Crisaborole Pickering emulsion as well as preparation method and application thereof

Abstract

The invention discloses a crisaborole Pickering emulsion as well as a preparation method and application of the crisaborole Pickering emulsion. The crisaborole Pickering emulsion prepared by the invention solves the problem of poor water solubility of crisaborole, improves the drug solubility, and has the characteristics of small particle size, high centrifugal stability, good long-term stability and the like. Compared with the traditional nanoemulsion, the Pickering emulsion disclosed by the invention shows certain advantages in the aspects of biocompatibility, cumulative transdermal amount and cutin retention, improves the curative effect of a local preparation and reduces skin toxicity, and can be used as an ideal local preparation for treating atopic dermatitis and other diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a crisaborole Pickering emulsion and a preparation method and application thereof. Background Art

[0002] Atopic dermatitis (AD) is a chronic, relapsing, inflammatory skin disease characterized by recurrent, chronic eczematous rashes, accompanied by significant skin dryness, itching, and pain. In severe cases, it can cause complications such as metabolic disorders and cardiovascular diseases, which have serious physical and psychological effects on patients. Furthermore, the high prices of marketed preparations also place a considerable financial burden on patients.

[0003] Phosphodiesterase 4 (PDE4) is a key member of the phosphodiesterase family. Its physiological function is to specifically hydrolyze cyclic adenosine monophosphate (cAMP). PDE4 inhibition leads to intracellular accumulation of cAMP, which acts as a second messenger to activate PKA, cyclic nucleotide-gated ion channels, and Epac1 / 2. These channels are involved in regulating physiological processes such as the synthesis of pro- and anti-inflammatory cytokines, T cell activation, neutrophil degranulation, antigen presentation, and epithelial integrity. A growing number of studies have shown that PDE4 expression is higher in patients with AD than in healthy individuals. Inhibiting excessive PDE4 expression can modulate the activity of immune cells and inhibit the production and release of multiple inflammatory cytokines in the skin of AD patients, potentially achieving the goal of treating AD. Crisaborole is a small molecule PDE4 inhibitor with a molecular weight of 251.05. It is poorly water-soluble, unstable in water, and easily hydrolyzed. It is also unstable under strong light irradiation.

[0004] The currently marketed crisaborole preparation is crisaborole ointment ( Specification: 2%), is an ointment based on white vaseline. The white vaseline ratio in the prescription is over 75%, which effectively prevents the drug from decomposing in water and ensures the stability of the drug. At the same time, white vaseline has good skin moisturizing properties, improving symptoms such as dry skin and pain in AD patients. However, it has a strong greasy and sticky feeling when applied, and the ointment adheres to the skin surface and is difficult to remove, which is not conducive to patient compliance with the medication. At the same time, After the ointment is opened, changes in external factors such as temperature and humidity may cause physical stability problems such as exudate and crystallization. The large amount of base materials such as white vaseline and paraffin may also cause The skin permeability is poor, so more ointment needs to be applied at one time. 2% ointment needs to be applied 5-10g at a time, twice a day, and one tube is used up in 2-4 days. The specification is 30g and the price is 150-200 yuan per tube. Ointment is a heavy economic burden for AD patients who need long-term medication. Ointments made directly from prototype drugs also have certain skin irritation problems. Direct contact of drugs with damaged skin may cause stinging. During the Phase III clinical trial of the ointment, approximately 5% of patients experienced adverse reactions such as stinging at the application site, and approximately 2% of patients were unable to complete the trial due to adverse reactions such as skin irritation.

[0005] An emulsion is a heterogeneous liquid dispersion system formed by mixing two immiscible liquids, with one phase of liquid dispersed in the other phase in the form of droplets. Emulsions can significantly improve the absorption and bioavailability of highly lipid-soluble drugs. Since crisaborole has good lipid solubility, it can be dissolved in an oil phase to prepare an emulsion. Emulsions can also significantly reduce the surface tension of the oil / water interface and exhibit excellent wettability, permeability, and spreadability, offering promising applications in cosmetics, pharmaceuticals, and other fields. Due to their small particle size and large surface area, they can successfully penetrate the stratum corneum and reach the dermis, improving transdermal efficiency while increasing drug concentration and retention time within the skin. This holds great potential for application in topical drug delivery.

[0006] Pickering emulsion is a new type of emulsion system in which solid particles replace surfactants. Unlike traditional nanoemulsions, the type of traditional emulsion is determined by the properties of the surfactant and the ratio of the oil and water phases, while the type of Pickering emulsion is determined by the wettability of the solid particles. Because it uses solid particles instead of surfactants, it not only has good biocompatibility, but also avoids the irritation caused by surfactants, and has higher safety and skin tolerance. At the same time, solid particles act as emulsifiers, making the Pickering emulsion have higher anti-agglomeration ability and show better stability. Many poorly soluble drugs also have problems such as poor stability in water. By preparing O / W Pickering emulsions, the active ingredients can be embedded in the oil phase, which not only improves water solubility but also enhances stability.

[0007] In recent years, a new type of drug nanocrystal self-stabilized Pickering emulsion (DNSPE) has garnered increasing attention. DNSPE, a novel Pickering emulsion, utilizes nanocrystals of poorly soluble drugs as stabilizers. It contains no excipients, such as surfactants or foreign solid particles, eliminating the potential safety risks associated with these excipients. Besides dissolving in the oil droplets, the poorly soluble drugs also adsorb onto the droplet surface as nanocrystals, increasing drug loading. These advantages make DNSPE promising for application in drug delivery systems.

[0008] Therefore, the present invention designs to prepare crisaborole into an O / W Pickering emulsion and verifies its potential in the topical treatment of atopic dermatitis. Summary of the Invention

[0009] One of the purposes of the present invention is to provide a crisoborole Pickering emulsion, which uses crisoborole nanocrystals or polymer nanoparticles as an emulsifier.

[0010] Furthermore, the crisoborole Pickering emulsion uses crisoborole nanocrystals as an emulsifier, and its preparation method includes the following steps:

[0011] Step 1, dissolving crisaborole in an organic solvent to obtain a drug-containing organic phase;

[0012] Step 2, dissolving the stabilizer in water to obtain an aqueous phase;

[0013] Step 3, adding the drug-containing organic phase of step 1 to the aqueous phase of step 2 under stirring, stirring and mixing, and then performing probe ultrasonication to obtain a suspension of creborone nanocrystals;

[0014] Step 4: adding the oil phase to the crisaborole nanocrystal suspension obtained in step 3, obtaining a coarse emulsion by high-speed shearing, and homogenizing the coarse emulsion by microfluidization to obtain the Pickering emulsion of crisaborole.

[0015] Furthermore, the stabilizer is selected from polyvinyl alcohol, poloxamer 188 or poloxamer 407, preferably polyvinyl alcohol; the oil phase is selected from medium chain triglycerides, castor oil, soybean oil or coconut oil, preferably medium chain triglycerides.

[0016] Furthermore, in step 1, the concentration of crisaborole is 2.5-37.5 mg / mL, preferably 2.7-25 mg / mL; in step 2, the concentration of the stabilizer is 0.5-2.5%, preferably 1-2%; in step 3, the volume ratio of the drug-containing organic phase to the aqueous phase is 1:5-1:15, preferably 1:10; and in step 4, the volume of the oil phase is 10-50% of the crude emulsion, preferably 30%.

[0017] Furthermore, the crisaborole Pickering emulsion uses polymer nanoparticles as an emulsifier, and its preparation method includes the following steps:

[0018] Step 1, dissolving the polymer in an organic solvent to obtain an organic phase;

[0019] Step 2, dissolving the stabilizer in water to obtain an aqueous phase;

[0020] Step 3, adding the organic phase from step 1 to the aqueous phase from step 2 under stirring, stirring and mixing, and then rotary evaporating to obtain a nanoparticle colloidal solution;

[0021] Step 4: dissolving crisaborole in the oil phase to obtain a drug-containing oil phase, adding the drug-containing oil phase to the nanoparticle colloidal solution obtained in step 3, obtaining a coarse emulsion by high-speed shearing, and homogenizing the coarse emulsion by microfluidization to obtain the Pickering emulsion of crisaborole.

[0022] Furthermore, the polymer is selected from polylactic acid-co-glycolic acid (PLGA), polylactic acid (PLA), polycaprolactone (PCL), polyglycolide (PGA) or polyhydroxybutyrate (PHB), preferably PLGA; the stabilizer is selected from polyvinyl alcohol, poloxamer 188 or poloxamer 407, preferably polyvinyl alcohol; the oil phase is selected from medium chain triglycerides, castor oil, soybean oil or coconut oil, preferably medium chain triglycerides.

[0023] Furthermore, in step 1, the concentration of the polymer is 2.5-37.5 mg / mL, preferably 2.7-25 mg / mL; in step 2, the concentration of the stabilizer is 0.5-2.5%, preferably 1-2%; in step 3, the volume ratio of the drug-containing organic phase to the aqueous phase is 1:5-1:15, preferably 1:10; in step 4, the concentration of crisaborole is 1-2 mg / mL, and the volume of the oil phase is 10-50% of the crude emulsion, preferably 30%.

[0024] Furthermore, in step 4, the high-speed shearing conditions are 2000-20000 rpm, 2-10 minutes, and the microfluidization homogenization conditions are 5000-25000 psi, 1-10 times.

[0025] A second object of the present invention is to provide the use of the above-mentioned crisaborole Pickering emulsion in the preparation of a drug for treating atopic dermatitis.

[0026] Furthermore, the therapeutic drug is an external preparation.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The crisaborole Pickering emulsion provided by the present invention, as a nanoemulsion, solves the problem of poor water solubility of crisaborole and improves the solubility of the drug. Due to its small particle size and large surface area, it can smoothly pass through the stratum corneum to the dermis, thereby improving the transdermal efficiency of the drug.

[0029] 2. Pickering emulsion has its unique advantages. It can greatly reduce the amount of traditional emulsifiers used and save costs. The toxic effect of solid particles on the human body is far less than that of surfactants. It is environmentally friendly and has strong interfacial stability.

[0030] 3. Drug nanocrystal self-stabilized Pickering emulsion further eliminates the safety risks brought by excipients and significantly increases the drug loading capacity of the pickering emulsion. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The particle size and potential results of the CRB nanocrystal suspension prepared with different stabilizer dosages in Example 1.

[0032] Figure 2 The particle size and potential results of CRB-DNSPE prepared with different stabilizer dosages in Example 1.

[0033] Figure 3 The particle size and potential results of the CRB nanocrystal suspension prepared with different types of stabilizers in Example 1.

[0034] Figure 4 The particle size and potential results of CRB-DNSPE prepared with different types of stabilizers in Example 1.

[0035] Figure 5 The particle size and potential results of the CRB nanocrystal suspension prepared at different particle concentrations in Example 1.

[0036] Figure 6 The particle size and potential results of CBR-DNSPE prepared with different particle concentrations in Example 1.

[0037] Figure 7 The particle size and potential results of the CRB nanocrystal suspension prepared at different ultrasonic powers in Example 1.

[0038] Figure 8 The particle size and potential results of CRB-DNSPE prepared at different ultrasonic powers in Example 1.

[0039] Figure 9 The particle size and potential results of CRB-DNSPE prepared at different homogenization pressures in Example 1.

[0040] Figure 10 The particle size and potential of CRB-DNSPE prepared with different homogenization times in Example 1.

[0041] Figure 11 This is the electron microscope image of CRB-DNSPE in test example 1.

[0042] Figure 12 The in vitro release results of the three preparations in Test Example 1 are shown.

[0043] Figure 13 The in vitro permeation results of the three preparations in Test Example 1 are shown.

[0044] Figure 14 This is the therapeutic effect of the preparation in Test Example 2 on atopic dermatitis. DETAILED DESCRIPTION

[0045] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0047] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0048] Example 1

[0049] A drug nanocrystal self-stabilized Pickering emulsion (CRB-DNSPE) with crisaborole as a stabilizer is prepared as follows:

[0050] 1. Dissolve crisaborole in dimethyl sulfoxide to obtain a drug-containing organic phase;

[0051] 2. Dissolve the stabilizer in an appropriate amount of purified water to obtain an aqueous phase;

[0052] 3. The drug-containing organic phase was added dropwise to the aqueous phase under magnetic stirring, and the generated nanocrystals were further crushed by probe ultrasound to obtain a nanocrystal suspension, which served as the aqueous phase of CRB-DNSPE.

[0053] 4. Blank medium-chain triglyceride was added as the oil phase to the aqueous phase of the emulsion, and a crude emulsion was prepared by high-speed shearing. The crude emulsion was homogenized by microfluidization to obtain a CRB-DNSPE emulsion.

[0054] The above prescription processes are screened below.

[0055] (1) Effect of stabilizer content on the preparation of CRB-DNSPE

[0056] Due to polyvinyl alcohol (PVA1788) and poloxamer 188 ( HS188) and Poloxamer 407 (Pluronic F-127) have similar stabilizing effects. Therefore, CRB-DNSPE was first prepared using PVA as a stabilizer to investigate the effect of the stabilizer content on CRB-DNSPE. The specific steps are as follows:

[0057] 1. Accurately weigh 10.8 mg of crisaborole and fully dissolve it in 0.5 mL of dimethyl sulfoxide to obtain the drug-containing organic phase;

[0058] 2. According to the prescription shown in Table 1, weigh the stabilizer and dissolve it in purified water to obtain an aqueous phase with the specified content of stabilizer;

[0059] 3. Under magnetic stirring, the drug-containing organic phase was added dropwise to 14 mL of the aqueous phase using a syringe (No. 5 needle) at a stirring speed of 800 rpm. After stirring for 10 min, the generated nanocrystals were further crushed using probe ultrasound to obtain a suspension of cristalin nanocrystals.

[0060] The particle size, PDI and Zeta potential of the samples were measured by nanoparticle size and zeta potential analyzer, and the measurements were repeated three times. The results are shown in Table 1 and Figure 1 shown.

[0061] Table 1. Particle size and potential statistics of CRB nanocrystal suspensions prepared with different stabilizer dosages ( n=3)

[0062]

[0063] The percentage in the table indicates the amount of stabilizer in 100 mL of aqueous solution (g / mL).

[0064] 4. The prepared nanocrystal suspensions of each batch were used as the aqueous phase, medium-chain triglycerides were used as the oil phase, and the oil phase content was 30%. 6 mL of blank medium-chain triglycerides were added to the aqueous phase of the emulsion as the oil phase. A crude emulsion was prepared by high-speed shearing. The crude emulsion was homogenized by microfluidization to prepare the corresponding CRB-DNSPE emulsions, and their particle sizes and Zeta potentials were measured. The results are shown in Tables 2 and Figure 2 shown.

[0065] Table 2. Particle size and potential statistics of CRB-DNSPE prepared with different stabilizer dosages ( n=3)

[0066]

[0067] The results in Tables 1 and 2 indicate that at 1% and 1.5% PVA solution concentrations, both the cristalborole nanocrystal suspension and CRB-DNSPE exhibited excellent stability, with smaller particle size and PDI, and larger zeta potential. At lower PVA concentrations (0.5%), the drug crystals formed in the system were unable to fully stabilize, resulting in an increase in particle size and the appearance of large particles exceeding 10,000 nm. Furthermore, as the PVA concentration increased, the system viscosity increased, potentially affecting the energy transfer during probe sonication and the complete dispersion of the drug crystals, leading to uneven drug concentration distribution within the system and, in turn, increased particle size and PDI. During the preparation of Pickering emulsions, increasing PVA concentrations generated a significant amount of foam during high-speed shearing, hindering shearing and mixing of the emulsion. Excessive foaming during microfluidization can also lead to inadequate homogenization, resulting in larger Pickering emulsion particle size. Therefore, based on the above evaluation criteria, a PVA concentration of 1.5% was selected as the stabilizer concentration in the aqueous phase of the formulation.

[0068] (2) Effect of stabilizer type on the preparation of CRB-DNSPE

[0069] Next, we investigate the effect of stabilizer type on the preparation of CRB-DNSPE. The specific steps are as follows:

[0070] 1. Accurately weigh 10.8 mg of crisaborole and fully dissolve it in 0.5 mL of dimethyl sulfoxide to obtain the drug-containing organic phase;

[0071] 2. According to the prescription shown in Table 3, weigh each stabilizer (mass concentration is 1.5%) and dissolve it in purified water to obtain an aqueous phase containing the specified type of stabilizer;

[0072] 3. Under magnetic stirring, the drug-containing organic phase was added dropwise to 14 mL of the aqueous phase using a syringe (No. 5 needle) at a stirring speed of 800 rpm. After stirring for 10 min, the generated nanocrystals were further crushed using probe ultrasound to obtain a suspension of cristalin nanocrystals.

[0073] The particle size, PDI and zeta potential of the creborone nanocrystal suspension were measured by nanoparticle size and zeta potential analyzer, and the measurements were repeated three times. The results are shown in Table 3 and Figure 3 shown.

[0074] Table 3. Particle size and potential statistics of CRB nanocrystal suspensions prepared with different stabilizer types n=3)

[0075]

[0076] 4. The prepared nanocrystal suspensions of each batch were used as the aqueous phase, medium-chain triglycerides were used as the oil phase, and the oil phase content was 30%. Blank medium-chain triglycerides were used as the oil phase and added to the aqueous phase of the emulsion. A crude emulsion was prepared by high-speed shearing. The crude emulsion was homogenized by microfluidization to prepare the corresponding CRB-DNSPE emulsions, and their particle sizes and Zeta potentials were measured. The results are shown in Tables 4 and Figure 4 shown.

[0077] Table 4. Particle size and potential statistics of CRB-DNSPE prepared with different stabilizer types ( n=3)

[0078]

[0079] The results in Tables 3 and 4 show that, compared to P188 and P407, the cresborole nanocrystal suspensions and CRB-DNSPE prepared with PVA as the stabilizer exhibited superior stability, smaller particle size and PDI, and higher zeta potential. However, when stabilized with P188 and P407, the cresborole nanocrystals were not effectively stabilized, exhibiting lower zeta potentials. The nanocrystals tended to aggregate into larger particles, hindering the preparation of emulsions. Therefore, based on the above evaluation criteria, PVA was selected as the stabilizer for cresborole nanocrystals.

[0080] (III) Effect of particle concentration on the preparation of CRB-DNSPE

[0081] 1. Accurately weigh crisaborole according to the prescription shown in Table 5 and fully dissolve it in 0.5 mL of dimethyl sulfoxide to obtain a drug-containing organic phase;

[0082] 2. Weigh 1.5% PVA by mass and dissolve it in 14 mL of purified water to obtain the aqueous phase of CRB-DNSPE;

[0083] 3. Under magnetic stirring, the drug-containing organic phase was added dropwise to 14 mL of the aqueous phase using a syringe (No. 5 needle) at a stirring speed of 800 rpm. After stirring for 10 min, the generated nanocrystals were further crushed using a probe ultrasound to obtain a criborol nanocrystal suspension.

[0084] The particle size, PDI and Zeta potential of the samples were measured by nanoparticle size and zeta potential analyzer, and the measurements were repeated three times. The test results of each group of nanocrystal suspension are shown in Table 5 and Figure 5 shown.

[0085] Table 5. Particle size and potential statistics of CRB nanocrystal suspensions prepared at different particle concentrations ( n=3)

[0086]

[0087] 4. The prepared nanocrystal suspensions of each batch were used as the aqueous phase, medium-chain triglycerides were used as the oil phase, and the oil phase content was 30%. Blank medium-chain triglycerides were used as the oil phase and added to the aqueous phase of the emulsion. A crude emulsion was prepared by high-speed shearing. The crude emulsion was homogenized by microfluidization to prepare the corresponding CRB-DNSPE emulsions, and their particle sizes and zeta potentials were measured. The results are shown in Tables 6 and Figure 6 shown.

[0088] Table 6. CBR-DNSPE particle size and potential statistics prepared at different particle concentrations ( n=3)

[0089]

[0090] From the results in Tables 5 and 6, it can be seen that when the particle concentration increases from 10.8 mg to 100 mg, the particle size, PDI and zeta potential of CRB-DNSPE do not change significantly, indicating that increasing the particle concentration within a certain range will not affect the properties of the emulsion. When the particle concentration increases to 150 mg, the particle size and PDI of CRB-DNSPE begin to increase. This may be because the particles reach saturation adsorption at the oil-water interface, and double-layer adsorption or bridging between emulsion droplets begins to appear, causing the particle size to increase. In subsequent experiments, although the particle size increases, the long-term stability of the emulsion is not significantly affected, showing that CRB-DNSPE has advantages in terms of drug loading. Considering that this preparation is a local preparation, a smaller particle size is required. Therefore, based on the above evaluation indicators and production costs, a particle concentration of 10.8 mg was selected to prepare CRB-DNSPE.

[0091] (IV) Effect of probe ultrasonic power on the preparation of CRB-DNSPE

[0092] 1. Accurately weigh 10.8 mg of crisaborole and fully dissolve it in 0.5 mL of dimethyl sulfoxide to obtain the drug-containing organic phase;

[0093] 2. Weigh 1.5% PVA by mass and dissolve it in 14 mL of purified water to obtain the aqueous phase of CRB-DNSPE;

[0094] 3. Under magnetic stirring, the drug-containing organic phase was added dropwise to 14 mL of the aqueous phase using a syringe (No. 5 needle) at a stirring speed of 800 rpm. After stirring for 10 min, the generated nanocrystals were further crushed using probe ultrasound to obtain a criborol nanocrystal suspension. The ultrasound power is shown in Table 7.

[0095] The particle size, PDI and zeta potential of the samples were measured by nanoparticle size and zeta potential analyzer, and the measurements were repeated three times. The test results of each group of nanocrystal suspension are shown in Table 7 and Figure 7 shown.

[0096] Table 7. Particle size and potential statistics of CRB nanocrystal suspensions prepared at different ultrasonic powers ( n=3)

[0097]

[0098] 4. The prepared nanocrystal suspensions of each batch were used as the aqueous phase, medium-chain triglycerides were used as the oil phase, and the oil phase content was 30%. Blank medium-chain triglycerides were used as the oil phase and added to the aqueous phase of the emulsion. A crude emulsion was prepared by high-speed shearing. The crude emulsion was homogenized by microfluidization to prepare the corresponding CRB-DNSPE emulsions, and their particle sizes and zeta potentials were measured. The results are shown in Table 8 and Figure 8 shown.

[0099] Table 8. Particle size and potential statistics of CRB-DNSPE prepared at different ultrasonic powers ( n=3)

[0100]

[0101] Within the appropriate range, the greater the ultrasonic power of the probe, the more evenly the cristalin nanocrystals are dispersed, the smaller the crystal volume, and the relatively larger the number, which is also more conducive to stabilizing the Pickering emulsion. As can be seen from the data in Tables 7 and 8, when the ultrasonic power is 800W, the particle size and PDI of the nanocrystal suspension and Pickering emulsion are both smaller, while the zeta potential is the highest. The other two groups can also form uniform and stable Pickering emulsions. However, because the microfluidization homogenization parameters, a key step in determining the emulsion particle size, have not yet been screened, the emulsion particle size has not yet reached the minimum range. Based on the above conditions, an ultrasonic power of 800W was selected as the optimal power for this preparation.

[0102] (V) Effect of homogenization pressure on the preparation of CRB-DNSPE

[0103] 1. Accurately weigh 10.8 mg of crisaborole and fully dissolve it in 0.5 mL of dimethyl sulfoxide to obtain the drug-containing organic phase;

[0104] 2. Weigh 1.5% PVA by mass and dissolve it in 14 mL of purified water to obtain the aqueous phase of CRB-DNSPE;

[0105] 3. Under magnetic stirring, the drug-containing organic phase was added dropwise to 14 mL of the aqueous phase using a syringe (No. 5 needle) at a stirring speed of 800 rpm. After stirring for 10 min, an 800 W probe ultrasound was used to further crush the generated nanocrystals to obtain a suspension of cristalin nanocrystals.

[0106] 4. The prepared nanocrystal suspensions of each batch were used as the aqueous phase, medium-chain triglycerides were used as the oil phase, and the oil phase content was 30%. Blank medium-chain triglycerides were used as the oil phase and added to the aqueous phase of the emulsion. A crude emulsion was prepared by high-speed shearing. The crude emulsion was homogenized by microfluidization. The homogenization pressure is shown in Table 9. The corresponding CRB-DNSPE emulsions were prepared respectively, and their particle size and zeta potential were measured. The results are shown in Table 9 and Figure 9 shown.

[0107] Table 9. CRB-DNSPE particle size and potential statistics prepared at different homogenization pressures ( n=3)

[0108]

[0109] The results in Table 9 show that when the homogenization pressure is between 10,000 and 25,000 msi, there is no significant difference in the particle size, PDI, and zeta potential of the prepared CRB-DNSPE. Therefore, considering the working pressure range and service life of the microfluidizer, the pressure for preparing the emulsion was determined to be 15,000 msi.

[0110] (6) Effect of homogenization times on the preparation of CRB-DNSPE

[0111] 1. Accurately weigh 10.8 mg of crisaborole and fully dissolve it in 0.5 mL of dimethyl sulfoxide to obtain the drug-containing organic phase;

[0112] 2. Weigh 1.5% PVA by mass and dissolve it in 14 mL of purified water to obtain the aqueous phase of CRB-DNSPE;

[0113] 3. Under magnetic stirring, the drug-containing organic phase was added dropwise to 14 mL of the aqueous phase using a syringe (No. 5 needle) at a stirring speed of 800 rpm. After stirring for 10 min, an 800 W probe ultrasound was used to further crush the generated nanocrystals to obtain a suspension of cristalin nanocrystals.

[0114] 4. The prepared nanocrystal suspensions of each batch were used as the aqueous phase, medium-chain triglycerides were used as the oil phase, and the oil phase content was 30%. Blank medium-chain triglycerides were used as the oil phase and added to the aqueous phase of the emulsion. A crude emulsion was prepared by high-speed shearing. The crude emulsion was homogenized by microfluidization at a homogenization pressure of 15000 msi. The homogenization times are shown in Table 10. The corresponding CRB-DNSPE emulsions were prepared respectively, and their particle size and zeta potential were measured. The results are shown in Table 10 and Figure 10 shown.

[0115] Table 10. Particle size and potential statistics of CRB-DNSPE prepared at different homogenization times n=3)

[0116]

[0117] The results in Table 10 show that when the homogenization pressure is between 10,000 and 25,000 msi, there is no significant difference in the particle size, PDI, and zeta potential of the prepared CRB-DNSPE. Therefore, considering the working pressure range and service life of the microfluidizer, the pressure for preparing the emulsion was determined to be 15,000 msi.

[0118] Example 2

[0119] A crisaborole-loaded Pickering emulsion (CRB-PE) using PLGA as an emulsifier is prepared as follows:

[0120] 1. Accurately weigh 10.8 mg of PLGA (PLGA75 / 25COOH, 1.8W) and dissolve it in 0.5 mL of dimethyl sulfoxide to obtain the organic phase;

[0121] 2. Accurately weigh 1.5% PVA and dissolve it in an appropriate amount of purified water to obtain an aqueous phase;

[0122] 3. Under magnetic stirring, add the organic phase dropwise to 14 mL of aqueous phase using a syringe (No. 5 needle). Continue stirring for 10 min after the addition is complete, then perform vacuum rotary evaporation to obtain a nanoparticle colloidal solution, which serves as the aqueous phase of CRB-PE.

[0123] 4. Accurately weigh 10.8 mg of crisaborole and dissolve it in 6 mL of medium-chain triglyceride as the drug-containing oil phase. Add it to the aqueous phase and prepare a crude emulsion by high-speed shearing at 10,000 rpm. The crude emulsion is homogenized by microfluidization at 15,000 msi for 8 to 10 times to obtain a CRB-PE emulsion.

[0124] Example 3

[0125] Using stearic acid polyether-21 as an emulsifier, crisaborole reference nanoemulsion (CRB-NE) was prepared as follows:

[0126] 1. Accurately weigh 10.8 mg of polystearate-21 and 10.8 mg of crisaborole and dissolve them in 6 mL of medium-chain triglycerides to obtain a drug-containing organic phase;

[0127] 2. Accurately weigh 1.5% PVA and dissolve it in an appropriate amount of purified water to obtain an aqueous phase;

[0128] 3. The drug-containing oil phase was added dropwise to the aqueous phase, and a crude emulsion was prepared by high-speed shearing at 10,000 rpm. The crude emulsion was homogenized by microfluidization at 15,000 msi for 8 to 10 times to obtain CRB-NE emulsion.

[0129] The particle size, PDI, and Zeta potential of the sample were measured using a nanoparticle size and zeta potential analyzer in triplicate. The particle size was 330-350 nm, the PDI was <0.3, and the Zeta potential was -17--19 mV.

[0130] Test Example 1

[0131] The following quality evaluation was performed on the crisaborole nanocrystal self-stabilized Pickering emulsion (CRB-DNSPE) and the PLGA-stabilized crisaborole Pickering emulsion (CRB-PE) prepared according to the optimal recipe of Example 1 and Example 2.

[0132] (1) Particle size, Zeta potential and drug loading

[0133] The final prepared CRB-DNSPE had a particle size of 320-330 nm, PDI < 0.3, a narrow particle size distribution, a zeta potential of -17--19 mV, and a drug loading of 0.5%.

[0134] The final prepared CRB-PE particle size is 320-330 nm, PDI < 0.3, narrow particle size distribution, zeta potential is -17--19 mV, and drug loading is 0.05%.

[0135] (2) Microscopic morphological observation

[0136] The micromorphology of CRB Pickering milk was observed using a transmission electron microscope. Figure 11 shown.

[0137] From the above results, it can be seen that the appearance of the two types of crisaborole Pickering emulsions is nearly spherical, with a size of about 300-350 nm, which is consistent with the particle size results measured by the nanoparticle size analyzer.

[0138] (III) In vitro release

[0139] The crisaborole nanoemulsion in Example 3 and the commercially available crisaborole The ointment was used as the reference preparation (except for the different types of emulsifiers, the three emulsions had the same formulation, particle size, and zeta potential). The Franz diffusion cell was used for the experiment. The organic filter membrane was selected and cut into 2.5×2.5 cm 2Circular, place the filter membrane between the supply pool and the receiving pool and fix it with a clamp. Accurately pipette 200μL of three types of crisaborole emulsions (CRB-NE, CRB-PE, CRB-DNSPE) and evenly apply them on the surface of the filter membrane. According to the drug loading, weigh an equal amount of control ointment and apply it on the surface of the filter membrane. Accurately pipette 4mL of 1% SDS PBS (pH 7.4) solution into the receiving pool (it has been confirmed in the pre-formulation study that the receiving solution can meet the sink conditions), keep the temperature at 32°C, stir at 600rpm / min, and take 1mL of samples at 0, 1, 2, 4, 6, 8, 12, and 24h (immediately add the same volume of fresh receiving solution). After the sample is filtered through a 0.22μm organic microporous filter membrane, the filtrate is subjected to HPLC analysis to determine the content. The cumulative permeation amount (Q) of the drug is calculated according to the following formula:

[0140]

[0141] Where, Q: Cumulative drug permeation per unit area at the nth sampling (μg / cm 2 ); V: receiving cell volume (mL); C n : concentration at the nth sampling point (μg / mL); V i : the volume of the i-th sampling (mL); C i : drug concentration of the i-th sampling (μg / mL); A: diffusion area (cm 2 ).

[0142] The in vitro release results of the three preparations are as follows Figure 12 shown.

[0143] (IV) In vitro transdermal ability

[0144] The crisaborole nanoemulsion in Example 3 and the commercially available crisaborole The ointment was used as the reference preparation (except for the different types of emulsifiers, the three emulsions had the same formulation, particle size, and zeta potential). Franz diffusion cells were used to conduct the experiment. The skin on the back of the pig's ear was cut with surgical scissors to obtain a 2.5×2.5 cm 2Place the skin with the stratum corneum facing up between the supply tank and the receiving tank, cut off the excess skin and fix it with a clip. Accurately pipette 200μL of three crisaborole emulsions (CRB-NE, CRB-PE, CRB-DNSPE) and evenly apply it on the stratum corneum of the skin. According to the drug loading, weigh an equal amount of control ointment and apply it on the surface of the stratum corneum of the skin. Accurately pipette 4mL of 1% SDS PBS (pH 7.4) solution into the receiving tank (it has been confirmed in the pre-formulation study that the receiving solution can meet the sink conditions), keep the temperature at 32°C, stir at 600rpm, and take 1mL of samples at 1, 2, 4, 6, 8, 10, 12, and 24h (immediately add the same volume of fresh receiving solution). After the sample is filtered through a 0.45μm organic microporous filter membrane, the filtrate is subjected to HPLC analysis to determine the content. The cumulative permeation amount (Q) of the drug is calculated according to the following formula:

[0145]

[0146] Where, Q: Cumulative drug permeation per unit area at the nth sampling (μg / cm 2 ); V: receiving cell volume (mL); C n : concentration at the nth sampling point (μg / mL); V i : the volume of the i-th sampling (mL); C i : drug concentration of the i-th sampling (μg / mL); A: diffusion area (cm 2 ).

[0147] After percutaneous penetration, the remaining preparation was scraped off, and the skin within the effective area was cut. The skin was rinsed three times with warm saline, dried with filter paper, and the stratum corneum was peeled off with tape (20 times). The tape was ultrasonically extracted with methanol twice, 15 mL each time, for 30 minutes. The extracts were combined and the solvent was evaporated (40°C, 50 rpm). The residue was accurately dissolved in 2 mL of methanol and centrifuged. 1 mL of the supernatant was filtered through a 0.45 μm organic filter membrane. The filtrate was then analyzed by HPLC to calculate the drug content in the stratum corneum.

[0148] After peeling the stratum corneum, approximately 0.1 g of skin was accurately weighed, minced, and ground into a homogenate with 10 times its weight of methanol. Ultrasonic extraction was then performed with 5 mL of methanol. This process was repeated three times. The combined extracts were then rotary evaporated (40°C, 50 rpm). The residue was accurately dissolved in 2 mL of methanol and centrifuged. 1 mL of the supernatant was filtered through a 0.45 μm organic filter membrane. The filtrate was then analyzed by HPLC to calculate the drug content in all layers of the skin except the stratum corneum.

[0149] The in vitro permeation results of the three preparations are as follows Figure 13 shown.

[0150] It can be seen from the results that within 24 hours, the cumulative transdermal amounts of CRB-PE and CRB-DNSPE were higher than those of CRB-NE, and there was a significant difference. Further investigation of the keratin retention and intradermal retention of each group of preparations after 24 hours showed that the keratin retention and intradermal retention of CRB-PE and CRB-DNSPE were significantly higher than those of CRB-NE. This result indicates that Pickering emulsions tend to form drug reservoirs in the stratum corneum and have certain sustained-release and controlled-release effects. When comparing the transdermal amounts of the three CRB emulsions, it was found that CRB-DNSPE had the highest cumulative transdermal amount, while CRB-NE had the lowest cumulative transdermal amount. This phenomenon may be related to the adhesion energy of the skin or the fluidity of stratum corneum lipids.

[0151] Test Example 2

[0152] The therapeutic effects of CRB-PE and CRB-DNSPE on atopic dermatitis were evaluated, and CRB-NE and commercially available crisaborole were compared. The ointment was used as a reference preparation. ICR mice were selected as experimental subjects, and an acne ICR mouse model was established using 2,4-dinitrofluorobenzene (DNFB). The therapeutic effects of several crisaborole preparations were investigated and compared.

[0153] (1) Atopic dermatitis mouse modeling method

[0154] This experiment was approved by the Experimental Animal Care Ethics Committee of China Pharmaceutical University. Eighteen 8-week-old male ICR mice were selected and acclimatized for 1 week in an environment with a temperature of (22±2)℃ and a relative humidity of (55±5)%. 2,4-Dinitrofluorobenzene (DNFB) dissolved in acetone-olive oil system was used to establish the AD model: after removing the hair on the back of the mice, they were randomly divided into 6 groups (n=6): negative control group, AD positive control group, Ointment group, CRB-NE group, CRB-PE group, and CRB-DNSPE group. Except for the negative control group, all other groups were sensitized with 2% DNFB solution (50 μL / mouse) applied to the shaved area on days 0 and 3. Starting on day 6, the negative control group was sensitized with an acetone-olive oil mixture (50 μL / mouse), while the other groups were challenged with 0.5% DNFB solution (50 μL / mouse). Model establishment was considered successful when mice in the model group developed significant erythema, edema, and hyperkeratosis (recorded as treatment day 0).

[0155] (2) Photos of mice during treatment

[0156] Starting from the 0th day of treatment, photos of the modeling area on the back of each group of mice were taken every two days to observe the degree of inflammation healing.

[0157] (III) Measurement of mouse scratching behavior

[0158] The number of scratches by the mice was recorded within 30 minutes of treatment. The starting point of the behavior was defined as the hind limbs raised and touching the head, neck, or trunk, and the end point was the hind limbs touching the ground. Short pauses or repeated movements during the period were not counted.

[0159] (IV) Cure rate of skin inflammation area

[0160] ImageJ software was used to analyze the area of inflammation on the back of mice in each group. The formula for the cure rate of skin inflammation area is as follows:

[0161]

[0162] Among them, S8 is the skin inflammation area of each group of mice on the 8th day, and S0 is the skin inflammation area of each group of mice on the 0th day.

[0163] (V) Scoring of inflammation degree of atopic dermatitis in mice

[0164] A 4-point scale (0-3) was assigned based on the degree of erythema, erosion, dryness, and lichenification, and the total score was calculated based on the percentage of lesion area. Lesion photographs were taken at a fixed viewing angle every 2 days during treatment and independently scored by two researchers in a blinded manner.

[0165] The experimental results are as follows Figure 14 shown.

[0166] After the model was established on day 0, the pathological condition of the back skin of mice in the CRB emulsion group was significantly improved after 8 days of treatment. During the entire treatment process, the number of scratching and weight changes of mice in each group 30 minutes after administration were counted. On the 8th day after the end of treatment, the percentage reduction of the inflammation area on the back of mice in each group and the atopic dermatitis score of the affected skin were counted. The results showed that compared with commercially available preparations, CRB emulsion performed better in all indicators, especially in reducing the area of inflamed skin and lowering the dermatitis score, indicating the significant advantage of nanoformulations in therapeutic effects.

[0167] Furthermore, compared with CRB-NE, mice in the CRB-PE and CRB-DNSPE groups significantly reduced scratching during treatment, alleviating the pruritus associated with AD. This result suggests that the emulsion possesses unique cooling and moisturizing properties, effectively alleviating itching and pain. There were no significant differences in body weight changes among the mice in the treatment groups, demonstrating the safety of the formulation.

Claims

1. A crisaborole Pickering emulsion, characterized in that: Prepared by one of the following methods: Method (1) comprises the following steps: Step 1, dissolving crisaborole in an organic solvent to obtain a drug-containing organic phase; Step 2, dissolving the stabilizer in water to obtain an aqueous phase; Step 3, adding the drug-containing organic phase of step 1 to the aqueous phase of step 2 under stirring, stirring and mixing, and then performing probe ultrasonication to obtain a suspension of creborone nanocrystals; Step 4, adding the oil phase to the crisoborole nanocrystal suspension obtained in step 3, obtaining a coarse emulsion by high-speed shearing, and homogenizing the coarse emulsion by microfluidization to obtain the crisoborole Pickering emulsion; Method (2) comprises the following steps: Step 1, dissolving the polymer in an organic solvent to obtain an organic phase; Step 2, dissolving the stabilizer in water to obtain an aqueous phase; Step 3, adding the organic phase from step 1 to the aqueous phase from step 2 under stirring, stirring and mixing, and then rotary evaporating to obtain a nanoparticle colloidal solution; In step 4, crisaborole is dissolved in the oil phase to obtain a drug-containing oil phase, and the drug-containing oil phase is added to the nanoparticle colloidal solution obtained in step 3, and a coarse emulsion is obtained by high-speed shearing, and the coarse emulsion is homogenized by microfluidization to obtain the crisaborole Pickering emulsion.

2. The crisaborole Pickering emulsion according to claim 1, characterized in that In method (1), the stabilizer is selected from polyvinyl alcohol, poloxamer 188 or poloxamer 407; and the oil phase is selected from medium-chain triglycerides, castor oil, soybean oil or coconut oil.

3. The crisaborole Pickering emulsion according to claim 2, characterized in that In method (1), the stabilizer is polyvinyl alcohol, and the oil phase is medium-chain triglyceride.

4. The crisaborole Pickering emulsion according to claim 1, characterized in that In method (1), the concentration of crisaborole in step 1 is 2.5-37.5 mg / mL; the concentration of the stabilizer in step 2 is 0.5-2.5%; the volume ratio of the drug-containing organic phase to the aqueous phase in step 3 is 1:5-1:15; and the volume of the oil phase in step 4 is 10-50% of the crude emulsion.

5. The crisaborole Pickering emulsion according to claim 1, characterized in that In method (2), the polymer is selected from polylactic acid-co-glycolic acid, polylactic acid, polycaprolactone, polyglycolide or polyhydroxybutyrate; the stabilizer is selected from polyvinyl alcohol, poloxamer 188 or poloxamer 407; and the oil phase is selected from medium-chain triglycerides, castor oil, soybean oil or coconut oil.

6. The crisaborole Pickering emulsion according to claim 5, characterized in that In method (2), the polymer is polylactic acid-glycolic acid copolymer, the stabilizer is polyvinyl alcohol, and the oil phase is medium-chain triglyceride.

7. The crisaborole Pickering emulsion according to claim 1, wherein In method (ii), the concentration of the polymer is 2.5-37.5 mg / mL; the concentration of the stabilizer in step 2 is 0.5-2.5%; the volume ratio of the drug-containing organic phase to the aqueous phase in step 3 is 1:5-1:15; and in step 4, the concentration of crisaborole is 1-2 mg / mL, and the volume of the oil phase is 10-50% of the crude emulsion.

8. The crisaborole Pickering emulsion according to claim 1, wherein In method (1) and method (2), in step 4, the high-speed shearing conditions are 2000-20000 rpm, 2-10 minutes, and the microfluidization conditions are 5000-25000 psi, 1-10 times.

9. Use of the crisaborole Pickering emulsion according to any one of claims 1 to 8 in the preparation of a drug for treating atopic dermatitis.

10. The use according to claim 9, characterized in that The therapeutic drug is an external preparation.

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