A transdermal microneedle patch for treating cutaneous lupus erythematosus and its preparation method and application

Abstract

The present invention relates to the field of biomedicine technology, and in particular to a transdermal microneedle patch for treating cutaneous lupus erythematosus, and a preparation method and application thereof. It includes: mixing polyvinyl pyrrolidone with water and (S, R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetate methyl ester solution, defoaming treatment, and obtaining a treatment liquid; injecting the treatment liquid into a PDMS microneedle mold, and then injecting a polyvinyl pyrrolidone solution with a mass percentage of 30% into the PDMS microneedle mold, and obtaining a transdermal microneedle patch after drying. The transdermal microneedle patch provided by the present invention realizes the transdermal administration of systemic drugs, and significantly reduces the dosage while ensuring the therapeutic effect.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a transdermal microneedle patch for treating cutaneous lupus erythematosus and a preparation method and application thereof. Background Art

[0002] Cutaneous lupus erythematosus (CLE) is a skin manifestation of systemic lupus erythematosus (SLE), which is seen in up to 80% of SLE patients. 20-25% of SLE patients present with CLE as the first symptom, and up to 42% of CLE patients may progress to SLE. Skin lesions are the most intuitive, and as the earliest and most commonly affected organ, they are of great significance for the early identification and early intervention of SLE. Preventing the recurrence of CLE rash and blocking the progression of CLE to SLE are of great value in improving disease prognosis and reducing socioeconomic burden. At the same time, considering the refractory, recurrent, disfiguring and potential systemic involvement risk of CLE, the development of new therapeutic targets and new treatment methods is of great clinical significance.

[0003] As the understanding of the disease deepens, more and more new targets continue to emerge. However, the current treatment is still mainly systemic medication, and there is a lack of effective means for local administration of drugs in the skin lesion area. Systemic administration helps the treatment of visceral damage, but also requires higher doses and brings more obvious side effects. For CLE patients with only skin manifestations but no systemic symptoms, local administration is still mainly based on traditional glucocorticoid ointments, and the clinical benefits brought are not clear. The present invention uses an inhibitor of macrophage migration inhibitory factor MIF targeting the key pathogenic factor of SLE, ISO-1 (Chinese name is (S, R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazoleacetic acid methyl ester), to construct a transdermal microneedle patch, which, on the one hand, achieves limited diffusion of drugs in the skin, and on the other hand, significantly reduces the effective dosage, while reducing the side effects that may be caused by systemic administration. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a transdermal microneedle patch for treating cutaneous lupus erythematosus and a preparation method and application thereof. The present invention can solve the problems of existing solutions that mainly rely on systemic administration and lack of local medication solutions. The patch is simple to use and easy to implement, has a stable construction effect, can be mass-produced, and has extremely high practical value.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing a transdermal microneedle patch for treating cutaneous lupus erythematosus, comprising the following steps:

[0007] 1) mixing polyvinyl pyrrolidone with water and (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazoleacetic acid methyl ester solution, and performing defoaming treatment to obtain a treatment solution;

[0008] 2) injecting the treatment solution obtained in step 1) into the PDMS microneedle mold, and then injecting a polyvinyl pyrrolidone solution with a mass percentage of 30% into the PDMS microneedle mold, and obtaining a transdermal microneedle patch after drying.

[0009] Preferably, in step 1), the volume ratio of the mass of polyvinyl pyrrolidone to the mass of water and the volume ratio of the (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazoleacetic acid methyl ester solution is 3g:9g:1mL.

[0010] Preferably, the concentration of the (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazoleacetic acid methyl ester solution is 23.54 μg / μL.

[0011] Preferably, the defoaming treatment in step 1) is performed for 20 minutes, and the defoaming treatment is performed under centrifugation at a speed of 4000 rpm.

[0012] Preferably, the specifications of the PDMS microneedle mold in step 2) are: needle distance 750 μm, needle height 850 μm, bottom size 350 μm, microneedle patch size 10*10 mm, and array number 10*10.

[0013] Preferably, the amount of the treatment liquid injected into the PDMS microneedle mold in step 2) is 150 μL, and the vacuum treatment is performed for 10 minutes to prepare the microneedle body part.

[0014] Preferably, in step 2), the amount of polyvinyl pyrrolidone solution injected into the PDMS microneedle mold is 200 μL to prepare the microneedle backing part.

[0015] Preferably, the drying conditions in step 2) include: drying overnight at a temperature of 30° C. and a humidity of 55%.

[0016] The present invention also provides a transdermal microneedle patch prepared by the preparation method described in the above technical solution.

[0017] The present invention also provides the use of the transdermal microneedle patch described in the above technical solution in the preparation of a drug for treating cutaneous lupus erythematosus.

[0018] The beneficial effects of the present invention are:

[0019] The transdermal microneedle patch provided by the present invention realizes the transdermal administration of systemic drugs, which significantly reduces the dosage while ensuring the therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0021] Figure 1 Flow chart for the construction of microneedles;

[0022] Figure 2 The molecular structure of ISO-1 is cited from Al-Abed Y, Van Patten S. MIF as a disease target: ISO-1 as a proof-of-concept therapeutic. Future Med Chem. 2011 Jan; 3(1): 45-63. doi: 10.4155 / fmc.10.281. PMID: 21428825.

[0023] Figure 3 Representative stereomicroscope images and scanning electron microscope (SEM) images of the microneedle patch;

[0024] Figure 4 The mechanical strength characterization results of the microneedle patches loaded with DMSO and ISO-1;

[0025] Figure 5 Methylene blue staining shows the skin insertion test images of DMSO and ISO-1 microneedle patches;

[0026] Figure 6 Images of degradation test results of the microneedle patch inserted into rat skin for 5, 10, 20 and 30 minutes;

[0027] Figure 7 Representative macroscopic images of the skin of MRL / lpr mice during microneedle patch administration and 12 hours after microneedle patch removal;

[0028] Figure 8 This is the irritation test result of the microneedle patch;

[0029] Fig. 9 Compared with DMSO microneedle patch (DMSO-MP), ISO-1 microneedle patch (ISO-MP) significantly improved the rash manifestations of lupus mice, and the pathological score was significantly reduced;

[0030] Fig.10 Compared with DMSO microneedle patch (DMSO-MP), ISO-1 microneedle patch (ISO-MP) significantly improved the rash manifestations of lupus mice;

[0031] Fig.11Compared with DMSO microneedle patch (DMSO-MP), ISO-1 microneedle patch (ISO-MP) significantly improved the expression levels of MIF, p-p38, and GSDMD-NT in the skin of lupus mice in the UVB-irradiated area;

[0032] Fig.12 Compared with DMSO microneedle patch (DMSO-MP), ISO-1 microneedle patch (ISO-MP) significantly improved the expression levels of COL I, MMP9, MMP2 and TNFA in the skin of lupus mice in the UVB irradiated area. DETAILED DESCRIPTION

[0033] The present invention provides a method for preparing a transdermal microneedle patch for treating cutaneous lupus erythematosus, comprising the following steps:

[0034] 1) mixing polyvinyl pyrrolidone with water and (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazoleacetic acid methyl ester solution, and performing defoaming treatment to obtain a treatment solution;

[0035] 2) injecting the treatment solution obtained in step 1) into the PDMS microneedle mold, and then injecting a polyvinyl pyrrolidone solution with a mass percentage of 30% into the PDMS microneedle mold, and obtaining a transdermal microneedle patch after drying.

[0036] The invention comprises mixing polyvinyl pyrrolidone with water and (S, R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester solution and performing defoaming treatment to obtain a treatment liquid.

[0037] In the present invention, the mass of the polyvinyl pyrrolidone, the mass of the water, and the volume ratio of the (S, R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetate methyl ester solution are preferably 3g:9g:1mL. In the present invention, the concentration of the (S, R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetate methyl ester solution is preferably 23.54μg / μL. In the prior art, the concentration of systemic administration ((S, R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetate methyl ester) is 40mg / kg, and the transdermal dosage of the present invention is set to 1 / 5-1 / 10 of the system dosage, and the transdermal microneedle patch can reduce the system dosage. In the present invention, the defoaming treatment time is preferably 20min, and the present invention is preferably defoamed under centrifugation, and the centrifugal speed is preferably 4000rpm.

[0038] The invention injects the obtained treatment liquid into a PDMS microneedle mold, then injects a polyvinyl pyrrolidone solution with a mass percentage of 30% into the PDMS microneedle mold, and obtains a transdermal microneedle patch after drying.

[0039] In the present invention, the specifications of the PDMS microneedle mold are preferably: needle distance 750μm, needle height 850μm, bottom size 350μm, microneedle patch size 10*10mm, array number 10*10. In the present invention, the amount of the treatment liquid injected into the PDMS microneedle mold is preferably 150μL, the time of vacuum treatment is preferably 10min, and the microneedle body part is prepared. In the present invention, the amount of the polyvinyl pyrrolidone solution injected into the PDMS microneedle mold is preferably 200μL, and the microneedle backing part is prepared. In the present invention, the drying conditions preferably include: drying overnight at a temperature of 30°C and a humidity of 55%.

[0040] The present invention provides a transdermal microneedle patch prepared by the preparation method described in the above technical solution. In the present invention, the transdermal microneedle patch is preferably used by directly applying it to the skin lesion area.

[0041] The present invention also provides the use of the transdermal microneedle patch described in the above technical solution in the preparation of a drug for treating cutaneous lupus erythematosus.

[0042] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0043] Example 1

[0044] Preparation of transdermal microneedle patches ( Figure 1 ):

[0045] (1) PDMS microneedle mold parameters, the specifications are: needle distance 750 μm, needle height 850 μm, bottom size 350 μm, microneedle patch size 10*10 mm, array number 10*10;

[0046] (2) 3 g of PVP powder was accurately weighed and fully dissolved in 9 ml of deionized water, and then 1 ml of 23.54 μg / μl ISO-1 solution was added, mixed evenly, and centrifuged at 4000 rpm for defoaming treatment for 20 min to obtain a mixed solution, wherein ISO-1, Chinese name: (S, R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazoleacetic acid methyl ester, Figure 2 , DOI:10.4155 / fmc.10.281;

[0047] (3) Pipette 150 μl of the mixed solution and inject it into the microneedle mold (at the mold needle body), and vacuum treat for 10 min to prepare the microneedle body;

[0048] (4) Prepare a 30% PVP solution (w / v), add 200 μl into the above-mentioned microneedle mold (the base of the mold), prepare the microneedle backing part, and dry it overnight in a constant temperature and humidity chamber at a temperature of 30°C and a humidity of 55%. After demolding, a transdermal microneedle patch is obtained for testing and animal evaluation.

[0049] Example 2

[0050] Performance characterization and biosafety evaluation of microneedles

[0051] (1) Morphology detection:

[0052] The uniformity of the needle body of the transdermal microneedle patch prepared in Example 1 was checked using an optical microscope, and the length and base size of the microneedles were measured. The microscopic morphology of the microneedles was examined by scanning electron microscopy. Figure 3 As shown, the microneedles are distributed in the same direction, with uniform needle distances and no bending or breakage.

[0053] (2) Mechanical properties inspection:

[0054] Use a universal testing machine to evaluate the mechanical properties of the microneedles. Before the test, observe each microneedle to be tested under a microscope to ensure that the needle shaft is not bent or broken and the backing is smooth and flat. Place the microneedle (tip facing up) horizontally in the center of the horizontal stage of the universal testing machine. The distance between the sensor and the stage is set the same in all tests. Then use the probe to apply an axial force at a speed of 0.2mm / min to compress the microneedle until the preset displacement of 450μm is reached. The trigger force is set to 0.05N, and the force-displacement curve of the microneedle is recorded. Figure 4 As shown, the microneedles can withstand loads up to 8N at a depth of 450μm without breakage, indicating that the microneedles can maintain their integrity during insertion into the skin.

[0055] (3) Preparation of methylene blue loaded microneedles for skin penetration study:

[0056] Take the ex vivo pig skin, remove the subcutaneous fat and store it in physiological saline. Spread the skin flat on a plastic plate with the stratum corneum facing outwards, and use absorbent paper to absorb the moisture on the skin surface. Press the methylene blue-loaded microneedle into the ex vivo pig skin with a force of 5-10N, remove it after 5 minutes, observe the needle hole in the skin and take pictures. Figure 5 As shown, a methylene blue-loaded microneedle patch was inserted into pig skin for 5 minutes, and after removal, a series of blue pinholes appeared at the insertion site, indicating that the needles could penetrate deep enough for drug delivery.

[0057] (4) Transdermal solubility study:

[0058] After the rats were anesthetized with isoflurane, the hair on the abdominal skin was shaved, and then the microneedle patch was pressed tightly on the abdominal skin and fixed with medical tape. The microneedles were removed after 5 min, 10 min, 20 min, and 30 min, and the changes in the length of the microneedles were observed with an optical microscope to measure the remaining length. Figure 6 As shown, the microneedle patch can be gradually released within 30 minutes after insertion.

[0059] (5) Skin irritation test:

[0060] The hair on the abdominal skin of the rats was shaved 24 hours before the experiment, and the location without obvious wounds or scars after hair removal was selected for the skin irritation experiment. During the experiment, the microneedle patch was first pressed vertically into the abdominal skin, and the microneedle patch was peeled off after 30 minutes of application. The erythema and edema on the skin were recorded at 0h, 1h, 6h, 12h and 24h. Figure 7-8 As shown, no erythema and edema occurred in the skin at different time points after microneedle insertion.

[0061] Example 3

[0062] The results of animal experiments demonstrated the effectiveness of ISO transdermal microneedle patch in treating lupus lesions.

[0063] UVB irradiation protocol: The UVB irradiator (SS-01, SIGMA, Shanghai) used for MRL / lpr lupus mice had a wavelength range of 290-320 nm, a peak of 311 nm, and an irradiation power of 11.00 mw / cm 2 In the animal experiment, the irradiation area of ​​each MRL / lpr lupus mouse was located in the hair-removing area on the back, with a diameter of 1.5 cm. The irradiation area was irradiated for 4 consecutive cycles, each cycle lasting 5 days. The irradiation energy in the first two cycles was 330 mJ / cm 2 The irradiation energy of the last two cycles is 110mJ / cm 2 This irradiation regimen can induce skin lesions in MRL / lpr mice.

[0064] After application of the ISO-1-loaded microneedle patch (ISO-1-MP) prepared in Example 1, the severity of skin lesions induced by UVB exposure in lupus mice was significantly improved, showing lower clinical scores and biopsy scores than those in the DMSO-MP group ( Fig. 9 Although the fluorescence intensity of MIF in each keratinocyte was comparable between the two groups, the ISO-1-loaded microneedle patch significantly reduced the overall number of MIF-positive keratinocytes ( Fig.10 ). In addition, ISO-1-MP treatment significantly reduced the levels of MIF, p-p38, and GSDMD-NT in lupus keratinocytes ( Fig.11), and reversed the upregulation of MMP9, MMP2, and TNFA by UVB exposure ( Fig.12 ). Taken together, these results suggest that MIF could not only be a therapeutic target for UVB-induced skin damage, but also that the microneedle patch system could be a promising treatment for CLE.

[0065] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing a transdermal microneedle patch for treating cutaneous lupus erythematosus, characterized in that: The following steps are involved: 1) mixing polyvinyl pyrrolidone with water and (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazoleacetic acid methyl ester solution, and performing defoaming treatment to obtain a treatment solution; 2) injecting the treatment solution obtained in step 1) into the PDMS microneedle mold, and then injecting a polyvinyl pyrrolidone solution with a mass percentage of 30% into the PDMS microneedle mold, and obtaining a transdermal microneedle patch after drying.

2. The preparation method according to claim 1, characterized in that: In the step 1), the volume ratio of the mass of polyvinyl pyrrolidone to the mass of water and the volume ratio of the (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazoleacetic acid methyl ester solution is 3g:9g:1mL.

3. The preparation method according to claim 1 or 2, characterized in that: The concentration of the (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazoleacetic acid methyl ester solution is 23.54 μg / μL.

4. The preparation method according to claim 1, characterized in that: The defoaming treatment time of step 1) is 20 minutes, and the defoaming treatment is carried out under centrifugation, and the rotation speed of the centrifuge is 4000 rpm.

5. The preparation method according to claim 1, characterized in that: The specifications of the PDMS microneedle mold in step 2) are: needle distance 750 μm, needle height 850 μm, bottom size 350 μm, microneedle patch size 10*10 mm, and array number 10*10.

6. The preparation method according to claim 1 or 5, characterized in that: In the step 2), the amount of the treatment liquid injected into the PDMS microneedle mold is 150 μL, and the vacuum treatment is performed for 10 minutes to prepare the microneedle body part.

7. The preparation method according to claim 1 or 5, characterized in that: In the step 2), the amount of polyvinyl pyrrolidone solution injected into the PDMS microneedle mold is 200 μL to prepare the microneedle backing part.

8. The preparation method according to claim 1, characterized in that: The drying conditions in step 2) include: drying overnight at a temperature of 30° C. and a humidity of 55%.

9. A transdermal microneedle patch prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the transdermal microneedle patch according to claim 9 in the preparation of a drug for treating cutaneous lupus erythematosus.

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