Microneedle patch for treating multiple sclerosis and use thereof

Abstract

This invention belongs to the field of pharmaceutical preparation technology, specifically relating to a microneedle patch for treating multiple sclerosis and its application. The microneedle patch preparation method includes: (1) adding a solution containing polypeptides to acryloyl polyethylene glycol NHS ester for mixing, followed by vortexing in the dark to dissolve the mixture and then filtering to sterilize it; wherein the polypeptides contain the amino acid sequence shown in SEQ ID NO:1; (2) mixing the mixture with a GelMA gel solution to obtain a drug-containing gel; (3) injecting the drug-containing gel into a PDMS microneedle mold, then removing air bubbles, drying, photocuring, and demolding to obtain the microneedle patch. This microneedle patch can avoid inhibiting normal immune system function and producing possible side effects; and through transdermal administration, it can avoid the first-pass effect of the liver and inactivation in the gastrointestinal tract, avoiding gastrointestinal irritation, and solving the problems of short half-life and low bioavailability of intravenously administered polypeptide drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, specifically relating to a microneedle patch for treating multiple sclerosis and its application. Background Technology

[0002] Multiple sclerosis (MS) is an autoimmune disease caused by myelin antigen-specific T cells migrating to the central nervous system (CNS) and attacking its own tissues, leading to chronic inflammatory demyelination of the CNS and resulting in neurocognitive impairment. Current treatments for MS, like most autoimmune diseases, primarily focus on immunosuppression and immunomodulation; for example, using non-antigen-specific immunomodulators such as interferon-β, fingolimod, dimethyl fumarate, and natetizumab. However, these non-specific immunomodulators may produce serious side effects such as progressive multifocal leukoencephalopathy.

[0003] Current research suggests that MS can be treated by inducing immune tolerance to target and eliminate autoreactive T cells in the immune system or to disable these T cells. This is because targeting pathogenic T cells is highly specific; non-antigen-specific lymphocytes are not reduced or activated, thus avoiding suppression of normal immune system function and potential side effects. However, this treatment typically uses autoreactive T cell-specific peptides / proteins as therapeutic drugs to treat T cell-mediated autoimmune diseases by inducing T cell tolerance. Soluble autoantigen peptide drugs have shown some efficacy in animal models of autoimmune diseases, but clinical trials have yielded poor results. Furthermore, systemic administration of high doses of soluble peptide tolerance agents may cause allergic reactions in mice and primates, raising safety concerns regarding soluble peptide strategies. To overcome these limitations of soluble peptide drugs, novel drug delivery technologies based on biomaterials have recently been applied to the design of peptide drug delivery systems.

[0004] As the largest immune organ, the skin possesses various types of antigen-presenting cells (APCs), including dendritic cells (DCs) and macrophages, thus holding great potential for utilizing the skin's immune system to regulate autoimmune diseases of the central nervous system. However, transdermal delivery of protein and peptide drugs remains a significant challenge due to their large molecular weight, high hydrophilicity, and the obstruction posed by the skin barrier structure.

[0005] Therefore, it is necessary to develop a formulation containing proteins and peptides that can be administered transdermally to treat MS. Summary of the Invention

[0006] To address the above problems, one of the objectives of this invention is to provide a microneedle patch for treating multiple sclerosis (MS). This microneedle patch can deliver protein and polypeptide drugs transdermally, thereby effectively treating MS.

[0007] To achieve the above objectives, the present invention can adopt the following technical solutions: The present invention provides a microneedle patch for treating multiple sclerosis, the preparation method of which includes: (1) adding a solution containing polypeptides to acryloyl polyethylene glycol NHS ester for mixing, and after mixing, dissolving in a light-protected vortex and filtering to sterilize to obtain a mixed solution; wherein, the polypeptides contain an amino acid sequence as shown in SEQ ID NO:1; (2) mixing the mixed solution with a GelMA gel solution to obtain a drug-containing gel; (3) injecting the drug-containing gel into a PDMS microneedle mold, then removing air bubbles, drying, photocuring and demolding to obtain a microneedle patch.

[0008] In another aspect, the present invention provides the application of the above-mentioned microneedle patch for treating multiple sclerosis in the preparation of a drug for developing a skin DC cell tolerance phenotype.

[0009] The beneficial effects of this invention include at least the following: (1) The microneedle patch provided by the present invention is a non-injection drug delivery method. It uses a microneedle transdermal drug delivery system to deliver myelin tolerance peptides, aiming to release self-antigens in situ under the skin and induce the tolerance phenotype of skin DC cells. The tolerance phenotype of DC is conducive to inducing the dysfunction of self-reactive T cells, thereby preventing T cells from migrating to the central nervous system to produce pathogenic effects, thus avoiding the inhibition of normal immune system function and the production of possible side effects.

[0010] (2) The microneedle patch provided by the present invention has sufficient breaking force to meet the requirements of skin penetration, enabling transdermal drug delivery, thereby avoiding pain caused by drug delivery via injection, and improving patient compliance with medication.

[0011] (3) The microneedle patch provided by the present invention delivers polypeptide drugs through the transdermal route, realizing the continuous and gradual release of polypeptide drugs, thereby prolonging the action time of polypeptide drugs, and the transdermal drug delivery will not result in a peak drug concentration, thereby reducing the toxic side effects of polypeptide drugs.

[0012] (4) The microneedle patch provided by the present invention can realize transdermal drug delivery of biological macromolecular polypeptides, which can avoid the first-pass effect of the liver and inactivation in the gastrointestinal tract, avoid gastrointestinal irritation, and solve the problems of short half-life and low bioavailability of polypeptide drugs when administered intravenously. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of a microneedle patch array; Figure 2 This is a side view of a microneedle patch. Figure 3 This is a front view of a microneedle patch. Figure 4Diagram of the microneedle attached to a four-sided pyramidal shape; Figure 5 The average breaking force of each needle in the microneedle patch; Figure 6 Schematic diagram of the tolerance phenotype induced by tolerance-generating microneedles in skin DC cells; Figure 7 MHCII expression in skin tissue DC cells; Figure 8 The expression of CD86 in DC cells of skin tissue. Detailed Implementation

[0014] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0015] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of a feature, number, operation, material, or combination thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0016] An embodiment of the present invention provides a microneedle patch for treating multiple sclerosis, the preparation method of which includes: (1) adding a solution containing polypeptides to acryloyl polyethylene glycol NHS ester for mixing, dissolving the mixture in a light-protected vortex and then filtering to sterilize the mixture; wherein the polypeptides contain an amino acid sequence as shown in SEQ ID NO:1; (2) mixing the mixture with a GelMA gel solution to obtain a drug-containing gel; (3) injecting the drug-containing gel into a PDMS microneedle mold, then removing air bubbles, drying, photocuring and demolding to obtain the microneedle patch.

[0017] It should be noted that improving penetration rate is one of the key issues in solving transdermal drug delivery for biological drugs; physical penetration-enhancing methods, such as microneedles that can penetrate the stratum corneum, have made significant research progress in the field of transdermal drug delivery for biological drugs. Microneedles (MN) are a novel transdermal drug delivery technology, typically consisting of multiple micrometer-sized needle tips arranged in an array on a carrier to form a microneedle patch. Microneedle transdermal drug delivery offers advantages such as painlessness, minimal invasiveness, and sustained-release of drugs; furthermore, it addresses the issue of short half-life in intravenous administration of peptide drugs through local skin delivery.

[0018] It should also be noted that the microneedle patch of this invention has advantages such as good biocompatibility, biodegradability, and non-toxicity; and it has sufficient mechanical strength to penetrate the stratum corneum of the skin, enabling controlled and sustained release of peptide drugs in skin tissue. Furthermore, the peptide containing the amino acid sequence shown in SEQ ID NO:1 (H-Met-Glu-Val-Gly-Trp-Tyr-Arg-Pro-Pro-Phe-Ser-Arg-Val-Val-His-Leu-Tyr-Arg-Asn-Gly-Lys-OH) is a tolerogen peptide of myelin oligodendrocyte glycoprotein (MOG).

[0019] In addition, it should be noted that skin tissue contains various types of antigen-presenting cells (APCs), including dendritic cells (DCs) and macrophages. Microneedles deliver tolerogens transdermally, inducing a tolerant phenotype in skin DCs, thereby promoting the dysfunction / apoptosis of autoreactive T cells and preventing autoreactive T cells from infiltrating to the lesion site to attack the body's own tissues, thus achieving the goal of preventing the progression of autoimmune diseases.

[0020] In some specific embodiments, the concentration of the peptide in the solution containing the peptide is 1.5 mg / ml-2.5 mg / ml, more preferably 2 mg / ml, at which the microneedle patch prepared has the best therapeutic effect; in addition, the mass concentration of the GelMA gel solution can be 15%-25% (w / v), preferably 20%, at which the microneedle patch prepared has the best performance. The preferred method for preparing the GelMA gel solution includes: weighing a certain amount of solid GelMA and dissolving it with 0.25% (w / v) photoinitiator solution (LAP) under stirring in a 50°C water bath to prepare a GelMA gel solution with a concentration of 20% (w / v).

[0021] In some specific embodiments, in step (3), after curing, a 15% (w / v) polyvinyl alcohol solution is added to the mold as the base layer of the microneedles, and the mold is returned to a 4°C environment to continue drying for 5 days.

[0022] In some specific embodiments, the microneedle patch body can be a square pyramid, the needle height can be 600μm-650μm, the bottom diameter can be 300μm-330μm x 300μm-330μm, and the needle tip distance can be 600μm-650μm.

[0023] In some specific embodiments, the microneedle patch described above can be arranged in a 10x10 array, and the patch size can be (8.7-8.9) mm x (8.7-8.9) mm.

[0024] It should be noted that the schematic diagram of the microneedle patch structure in this invention is as follows: Figure 1As shown, a photocurable gel and other water-soluble polymers carrying peptide drugs form the needle body of the transdermal drug delivery microneedle, with low-polymerization polyvinyl alcohol serving as the base layer. The microneedle patch can be arranged in a 10x10 array, with patch dimensions of (8.7-8.9) x (8.7-8.9) mm. The needle body of the microneedle patch can be a square pyramid, with a needle height of 600μm-650μm, a base diameter of 300μm-330μm x 300μm-330μm, and a needle tip distance of 600μm-650μm. After application to the skin, the microneedle patch is fixed with medical tape and remains on the skin for 3-5 days. It should also be noted that the shape of the mold can be designed according to the shape of the microneedle patch.

[0025] In some specific embodiments, in step (3) above, vacuum and / or centrifugation methods can be used to remove air bubbles. It should be noted that air bubbles will exist when the drug-containing gel is injected into the PDMS microneedle mold, and it is necessary to remove the air bubbles to improve the hardness of the subsequent needle body; the method for removing air bubbles can be selected from those known in the art, preferably vacuum and / or centrifugation, which have higher efficiency and better removal effect.

[0026] In some specific embodiments, the drying in step (3) above includes drying in an environment of 0℃-4℃ for 0-10 hours. It should be noted that the PDMS microneedle mold containing the drug-eluting gel needs to be dried after removing air bubbles; in addition, low-temperature drying is beneficial to maintaining the biological activity of the peptides, so the 0℃-4℃ environment is selected for drying in the embodiments of the present invention, and 4℃ is generally selected, which consumes less energy and can also maintain the biological activity of the peptides well.

[0027] In some specific embodiments, the photocuring time in step (3) above can be 0-10 minutes. It should be noted that the above photocuring method can be performed using a photocuring device, and the photocuring time can be within 10 minutes, preferably 10 minutes, at which point a solid can be formed.

[0028] In some specific embodiments, step (3) above, demolding may include: adding a polyvinyl alcohol solution to the mold after light curing, returning it to a 4°C environment, and continuing to dry for 0-12 days; after drying, performing microneedle patch demolding. It should be noted that before demolding, a polyvinyl alcohol solution is added for pretreatment, and then the mold is dried at 4°C for a period of time before demolding.

[0029] Another embodiment of the present invention provides the application of the above-mentioned microneedle patch for treating multiple sclerosis in the preparation of a drug that induces a tolerance phenotype in skin DC cells.

[0030] It should be noted that the interaction between antigen-presenting cells and immature T lymphocytes in peripheral lymphoid organs determines the fate of T lymphocytes and their immunological effects; Tolerant DCs (TolDCs) are a type of cell that lacks or expresses low levels of T cell co-stimulatory molecules, such as CD80, CD86, or CD40; the tolerant phenotype of DCs is conducive to inducing the inactivation of self-reactive T cells.

[0031] It should also be noted that the skin is the largest immune organ, containing various types of antigen-presenting cells (APCs), including dendritic cells (DCs) and macrophages. This invention discovers that a transdermal delivery method using microneedles loaded with tolerogens can induce a tolerogenic phenotype in local skin DCs, thereby promoting the dysfunction / apoptosis of autoreactive T cells, affecting the composition and function of immune cells in the central nervous system, and ultimately achieving the treatment of autoimmune diseases.

[0032] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0033] I. Preparation and Characterization of Microneedle Patches (I) Preparation of microneedle patches (1) Weigh a certain amount of GelMA solid, add 0.25% (w / v) photoinitiator solution (LAP, solvent is PBS phosphate buffer), and stir to dissolve it under water bath heating at 50℃ to prepare a 20% (w / v) GelMA gel solution (the mass concentration of GelMA in the photoinitiator solution is 20%). (2) Weigh a certain amount of polypeptide drug (polypeptide sequence SEQ ID NO:1: H-Met-Glu-Val-Gly-Trp-Tyr-Arg-Pro-Pro-Phe-Ser-Arg-Val-Val-His-Leu-Tyr-Arg-Asn-Gly-Lys-OH) and use PBS solution (phosphate buffer with pH value of 7.4) to prepare a polypeptide drug solution of 2 mg / ml; (3) Add 3.1 mg of acrylamide polyethylene glycol NHS ester to a solution containing 2 mg of polypeptide drug, mix and dissolve in a light-protected vortex, then filter to sterilize to obtain a mixed drug solution; (4) Mix the GelMA gel solution with the above polypeptide drug solution at a volume ratio of 1:1 to make the polypeptide concentration 1 mg / ml. The polypeptide drug and the photocrosslinking material are linked together through NHS ester double bonds to form a drug-containing gel. (5) Inject the drug-containing gel into the PDMS microneedle mold and remove air bubbles from the drug-containing gel using vacuum; (6) After drying the above microneedle mold in an environment of 4°C for 5 hours, use a light curing instrument to perform light curing for 5 minutes; (7) After curing, add 15% (w / v) polyvinyl alcohol solution to the mold and return it to a 4°C environment to continue drying for 5 days; (8) After drying, the microneedle patch is demolded to obtain the microneedle patch.

[0034] (II) Characterization of microneedle patches The morphology of the prepared microneedle patch is as follows Figure 2 and Figure 3 As shown, the microneedle patch is arranged in a 10x10 array, with a patch size of 8.7 mm x 8.7 mm. The needle body is a four-sided pyramid with a height of 600 μm, a bottom diameter of 330 μm x 330 μm, and a needle tip distance of 600 μm.

[0035] In addition, the microneedle attachments were labeled with rhodamine dye and then observed using a laser confocal microscope. The structure of a single microneedle was as follows: Figure 4 As shown, it has a square pyramid shape and a complete structure.

[0036] Using a computer-controlled electronic universal testing machine, the average breaking force of each needle in the microneedle patch was measured to be approximately 0.5 N using the compression method (reference). Figure 5 This indicates that the microneedle patch has sufficient hardness and strength to facilitate skin penetration (Reference: Chen W, Tian R, Xu C, Yung BC, Wang G, Liu Y, Ni Q, Zhang F, Zhou Z, Wang J, Niu G, Ma Y, Fu L, Chen X. Microneedle-array patches loaded with dualmineralized protein / peptide particles for type 2 diabetes therapy. Nat. Commun. 2017 Nov 24;8(1):1777. doi:10.1038 / s41467-017-01764-1.).

[0037] II. Verification of the efficacy of microneedle patch application (I) Establishment of a mouse model of multiple sclerosis (EAE) (1) Preparation of MOG / CFA emulsifier: Add 100 mg of tuberculin (Mtb) to 25 ml of Freund's complete adjuvant and mix well (final Mtb concentration is 5 mg / ml); MOG 35-55The peptide was dissolved in PBS to a concentration of 2 mg / ml; MOG and CFA / Mtb were mixed 1:1 (final MOG concentration was 1 mg / ml); emulsification was performed using an ultrasonic homogenizer at a frequency of (output control 7, % duty cycle 50) on ice; after complete emulsification, the tube was inverted and the emulsion did not flow, and it was stored at 4°C and used within one week.

[0038] (2) Immunization of mice: Ten 10-week-old C57BL / 6 female mice were prepared. On day 0, 100ul MOG / CFA emulsifier was injected subcutaneously at two points on the back of each mouse. Five hours later, 100ul pertussis toxin PTX (2ug / ml) was injected intraperitoneally. On day 1, 100ul PTX was injected intraperitoneally per mouse to construct EAE mice.

[0039] (3) Scoring of mice began on day 10 post-immunization. This experiment used a double-blind scoring method, meaning that all EAE mice used in the experiment were scored by other researchers who were not involved in the drug treatment of this study. The scoring used a 5-point scale (HookeLab): 1, drooping and weak tail; 2, tail paralysis; 2.5, unilateral hind limb weakness; 3, unilateral hind limb paralysis; 3.5, bilateral hind limb paralysis; 4, complete paralysis of both hind limbs and weakness of the forelimbs; mice with a score of 4 or higher were euthanized. The above symptoms appeared in EAE mice from day 10-12 post-immunization, and the course of the disease usually progressed to a score of 3-3.5 (average) on day 14-16 post-immunization, indicating successful modeling.

[0040] (ii) Application of microneedle patches On day 14 post-immunization (with a score of 3-3.5 as described above), EAE mice were treated with the microneedle patch prepared in Example 1. The patch was inserted into the dorsal skin area of ​​the EAE mice, secured with medical tape, and remained in place for 3 days. Starting from day 9 post-immunization, the mice were scored daily according to the evaluation method described above to assess the treatment effect.

[0041] (III) Validation of the induced skin DC cell tolerance phenotype A schematic diagram of the tolerance phenotype induced by tolerogen microneedling in skin DC cells is shown below. Figure 6 As shown, the microneedle patch implanted in the skin releases autoantigens in a controlled manner, inducing a tolerance phenotype in local dendritic cells (DCs) and apoptosis or dysfunction of autoreactive T cells, thereby preventing autoreactive T cells from entering the central nervous system and exerting pathogenic effects. Furthermore, the microneedle patch prepared above was implanted into the dorsal skin of the aforementioned EAE mice, fixed with medical tape for 3 days, and skin tissue samples were taken for immunofluorescence analysis.

[0042] Analysis of DC cell phenotype in skin tissue at microneedle implantation site using flow cytometry: Preparation of single-cell suspensions of skin tissue (specific method reference: CJ Benck, T. Martinov, BT Fife, D. Chatterjea, Isolation of infiltrating leukocytes from mouse skin using enzymatic digest and gradient separation). J. Vis. Exp. (2016), doi:10.3791 / 53638), cells were stained using flow cytometry; the monoclonal antibodies used were as follows: anti-CD4 (eBioscience, GK1.5), anti-CD45.2 (Invitrogen, 104), anti-CD3 (eBioscience, 145-2C11), anti-CD11b (eBioscience, M1 / 70), anti-CD11c (eBioscience, N418), anti-F4 / 80 (eBioscience, BM8), anti-MHCClass II (eBioscience, M5 / 114.15.2), anti-CD80 (eBioscience, 16-10A1), and anti-CD86 (eBioscience, GL1). Results are as follows. Figure 7 and Figure 8 As shown, compared with the skin tissue of the group without toleranceogen microneedles, the expression levels of MHCII and CD86 in skin DC cells of the toleranceogen microneedle implantation group were significantly lower than those in untreated mice.

[0043] In summary, the tolerogen microneedle patch of the present invention can recruit dendritic cells (DCs) in local skin areas and induce a tolerance phenotype of DCs. The tolerance phenotype of DCs is conducive to inducing the inactivation of autoreactive T cells, thereby preventing T cells from migrating to the central nervous system and producing pathogenic effects.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A microneedle patch for treating multiple sclerosis, characterized in that, The preparation method includes: (1) adding a solution containing polypeptides to acrylamide polyethylene glycol NHS ester for mixing, dissolving the mixture in a light-protected vortex, and then filtering and sterilizing to obtain a mixed solution; wherein, the polypeptides contain the amino acid sequence shown in SEQ ID NO:1; (2) mixing the mixed solution with GelMA gel solution to obtain a drug-containing gel; (3) injecting the drug-containing gel into a PDMS microneedle mold, then removing air bubbles, drying, photocuring, and demolding to obtain a microneedle patch; wherein, the concentration of the polypeptide solution is 1.5 mg / ml-2.5 mg / ml, and the mass concentration of the GelMA gel solution is 15%-25%.

2. The microneedle patch for treating multiple sclerosis according to claim 1, characterized in that, The microneedle patch has a four-sided pyramidal body with a height of 600μm-650μm, a bottom diameter of (300μm-330μm) x (300μm-330μm), and a needle tip distance of 600μm-650μm.

3. The microneedle patch for treating multiple sclerosis according to claim 2, characterized in that, The microneedle patch is arranged in a 10x10 array, and the patch size is (8.7-8.9) mm x (8.7-8.9) mm.

4. The microneedle patch for treating multiple sclerosis according to any one of claims 1 to 3, characterized in that, In step (3), air bubbles are removed by vacuum and / or centrifugation.

5. The microneedle patch for treating multiple sclerosis according to any one of claims 1 to 3, characterized in that, In step (3), drying includes drying in an environment of 0℃-4℃ for 0-10 hours.

6. The microneedle patch for treating multiple sclerosis according to any one of claims 1 to 3, characterized in that, In step (3), the light curing time is 0-10 minutes.

7. The microneedle patch for treating multiple sclerosis according to any one of claims 1 to 3, characterized in that, In step (3), demolding includes: after light curing, adding polyvinyl alcohol solution to the mold and returning it to a 4°C environment to continue drying for 0-12 days; after drying, demolding the microneedle patch is performed.

8. The use of the microneedle patch for treating multiple sclerosis as described in any one of claims 1 to 7 in the preparation of a medicament for inducing a skin dendritic cell tolerance phenotype.

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