Simvastatin external hydrogel for treating vitiligo

Abstract

The application discloses a simvastatin external-use hydrogel for treating vitiligo, and relates to the technical field of medicines; the hydrogel is formed by in-situ cross-linking of a precursor solution containing simvastatin, propylene glycol, phosphate buffer and a cross-linking matrix; the cross-linking matrix is composed of 4-arm polyethylene glycol-succinimidyl ester and 4-arm polyethylene glycol-amino, the molecular weight of both is independently 10 kDa, the A component and the B component are mixed in a mass ratio of 1:1, and the hydrogel is formed in-situ by amidation cross-linking reaction at 20-40 DEG C for 10-30 s. The 4ArmPEG-NHS / 4ArmPEG-NH2 is used to complete the amidation cross-linking in 10-30 s under physiological conditions, the precursor solution is a flowable liquid, can be coated on irregular skin lesions and wrinkle parts, the storage modulus of the formed gel is 800-1200 Pa, the gel can be closely attached to the skin and will not fall off due to clothes rubbing, and no additional cross-linking agent is needed, and the biocompatibility is good.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more particularly to a topical hydrogel of simvastatin for the treatment of vitiligo. Background Technology

[0002] Vitiligo is a common acquired depigmenting skin disease characterized by the loss of functional melanocytes in the skin and / or hair, resulting in characteristic white patches. Its pathological mechanism is complex, involving multiple factors such as autoimmune attack, oxidative stress, melanocyte dysfunction, or apoptosis. Current clinical treatments mainly include topical corticosteroids, calcineurin inhibitors, phototherapy (such as narrowband UVB), and surgical treatment. However, existing therapies suffer from inconsistent efficacy, high recurrence rates, potential side effects with long-term use (such as skin atrophy and telangiectasia), and inconvenience. Therefore, the development of novel, highly effective, convenient-to-use, and low-side-effect topical medications is of significant clinical importance.

[0003] Simvastatin is an hydroxymethylglutaryl-CoA reductase inhibitor widely used clinically to regulate blood lipids. Recent studies have found that in addition to its lipid-lowering effect, simvastatin also possesses pleiotropic properties, including anti-inflammatory, immunomodulatory, antioxidant, and cell differentiation-promoting functions. Existing research suggests that simvastatin may have protective or stimulatory effects on melanocytes, but its application in the treatment of vitiligo, particularly through topical formulations, has not yet been reported.

[0004] Traditional topical preparations (such as creams, ointments, gels, etc.) have a short retention time on the skin surface and are easily washed away by clothing, sweat, or personal activities, resulting in insufficient actual drug action time and affecting efficacy.

[0005] Although existing PEG in situ crosslinked hydrogels can be used as local drug delivery carriers to load hydrophobic drugs, the following problems still exist when used for the treatment of vitiligo: (1) The crosslinking sites of conventional PEG hydrogels are mostly non-degradable chemical bonds, which cannot respond to the pathological characteristics of vitiligo lesions; (2) The storage modulus and porosity of general PEG hydrogels have not been optimized according to the needs of skin drug delivery. If they are too soft, they are easy to flow and fall off. If they are too hard, they have poor air permeability and poor adhesion to the skin; (3) Simvastatin is a strongly hydrophobic drug that is easy to aggregate and precipitate in hydrogel systems. Conventional methods require the use of surfactants to solubilize it, which can easily cause skin irritation; (4) During the active phase of vitiligo, the expression of matrix metalloproteinase MMP-9 around the hair follicles of the lesions is significantly upregulated, but existing PEG hydrogels do not utilize this pathological characteristic to achieve lesion-responsive drug release.

[0006] Although the simvastatin gelatin microsphere ointment disclosed in CN112791049A can be applied topically, its petrolatum matrix has strong occlusive properties and is prone to clogging hair follicles. The gelatin microspheres need to be prepared through multiple steps such as emulsification and cross-linking. Furthermore, simvastatin has insufficient storage stability in ointments. These defects mean that this dosage form still cannot meet the needs of topical treatment for vitiligo. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies by proposing a topical hydrogel for the treatment of vitiligo using simvastatin.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A topical hydrogel for the treatment of vitiligo, comprising simvastatin, propylene glycol, phosphate buffer and crosslinking matrix, is formed by in-situ crosslinking of a precursor solution.

[0010] The crosslinking matrix is ​​composed of 4-arm polyethylene glycol-succinimide ester (4ArmPEG-NHS) and 4-arm polyethylene glycol-amino (4ArmPEG-NH2), both of which have an independent molecular weight of 10 kDa. Component A and component B are mixed in a 1:1 mass ratio and an amidation crosslinking reaction is carried out at 20-40°C for 10-30 seconds to form a hydrogel in situ.

[0011] The hydrogel has a storage modulus of 800–1200 Pa, a porosity of 65%–75%, a pore size of 20–50 nm, and the amide bonds formed by cross-linking can be degraded by matrix metalloproteinase MMP-9.

[0012] The simvastatin is dispersed in the precursor solution in the form of nanocrystals, the propylene glycol has a volume fraction of 2% to 5% in the phosphate buffer, the final concentration of simvastatin in the hydrogel is 0.01% to 1% w / v, and the pH of the phosphate buffer is 7.2 to 7.4.

[0013] Preferably, the final concentration of simvastatin in the hydrogel is 0.1% w / v, and the volume fraction of propylene glycol in phosphate buffer is 3.3%.

[0014] Preferably, the simvastatin nanocrystals have a particle size of 100–300 nm and a zeta potential of -15–-25 mV.

[0015] Preferably, the oxygen permeability of the hydrogel is 800-1200 Barrer.

[0016] Preferably, the simvastatin is in the α-crystal form.

[0017] A method for preparing the simvastatin topical hydrogel includes the following steps:

[0018] S1: Simvastatin was dissolved in propylene glycol and ultrasonically treated to prepare simvastatin stock solution;

[0019] S2: Dilute the stock solution obtained from S1 with phosphate buffer solution at pH 7.2–7.4 to obtain the drug loading buffer;

[0020] S3: Dissolve 4ArmPEG-NHS and 4ArmPEG-NH2 in the drug loading buffer obtained in S2 to prepare precursor solutions of component A and component B, respectively. The concentrations of components A and B in the precursor solutions are independently 2% to 15% w / v.

[0021] S4: Mix the precursor solution of component A with the precursor solution of component B, and crosslink them in situ at the administration site to form a hydrogel.

[0022] Preferably, the ultrasonic power in S1 is 200-300W, the ultrasonic time is 5-10min, and the simvastatin stock solution obtained contains simvastatin in the form of nanocrystals.

[0023] Preferably, in step S3, the concentrations of both component A and component B in the precursor solution are 6% w / v; in step S4, the precursor solutions of components A and B are mixed at a volume ratio of 1:1, and after mixing, they are cross-linked in situ within 10 to 30 seconds to form a hydrogel membrane with a thickness of 0.1 to 0.3 mm.

[0024] The use of the simvastatin topical hydrogel in the preparation of a medicament for treating vitiligo.

[0025] Preferably, the drug exerts its effect by upregulating the protein expression of microphthalmia-associated transcription factor MITF, tyrosinase TYR, tyrosinase-associated protein 1 TYRP1, and dopachrome isomerase DCT in skin tissue.

[0026] The beneficial effects of this invention are as follows:

[0027] 1. This invention uses 4ArmPEG-NHS / 4ArmPEG-NH2 to complete amidation crosslinking in 10-30s under physiological conditions. The precursor solution is a fluid liquid that can be applied to irregular skin lesions and wrinkled areas. After gelation, the storage modulus is 800-1200Pa. It can adhere tightly to the skin and will not fall off due to friction from clothing. No external crosslinking agent is required, and it has good biocompatibility.

[0028] 2. In this invention, propylene glycol is used both as a solvent for simvastatin to stably disperse it in the aqueous phase in nanocrystal form and as a penetration enhancer to assist the drug in transdermal delivery. This eliminates the need for adding surfactants for solubilization and reduces the risk of skin irritation.

[0029] 3. During the active phase of vitiligo, MMP-9 expression is significantly upregulated in skin lesions, especially around hair follicles, compared to normal skin. The cross-linked amide bonds of the hydrogel of this invention can be specifically degraded by MMP-9, allowing simvastatin to be released near the melanocyte nests in hair follicles, thereby increasing the local concentration of the drug at the target site.

[0030] 4. The present invention uses a hydrogel with a porosity of 65% to 75%, a pore size of 20 to 50 nm, and an oxygen permeability of 800 to 1200 Barrer, which allows nutrients and oxygen to diffuse to the hair follicle while avoiding hair follicle hypoxia caused by petrolatum-like matrix, thus promoting the recovery of melanocyte function. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the decolorization phenotype of the back of C57BL / 6 mice induced by monobenzone combined with retinoic acid according to the present invention;

[0032] Figure 2 This is a schematic diagram of the focal depigmentation phenotype of the tail of a vitiligo-like mouse according to the present invention;

[0033] Figure 3 This is a schematic diagram showing the representative appearance of the back lesions in groups W0, W2, W3, and W4 of this invention, the corresponding NPI dynamic change curves, and statistical comparisons.

[0034] Figure 4 This is a schematic diagram showing the comparison of the appearance of the tail hair / skin repigmentation after 4 weeks of continuous treatment according to the present invention.

[0035] Figure 5 This is a schematic diagram illustrating the differences in epidermal structure and inflammatory infiltration of the back skin in each group of the present invention, obtained by HE staining.

[0036] Figure 6 This is a diagram showing the differences in melanin deposition in the back skin of each group according to the Masson-Fontana melanin staining method of the present invention;

[0037] Figure 7 Masson-Fontana melanin staining images of the tail skin of each group in this invention;

[0038] Figure 8 This is a comparison image of immunohistochemical staining of MITF, TYR, TYRP1, and DCT in the tail skin of each group in this invention.

[0039] Figure 9 This is a comparison image of MITF immunohistochemical staining of the hair follicle area on the back of each group in this invention.

[0040] Figure 10 This is an MITF immunofluorescence image of the tail skin and a semi-quantitative analysis of fluorescence intensity from the present invention.

[0041] Figure 11This is a Western blot image and grayscale quantitative analysis of MITF, TYR, TYRP1, and DCT on the back skin of the present invention. Detailed Implementation

[0042] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0043] Preparation of simvastatin topical hydrogel and its application in the treatment of vitiligo

[0044] I. Plan

[0045] Simvastatin was dispersed / dissolved in a propylene glycol-buffer system and then mixed with two components, 4-arm PEG-NHS (10k) and 4-arm PEG-NH2 (10k), to form an adhesive topical hydrogel by in-situ cross-linking A:B=1:1 on the skin surface (or administration site). In a monobenzone + retinoic acid-induced vitiligo-like mouse model, topical application of this gel promoted repigmentation of skin lesions and hair, upregulated the MITF-TYR / TYRP1 / DCT melanin production axis, and increased melanin deposition.

[0046] II. Materials

[0047] 2.1 Key Raw Materials and Reagents

[0048] Simvastatin: Sigma-Aldrich, PHR1438.

[0049] 1,2-Propanediol (PG): Used to dissolve simvastatin and aid in dispersion.

[0050] PBS buffer: Used for diluting and preparing drug-loaded working solutions.

[0051] Dual-mix ready-to-use PEG hydrogel kit: includes

[0052] 4ArmPEGNHS10K (Component A)

[0053] 4ArmPEGNH210K (component B) is used with a dual mixing head for injection, mixing, and in-situ crosslinking into a gel.

[0054] Carrier gel (Veh@Gel) control: Except for the absence of simvastatin, all other aspects (PEG matrix, PG concentration, process) are completely identical, used to eliminate matrix / solvent interference.

[0055] 2.2 Animals and Modeling-Related Materials

[0056] SPF-grade female C57BL / 6 mice, 6-8 weeks old.

[0057] 4-Benzyloxyphenol (Monobenzone, MBEH): Formulated with petrolatum to form a 40% cream.

[0058] 0.1% retinoic acid cream: used in combination with MBEH to enhance depigmentation-induced stability.

[0059] 2.3 Testing related materials

[0060] HE staining and Masson-Fontana melanin staining kit.

[0061] Antibodies: MITF, TYR, TYRP1, DCT, etc. (IHC / IF / WB).

[0062] Western blot, imaging systems, fluorescence microscopes, etc.

[0063] III. Methods

[0064] 3.1 Example 1: Preparation method of 0.1% simvastatin PEG hydrogel (Sim@Gel)

[0065] Simvastatin stock solution: Weigh simvastatin, dissolve it in PG and vortex to obtain a 30 mg / mL stock solution.

[0066] Drug-loaded working solution (SimPBS): Dilute with PBS before use to achieve a final simvastatin concentration of 0.1% (w / v). If slight opalescence / microparticles are observed, consider it a uniform dispersion system and vortex resuspend thoroughly before gel preparation to ensure homogeneity.

[0067] PEG precursor solutions: After fraction A (4ArmPEGNHS10K) and fraction B (4ArmPEGNH210K) were brought to room temperature, they were prepared into **6% (w / v, 60 mg / mL)** precursor solutions with SimPBS.

[0068] In-situ cross-linking to form a gel: The gel is mixed by injecting it through a dual mixing head at a ratio of **A:B=1:1 (volume ratio)** and used within 30 minutes. It forms a gel layer in-situ at the administration site through cross-linking.

[0069] Vector control (Veh@Gel): Except for the absence of simvastatin, the other steps were the same (to ensure consistent PG / PEG exposure and reduce confounding bias).

[0070] 3.2 Example 2: Establishment of a vitiligo-like mouse model

[0071] Female C57BL / 6 mice were selected, and the back hair was shaved to a depth of approximately 2cm × 3cm.

[0072] Except for the normal control group, the simulated mice were given topical application on their backs every other day:

[0073] 40% Monobenzone Cream (approximately 50mg) (Monobenzone: Vaseline = 2:3)

[0074] Apply approximately 20 mg of 0.1% retinoic acid cream for 4 consecutive weeks to induce depigmentation.

[0075] The successful modeling phenotype was defined by visible discoloration of the back, lightening / whitening of the fur, and focal discoloration of the tail; the baseline W0 was set at the end of the 4th week.

[0076] Animals that successfully established the model and exhibited stable phenotypes were selected from the simulated animals for intervention.

[0077] 3.3 Example 3: Dosing regimen and efficacy evaluation

[0078] Grouping:

[0079] Control group: No modeling was performed, and a blank hydrogel matrix was applied to the back.

[0080] Vitiligo-Veh group: After successful modeling, the carrier hydrogel was applied externally.

[0081] Vitiligo-Sim group: Apply 0.1% Sim@Gel externally after successful modeling.

[0082] Dosage

[0083] Starting from W0, apply the medication once daily for four consecutive weeks, uniformly coating the modeling area to form a thin layer, ensuring consistent dosage. After administration, keep the animals in individual cages for a short period to reduce licking / contamination.

[0084] Primary outcome measure: Standardized Pigment Index (NPI)

[0085] W0, W2, W3, and W4 are used to take photos under the same conditions; ImageJ defines the desaturated area and the adjacent normal area (ROI).

[0086] The NPI was obtained by normalizing the average pigment index of the control group to 100% and then performing dynamic statistics.

[0087] Histological and Molecular Detection

[0088] HE: Assess epidermal structure, inflammatory infiltration, hair follicle appendages, etc.

[0089] Masson-Fontana: Evaluation of melanin granule deposition in the epidermal basal layer / hair follicle.

[0090] IHC: MITF, TYR, TYRP1, DCT (epidermal basal layer and hair follicle related area).

[0091] IF: Semi-quantitative MITF fluorescence intensity.

[0092] WB: Expression of MITF, TYR, TYRP1, and DCT proteins (β-actin normalized).

[0093] statistics

[0094] Three comparison groups: single-factor ANOVA + Tukey.

[0095] NPI variation over time: Two-factor repeated measures ANOVA (time × treatment) + Tukey correction

[0096] IV. Results

[0097] 4.1 Model construction successful (stable depigmentation resembling vitiligo appears)

[0098] Back: After 4 weeks, relatively well-defined depigmented patches form, and the hair turns noticeably whiter.

[0099] Tail: Scattered, localized patchy decolorization is observed, exhibiting the characteristic of "large patches on the back and scattered patches on the tail".

[0100] 4.2 Sim@Gel promotes repigmentation of skin lesions and hair (consistent with skin phenotype and NPI quantification)

[0101] Visual observation: In the Vitiligo-Veh group, depigmentation was basically continuous; in the Vitiligo-Sim group, the edge pigmentation deepened starting from W2, the repigmentation range expanded from W3 to W4, and the white patches shrank; the depigmentation of the tail also showed a recovery trend.

[0102] NPI dynamic analysis: There was an interaction between time and treatment; compared with Veh, the NPI of the Sim group increased from W3, and the difference between W3 and W4 was statistically significant (P < 0.05).

[0103] 4.3 HE suggests that Sim@Gel improves histopathological changes (with a trend towards reduced inflammation and structural damage).

[0104] Vitiligo group: mild irregular thickening of the epidermis, loose collagen in the dermis, and increased inflammatory cell infiltration around blood vessels / hair follicles.

[0105] Sim group: The above changes were alleviated, inflammatory infiltration was reduced, and the structure was closer to that of the control group.

[0106] 4.4 Masson-Fontana confirmed the restoration of melanin deposition (both the epidermal basal layer and hair follicles were enhanced).

[0107] Vitiligo group: melanin granules were significantly reduced / absent.

[0108] Sim group: Scattered band-like deposits appeared in the basal layer of the epidermis, and the melanin signal in and around the hair follicles increased, with the overall result falling between the control and Vitiligo; the tail results were consistent.

[0109] 4.5 Immunohistochemistry / immunofluorescence / WB collectively indicate that the "MITF-melanin synthesis axis" has been restarted.

[0110] IHC: The number and intensity of MITF, TYR, TYRP1, and DCT positive cells in the basal layer of the tail epidermis in the Sim group increased compared with the Veh group; the MITF positive rings in the hair follicles on the back showed a recovery trend.

[0111] IF: MITF fluorescence almost disappeared in the Vitiligo group; MITF fluorescence recovered in the Sim group, and the quantification was significantly higher than that in the Vitiligo group (P < 0.05).

[0112] WB: MITF, TYR, TYRP1, and DCT were significantly decreased in the Vitiligo group; all were significantly increased in the Sim group, with some indicators approaching or returning to control levels.

[0113] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A topical hydrogel of simvastatin for the treatment of vitiligo, characterized in that, It is formed by in-situ crosslinking of a precursor solution containing simvastatin, propylene glycol, phosphate buffer, and a crosslinking matrix; The crosslinking matrix is ​​composed of 4-arm polyethylene glycol-succinimide ester and 4-arm polyethylene glycol-amino, both of which have an independent molecular weight of 10 kDa. Component A and component B are mixed in a 1:1 mass ratio and an amidation crosslinking reaction is carried out at 20-40°C for 10-30 seconds to form a hydrogel in situ. The hydrogel has a storage modulus of 800–1200 Pa, a porosity of 65%–75%, a pore size of 20–50 nm, and the amide bonds formed by cross-linking can be degraded by matrix metalloproteinase MMP-9. The simvastatin is dispersed in the precursor solution in the form of nanocrystals, the propylene glycol has a volume fraction of 2% to 5% in the phosphate buffer, the final concentration of simvastatin in the hydrogel is 0.01% to 1% w / v, and the pH of the phosphate buffer is 7.2 to 7.

4.

2. The simvastatin topical hydrogel for treating vitiligo according to claim 1, characterized in that, The final concentration of simvastatin in the hydrogel is 0.1% w / v, and the volume fraction of propylene glycol in phosphate buffer is 3.3%.

3. The simvastatin topical hydrogel for treating vitiligo according to claim 2, characterized in that, The simvastatin nanocrystals have a particle size of 100–300 nm and a zeta potential of -15–-25 mV.

4. The simvastatin topical hydrogel for treating vitiligo according to claim 1, characterized in that, The oxygen permeability of the hydrogel is 800–1200 Barrer.

5. A simvastatin topical hydrogel for treating vitiligo according to claim 4, characterized in that, The simvastatin is in the α-crystal form.

6. A method for preparing the simvastatin topical hydrogel according to any one of claims 1-5, characterized in that, Includes the following steps: S1: Simvastatin was dissolved in propylene glycol and ultrasonically treated to prepare simvastatin stock solution; S2: Dilute the stock solution obtained from S1 with phosphate buffer solution at pH 7.2–7.4 to obtain the drug loading buffer; S3: Dissolve 4ArmPEG-NHS and 4ArmPEG-NH2 in the drug loading buffer obtained in S2 to prepare precursor solutions of component A and component B, respectively. The concentrations of components A and B in the precursor solutions are independently 2% to 15% w / v. S4: Mix the precursor solution of component A with the precursor solution of component B, and crosslink them in situ at the administration site to form a hydrogel.

7. The method according to claim 6, characterized in that, The ultrasonic power described in S1 is 200-300W, the ultrasonic time is 5-10min, and the simvastatin stock solution obtained contains simvastatin in the form of nanocrystals.

8. The method according to claim 6, characterized in that, In S3, the concentrations of both component A and component B in the precursor solution are 6% w / v; in S4, the precursor solutions of components A and B are mixed at a volume ratio of 1:1, and after mixing, they are cross-linked in situ within 10 to 30 seconds to form a hydrogel film with a thickness of 0.1 to 0.3 mm.

9. The use of the simvastatin topical hydrogel according to any one of claims 1-5 in the preparation of a medicament for treating vitiligo.

10. The application according to claim 9, characterized in that, The drug exerts its effects by upregulating the protein expression of microphthalmia-associated transcription factor MITF, tyrosinase TYR, tyrosinase-associated protein 1 TYRP1, and dopachrome isomerase DCT in skin tissue.

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