A recombinant humanized collagen type XVII composition for treating psoriasis and a preparation method and application thereof

Abstract

The present application relates to the technical field of medicine, in particular to a recombinant type XVII humanized collagen composition for treating psoriasis and a preparation method and application thereof.The present application provides a composition based on choline carboxylate and recombinant type XVII humanized collagen (COL17 / COL XVII). The composition utilizes the unique penetration performance of choline carboxylate to effectively overcome the stratum corneum barrier and deliver COL17 to the skin lesion site efficiently; meanwhile, choline carboxylate can maintain the structural stability of COL17, prolong the local action time, and achieve precise and long-acting skin barrier repair and anti-inflammatory regulation. The drug for treating psoriasis provided by the present application can be used externally at home, does not need physical damage, avoids the risk of pain and infection, and has a simple preparation process and easy continuous production, thereby providing a safe, convenient and highly compliant new local treatment scheme for psoriasis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a recombinant type XVII humanized collagen composition for treating psoriasis, its preparation method, and its application. Background Technology

[0002] Psoriasis is a common, chronic autoimmune inflammatory skin disease driven by a combination of genetic, immune, and environmental factors. Its typical clinical manifestations include well-defined red plaques covered with silvery-white scales, often accompanied by itching, scaling, and even pain. In severe cases, the plaques can spread throughout the body, significantly impacting the patient's appearance and quality of life. The core pathological mechanism of this disease lies in a vicious cycle of impaired skin barrier function and immune system dysfunction, and it is characterized by recurrent flare-ups and difficulty in achieving a complete cure. Even if patients experience temporary symptom relief after using conventional medications, relapse is highly likely after discontinuation, especially for moderate to severe cases, where current treatments often face limitations in efficacy, significant side effects, and high relapse rates.

[0003] Currently, clinical treatment techniques for psoriasis are mainly divided into the following three categories:

[0004] Topical medication: As a basic treatment method, this involves applying preparations directly to the affected area. However, traditional topical preparations suffer from low transdermal absorption efficiency when delivering large molecular repair agents, while some small molecule drugs with clear anti-inflammatory activity may be accompanied by local or systemic side effects.

[0005] Systemic drug therapy: This involves administering medication orally or by injection to systemically suppress the activity of the immune system and block the inflammatory cascade. While this approach is effective for moderate to severe psoriasis, it carries the risk of serious side effects from systemic immunosuppression (such as increased risk of infection and tumors), requires regular hematological monitoring, and is expensive. Furthermore, it cannot effectively repair the damaged skin barrier.

[0006] Physical therapy mainly includes ultraviolet radiation (phototherapy) and minimally invasive physical techniques (such as microneedling and laser therapy). Phototherapy requires patients to frequently visit medical institutions for treatment, and long-term use can accumulate the risk of photoaging and skin cancer; while invasive or minimally invasive methods such as microneedling and laser therapy can promote drug penetration, but there is a possibility of skin damage, pain, and secondary infection.

[0007] To address these issues, researchers are actively exploring transdermal delivery technologies for macromolecular drugs to achieve non-invasive and highly effective local treatment. For example, melittin (Mel) has been studied for its anti-inflammatory and antioxidant activities in the treatment of psoriasis. However, the mechanism of action of melittin in current technologies leans towards broad-spectrum immune regulation. Its core deficiency lies in its failure to target key targets in skin structure repair during psoriasis pathogenesis, focusing on short-term symptom relief while neglecting the long-term restoration of skin barrier function. Given that psoriasis recurrence is closely related to incomplete repair of skin barrier function, existing strategies based on melittin or similar broad-spectrum anti-inflammatory molecules still fall short of meeting the clinical need for long-term stable, low-recurrence treatment.

[0008] In addition, existing topical treatments may have insufficient biocompatibility issues with their excipients or active ingredients during long-term use, such as causing skin irritation, sensitization, or even hemolysis, which limits their long-term and safe application in chronic skin diseases.

[0009] Therefore, there is an urgent need in this field to develop a novel topical treatment technology for psoriasis that is naturally more biocompatible, free from potential risks such as hemolysis and sensitization, and can simultaneously achieve efficient transdermal drug delivery, repair of skin barrier function, and is suitable for long-term home use by patients. Summary of the Invention

[0010] In view of this, the technical problem to be solved by the present invention is to provide a composition.

[0011] The present invention provides a composition comprising a transdermal agent and type XVII collagen.

[0012] Compared to other traditional anti-inflammatory drugs or broad-spectrum active peptides, type XVII collagen can directly improve psoriasis-related symptoms such as skin fragility, scaling, and cracking, thus enhancing overall skin health. Specifically, type XVII collagen can maintain the integrity of the epidermal-dermal junction, regulate the differentiation and proliferation of keratinocytes, participate in cell signaling and inflammation regulation, and effectively repair damaged basement membrane structures.

[0013] In some embodiments, the type XVII collagen is recombinant type XVII collagen.

[0014] Preferably, the type XVII collagen is recombinant type XVII humanized collagen.

[0015] In some embodiments, the collagen is dissolved in an acidic solution; the acidic solution includes hydrochloric acid, citric acid, acetic acid, phosphoric acid, sulfuric acid, formic acid, lactic acid, malic acid, oxalic acid, or tartaric acid.

[0016] In some embodiments, the composition comprises choline carboxylate and type XVII collagen. Through experimental screening and optimization, compared with other types of transdermal agents, choline carboxylate exerts a permeation-enhancing effect, significantly improving the transdermal delivery efficiency of type XVII collagen; on the other hand, it protects the bioactivity of type XVII collagen and prolongs its effective retention time at the lesion site through a stabilizing effect.

[0017] In some embodiments, the choline carboxylate is at least one selected from geraniol choline, citrate choline, lactate choline, malate choline, acetylpropionic acid choline, or sorbate choline. Specifically, the choline carboxylate is geraniol choline.

[0018] In some embodiments, the choline carboxylate is prepared by reacting choline compounds and carboxylic acid compounds.

[0019] In some embodiments, the choline compound is selected from at least one of choline hydroxide, choline chloride, or choline bicarbonate. These compounds exhibit high biocompatibility and low toxicity, which helps reduce the risk of skin irritation from long-term topical application and improves the safety of the formulation.

[0020] In some specific embodiments, the choline compound is bicarbonate choline. Compared to other choline salts, bicarbonate choline has a milder pH-regulating ability, which can reduce irritation to damaged skin while maintaining the stability of transdermal agents.

[0021] In some embodiments, the carboxylic acid compound is selected from at least one of geranilic acid, citric acid, lactic acid, malic acid, levulinic acid, or sorbic acid. These carboxylic acid compounds can form stable ionic liquids with choline compounds through ion pairing, and impart good skin compatibility and penetration-enhancing properties to the formulation.

[0022] In some specific embodiments, the carboxylate compound is geranilic acid or citric acid. Preferably, the carboxylate compound is geranilic acid. Experimental verification has shown that, compared to other compounds, geranilic acid not only significantly enhances skin permeability and promotes transdermal absorption of active ingredients such as collagen, but also possesses anti-inflammatory activity. When used in combination with type XVII collagen, it can further alleviate the inflammatory response of psoriatic lesions, improve local redness, swelling, and desquamation, producing a synergistic effect.

[0023] As a feasibility example, the composition comprises type XVII collagen and geraniol choline. Geraniol choline is prepared from carbohydrate choline and geranilic acid. Through experimental screening, compared to other compositions, type XVII collagen and geraniol choline prepared from carbohydrate choline exhibit a significant synergistic effect. Geranilic acid not only promotes the transdermal absorption of type XVII collagen, but its combined use with type XVII collagen also inhibits the release of inflammatory factors such as IL-17A, IL-6, and TNF-α in psoriatic lesions, forming a dual "anti-inflammatory + repair" effect with the barrier repair function of type XVII collagen.

[0024] In some embodiments, the molar ratio of the choline carboxylate to the choline compound is (0.5~2.5):(0.5~3.5); for example, the molar ratio of the choline compound to the carboxylate is 1:1, 1:2, 2:3, 1.5:1, 2:1, 1.5:2 or 2.5:3.5.

[0025] In some embodiments, the molar ratio of the choline carboxylate to the choline compound is (0.5~2.5):1; for example, the molar ratio of the choline compound to the carboxylate is 1:1, 1.5:1, 2:1 or 2.5:1.

[0026] In some specific embodiments, the molar ratio of choline compounds to carboxylic acid compounds in the choline carboxylate is 1:1, 1.5:1, and 2:1. Preferably, the molar ratio of choline compounds to carboxylic acid compounds in the choline carboxylate is 1.5:1. Experimental results show that, compared with other ratios, the choline carboxylate at this ratio has the best transdermal penetration-enhancing effect on type XVII collagen, significantly increasing the cumulative penetration of the drug in the local skin while reducing the retention and loss of collagen in the epidermis, thereby achieving a more efficient and longer-lasting local treatment effect for psoriasis.

[0027] As a feasible example, the composition comprises type XVII collagen and geraniol choline; the geraniol choline is prepared by reacting carbohydrate choline and geraniol, wherein the molar ratio of carbohydrate choline to geraniol is (0.5~2.5):1. For example, the molar ratio of the choline compound to geraniol is 1:1, 1.5:1, 2:1, or 2.5:1.

[0028] Preferably, the molar ratio of hydrocarbon choline to geraniol is 1.5:1.

[0029] The components and proportions were determined through experimental screening. Compared with other components and proportions, the advantages of this composition are as follows: Germicidal acid and choline exert a dual effect of promoting penetration and stabilizing, achieving precise, efficient, and long-lasting delivery of type XVII collagen to psoriatic lesions, reducing its retention and loss in the epidermis; secondly, germicidal acid itself has anti-inflammatory activity, forming a synergistic "anti-inflammatory + repair" effect with the barrier repair function of type XVII collagen; thus, this composition achieves a more efficient and longer-lasting local treatment effect for psoriasis.

[0030] In some embodiments, the molar ratio of the transdermal agent to type XVII collagen in the composition is (100~400):(1~5); for example, the molar ratio of the transdermal agent to type XVII collagen is 100:1, 200:1, 300:1, 300:2, 350:3, or 400:1. At this ratio, the transdermal agent can better promote the transdermal absorption of type XVII collagen, and the complex system formed by collagen and transdermal agent is homogeneous and stable, less prone to phase separation or decreased activity, which is beneficial to the storage and use of the product.

[0031] In some embodiments, the molar ratio of the choline carboxylate to type XVII collagen in the composition is (100-400):(1-5); for example, the molar ratio of the choline carboxylate to type XVII collagen is 100:1, 200:1, 300:1, 300:2, 350:3, or 400:1. Experimental results show that, compared to other molar ratio ranges, choline carboxylate has the best transdermal penetration enhancement effect on type XVII collagen within this range, enabling type XVII collagen to penetrate the stratum corneum in a dispersed and stable form, achieving an effective therapeutic concentration at the lesion site, while reducing non-specific adsorption and waste, thereby achieving "low-dose, high-efficiency" local drug delivery.

[0032] In some embodiments, the molar ratio of the choline carboxylate to type XVII collagen is (100~400):1; for example, the molar ratio of the choline carboxylate to type XVII collagen is 100:1, 200:1, 300:1 or 400:1.

[0033] In some specific embodiments, the molar ratio of choline carboxylate to type XVII collagen is 100:1, 200:1, 300:1 and 400:1; preferably, the molar ratio of choline carboxylate to type XVII collagen is 300:1.

[0034] As a feasible example, in the composition, the molar ratio of geraniol choline to type XVII collagen is 300:1.

[0035] In some embodiments, the volume fraction of geraniol choline in the composition is 20% to 80%; for example, the volume fraction of geraniol choline is 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70% or 80%.

[0036] In some embodiments, the volume fraction of geraniol choline in the composition is 20% to 40%; for example, the volume fraction of geraniol choline is 20%, 25%, 30%, 35%, or 40%. Experimental results show that the compositions in this range have the best transdermal effect compared to other ranges.

[0037] In some specific embodiments, the volume fraction of geraniol choline is 20%, 40%, 60%, or 80%.

[0038] Preferably, the volume fraction of geraniol choline is 20%. Quantitative detection results show that, compared with other conditions, the composition has the best transdermal effect under these conditions, with type XVII collagen exhibiting higher permeability and faster diffusion rate in the first six hours.

[0039] The present invention also provides a method for preparing the composition as described above, comprising: reacting a choline-based compound with a carboxylic acid compound in an organic solvent, mixing the resulting product with type XVII collagen, and obtaining the composition.

[0040] In this invention, the organic solvent includes, but is not limited to, at least one of anhydrous ethanol, methanol, isopropanol, or acetonitrile. In some specific embodiments, the organic solvent is anhydrous ethanol. Experiments show that using anhydrous ethanol as a reaction reagent can more effectively ensure sufficient contact between cations and anions, promote complete reaction, and form a homogeneous reaction product.

[0041] In this invention, the reaction conditions include: ambient temperature and pressure, stirring at 200-300 r / min for 4-10 h. Ambient temperature and pressure refer to a temperature of 25℃ ± 5℃ and a pressure of 1 atmosphere ± 0.05 atmospheres.

[0042] In this invention, the reaction is followed by a step of evaporating the organic solvent and / or drying. This is achieved through rotary evaporation. The conditions for rotary evaporation include: a temperature of 45℃±5℃, a rotation speed of 40~60 r / min, and vacuum distillation.

[0043] In some embodiments, the stirring reaction time is 4–10 hours, and the stirring speed is 200–300 r / min. For example, the stirring reaction time is 4, 6, 8, or 10 hours, and the stirring speed is 200, 250, or 300 r / min. The stirring conditions were determined through experimental screening. Compared to other conditions, these conditions enable thorough and uniform mixing of choline carboxylate and type XVII collagen, preventing collagen aggregation or denaturation, thereby ensuring the homogeneity and stability of the composition and facilitating the effective penetration of type XVII collagen during subsequent transdermal administration.

[0044] In some embodiments, the rotary evaporation temperature is 45±5°C, and the rotary evaporation rate is 40~60 r / min. For example, the rotary evaporation temperature is 40, 43, 45, 48, or 50°C, and the rotary evaporation rate is 40, 45, 50, 55, or 60 r / min. The rotary evaporation conditions were determined through experimental screening, and compared with other conditions, these conditions can better remove anhydrous ethanol and trace amounts of water that may be generated during the reaction.

[0045] Furthermore, the present invention provides the use of type XVII collagen, the composition as described above, or the composition obtained by the preparation method as described above, in the preparation of a medicament for treating psoriasis.

[0046] In some embodiments, the psoriasis treatment drug has the function of at least one of the following: maintaining the integrity of the epidermal-dermal junction; regulating keratinocyte differentiation and proliferation; inhibiting the release of inflammatory factors; repairing damaged basement membrane structures; or improving skin barrier function.

[0047] In some embodiments, the improvement of skin barrier function includes at least one of the following: reducing psoriasis and redness, reducing epidermal thickening and hyperkeratosis, or improving symptoms such as skin fragility, peeling, and cracking.

[0048] Furthermore, the present invention also provides a medicament for treating psoriasis, comprising type XVII collagen as described above, or a composition as described above, or a composition obtained by the preparation method described above.

[0049] In some embodiments, the mass fraction of choline carboxylate in the drug is 50% to 80%, and the mass fraction of type XVII collagen is 20% to 50%.

[0050] In some embodiments, the dosage form of the drug includes a gel, patch, liquid, emulsion, or suspension.

[0051] In some embodiments, the drug further includes pharmaceutically acceptable excipients; the excipients include thickeners, humectants, pH adjusters, preservatives, antibacterial agents, antioxidants, emulsifiers, or stabilizers.

[0052] The drug of the present invention is administered transdermally for the improvement and / or treatment of skin diseases, including but not limited to psoriatic arthritis, plaque psoriasis, atopic dermatitis, and inflammatory skin diseases.

[0053] Furthermore, the present invention also provides a method of using the drug as described above, comprising applying an effective amount of the drug to the affected skin of an individual in need.

[0054] The beneficial effects of this invention are:

[0055] 1. The strategy of delivering COL17 protein via choline geranium ionic liquid has the core advantage of repair and regeneration, which improves the skin barrier function from the root and may bring long-term relief.

[0056] 2. Potential for long-term remission: The COL17 strategy targets the structural defects and stem cell dysfunction in psoriatic skin lesions, the very conditions that breed the disease. Repairing this foundation could potentially restore normal skin homeostasis, rather than merely suppressing surface inflammation. Theoretically, this could potentially induce longer periods of remission and reduce relapses.

[0057] 3. Improve skin barrier function: A repaired skin barrier is a natural physical and immune defense line that can resist external irritants, allergens, and microbial invasion, which are common triggers or aggravating factors for psoriasis. This is a proactive defense strategy;

[0058] 4. Wider range of effects: In addition to anti-inflammatory and anti-proliferative effects, COL17 can directly improve symptoms such as skin fragility, peeling, and cracking, and improve the overall health quality of the skin.

[0059] This invention relates to a recombinant type XVII humanized collagen composition for treating psoriasis, its preparation method, and its application. This technical solution addresses the core pain points of existing psoriasis treatments, such as low transdermal delivery efficiency, easy loss of bioactivity, and significant side effects, by employing a composite formulation design of "choline carboxylate + recombinant type XVII humanized collagen." Through the dual effects of choline carboxylate in promoting penetration and stabilizing the skin, precise, efficient, and long-lasting delivery of recombinant type XVII humanized collagen to psoriatic lesions is achieved, while simultaneously ensuring treatment safety and patient compliance. Attached Figure Description

[0060] Figure 1 Infrared spectra of bicarbonate choline (Ch), geranilic acid (Ger), and bicarbonate choline:geranilic acid = 1.5:1 (Ch:Ger = 1.5:1);

[0061] Figure 2NMR spectra of bicarbonate choline (Ch), geranilic acid (Ger), and bicarbonate choline:geranilic acid = 1.5:1 (Ch:Ger = 1.5:1);

[0062] Figure 3 The results are for the stability test of type XVII collagen;

[0063] Figure 4 Results of qualitative fluorescence analysis in the Franz diffusion cell (at different scales);

[0064] Figure 5 The results of qualitative fluorescence analysis in the Franz diffusion cell (for different viscosities);

[0065] Figure 6 Results of quantitative determination of the permeability of type XVII collagen in ionic liquids of different viscosities using a Franz diffusion cell;

[0066] Figure 7 The results are from the skin transdermal assay in KM mice.

[0067] Figure 8 These are the results of cytotoxicity assays.

[0068] Figure 9 These are the results of in vitro anti-inflammatory performance tests;

[0069] Figure 10 These are representative images from the seventh day of psoriasis treatment;

[0070] Figure 11 Figure 1 shows the results of the detection of psoriasis area and severity index (a is the mouse's weight change graph, b is the PASI score index, c is a representative image of the mouse's spleen on day 7, and d is the spleen weight index).

[0071] Figure 12 Representative images of skin stained with HE and quantitative statistical charts of epidermal thickness in sections (A is a representative image of skin stained with HE, B is a quantitative statistical chart of epidermal thickness in sections).

[0072] Figure 13 The images show the appearance of choline-germicolic acid ionic liquids with different molar ratios (left bottle: Ch:Ger=1:1, i.e., choline:germicolic acid=1:1; right bottle: Ch:Ger=3:2, i.e., choline:germicolic acid=1.5:1). Detailed Implementation

[0073] This invention provides a recombinant type XVII humanized collagen composition for treating psoriasis, its preparation method, and its application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0074] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0075] The terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” herein also provides for schemes “consisting of…”.

[0076] When used herein, the term “and / or” includes the meaning of “and,” “or,” and “all or any other combination of elements linked by the term.”

[0077] The term "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or plural items.

[0078] The term "ionic liquid" refers to an organic salt formed by the combination of cations and anions, which exists in a liquid state at or near room temperature. Ionic liquids include ion-pair compositions generated by acid-base neutralization reactions, such as choline carboxylates formed by choline cations and carboxylate anions, including but not limited to geraniol choline, citrate choline, lactate choline, malate choline, levulinate choline, sorbate choline, or any combination thereof.

[0079] The term "viscosity" refers to the volume fraction of geraniol choline in a composition. For example, "20% to 40% viscosity" means that the volume fraction of geraniol choline is 20% to 40%.

[0080] The terms “recombinant humanized collagen type XVII”, “COL17”, and “COL17” are used interchangeably in this article. They can be obtained by expressing them in host cells through genetic engineering methods and have biological activities such as promoting skin repair and improving psoriasis-related symptoms.

[0081] In the composition of this invention, the transdermal agent and type XVII collagen can exist mixed or independently, and this invention does not limit this. If they are mixed, the composition can be in a solution or gel state. If they exist independently, the transdermal agent (such as choline carboxylate) exists in a liquid form at or near room temperature, and the type XVII collagen can be in a solution or powder form.

[0082] When the composition is in solution, type XVII collagen and choline carboxylate can be dissolved together in a suitable solvent, such as water, phosphate buffer, or other physiologically compatible solvents, to form a homogeneous solution. In such a solution, choline carboxylate acts as a transdermal agent, fully contacting type XVII collagen molecules to promote their transdermal absorption. When the composition is in gel state, the addition of appropriate gel matrix, such as carbomer or hydroxypropyl methylcellulose, can maintain good biocompatibility while imparting suitable viscosity and spreadability, making it more suitable for topical application to the skin.

[0083] When the transdermal agent and type XVII collagen exist independently, the transdermal agent is provided in solution form, which can be formulated into an aqueous solution, salt solution, or alcohol solution of a specific concentration; the type XVII collagen can be provided in solution form or stored separately in powder form. In use, the transdermal agent solution can be applied to the skin surface first to pre-treat the stratum corneum, followed by the application of the solution or powder containing type XVII collagen; alternatively, the type XVII collagen powder can be dissolved in a solvent before use and then applied in conjunction with the transdermal agent solution, either sequentially or simultaneously. Similarly, when type XVII collagen is in solution, it can be prepared separately into a solution formulation of a certain concentration and used in conjunction with the transdermal agent solution; when in powder form, it can be stored separately and used in combination with the transdermal agent solution as needed. This flexible form makes the compositions of the present invention more convenient and adaptable in practical applications, allowing for selection and adjustment according to different usage scenarios and needs.

[0084] In this invention, "choline carboxylate" refers to a salt compound formed by the ionic bonding of a choline-like compound (cationic moiety) and a carboxylic acid compound (anionic moiety). The chemical formula of the choline-like compound is [HOCH2CH2N]. + [CH3)3]OH - Or it may be written as ([Ch][OH]·H2O), and carboxylic acid compounds provide carboxylate anions (RCOO). -The R group can be various alkyl, alkenyl, aromatic, etc. The choline carboxylate obtained from the reaction of geranilic acid (denoted as H[Ger]) and choline is geranilic acid choline, or "choline geraniyl bicarbonate," with the reaction formula [Ch][OH] + H[Ger] → [Ch][Ger] + H₂O + CO₂, which is a eutectic solvent. In this invention, geranilic acid choline not only acts as a transdermal agent but also further enhances the therapeutic effect of type XVII collagen on psoriasis, producing a synergistic effect in combination with type XVII collagen.

[0085] In this invention, the "Type XVII collagen" is a key component of the skin's basement membrane, mainly located at the junction of the epidermis and dermis, responsible for maintaining skin structural stability and cell connections. It consists of over 1500 amino acid residues, and its molecular structure comprises three main parts: an extracellular domain, a transmembrane domain, and an intracellular domain. The type XVII collagen used in this invention can be naturally extracted or recombinantly expressed through genetic engineering technology. Its amino acid sequence is shown in SEQ ID NO.1, specifically GSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEA, exhibiting good biological activity and safety.

[0086] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0087] This technical solution addresses the core pain points of existing psoriasis treatments, such as low transdermal delivery efficiency, easy loss of bioactivity, and significant side effects. It adopts a compound formulation design of "choline carboxylate + recombinant XVII type humanized collagen". Through the dual effects of choline carboxylate in promoting penetration and stabilizing, it achieves precise, efficient, and long-lasting delivery of recombinant XVII type humanized collagen (COL17) to psoriatic lesions, while taking into account treatment safety and patient compliance.

[0088] This invention provides a more convenient and safer alternative for topical treatment of psoriasis: it does not interfere with the systemic immune system, thus reducing systemic side effects; it can be used at home, is easy to operate, and requires no professional equipment; it utilizes choline carboxylates to enhance penetration, eliminating the need for physical damage and avoiding the risks of pain and infection; simultaneously, COL17 can accelerate skin healing and reduce inflammatory responses after physical therapy. Furthermore, the preparation process of this invention is simple, has a short cycle, and can be continuously produced; by optimizing the ion combination (such as amino acid groups / choline groups), skin irritation and sensitization are reduced, improving skin suitability.

[0089] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have a reasonable deviation within a certain range, such as ±10%, ±5%, ±1%, or ±0.5%.

[0090] All test materials used in this invention are commercially available products. The recombinant XVII humanized collagen was purchased from Jinbo Biotechnology.

[0091] The present invention will be further illustrated below with reference to the embodiments:

[0092] Example 1: Construction and Characterization of the Delivery System

[0093] 1. Preparation of ionic liquids (ILs)

[0094] 1.1 Raw material pretreatment: Select the target cationic compound and anionic compound respectively, and weigh them according to a variety of preset molar ratios (such as 1:1, 1.5:1, 1:2, 2:1, etc., and the gradient molar ratio can be adjusted according to actual needs);

[0095] 1.2 Raw material feeding and mixing: The pretreated cationic compound and anionic compound are added sequentially to a dry and clean round-bottom flask, ensuring that the mixture occupies 1 / 3 to 1 / 2 of the flask volume to facilitate subsequent stirring operations.

[0096] 1.3 Solvent addition and room temperature stirring: Add 5-15 mL of anhydrous ethanol to the round-bottom flask as a reaction medium to promote the interaction between cations and anions. Then place the round-bottom flask on a magnetic stirrer and stir continuously at 200-300 r / min for 4 h at room temperature and atmospheric pressure to ensure that the cations and anions are in full contact, the reaction is complete, no carbon dioxide bubbles are formed, and a homogeneous reaction system is formed.

[0097] 1.4 Removal of organic solvents and water: After the reaction is completed by stirring, the round-bottom flask is connected to a rotary evaporator. The rotary evaporation parameters are set as follows: water bath temperature 45±5℃, rotation speed 40~60 r / min. The anhydrous ethanol and trace amounts of water that may be generated during the reaction are completely removed from the system by vacuum distillation until there is no obvious liquid residue in the flask, and a viscous intermediate product is obtained.

[0098] 1.5 Vacuum Drying and Purification: The viscous intermediate product after rotary evaporation is transferred to a vacuum drying oven to further remove residual trace solvents and moisture, ensuring product purity. After drying, the product, which is the target ionic liquid (IL), is removed and stored in a dry, sealed container away from light for later use.

[0099] Among them, ionic liquids (ILs) are eutectic solvents that exist in liquid form at room temperature.

[0100] In addition, during the synthesis of IL-COL17, COL17 is added to IL at the desired concentration and mixed thoroughly to obtain the final product IL-COL17.

[0101] 2. Characterization: Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (NMR) were used. 1 The structure of ionic liquids (ILs) was analyzed by 1H NMR.

[0102] 3. COL17 Stability: To investigate the effect of ionic liquids on the stability of collagen, the stability of collagen in the prepared materials was tested. Collagen was treated with pure water and ionic liquid respectively, and after 24 hours, the peak time was determined by high-performance liquid chromatography (HPLC). The stability of collagen was evaluated by comparing the changes in peak time, with three parallel groups. Representative results are shown below. Figure 2 As shown.

[0103] 4. In vitro release behavior: Franz diffusion cell experiment, investigation of qualitative and quantitative fluorescence.

[0104] The specific steps are as follows: Qualitative analysis: The pre-treated pigskin skin (skin thickness approximately 0.5 mm, area approximately 2 cm²) 2 The skin was fixed between the donor and receiver cells in the Franz diffusion cell. 400 μL of IL or PBS and 100 μL of FITC-COL17 (1 mg / mL) were added to the donor cell, and the receiver cell was filled with PBS (pH=7.4). After incubation at 37°C for 12 hours, the skin was removed and gently washed with PBS. After freezing and sectioning, DAPI staining was performed and the samples were analyzed using a fluorescence microscope.

[0105] Quantitative analysis: Similarly, the treated pig skin was placed between the donor and receiver pools. The sample to be tested was added to the donor pool, and the receiver pool was filled with PBS. The water bath temperature was maintained at 37°C, and the stirring speed was 500 r / min. At preset time points (1, 2, 4, 6, 8, 12, and 24 hours), 0.5 mL of the receiving liquid was quantitatively aspirated from the sampling port of the receiver pool, and an equal volume of fresh receiving liquid was immediately added at the same temperature. After the sample was filtered, the collagen content was analyzed by high-performance liquid chromatography (HPLC). The cumulative permeability was calculated using Formula 1:

[0106] Q n =(VC n + ) / A (Formula 1)

[0107] Q n It is the cumulative permeation at the nth sampling point (μg / cm³). 2 V is the volume of the receiving cavity. i C is the volume of the received solution sampled at the i-th time point. i The concentration of collagen in the solution is received at the i-th sampling point C. n is the collagen concentration at the nth sampling point, and A is the diffusion area.

[0108] 5. In vivo release behavior: Healthy KM mice were used, and after hair removal on their backs, samples of different groups were evenly applied to the hair-removed areas, with 3 mice in each group. The animals were sacrificed 12 hours after application, and the skin from the treatment area was excised, the subcutaneous tissue was removed, and the samples were frozen and sectioned. The fluorescence distribution was observed under an inverted fluorescence microscope.

[0109] Results analysis:

[0110] Figure 1 The infrared spectra of Ch, Ger, and Ch:Ger = 1.5:1 show that choline is present at 3300 cm⁻¹. -1 1085 cm -1 955 cm -1 The characteristic absorption peak of choline cations is observed at 2920 cm⁻¹; geraniol shows an absorption peak at 2920 cm⁻¹. -1 1680 cm -1 The peaks at these locations correspond to the characteristic absorption peaks of alkyl CH stretching vibration and carboxyl C=O stretching vibration, respectively; after the ionic liquid is prepared by combining the two, geraniol originally had a peak at 1680 cm⁻¹. -1 The characteristic absorption peak of the carboxyl carbonyl group completely disappeared, and at 1550 cm⁻¹... -1 The generation of a new characteristic absorption peak of carboxylate anion at wavenumber confirms that choline cations and geranate anions undergo acid-base proton transfer to form an ionic liquid with ionic bonds. Figure 2The NMR spectra are for Ch, Ger, and Ch:Ger = 1.5:1. The graph shows the NMR of choline (Ch). 1 In the 1H-NMR spectrum, a characteristic singlet of -CH3 appears at 3.19 ppm, and a multiplet of CH2-OH appears at 3.90 ppm; geranic acid (Ger) 1 In the H-NMR spectrum, a characteristic singlet of CH3 appeared at 1.60 ppm, and a characteristic multiplet of CH2 appeared at 2.10 ppm. After the two reacted to obtain the ionic liquid (Ch-Ger), the characteristic chemical shift signals corresponding to choline and geranium were retained in the product spectrum. The peak shape and elution position matched the precursor structural characteristics, proving that the Ch-Ger ionic liquid contains the characteristic structures of both choline cation and geranium anion, indicating that the ionic liquid was successfully synthesized.

[0111] Figure 3 The results showed that, based on the peak times obtained by high-performance liquid chromatography (HPLC) after treating collagen with pure water and ionic liquids, the peak time of H2O-COL17 was 20.606±0.022, and the peak time of IL-COL17 was 20.605±0.012. This indicates that dissolving collagen in ionic liquids did not alter the collagen structure and demonstrated good stability.

[0112] Figure 4 The transdermal effects of choline (Ch) with citric acid (Cit) and geranilic acid (Ger) at different ratios were compared. The results showed that the transdermal effect was best when the molar ratio of choline to geranilic acid was 1.5:1. Figure 13 The images show the appearance of choline-germicolic acid ionic liquids with different molar ratios (left bottle: Ch:Ger=1:1, i.e., choline:germicolic acid=1:1; right bottle: Ch:Ger=3:2, i.e., choline:germicolic acid=1.5:1).

[0113] Figure 5 The effect of different viscosities on transdermal absorption was investigated when the molar ratio of choline to geraniol was 1.5:1. The results showed that the transdermal absorption effect was better when the viscosity was between 20% and 40%. The viscosity of 20% to 40% means that 20% to 40% of the total volume of the ionic liquid was dispersed in PBS, dissolved, and then the corresponding COL17 powder was weighed and dissolved in it to form an IL-COL17 complex.

[0114] Figure 6 The permeability of ionic liquid type XVII collagen with different viscosities was quantitatively detected. The results showed that type XVII collagen diffused faster in the first six hours and then tended to stabilize. At a viscosity of 20%, type XVII collagen permeated more.

[0115] Figure 7The results of the skin transdermal assay in KM mice showed that IL-COL17 had a better transdermal effect than PBS-COL17, with COL17 reaching the dermis layer, while the control group showed no fluorescent penetration in the epidermis.

[0116] Example 2 In vitro cell experiments

[0117] 1. Biocompatibility (cellular level) – Verifying whether it is toxic and safe;

[0118] The prepared ionic liquid material was subjected to cytotoxicity tests. The cytotoxic effect on HaCaT (human immortalized keratinocytes) was investigated by co-culturing the ionic liquid material with the material. First, the ionic liquid material was sterilized by UV light and then placed in DMEM medium, diluted to 5, 2, 1, 0.5, 0.25, and 0.125 mg / mL. Then, healthy HaCaT cells were selected and plated in 96-well plates, with 5 × 10⁶ cells per well. 3 Cells were cultured in a 5% CO2 incubator at 37°C until they adhered to the culture vessel. The medium was then changed using DMEM medium containing the distributed material and fresh DMEM medium (control group), and the cells were cultured for another 24 h. CCK-8 solution was added to each well, and the cells were cultured for another 1 to 2 h. After culture, the absorbance (OD value) of each group was measured at 450 nm using a microplate reader. Cell viability was calculated using Formula 2. Each group was divided into five replicates.

[0119] Cell viability (%) = (ODtest - ODblank) / (ODcontrol - ODblank) (Formula 2)

[0120] Wherein, ODtest: OD value of the sample group; ODblank: OD value of the blank group; ODcontrol: OD value of the control group. Results are as follows: Figure 8 As shown.

[0121] Analysis: The ionic liquid exhibited a concentration-dependent, bidirectional effect on cell survival: high concentrations (≥0.5 mg / mL) significantly inhibited cell survival, while low concentrations (≤0.5 mg / mL) promoted cell survival, with the strongest promoting effect observed at a concentration of 0.125 mg / mL. Based on the cytotoxicity assay results, an IL concentration of 0.5 mg / mL was selected as the concentration for subsequent experiments, demonstrating both no cytotoxicity and effective inhibition of HaCaT cell proliferation.

[0122] 2. In vitro anti-inflammatory properties

[0123] The anti-inflammatory properties of the prepared material were tested. Specifically, HaCaT cells in the logarithmic growth phase were digested, resuspended, and then subjected to a 1.5 × 10⁻⁶ ppm solution. 5Cells were seeded at a density of 1 cell / well in 6-well plates and cultured for 24 hours until adherence. The experiment was divided into five groups: control group, LPS model group, LPS+COL17 group, LPS+IL group, and LPS+IL-COL17 group (LPS 500 ng / mL, IL 0.5 mg / mL, COL17 0.2 mg / mL). Old culture medium was discarded, and fresh culture medium containing the optimal concentration of LPS was added to the model group. The experimental groups were simultaneously treated with LPS and the test material. The culture plates were returned to the incubator and cultured for another 24 hours. Cell supernatant was collected, centrifuged to remove impurities, aliquoted, and stored at -80℃ for later use. The expression level of the IL-6 inflammatory factor was detected using an ELISA kit. Simultaneously, the same groups were seeded in 96-well plates and induced for 24 hours. CCK-8 reagent was added to the cells, and incubation was continued for 1-2 hours. Absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated to assess the effect of the test material on LPS-induced cell proliferation. Results are as follows: Figure 9 As shown.

[0124] Analysis: This invention, through the combined application of IL and COL17 in the LPS-induced HaCaT cell model, shows, as indicated by the cell viability bar chart, that the increased cell viability due to LPS can be restored to near-normal levels, maintaining cellular homeostasis. ELISA detection of IL-6 factor results shows that it significantly reduces the release of LPS-induced IL-6 inflammatory factors, and the combined treatment group (LPS+IL-COL17) exhibits better anti-inflammatory effects than IL or COL17 alone, demonstrating a synergistic anti-inflammatory effect.

[0125] Example 3: Establishment of an animal model

[0126] IMQ-induced mouse psoriasis model:

[0127] Procedure: Take 6-8 week old C57BL / 6 mice and shave the fur on their backs (approximately 2×2cm area). 2 Apply 62.5 mg of 5% imiquimod (IMQ) cream daily for 7 consecutive days. Obvious skin lesions began to appear on the 3rd day.

[0128] Experimental grouping and drug administration

[0129] Grouping (n=6 animals / group, meeting statistical requirements):

[0130] (1) Normal control group;

[0131] (2) Model control group;

[0132] (3) PBS-COL17 group;

[0133] (4) IL-COL17 group;

[0134] Administration method: COL17 2 mg / mL, 100 μL each time, corresponding to a COL17 dose of about 0.2 mg / animal and an IL dose of 0.5 mg / animal, applied topically (at the same site as IMQ application), once a day, starting from day 1 of modeling, and treatment was initiated 6 hours after application of the cream, for a period of 7 days.

[0135] Detection methods

[0136] 1. The Psoriasis Area and Severity Index (PASI) was used to score the severity of skin lesions daily using the following scale: 0-4 points for erythema from disappearance to present, color from light to dark, and plaque from large to small; 0-4 points for scales from none, small to large, and partially to completely covered; and 0-4 points for plaques from none, thin to thick, and slightly elevated to significantly elevated. The scores for each of these indices were weighted to obtain the final daily PASI score. On day eight, mice were euthanized, and skin, blood, heart, liver, spleen, lung, and kidney tissues were collected.

[0137] 2. H&E staining

[0138] Mouse skin was preserved in a solution containing 4% paraformaldehyde, and the skin samples were then subjected to H&E staining tests, and the results were statistically analyzed.

[0139] Results analysis:

[0140] Figure 10 These are representative images from the four groups treated for psoriasis on day seven. It can be seen that on day seven, IL-COL17 showed significant improvement, with less psoriasis and redness in the skin compared to the IMQ group.

[0141] Figure 11 The results show the psoriasis area and severity index. Image a shows the mouse's weight change, indicating a sharp decrease in weight in the first two days followed by a recovery on the third day. Image b shows the PASI score, which, in cases resembling psoriasis, shows symptom changes starting on the third day, with repair beginning after the fifth day. The IL-COL17 group showed more significant repair, indicating more successful transdermal treatment and a certain therapeutic effect of COL17 on psoriasis. Image c shows a representative image of the mouse spleen on day 7, and image d shows the spleen weight index. Since psoriasis causes splenomegaly, this represents an inflammatory response. These two images show that the IL-COL17 treatment group showed anti-inflammatory effects and a milder inflammatory response.

[0142] Figure 12 The representative image of skin stained with HE and the quantitative statistical diagram of epidermal thickness in the sections show that IL-COL17 treatment reduced epidermal thickening and hyperkeratosis.

[0143] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A composition, characterized in that, It is composed of choline carboxylates and type XVII collagen; The choline carboxylate is at least one of geraniol choline, citrate choline, lactate choline, malate choline, acetylpropionic acid choline, or sorbate choline.

2. The composition according to claim 1, characterized in that, It is composed of type XVII collagen and geraniol choline.

3. The composition according to claim 2, characterized in that, Geraniol choline is prepared by reacting carbolic acid choline and geraniol, wherein the molar ratio of carbolic acid choline to geraniol is (0.5~2.5):

1.

4. The composition according to any one of claims 1 to 3, characterized in that, In the composition, the molar ratio of choline carboxylate to type XVII collagen is (100~400):

1.

5. A method for preparing the composition according to any one of claims 1 to 4, characterized in that, include: Choline compounds and carboxylic acid compounds are reacted in anhydrous ethanol, and the mixture is dried by rotary evaporation. The resulting product is then mixed with type XVII collagen to obtain the composition.

6. The preparation method according to claim 5, characterized in that, The reaction conditions include: ambient temperature and pressure, stirring at 200~300 r / min for 4~10 h.

7. The use of type XVII collagen, the composition according to any one of claims 1 to 4, or the composition obtained by the preparation method according to claim 5 or 6, in the preparation of a medicament for treating psoriasis.

8. The application according to claim 7, characterized in that, The functions of the psoriasis treatment drug include at least one of the following: maintaining the integrity of the epidermal-dermal junction; regulating keratinocyte differentiation and proliferation; inhibiting the release of inflammatory factors; repairing damaged basement membrane structure; or improving skin barrier function.

9. A medicine for treating psoriasis, characterized in that, Includes type XVII collagen, or the composition according to any one of claims 1 to 4, or the composition obtained by the preparation method according to claim 5 or 6.

10. The medicament according to claim 9, characterized in that, The drug contains 50% to 80% choline carboxylate and 20% to 50% type XVII collagen.

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