Crisaborole nano-cream as well as preparation method and application thereof

Abstract

The invention discloses a crisaborole nano-cream and a preparation method and application thereof, and the crisaborole nano-cream comprises the following substances in parts by weight: 1-100 parts of crisaborole, 4.5-450 parts of solid lipid, 0.5-50 parts of liquid lipid, 0.5-50 parts of a main emulsifier, 0-50 parts of an auxiliary emulsifier, and 10-1000 parts of deionized water. The preparation method comprises the following steps: heating and melting crisaborole, liquid lipid and solid lipid, heating and uniformly stirring in a water bath to obtain an oil phase, uniformly dispersing an emulsifier in deionized water, and heating and uniformly stirring in the water bath to obtain a water phase; under a constant-temperature stirring condition, transferring the oil phase into a water phase, and shearing to obtain primary emulsion; ultrasonically crushing the primary emulsion at constant temperature, and cooling; and carrying out high-pressure homogenization treatment on the obtained product to obtain primary emulsion, and cooling. The invention provides an application of crisaborole nano-cream in preparation of a medicine for treating dog atopic dermatitis. The crisaborole nano-cream prepared by the preparation method disclosed by the invention is good in characterization performance and stability and relatively good in percutaneous absorption effect.

Description

Technical Field

[0001] This invention pertains to creams, their preparation methods and applications, specifically a criborone nano-cream, its preparation method and application. Background Technology

[0002] Criborone belongs to the benzoborazole class of compounds and is a small molecule compound containing a five-membered heterocyclic ring with boron atoms. In animals, criborone competitively and reversibly inhibits PDE-4 activity by mimicking cAMP phosphate through its boron atoms, reducing cAMP degradation, increasing intracellular cAMP levels, and promoting the interaction between cAMP and protein kinase A. This inhibits the activation and release of inflammatory factors, effectively suppressing inflammatory responses and showing good therapeutic effects on various inflammatory skin diseases, primarily atopic dermatitis. Studies have also shown that criborone has good safety, with no significant gastrointestinal reactions. It has a high plasma protein binding rate of 97%, reaching its maximum plasma concentration 3 hours after administration. Its metabolites are 5-(4-cyanophenoxy)-2-hydroxybenzyl alcohol and its further metabolite, 5-(4-cyanophenoxy)-2-hydroxybenzoic acid, which is metabolized through renal excretion. However, since creborol itself is insoluble in water and slightly soluble in dimethyl sulfoxide and methanol, it is necessary to find more suitable solvents to increase its solubility and transdermal absorption efficiency.

[0003] Nanostructured lipid carriers consist of bioactive and biodegradable lipids with low toxicity, offering numerous beneficial properties such as adhesion, occlusion, skin hydration, lubrication, smoothness, emollient properties, enhanced skin penetration, sustained release, improved formulation appearance, whitening effects, and protection of active ingredients from degradation. These key characteristics of nanostructured lipid carriers provide significant advantages for transdermal drug delivery.

[0004] Nanostructured lipid carriers consist of lipids (solid and liquid), emulsifiers, surface modifiers, and water. By replacing part of the solid lipid core with liquid lipids, the recrystallization ability of the solid lipids is reduced, which facilitates disordered arrangement, improves drug loading capacity and stability, and thus enhances the solubility of poorly soluble drugs and improves transdermal drug absorption. Due to their unique structure, nanostructured lipid carriers are currently commonly used to improve drug stability, prolong drug action time, target drug delivery, and enhance transdermal drug absorption.

[0005] Existing criborone cream technology suffers from the problem of balancing stability and drug loading. Systems that rely on oil-phase solubility are simple to process but have poor stability. While novel carriers such as Pickering emulsions and solid lipid nanoparticles can improve stability to some extent, they introduce new problems such as complex processes, high equipment requirements, and limited drug loading.

[0006] Therefore, the technical problem to be solved by this application is to provide a criborone nanoemulsion with strong anti-crystallization ability, high drug loading capacity, simple preparation process and easy industrialization. It constructs a nanostructured lipid carrier (NLC) by compounding solid lipids and liquid lipids, realizes stable drug loading by utilizing lattice defects, and simplifies the emulsification process by using surfactants. Summary of the Invention

[0007] Purpose of the invention: In order to overcome the shortcomings of the existing technology, the purpose of this invention is to provide a criborone nanoemulsion with high solubility, good absorption activity and good stability. Another purpose of this invention is to provide a feasible, non-toxic and safe method for preparing criborone nanoemulsion. A further purpose of this invention is to provide the role of criborone nanoemulsion in the preparation of drugs for canine atopic dermatitis.

[0008] Technical solution: The present invention provides a criborone nano-emulsion, comprising the following components in parts by weight: criborone 1-100 parts, solid lipids 4.5-450 parts, liquid lipids 0.5-50 parts, primary emulsifier 0.5-50 parts, secondary emulsifier 0-50 parts, and deionized water 10-1000 parts.

[0009] The main active ingredient is criborone. The solid lipids act as a lipid carrier framework, increasing drug loading. The liquid lipids enhance skin permeability. The primary emulsifier provides steric stability and prevents nanoparticle aggregation, while the co-emulsifier aids emulsification and synergistically stabilizes the interface with the primary emulsifier. Deionized water serves as the dispersion medium.

[0010] Furthermore, the solid lipid is one or more of glyceryl behenate, glyceryl distearate, and glyceryl monostearate. Preferably, the solid lipid is glyceryl monostearate, which has a smaller particle size and more uniform distribution.

[0011] Furthermore, the liquid lipid is one or more of isopropyl myristate and oleic acid. Preferably, the liquid lipid is oleic acid, which has a smaller particle size and more uniform distribution.

[0012] Furthermore, the primary emulsifier is one or more of poloxamer 407, Tween 80, and Tween 85, and the secondary emulsifier is one or more of Span 80 and Span 85. Preferably, the primary emulsifier is poloxamer 407, and the secondary emulsifier is Span 85, which has smaller particle size, narrower distribution, and better stability.

[0013] Preferably, the mass ratio of solid lipid to liquid lipid is 1:7 to 10, more preferably, the mass ratio is 1:9. Preferably, the mass ratio of criborone to the sum of solid lipid and liquid lipid is 1:3 to 9, more preferably, the mass ratio is 1:5. Preferably, the mass ratio of criborone to emulsifier is 1:1 to 3, more preferably, the mass ratio is 1:1.

[0014] This invention provides a method for preparing criborone nano-emulsion, comprising the following steps:

[0015] Step 1: Take creborol, liquid lipid, and solid lipid, heat and melt them, and stir evenly in a water bath to obtain the oil phase. Take the emulsifier, disperse it evenly in deionized water, and stir evenly in a water bath to obtain the aqueous phase.

[0016] Step 2: Under constant temperature and stirring conditions, the oil phase is transferred into the aqueous phase and sheared to obtain the primary emulsion;

[0017] Step 3, constant temperature ultrasonic disruption of colostrum or step two The resulting material was subjected to high-pressure homogenization of the primary emulsion and then cooled to obtain criborone nano-emulsion.

[0018] Furthermore, in step one, the water bath heating and stirring temperature is 65~95℃, the rotation speed is 400~800 rpm, and the time is 20~30 min. The oil phase also includes a co-emulsifier. Preferably, the water bath heating and stirring temperature is 95℃, the rotation speed is 500 rpm, and the time is 20 min.

[0019] Further, in step two, the constant-temperature stirring temperature is 65~95℃, the stirring speed is 400~800 rpm, the shearing speed is 8000~15000 rpm, and the shearing time is 5~10 min. Preferably, the constant-temperature stirring temperature is 95℃, the stirring speed is 500 rpm, the shearing speed is 11000 rpm, and the shearing time is 5 min.

[0020] Furthermore, in step three, the temperature for isothermal ultrasonic fragmentation is 65~95℃, the power for ultrasonic fragmentation is 200~500W, and the time is 10~20 min. Preferably, the temperature for isothermal ultrasonic fragmentation is 95℃, the power for ultrasonic fragmentation is 300 W, and the time is 20 min.

[0021] Furthermore, in step three, the pressure of the high-pressure homogenization process is 400-700 bar, and the number of cycles is 5-10. Preferably, the pressure of the high-pressure homogenization process is 500 bar, and the number of cycles is 5.

[0022] This invention provides the application of criborone nano-emulsion in the preparation of drugs for canine atopic dermatitis.

[0023] Preparation Principle: Crizotinol is insoluble in water and slightly soluble in dimethyl sulfoxide and methanol, exhibiting poor solubility. This invention disperses drug molecules in a nanostructured lipid carrier, increasing the solubility of crizotinol while facilitating transdermal absorption, thus promoting targeted drug delivery and improving its therapeutic effect on canine atopic dermatitis. This invention uses a high-shear method to prepare the crizotinol nanoemulsion pre-emulsion, which is then further processed into a nanoparticle emulsion using ultrasonic disruption and high-pressure homogenization methods. After cooling at room temperature, the nanostructured lipid carrier is formed. Unlike existing crizotinol creams that rely on ordinary hydrogen bonds and van der Waals forces to maintain drug solubility, this invention's crizotinol nanoemulsion significantly enhances the binding strength between the drug and the carrier through a strong and specific hydrogen bond network formed between the boric acid hydroxyl groups in the crizotinol molecule and the ether oxygen atoms in the Tween 80 and poloxamer 407 polyoxyethylene chains. This results in superior physical stability and anti-crystallization ability of the cream.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following significant features:

[0025] 1. The prepared criborone nano-emulsion exhibited good performance and stability.

[0026] 2. Crizotinol nano-emulsion has good transdermal absorption effect;

[0027] 3. It is highly feasible, non-toxic and safe, does not affect the original properties of criborone, can be targeted for drug delivery, and is easy to mass-produce. The resulting criborone nano-emulsion has great application potential in the treatment of canine atopic dermatitis. Attached Figure Description

[0028] Figure 1 This is a DLS hydrodynamic size distribution diagram of the criborone nanostructured lipid carrier prepared in Example 1;

[0029] Figure 2 This is a zeta potential distribution diagram of the criborone nanostructured lipid carrier prepared in Example 1;

[0030] Figure 3 This is a DLS hydrodynamic size distribution diagram of the criborone nanostructured lipid carrier prepared in Example 2;

[0031] Figure 4 This is a zeta potential distribution diagram of the criborone nanostructured lipid carrier prepared in Example 2;

[0032] Figure 5 This is a DLS hydrodynamic size distribution diagram of the criborone nanostructured lipid carrier prepared in Example 3;

[0033] Figure 6 This is a zeta potential distribution diagram of the criborone nanostructured lipid carrier prepared in Example 3;

[0034] Figure 7 This is a DLS hydrodynamic size distribution diagram of the criborone nanostructured lipid carrier prepared in Example 4;

[0035] Figure 8 This is a zeta potential distribution diagram of the criborone nanostructured lipid carrier prepared in Example 4;

[0036] Figure 9 This is a DLS hydrodynamic size distribution diagram of the criborone nanostructured lipid carrier prepared in Example 5;

[0037] Figure 10 This is a zeta potential distribution diagram of the criborone nanostructured lipid carrier prepared in Example 5;

[0038] Figure 11 This is a DLS hydrodynamic size distribution diagram of the criborone nanostructured lipid carrier prepared in Example 6;

[0039] Figure 12 This is a zeta potential distribution diagram of the criborone nanostructured lipid carrier prepared in Example 6;

[0040] Figure 13 This is a transmission electron microscope image of a criborone nanostructured lipid carrier;

[0041] Figure 14 This is a DLS hydrodynamic size distribution diagram of the criborone nanostructured lipid carrier prepared in Example 7;

[0042] Figure 15 This is a zeta potential distribution diagram of the criborone nanostructured lipid carrier prepared in Example 7;

[0043] Figure 16 This is a DLS hydrodynamic size distribution diagram of the criborone nanostructured lipid carrier prepared in Example 8;

[0044] Figure 17 This is a zeta potential distribution diagram of the criborone nanostructured lipid carrier prepared in Example 8;

[0045] Figure 18 The image shows the pruritus score of dogs with hydrocortisone ointment and criborone nanostructured lipid carrier group.

[0046] Figure 19 Scoring chart of atopic dermatitis in dogs treated with hydrocortisone ointment and criborone nanostructured lipid carrier group. Detailed Implementation

[0047] Unless otherwise specified, all materials and reagents used in the following embodiments are commercially available. Experimental methods not specifically described in the embodiments are generally performed under standard conditions or as recommended by the manufacturer.

[0048] In the following embodiments, glyceryl monolaurate was purchased from Henan Zhengtong Technology Co., Ltd.; stearic acid, palmitic acid, glyceryl monostearate, glyceryl distearate, myristic acid, glyceryl tristearate, polyglycerol fatty acid ester, glyceryl behenate, Span 85, Span 80, Span 20, Tween 80, Tween 20, and isopropyl myristate were purchased from Shanghai Aladdin Biotechnology Co., Ltd.; Tween 85 was purchased from Sinopharm Chemical Reagent Co., Ltd.; oleic acid and phosphotungstic acid were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; poloxamer 188 was purchased from Shanghai Yuanhong Chemical Preparations and Excipients Co., Ltd.; and poloxamer 407 was purchased from Shandong Yousuo Chemical Co., Ltd.

[0049] The UV-Vis spectrophotometer was purchased from Shimadzu Corporation, Japan (UV-1900); the laser particle size potentiometer was purchased from Malvern, UK (zetasizer Nano ZSE); the freeze dryer was purchased from Ningbo Xinzhi Biotechnology Co., Ltd. (Scientz-18N); the Fourier transform infrared spectrometer was purchased from Nicolet, USA (IS5&N380); the X-ray diffraction spectrometer was purchased from Bruker-AXS, Germany (D8 Advance); the high shear apparatus was purchased from IKA Instruments, USA (FM200); the ultrasonic disruptor was purchased from Ningbo Xinzhi Biotechnology Co., Ltd. (JY96-II); the differential scanning calorimeter was purchased from PerkinElmer, USA (DSC 8500); and the far-infrared rapid dryer was purchased from Taizhou Chemical Reagent Co., Ltd. (WS70-1).

[0050] Example 1

[0051] A method for preparing a criborone nano-emulsion includes the following steps:

[0052] (1) Weigh 0.3 g of the primary emulsifier Tween 80 into a clean vial and add 10 mL of deionized water as the aqueous phase. Weigh 0.2 g of criborone raw material, 0.1 g of liquid lipid oleic acid, and 0.9 g of solid lipid glyceryl monostearate into another clean vial as the oil phase. Place both in a 95°C water bath and heat with magnetic stirring at 500 rpm for 20 min. Wait until both phases are dissolved and the texture is clear and transparent.

[0053] (2) Use a pipette to draw up the oil phase and inject it into the water phase under magnetic stirring. Continue stirring at a constant temperature for 20 min to ensure thorough mixing.

[0054] (3) Then continue to use a high shear apparatus at 11,000 rpm for 5 min under the previous water bath conditions to obtain colostrum.

[0055] (4) Use an ultrasonic disruptor at a power of 300 W for 20 min to disrupt the initial emulsion temperature at 95℃. After cooling, obtain creborol nano-emulsion.

[0056] The hydrodynamic size, zeta potential, and PDI of the criborone nanostructured lipid carrier were determined using a Malvern laser particle size analyzer. The instrument equilibration time was set to 120 s, the measurement temperature to 25℃, and the detection angle to 90°. Before testing, the criborone nanostructured lipid carrier stock solution was diluted with deionized water until transparent, and then 1 mL was taken and placed in the hydrodynamic size and PDI sample cells for analysis.

[0057] Testing: The hydrodynamic size of the criborone nanostructured lipid carrier obtained in this embodiment is 445.7±64.66 nm, the zeta potential is -16.8±0.513 mV, the polydispersity index is 0.664±0.153, and it remains stable without stratification after standing for 48 hours. Figure 1 As shown, the particle size distribution exhibits a bimodal pattern with one extraneous peak. The main peak is located at approximately 400 nm and has a wide distribution range, suggesting the possibility of large particle aggregation. Figure 2 As shown, the zeta potential distribution exhibits a single peak, with the main peak located at approximately -16.8 mV. The negative charge provides electrostatic repulsion, which is beneficial to the stability of the system.

[0058] Example 2

[0059] A method for preparing a criborone nano-emulsion includes the following steps:

[0060] (1) Weigh 0.15 g of the primary emulsifier Tween 80 into a clean vial and add 10 mL of deionized water as the aqueous phase. Weigh 0.2 g of criborone raw material, 0.1 g of liquid lipid oleic acid, 0.9 g of solid lipid glyceryl monostearate, and 0.15 g of co-emulsifier Span 85 into another clean vial as the oil phase. Place both in a 95°C water bath and heat with magnetic stirring at 500 rpm for 20 min. Wait until both phases are dissolved and the texture is clear and transparent.

[0061] (2) Use a pipette to draw up the oil phase and inject it into the water phase under magnetic stirring. Continue stirring at a constant temperature for 20 min to ensure thorough mixing.

[0062] (3) Then continue to use a high shear apparatus at 11,000 rpm for 5 min under the previous water bath conditions to obtain colostrum.

[0063] (4) Use an ultrasonic disruptor at a power of 300 W for 20 min to disrupt the initial emulsion temperature at 95℃. After cooling, obtain creborol nano-emulsion.

[0064] Testing: The hydrodynamic size of the creborol nanostructured lipid carrier obtained in this embodiment is 237.5±2.701 nm, the zeta potential is -20.4±0.874 mV, the polydispersity index is 0.518±0.061, and it remains stable without stratification after standing for 48 hours. Figure 3 As shown, the particle size distribution exhibits a bimodal and broad distribution, indicating that the system is not uniformly dispersed; Figure 4 As shown, the zeta potential distribution has a single peak and a large absolute value, indicating that the electrostatic repulsion is sufficient and the system has good stability.

[0065] Example 3

[0066] A method for preparing a criborone nano-emulsion includes the following steps:

[0067] (1) Weigh 0.1 g of the primary emulsifier poloxamer 407 into a clean vial and add 10 mL of deionized water as the aqueous phase. Weigh 0.2 g of criborone raw material, 0.1 g of liquid lipid oleic acid, 0.9 g of solid lipid glyceryl monostearate, and 0.1 g of co-emulsifier Span 85 into another clean vial as the oil phase. Place both in a 95°C water bath and heat with magnetic stirring at 500 rpm for 20 min. Wait until both phases are dissolved and the texture is clear and transparent.

[0068] (2) Use a pipette to draw up the oil phase and inject it into the water phase under magnetic stirring. Continue stirring at a constant temperature for 20 min to ensure thorough mixing.

[0069] (3) Then continue to use a high shear apparatus at 11,000 rpm for 5 min under the previous water bath conditions to obtain colostrum.

[0070] (4) Use an ultrasonic disruptor at a power of 300 W for 20 min to disrupt the initial emulsion temperature at 95℃. After cooling, obtain creborol nano-emulsion.

[0071] Testing: The hydrodynamic size of the creborol nanostructured lipid carrier obtained in this embodiment is 314.3±4.646 nm, the zeta potential is -20.0±0.721 mV, the polydispersity index is 0.301±0.020, and it remains stable without stratification after standing for 48 hours. Figure 5 As shown, the particle size distribution exhibits a bimodal pattern, but the impurity peaks are small and narrowly distributed, indicating that the nanoparticles are uniformly distributed; Figure 6As shown, the zeta potential distribution has a single peak and a large absolute value (-20.0 mV), indicating that the electrostatic repulsion is sufficient and the system has good stability.

[0072] Example 4

[0073] A method for preparing a criborone nano-emulsion includes the following steps:

[0074] (1) Weigh 0.1 g of the primary emulsifier poloxamer 407 into a clean vial and add 4 mL of deionized water as the aqueous phase. Weigh 0.2 g of criborone raw material, 0.1 g of liquid lipid oleic acid, 0.9 g of solid lipid glyceryl monostearate, and 0.1 g of co-emulsifier Span 85 into another clean vial as the oil phase. Place both in a 95°C water bath and heat with magnetic stirring at 500 rpm for 20 min. Wait until both phases are dissolved and the texture is clear and transparent.

[0075] (2) Use a pipette to draw up the oil phase and inject it into the water phase under magnetic stirring. Continue stirring at a constant temperature for 20 min to ensure thorough mixing.

[0076] (3) Then continue to use a high shear apparatus at 11,000 rpm for 5 min under the previous water bath conditions to obtain colostrum.

[0077] (4) Use an ultrasonic disruptor at a power of 300 W for 20 min to disrupt the initial emulsion temperature at 95℃. After cooling, obtain creborol nano-emulsion.

[0078] Testing: The hydrodynamic size of the criborone nanostructured lipid carrier obtained in this embodiment is 334.4±8.9 nm, the zeta potential is -16.8±0.4 mV, the polydispersity index is 0.175±0.172, and it remained stable without stratification after standing for 48 hours. Figure 7 As shown, the particle size distribution exhibits a single peak and a narrow distribution, indicating that the system is relatively uniformly distributed; Figure 8 As shown, the zeta potential distribution has a single peak and a large absolute value, indicating that the electrostatic repulsion is sufficient and the system has good stability.

[0079] Example 5

[0080] A method for preparing a criborone nano-emulsion includes the following steps:

[0081] (1) Weigh 0.1 g of the primary emulsifier poloxamer 407 into a clean vial and add 4 mL of deionized water as the aqueous phase. Weigh 0.2 g of criborone raw material, 0.1 g of liquid lipid oleic acid, 0.9 g of solid lipid glyceryl monostearate, and 0.1 g of co-emulsifier Span 85 into another clean vial as the oil phase. Place both in a 95°C water bath and heat with magnetic stirring at 500 rpm for 20 min. Allow both phases to dissolve until they are clear and transparent.

[0082] (2) Use a pipette to draw up the oil phase and inject it into the water phase under magnetic stirring. Continue stirring at a constant temperature for 20 min to ensure thorough mixing.

[0083] (3) Then continue to use a high shear apparatus at 11,000 rpm for 5 min under the previous water bath conditions to obtain colostrum.

[0084] (4) The product obtained in step (3) is subjected to high pressure homogenization at 500 bar using a high pressure homogenizer for 5 cycles, and then cooled to obtain criborone nano-emulsion.

[0085] Testing: In this embodiment, the hydrodynamic size of the criborone nanoemulsion was measured to be 321.7±4.5 nm, the zeta potential was -16.63±0.06 mV, and the polydispersity index was 0.282±0.056. It remained stable without stratification after standing for 48 hours. Figure 9 As shown, the particle size distribution exhibits a single peak and a narrow distribution, indicating that the nanoparticles are of uniform size; Figure 10 As shown, the zeta potential distribution exhibits a single peak, with the negative charge providing electrostatic repulsion, indicating good system stability.

[0086] Example 6

[0087] A method for preparing a criborone nano-emulsion includes the following steps:

[0088] (1) Weigh 0.1 g of the primary emulsifier poloxamer 407 into a clean vial and add 4 mL of deionized water as the aqueous phase. Weigh 0.2 g of criborone raw material, 0.1 g of liquid lipid oleic acid, 0.9 g of solid lipid glyceryl monostearate, and 0.1 g of co-emulsifier Span 85 into another clean vial as the oil phase. Place both in a 95°C water bath and heat with magnetic stirring at 500 rpm for 20 min. Allow both phases to dissolve until they are clear and transparent.

[0089] (2) Use a pipette to draw up the oil phase and inject it into the water phase under magnetic stirring. Continue stirring at a constant temperature for 20 min to ensure thorough mixing.

[0090] (3) Then continue to use a high shear apparatus at 11,000 rpm for 5 min under the previous water bath conditions to obtain colostrum.

[0091] (4) The product obtained in step (3) is subjected to high pressure homogenization at 700 bar using a high pressure homogenizer for 5 cycles, and then cooled to obtain creborol nano-emulsion.

[0092] Detection: In this embodiment, the hydrodynamic size of the criborone nanoemulsion was measured to be 274.7 ± 1.5 nm, the zeta potential was -14.3 ± 0.5 mV, and the polydispersity index was 0.206 ± 0.035. (The last sentence appears to be incomplete and possibly refers to a different example.) Figure 11 As shown, the particle size distribution exhibits a single peak and a narrow distribution, indicating that the nanoparticles are highly uniform in size; Figure 12 As shown, the zeta potential distribution exhibits a single peak (-16.6 mV), with the negative charge providing electrostatic repulsion, indicating good system stability.

[0093] After the above formulation screening, the optimal formulation was obtained with the smallest possible hydrodynamic size and polydispersity index (PDI), and the largest possible absolute zeta potential. The performance of the prepared nanostructured lipid carriers is often evaluated by hydrodynamic size, polydispersity index (PDI), and zeta potential. Typically, the particle size needs to be between 50 and 1000 nm. Particle distribution is measured by the polydispersity index; a PDI < 0.3 can be used as an indicator of uniform particle distribution, and an absolute zeta potential > 30 mV indicates strong repulsive forces between particles and relative stability in solution. Figure 11 and Figure 12 The figures show the hydrodynamic size distribution and zeta potential distribution of the nanostructured lipid carrier prepared by DLS method, respectively, for the optimal formulation (Example 6). The average hydrodynamic size of the optimal formulation is 274.7 ± 1.5 nm, and its PDI is 0.206 ± 0.035, which is unimodal and conforms to a normal distribution, indicating that its particle size distribution is relatively uniform. Its zeta potential is -14.3 ± 0.5 mV, indicating that the particles have good stability. Therefore, the creborol nanostructured lipid carrier has good stability.

[0094] To observe the morphology of the prepared criborone nanostructured lipid carrier, the criborone nanostructured lipid carrier prepared in Example 6 was diluted several times and dropped onto a copper grid with a carbon support film. The excess liquid was absorbed with filter paper, and then 2% (w / v) phosphotungstic acid solution was added for negative staining for 2 min. The excess staining solution was absorbed, and after the sample was allowed to air dry naturally, it was recorded and observed using TEM.

[0095] Transmission electron microscopy allows for direct observation of the morphology and hydrodynamic size of criborone nanostructured lipid carriers. Figure 13It was observed that the criborone nanostructured lipid carrier exhibited a spherical shape and uniform morphology under TEM, with a size of approximately 500 nm, which is not significantly different from the hydrodynamic size measured by DLS. Furthermore, the coating of criborone by the nanostructured lipid carrier was clearly visible. Therefore, it can be concluded that the criborone nanostructured lipid carrier prepared by this method has a spherical morphology, uniform size, homogeneous structure, and no aggregation.

[0096] Example 7

[0097] A method for preparing a criborone nano-emulsion includes the following steps:

[0098] (1) Weigh 0.05 g of the primary emulsifier Tween 85 into a clean vial and add 1 mL of deionized water as the aqueous phase. Weigh 0.1 g of criborone raw material, 0.05 g of liquid lipid isopropyl myristate, 0.45 g of solid lipid glyceryl behenate, and 0.05 g of co-emulsifier Span 80 into another clean vial as the oil phase. Place both in an 80°C water bath and heat, stirring magnetically at 400 rpm for 30 min. Allow both phases to dissolve until the texture is clear and transparent.

[0099] (2) Use a pipette to draw up the oil phase and inject it into the water phase under magnetic stirring. Continue stirring at a constant temperature for 20 min to ensure thorough mixing.

[0100] (3) Then continue to use a high shear apparatus at 8000 rpm for 10 min under the previous water bath conditions to obtain colostrum.

[0101] (4) The product obtained in step (3) is subjected to high pressure homogenization at 700 bar using a high pressure homogenizer for 5 cycles, and then cooled to obtain creborol nano-emulsion.

[0102] Detection: The hydrodynamic size of the criborone nanoemulsion measured in this embodiment was 377.2±116.4 nm, the zeta potential was -13.93±0.81 mV, and the polydispersity index was 0.570±0.215-0.793. For example... Figure 14 As shown, the particle size distribution exhibits a broad peak and a wide distribution range, indicating that the nanoparticle size distribution in the system is uneven, with the presence of some large particles or aggregation; for example... Figure 15 As shown, the zeta potential distribution has a single peak, and the negative charge can provide a certain electrostatic repulsion effect, but the large PDI suggests that the stability of the system needs to be optimized.

[0103] Example 8

[0104] A method for preparing a criborone nano-emulsion includes the following steps:

[0105] (1) Weigh 5 g of the primary emulsifier Tween 85 into a clean vial and add 100 mL of deionized water as the aqueous phase. Weigh 10 g of criborone raw material, 5 g of liquid lipid isopropyl myristate, 45 g of solid lipid glyceryl distearate, and 5 g of co-emulsifier Span 80 into another clean vial as the oil phase. Place both in a 65°C water bath and heat with magnetic stirring at 800 rpm for 25 min. Wait until both phases are dissolved and the texture is clear and transparent.

[0106] (2) Use a pipette to draw up the oil phase and inject it into the water phase under magnetic stirring. Continue stirring at a constant temperature for 20 min to ensure thorough mixing.

[0107] (3) Then continue to use a high shear apparatus at 15,000 rpm for 7 min under the previous water bath conditions to obtain colostrum.

[0108] (4) The product obtained in step (3) is subjected to high pressure homogenization at 700 bar using a high pressure homogenizer for 5 cycles, and then cooled to obtain creborol nano-emulsion.

[0109] Detection: The hydrodynamic size of the criborone nanoemulsion measured in this example was 392.4 ± 162.5 nm, and the zeta potential was -16.5 ± 2.5 mV. The polydispersity index was 0.455 ± 0.141. Figure 16 As shown, the particle size distribution exhibits a single peak with a moderately wide distribution range, indicating that the nanoparticle size distribution in the system is uneven; Figure 17 As shown, the zeta potential distribution has a single peak, with the main peak located at approximately -13.9 mV. The negative potential can provide a certain electrostatic repulsion effect, which helps maintain the stability of the system.

[0110] Comparative Example 1

[0111] The remaining steps of this comparative example are the same as those of Example 6, except that the co-emulsifier is replaced by Span 20 instead of Span 85.

[0112] The results showed that the obtained criborone nanoemulsion had a particle size of 311.9 ± 2.4 nm, a PDI of 0.401 ± 0.062, and a zeta potential of -6.15 ± 0.93 mV. Compared with Example 6, it exhibited uneven distribution and poor stability.

[0113] The reason for this is that after replacing the co-emulsifier from Span 85 to Span 20, the HLB value was mismatched and the molecular chain length was not suitable, which led to an increase in the interfacial tension between oil and water. The resulting interfacial film was thin and weak with insufficient steric hindrance, which could not effectively prevent the coalescence and aggregation of nanodroplets. Ultimately, this resulted in a significant increase in particle size and a decrease in stability.

[0114] Comparative Example 2

[0115] The remaining steps of this comparative example are the same as those of Example 6, except that the water bath temperature is adjusted from 95°C to 80°C.

[0116] The results showed that the hydrodynamic size of the obtained criborone nanoemulsion was 372.5 ± 6.4 nm, the polydispersity index was 0.494 ± 0.033, and the zeta potential was -10.17 ± 1.01 mV. Compared with Example 6, the particle size was larger, the distribution was uneven, and the stability was poor.

[0117] The reason for this is that the melting point of solid lipid glyceryl monostearate is 55-60℃, but in order to ensure that it is fully mixed with liquid lipid and forms a uniform lipid melt, it needs to be heated to 95℃; when heated to 80℃, the lipid melts incompletely, the drug does not completely dissolve, and the quality of nanoparticle formation is affected.

[0118] Test case

[0119] A comparative trial of the therapeutic effects of boronic acid cream and hydrocortisone ointment on canine atopic dermatitis.

[0120] (1) Animal grouping

[0121] The diagnostic criteria for selecting experimental animals were based on the diagnostic criteria for canine atopic dermatitis (2010):

[0122] a. The first onset of the disease occurred before the age of 3;

[0123] B-type dogs spend most of their time indoors;

[0124] c. Glucocorticoid-responsive skin itching;

[0125] d. Itching but no substantial lesions;

[0126] The forefoot was affected.

[0127] f. Disease of the external auricle;

[0128] The edge of the ear was unaffected;

[0129] The back and waist were not affected.

[0130] If five of the above eight criteria are met, the sensitivity is 85% and the specificity is 79% to distinguish between CAD and recurrent pruritus without CAD in dogs. If all six criteria are met, the specificity increases to 89%, but the sensitivity decreases to 58%.

[0131] When selecting experimental animals, their breed, size, weight, sex, and age should be noted. Blood routine tests and blood glucose levels should be checked to exclude animals with zoonotic diseases and highly contagious diseases. Animals that have received medication within two weeks prior to the experiment, animals that have had infectious diseases, animals with a white blood cell count below the normal range, and animals with a fasting blood glucose level above the normal range should not be included.

[0132] Dogs meeting the inclusion criteria for inflammation were randomly divided into two groups of 30 each. One group was the experimental group (criborone nanostructure lipid carrier group), and the other group was the control group (hydrocortisone cream group). The dogs were treated according to the corresponding drug administration method, dosage, and course of treatment.

[0133] According to the order in which the dogs were selected, they were assigned trial numbers from 1 to 60. The trial personnel recorded and saved the trial number and random group number of each selected dog.

[0134] (2) Experimental process

[0135] Apply a thin layer of the cream described in Example 6 to the affected area twice daily, with each application spaced 10–14 hours apart. Avoid application within 2 hours before bathing or 30 minutes after strenuous exercise. Do not apply the cream within 2 hours of a physical examination. If symptoms resolve within one week, continue using the ointment for a total of two weeks. In all other cases, continue use until one week after symptom resolution or until three weeks of treatment have been completed, with a total treatment duration not exceeding three weeks.

[0136] A recovery period check was performed 7 days after the test, and the results were recorded.

[0137] Detailed observation and recording were conducted before administration (D0), one week after administration (D7), two weeks after administration (D14), and three weeks after administration (D21). The physiological state, clinical symptoms, and occurrence, development, disappearance, and outcome of clinical symptoms of the test animals were assessed. Clinical responses of the animals, such as diet, defecation, and mental state, were observed and recorded daily.

[0138] Clinical responses in the dogs during the experiment were observed and scored, categorized into two groups: itching and skin lesions, based on the CADESI-4 score and the itching severity scoring criteria. The scores for the two drugs were obtained as follows: Figure 18 As shown.

[0139] like Figure 19 Based on clinical symptoms, pruritus scores, and atopic dermatitis scores, both hydrocortisone ointment and criborone nano-emulsion alleviated clinical symptoms in animals such as pruritus, erythema, and hair loss, and had a certain therapeutic effect on atopic dermatitis.

[0140] Of the above embodiments, the preferred embodiment is Embodiment 6.

Claims

1. A criborone nano-emulsion, characterized in that: The substance comprises the following components in parts by weight: 1-100 parts of creborol, 4.5-450 parts of solid lipids, 0.5-50 parts of liquid lipids, 0.5-50 parts of primary emulsifier, 0-50 parts of secondary emulsifier, and 10-1000 parts of deionized water.

2. The criborone nano-emulsion according to claim 1, characterized in that: The solid lipid is one or more of behenicol, distearate, and monostearate.

3. The criborone nano-emulsion according to claim 1, characterized in that: The liquid lipid is one or more of isopropyl myristate and oleic acid.

4. The criborone nano-emulsion according to claim 1, characterized in that: The primary emulsifier is one or more of poloxamer 407, Tween 80, and Tween 85, and the secondary emulsifier is one or more of Span 80 and Span 85.

5. A method for preparing the criborone nano-emulsion according to claim 1, characterized in that, Includes the following steps: Step 1: Take creborol, liquid lipid, and solid lipid, heat and melt them, and stir evenly in a water bath to obtain the oil phase. Take the main emulsifier, disperse it evenly in deionized water, and stir evenly in a water bath to obtain the aqueous phase. Step 2: Under constant temperature and stirring conditions, the oil phase is transferred into the aqueous phase and sheared to obtain the primary emulsion; Step 3: The colostrum is broken up by constant temperature ultrasonication or the colostrum obtained in Step 2 is homogenized under high pressure and then cooled to obtain criborone nano-emulsion.

6. The method for preparing the criborone nano-emulsion according to claim 5, characterized in that: In step one, the water bath heating and stirring temperature is 65~95℃, the rotation speed is 400~800 rpm, and the time is 20~30 min. The oil phase also includes a co-emulsifier.

7. The method for preparing the criborone nano-emulsion according to claim 5, characterized in that: In step two, the constant temperature stirring temperature is 65~95℃, the rotation speed is 400~800 rpm, the shearing speed is 8000~15000 rpm, and the shearing time is 5~10 min.

8. The method for preparing the criborone nano-emulsion according to claim 5, characterized in that: In step three, the temperature of the isothermal ultrasonic crushing is 65~95℃, the power of the ultrasonic crushing is 200~500 W, and the time is 10~20 min.

9. The method for preparing the criborone nano-emulsion according to claim 5, characterized in that: In step three, the pressure of the high-pressure homogenization process is 400~700 bar, and the number of cycles is 5~10.

10. The use of the criborone nano-emulsion according to claim 1 in the preparation of a drug for canine atopic dermatitis.

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