A patch for treating fungal skin infection and preparation method thereof

Abstract

The present invention discloses a patch for treating fungal skin infection and a preparation method thereof, and belongs to the field of biomedical technology and pharmaceutical preparations. The soluble microneedle patch of fluconazole combined with doxycycline of the present invention comprises a base layer and a needle tip layer, wherein the needle tip layer comprises microneedles arranged in an array on the base layer, and fluconazole and doxycycline are loaded in the microneedles, thereby solving the problems of fluconazole drug instability and poor antibacterial effect. At the same time, the base material of the patch of the present invention is mainly chitosan, supplemented by other biocompatible organic high molecular polymers, and has the advantages of high mechanical strength and good formability compared with chitosan alone.

Description

Technical Field

[0001] The invention relates to a patch for treating fungal skin infection and a preparation method thereof, belonging to the field of biomedical technology and pharmaceutical preparations. Background Art

[0002] Skin fungal infection is a common skin disease, also known as fungal skin disease, which belongs to a class of multiple infections clinically, generally including tinea manuum, tinea pedis and tinea cruris. Skin fungi Skin fungal infection (fungal disease) currently affects 20-25% of the world's population. If there is no timely and effective treatment, it is easy to relapse. Over time, the infected part will become rough and thickened, and the skin lines will widen and deepen, which is not conducive to the local treatment effect and also affects the patient's daily life. The traditional way of administration is to use ketofluconazole for local treatment in the form of ointment, etc. It is an azole antifungal drug with a wide antifungal spectrum. It is effective for fungal infections in humans and various animals, and can relieve the patient's symptoms to a certain extent. However, the treatment effect is general, and long-term and extensive use will cause fungal resistance to increase. For example, when the patient's skin thickens and drug resistance is enhanced, oral administration is often required, resulting in certain adverse consequences. Therefore, it is considered to adopt a combined medication method, and improve the penetration ability of the stratum corneum in the form of local administration to improve the treatment effect.

[0003] Studies have shown that a certain concentration of tetracycline antibiotic doxycycline combined with azole drugs has a synergistic effect on fungal infections such as Candida albicans (Int J Antimicrob Agents 2009, 34(4): 326-332.), and even has a good resistance to biofilms caused by fungal infections (FEMS Yeast Research, 2013, 13(5): 453-462.; Journal of medical microbiology 63.7(2014): 956-961.).

[0004] In addition, with the development of polymer microneedle technology, polymer microneedle drug delivery combines the advantages of subcutaneous injection and transdermal patch drug delivery, and is currently the most widely studied and most promising type of microneedle product. Polymer microneedles are used to treat fungal skin infections and have unique advantages such as local drug concentration enrichment, enhanced ability to penetrate the stratum corneum, and improved drug delivery efficiency. However, fluconazole has weak water solubility and chemical instability. When it is combined with soluble microneedle technology, there is still the problem of demanding manufacturing process requirements. In addition, due to the poor solubility of fluconazole in water, how to improve the drug loading capacity in the microneedle is also a difficulty. Summary of the invention

[0005] [Technical issues]

[0006] The technical problem to be solved by the present invention is to provide a polymer microneedle patch which can ensure the drug loading capacity and stability of the drug and has a high-efficiency antifungal effect and a preparation method thereof.

[0007] [Technical solution]

[0008] In order to solve the above technical problems, the present invention provides a patch for treating fungal skin infections and a preparation method thereof. The patch has a structure in the form of polymer microneedles, including a base layer and a needle tip layer; the needle tip layer includes microneedles (needle tips) arranged in an array on the base layer, and the microneedles are loaded with fluconazole and doxycycline.

[0009] The microneedle is conical, with a bottom diameter of 40-400 μm, a height of 200-100 μm, and a needle tip diameter of 1-20 μm.

[0010] The molar ratio of fluconazole to doxycycline is 1:2-2:1, preferably 1:1.

[0011] The material of the base layer is one of PE film, PVC film, PET film, EVA film, PU film, PSA film, PVA film and PVA / PVP composite film.

[0012] The substrate of the microneedle is an organic high molecular polymer with biocompatibility, and fluconazole and doxycycline can be uniformly dispersed in the organic high molecular polymer.

[0013] In some embodiments of the present invention, the substrate of the microneedle (needle tip) is a compound of chitosan and other biocompatible polymers, and the other biocompatible polymers include maltose, cellulose and its derivatives, starch and its derivatives, polyvinyl alcohol, polyamino acids, dextran, sodium alginate, polyvinyl pyrrolidone, sodium hyaluronate, and one of polyethylene glycol.

[0014] In one embodiment of the present invention, the substrate of the microneedle is a composite of chitosan and polyvinyl pyrrolidone. Preferably, the mass ratio of chitosan to polyvinyl pyrrolidone is 1:5-1:50.

[0015] The present invention provides a method for preparing a patch for treating skin fungal infections, comprising the following steps:

[0016] (1) adding doxycycline to a solvent of pure water, stirring and dissolving at room temperature to obtain a dispersion system A with a drug concentration of 0.1 M, and adding fluconazole to ethanol to dissolve to obtain a dispersion system B with a drug concentration of 0.1 M;

[0017] (2) mixing dispersion system A and dispersion system B in equal volume ratio to obtain dispersion system C, adding the microneedle substrate to dispersion system C, stirring and dispersing the dispersion evenly, and removing bubbles to obtain dispersion system D;

[0018] (3) filling the dispersed system D into a microneedle mold to form a liquid layer of uniform thickness in the mold;

[0019] (4) leaving the mold at room temperature for 30-40 minutes, and then placing it in an oven for baking;

[0020] (5) The microneedle mold is removed, the base layer is covered, and the patch for treating fungal skin infection is slowly peeled off.

[0021] In one embodiment of the present invention, in step (2), the ratio of the microneedle substrate to the dispersion system C is 0.05-5 g / mL.

[0022] In one embodiment of the present invention, in step (4), the oven baking temperature is 30-60°C, preferably 40-50°C; the baking time is 3-12h, preferably 6-8h.

[0023] In one embodiment of the present invention, the substrate of the microneedle is a composite of chitosan and polyvinyl pyrrolidone. Preferably, the mass ratio of chitosan to polyvinyl pyrrolidone is 1:5-1:50.

[0024] The patch for treating fungal skin infections of the present invention can be used to prepare products for treating deep fungal skin infections, including products for treating fungal skin infections based on Candida albicans.

[0025] [Beneficial Effects]

[0026] The soluble microneedle patch of fluconazole combined with doxycycline of the present invention comprises a base layer and a needle tip layer, wherein the needle tip layer comprises microneedles arranged in an array on the base layer, and fluconazole and doxycycline are loaded in the microneedles. The problem of fluconazole drug instability and poor antibacterial effect is solved, fluconazole is combined with doxycycline and polymer microneedle technology, and drug stability and drug loading capacity are improved by optimizing the preparation process, thereby greatly improving the antifungal effect.

[0027] The substrate of the microneedle of the patch of the present invention is mainly chitosan, supplemented by other biocompatible organic polymers, and has the advantages of high mechanical strength and good formability compared to using chitosan alone. Compared to using chitosan or polyvinyl pyrrolidone alone, when the composite of chitosan and polyvinyl pyrrolidone is used as the substrate of the microneedle, it only needs to be baked at a relatively low temperature of 30-60°C to form, which has the advantage of low-temperature forming and avoids the problem of drug degradation caused by high temperature.

[0028] In the patch of the present invention, the content ratio of fluconazole to doxycycline is 1:2-2:1, which is beneficial to improving the therapeutic effect of fungal infection.

[0029] When preparing the patch, the present invention dissolves doxycycline in pure water and fluconazole in ethanol, and then mixes them, which is conducive to uniform mixing of the two. Compared with using pure water as a solvent, it is conducive to reducing the required baking temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a structural diagram of a patch for treating fungal skin infections according to the present invention;

[0031] Figure 2 A schematic diagram of a patch for treating fungal skin infections according to the present invention;

[0032] Figure 3 This is a comparison of the in vitro antibacterial effect of a patch for treating skin fungal infections according to the present invention;

[0033] Figure 4 is the average score of skin lesions. DETAILED DESCRIPTION

[0034] The centrifuge used in the following examples is a Centrifuge 5810-microcentrifuge (Eppendorf); doxycycline hydrochloride and fluconazole were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; polyvinylpyrrolidone K30 (PVPK30) was purchased from Shenggong Biotechnology (Shanghai) Co., Ltd.; chitosan was purchased from Jinhu Crust Products Co., Ltd.

[0035] Example 1 Preparation and Optimization of a Patch for Treating Skin Fungal Infections

[0036] like Figure 1 As shown, the patch for treating fungal skin infection of this embodiment includes a base layer 1 and a needle tip layer 2. The needle tip layer includes microneedles (needle tips) arranged in an array on the base layer, and the needle tip layer 2 contains doxycycline and fluconazole 21, and the needle tip layer 2 finally acts on and stays in the subcutaneous tissue.

[0037] like Figure 2 As shown, the entire patch is a square with a side length of 1.2 cm to 1.5 cm. The polymer microneedles are distributed in an array of 11×11 on the base layer 1. The microneedle height is 500 μm, the tip diameter of each microneedle is 5-10 μm, the needle seat diameter is 200 μm, and the distance between the needle tips is 800 μm.

[0038] The base layer of the patch is made of PET film, the base material of the microneedle is chitosan / polyvinyl pyrrolidone PVPK30, and doxycycline and fluconazole are uniformly dispersed inside the base material of the needle tip layer; wherein the molar concentration ratio of doxycycline to fluconazole is 1:1.

[0039] The preparation method of the patch for treating skin fungal infection of this embodiment comprises the following steps:

[0040] (1) Weigh 48.1 mg of doxycycline hydrochloride and dissolve it in 1 mL of H 2 O to obtain dispersion system A (final drug concentration: 0.1 M); weigh 30.6 mg of fluconazole and dissolve it in 1 mL of anhydrous ethanol to obtain dispersion system B (final drug concentration: 0.1 M).

[0041] (2) Dispersion system A and dispersion system B were directly mixed in equal volumes to obtain dispersion system C; PVPK30 powder was slowly added to 1 ml dispersion system C and stirred evenly, followed by slow addition of 1 ml 4% chitosan aqueous solution, and stirring was continued for 1-2 h until the solution was uniformly dispersed, and air bubbles were removed by vacuum to obtain dispersion system D;

[0042] (3) A certain amount of the above dispersion system D is filled into the PDMS female mold, the surface is sealed with a sealing film, and centrifuged at 4000 rpm for 10 min to allow the dispersion system D to fully enter the groove of the mold;

[0043] (4) leaving the mold at room temperature for 10 minutes, then placing it in an oven and baking it at 30-60° C. for 3-12 hours to form the needle tip;

[0044] (5) Take out the microneedle mold, cover it with the cut PET film, and slowly peel it off to obtain a patch for treating fungal skin infections ( Figure 2 ).

[0045] In this embodiment, since the volume of the drug-containing microneedles in each patch is about 20 μl, and the concentrations of doxycycline and fluconazole are both 0.1 M, the drug loading of the two drugs in the patch is 2 μmol.

[0046] This embodiment also compares and optimizes the amount of PVPK30 added in step (2) and the baking temperature and time in step (4) by two parameters: the microneedle forming rate (i.e., the proportion of the final formed patch to the total number of patches when 50 patches of the same type are made at the same time) and the average drug loading (i.e., the content of fluconazole in the microneedle tip layer, taking the average value of 10 formed patches). The patch of this embodiment has 121 microneedles with an array of 11×11 distribution. The patch that can form a complete 121 independent microneedles is formed or qualified, otherwise it is a non-formed or unqualified patch.

[0047] The results are shown in Table 1. First, the amount of PVPK30 added has an effect on the forming rate and the drug loading of fluconazole. Due to the poor solubility of fluconazole, the addition of PVPK30 helps the dispersion of fluconazole in the system. Therefore, the higher the amount of PVPK30 added, the higher the drug loading. However, due to the increase in the polymer concentration in the dispersion system, the viscosity of the dispersion system increases and the patch forming rate decreases. Therefore, after optimization, the optimal amount of PVPK30 added is 500-1000 mg / ml; then, the baking temperature also has an important effect on the drug loading and forming rate. If the temperature is too low, the patch forming rate is not good. If the temperature is too high, the drug is easily denatured and inactivated. Through optimization experiments, the optimal baking temperature is 40-50 ° C; finally, the baking time also affects the drug loading and forming rate of the patch. The longer the baking time, the higher the forming rate, but it will cause the drug to denature and inactivate. Through optimization, the optimal baking time is 6-8h.

[0048] Table 1

[0049]

[0050] Example 2 Comparison of Storage Stability of Fluconazole Powder and Polymer Microneedle Patch

[0051] The patch for treating fungal skin infection prepared in Example 1 (PVPK30 addition amount 500mg / ml, baking temperature 50°C, time 6h) and fluconazole powder were placed in a cool and dry room temperature. The content of fluconazole in the microneedle patch was 2μmol. One microneedle patch and an appropriate amount of fluconazole powder were dissolved in distilled water at 12 hours, 24 hours, 2 days, 5 days, 7 days, 30 days, and 60 days to obtain a sample to be tested, and the final concentration was 2mM. The actual content of the drug was measured by HPLC, and each sample was tested three times, and the average value was finally taken.

[0052] The results are shown in Table 2. Fluconazole loaded in the microneedle patch can maintain a basically consistent content under room temperature conditions for 2 months, while the powder state is reduced by 35.9% after being placed at room temperature for one week. After one month, the fluconazole content is reduced from the original 2mM to 0.88mM, a decrease of 56%. In the patch state, the fluconazole content is 95% of the original. This result shows that the drug storage in the patch state is more stable.

[0053] Table 2 Storage stability

[0054] time Powder(mM) Patch(mM) 12 hours 1.98 1.98 24 hours 1.85 1.98 2 days 1.50 1.98 5 days 1.29 1.98 7 days 1.27 1.95 14 days 1.08 1.95 30 days 0.88 1.90 60 days 0.75 1.90

[0055] Example 3 In vitro antifungal effect

[0056] The microneedle patch for treating skin fungal infection obtained in Example 1 (PVPK30 addition amount 500 mg / ml, baking temperature 50°C, time 6 h) was added to the liquid culture medium inoculated with Candida albicans (the content of fluconazole and doxycycline was 2 μmol), and cultured in a constant temperature oscillator (37°C, 150 rpm). At 0 h, 1 h, 3 h, 5 h, 7 h, 9 h, 11 h, and 24 h, a certain amount of bacterial solution was taken out and its OD was measured by an ELISA instrument. 600 The values ​​were repeated three times in parallel, and the growth curve of Candida albicans was calculated and prepared for comparative analysis. The grouping method is as follows:

[0057] Experimental group 1: chitosan-PVPK30 microneedle patch containing 2 μmol fluconazole and 2 μmol doxycycline prepared in Example 1;

[0058] Experimental group 2: based on the experimental group 1, fluconazole and doxycycline were not added to obtain a drug-free chitosan-PVPK30 microneedle patch;

[0059] Experimental group 3: PVPK30 microneedle patch without drug (except that two drugs and chitosan were not added, the rest was the same as Example 1);

[0060] Experimental group 4: 2 μmol fluconazole combined with 2 μmol doxycycline;

[0061] Negative control group: no treatment.

[0062] Inhibition rate (%) = (OD of negative control group 600 Value-experimental group OD 600 (value of inhibition zone diameter) / experimental group OD 600 value × 100%.

[0063] The results are as follows Figure 3 As shown in the figure, the antibacterial rate of the fluconazole combined with doxycycline polymer microneedle patch after 24 hours of action was 96.42%, and the antibacterial rate of the drug-free microneedle patch after action was 18.88%, which may be because the chitosan contained in the patch has a certain antibacterial effect. The antibacterial rate was 79.97% when treated with the same amount of fluconazole and doxycycline, which was lower than that of the patch with the same content, mainly because the chitosan in the microneedle had a certain antibacterial effect. In summary, the antifungal effect is best in the form of microneedles, and the chitosan component in the microneedle can work synergistically with the drug to improve the antibacterial effect.

[0064] Example 4 Effect of the patch on inhibiting fungal infection in rabbits

[0065] A. Preparation of bacterial suspension: Take 0.5 mL of Trichophyton rubrum suspension and place it in a culture bottle containing potato dextrose broth liquid medium. After continuous culture and activation at 28°C for 7 days, gently aspirate the bacterial suspension with a pipette to adjust the final concentration of the bacterial suspension to about 2×10 8 / mL, and then dilute the bacterial solution step by step to 2×10 5 / mL for future use.

[0066] B. Modeling method: 42 healthy rabbits were randomly divided into (1) experimental group 1: chitosan-PVPK30 microneedle patch containing 2 μmol fluconazole and 2 μmol doxycycline prepared in Example 1; (2) experimental group 2: chitosan-PVPK30 microneedle patch containing 2 μmol fluconazole and 4 μmol doxycycline; (3) experimental group 3: chitosan-PVPK30 microneedle patch containing 4 μmol fluconazole and 2 μmol doxycycline; (4) experimental group 4: chitosan-PVPK30 microneedle patch containing 2 μmol doxycycline. PK30 microneedle patch; (5) Experimental group 5: 2 μmol fluconazole chitosan-PVPK30 microneedle patch; The patches of experimental groups 1 to 5 were prepared according to the preparation method of Example 1, with a PVPK30 addition amount of 500 mg / ml, a baking temperature of 50°C, and a time of 6 h; (6) Negative control group: chitosan-PVPK30 microneedle patch without drug; (7) Positive control: 2.0 μmol doxycycline-2.0 μmol fluconazole, subcutaneous injection; (8) Normal control group; 6 females in each group. Before modeling, the rabbits in each group were depilated with a depilatory agent on the back area on both sides of the spine, and the depilatory area was about 10×10 cm 2 , prepare a smooth and hairless skin area. After depilation, wash and dry the rabbit back skin with sodium chloride injection (0.9%), and then gently polish the skin with fine sandpaper to prepare a rough surface with slight bleeding. Except for the normal control group, the prepared bacterial suspension was injected into the rabbit back skin by intradermal injection, about 0.5mL / rabbit. The rabbit skin was continuously inoculated with bacterial suspension for 3 days to prepare the fungal infection model control group. The normal control group was given an equal amount of sodium chloride injection (0.9%) under the same conditions.

[0067] C. Administration: 3 days after the rabbits were infected with the bacteria, the drug was administered the next day. The normal control group did not receive any treatment. The positive control group was administered subcutaneously, and the experimental group and other groups were administered with microneedles according to the above dosage. The drug was administered once a day for 7 consecutive days.

[0068] D.Evaluation method:

[0069] 1) Visually observe the changes of skin lesions on the back of rabbits. The degree of skin infection damage of rabbits is used as the judgment standard, which is divided into 6 grades, see Table 3. The efficacy of the drug is judged according to the change of the score.

[0070] Table 3 Grading and scoring criteria for the degree of damage in rabbit skin fungal infection model

[0071]

[0072]

[0073] The results are as follows Figure 4 As shown in the figure: The back skin of rabbits in the normal control group had no redness, swelling, or bruises, and the structure was normal. Compared with the normal control group, the back skin of rabbits in the model control group was red and swollen after infection, and there was an obvious inflammatory reaction. The average score of back skin lesions was significantly different (P<0.01). Compared with the model control group, the back skin of rabbits in the experimental groups improved to varying degrees; among them, the average scores of back skin lesions in experimental groups 1, 2, and 3 were significantly different from those in the model control group (P<0.01), and there was no significant difference between experimental group 1 and the positive control group (P>0.05). The results showed that the chitosan-PVPK30 microneedle patch with 2μmol fluconazole and 2μmol doxycycline was the best in inhibiting fungal infection in rabbits.

[0074] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A patch for treating fungal skin infections, characterized in that: It comprises a base layer and a needle tip layer, wherein the needle tip layer comprises microneedles arranged in an array on the base layer, and the microneedles are loaded with fluconazole and doxycycline, and the molar ratio of fluconazole to doxycycline is 1:1; The material of the base layer is one of PE film, PVC film, PET film, EVA film, PU film, PSA film, PVA film and PVA / PVP composite film; The substrate of the microneedle is a composite of chitosan and polyvinyl pyrrolidone, and the mass ratio of chitosan to polyvinyl pyrrolidone is 1:5-50.

2. A patch for treating fungal skin infections according to claim 1, characterized in that: The microneedle is conical, with a bottom diameter of 40-400 μm, a height of 200-100 μm, and a needle tip diameter of 1-20 μm.

3. A method for preparing the patch for treating skin fungal infection according to claim 1 or 2, characterized in that: The following steps are involved: (1) Doxycycline was added to pure water as a solvent, and the mixture was stirred and dissolved at room temperature to obtain a dispersion system A with a drug concentration of 0.1 M. Fluconazole was added to ethanol and dissolved to obtain a dispersion system B with a drug concentration of 0.1 M. (2) Dispersion system A and dispersion system B are mixed in equal volume ratios to obtain dispersion system C, and the substrate of the microneedle is added to the dispersion system C, stirred and dispersed evenly, and bubbles are removed to obtain dispersion system D. In dispersion system D, the concentration of polyvinyl pyrrolidone is 500-1000 mg / mL; the ratio of the substrate of the microneedle to dispersion system C is 0.05-5 g / mL; (3) Filling the dispersed system D into the microneedle mold to form a liquid layer of uniform thickness in the mold; (4) Let the mold stand at room temperature for 30-40 min, then place it in an oven to bake it at a temperature of 40-50 °C for 6-8 h. (5) Take out the microneedle mold, cover the base layer, and peel it off to obtain the patch.

4. Use of the patch for treating fungal skin infections according to claim 1 or 2 in the preparation of a medicament for treating deep fungal skin infections.

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