WO2025185660A1 - High-drug-loading molecular state simvastatin skin ointment, preparation therefor, and use thereof - Google Patents

Abstract

The present disclosure relates to a high-drug-loading molecular state simvastatin skin ointment and a preparation process therefor. The ointment comprises simvastatin and a basic matrix composition. The basic matrix composition comprises a basic matrix, a solubilizer, a consistency regulator, a stabilizer, and the like. According to the ointment of the present disclosure, simvastatin is stably present in the basic matrix composition in the form of molecules. Phospholipid and cholesterol, as the solubilizer and the stabilizer, are self-assembled in a non-aqueous solution to form a reverse micelle structure, thereby enhancing the stability of a drug. Meanwhile, the solubility of a weak-polarity drug is increased by means of the reverse micelle system, so that the skin retention amount can be greatly increased when the reverse micelle system is used as a percutaneous administration carrier, thereby enhancing the percutaneous absorption of the drug. The formulation of the present disclosure is easy to prepare, good in storage stability, high in drug loading, high in drug accumulation amount in skin, easy to permeate, and quick to take effect, thereby avoiding side effects caused by oral simvastatin.

Description

A high-drug-loading molecular simvastatin skin ointment and its preparation and application Technical Field

The present disclosure relates to the field of medical technology, and in particular to a high-drug-loading molecular simvastatin skin ointment and its preparation and application.

Background Art

Autosomal recessive ichthyosis is a heterogeneous group of nonsyndromic ichthyoses characterized by erythematous, keratotic, and scaly skin. Clinical manifestations and severity vary widely. They are caused by mutations in genes encoding enzymes involved in cholesterol synthesis, leading to impaired synthesis of the end product cholesterol and accumulation of toxic lipid metabolic intermediates. Currently, moisturizers and topical keratolytics are the preferred treatment options for autosomal recessive ichthyosis. These agents can improve skin barrier function and promote desquamation. However, these treatments are associated with significant and numerous side effects, necessitating the development of safe, effective, and patient-friendly pharmacological agents.

For patients with autosomal recessive ichthyosis, since the pathogenesis of the disease is due to genetic mutations in enzymes involved in cholesterol synthesis, topical proximal enzyme (HMG-CoA reductase) inhibitors plus cholesterol are used to target the pathogenic mechanism, reduce the accumulation of toxic lipid intermediates, and supplement cholesterol to promote the formation of a normal skin barrier. In recent years, new treatments for autosomal recessive ichthyosis have emerged. For example, the use of 2.5% and 5% simvastatin ointment applied to patients has shown good results. It has also been found that the accumulation of toxic products plays a more important role in the pathogenesis of autosomal recessive ichthyosis than a lack of cholesterol.

Vitiligo is an acquired, localized or generalized skin depigmentation disorder caused by the functional loss or decrease in number of melanocytes in the skin. To date, its etiology and pathogenesis remain largely unexplained. International and domestic analyses suggest that the pathogenesis of vitiligo is multifactorial, primarily involving genetics, immunity, oxidative stress, and melanocyte self-destruction. Due to this lack of clarity, no single treatment method has been found that is consistently effective and minimizes side effects.

Studies have found that patients with hypercholesterolemia who took simvastatin experienced regression and re-pigmentation of their existing vitiligo lesions, leading researchers to investigate the therapeutic effects of simvastatin on vitiligo. Oxidative stress in melanocytes can induce local inflammatory responses and innate immune responses, leading to melanocyte destruction and is considered a key pathogenic factor in the development and progression of vitiligo. Simvastatin possesses antioxidant properties and has demonstrated protective effects in a variety of oxidative stress-related diseases, protecting vitiligo melanocytes from oxidative stress damage.

Autosomal recessive ichthyosis and vitiligo cannot be cured for a long time, which brings double burden of economic and mental health to patients, seriously affecting their appearance, physical and mental health and even passing on to the next generation. They have troubled humanity for many years. Therefore, there is a need for a drug formulation that can provide safe and effective treatment and has good patient adaptability.

Simvastatin is a synthetic product of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors fermented from Aspergillus terreus. It is a white or off-white crystalline powder. It is easily soluble in ethanol, acetone or acetonitrile, but insoluble in water. It belongs to BCS Class II drugs. At the same time, in the presence of water, the lactone ring of simvastatin will be hydrolyzed into hydroxy acid.

Patent CN110368358A discloses a preparation method of simvastatin external preparation for treating autosomal recessive ichthyosis and xanthomas, which is made from the following weight ratio of raw materials and excipients: simvastatin 25-500g, stearic acid 550-600g, glyceryl monostearate 300-350g, light liquid paraffin 100-110ml, ethyl hydroxybenzoate 4-6g, glycerol 600-650g, sodium lauryl sulfate 8-12g, triethanolamine 8-12ml, laurocapram 20-30ml, water is supplemented to 5000g. In the preparation method, it is necessary to prepare the aqueous phase and the oil phase separately, and the two are mixed to make the preparation. Involving a large amount of water in the preparation process, alkaline water further accelerates the decomposition of the drug, making it difficult for the drug to exist in a molecular state, difficult to absorb, and the therapeutic effect is greatly reduced. It is not possible to achieve the purpose of continuous administration, and the drug effect time is short.

Patent CN112791049 A discloses a method for preparing simvastatin ointment for treating vitiligo. The raw materials include 1-6% simvastatin, 2-2.5% triethanolamine, 3.5-4.5% glycerol, 2-2.5% soft soap, 0.2-0.4% ethylparaben, 20-30% vaseline, 3-5% anhydrous lanolin, 20-30% octadecyl alcohol, 3-5% stearic acid, and the balance is distilled water. The preparation method separately prepares an aqueous phase and an oil phase, adds the aqueous phase to the oil phase, heats to 40-45°C, and stirs to form an emulsion to obtain simvastatin ointment. The preparation method is very cumbersome, difficult to scale up production, and has poor transdermal effect. In addition, the water involved in the preparation process can easily degrade simvastatin, making it difficult to prepare as a drug, greatly reducing the therapeutic effect, lowering the drug accumulation concentration, and extending the treatment cycle.

Summary of the Invention

The present invention aims to provide a highly drug-loaded molecular simvastatin skin ointment, its preparation, and use. By administering the drug via application, the adverse side effects of oral simvastatin can be effectively avoided, significantly enhancing its safety. Furthermore, the present invention utilizes an anhydrous preparation and storage process, and the drug is dissolved in the matrix in molecular form, which not only increases the drug's solubility but also its retention in the skin, significantly improving its efficacy.

In order to solve the above technical problems, the present disclosure provides the following technical solutions:

A molecular simvastatin ointment for skin use with a high drug loading content comprises simvastatin as an active ingredient and a pharmaceutically acceptable non-aqueous semisolid material.

Furthermore, the simvastatin ointment for skin use comprises simvastatin as an active ingredient and a basic matrix composition; the basic matrix composition comprises a basic matrix, a solubilizer, a consistency regulator and a stabilizer.

Preferably, the simvastatin skin ointment is prepared from the following raw materials in the following weight ratios:

0.5-5 parts of simvastatin, 5-30 parts of solubilizer, 0.5-5 parts of stabilizer, 5-20 parts of consistency regulator, and 40-89 parts of basic matrix.

Furthermore, the basic matrix composition is selected from one or more of white petrolatum, yellow petrolatum, solid paraffin, vegetable oil, stearic acid and beeswax.

Wherein, the solubilizing agent is selected from one or more of Tween-80, Span-80 and phospholipids.

Wherein, the consistency regulator is selected from one or more of anhydrous lanolin and liquid paraffin.

Wherein, the stabilizer is selected from one or more of glyceryl monostearate, cholesterol and caprylic/capric triglyceride.

On the other hand, the present disclosure further provides a method for preparing simvastatin skin ointment:

The basic matrix composition, i.e., the solubilizer, stabilizer, consistency regulator and basic matrix are mixed, heated to 60-90° C. to melt, cooled to 30-50° C. after melting, simvastatin is added to the basic matrix composition, stirred evenly and cooled to room temperature to obtain simvastatin ointment.

On the other hand, the present disclosure also provides use of the aforementioned simvastatin skin ointment in treating autosomal recessive ichthyosis and vitiligo.

On the other hand, the present disclosure also provides the use of the aforementioned simvastatin skin ointment in the preparation of a medicament for treating autosomal recessive ichthyosis and vitiligo.

The present disclosure has the following beneficial effects:

The disclosed simvastatin skin ointment has a high drug loading capacity, with simvastatin molecularly dispersed in the matrix, resulting in improved stability and significantly enhanced efficacy. Conventional simvastatin topical preparations can have a maximum drug loading of 3%, but the disclosed ointment can increase the drug loading to 5%, allowing for a higher concentration of simvastatin in the preparation and improving efficacy.

The preparation process of the invention is simple and easy to produce in large quantities.

By applying simvastatin externally, the adverse side effects of oral administration can be overcome, greatly improving the safety of medication.

The simvastatin skin ointment disclosed herein contains a reverse micelle structure. Cholesterol and phospholipids can self-assemble to form reverse micelles in non-aqueous solutions, enhancing drug stability and the solubility of weakly polar drugs. When used as a transdermal drug delivery vehicle, the reverse micelle system can promote transdermal transport and enhance drug absorption.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the in vitro release test results of Test Example 1.

FIG2 shows the in vitro permeation test results of Test Example 2.

FIG3 shows a thin layer chromatogram of the stability of the preparation of Test Example 3.

FIG4 shows the drug efficacy study of Test Example 4 - changes in skin and fur color before and after administration.

FIG5A shows the HE-stained pathological section of the blank group in the drug efficacy study of Test Example 4. ...

FIG5B shows the HE-stained pathological sections of the drug efficacy study of the model group of Test Example 4. FIG5B shows the HE-stained pathological sections of the model group of Test Example 4.

FIG5C shows the efficacy study of the UV group of Test Example 4 - HE-stained pathological sections.

FIG5D shows the HE-stained pathological section of the drug efficacy study of the natural recovery group of Test Example 4.

FIG5E shows the HE-stained pathological section of the low-dose+UV group efficacy study of Test Example 4. FIG5E shows the HE-stained pathological section of the low-dose+UV group.

FIG5F shows the HE-stained pathological section of the efficacy study of the medium dose + UV group of Test Example 4.

FIG5G shows the HE-stained pathological section of the high-dose+UV group efficacy study of Test Example 4.

FIG5H shows the efficacy study of the tacrolimus group in Test Example 4 - HE-stained pathological sections.

FIG6 shows the results of a single skin irritation test of Test Example 5.

FIG. 7 shows the results of multiple skin irritation experiments in Test Example 5.

FIG8 is a graph showing the differential scanning calorimetry (DSC) results of Test Example 6. FIG.

FIG. 9 shows the therapeutic effect of Test Example 1 on autosomal recessive ichthyosis.

FIG. 10 shows the therapeutic effect of vitiligo in Test Example 2 after 80 days of treatment.

Figure 11 shows the thin layer chromatogram of phospholipid-modified simvastatin nanocrystals of Comparative Example 3

DETAILED DESCRIPTION

In order to more clearly illustrate the embodiments of the present disclosure or the technical solutions in the prior art, the terms and implementation methods required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the implementation method described below is only one implementation method of the present disclosure. For ordinary technicians in this field, other implementation methods can also be obtained based on these drawings.

I. Terminology

In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the present disclosure belongs.

As used herein, the articles "a" and "an" refer to one or more than one (ie, to at least one) of the grammatical object to which the article refers. For example, "an element" means one element or more than one element.

As used herein, the term "about" refers to and encompasses a specified value and a range greater than or less than that value. In certain embodiments, the term "about" can refer to a variation of ±0.1%, ±0.5%, ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, or ±10%. In certain embodiments, where applicable, the term "about" refers to a specified value ± one standard deviation of that value.

The endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

The terms "comprising," "consisting essentially of," or variations thereof, used throughout the specification and claims mean that all recited elements or groups of elements are included, and optionally, other elements of similar or different properties to the recited elements that do not significantly alter the basic or novel properties of a specified dosage regimen, method, or composition.

"Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and where it does not.

II. Detailed description of specific implementation plan

Simvastatin is a semisynthetic derivative/analog of lovastatin. While insoluble in water, simvastatin is soluble in polar organic solvents. For lipid-soluble drugs, solubility must be addressed when formulating them into pharmaceutical formulations. However, due to simvastatin's readily hydrolyzed nature, carrier solubilization technology is preferred to address this poor solubility issue.

Reverse micelles have attracted considerable attention as a novel drug carrier for drug delivery and solubility enhancement. Unlike traditional micelles, reverse micelles possess a relatively low internal polarity, which facilitates the solubility and stabilization of non-polar or hydrophobic drugs. This property enables reverse micelles to effectively enhance the solubility of hydrophobic drugs, thereby increasing their bioavailability.

On the one hand, the unique structure of reverse micelles provides a "refuge"-like environment, allowing hydrophobic drugs to exist stably within it. This environment reduces interactions between drug molecules, thereby reducing aggregation and precipitation during drug delivery. Furthermore, this sanctuary effect of reverse micelles protects drugs from environmental influences, such as enzymatic degradation.

On the other hand, reverse micelles, as drug carriers, can improve drug solubility by changing the physical state of the drug, such as from a crystalline to an amorphous state. Furthermore, reverse micelles can stabilize drug molecules through interactions with drug molecules, such as hydrogen bonds and hydrophobic interactions, thereby increasing their solubility in physiological environments.

In practical applications, reverse micelles as drug carriers have been shown to significantly improve the solubility and bioavailability of hydrophobic drugs. This advantage makes reverse micelles promising for future drug delivery systems, providing new strategies and tools for improving drug solubility, stability, and bioavailability.

This structure can increase drug stability and bioavailability. Upon contact with body fluids, it transforms into a liquid crystal structure, retarding drug dissolution and thus achieving sustained-release drug delivery. It also has particular advantages as a carrier for transdermal drug delivery systems, promoting transdermal drug transport, enhancing drug stability, and reducing skin irritation.

Materials commonly used to form reverse micelle structures include anionic surfactants, cationic surfactants, nonionic surfactants and amphoteric surfactants.

1. Anionic surfactants: This type of surfactant has good foaming properties and can quickly produce a large amount of foam. The foam is large and stable and not easy to break. It has little irritation to the skin and eyes, good biodegradability, strong antistatic properties, and is easy and safe to use.

2. Cationic surfactants: This type of surfactant has good bactericidal, antistatic and softening properties, but it is highly irritating, easily soluble in water, and has poor oxidation resistance, and can generally only be used as a detergent.

3. Non-ionic surfactants: These surfactants are resistant to strong electrolytes, hard water, acids and alkalis. They are colorless, odorless, non-toxic, non-irritating, and non-allergenic. They have excellent wetting, cleaning, rust prevention, and softening properties, and are particularly miscible with various solvents.

4. Amphoteric surfactants: These surfactants contain both anionic and cationic hydrophilic groups within the same molecule. Their greatest characteristic is their ability to both donate and accept protons. They exhibit excellent emulsification and dispersibility during use.

Since simvastatin is a drug with a higher polarity than vaseline, reverse micelles are needed to increase its solubility. The reverse micelle structure in the preparation utilizes a solubilizing agent (one or more of Tween-80, Span-80, and phospholipids) and a stabilizer (one or more of glyceryl monostearate, cholesterol, and caprylic/capric triglyceride) to self-assemble into reverse micelles in a non-aqueous solution. The solubilizing agent and stabilizer are dissolved in a non-aqueous solution such as yellow vaseline, stirred at high temperature to mix them evenly, and then cooled and added to the basic matrix composition. The reverse micelle system can enhance the stability of the drug and increase the solubility of weakly polar drugs. When used as a transdermal drug delivery carrier, the skin retention can be greatly increased, thereby enhancing the transdermal absorption of the drug.

In order to investigate the solubility of simvastatin in different media, the following experiments were conducted.

The specific steps of the experiment on the solubility of simvastatin disclosed in the present invention are as follows:

Yellow vaseline, liquid paraffin, yellow vaseline + phospholipid + cholesterol, and liquid paraffin + phospholipid + cholesterol were melted completely at 70°C, cooled to 37°C, and then an appropriate amount of simvastatin was added and stirred thoroughly. The mixture was ultrasonicated for 2 hours and equilibrated on a shaker at 37°C for 24 hours. The absorbance was measured at 238 nm by UV spectrophotometry, and the solubility was calculated (Table 1).

Table 1: Solubility of simvastatin in different media

Conclusion: As shown in Table 1, simvastatin is almost insoluble in yellow vaseline and liquid paraffin. After adding phospholipids and cholesterol, the drug can exist in the form of molecules, forming reverse micelles, which significantly improves the solubility.

Based on the above research, the present invention prepares a simvastatin skin ointment with high drug loading, high drug accumulation in the skin, easy penetration, rapid onset and significant efficacy.

In one aspect, the present disclosure provides a high-drug-loaded molecular simvastatin skin ointment, comprising simvastatin as an active ingredient and a pharmaceutically acceptable non-aqueous semi-solid material. To prevent hydrolysis of simvastatin, the present disclosure requires a non-aqueous semi-solid material, including a base matrix, a solubilizer, a consistency regulator, and a stabilizer.

The non-aqueous semi-solid material includes a basic matrix, a solubilizer, a consistency regulator, a stabilizer, etc. or a combination thereof.

Furthermore, the simvastatin ointment for skin use comprises simvastatin as an active ingredient and a basic matrix composition; the basic matrix composition comprises a basic matrix, a solubilizer, a consistency regulator and a stabilizer.

Preferably, the simvastatin skin ointment is prepared from the following raw materials in the following weight ratios:

0.5-5 parts of simvastatin, 5-30 parts of solubilizer, 0.5-5 parts of stabilizer, 5-20 parts of consistency regulator, and 40-89 parts of basic matrix.

Furthermore, the basic matrix composition is selected from one or more of white petrolatum, yellow petrolatum, solid paraffin, vegetable oil, stearic acid and beeswax.

Wherein, the solubilizing agent is selected from one or more of Tween-80, Span-80 and phospholipids.

Wherein, the consistency regulator is selected from one or more of anhydrous lanolin and liquid paraffin.

Wherein, the stabilizer is selected from one or more of glyceryl monostearate, cholesterol and caprylic/capric triglyceride.

The present disclosure further provides a method for preparing simvastatin skin ointment:

The basic matrix composition, i.e., the solubilizer, stabilizer, consistency regulator and basic matrix are mixed, heated to 60-90° C. to melt, cooled to 30-50° C. after melting, simvastatin is added to the basic matrix composition, stirred evenly and cooled to room temperature to obtain simvastatin ointment.

The present disclosure also provides use of the aforementioned simvastatin skin ointment in treating autosomal recessive ichthyosis and vitiligo.

The present disclosure also provides use of the aforementioned simvastatin skin ointment in the preparation of a medicament for treating autosomal recessive ichthyosis and vitiligo.

Example

The present invention is further described below with reference to the following examples. The simvastatin skin ointment provided by the present invention and the raw materials and excipients used in its application can all be purchased from the market.

Example 1

125 parts of Tween-80, 25 parts of glyceryl monostearate, 50 parts of anhydrous lanolin and 275 parts of white vaseline were weighed and mixed, heated to 85°C to melt, and then cooled to 45°C. 25 parts of simvastatin were added to the molten phase, stirred evenly, and cooled to room temperature to obtain a simvastatin skin ointment.

Example 2

25 parts of Span-80, 25 parts of cholesterol, 100 parts of liquid paraffin, 118 parts of beeswax and 207 parts of vegetable oil were weighed and mixed, heated to 85°C to melt, and then cooled to 45°C. 25 parts of simvastatin was added to the molten phase, stirred evenly, and cooled to room temperature to obtain a simvastatin skin ointment.

Example 3

125 parts of phospholipid, 25 parts of caprylic/capric triglyceride, 50 parts of liquid paraffin, 175 parts of solid paraffin and 100 parts of vegetable oil were weighed and mixed, heated to 85°C to melt, and then cooled to 45°C. 25 parts of simvastatin was added to the molten phase, stirred evenly, and cooled to room temperature to obtain a simvastatin skin ointment.

Example 4

125 parts of Tween-80, 25 parts of glyceryl monostearate, 50 parts of anhydrous lanolin and 275 parts of yellow vaseline were weighed and mixed, heated to 85°C to melt, and then cooled to 45°C. 25 parts of simvastatin were added to the molten phase, stirred evenly, and cooled to room temperature to obtain a simvastatin skin ointment.

Example 5

125 parts of phospholipid, 15 parts of glyceryl monostearate, 50 parts of liquid paraffin and 285 parts of yellow vaseline were weighed and mixed, heated to 85°C to melt, and then cooled to 45°C. 25 parts of simvastatin were added to the molten phase, stirred evenly, and cooled to room temperature to obtain a simvastatin skin ointment.

Example 6

Weigh 50 parts of Span-80, 25 parts of glyceryl monostearate, 50 parts of anhydrous lanolin and 350 parts of white vaseline, mix them, heat to 85°C to melt, and then cool to 45°C after melting. Add 25 parts of simvastatin to the molten phase, stir evenly, and then cool to room temperature to obtain a simvastatin skin ointment.

Example 7

125 parts of phospholipid, 2.5 parts of cholesterol, 50 parts of liquid paraffin and 297.5 parts of white vaseline were weighed and mixed, heated to 85°C to melt, and then cooled to 45°C. 25 parts of simvastatin was added to the molten phase, stirred evenly, and cooled to room temperature to obtain a simvastatin skin ointment.

Example 8

125 parts of Tween-80, 25 parts of glyceryl monostearate, 50 parts of anhydrous lanolin and 275 parts of stearic acid were weighed and mixed, heated to 85°C to melt, and then cooled to 45°C. 25 parts of simvastatin was added to the molten phase, stirred evenly, and cooled to room temperature to obtain a simvastatin skin ointment.

Example 9

125 parts of phospholipid, 25 parts of cholesterol, 50 parts of liquid paraffin and 275 parts of yellow vaseline were weighed and mixed, heated to 85°C to melt, and then cooled to 45°C. 25 parts of simvastatin were added to the molten phase, stirred evenly, and cooled to room temperature to obtain a simvastatin skin ointment.

The physical parameters of the simvastatin skin ointment prepared according to the respective prescriptions of Examples 1-9 are shown in Table 2 below.

Test Example 1 In vitro release test

The simvastatin skin ointment prepared in Examples 1-9 was subjected to an in vitro release test as follows:

0.004 g of the ointment of Examples 1-9 was accurately weighed and evenly coated on a piece of cellophane fixed to a Franz diffusion cell for release experiments. 2 ml of the receiving solution was collected at 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 hours, and fresh receiving medium was added. The initial filtrate was filtered through a microporous filter membrane with a pore size of 0.45 μm, and the peak area was measured by high performance liquid chromatography. The in vitro release results are shown in Figure 1.

As can be seen from FIG1 , the preparations prepared in Examples 1 to 9 can all be slowly released from the preparations and can meet the expected release requirements of the preparations.

Test Example 2 In vitro transdermal test

An in vitro transdermal test was conducted on the high drug loading molecular simvastatin skin ointment of the present invention.

A 0.004 g portion of the ointment formulation from Example 9 was precisely weighed and evenly applied to pigskin and ratskin mounted in a Franz diffusion cell for transdermal permeation testing. 2 ml of the receiving medium was collected at 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 hours, and fresh receiving medium was added. The initial filtrate was filtered through a 0.45 μm microporous membrane, and the peak area was determined by high-performance liquid chromatography. Over a 24-hour period, the cumulative drug permeation rate through pigskin was significantly lower than that through ratskin at each time point. The 24-hour cumulative permeation rate was 13.45% for pigskin vs. 27.8% for ratskin. The final skin retention was greater in pigskin than in ratskin: 35.7% for pigskin vs. 11.4% for ratskin. The experimental results are shown in Figure 2.

Test Example 3: Stability Experiment

The sample prepared in Example 9 was placed under accelerated conditions (30±2°C, relative humidity 65±5%) for 3 months. The properties, content uniformity and related substances were used as key indicators. The results are shown in Table 3 below:

Table 3: Accelerated 3-month stability test results

The sample prepared in Example 9 was placed under long-term stability conditions (5°C ± 3°C) for 6 months, with properties, content uniformity and related substances as key indicators. The results are shown in Table 4 below:

Table 4: Results of 6-month long-term stability test

Conclusion: The ointment prepared according to Example 9 showed little change in drug content and related substance content in the accelerated stability test and the long-term stability test, and no discoloration or stratification occurred.

Three batches of samples prepared in Example 9 were placed under accelerated conditions (30±2°C, relative humidity 65±5%), and the drug stability was observed using TLC. The results are shown in Figure 3. In Figure 3, 1 represents simvastatin, 2 represents simvastatin acid, 3 represents the preparation of Example 9 at 0 months, 4 represents the preparation of Example 9 at 1 month, and 5 represents the preparation of Example 9 at 3 months.

Conclusion: No drug degradation products were found in the preparation after storage for 0, 1, and 3 months.

Test Example 4: Pharmacodynamic Study of High-Drug-Loaded Molecular Simvastatin Skin Ointment

Grouping: 40 mice were randomly divided into 8 groups, 5 mice in each group, and numbered within each group. They were divided into blank control group, vitiligo model group, natural recovery group, UV group, low-dose + UV group, medium-dose + UV group, high-dose + UV group, and tacrolimus + UV group.

Dosage method: C57BL/6 mice were selected and the animals were allowed to adapt to the environment for 3 days. On the 4th day, 2×2 square centimeters of dorsal hair were removed from each mouse with a depilatory cream, and modeling was started 24 hours later. The modeling period was 50 days. The blank control group was coated with 0.5 ml/day of sterile distilled water; the model control group, natural recovery group, UV group, low dose + UV group, medium dose + UV group, high dose + UV group, tacrolimus + UV group were smeared on the test area with 3% hydroquinone glycerol solution (1 g of glycerol and 3 g of hydroquinone dissolved in 100 mL of deionized water), with a dose of 100 mg/kg·d, bid; starting from the 30th day, the tacrolimus group applied a thin layer of the ointment of Example 9 to the mouse skin, gently rubbed it evenly, and completely covered it, twice a day in the morning and evening, plus UV therapy twice a week; UV group: UV therapy twice a week The initial dose was 350 mj/cm², adjusted based on the presence of erythema or varicella. If no change was observed, the dose was increased by 50 mj/cm² the next time. If skin damage was observed, the dose was temporarily discontinued the next time. In the low-dose + UV group, 10 mg/kg was applied to the mouse skin and evenly spread once daily, followed by UV phototherapy twice weekly. In the medium-dose + UV group, 18 mg/kg was applied to the mouse skin and evenly spread once daily, followed by UV phototherapy twice weekly. In the high-dose + UV group, 25 mg/kg was applied to the mouse skin and evenly spread once daily, followed by UV phototherapy twice weekly. Skin images were taken every other day. Changes in mouse skin color were closely observed throughout the experiment. The results are shown in Figure 4.

Conclusion: As shown in Figure 4, the skin and hair color restored by high-dose simvastatin skin ointment + UV treatment was the best, and the time to complete healing in each group was: natural recovery group: 40 days; UV group: 35 days; low-dose + UV group: 30 days; medium-dose + UV group: 26 days; high-dose + UV group: 22 days; tacrolimus group + UV group: 28 days.

Pharmacological efficacy study of Test Example 4 - HE-stained pathological sections are shown in FIG5 .

Conclusion: Pathological sections showed that in the model group, the epidermal spinous layer was significantly thickened, the stratum corneum was significantly proliferated, almost no melanin was found in the basal cells and spinous cells, and the melanin in the hair follicles was significantly reduced; in the simvastatin group, the melanin in the basal cells and spinous cells was significantly increased, the melanin in the hair follicles was significantly increased, and the thickening of the skin epidermis was alleviated.

The results of the efficacy study-biochemical factors of Test Example 4 are shown in Table 5.

Each animal was sacrificed by collecting blood from its orbital cavity. The blood was kept at 4°C for 3 h and then centrifuged at 3500 r/min in a refrigerated centrifuge at 4°C for 10 min. The serum of each mouse was drawn and the tyrosinase (TYR) content, cholinesterase (CHE) activity and malondialdehyde (MDA) content of each group were determined by ELISA using a microplate reader according to the instructions of the kit.

Table 5: Efficacy study - biochemical factor test results

Conclusion: Compared with the model control group, the TYR activity of the low, medium and high dose groups of simvastatin skin ointment was increased (P＜0.01), and the MDA and CHE activities were inhibited (P＜0.01), and the results were positively correlated with the dosage.

In conclusion, simvastatin skin ointment has a therapeutic effect on the hydroquinone-induced vitiligo model, with better therapeutic effect and faster recovery compared with the tacrolimus group.

Test Example 5: Skin Safety Study of High-Drug-Loaded Molecular Simvastatin Skin Ointment

Single-dose skin irritation test

Japanese white rabbits were subjected to a skin irritation test using a self-comparison method using the left and right skin sides of the same rabbit. Twenty-four hours before the experiment, hair was removed along both sides of the spine. The depilated area was 3 cm × 3 square centimeters, and no skin damage was allowed to ensure adequate drug-skin contact. On the second day, the low-dose, medium-dose, and high-dose groups of the simvastatin skin ointment of Example 9 were applied to the intact skin on the left side of the spine at a thickness of approximately 1 mm. An equal amount of a blank formulation was applied to the right side and covered with gauze. Twelve hours later, the application site was cleaned with warm physiological saline. The application site was observed for erythema, edema, and other conditions 24, 48, and 72 hours after drug withdrawal, and the experimental results were recorded.

Repeated administration skin irritation test

The pre-experimental treatment process was the same as that of the single-dose skin irritation experiment. The low-dose, medium-dose and high-dose simvastatin topical preparations were applied to the depilated area on the left side of the spine with a thickness of about 1 mm, and the same amount of blank preparation was applied to the right side and wrapped with gauze. The medication was used once a day for seven consecutive days. The medication site was cleaned with warm saline the next day. The erythema and edema of the medication site were observed 24 hours, 48 hours and 72 hours after stopping the medication.

The skin irritation scoring criteria are shown in Table 6. Irritation scores were calculated using the evaluation method in Table 6. The average value was then calculated and evaluated according to the following evaluation criteria: Non-irritant: 0-0.49; Mild Irritation: 0.50-2.99; Moderate Irritation: 3.00-5.99; Severe Irritation: 6.00-8.00.

Table 6: Rating criteria for skin irritation reactions

According to Table 6 (Skin Irritation Scoring Criteria), the skin erythema and edema of Japanese white rabbits at each time period were assessed, and the total reaction score was calculated to determine the skin irritation status. The total reaction score is the sum of the total erythema and edema scores. The specific results of the individual skin irritation tests are shown in Figure 6 and Table 7.

Table 7: Total score results of single skin irritation reaction

Compared with the blank control group, the low and medium dose groups of simvastatin skin ointment did not show irritation phenomena such as erythema and edema. The high dose group had a mild erythema reaction with total scores of 0, 0, and 0.3 respectively, indicating that in the single skin irritation experiment, the low, medium, and high doses of simvastatin skin ointment were non-irritating to intact skin.

The results of multiple skin irritation tests are shown in Figure 7 and Table 8.

Table 8: Results of multiple skin irritation tests

The results in Table 8 show that in multiple skin irritation tests on Japanese white rabbits, the total reaction scores for the low, medium, and high dose groups of the simvastatin topical preparation were all 0, indicating no skin irritation. As shown in Figure 4, compared with the blank control group, none of the three test groups showed irritation such as erythema or edema on the skin surface. This experiment demonstrates that the simvastatin skin ointment is non-irritating to the intact skin surface of rabbits.

Test Example 6: Differential Scanning Calorimetry (DSC) Determination of Thermal Effects of Different Samples

Differential scanning calorimetry (DSC) is used to observe the heat flow power difference between the sample end and the reference end as a function of temperature or time, thereby obtaining relevant thermal effect information such as heat absorption and heat release of the sample during the temperature program process to determine the presence of the drug. Take about 5 mg of simvastatin raw material, blank ointment base, and 5% simvastatin ointment (Example 9) and place them in an aluminum crucible dedicated to the differential scanning calorimeter. Cover the aluminum crucible with a lid, seal it, and use a needle tip to poke a hole in the aluminum lid. Each sample is dynamically scanned at a heating rate of 10°C/min under a nitrogen flow, and the scanning temperature range is -20°C-160°C. As shown in Figure 8, pure simvastatin shows a sharp exothermic peak at 140.3°C.

Those skilled in the art are aware that drugs can interact with the matrix, transforming the drug from a crystalline state to a molecular state. For poorly soluble drugs and suspension-type semisolid preparations, only drugs that dissolve into a molecular state, thus possessing enhanced lipid solubility, can penetrate the skin lesions and exert their therapeutic effects. As shown in Figure 8, the 5% simvastatin ointment of Example 9 exhibits no drug peak at the same location. The disappearance of the simvastatin melting peak indicates that the drug interacts with the matrix, transforming the drug from a crystalline state to an amorphous state or a uniformly dispersed molecular state.

Test Example 1: Efficacy trial for autosomal recessive ichthyosis

To investigate the efficacy of ointment in the treatment of autosomal recessive ichthyosis.

Two patients with clinically, pathologically, and genetically confirmed autosomal recessive ichthyosis were recruited.

Experimental drug: The formula composition of the 2% preparation is: 125 parts phospholipid, 25 parts cholesterol, 50 parts liquid paraffin, 290 parts yellow vaseline and 10 parts simvastatin

The prescription composition of the 4% preparation is: 125 parts of phospholipids, 25 parts of cholesterol, 50 parts of liquid paraffin, 280 parts of yellow vaseline and 20 parts of simvastatin.

Test method: 2% simvastatin ointment was given for the first month, followed by 4% simvastatin skin ointment for external use. Apply it twice a day, applying a thin layer and massaging.

The experimental results are shown in Figure 9. Figures 9-1(a), 9-2(a), and 9-3(a) show the skin lesions before treatment; Figures 9-1(b), 9-2(b), and 9-3(b) show the effects of 2% simvastatin skin ointment for one month. While the study drug was effective, the local lesions remained stubborn. Figures 9-1(c), 9-2(c), and 9-3(c) show the effects of 4% simvastatin skin ointment for three months, showing accelerated improvement and significant regression of stubborn lesions.

As shown in Figure 9, the simvastatin skin ointment can significantly improve the symptoms of autosomal recessive ichthyosis after treatment. The prepared 4% simvastatin skin ointment is more effective than the prepared 2% simvastatin ointment.

Test Example 2 Vitiligo Efficacy Test

The simvastatin skin ointment prepared in Example 9 was subjected to a drug efficacy test to investigate the efficacy of the ointment on vitiligo.

Trial subject: One patient clinically diagnosed with vitiligo was recruited.

The trial drug was applied topically twice daily, with massage, and supplemented with narrow-band ultraviolet B phototherapy for 80 days. Patients and evaluators evaluated the efficacy and safety of the treatment.

The experimental results are shown in Figure 10. As shown in Figure 10, the symptoms of vitiligo can be significantly improved after treatment with the simvastatin skin ointment.

Comparative Example 1 Simvastatin liposome preparation for external use

The simvastatin liposome preparation for external use is prepared using a thin film dispersion method, which involves dissolving simvastatin in an organic solvent, evaporating the solvent, and leaving a thin film of the drug. This film is then added to an appropriate amount of water and ultrasonically or stirred to form liposomes.

The prepared simvastatin liposomes were subjected to a storage stability test. Specifically, three batches of samples were placed at room temperature (25±2°C, relative humidity 65±5%) for 10 days, with properties and content as key indicators. The results are shown in Table 9:

Table 9: Simvastatin liposome stability test results

Conclusion: Calculations show that the encapsulation efficiency of simvastatin liposomes is over 80%, but the drug loading is around 3%, which falls short of therapeutic requirements. Furthermore, the preparation process involves water, and alkaline water accelerates the hydrolysis of simvastatin, making it difficult for the drug to remain in a molecular state and difficult to absorb.

Comparative Example 2 Simvastatin liposome emulsion composite external preparation

The following method was used to prepare a simvastatin liposome composite external preparation: water and a surfactant (Tween 80) were mixed at 45°C to obtain an aqueous phase; simvastatin was dissolved in a medium-chain oil at 45°C, phospholipids and cholesterol were added thereto, and the mixture was stirred to obtain an oil phase; the aqueous phase was slowly injected into the oil phase in a 60°C water bath and the mixture was stirred for 45 minutes; the mixed liquid was sheared at 10,000 rpm for 3 minutes, and then allowed to stand for defoaming after shearing; the defoamed solution was circulated once at 500 bar and 6 times at 800 bar to obtain a final emulsion.

The prepared simvastatin liposomal emulsion composite external preparation was subjected to a shelf stability test. Specifically, three batches of samples were placed at room temperature (25±2°C, relative humidity 65±5%) for 10 days. The properties and content were used as key indicators. The results are shown in Table 10:

Table 10: Stability test results of simvastatin liposome emulsion composite external preparation

Conclusion: The addition of ethanol to the liposome emulsion complex prepared with a high drug loading was attempted to improve the stability and homogeneity of the formulation. However, due to the low solubility of the drug in oil, the excessive amount of oil used resulted in an extremely thick, gel-like formulation, instability, and precipitation, resulting in poor formulation efficacy and difficulty in drug development.

Comparative Example 3 Phospholipid-modified simvastatin nanocrystal external preparation

The following method was used to prepare a phospholipid-modified simvastatin nanocrystal topical preparation: the simvastatin raw material was subjected to air flow milling at a pressure combination of Venturi pressure: annular pressure = 3:2, ultimately obtaining a simvastatin raw material powder with a smaller particle size; HPMC-E5 was dissolved in boiling water, cooled until clear, and then SDS was added and mixed evenly; SV was added at 40°C and stirred for 40 minutes to obtain a coarse suspension, which was then milled at a speed of 2500 r/min for 15 minutes to obtain an SV nanocrystal suspension; the prescribed amount of phospholipid and cholesterol were ultrasonically dissolved in an appropriate amount of chloroform, and the organic solvent was rotary evaporated to form a uniform film at the bottom of a pear-shaped flask; the SV nanocrystal suspension was added to the pear-shaped flask, the film was ultrasonically dissolved, and then hydrated at 40°C for 45 minutes; the hydrated solution was milled at a speed of 2500 r/min for 10 minutes to obtain a phospholipid-modified SV nanocrystal solution.

The prepared phospholipid-modified simvastatin nanocrystal topical preparation was subjected to a shelf stability test. Specifically, three batches of samples were placed at room temperature (25±2°C, relative humidity 65±5%) for 10 days. Properties and content were used as key indicators. The results are shown in Table 11:

Table 11: Stability test results of phospholipid-modified simvastatin nanocrystals for external use

In addition, the prepared samples were also used as a standard to observe the drug stability, and the results are shown in Figure 11. In Figure 11, 1 represents simvastatin, 2 represents simvastatin acid, 3 represents nanocrystals (stored at room temperature for 5 days), and 4 represents phospholipid-modified nanocrystals (stored at room temperature for 5 days).

According to thin layer chromatography:

Developing solvent: n-hexane: ethyl acetate: diethyl ether: glacial acetic acid = 10:8:2:1, volume ratio;

Thin layer board: GF254 silicone prefabricated board

Color developer: iodine vapor color development

Control solution: Dissolve 0.05 g of simvastatin in 5 ml of methanol and shake well.

Test solution: Take 1 ml of simvastatin nanocrystals and dilute it to 5 ml with methanol; take 1.5 ml of phospholipid-modified simvastatin nanocrystals and dilute it to 5 ml with methanol.

Simvastatin acid solution: Take 0.02g of simvastatin and place it in a 50ml volumetric flask. Add 5ml of a mixed solution of 0.2mol/L sodium hydroxide solution and acetonitrile (1:1) and shake to dissolve it. Let it stand for 5 minutes. After neutralization with dilute hydrochloric acid, add methanol to dilute to the scale to obtain a simvastatin acid solution containing open-ring degradation products.

Test method:

Take 10 μL each of the control solution, test solution and simvastatin acid solution, spot them on the thin layer plate respectively, develop them for 10 cm with a developing agent (about 10 ml), dry them, and develop them with iodine vapor. If spots appear in the test solution, they should be consistent with the main spots in the control solution, and no other spots should be present.

Conclusion: Thin layer chromatography was used to detect whether the final preparation was hydrolyzed. The results showed that the simvastatin in the prepared simvastatin nanocrystals and phospholipid-modified simvastatin nanocrystal gel had been hydrolyzed and lost its efficacy. At the same time, the storage stability test showed that the drug content had decreased significantly.

The above description is only an embodiment of the present disclosure and does not limit the present disclosure in any form or substance. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present disclosure, and these improvements and supplements should also be regarded as the scope of protection of the present disclosure. Any equivalent changes, modifications and evolutions made by technicians familiar with this profession without departing from the spirit and scope of the present invention by using the technical content disclosed above are all equivalent embodiments of the present disclosure; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present disclosure are still within the scope of the technical solution of the present disclosure.

Claims (10)

A high-drug-loaded molecular simvastatin skin ointment comprises simvastatin and a pharmaceutically acceptable non-aqueous semisolid material. The ointment according to claim 1, comprising a basic matrix composition and simvastatin; the basic matrix composition comprises a basic matrix, a solubilizer, a consistency regulator and a stabilizer. The ointment according to claim 2, wherein the basic matrix comprises one or more of white petrolatum, yellow petrolatum, solid paraffin, vegetable oil, stearic acid and beeswax. The ointment according to claim 2 or 3, wherein the solubilizer comprises one or more of Tween-80, Span-80 and phospholipids. The ointment according to claim 2 or 3, wherein the consistency regulator comprises one or more of anhydrous lanolin and liquid paraffin. The ointment according to claim 2 or 3, wherein the stabilizer comprises one or more of glyceryl monostearate, cholesterol and caprylic/capric triglyceride. The ointment according to any one of claims 2 to 6, characterized in that the weight proportion of simvastatin in the ointment is 0.5-5 parts, the solubilizer is 5-30 parts, the stabilizer is 0.5-5 parts, the consistency regulator is 5-20 parts, and the basic matrix is 40-89 parts. The method for preparing the simvastatin skin ointment according to any one of claims 1 to 7, comprising: The basic matrix composition is mixed, heated to 60-90° C. to melt, cooled to 30-50° C. after melting, simvastatin is added to the basic matrix composition, stirred evenly, and cooled to room temperature to obtain simvastatin ointment. Use of the skin ointment according to any one of claims 1 to 7 or the ointment obtained by the preparation method according to claim 8 for treating autosomal recessive ichthyosis. Use of the skin ointment according to any one of claims 1 to 7 or the ointment obtained by the preparation method according to claim 8 for treating vitiligo.