A compound ophthalmic in situ gel composition for treating ocular Demodex mite infection, its preparation method and application.

Abstract

This invention belongs to the field of pharmaceutical formulations and relates to a compound ophthalmic in-situ gel composition for treating ocular Demodex mite infection, its preparation method, and its application. By weight percentage, the compound ophthalmic in-situ gel composition for treating ocular Demodex mite infection comprises 0.1-2.0% of a nitroimidazole compound, 0.05-3.0% of an isoxazoline compound, 0.1-1.5% of a gelling polymer, 1.0-8.0% of a complex solubilizing system, 0.1-3.0% of pharmaceutically acceptable excipients, and the balance being water. The complex solubilizing system includes polyvinylpyrrolidone and cyclodextrin derivatives. This invention effectively solves the problem of poor solubility of isoxazoline drugs through a complex solubilizing system, and the combination of the two drugs has a significant synergistic acaricidal effect. Combined with a gelling polymer, it can achieve in-situ gelation on the ocular surface, prolong retention, and sustained drug release, with low ocular irritation, providing a highly effective, long-lasting, and safe new local treatment option for Demodex mite-related eye diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, specifically to a compound ophthalmic in-situ gel composition for treating Demodex mite infection in the eye, its preparation method, and its application. Background Technology

[0002] Demodex mite infestation is a common chronic inflammatory disease of the ocular surface, mainly caused by Demodex folliculorum and Demodex brevis. Its pathological mechanism is complex, involving not only the mechanical stimulation of the mites but also the immune inflammatory response triggered by the bacteria they carry, such as Staphylococcus aureus and Propionibacterium acnes. Clinical manifestations include eyelid swelling and redness, eyelash loss, meibomian gland dysfunction, and secondary dry eye syndrome. In severe cases, it can induce keratoconjunctivitis, significantly impacting the patient's visual quality and quality of life.

[0003] Currently, clinical treatment for Demodex mite infections mainly involves single-drug topical preparations, such as tea tree oil, metronidazole ointment, or ivermectin cream. However, these regimens suffer from inconsistent efficacy, high irritation, viscous formulations causing blurred vision, and poor patient compliance. While oral ivermectin or metronidazole has some efficacy, it carries the risk of systemic exposure and potential side effects. Nitroimidazoles possess broad-spectrum anti-anaerobic and antiprotozoal activity, but their direct killing effect on Demodex mites is limited. Overall, existing single-drug treatments struggle to simultaneously address the multiple pathological aspects of highly effective acaricidal, antibacterial, and anti-inflammatory effects, and their clinical efficacy needs improvement.

[0004] Isoxazoline drugs, such as loteranal, saloranal, and fleraclodal, are a novel class of GABA chloride channel inhibitors for insects and mites. In vitro studies have confirmed their potent killing effect on Demodex mites, with significantly superior acaricidal activity compared to tea tree oil, metronidazole, or ivermectin. However, the pathological process of Demodex mite infection involves a vicious cycle of mite parasitism, secondary bacterial infection, and immune inflammatory response. Relying solely on acaricidal action is insufficient to fully control the disease and may lead to prolonged inflammation or recurrence. Furthermore, isoxazoline drugs such as loteranal have extremely high hydrophobicity (LogP>5), are almost insoluble in water, and are difficult to dissolve in the hydrophilic media of conventional ophthalmic preparations. This presents a challenge for developing stable and highly effective compound topical ophthalmic preparations.

[0005] Therefore, there is a need for an ophthalmic formulation that is more effective in treating Demodex mite infections and has a higher drug delivery efficiency. Summary of the Invention

[0006] (a) Technical problems to be solved

[0007] To address the limitations of existing technologies where single drugs cannot simultaneously achieve efficient acaricidal and anti-inflammatory / antibacterial synergistic treatment, and the low delivery efficiency and difficulty in stable loading of strongly hydrophobic drugs in ophthalmic formulations, this invention provides a compound ophthalmic in-situ gel composition for treating ocular Demodex mite infections, its preparation method, and its application.

[0008] (II) Technical Solution

[0009] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0010] In a first aspect, the present invention provides a compound ophthalmic in-situ gel composition for treating ocular Demodex mite infection, comprising, by weight percentage, 0.1-2.0% of a nitroimidazole compound, 0.05-3.0% of an isoxazoline compound, 0.1-1.5% of a gelling polymer, 1.0-8.0% of a complex solubilizing system, 0.1-3.0% of pharmaceutically acceptable excipients, and the balance being water;

[0011] The composite solubilizing system includes polyvinylpyrrolidone and cyclodextrin derivatives.

[0012] In the compound ophthalmic in situ gel composition described above, preferably, the nitroimidazole compound is selected from metronidazole, tinidazole, ornidazole and their pharmaceutically acceptable salts, and the isoxazoline compound is selected from loteranal, saloranal, fleroranal and their pharmaceutically acceptable salts or crystal forms.

[0013] In the compound ophthalmic in-situ gel composition described above, preferably, the gelling polymer is carbomer or low-acyl gellan gum.

[0014] In the compound ophthalmic in-situ gel composition described above, preferably, the polyvinylpyrrolidone has a K value of 30, and the cyclodextrin derivative is sulfobutyl-β-cyclodextrin or hydroxypropyl-β-cyclodextrin;

[0015] The mass ratio of polyvinylpyrrolidone to cyclodextrin derivative is 1:(0.5-2).

[0016] The compound ophthalmic in situ gel composition described above preferably includes an isotonic adjuster, a pH adjuster, and a preservative as its excipients.

[0017] The compound ophthalmic in situ gel composition further comprises 0.2-2.0% by weight of an auxiliary thickener, wherein the auxiliary thickener is at least one selected from hydroxypropyl methylcellulose and sodium hyaluronate.

[0018] Preferably, the compound ophthalmic in-situ gel composition described above has a pH value of 5.5-7.0, an osmotic molar concentration of 280-320 mOsmol / kg, and is suitable for use at 25°C and 0.1 s. -1 At a shear rate of , the viscosity of the gel after formation is 3000-15000 cP.

[0019] The compound ophthalmic in-situ gel composition described above preferably further comprises, by weight percentage, 0.2-2.0% of cyclodextrin metal-organic framework nanocarriers and 0.1-0.5% of functional ionic liquids;

[0020] The cyclodextrin metal-organic framework nanocarrier is a cyclodextrin metal-organic framework nanocarrier with potassium ions as the metal center.

[0021] The functional ionic liquid is either a choline-geranilic acid ionic liquid or a choline-cinnamic acid ionic liquid.

[0022] Secondly, the present invention provides a method for preparing the above-mentioned compound ophthalmic in-situ gel composition, comprising the following steps:

[0023] S1: Isoxazoline compounds and polyvinylpyrrolidone are prepared into a solid dispersion, and then the solid dispersion and cyclodextrin derivative are dissolved in water to obtain a solubilized drug solution;

[0024] S2: Dissolve nitroimidazole compounds in water to obtain nitroimidazole solution;

[0025] S3: Disperse the gelling polymer in water, add excipients, and obtain a gel matrix;

[0026] S4: Add the solubilizing drug solution and nitroimidazole drug solution to the gel matrix, mix well, adjust the pH, then add the remaining water, and post-process to obtain the compound ophthalmic in-situ gel composition.

[0027] In the preparation method described above, preferably, in step S1, the first part of the isoxazoline compound and polyvinylpyrrolidone are made into a solid dispersion, and then the solid dispersion and cyclodextrin derivative are dissolved in water to obtain a solubilized drug solution.

[0028] Between step S1 and step S2, the process further includes: loading the second part of isoxazoline compounds into a cyclodextrin metal-organic framework nanocarrier to obtain drug-loaded nanoparticles; then dissolving the third part of isoxazoline compounds in a functional ionic liquid to obtain an ionic liquid complex.

[0029] In step S4, the solubilizing drug solution, drug-loaded nanoparticles, ionic liquid complex and nitroimidazole drug solution are added to the gel matrix, mixed and the pH is adjusted, and then the remaining water is added. The post-processing yields the compound ophthalmic in-situ gel composition.

[0030] The first part consists of isoxazoline compounds, which account for 50-90% of the total mass of isoxazoline compounds; the second part consists of isoxazoline compounds, which account for 5-30% of the total mass of isoxazoline compounds; and the third part consists of isoxazoline compounds, which account for 5-20% of the total mass of isoxazoline compounds.

[0031] Thirdly, the present invention also provides the use of the above-mentioned compound ophthalmic in situ gel composition or the compound ophthalmic in situ gel composition prepared by the above-mentioned preparation method in the preparation of a medicament for treating or preventing ocular Demodex mite infection;

[0032] The ocular Demodex mite infection is Demodex blepharitis, Demodex-associated meibomian gland dysfunction, Demodex-associated keratoconjunctivitis, or rosacea-associated ophthalmopathy.

[0033] (III) Beneficial Effects

[0034] First, this invention combines nitroimidazole and isoxazoline compounds. The isoxazoline compounds exert a highly effective acaricidal effect by inhibiting the GABA chloride channels of mites, while the nitroimidazole compounds exhibit anti-anaerobic and antiprotozoal activity. The mechanisms of the two are complementary. Experiments show that the graded inhibition concentration index of the two drugs used in combination is less than 0.5, demonstrating a significant synergistic effect. This combination can simultaneously act on multiple pathological processes, including mite parasitism, secondary bacterial infection, and immune inflammatory responses, overcoming the limitations of single-drug treatment.

[0035] Secondly, this invention significantly improves the solubility of isoxazoline compounds in aqueous media through a composite solubilizing system containing polyvinylpyrrolidone and cyclodextrin derivatives, enabling them to exist stably in the composition in a clear or microemulsion state. Simultaneously, the gelling polymer in the composition, upon instillation into the ocular surface, rapidly forms an in-situ gel in response to the tear environment, prolonging the drug's residence time on the ocular surface and achieving slow drug release, thereby improving drug delivery efficiency and therapeutic durability.

[0036] Third, the composition of this invention is water-based, and all components are ophthalmic preparations acceptable reagents. Eye irritation tests have verified that the composition of this invention does not significantly irritate the ocular surface, does not affect patient vision, and exhibits good patient compliance. Therefore, this invention provides a highly effective, long-lasting, and safe new local treatment option for ocular Demodex mite infection-related diseases such as Demodex blepharitis. Attached Figure Description

[0037] Figure 1 The average physical signs of rabbit eyelids are statistically analyzed in the negative control group, model control group, Examples 1-7 and Comparative Examples 1-3 14 days after drug administration. Detailed Implementation

[0038] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to specific embodiments.

[0039] This invention provides a compound ophthalmic in-situ gel composition for treating ocular Demodex mite infection. By weight percentage, it comprises 0.1-2.0% of a nitroimidazole compound, 0.05-3.0% of an isoxazoline compound, 0.1-1.5% of a gelling polymer, 1.0-8.0% of a complex solubilizing system, 0.1-3.0% of pharmaceutically acceptable excipients, and the balance being water. All water used in this invention is water for injection. The complex solubilizing system of this invention includes polyvinylpyrrolidone and cyclodextrin derivatives.

[0040] Nitroimidazole compounds possess broad-spectrum anti-anaerobic and antiprotozoal activity, inhibiting bacteria carried by Demodex mites, such as Staphylococcus and Propionibacterium acnes, and reducing inflammatory responses. Isoxazoline compounds are highly effective and selective GABA chloride channel inhibitors for mites, directly killing Demodex mites. The combination of these two compounds achieves synergistic treatment with both acaricidal and anti-inflammatory / antibacterial effects.

[0041] Due to the extremely high hydrophobicity (LogP>5) of isoxazoline compounds, this invention employs a composite solubilization system comprising polyvinylpyrrolidone and cyclodextrin derivatives. Polyvinylpyrrolidone and isoxazoline compounds can form an amorphous solid dispersion, inhibiting drug crystallization. The cyclodextrin derivatives further encapsulate drug molecules, forming inclusion complexes. Together, they solubilize the drug to a therapeutically effective concentration while maintaining physical stability. The gelling polymer enables the composition to respond to the tear environment after instillation, such as under conditions of increased pH or the presence of cations, undergoing a sol-gel phase transition to form an in-situ gel, thereby prolonging the ocular surface retention time and achieving sustained drug release. Water serves as the primary solvent carrier. The composition is a low-viscosity liquid during storage and instillation, with a viscosity around 500 cP, facilitating dispensing. After instillation into the ocular surface, it rapidly forms a gel with a viscosity of 3000-15000 cP, combining good ease of use with long-lasting therapeutic effect.

[0042] Preferably, the compound ophthalmic in-situ gel composition of the present invention further comprises 0.2-2.0% cyclodextrin metal-organic framework nanocarrier and 0.1-0.5% functional ionic liquid by weight percentage. The raw materials for preparing the cyclodextrin metal-organic framework nanocarrier include cyclodextrin and potassium hydroxide, and the functional ionic liquid is choline-geraniol ionic liquid or choline-cinnamic acid ionic liquid.

[0043] The preparation method of cyclodextrin metal-organic framework nanocarriers is as follows: γ-cyclodextrin and potassium hydroxide are dissolved in water at a molar ratio of 1:8 to 1:12, and stirred until completely dissolved. Methanol is added, with a volume ratio of methanol to water of 1:1 to 3:1. The mixed solution is transferred to a sealed container and allowed to react at 40-50°C for 12-24 hours. After the reaction, the precipitated crystals are collected by filtration, washed 2-3 times with methanol, vacuum dried at room temperature, and then ball-milled or sonicated to obtain cyclodextrin metal-organic framework nanocarriers with an average particle size of 100-300 nm. The cyclodextrin metal-organic framework nanocarriers prepared by the above method have a porous structure, which can efficiently load isoxazoline compounds, further prolonging the drug release time. At the same time, the cyclodextrin metal-organic framework nanocarriers themselves can slowly degrade on the ocular surface, achieving long-acting sustained release. The cyclodextrin metal-organic framework nanocarriers of this invention have an average particle size of 100-300 nm, which is far below the foreign body perception threshold of the human eye, so patients will not experience a foreign body sensation during use. The nanoparticles are uniformly dispersed in a soft hydrogel matrix, and the three-dimensional network structure of the gel effectively anchors them, preventing the nanoparticles from rolling freely and rubbing against the ocular surface. Furthermore, this nanocarrier is composed of γ-cyclodextrin and potassium ions, both of which are biodegradable and biocompatible materials. γ-cyclodextrin can be gradually degraded into oligosaccharides and small-molecule sugars by enzymes in tears, while potassium ions are electrolytes in the body and can be absorbed or excreted with tears.

[0044] The preparation method of functional ionic liquid is as follows: choline bicarbonate is mixed with geranium acid or cinnamic acid in an equimolar ratio, and the mixture is stirred at 50-70℃ for 2-4 hours. The water is removed by vacuum drying to obtain choline-geranium acid ionic liquid or choline-cinnamic acid ionic liquid.

[0045] Ionic liquids play multiple roles in this invention: First, as highly efficient solubilizers, their solubility for isoxazoline drugs far exceeds that of traditional cyclodextrins, significantly reducing the amount of complex solubilizing systems required. Second, the geraniol or cinnamate anions in the ionic liquids themselves possess anti-mite, antibacterial, and anti-inflammatory activities, forming a triple synergistic acaricidal effect with nitroimidazoles and isoxazoline drugs. Experiments show that the fractional inhibitory concentration index (FIC Index) of the three combined can be reduced to below 0.3. Third, ionic liquids can also act as penetration enhancers, temporarily regulating the tight junctions between corneal epithelial cells and helping drugs penetrate deep into the hair follicle. Fourth, ionic liquids can also adsorb onto the surface of cyclodextrin metal-organic framework nanocarriers, forming a hydrophilic modification layer, improving the dispersion stability of nanoparticles in aqueous gel matrices, and preventing aggregation and precipitation. It should be noted that the in-situ gelation properties of the present invention are dominated by the gelling polymer, and the content of the functional ionic liquid in the composition is low. Its ionic strength is much lower than the cation concentration required to trigger gelling gelation and also much lower than the concentration required to affect the pH response of carbomer. Therefore, it will not cause the composition to gel prematurely in the bottle, and the in-situ gelation properties are completely preserved.

[0046] Preferably, the nitroimidazole compounds in this invention are selected from metronidazole, tinidazole, ornidazole and their pharmaceutically acceptable salts, more preferably metronidazole, and the content is more preferably 0.5-1.0%. The isoxazoline compounds are selected from loteranal, saloranal, fleraranal and their pharmaceutically acceptable salts or crystal forms, more preferably loteranal, and the content is more preferably 0.25-1.5%.

[0047] Preferably, the gelling polymer in this invention is carbomer 974P or low-acyl gellan gum, with a more preferably content of 0.2-0.8%. Carbomer, such as carbomer 974P, is a pH-sensitive polymer that is liquid at low pH levels and forms a gel when the pH rises above 5.5. Therefore, the pH of the composition can be adjusted to keep it liquid in the bottle, and gelling occurs upon contact with tears after eye drops. Low-acyl gellan gum is an ion-sensitive polysaccharide that is liquid at low cation concentrations and forms a gel upon contact with potassium and calcium ions in tears. Both can be used alone or in combination to obtain faster gelation speed and higher gel strength.

[0048] Preferably, the K value of polyvinylpyrrolidone (PVP) is 30, i.e., PVP K30. This specification has a moderate molecular weight, good solubilizing effect, and is easy to process. The cyclodextrin derivative is sulfonyl-β-cyclodextrin or hydroxypropyl-β-cyclodextrin. The mass ratio of PVP to cyclodextrin derivative is 1:(0.5-2). Within this ratio range, the two can form a synergistic solubilizing effect. When the ratio is lower than 1:0.5, the amount of cyclodextrin is insufficient, and the inclusion is inadequate. When the ratio is higher than 1:2, excessive cyclodextrin may lead to increased system viscosity or increased cost.

[0049] Preferably, the excipients include isotonic adjusters, pH adjusters, and preservatives. Furthermore, the compound ophthalmic in-situ gel composition also contains 0.2-2.0% by weight of an auxiliary thickener, preferably at least one of hydroxypropyl methylcellulose and sodium hyaluronate.

[0050] The role of thickeners is to adjust the base viscosity of the composition, help suspend insoluble microparticles, and improve comfort during eye drops. Isotonic adjusters, such as glycerin, sodium chloride, and mannitol, are used to adjust the osmotic pressure to be isotonic with tears, reducing irritation. pH adjusters, such as borate-borax buffer, sodium hydroxide solution, and phosphate buffer, are used to adjust the pH of the composition to 5.5-7.0, ensuring the gel polymer remains liquid in the bottle while avoiding irritation to the ocular surface. Preservatives, such as benzalkonium chloride and methylparaben, are used to inhibit microbial growth and ensure the sterility of the formulation.

[0051] Preferably, the compound ophthalmic in-situ gel composition of the present invention has a pH value of 5.5-7.0, an osmotic molar concentration of 280-320 mOsmol / kg, and is suitable for use at 25°C and 0.1 s. -1 At the given shear rate, the viscosity of the gel after in-situ gel formation is 3000-15000 cP. When stored in the bottle, its viscosity is approximately 500 cP. Within this viscosity range, the composition can still be easily dispensed from the bottle. After being applied to the ocular surface, the viscosity increases, forming an in-situ gel. If the viscosity is too low, the gelling ability is insufficient; if the viscosity is too high, it will lead to difficulty in dispensing.

[0052] The present invention also provides a method for preparing the above-mentioned compound ophthalmic in-situ gel composition, comprising the following steps:

[0053] S1: The isoxazoline compound from the first part is prepared into a solid dispersion with polyvinylpyrrolidone. Then, the solid dispersion is dissolved in water with a cyclodextrin derivative to obtain a solubilized solution. Specifically, the isoxazoline compound and polyvinylpyrrolidone can be dissolved in an organic solvent such as ethanol or acetone, and the solvent can be removed by rotary evaporation or spray drying to obtain an amorphous solid dispersion. The solid dispersion and the cyclodextrin derivative are then dissolved together in water and stirred until clear or microemulsion formed. This step achieves the initial solubilization of the isoxazoline compound.

[0054] S2: The second part of the isoxazoline compounds is loaded into cyclodextrin metal-organic framework nanocarriers to obtain drug-loaded nanoparticles. Specifically, the cyclodextrin metal-organic framework nanocarriers can be dispersed in an organic solvent containing isoxazoline compounds, such as ethanol, and stirred for 12-24 hours to allow the drug to be adsorbed into the carrier. After centrifugation, washing, and drying, drug-loaded nanoparticles are obtained.

[0055] S3: Dissolve the isoxazoline compound from the third part in a functional ionic liquid to obtain an ionic liquid complex. Specifically, the isoxazoline compound can be added to the ionic liquid and sonicated at 40-60℃ for 15-30 min to obtain a clear ionic liquid-drug complex solution.

[0056] S4: Dissolve nitroimidazole compounds in water to obtain nitroimidazole solution.

[0057] S5: Disperse the gel polymer in water, add excipients, stir to disperse or dissolve it, and let it stand to fully hydrate and swell to obtain a gel matrix.

[0058] S6: Add the solubilizing drug solution, drug-loaded nanoparticles, ionic liquid complex and nitroimidazole drug solution to the gel matrix, mix well and adjust the pH, then add the remaining water, and after homogenization, sterilization and filling, the compound ophthalmic in-situ gel composition is obtained.

[0059] This invention divides isoxazoline compounds into three parts and processes them separately, each forming a different solubilization / loading form, and finally combines them into the same gel matrix, thus achieving the integration of multiple release behaviors.

[0060] More preferably, the first part of the isoxazoline compounds accounts for 50-90% of the total mass of the isoxazoline compounds, the second part of the isoxazoline compounds accounts for 5-30% of the total mass of the isoxazoline compounds, and the third part of the isoxazoline compounds accounts for 5-20% of the total mass of the isoxazoline compounds.

[0061] This invention presents isoxazoline compounds in three different forms in the composition, with the three forms exhibiting complementary functions and synergistic effects:

[0062] The first part consists of isoxazoline compounds that form a solid dispersion with PVP and cyclodextrin, providing rapid release and ensuring quick onset of action after eye drops, rapidly killing Demodex mites on the ocular surface.

[0063] The second part, isoxazoline compounds, provides ultra-long-lasting release, forming a drug reservoir on the ocular surface to maintain a sustained and effective acaricidal concentration, reducing the frequency of administration.

[0064] The third part, isoxazoline compounds, provides highly efficient solubilization and penetration enhancement, helping the drug penetrate the hair follicle opening. At the same time, the geraniol / cinnamic acid component of the ionic liquid forms a triple synergistic acaricidal effect with the two drugs, enhancing the overall efficacy.

[0065] By combining the above three forms, this invention achieves a triple therapeutic goal of rapid onset, long-lasting effect, and deep clearance, a technical effect that cannot be achieved simultaneously by any one or two forms. Compared with solutions using only solid dispersions, this invention offers more sustained release; compared with solutions using only cyclodextrin metal-organic framework nanocarriers, this invention offers more rapid onset; and compared with solutions using only ionic liquids, this invention offers better physical stability and lower irritation.

[0066] This invention also provides the application of the above-mentioned compound ophthalmic in-situ gel composition in the preparation of pharmaceuticals. Specifically, the above composition can be used to prepare pharmaceuticals for the treatment or prevention of ocular Demodex mite infections. These ocular Demodex mite infections include, but are not limited to: Demodex blepharitis, Demodex-associated meibomian gland dysfunction, Demodex-associated keratoconjunctivitis, and rosacea-associated ophthalmopathy. Because the composition of this invention has synergistic acaricidal, anti-inflammatory, antibacterial, long-acting sustained-release, and low-irritant properties, it is particularly suitable for the above-mentioned chronic ocular surface diseases requiring long-term topical administration.

[0067] To further clarify the present invention and its technological advancements, the following description is provided in conjunction with specific embodiments and technical effects.

[0068] Example 1

[0069] This embodiment provides a compound ophthalmic in-situ gel composition for treating Demodex folliculorum infection of the eye, comprising, by weight percentage: 0.75% metronidazole, 1% loteranal, 0.5% carbomer 974P, 2% PVP K30, 3% sulfobutyl-β-cyclodextrin, 0.3% hydroxypropyl methylcellulose, 2% glycerol, 0.1% benzalkonium chloride at a concentration of 10%, 0.45% sodium chloride, with the balance being water.

[0070] In this embodiment, the preparation method of the compound ophthalmic in-situ gel composition includes the following steps:

[0071] S1: Loteranar and PVP K30 are dissolved in an ethanol-acetone mixture, the solvent is removed by rotary evaporation, and the mixture is ground and sieved to obtain an amorphous solid dispersion. The solid dispersion is then added to an aqueous solution containing sulfobutyl-β-cyclodextrin, stirred and sonicated to obtain a clear and solubilized solution.

[0072] S2: Sprinkle Carbomer 974P into water under high-speed shearing, stir, and let stand at 4°C overnight to hydrate.

[0073] S3: Dissolve metronidazole in water to obtain metronidazole solution.

[0074] S4: Add the solubilizing drug solution and metronidazole solution to the gel matrix dispersion in sequence and stir until homogeneous.

[0075] S5: Add hydroxypropyl methylcellulose, glycerin, sodium chloride, and benzalkonium chloride solution, and stir to dissolve. Then add 1M sodium hydroxide solution dropwise, and adjust the pH to 6.0 using borate-borax buffer to form a transparent gel. Add the remaining water. After homogenization, sterilization, and filling, fill into ophthalmic gel tubes to obtain the compound ophthalmic in-situ gel composition.

[0076] Example 2

[0077] This embodiment provides a compound ophthalmic in-situ gel composition for treating ocular Demodex mite infection, comprising, by weight percentage: 1.0% ornidazole, 0.5% saloranaline, 0.3% low-acyl gellan gum, 1.5% PVP K30, 3.0% hydroxypropyl-β-cyclodextrin, 0.5% hydroxypropyl methylcellulose, 2.5% mannitol, 0.05% disodium edetate, 0.02% methylparaben, and the balance being water.

[0078] In this embodiment, the preparation method of the compound ophthalmic in-situ gel composition includes the following steps:

[0079] S1: Dissolve salorana and PVP K30 in an ethanol-acetone mixture, remove the solvent by rotary evaporation, and grind and sieve to obtain an amorphous solid dispersion. Then add the solid dispersion to an aqueous solution containing hydroxypropyl-β-cyclodextrin, stir and sonicate to obtain a clear and solubilized solution.

[0080] S2: Disperse low-acyl gellan gum in an appropriate amount of water for injection, heat to 80°C and stir to completely dissolve it, then cool to below 40°C to obtain a gellan gum solution.

[0081] S3: Dissolve ornidazole in an appropriate amount of water for injection, add hydroxypropyl methylcellulose, mannitol, disodium edetate and methylparaben, stir to dissolve, and obtain ornidazole solution.

[0082] S4: Add the solubilizing solution and ornidazole solution to the gellan gum solution in sequence and stir until well mixed.

[0083] S5: Adjust the pH of the mixture to 6.8 with phosphate buffer, then fine-tune the pH with 1M sodium hydroxide solution, add the remaining water for injection, and stir at low speed below 30°C until homogeneous. Then, homogenize the resulting mixture, filter to remove bacteria, and fill into sterile ophthalmic dropper bottles to obtain the compound ophthalmic in-situ gel composition.

[0084] Example 3

[0085] This embodiment provides a compound ophthalmic in-situ gel composition for treating ocular Demodex mite infection, comprising, by weight percentage: 0.1% metronidazole, 0.05% loteranal, 0.1% carbomer 974P, 1.0% PVP K30, 0.5% sulfobutyl-β-cyclodextrin, 0.2% cyclodextrin metal-organic framework nanocarrier, 0.1% choline-germicolic acid ionic liquid, 0.2% hydroxypropyl methylcellulose, 0.05% sodium chloride, 0.02% boric acid, 0.03% benzalkonium chloride, with the balance being water.

[0086] In this embodiment, the preparation method of the compound ophthalmic in-situ gel composition includes the following steps:

[0087] S1: Dissolve 50% of loteranal and PVP K30 in an ethanol-acetone mixture, remove the solvent by rotary evaporation, and grind and sieve to obtain an amorphous solid dispersion. Then, dissolve the solid dispersion together with sulfobutyl-β-cyclodextrin in water, stir and sonicate to obtain a solubilized solution.

[0088] S2: Cyclodextrin metal-organic framework nanocarriers were dispersed in an ethanol solution containing 30% loteranal, stirred for 12 h, centrifuged, washed, and dried to obtain drug-loaded nanoparticles. The cyclodextrin metal-organic framework nanocarriers were prepared by reacting γ-cyclodextrin and potassium hydroxide at a molar ratio of 1:8 at 40 °C for 12 h, and then ball-milled to an average particle size of 100 nm.

[0089] S3: Add 20% loteranal to the choline-germicolic acid ionic liquid and sonicate at 40°C for 15 min to obtain a clear ionic liquid-drug complex solution.

[0090] S4: Dissolve metronidazole in water to obtain nitroimidazole solution.

[0091] S5: Disperse Carbomer 974P in water, add hydroxypropyl methylcellulose, sodium chloride, boric acid, and benzalkonium chloride, stir to disperse, and let stand overnight at 4°C to hydrate, thus obtaining a gel matrix.

[0092] S6: Add the solubilizing drug solution, drug-loaded nanoparticles, ionic liquid complex and nitroimidazole drug solution to the gel matrix, mix well, adjust the pH to 5.5 with 1M sodium hydroxide solution, add water to the total volume, homogenize, filter for sterilization, and fill to obtain the final product.

[0093] Example 4

[0094] This embodiment provides a compound ophthalmic in-situ gel composition for treating Demodex folliculorum infection of the eye, comprising, by weight percentage: 2.0% tinidazole, 3.0% saloranol, 1.5% low-acyl gellan gum, 8.0% a complex solubilizing system, wherein the mass ratio of PVP K30 to hydroxypropyl-β-cyclodextrin is 1:2, i.e., approximately 2.67% PVP K30 and approximately 5.33% hydroxypropyl-β-cyclodextrin, 2.0% cyclodextrin metal-organic framework nanocarrier, 0.5% choline-cinnamic acid ionic liquid, 2.0% sodium hyaluronate, 2.0% mannitol, 0.5% phosphate buffer, 0.5% methylparaben, and the balance being water.

[0095] In this embodiment, the preparation method of the compound ophthalmic in-situ gel composition includes the following steps:

[0096] S1: Dissolve 90% of saloranax and PVP K30 in acetone, remove the solvent by spray drying to obtain an amorphous solid dispersion. Then, dissolve the solid dispersion together with hydroxypropyl-β-cyclodextrin in water, stir and sonicate to obtain a solubilized drug solution.

[0097] S2: Cyclodextrin metal-organic framework nanocarriers were dispersed in an ethanol solution containing 5% saloranal, stirred for 24 h, centrifuged, washed, and dried to obtain drug-loaded nanoparticles. The cyclodextrin metal-organic framework nanocarriers were prepared by reacting γ-cyclodextrin and potassium hydroxide at a 1:12 molar ratio at 50 °C for 24 h, and then ultrasonically treated to achieve an average particle size of 300 nm.

[0098] S3: Add 5% saloranaline to the choline-cinnamic acid ionic liquid and sonicate at 60°C for 30 min to obtain a clear ionic liquid-drug complex solution.

[0099] S4: Dissolve tinidazole in water to obtain nitroimidazole solution.

[0100] S5: Disperse low-acyl gellan gum in water, heat to 80°C and stir to dissolve, cool to below 40°C, add sodium hyaluronate, mannitol, phosphate buffer, and methylparaben, stir evenly to obtain a gel matrix.

[0101] S6: Add the solubilizing drug solution, drug-loaded nanoparticles, ionic liquid complex and nitroimidazole drug solution to the gel matrix, mix well, adjust the pH to 7.0 with 1M sodium hydroxide solution, add water to the total volume, stir at low speed below 30℃, homogenize, irradiate sterilize, and fill to obtain the final product.

[0102] Example 5

[0103] This embodiment provides a compound ophthalmic in-situ gel composition for treating ocular Demodex mite infection, comprising, by weight percentage: 1.0% ornidazole, 1.5% fluranal, 0.5% carbomer 974P, 0.3% low-acyl gellan gum, and 5.0% a complex solubilizing system, wherein the mass ratio of PVP K30 to sulfobutyl-β-cyclodextrin is 1:1, i.e., PVP K30 2.5%, sulfobutyl-β-cyclodextrin 2.5%, 1.0% cyclodextrin metal-organic framework nanocarrier, 0.3% choline-geraniol ionic liquid, 1.0% hydroxypropyl methylcellulose, 0.5% glycerol, 0.3% borate-borax buffer, 0.2% disodium edetate, and the balance being water.

[0104] In this embodiment, the preparation method of the compound ophthalmic in-situ gel composition includes the following steps:

[0105] S1: Dissolve 70% of fluranaridine and PVP K30 in ethanol, remove the solvent by rotary evaporation, and grind and sieve to obtain an amorphous solid dispersion. Then, dissolve the solid dispersion together with sulfobutyl-β-cyclodextrin in water, stir and sonicate to obtain a solubilized drug solution.

[0106] S2: Cyclodextrin metal-organic framework nanocarriers were dispersed in an ethanol solution containing 20% ​​freranil, stirred for 18 h, centrifuged, washed, and dried to obtain drug-loaded nanoparticles. The cyclodextrin metal-organic framework nanocarriers were prepared by reacting γ-cyclodextrin and potassium hydroxide at a 1:10 molar ratio at 45 °C for 18 h, and then ball-milled to an average particle size of 260 nm.

[0107] S3: Add 10% flurana to the choline-germic acid ionic liquid and sonicate at 50°C for 20 min to obtain a clear ionic liquid-drug complex solution.

[0108] S4: Dissolve ornidazole in water to obtain nitroimidazole solution.

[0109] S5: Disperse carbomer 974P and low-acyl gellan gum in water, add hydroxypropyl methylcellulose, glycerol, borate-borax buffer, and disodium edetate, stir and disperse. The carbomer part needs to be hydrated overnight at 4°C, and the gellan gum part needs to be heated to dissolve and then cooled and mixed to obtain the gel matrix.

[0110] S6: Add the solubilizing drug solution, drug-loaded nanoparticles, ionic liquid complex and nitroimidazole drug solution to the gel matrix, mix well, adjust the pH to 6.2 with 1M sodium hydroxide solution, add water to the total volume, homogenize, filter for sterilization, and fill to obtain the final product.

[0111] Example 6

[0112] This embodiment provides a compound ophthalmic in-situ gel composition for treating Demodex folliculorum infection of the eye. By mass percentage, it comprises: 0.75% metronidazole, 1.0% loteranal, 0.6% carbomer 974P, and 4.0% a complex solubilizing system. The mass ratio of PVP K30 to hydroxypropyl-β-cyclodextrin is 1:1.5, i.e., PVP K30 1.6%, hydroxypropyl-β-cyclodextrin 2.4%, cyclodextrin metal-organic framework nanocarrier 1.5%, choline-cinnamic acid ionic liquid 0.2%, hydroxypropyl methylcellulose 1.2%, sodium hyaluronate 0.8%, mannitol 0.5%, phosphate buffer 0.3%, benzalkonium chloride 0.2%, and the balance being water.

[0113] In this embodiment, the preparation method of the compound ophthalmic in-situ gel composition is the same as in Examples 3 and 5, and the specific steps are as follows:

[0114] S1: Dissolve 80% of loteranal and PVP K30 in an ethanol-acetone mixture, and rotary evaporate to obtain a solid dispersion. Then dissolve the dispersion in water with hydroxypropyl-β-cyclodextrin to obtain a solubilized solution.

[0115] S2: Cyclodextrin metal-organic framework nanocarriers were dispersed in an ethanol solution containing 15% loteranal, stirred for 18 h, centrifuged, washed, and dried to obtain drug-loaded nanoparticles. The cyclodextrin metal-organic framework nanocarriers were prepared by reacting γ-cyclodextrin and potassium hydroxide at a molar ratio of 1:11 at 45 °C for 18 h, and then ball-milled to an average particle size of 220 nm.

[0116] S3: Dissolve 5% loteranar in choline-cinnamic acid ionic liquid to obtain an ionic liquid complex.

[0117] S4: Dissolve metronidazole in water to obtain nitroimidazole solution.

[0118] S5: Disperse Carbomer 974P in water, add hydroxypropyl methylcellulose, sodium hyaluronate, mannitol, phosphate buffer, and benzalkonium chloride, hydrate overnight at 4°C to obtain a gel matrix.

[0119] S6: Mix all components, adjust pH to 6.5, add water, homogenize, sterilize, and fill.

[0120] Example 7

[0121] This embodiment provides a compound ophthalmic in-situ gel composition for treating Demodex folliculorum infection of the eye. By mass percentage, it comprises: 1.5% tinidazole, 2.0% loteranal, 0.8% low-acyl gellan gum, and 6.0% a complex solubilizing system. The mass ratio of PVP K30 to sulfobutyl-β-cyclodextrin is 1:0.8, i.e., PVP K30 3.33%, sulfobutyl-β-cyclodextrin 2.67%, 2.0% cyclodextrin metal-organic framework nanocarrier, 0.4% choline-geraniol ionic liquid, 2.0% sodium hyaluronate, 1.0% glycerol, 0.5% borate-borax buffer, 0.5% methylparaben, and the balance being water.

[0122] In this embodiment, the preparation method of the compound ophthalmic in-situ gel composition includes the following steps:

[0123] S1: Dissolve 60% loteranal and PVP K30 in acetone, spray dry to obtain a solid dispersion, and then dissolve it in water with sulfobutyl-β-cyclodextrin to obtain a solubilized solution.

[0124] S2: Cyclodextrin metal-organic framework nanocarriers were dispersed in an ethanol solution containing 25% loteranal, stirred for 18 h, centrifuged, washed, and dried to obtain drug-loaded nanoparticles. The cyclodextrin metal-organic framework nanocarriers were prepared by reacting γ-cyclodextrin and potassium hydroxide at a 1:10 molar ratio at 46 °C for 16 h, and then ball-milled to an average particle size of 240 nm.

[0125] S3: Dissolve 15% loteranar in choline-germic acid ionic liquid to obtain an ionic liquid complex.

[0126] S4: Dissolve tinidazole in water to obtain nitroimidazole solution.

[0127] S5: Disperse low-acyl gellan gum in water, heat to 80°C to dissolve, cool to below 40°C, add sodium hyaluronate, glycerin, borate-borax buffer, and methylparaben to obtain the gel matrix.

[0128] S6: Add the solubilizing drug solution, drug-loaded nanoparticles, ionic liquid complex and nitroimidazole solution to the gel matrix, mix well, adjust the pH to 6.8 with phosphate buffer, add water to the total volume, stir at low speed below 30°C, homogenize, sterilize by irradiation, and fill into the gel to obtain the final product.

[0129] Comparative Example 1

[0130] This comparative example provides a compound ophthalmic in situ gel composition for treating ocular Demodex mite infection, which differs from Example 1 in that the amount of PVP K30 added is 0.

[0131] Comparative Example 2

[0132] This comparative example provides a compound ophthalmic in-situ gel composition for treating ocular Demodex mite infection, which differs from Example 1 in that the amount of cyclodextrin derivative added is 0.

[0133] Comparative Example 3

[0134] This comparative example provides a metronidazole-loteranal suspension eye drop containing 0.75 wt% metronidazole, 1.0 wt% micronized loteranal, 0.5 wt% sodium carboxymethyl cellulose, and the balance being conventional eye drop excipients.

[0135] To verify the technical efficacy of the compound ophthalmic in-situ gel composition of the present invention, the samples prepared in Examples 1-7 and Comparative Examples 1-3 were subjected to the following performance tests. All tests were repeated three times, and the results are expressed as mean ± standard deviation.

[0136] I. Solubilizing ability and physical stability:

[0137] Test methods: The apparent solubility of isoxazoline drugs in each example and comparative example was determined. An appropriate amount of sample was taken, stirred at 25°C for 24 hours, filtered, and the drug concentration was determined by HPLC. Simultaneously, the sample was placed under accelerated conditions, specifically at 40°C and 75% RH for 30 days, and the particle size change was measured and the appearance observed. See Table 1 for details.

[0138] Table 1. Comparison of particle size changes after 30 days of storage for products from Examples 1-7 and Comparative Examples 1-3.

[0139]

[0140] As shown in Table 1, Examples 1-2, using only the PVP + cyclodextrin composite solubilization system, achieved a solubility of approximately 450-480 μg / mL with good stability. Examples 3-7, by further introducing CD-MOF and ionic liquids, showed a significant increase in solubility, stable particle size, and no precipitation. Comparative Examples 1 and 2 exhibited poor solubilization effects and were unstable.

[0141] II. Rheological properties and in-situ gelation behavior

[0142] Test methods: The rheological properties of each embodiment were determined using a rheometer. The viscosity of the composition in its ungelled state in the bottle and the viscosity of the gel formed after being dropped with simulated tears were measured. The shear thinning behavior of the gel was also measured.

[0143] The results showed that the compositions of Examples 1-7, when not in contact with tears in the bottle, were low-viscosity, transparent liquids that were easy to dispense smoothly from the ophthalmic dropper without problems such as residue sticking to the bottle walls or difficulty in dispensing. The compositions of Examples 1-7, when stored in the bottle at 25°C and a shear rate of 100 s... -1 When simulating the shear force during extrusion and dripping, the viscosity is 180-550 cP.

[0144] When the compositions of Examples 1-7 were mixed with simulated tears, a sol-gel phase transition occurred rapidly, with a gelation time of 30-60 seconds. This allowed for the rapid formation of a stable gel on the ocular surface, preventing the drug from being lost rapidly with the tears and achieving long-lasting retention.

[0145] Examples 1-7 show gels formed after instilling simulated tears, which were then subjected to temperature changes at 25°C for 0.1 seconds. -1 At low shear rates, the viscosity ranges from 3000 to 15000 cP. Gels in this viscosity range exhibit good viscoelasticity and stability, adhering closely to the ocular surface and resisting detachment, while avoiding foreign body sensation due to excessive viscosity. However, as the shear rate gradually increases to 100 s⁻¹... -1 When simulating the shear force during human blinking, the gel exhibits significant shear-thinning behavior, with the viscosity rapidly decreasing to around 500 cP. It can adapt to the deformation of the blinking action, avoiding impact on vision and eye comfort. After the blinking action ends, the viscosity rapidly rises to 1200-1350 cP within 20 seconds and remains stable, forming a gel within 60 seconds, demonstrating excellent in-situ gelling ability.

[0146] III. In vitro drug release

[0147] Test method: The Franz diffusion cell method was used to measure the cumulative release percentage of isoxazoline drugs and nitroimidazole drugs in each example and comparative example using simulated tears as the release medium, and the time required for 50% release, i.e., T50, was calculated.

[0148] Table 2. Comparison of in vitro drug release in Examples 1-7 and Comparative Examples 1-3

[0149]

[0150] As shown in Table 2, Examples 1-2, containing only solid dispersions for solubilization, exhibited a gradual release but a relatively slow onset of action. Examples 3-7, containing solid dispersions, CD-MOF support, and ionic liquid complexes, displayed an ideal dual-phase release characteristic of rapid onset and long-lasting effect, with moderate T50 values.

[0151] IV. In vitro synergistic acaricidal pharmacodynamics

[0152] Test method: An in vitro Demodex folliculorum model was used to determine the half-maximal effective concentration (EC50) of each sample against Demodex mites, and the fractional inhibitory concentration index (FIC Index) was calculated. FIC < 0.5 indicates synergistic effect.

[0153] The pharmacodynamic evaluation results based on the in vitro Demodex folliculorum model are as follows: Metronidazole alone had an EC50 of 12.8 μg / mL; Loteranal alone had an EC50 of 1.85 μg / mL. In Example 1, the EC50 of Loteranal decreased to 0.42 μg / mL, and the FIC index was 0.25, indicating a strong synergistic acaricidal effect. The FIC index of Example 2 was 0.28, also showing strong synergy. Examples 3-7 further introduced functional ionic liquids with anti-mite activity, further reducing the EC50 of Loteranal to 0.18-0.22 μg / mL and the FIC index to 0.12-0.15, showing a highly significant triple synergistic acaricidal effect. The FIC indices of Comparative Examples 1 and 2 were 0.29 and 0.28, respectively, still showing a synergistic effect, but the instability of solubilization led to large fluctuations in the actual pharmacodynamic data. Comparative Example 3 showed an FIC index of 0.65, indicating only an additive effect with no significant synergistic effect. These results demonstrate that the present invention, through the combination of nitroimidazole and isoxazoline drugs, particularly with the further introduction of functional ionic liquids, can produce a significant synergistic acaricidal effect, with efficacy significantly superior to single drugs and conventional formulations.

[0154] V. Evaluation of eye irritation (HET-CAM test)

[0155] Test method: Following OECD guidelines, the chicken embryo chorioallantoic membrane test was used to calculate the Intense Stimulation Score (IS). The judgment criteria are as follows: IS < 1 indicates no irritation, 1-5 indicates mild irritation, 5-10 indicates moderate irritation, and > 10 indicates severe irritation.

[0156] The results of the HET-CAM eye irritation test are as follows: the irritation score of the negative control (physiological saline) was 0, and the irritation score of the positive control (1% SDS) was 16.8, indicating that the test system is reliable. The irritation scores of Examples 1 and 2 were 0.6±0.2 and 0.7±0.3, respectively, both showing no irritation. The irritation scores of Examples 3-7 were between 0.8 and 0.9, with Examples 3, 5, 6, and 7 showing no irritation. The irritation scores of Comparative Examples 1 and 2 were both between 0.6 and 0.9, showing no irritation. The irritation score of Comparative Example 3 was 2.4, indicating mild irritation, demonstrating that the gel formulation of this invention has better ocular safety compared to ordinary suspension eye drops. These results indicate that the compound ocular in-situ gel composition prepared by this invention has low ocular irritation and good safety.

[0157] VI. Preliminary Stability Study

[0158] Test method: The samples of each embodiment were placed under accelerated conditions, specifically 40℃±2℃ and 75%RH±5%RH, for 3 months, and their appearance, pH, viscosity, content, and related substances were measured. Example 1 is used as a representative example below; the trends of change in the other examples are similar to those in Example 1.

[0159] Table 3 Comparison of Long-Term Performance Changes of the Product in Example 1

[0160]

[0161] The ophthalmic in-situ gel prepared in Example 1, after being tested under accelerated conditions for 3 months, showed no significant changes in its key quality attributes, including appearance, pH value, viscosity, active pharmaceutical ingredient content, and related substances, and all indicators met the predetermined standards. The results indicate that this formulation possesses excellent physical and chemical stability, suggesting good shelf-life potential. Furthermore, statistical analysis showed that all examples met quality standards within 3 months under accelerated conditions, with no significant changes in appearance, pH, viscosity, content, and impurities, indicating that the compositions of this invention possess excellent physical and chemical stability and suggest good shelf-life potential.

[0162] The compound ophthalmic in-situ gel compositions prepared in Examples 1-7 of this invention successfully overcome the poor solubility of isoxazoline drugs through a composite solubilizing system, achieving long-term retention and sustained release on the ocular surface in conjunction with the in-situ gel matrix. Examples 3-7, which further incorporate CD-MOF and ionic liquids, are significantly superior to Examples 1-2, which only contain a composite solubilizing system, in terms of solubilizing ability, biphasic release, synergistic acaricidal effect, and physical stability, and are even superior to the comparative examples. Simultaneously, all examples exhibit low ocular irritation and good stability. Therefore, this invention provides a highly effective, long-lasting, and safe new local treatment option for ocular Demodex mite infection-related diseases.

[0163] VII. In vivo pharmacodynamic evaluation of Demodex mite infection

[0164] Experimental animals: Healthy SPF-grade New Zealand white rabbits, weighing 2.0-2.5 kg, were selected, with half males and half females. Before the experiment, slit-lamp examination confirmed that there was no inflammation in both eyelids, no scales or secretions at the base of the eyelashes, and the microscopic examination for mites was negative.

[0165] Model establishment: Demodex folliculorum was isolated from eyelash samples collected from the eyelid margins of Demodex-positive patients and stored in culture medium at 4°C for later use. For model establishment, approximately 50-100 live mites were suspended in 20 μL of sterile saline and inoculated into the base of the eyelashes and the skin of the eyelid margin of both eyes of rabbits, once daily for 7 consecutive days. Eyelid manifestations were observed daily during the inoculation period. On day 8, eyelashes were randomly selected from model rabbits and plucked. Successful model establishment was defined as when the mite count per eyelash was ≥3, microscopic examination confirmed successful mite colonization, and inflammatory manifestations such as eyelid margin congestion and scaling appeared. The negative control group was not inoculated with mites but only received an equal volume of saline solution as a normal control.

[0166] The successfully modeled rabbits were randomly divided into 12 groups, with 6 rabbits (12 eyes) in each group. These groups were Example 1-7, Comparative Example 1-3, Model Control Group, and Negative Control Group.

[0167] Rabbits in each group were administered the drug once daily, with 50 μL dripped into the upper and lower eyelids and the base of the eyelashes in both eyes, for 14 consecutive days. The model control group received no drug treatment, while the negative control group received an equal volume of physiological saline. Various indicators were measured on day 0 before drug administration and on day 14 after drug administration.

[0168] Evaluation indicators:

[0169] (a) Demodex folliculorum count in eyelashes. Before and on day 14 after drug administration, two eyelashes were plucked from each eyelid using sterile microforceps under a slit-lamp microscope. Glycerin was added to each eyelash, and a coverslip was placed on the slide. The number of Demodex folliculorum mites on each eyelash was observed and counted under an optical microscope. The average number of mites per eyelash was calculated, and the mite reduction rate was also calculated. Mite reduction rate (%) = (Average mite count before drug administration - Average mite count after drug administration) / Average mite count before drug administration × 100%.

[0170] (II) Eyelid Signs Scoring. Eyelid signs were assessed using a 0-3 scoring system, where 0 = no abnormalities; 1 = mild, characterized by slight eyelid congestion and a small amount of scaling; 2 = moderate, characterized by significant eyelid congestion, cylindrical scales or sleeve-like secretions at the base of the eyelashes, and sparse eyelashes; and 3 = severe, characterized by severe eyelid congestion and thickening, abundant cylindrical scales, significant eyelash loss, and possibly meibomian gland obstruction. Each eye was scored independently, and the average score for both eyes was used. The negative control group, as no model was established, had normal rabbit eyelids without inflammation, and their baseline and overall score during the experiment was 0.

[0171] Experimental results:

[0172] (a) Changes in the number of mites

[0173] Before administration, there was no significant difference in mite count at the base of the eyelashes of rabbits in the model control group and each administered group, and no mites were detected in the negative control group. After administration, all groups in the examples showed significant acaricidal effects. In Example 1, the reduction rate reached 82.5% on day 14 after administration, and the mite count was significantly lower than that in the model control group (P<0.01). Example 2 showed comparable effects to Example 1, with a reduction rate of 80.3% on day 14. Examples 3-7 showed even more significant acaricidal effects, with reduction rates of 92.8%-96.5% on day 14, demonstrating extremely significant mite eradication (P<0.001). Example 3 showed the best effect, with a reduction rate of 96.5% on day 14. Comparative Example 1 and Comparative Example 2 showed mite reduction rates of 68.3% and 66.7% on day 14, respectively, which were better than the model control group but weaker than Examples 1-2, possibly due to uneven drug release caused by instability during solubilization. Comparative Example 3: The mite reduction rate on day 14 after administration was only 41.2%, significantly lower than all example groups (P<0.01), indicating that the efficacy of conventional preparations without in-situ gel retention is limited. Model control group: The mite reduction rate on day 14 was 6.8%. Negative control group: No mites were detected throughout the entire process.

[0174] (II) Changes in eyelid physical signs score

[0175] Before administration, the eyelids of rabbits in the model control group and each administration group showed varying degrees of inflammation such as congestion and scaling, with an average physical examination score of 2.2-2.5 points, while the negative control group had a score of 0 points.

[0176] On day 14 after administration, the average physical examination scores of groups 1 and 2 decreased to 0.9 and 1.0, respectively, with significant reduction in eyelid congestion and scaling. The average physical examination scores of groups 3-7 further decreased to 0.3-0.6, with group 3 decreasing to 0.3. The appearance of the eyelids essentially returned to normal, with no obvious congestion or scaling. The physical examination scores of comparative groups 1-2 were 1.4 and 1.5, respectively, showing less improvement than in groups 1-2. The physical examination score of comparative group 3 was 1.7. The physical examination score of the model control group remained at 2.2, with no significant improvement. The negative control group remained at 0. Figure 1 As shown, the AL group corresponds to the negative control group, the model control group, the Example 1 group, the Example 2 group, the Example 3 group, the Example 4 group, the Example 5 group, the Example 6 group, the Example 7 group, the Comparative Example 1 group, the Comparative Example 2 group, and the Comparative Example 3 group, respectively. Figure 1 In the above, if the letters a, b, and c are the same, it means there is no statistically significant difference between the corresponding groups (P > 0.05), while if the letters are different, it means there is a statistically significant difference between the corresponding groups (P < 0.05).

[0177] In addition, on day 14 after drug administration, eyelid tissue from each group of rabbits was collected for HE staining and microscopic examination. The results showed:

[0178] In the model control group, numerous Demodex mites and their eggs were observed in the eyelid hair follicles. The hair follicles were significantly dilated, with abundant lymphocytes and plasma cells infiltrating the surrounding area and dermis. The meibomian gland ducts were dilated with inflammatory cell infiltration, and the epidermis was thickened. In Examples 1-2, the number of mites in the hair follicles was significantly reduced, the hair follicle structure tended to be normal, inflammatory cell infiltration was reduced, and the meibomian gland structure improved. In Examples 3-7, mites and their eggs were rarely seen or absent in the hair follicles, the hair follicle structure was intact, inflammatory cell infiltration was essentially eliminated, the meibomian gland structure was close to normal, and the epidermal thickness returned to normal. In Comparative Examples 1-2, the number of mites was reduced but not completely; the hair follicles were still slightly dilated, and inflammatory infiltration was reduced compared to the model control group but still visible. In Comparative Example 3, a large number of mites were still visible, hair follicle dilation and inflammatory infiltration were significant, and the meibomian gland structure was disordered. In the negative control group, the hair follicle structure was normal, there were no mites, no inflammatory cell infiltration, and the meibomian glands were normal.

[0179] The in vivo pharmacodynamic experiments against Demodex mite infection described above demonstrate that the compound ophthalmic in-situ gel compositions prepared in Examples 1-7 of this invention have significant therapeutic effects against Demodex mite infection. Specifically, Examples 1-2, through the synergistic effect of nitroimidazole and isoxazoline, effectively kill mites and reduce inflammation. Examples 3-7, by further introducing functional ionic liquids, significantly improved both the acaricidal and anti-inflammatory effects, and pathological histology showed that the skin tissue essentially returned to normal. Comparative Example 3, lacking in-situ gel retention and sustained-release properties, showed significantly lower efficacy than the examples of this invention. These results prove that this invention, through a composite solubilization system, in-situ gel technology, and the synergistic effect of CD-MOF and ionic liquids, achieves a highly efficient, long-lasting, and safe therapeutic effect against Demodex mite infection.

[0180] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A compound ophthalmic in-situ gel composition for treating ocular Demodex mite infection, characterized in that, By weight percentage, it contains 0.1-2.0% nitroimidazole compounds, 0.05-3.0% isoxazoline compounds, 0.1-1.5% gelling polymers, 1.0-8.0% complex solubilizing systems, 0.1-3.0% pharmaceutically acceptable excipients, and the balance being water; The composite solubilizing system includes polyvinylpyrrolidone and cyclodextrin derivatives.

2. The compound ophthalmic in-situ gel composition according to claim 1, characterized in that, The nitroimidazole compounds are selected from metronidazole, tinidazole, ornidazole and their pharmaceutically acceptable salts, and the isoxazoline compounds are selected from loteranal, saloranal, fleraranal and their pharmaceutically acceptable salts or crystal forms.

3. The compound ophthalmic in-situ gel composition according to claim 1, characterized in that, The gelling polymer is carbomer or low-acyl gelling gel.

4. The compound ophthalmic in-situ gel composition according to claim 1, characterized in that, The K value of the polyvinylpyrrolidone is 30, and the cyclodextrin derivative is sulfobutyl-β-cyclodextrin or hydroxypropyl-β-cyclodextrin; The mass ratio of polyvinylpyrrolidone to cyclodextrin derivative is 1:(0.5-2).

5. The compound ophthalmic in-situ gel composition according to claim 1, characterized in that, The excipients include isotonicity regulators, pH regulators, and preservatives; The compound ophthalmic in situ gel composition further comprises 0.2-2.0% by weight of an auxiliary thickener, wherein the auxiliary thickener is at least one selected from hydroxypropyl methylcellulose and sodium hyaluronate.

6. The compound ophthalmic in-situ gel composition according to claim 1 or 5, characterized in that, The compound ophthalmic in-situ gel composition has a pH of 5.5-7.0 and an osmotic molar concentration of 280-320 mOsmol / kg, and is effective at 25°C and 0.1 s. -1 At a shear rate of , the viscosity of the gel after formation is 3000-15000 cP.

7. The compound ophthalmic in-situ gel composition according to claim 1, characterized in that, It also contains 0.2-2.0% cyclodextrin metal-organic framework nanocarriers and 0.1-0.5% functional ionic liquids by weight percentage; The cyclodextrin metal-organic framework nanocarrier is a cyclodextrin metal-organic framework nanocarrier with potassium ions as the metal center. The functional ionic liquid is either a choline-geranic acid ionic liquid or a choline-cinnamic acid ionic liquid.

8. A method for preparing a compound ophthalmic in-situ gel composition, characterized in that, Includes the following steps: S1: Isoxazoline compounds and polyvinylpyrrolidone are prepared into a solid dispersion, and then the solid dispersion and cyclodextrin derivative are dissolved in water to obtain a solubilized drug solution; S2: Dissolve nitroimidazole compounds in water to obtain nitroimidazole solution; S3: Disperse the gelling polymer in water, add excipients, and obtain a gel matrix; S4: Add the solubilizing drug solution and nitroimidazole drug solution to the gel matrix, mix well, adjust the pH, then add the remaining water, and post-process to obtain the compound ophthalmic in-situ gel composition.

9. The preparation method according to claim 8, characterized in that, In step S1, the first part of isoxazoline compounds and polyvinylpyrrolidone are prepared into a solid dispersion, and then the solid dispersion and cyclodextrin derivative are dissolved in water to obtain a solubilized drug solution; Between step S1 and step S2, the process further includes: loading the second part of isoxazoline compounds into a cyclodextrin metal-organic framework nanocarrier to obtain drug-loaded nanoparticles; then dissolving the third part of isoxazoline compounds in a functional ionic liquid to obtain an ionic liquid complex. In step S4, the solubilizing drug solution, drug-loaded nanoparticles, ionic liquid complex and nitroimidazole drug solution are added to the gel matrix, mixed and the pH is adjusted, and then the remaining water is added. The post-processing yields the compound ophthalmic in-situ gel composition. The first part consists of isoxazoline compounds, which account for 50-90% of the total mass of isoxazoline compounds; the second part consists of isoxazoline compounds, which account for 5-30% of the total mass of isoxazoline compounds; and the third part consists of isoxazoline compounds, which account for 5-20% of the total mass of isoxazoline compounds.

10. The use of a compound ophthalmic in situ gel composition according to any one of claims 1-7 or a compound ophthalmic in situ gel composition prepared by the preparation method according to any one of claims 8-9 in the preparation of a medicament for treating or preventing ocular Demodex mite infection; The ocular Demodex mite infection is Demodex blepharitis, Demodex-associated meibomian gland dysfunction, Demodex-associated keratoconjunctivitis, or rosacea-associated ophthalmopathy.

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