS), after 24h, the luciferase substrate was injected through the tail vein of the mouse, using Spectrum In Vivo Imaging System performs fluorescence imaging.
  • the principle of fluorescence imaging is that after the mRNA encoding luciferase is expressed in mice, the product luciferase can catalyze the oxidation of luciferin to oxidized luciferin.
  • the mouse will emit Bioluminescence, the fluorescence intensity is directly proportional to the expression intensity of luciferase, and then the bioluminescence released during the oxidation of luciferin is measured by a fluorescence analyzer.
  • the average fluorescence intensity detected by the LUC mRNA-CCC preparation of the present invention is 6.093e 6 p/sec/cm 2 /sr, which is significantly higher than other control groups.
  • the provided preparation can effectively deliver the nucleic acid drug through the skin to the subcutaneous tissue, and significantly enhance the efficiency of mRNA transdermal delivery.
  • balb/c mice aged 6-8 weeks were raised in ventilated cages under SPF conditions and a 12-h light and 12-h dark cycle.
  • Use a depilatory cream to remove the back hair apply 200 ⁇ l of the LUC mRNA-CCC preparation prepared in Example 4 in the range of 1 cm ⁇ 1 cm, observe the skin condition after 48 hours, take the skin tissue of the mouse, and extract the protein for enzyme-linked immunosorbent assay.
  • Mouse TNF alpha ELISA kit (abcam, ab46105) was used to detect the content of inflammatory factor TNFa. The results are shown in Figure 4 and Table 2, the preparation material provided by the present invention does not cause the level of immune factors to increase, and has a relative degree of safety.
  • EGF mRNA-CCC preparation Preparation of EGF mRNA-CCC preparation: Mix 2.5mL CAPA and 2.5mL CAGE, add 5mL EGF mRNA solution (the concentration of EGF mRNA is 1 ⁇ g/ ⁇ l), shake gently to dissolve, and then place it in a dialysis bag (molecular weight cut-off: 8000 ⁇ 10000D), placed in 10mM PBS and dialyzed for 72h to obtain the EGF mRNA-CCC preparation.
  • EGF mRNA-CCC preparation promotes skin wound healing in mice
  • balb/c mice aged 6-8 weeks purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.
  • Use depilatory cream to remove the back hair use a hole punch to create two symmetrical circular wounds on the back of the mouse, and apply 200 ⁇ l of EGF mRNA-CCC preparation in the range of 1cm ⁇ 1cm at the same time, apply once every 12h, and observe the skin condition after 10 days .
  • the EGF mRNA-CCC treatment group has a high degree of wound healing and a good healing effect, indicating that the EGF mRNA-CCC provided by the present invention has the ability to deliver across the skin and promotes repair of damage.
  • the preparation material provided by the present invention is a kind of preparation material for general transdermal administration, which can be used for local transdermal administration of different nucleic acid molecules, can significantly enhance the penetration of nucleic acid molecules to the skin, and can stabilize The delivery of mRNA is effectively realized, and the safety is good.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Dermatology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Molecular Biology (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

本发明提供了一种经皮给药的制剂材料、实现核酸皮肤递送的药物制剂及其制备方法和应用，属于药物制剂技术领域，所述经皮给药的制剂材料，包括碳酸氢胆碱-香叶酸低共熔混合物和/或碳酸氢胆碱-苯丙酸低共熔混合物；当所述制剂材料包括碳酸氢胆碱-香叶酸低共熔混合物和碳酸氢胆碱-苯丙酸低共熔混合物时，所述碳酸氢胆碱-香叶酸低共熔混合物和碳酸氢胆碱-苯丙酸低共熔混合物的体积比为（0.8～1.2）：（0.8～1.2）。

Description

一种经皮给药的制剂材料、实现核酸皮肤递送的药物制剂及其制备方法和应用 技术领域
本发明属于药物制剂技术领域，尤其涉及一种经皮给药的制剂材料、实现核酸皮肤递送的药物制剂及其制备方法和应用。
背景技术
某些遗传学或免疫性皮肤疾病，并没有很好的治愈手段，核酸药物介导的基因治疗可能是有效的解决办法之一。目前，经局部皮肤递送核酸药物的技术尚不成熟，因为核酸的分子量很高，并且容易降解，很难实现稳定、有效的递送。一些报告已经展示了使用球形核酸和自组装框架核酸等技术局部递送小RNA(El-Sagheer A H,Brown T.Click Nucleic Acid Ligation:Applications in Biology and Nanotechnology[J].Accounts of Chemical Research,2012,45(8):1258；Mokhtarzadeh A,Vahidnezhad H,Youssefian L,Mosafer J,Baradaran B,Uitto J.Applications of Spherical Nucleic Acid Nanoparticles as Delivery Systems.Trends Mol Med.2019Dec；25(12):1066-1079.doi:10.1016/j.molmed.2019.08.012.Epub 2019Nov 6.PMID:31703931)。微针也被用于局部递送核酸药物。电穿孔法和肽载体法也在探索中(Xt B,Fei J B,Ping W A,et al.Nucleic acid-based drug delivery strategies-ScienceDirect[J].Journal of Controlled Release,2020,323:240-252)。
银屑病俗称牛皮癣，是一种慢性炎症性皮肤病，病程较长，有易复发倾向，有的病例几乎终生不愈，对患者的身体健康和精神状况影响较大。大量研究证明银屑病是免疫介导的炎症性皮肤病，其发病与炎症细胞浸润和炎症因子有关。目前的治疗手段为口服维生素D3类似物，此药易引起高血钙症状；另外一种治疗方法为外用糖皮质激素，但是糖皮质激素对皮损的作用是暂时的，易反复发作。有研究发现，通过基因治疗或者抗体治疗等手段，对免疫因子进行调控可以有效地治疗银屑病(Dolgin E.New anti-IL-23drugs raise hopes for psoriasis plaque clearance[J].Nature Biotechnology,2016,34(12):1218-1219)。然而对这些多效性蛋白的全身抑制可能会导致严重的副作用，通过局部应用mRNA药物是一种有效且副作用低的治疗选择，具有安全、无创、患者依从性好等优势。但是这一方法的最大挑战是，只有少数满足低分子量(<500dal)及高辛醇-水分配系数的药物才能成功局部给药；包括核酸在内的生物大分子的经皮局部递送仍然具有挑战性。
发明内容
有鉴于此，本发明的一个目的在于提供一种经皮给药的制剂材料。
本发明的另一目的在于提供所述经皮给药的制剂材料的制备方法。
本发明的另一目的在于提供所述经皮给药的制剂材料的应用。
本发明的另一目的在于提供一种实现核酸皮肤递送的药物制剂。
本发明的另一目的在于提供所述药物制剂的制备方法。
本发明的另一目的在于提供所述药物制剂的应用。
本发明的另一目的在于提供一种治疗皮肤疾病的方法。
本发明提供的药物制剂能够稳定、有效地将核酸药物透过皮肤递送至皮下组织。
为了实现上述发明目的，本发明提供了以下技术方案：
本发明提供了一种经皮给药的制剂材料，包括碳酸氢胆碱-香叶酸低共熔混合物和/或碳酸氢胆碱-苯丙酸低共熔混合物；
当所述制剂材料包括碳酸氢胆碱-香叶酸低共熔混合物和碳酸氢胆碱-苯丙酸低共熔混合物时，所述碳酸氢胆碱-香叶酸低共熔混合物和碳酸氢胆碱-苯丙酸低共熔混合物的体积比为(0.8～1.2)：(0.8～1.2)。
优选的，所述碳酸氢胆碱-香叶酸低共熔混合物和碳酸氢胆碱-苯丙酸低共熔混合物的体积比为(0.9～1.1)：(0.9～1.1)。
本发明提供了所述的制剂材料的制备方法，包括以下步骤：制备碳酸氢胆碱-香叶酸低共熔混合物或碳酸氢胆碱-苯丙酸低共熔混合物，透析即得；
当所述制剂材料包括碳酸氢胆碱-香叶酸低共熔混合物和碳酸氢胆碱-苯丙酸低共熔混合物时，分别制备碳酸氢胆碱-香叶酸低共熔混合物和碳酸氢胆碱-苯丙酸低共熔混合物，将制备获得的碳酸氢胆碱-香叶酸低共熔混合物和碳酸氢胆碱-苯丙酸低共熔混合物混合、透析即得。
优选的，所述碳酸氢胆碱-香叶酸低共熔混合物通过将香叶酸乙醇溶液和碳酸氢胆碱水溶液混合反应而成；所述香叶酸乙醇溶液中香叶酸的质量百分含量为65％～75％，所述碳酸氢胆碱水溶液中碳酸氢胆碱的质量百分含量为75％～85％。
优选的，碳酸氢胆碱-苯丙酸低共熔混合物通过苯丙酸乙醇溶液和碳酸氢胆碱水溶液混合反应而成；所述苯丙酸乙醇溶液中苯丙酸的质量百分含量为65％～75％，所述碳酸氢胆碱水溶液中碳酸氢胆碱的质量百分含量为75％～85％。
优选的，所述混合反应的温度为20～25℃，所述混合反应的过程中伴随搅拌，所 述混合反应至不再产生二氧化碳气体时结束，所述混合反应后还包括依次进行的静置、去溶剂和烘干步骤。
优选的，所述透析所用的透析材料的截留分子量为8000～10000D，所述透析的时间为65～80h。
本发明提供了一种实现核酸皮肤递送的药物制剂，包括所述的制剂材料和核酸分子；所述制剂材料和核酸分子的比例为(0.8～1.2)mL：(0.8～1.2)mg。
优选的，所述核酸分子为mRNA。
本发明还提供了所述的制剂材料、所述的药物制剂在制备治疗皮肤病的药物中的应用，所述皮肤病包括银屑病和皮肤损伤。
本发明的有益效果：本发明提供的制剂材料是一类通用经皮给药的制剂材料，用于局部经皮给药，能够显著增强药物活性成分，尤其是核酸，对皮肤的渗透作用；所述制剂材料的组成原料仅包括碳酸氢胆碱、香叶酸和苯丙酸，具有可调性、广泛的适用性和卓越的安全性等许多潜在的优点。
根据实施例记载，本发明提供的制剂材料不引起免疫因子水平升高，安全性好，同时能够显著增强mRNA对皮肤的渗透作用；外敷本发明实施例中提供的EGF mRNA制剂能够显著促进小鼠皮肤伤口愈合。
附图说明
图1为本发明提供的实现核酸皮肤递送的药物制剂的结构示意图。
图2为定量PCR实验检测本发明提供的制剂材料CCC对mRNA的递送效率结果。
图3为实施例4中涂抹luc mRNA-CCC制剂，24h后荧光成像结果。
图4为实施例5中涂抹luc mRNA-CCC制剂，48h后炎症因子TNFa含量检测结果。
图5为实施例6中涂抹EGF mRNA-CCC制剂，10天后观察皮肤损伤修复情况。
具体实施方式
本发明提供了一种经皮给药的制剂材料，包括碳酸氢胆碱-香叶酸低共熔混合物和/或碳酸氢胆碱-苯丙酸低共熔混合物。在本发明中，所述碳酸氢胆碱-香叶酸低共熔混合物和/或碳酸氢胆碱-苯丙酸低共熔混合物能够促进药物活性成分对皮肤的渗透作用，尤其是能够促进mRNA对皮肤的渗透作用。
在本发明中，当所述制剂材料包括碳酸氢胆碱-香叶酸低共熔混合物和碳酸氢胆碱-苯丙酸低共熔混合物时，所述碳酸氢胆碱-香叶酸低共熔混合物和碳酸氢胆碱-苯丙酸低共熔混合物的体积比为(0.8～1.2)：(0.8～1.2)，优选为(0.9～1.1)：(0.9～1.1)，更优选为1：1。
本发明提供了所述的制剂材料的制备方法，包括以下步骤：制备碳酸氢胆碱-香叶酸低共熔混合物或碳酸氢胆碱-苯丙酸低共熔混合物，透析即得。
在本发明中，所述碳酸氢胆碱-香叶酸低共熔混合物通过将香叶酸乙醇溶液和碳酸氢胆碱水溶液混合反应而成。在本发明中，所述香叶酸乙醇溶液中香叶酸的质量百分含量优选为65％～75％，更优选为68％～72％，最优选为70％；所述碳酸氢胆碱水溶液中碳酸氢胆碱的质量百分含量优选为75％～85％，更优选为78％～82％，最优选为80％。在本发明具体实施过程中，将所述香叶酸溶解于无水乙醇中制备获得香叶酸乙醇溶液，将碳酸氢胆碱溶解于水中制备获得碳酸氢胆碱水溶液；然后将制备获得的香叶酸乙醇溶液和碳酸氢胆碱水溶液混合反应。在本发明中，所述香叶酸和碳酸氢胆碱的摩尔比优选为(1.5～2.5)：1，更优选为(1.6～2)：1。在本发明具体实施过程中，根据确定的摩尔比、以及香叶酸乙醇溶液、碳酸氢胆碱水溶液中的香叶酸和碳酸氢胆碱的质量百分比计算混合时的体积比。所述混合反应的温度优选为20～25℃，所述混合反应的过程中伴随搅拌。本发明对所述混合反应的时间没有特殊限定，优选的在所述混合反应至不再产生二氧化碳气体时结束。混合反应后得到本发明的碳酸氢胆碱-香叶酸低共熔混合物。
在本发明中，所述混合反应后，优选的还包括依次进行的静置、去溶剂和烘干步骤。在本发明中，所述静置的温度优选为35～45℃，更优选为38～42℃，最优选为40℃，所述静置的时间优选为20～30h，更优选为22～26h，最优选为24h；所述静置的作用是使反应物充分反应、沉降。本发明在所述静置结束后，优选的进行去溶剂的步骤，所述去溶剂优选的采用旋转蒸发的方法进行，所述旋转蒸发的温度优选为55～65℃，更优选为58～62℃，最优选为60℃，所述旋转蒸发的时间优选为1.5～2.5h，更优选为2h。本发明在所述去溶剂步骤结束后，优选的进行干燥；所述干燥的温度优选为55～65℃，更优选为58～63℃，最优选为60℃，所述干燥的时间优选为45～55h，更优选为48h。在本发明中，所述碳酸氢胆碱-香叶酸低共熔混合物干燥后呈粘稠状。
在本发明中，所述碳酸氢胆碱-苯丙酸低共熔混合物通过将苯丙酸乙醇溶液和碳酸氢胆碱水溶液混合反应而成；在本发明中，所述苯丙酸乙醇溶液中苯丙酸的质量百分 含量优选为65％～75％，更优选为68％～72％，最优选为70％；所述碳酸氢胆碱水溶液中碳酸氢胆碱的质量百分含量优选为75％～85％，更优选为78％～82％，最优选为80％。在本发明具体实施过程中，将所述苯丙酸溶解于无水乙醇中制备获得苯丙酸乙醇溶液，将碳酸氢胆碱溶解于水中制备获得碳酸氢胆碱水溶液；然后将制备获得的苯丙酸乙醇溶液和碳酸氢胆碱水溶液混合反应。在本发明中，所述苯丙酸和碳酸氢胆碱的摩尔比优选为(1.5～2.5)：1，进一步优选为(1.6～2)：1，更优选为1.77：1。在本发明具体实施过程中，根据确定的摩尔比、以及苯丙酸乙醇溶液、碳酸氢胆碱水溶液中的苯丙酸和碳酸氢胆碱的质量百分比计算混合时的体积比。所述混合反应的温度优选为20～25℃。所述混合反应的过程中伴随搅拌，本发明对所述搅拌的方法和转速没有特殊限定。本发明对所述混合反应的时间没有特殊限定，优选的在所述混合反应至不再产生二氧化碳气体时结束。混合反应后得到本发明的碳酸氢胆碱-苯丙酸低共熔混合物。
本发明在所述混合反应后，优选的还包括依次进行的静置、去溶剂和烘干步骤。在本发明中，所述静置的温度优选为35～45℃，更优选为38～42℃，最优选为40℃，所述静置的时间优选为20～30h，更优选为22～26h，最优选为24h；所述静置的作用是使反应物充分反应、沉降。本发明在所述静置结束后，优选的进行去溶剂的步骤，所述去溶剂优选的采用旋转蒸发的方法进行，所述旋转蒸发的温度优选为55～65℃，更优选为58～62℃，最优选为60℃，所述旋转蒸发的时间优选为1.5～2.5h，更优选为2h。本发明在所述去溶剂步骤结束后，优选的进行干燥；所述干燥的温度优选为55～65℃，更优选为58～63℃，最优选为60℃，所述干燥的时间优选为45～55h，更优选为48h。在本发明中，所述碳酸氢胆碱-苯丙酸低共熔混合物干燥后呈粘稠状。
在本发明中，当所述制剂材料包括碳酸氢胆碱-香叶酸低共熔混合物和碳酸氢胆碱-苯丙酸低共熔混合物时，分别制备碳酸氢胆碱-香叶酸低共熔混合物或碳酸氢胆碱-苯丙酸低共熔混合物，将制备获得的碳酸氢胆碱-香叶酸低共熔混合物和碳酸氢胆碱-苯丙酸低共熔混合物混合、透析即得。
在本发明中，所述透析所用的透析材料的截留分子量优选为8000～10000D，所述透析材料优选为透析袋，所述透析的时间优选为65～80h，更优选为70～75h，最优选为72h。本发明在所述透析过程中，优选的24h更换一次透析液。所述透析袋外的透析液优选为PBS，所述PBS的溶度优选为80～120mM，更优选为90～110mM，最优选为100mM。在本发明中，所述透析的作用是去除乙醇等小分子杂质。
本发明还提供了一种实现核酸皮肤递送的药物制剂，包括所述的制剂材料和核酸分子；所述制剂材料和核酸分子的体积比为(0.8～1.2)：(0.8～1.2)，优选为(0.9～1.1)：(0.9～1.1)，最优选为1：1。在本发明中，所述核酸分子优选为mRNA，本发明对所述mRNA的具体序列没有特殊限定，任意序列的mRNA均可，在本发明具体实施过程中，以表皮生长因子DGF、甘油醛-3-磷酸脱氢酶GAPDH以及荧光素酶LUC为例进行说明。在本发明中，所述核酸分子优选的以液体形式与制剂材料混合；本发明对所述核酸分子的浓度没有特殊限定，任意浓度均可；具体的mRNA浓度根据mRNA的作用以及药物药效发挥的需求所定。在本发明中，所述药物制剂的制备方法包括以下步骤：将制剂材料和核酸分子混合、透析。在本发明具体实施过程中，将制剂材料和核酸分子混合后震荡溶解，随后进行透析。所述透析所用的透析材料优选为透析袋，所述透析袋的截留分子量优选为8000～10000D，所述透析的时间优选为65～80h，更优选为70～75h，最优选为72h。本发明在所述透析过程中，优选的24h更换透一次析液。所述透析袋外的透析液优选为PBS，所述PBS的溶度优选为80～120mM，更优选为90～110mM，最优选为100mM。
本发明还提供了所述的制剂材料、所述的药物制剂在制备治疗皮肤病的药物中的应用。在本发明中，所述皮肤病包括但不限于银屑病和/或皮肤损伤。
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。本发明对实验中所使用到的材料以及实验方法进行一般性和/或具体的描述。虽然为实现本发明目的所使用的许多材料和操作方法是本领域公知的，但是本发明仍然在此作尽可能详细描述。未详细注明的方法操作，按照所属领域现有技术的常规操作或商厂说明书建议的操作进行。
实施例1
碳酸氢胆碱-香叶酸低共熔混合物CAGE制备
9.88g纯香叶酸溶解在5.36mL无水乙醇中制备获得70wt％香叶酸乙醇溶液；
6.06g碳酸氢胆碱溶解在1.52mL水中制备获得80wt％碳酸氢胆碱溶液；
将香叶酸乙醇溶液和碳酸氢胆碱溶液在500mL的圆底烧瓶中混匀。在20～25℃下，以200rpm的转速搅拌混合物直到不再产生二氧化碳为止，时间为15min，随后40℃静置反应24h。60℃旋转蒸发2h除去溶剂，60℃真空烘干48h，获得碳酸氢胆碱-香叶酸低共熔混合物(或称低共熔溶剂或深共熔溶剂，不排除其中部分碳酸氢胆碱与香叶酸聚合为聚合物)CAGE，此时CAGE呈粘稠状。
碳酸氢胆碱-苯丙酸低共熔混合物CAPA制备
9.75g苯丙酸溶解在5.29mL无水乙醇中制备获得70wt％苯丙酸乙醇溶液；
6.06g碳酸氢胆碱溶液溶于1.52mL水中制备获得80wt％碳酸氢胆碱溶液；
将苯丙酸乙醇溶液和碳酸氢胆碱溶液二者在500mL的圆底烧瓶中混匀。20～25℃下，以200rpm的转速搅拌混合物直到不再产生二氧化碳为止，时间为15min，随后40℃静置反应24h。60℃旋转蒸发2h除去溶剂，60℃真空烘干48h；获得碳酸氢胆碱-苯丙酸低共熔混合物(或称低共熔溶剂或深共熔溶剂，不排除其中部分碳酸氢胆碱与苯丙酸聚合为聚合物)CAPA，此时CAPA呈粘稠状。
制剂材料CAGE+CAPA的制备
将上述制备获得的CAGE和CAPA按照体积比1:1的比例混合获得制剂材料CAGE+CAPA。
实施例2
实现核酸皮肤递送的药物制剂的制备
CAGE+mRNA制备：
向2.5mL CAGE中加入2.5mL mRNA溶液(mRNA的浓度为1μg/μl)，轻轻震荡溶解，随后置于透析袋(截留分子量：8000～10000D)中，置于10mM PBS中透析72h。
CAPA+mRNA制备：
向2.5mL CAPA中加入2.5mL mRNA溶液(mRNA的浓度为1μg/μl)，轻轻震荡溶解，随后置于透析袋(截留分子量：8000～10000D)中，置于10mM PBS中透析72h。
CAGE+CAPA+mRNA制备
将2.5mL CAPA和2.5mL CAGE混合，加入5mL mRNA溶液(mRNA的浓度为1μg/μl)，轻轻震荡溶解，随后置于透析袋(截留分子量：8000～10000D)中，置于10mM PBS中透析72h。
图1为本发明提供的实现核酸皮肤递送的药物制剂mRNA-CCC的结构示意图。
实施例3
制剂材料经皮递送mRNA效率检测
按照实施例1和实施例2中记载的方法分别制备CAGE、CAPA、CAGE+mRNA(GAPDH)、CAPA+mRNA(GAPDH)、CAGE+CAPA+mRNA(GAPDH)。
mRNA为具体的GAPDH mRNA。
将6-8周龄的balb/c小鼠(购自北京维通利华实验动物技术有限公司)在SPF条件下，并且保持12h光亮和12h黑暗循环下的通气笼中饲养。使用脱毛膏脱去背毛，在1cm×1cm范围内涂抹200μl GAPDH mRNA-CCC制剂，24h后取小鼠皮肤组织，放入液氮中速冻，将速冻皮肤组织粉碎成粉末，匀浆于QIAzol裂解剂中(每1g组织粉末加入1ml裂解剂)，制备用于qPCR的组织裂解液。进行定量PCR实验检测CCC对mRNA的递送效率，β-actin作为内参。
设置7个处理组，不同处理组分别按照上述涂抹方式涂抹200μl的PBS、mRNA(GAPDH)、CAGE、CAPA、CAGE+mRNA(GAPDH)、CAPA+mRNA(GAPDH)、CAGE+CAPA+mRNA(GAPDH)。
逆转录PCR实验步骤如下：
①反应体系
Figure PCTCN2022131486-appb-000001
轻弹管底将溶液混合，6000rpm短暂离心。
②混合液在加入逆转录酶之前先70℃干浴3min，取出后立即冰水浴至管内外温度一致，然后加逆转录酶0.5μl，37℃水浴60min。
③取出后立即95℃干浴3min，得到逆转录终溶液即为cDNA溶液，保存于-80℃。
待测样品的实时定量PCR
①β-actin阳性模板的标准梯度制备：阳性模板的浓度为10 11copies/ml，反应前取3μl按10倍稀释(加水27μl并充分混匀)为10 10，依次稀释至10 9、10 8、10 7、10 6、10 5、10 4copies/ml，以备用。
②反应体系如下：
标准品反应体系
Figure PCTCN2022131486-appb-000002
轻弹管底将溶液混合，6000rpm短暂离心。
反应条件为：93℃2min，然后93℃1min，55℃2min，共40个循环。
GADPH上游引物F：AGGTCGGTGTGAACGGATTTG(SEQ ID No.1)；GADPH下游引物R：TGTAGACCATGTAGTTGAGGTCA(SEQ ID No.2)
β-actin上游引物：GGAGATTACTGCCCTGGCTCCTA(SEQ ID No.3)，β-actin下游引物：GACTCATCGTACTCCTGCTTGCTG(SEQ ID No.4)
结果如表1和图2所示，可见本发明提供的制剂材料能有显著增强mRNA经皮递送效率。(*：p<0.05；**:p<0.001。)
表1不同处理组的皮肤组织中的mRNA相对含量
Figure PCTCN2022131486-appb-000003
实施例4
LUC mRNA-CCC制剂的制备：将2.5mL CAPA和2.5mL CAGE混合，加入5mL LUC mRNA溶液(LUC mRNA的浓度为1μg/μl)，轻轻震荡溶解，随后置于透析袋(截留分子量：8000～10000D)中，置于10mM PBS中透析72h，获得LUC mRNA-CCC制剂。
将6-8周龄的balb/c小鼠(购自北京维通利华实验动物技术有限公司)在SPF条件下，并且保持12h光亮和12h黑暗循环下的通气笼中饲养。使用脱毛膏脱去背毛， 在1cm×1cm范围内涂抹200μl LUC mRNA-CCC制剂(LUC mRNA-CCC制剂即为CAGE+CAPA+mRNA，同时设置对照，分别涂抹CAGE+mRNA、CAPA+mRNA、mRNA和PBS)，24h后经小鼠尾静脉注射荧光素酶底物，利用
Figure PCTCN2022131486-appb-000004
Spectrum In Vivo Imaging System进行荧光成像，其荧光成像的原理为编码luciferase的mRNA在小鼠体内表达之后，产物luciferase可以催化荧光素氧化成氧化荧光素，在荧光素氧化的过程中，小鼠会发出生物荧光，荧光强度和luciferase表达强度成正比，然后通过荧光测定仪测定荧光素氧化过程中释放的生物荧光，荧光检测结果如图3所示(图3中右侧荧光卡标最大值为1.93e 7p/sec/cm 2/sr，最小值为1.05e 6p/sec/cm 2/sr)，其中A为涂抹CAGE+mRNA，B为CAPA+mRNA，C为CAGE+CAPA+mRNA，D为mRNA，E为PBS；A中区域1，B中区域2以及C中区域3依次为涂抹CAGE+mRNA组、CAPA+mRNA组和CAGE+CAPA+mRNA组荧光检测显色区域，区域1荧光分析结果为：ROI 34[12％]＝8.112e 5p/sec/cm 2/sr；区域2荧光分析结果为ROI 33[12％]＝1.131e 6p/sec/cm 2/sr；区域3荧光分析结果为ROI 32[12％]＝6.093e 6p/sec/cm 2/sr(12％是利用
Figure PCTCN2022131486-appb-000005
Spectrum In Vivo Imaging System检测荧光过程中的自动参数设置)；D处理组显色面积较小，难以进行定量分析；E处理组无显色区域。通过对比区域1、2和3荧光强度可以看出，利用本发明LUC mRNA-CCC制剂检测到的荧光强度平均为6.093e 6p/sec/cm 2/sr，显著高于其他对照组，本发明提供的制剂能够有效地将核酸药物透过皮肤递送至皮下组织，显著增强mRNA经皮递送效率。
实施例5
将6-8周龄的balb/c小鼠(购自北京维通利华实验动物技术有限公司)在SPF条件下，并且保持12h光亮和12h黑暗循环下的通气笼中饲养。使用脱毛膏脱去背毛，在1cm×1cm范围内涂抹200μl实施例4中制备的LUC mRNA-CCC制剂，48h后观察皮肤状态，取小鼠皮肤组织，提取蛋白进行酶联免疫吸附实验，采用小鼠TNF alpha ELISA试剂盒(abcam，ab46105)检测炎症因子TNFa含量。结果如图4和表2所示，本发明提供的制剂材料不引起免疫因子水平升高，具有相对程度的安全性。
表2不同处理组皮肤组织中炎症因子TNFa的含量
Figure PCTCN2022131486-appb-000006
实施例6
EGF mRNA-CCC制剂的制备：将2.5mL CAPA和2.5mL CAGE混合，加入5mL EGF mRNA溶液(EGF mRNA的浓度为1μg/μl)，轻轻震荡溶解，随后置于透析袋(截留分子量：8000～10000D)中，置于10mM PBS中透析72h，获得EGF mRNA-CCC制剂。
外敷EGF mRNA-CCC制剂促进小鼠皮肤伤口愈合
实验方法：
将6-8周龄的balb/c小鼠(购自北京维通利华实验动物技术有限公司)在SPF条件下，并且保持12h光亮和12h黑暗循环下的通气笼中饲养。使用脱毛膏脱去背毛，使用打孔器在小鼠背部造成对称的两个圆形伤口，同时在1cm×1cm范围内涂抹200μl EGF mRNA-CCC制剂，每12h涂抹一次，10天后观察皮肤状态。结果如图5所示，EGF mRNA-CCC处理组伤口愈合度高，愈合效果好，说明本发明提供的EGF mRNA-CCC具有跨皮肤递送的能力，促进了损伤修复。
由上述实施例可知，本发明提供的制剂材料是一类通用经皮给药的制剂材料，能够用于不同核酸分子的局部经皮给药，能够显著增强核酸分子对皮肤的渗透作用，能够稳定有效的实现mRNA的递送，安全性好。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (14)

  1. 一种经皮给药的制剂材料，其包括碳酸氢胆碱-香叶酸低共熔混合物和/或碳酸氢胆碱-苯丙酸低共熔混合物。
  2. 根据权利要求1所述的制剂材料，其中，所述制剂材料包括碳酸氢胆碱-香叶酸低共熔混合物和碳酸氢胆碱-苯丙酸低共熔混合物，所述碳酸氢胆碱-香叶酸低共熔混合物和碳酸氢胆碱-苯丙酸低共熔混合物的体积比为(0.8～1.2)：(0.8～1.2)；
    优选地，所述碳酸氢胆碱-香叶酸低共熔混合物和碳酸氢胆碱-苯丙酸低共熔混合物的体积比为(0.9～1.1)：(0.9～1.1)。
  3. 权利要求1或2所述的制剂材料，其中：
    所述碳酸氢胆碱-香叶酸低共熔混合物中，香叶酸与碳酸氢胆碱的摩尔比为(1.5～2.5)：1，优选为(1.6～2)：1；
    所述碳酸氢胆碱-苯丙酸低共熔混合物中，苯丙酸与碳酸氢胆碱的摩尔比为(1.5～2.5)：1，优选为(1.6～2)：1。
  4. 权利要求1-3任一项所述的制剂材料的制备方法，其包括步骤：
    制备碳酸氢胆碱-香叶酸低共熔混合物或碳酸氢胆碱-苯丙酸低共熔混合物，透析；或者
    所述制剂材料包括碳酸氢胆碱-香叶酸低共熔混合物和碳酸氢胆碱-苯丙酸低共熔混合物，分别制备碳酸氢胆碱-香叶酸低共熔混合物和碳酸氢胆碱-苯丙酸低共熔混合物，将制备获得的碳酸氢胆碱-香叶酸低共熔混合物和碳酸氢胆碱-苯丙酸低共熔混合物混合、透析。
  5. 根据权利要求4所述的制备方法，其中，所述碳酸氢胆碱-香叶酸低共熔混合物通过将香叶酸乙醇溶液和碳酸氢胆碱水溶液混合反应而成；所述香叶酸乙醇溶液中香叶酸的质量百分含量为65％～75％，所述碳酸氢胆碱水溶液中碳酸氢胆碱的质量百分含量为75％～85％。
  6. 根据权利要求4所述的制备方法，其中，碳酸氢胆碱-苯丙酸低共熔混合物通过苯丙酸乙醇溶液和碳酸氢胆碱水溶液混合反应而成；所述苯丙酸乙醇溶液中苯丙酸的质量百分含量为65％～75％，所述碳酸氢胆碱水溶液中碳酸氢胆碱的质量百分含量为75％～85％。
  7. 根据权利要求5或6所述的制备方法，其中，所述混合反应的温度为20～25℃，所述混合反应的过程中伴随搅拌，所述混合反应至不再产生二氧化碳气体时结束，所述混合反应后，还包括依次进行的静置、去溶剂和烘干步骤。
  8. 根据权利要求4所述的制备方法，其中，所述透析所用的材料的截留分子量为8000～10000D，所述透析的时间为65～80h。
  9. 一种实现核酸皮肤递送的药物制剂，其包括权利要求1或2所述的制剂材料和核酸分子；所述制剂材料和核酸分子的比例为(0.8～1.2)mL：(0.8～1.2)mg。
  10. 根据权利要求9所述的药物制剂，其特征在于，所述核酸分子为mRNA。
  11. 一种制备权利要求9或10所述的药物制剂的方法，该方法包括：
    将制剂材料和核酸分子混合、透析。
  12. 权利要求1-3任一项所述的制剂材料、权利要求9或10所述的药物制剂在制备治疗皮肤病的药物中的应用。
  13. 根据权利要求12所述的应用，其中，所述皮肤病包括银屑病和/或皮肤损伤。
  14. 一种治疗皮肤病的方法，该方法包括给予受试者有效量的权利要求9或10所述的药物制剂。
PCT/CN2022/131486 2021-11-11 2022-11-11 一种经皮给药的制剂材料、实现核酸皮肤递送的药物制剂及其制备方法和应用 WO2023083322A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111332497.3 2021-11-11
CN202111332497.3A CN114042032B (zh) 2021-11-11 2021-11-11 一种实现核酸皮肤递送的药物制剂及其制备方法和应用

Publications (1)

Publication Number Publication Date
WO2023083322A1 true WO2023083322A1 (zh) 2023-05-19

Family

ID=80208388

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/131486 WO2023083322A1 (zh) 2021-11-11 2022-11-11 一种经皮给药的制剂材料、实现核酸皮肤递送的药物制剂及其制备方法和应用

Country Status (2)

Country Link
CN (1) CN114042032B (zh)
WO (1) WO2023083322A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114042032B (zh) * 2021-11-11 2024-04-26 武汉瑞佶生物科技有限公司 一种实现核酸皮肤递送的药物制剂及其制备方法和应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200289421A1 (en) * 2017-11-17 2020-09-17 President And Fellows Of Harvard College Ionic liquids for internal delivery
WO2021102084A1 (en) * 2019-11-22 2021-05-27 President And Fellows Of Harvard College Ionic liquids for drug delivery
CN114042032A (zh) * 2021-11-11 2022-02-15 武汉瑞佶生物科技有限公司 一种实现核酸皮肤递送的药物制剂及其制备方法和应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA3094445A1 (en) * 2018-03-19 2019-09-26 Cage Bio Inc. Ionic liquid compositions for treatment of rosacea

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200289421A1 (en) * 2017-11-17 2020-09-17 President And Fellows Of Harvard College Ionic liquids for internal delivery
WO2021102084A1 (en) * 2019-11-22 2021-05-27 President And Fellows Of Harvard College Ionic liquids for drug delivery
CN114042032A (zh) * 2021-11-11 2022-02-15 武汉瑞佶生物科技有限公司 一种实现核酸皮肤递送的药物制剂及其制备方法和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
AMRITA. B ET AL.: "Transdermal Protein Delivery Using Choline and Geranate (CAGE) Deep Eutectic Solvent", ADV HEAL THE MATER, vol. 6, no. 15, 31 August 2017 (2017-08-31), XP055611365, DOI: 10.1002/adhm.201601411 *

Also Published As

Publication number Publication date
CN114042032B (zh) 2024-04-26
CN114042032A (zh) 2022-02-15

Similar Documents

Publication Publication Date Title
JP6140120B2 (ja) 挫瘡およびその他の状態を治療するためのビスファチン治療薬
JP2002537343A (ja) 多重粒子製剤
WO2023083322A1 (zh) 一种经皮给药的制剂材料、实现核酸皮肤递送的药物制剂及其制备方法和应用
WO2001091728A2 (en) Nanoemulsion formulations
Shi et al. Promoting Re-epithelialization in an oxidative diabetic wound microenvironment using self-assembly of a ROS-responsive polymer and P311 peptide micelles
CN111407721B (zh) 一种促进活性成分渗透皮肤的体系
Yang et al. Co-delivery of methotrexate and nicotinamide by cerosomes for topical psoriasis treatment with enhanced efficacy
EP3532071A2 (en) Pharmaceutical compositions and methods of use for activation of human fibroblast and myofibroblast apoptosis
US20110097391A1 (en) Phospholipid Emulsion Containing Dihydroquercetin, and Method of Producing Thereof
Vallorz et al. Formoterol PLGA-PEG nanoparticles induce mitochondrial biogenesis in renal proximal tubules
Huang et al. An mTOR siRNA‐Loaded Spermidine/DNA Tetrahedron Nanoplatform with a Synergistic Anti‐Inflammatory Effect on Acute Lung Injury
CN108066279A (zh) 一种含有苯烯莫德的外用乳膏组合物
Zhang et al. Combined intramyocardial injectable hydrogel and pericardial adhesive hydrogel patch therapy strategy to achieve gene/ion/gas delivery for improving cardiac function
Fan et al. Antioxidant‐engineered milk‐derived extracellular vesicles for accelerating wound healing via regulation of the pi3k‐akt signaling pathway
Cao et al. Turning gray selenium into a nanoaccelerator of tissue regeneration by PEG modification
CN102657602A (zh) 3,5-二羟基-4-异丙基二苯乙烯壳聚糖凝胶剂及其制备方法
Tanga et al. RETRACTED: Centella asiatica extract in carboxymethyl cellulose at its optimal concentration improved wound healing in mice models
Xu et al. Emerging application of nanomedicine-based therapy in acute respiratory distress syndrome
WO2019241985A1 (zh) 一种水凝胶、含其的药物组合物及其应用
Jin et al. Co enhances agomir transfection under pathological conditions to inhibit mmp overexpression
WO2013139918A1 (en) Topical application of ingenol mebutate with occlusion
CN110511930B (zh) Sal-miR-58及其在抑制血管炎性反应和动脉瘤形成中的用途
FR2686251A1 (fr) Composition pour la cicatrisation d&#39;une plaie.
Shaik et al. Cordycepin-melittin nanoconjugate intensifies wound healing efficacy in diabetic rats
US20210161816A1 (en) Methods and compositions for treating skin afflictions

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22892115

Country of ref document: EP

Kind code of ref document: A1