A long-acting ophthalmic preparation for improving symptoms of blepharitis

Abstract

The present application relates to the field of pharmaceutical preparations, in particular to a long-acting eye composite preparation for improving the symptoms of blepharitis. The preparation comprises a combination of two active ingredients in a therapeutically effective amount, and one or more ion-sensitive polymers or their complex systems, which are used to treat the symptoms of blepharitis caused by Demodex through a special double-cavity drug delivery bottle. By using a specific ion gel matrix concentration and regulating the viscosity of the drug solution before it reaches the eye, the limitations of traditional eye preparations are overcome, the retention time of the drug in the eye is prolonged, the bioavailability is improved, the probability of adverse events is reduced, and the medication compliance is increased.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical preparations, specifically to a long-acting ophthalmic preparation that can improve symptoms of blepharitis. Background Technology

[0002] Demodex mites were first discovered in earwax in 1842. In 1967, they were found to be a contributing factor to blepharitis. In 1984, the first report in China detailed Demodex mite infestation of eyelashes. Demodex mites are common, permanent, small parasitic mites. There are two types that parasitize the human eye: *Demodex folliculorum*, which lives in the hair follicles of the eyelashes, and *Demodex brevis*, which lives in the sebaceous glands and meibomian glands associated with the eyelashes. The parasitic life cycle of Demodex mites has five stages, with a relatively short lifespan of about 15 days. Due to this short lifespan, mating between males and females plays a significant role in the infection process. Demodex mites feed on keratinocytes and sebum secretions. Mating usually occurs at night at the hair follicle openings. Reproduction peaks after a sufficient food supply is obtained, and during this period, Demodex mites on the face can also spread to the eyes, exacerbating the eye infection.

[0003] Demodex folliculorum feeds on hair follicle epithelial cells and metabolic products, leading to follicle dilation, hyperplasia, excessive keratinization, and scaling at the base of the eyelashes, causing eyelash loss, trichiasis, and irregular eyelashes—symptoms known as anterior blepharitis. Demodex sesquiterpenes reside in the meibomian glands and their openings, causing blockage and dysfunction, which in turn affects tear film stability. Some scholars believe that meibomian gland dysfunction caused by Demodex sesquiterpenes is one of the causes of dry eye syndrome. Besides dry eye syndrome, meibomian gland dysfunction can also cause meibomian cysts. Furthermore, the hard exoskeleton surrounding the Demodex mites, which causes a foreign body sensation in the eye, can trigger a granulomatous reaction, leading to chalazion. The above symptoms are known as posterior blepharitis; when the inflammation of the eyelid margin caused by Demodex mites spreads to the conjunctiva, it can cause palpebral conjunctivitis; and trichiasis caused by Demodex mite infection can lead to punctate loss of corneal epithelium, which can eventually form corneal ulcers and potentially cause keratitis. Besides the pathogenicity of the Demodex mites themselves, the mites can also act as vectors for other pathogens. Typical clinical manifestations include itchy eyes, foreign body sensation, dry eyes, eyelid congestion, scaling, and cuff-like discharge at the base of the eyelashes. The disease causes significant damage to the eyelid margin and is prone to recurrence, leading to reduced lipid secretion, decreased tear film stability, and consequently, various other diseases.

[0004] The main transmission routes of Demodex mites are direct contact with the skin of an infected person or indirect contact with clothing, towels, and dust containing Demodex mites. Infection is also closely related to personal hygiene, sebum secretion, and the use of cosmetics. The incidence of Demodex mites increases when the host's immunity is weakened. Because of its wide transmission routes, the fact that it does not cause disease for most of its parasitic period, and its close association with the host's personal hygiene habits and immunity, it can be concluded that diseases caused by Demodex mites cannot be cured.

[0005] In light of the above, it is essential to develop a formulation for improving the symptoms of blepharitis caused by Demodex mites. The eye is a vital and sensitive organ. Currently, the most common topical treatment for eye diseases is eye drops. However, the unique structure of the eye makes it difficult for drugs to reach the ideal therapeutic concentration at the target site. Factors such as the blink reflex (BR), tear film dilution, and nasolacrimal duct drainage result in drug bioavailability typically below 5%. Furthermore, conventional eye drops usually require multiple daily administrations, which often leads to poor convenience. In addition, according to incomplete statistics, some people experience anxiety when using eye drops due to eye sensitivity, leading to administration failure. This situation, coupled with the need for multiple daily administrations, often causes patient compliance problems, thus affecting the treatment effect.

[0006] In existing technologies, tea tree oil of different concentrations is commonly used to treat blepharitis symptoms caused by Demodex mites. A study published by the Eye Hospital Affiliated to Nanchang University, which recorded and statistically analyzed clinical data of 120 patients with blepharitis caused by Demodex mite infection who visited the hospital between 2017 and 2019, showed that tea tree oil has the function of treating Demodex mite symptoms, and the therapeutic effect of tea tree oil is directly proportional to its concentration.

[0007] However, the treatment method in this study has a flaw: the concentration of tea tree oil needs to be controlled. Existing literature shows that tea tree oil with a concentration of 15% or higher can effectively kill Demodex mites, but a concentration of 10% tea tree oil can burn the eyes and damage the cornea. In the above-mentioned experiments, experimental groups using 20% ​​and 25% concentrations both produced different adverse reactions. Therefore, the concentration of tea tree oil reagents on the market usually does not exceed 5%, which leads to a significant reduction in its mite-killing efficiency.

[0008] A study published by Wuhan Aige Eye Hospital investigated 283 patients with Demodex blepharitis, using three different treatment methods. Data recording and analysis revealed that the patients were divided into three groups: Group A, treated with optimized intense pulsed light (IPL); Group B, treated with tea tree oil eye patches; and Group C, treated with a combination of optimized IPL and tea tree oil eye patches. The results showed that Group C, the patients treated with the optimized IPL combined with tea tree oil eye patches, experienced significant improvement in their blepharitis symptoms.

[0009] Although this study represents a breakthrough in treatment, the widespread transmission routes of Demodex mites, the fact that they remain asymptomatic for most of their lives, and their close association with the host's personal hygiene habits and immune system mean that patients often do not seek aggressive treatment at the onset of symptoms. Furthermore, the cost of using optimized intense pulsed light therapy is a significant financial burden for patients, which can lead to resistance to treatment in some patients in the early stages of the disease, thus exacerbating their condition.

[0010] Existing technology discloses a hospital-prepared formulation that combines tea tree oil and metronidazole. Specifically, 5% tea tree oil is first applied, followed by metronidazole injection solution applied evenly to the eyelashes and facial margins using a cotton swab. Experimental results have shown its effectiveness, improving symptoms of blepharitis caused by Demodex mites. However, due to the very small volume of the conjunctival sac, there are limitations on the dosage for each administration. Once the eye drops enter the conjunctival sac, they can pass through the lacrimal punctum into the lacrimal canaliculi, lacrimal sac, and nasolacrimal duct, and then through the inferior turbinate into the nasal cavity and pharyngeal wall. Therefore, patients often report a bitter taste in their mouth during use. Furthermore, because it is a hospital-prepared formulation, this treatment method is undoubtedly unfavorable for the promotion and use of the formulation.

[0011] Chinese Patent 2022104012873 discloses a metronidazole ophthalmic gel formulation for treating eye infections caused by mites and Acanthamoeba. However, it suffers from the defects of traditional ophthalmic formulations, namely, short drug residence time in the eye, poor stability, easy to be affected by tears leading to low bioavailability, long treatment cycle, need to be administered multiple times a day, and poor patient compliance.

[0012] Currently, novel formulations researched both domestically and internationally, such as ointments, suspensions, and implants, have extended the residence time to varying degrees, but still have some drawbacks. For example, petrolatum-based ointments are greasy and can easily cause blurred vision after use. In clinical practice, patients' compliance with implants is currently poor, and the above-mentioned improvements still cannot effectively solve the defects of traditional ophthalmic formulations.

[0013] Chinese Patent 021095035 discloses an ophthalmic formulation using poloxamer 188 and poloxamer 407 as a temperature-sensitive matrix. The claims mention that the active ingredient is selected from one or a combination of anti-glaucoma drugs, antibiotics, antibacterial drugs, and antiviral drugs. However, no examples are provided to support a specific formulation combining the drug with the temperature-sensitive matrix. Those skilled in the art cannot reproduce the technology based on the specification, presenting a technical obstacle. The specification fails to demonstrate the effect of combining the drug with the temperature-sensitive matrix, either because a feasible formulation cannot be designed, or because there are technological difficulties in its preparation. These points represent the technical challenges in the development of in-situ formulations.

[0014] Therefore, it is essential to develop a long-acting ophthalmic formulation that can effectively improve blepharitis symptoms and overcome the shortcomings of traditional formulations. Summary of the Invention

[0015] The inventors carefully studied and attempted to combine the existing therapeutic agent tea tree oil with metronidazole, and then add a temperature-sensitive in-situ gel matrix to form a compound in-situ gel formulation to solve the above problems. However, during multiple trials, due to the properties of tea tree oil itself, a satisfactory formulation could not be selected. Furthermore, the method of administering the two drugs separately only appeared in the prescriptions of hospital-prepared formulations, which also prevented patients from using them on their own.

[0016] After continuous experimentation and optimization, the inventors decided to use a dual-cavity composite eye drop bottle structure protected in authorized patent CN219271598U for drug delivery, placing tea tree oil separately in one cavity, thus overcoming its instability. To better illustrate the technical solution of this application, parts of the aforementioned utility model are incorporated herein by reference.

[0017] After solving the above problems, the inventors combined the temperature-sensitive gel matrix disclosed in the prior art with metronidazole to form a gel formulation, placed it on one side of a dual-chamber composite drug delivery bottle, and applied it to the affected area together with tea tree oil on the other side. However, during the experiment, a serious defect was found in the temperature-sensitive metronidazole gel: due to the special properties of the temperature-sensitive gel matrix poloxamer, it takes a certain amount of time for it to change from a liquid to a solid state, which is not conducive to drug delivery. To solve the above problem, the inventors conducted continuous screening and research, and finally abandoned the temperature-sensitive gel matrix. While researching in-situ gel technology, the inventors accidentally discovered that using an ion-sensitive in-situ gel matrix could improve the above problem. The matrix contains ion-sensitive polymers, and when administered in solution form, it can quickly form a gel after contacting the anterior tear film of the cornea. During the experiment, it was unexpectedly discovered that when the metronidazole component with the ion-sensitive gel matrix deforms upon binding with the tear film, both the tea tree oil and metronidazole therapeutic components can be retained simultaneously, and there is no need to strictly control the temperature and pH conditions in the application environment.

[0018] The present invention is achieved through the following scheme.

[0019] A long-acting ophthalmic formulation for improving symptoms of blepharitis, comprising a therapeutically effective amount of a combination of two active ingredients, one or more ion-sensitive polymers or their complex systems, and a dual-chamber delivery bottle, characterized in that:

[0020] Active ingredient 1 is metronidazole; active ingredient 2 is tea tree oil; the drug is administered via a double-chambered drug delivery bottle.

[0021] In some implementations, ophthalmic preparations are used to treat blepharitis symptoms caused by Demodex mites.

[0022] In some implementations, metronidazole is present in the formulation at a concentration of 1-2%; tea tree oil is present in the formulation at a concentration of 5%.

[0023] In some embodiments, the ion-sensitive gel matrix component, including metronidazole, is located in one cavity of the dual-cavity vial; tea tree oil is placed in the other cavity.

[0024] In some implementations, a side cavity of an ion-sensitive gel matrix including metronidazole is included, but tea tree oil is excluded.

[0025] In some implementations, a side cavity including tea tree oil is included, but metronidazole or its gel matrix component is excluded.

[0026] In some implementations, the ion-sensitive polymer is one or more of gellan gum, xanthan gum, alginate, or a combination thereof.

[0027] In some implementations, the gellan gum in the ion-sensitive polymer may specifically be a high-acyl gellan gum or a deacetylated gellan gum.

[0028] In some implementations, the compounding system in the ion-sensitive polymer may be gellan gum-xanthan gum, gellan gum-carbomer, gellan gum-hydroxypropyl methylcellulose, alginate-deacetylated gellan gum, deacetylated gellan gum-xanthan gum, or other compounding systems.

[0029] In some implementations, the preferred compounding system in the ion-sensitive polymer is a deacetylated gellan gum-xanthan gum compounding system or a sodium alginate-deacetylated gellan gum compounding system.

[0030] In some implementations, xanthan gum has been found to improve the comfort of the administration process and reduce irritation from the combined use of metronidazole and tea tree oil.

[0031] In some implementation schemes, it has been found that the deacetylated gellan gum-xanthan gum blend system has significant advantages in gelling properties, cohesiveness, and water retention compared to the single deacetylated gellan gum system.

[0032] In some embodiments, the compound composition may also include an osmotic pressure regulator.

[0033] In some implementations, the osmotic pressure regulator cannot be sodium chloride.

[0034] Beneficial effects: 1. Optimizes existing treatment methods for blepharitis caused by Demodex mites; 2. The preparation process of the ion-sensitive metronidazole in-situ gel component is simple, convenient, low-cost, and easy for large-scale production; 3. Achieving unexpected therapeutic effects by simultaneously applying metronidazole and tea tree oil, two active ingredients, through a special double-chamber administration bottle; 4. Overcomes the limitations of traditional ophthalmic preparations, prolongs the drug's residence time in the eye, improves bioavailability, reduces the burden on patients, and increases medication adherence. Attached Figure Description

[0035] Figure 1 Symptom 1 of Demodex mite infestation of the eyelid area in Example 2.

[0036] Figure 2 Symptom 2 of Demodex mite infestation of the eyelid area in Example 2.

[0037] Figure 3 Symptom 3 is the eyelid infection caused by Demodex mites in Example 2.

[0038] Figure 4 This is the improvement in the eyelid area after the application of metronidazole in Example 2.

[0039] Figure 5This is a front view of the dual-chamber ophthalmic solution administration bottle in Example 4.

[0040] Figure 6 This is a front view of the dual-chamber ophthalmic solution administration bottle in Example 4.

[0041] Figure 7 This is a front view of the cap of the dual-chamber ophthalmic solution administration bottle in Example 4.

[0042] Figure 8 This is a top view of the dual-chamber ophthalmic solution administration bottle in Example 4.

[0043] Figure 9 This study compares the drug release results of a standard metronidazole solution formulation with that of a metronidazole ion-sensitive in-situ gel formulation.

[0044] In the attached diagram, 1 is outlet a; 2 is outlet b; 3 is bottle mouth (with threads); 4 is bottle body; 5 is squeeze pad; 6 is bottle body divider; 7 is bottle mouth divider; 11 is retainer; 12 is bottle cap (with threads); 13 is outlet b cap; 14 is outlet a cap. Detailed Implementation

[0045] Those skilled in the art should understand that these embodiments are merely for explaining the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0046] Example 1: Concentration screening of gel matrix (compound system)

[0047] Simulated tear preparation: Dissolve 2.18g Na2CO3, 6.78g NaCl, 0.042g CaCl2·2H2O, and 1.38g KCl in 1L of water. To prevent CaCl2 from reacting with Na2CO3 and precipitating out, prepare them separately and then mix them slowly. Adjust the pH of the solution to 7.3.

[0048] Preparation of gellan gum: Take an appropriate amount of deacetylated gellan gum and add it to deionized water. Heat the mixture in a water bath at 90°C and stir it magnetically to prepare an aqueous solution of deacetylated gellan gum.

[0049] Preparation of xanthan gum: Take an appropriate amount of xanthan gum and add it to deionized water. Heat in a water bath at 90℃ for 30 minutes to prepare xanthan gum aqueous solution.

[0050] Screening of deacetylated gel solution concentrations: Deacetylated gel solutions with concentrations of 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, and 0.8% were prepared, and their viscosity changes were measured at room temperature (25℃) and under physiological ocular conditions (34℃). The deacetylated gel solution and simulated tear fluid were mixed in a ratio of 25:7, and the viscosity of the deacetylated gel was measured at 34℃ using rotor No. 2 at 12 r / min. The gelation process was observed, and the results are shown in the table below.

[0051] Table 1. Gelation ability of deacetylated gel solutions at different concentrations

[0052] concentration(%) 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Gel ability solution Weak gel Weak gel Weak gel gel gel gel

[0053] Table 2 Viscosities (mPa.s) of deacetylated gellan gel solutions of different concentrations

[0054] concentration(%) 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Room temperature (25℃) 7.50 10.00 12.60 63.50 601.00 1301.00 2105.00 Eye temperature (34℃) 2.30 5.10 7.15 10.10 10.55 12.50 15.00 Tears mixed (34℃) 11.30 66.35 530.10 916.20 1755.20 2398.00 /

[0055] " / " indicates that the measurement range is exceeded.

[0056] Xanthan gum solution concentration screening: Xanthan gum solutions of 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% were prepared, and their viscosity changes were measured at room temperature (25℃) and under physiological conditions of the eye (34℃). The measurement conditions were rotor No. 2, 12 r / min, and the gelation was observed. The results are shown in the table below.

[0057] Table 3. Gel-forming ability of xanthan gum solutions at different concentrations

[0058] concentration(%) 0.1 0.2 0.3 0.4 0.5 Gel ability solution solution solution viscous solution viscous solution

[0059] Table 4. Viscosities (mPa.s) of xanthan gum solutions at different concentrations

[0060] concentration(%) 0.1 0.2 0.3 0.4 0.5 Room temperature (25℃) 135.00 322.00 541.00 800.00 932.00 Eye temperature (34℃) 123.00 272.00 500.00 781.00 1106.00

[0061] Conclusions: At 25℃, the viscosity of deacetylated gellan gum solution increased with increasing concentration, while at 34℃, the viscosity of different concentrations of deacetylated gellan gum solution showed little change. The viscosity of deacetylated gellan gum solution of the same concentration at room temperature was significantly greater than that at 34℃. Furthermore, the viscosity of deacetylated gellan gum solution increased significantly after mixing with STF simulated tears (25:7), indicating that under the influence of cations in the tears, the deacetylated gellan gum solution undergoes a phase transition and forms a gel. Xanthan gum solution showed little viscosity change at different temperatures and, when existing alone, does not interact with ions in STF to form a gel; it primarily functions as a bioadhesive.

[0062] The advantages of the deacetylated gellan gum-xanthan gum gel matrix system are: 1. Improved mucosal adhesion of the formulation; 2. Increased stability and viscoelasticity of the gel formed after phase transition. Considering the dropability of ion-sensitive in-situ gels, it is necessary to meet the requirements of low viscosity at room temperature and rapid formation of a gel of suitable viscosity upon contact with ocular tears. Therefore, the concentration range of deacetylated gellan gum in the gel matrix (compound system) is 0.4%-0.6%, and the concentration range of xanthan gum is 0.1%-0.3%.

[0063] Example 2: Treatment trial for Demodex blepharitis

[0064] A prospective clinical trial included 10 patients with blepharitis and keratitis, aged 23 to 78 years (mean age 48.7 ± 13.7 years), with a disease duration of 1 month to 4 years (mean 2.11 ± 0.54 years). Routine ophthalmic examinations were conducted, including visual acuity testing. Under a slit-lamp microscope, the eyelid skin, eyelashes and their roots, eyelid margins, meibomian gland morphology and openings, bulbar conjunctiva, and cornea were examined sequentially. Eyelash sampling, Demodex folliculorum examination, and corneal fluorescein staining were also performed. For the Demodex folliculorum examination, eyelash sampling was performed under a slit-lamp microscope, prioritizing eyelashes with scales or cuff-like structures. Two eyelashes were collected from the upper eyelid and one from the lower eyelid, typically 3-4 from each eye. The plucked eyelashes were placed on a slide; for eyelashes with more cuff-like structures, one drop of 0.9% sodium chloride solution was slowly applied to the side edge of a coverslip. A 0.9% sodium chloride solution was slowly diffused into the slide, and the entire process was observed, recording the total number of Demodex mites on the eyelashes. A combined wiping method using metronidazole solution and 5% tea tree oil was also used. After 5-10 days of treatment, conjunctival hyperemia and discomfort symptoms disappeared in 10 patients, eyelid margin hyperemia and thickening significantly improved, eyelashes became cleaner, and scales or cuff-like growths disappeared in some patients.

[0065] Another study, Wacker, T.; Lang, G. (2014). Demodex folliculorum: Diagnostik und Therapie im klinischen Alltag. Klinische Monatsblätter für Augenheilkunde, 231(3), 241–245, investigated trials of treating Demodex mites with therapeutically effective doses of metronidazole, which is incorporated herein by reference. The symptoms of Demodex mite infestation on the eyelids before the trial were as follows: Figures 1-3 As shown, the effects after applying metronidazole are as follows: Figure 4As shown, Demodex mites cause hair follicle dilation, hyperplasia, excessive keratinization, and scaling at the base of the eyelashes, leading to eyelash loss, trichiasis, and irregular eyelashes, thus improving blepharitis symptoms. The above research results indicate that using 1-2% metronidazole preparations can effectively improve blepharitis symptoms caused by Demodex mites.

[0066] The results of the two sets of experiments above show that the use of therapeutically effective doses of metronidazole can effectively improve the symptoms of blepharitis caused by Demodex mites. Furthermore, the effect of symptom improvement is more pronounced when metronidazole is used in combination with a certain proportion of tea tree oil.

[0067] Example 3: Screening of Metronidazole Ion-Sensitive In-Situ Gel Formulation

[0068] To ensure good flowability of the ion-sensitive ophthalmic gel at room temperature (25°C), its viscosity should be below 1500 mPa·s. After mixing with STF (25:7, 34°C), to ensure good retention in the eye, the viscosity of the resulting gel should not be below 2500 mPa·s. Preliminary experiments revealed the effect of the solution on the viscosity of the gel matrix. Using a star-point design-response surface methodology, experiments were conducted on the gel matrix at room temperature (25°C) and after mixing with tears (34°C). The final determined optimal gel combination consisted of 0.50-0.60% deacetylated gellan gum and 0.20-0.25% xanthan gum.

[0069] Screening of excipients: Common osmotic pressure regulators include sodium chloride and mannitol. Since the cations in sodium chloride will cause the deacetylated gel in the formulation to change from solution to gel state, 2.5% (w / v) mannitol is selected to adjust the osmotic pressure of the formulation. Tris solution is selected as the pH regulator to adjust the pH of the solution to between 7.0 and 7.5.

[0070] The composition of the formulation is shown in the table below.

[0071] Table 5. Metronidazole Ion-Sensitive In-Situ Gel Formulation

[0072] Element Dosage Metronidazole 1、1.5、1.75、2% Deacetylated gel 0.5、0.55、0.6% Xanthan Gum 0.2、0.25% Mannitol 2.5% Tris solution Appropriate amount (adjust pH to 7.0-7.5)

[0073] Add water for injection to a final volume of 100 ml.

[0074] Preparation process: (1) Weigh the prescribed amount of metronidazole, add an appropriate amount of sterile deionized water, sonicate to dissolve, then weigh the prescribed amount of mannitol and add it to the metronidazole solution, mix it evenly, and adjust the pH to the above range with 2% Tris solution; (2) Weigh the prescribed amount of gellan gum (deacetylated gellan gum) and xanthan gum, add sterile deionized water, the amount of which is 75% of the total weight of water, stir in a water bath at 90°C for 30 min, and cool to room temperature for later use; under sterile conditions, combine the products of steps (1) and (2), then adjust the pH of the mixture to the above range with 2% Tris solution, add the remaining amount of water for injection, sonicate for 20 min to mix evenly, dispense, sterilize, and the metronidazole ion-sensitive in situ gel is obtained.

[0075] This embodiment provides 24 different metronidazole ion-sensitive in-situ gel formulations.

[0076] Example 4: Dual-chamber eye solution administration bottle

[0077] The structure of the dual-chamber ophthalmic solution administration bottle is as follows: Figures 5-8 As shown, the features are: it can store two therapeutic agents simultaneously, and the two therapeutic agents are independent of each other; it can apply two therapeutic agents simultaneously; the bottle body is an elliptical cylinder; the bottle body has a compression pad; the thread at the bottle cap matches the thread at the bottle mouth.

[0078] The bottle has a rubber divider inside to separate the two treatment agents; there is also a rubber divider at the bottle opening to separate the two treatment agents. The squeeze pad on the bottle body has striped protrusions to make pressing the bottle easier.

[0079] The rubber divider inside the bottle forms a 90-degree angle with the squeeze pad on the bottle body, and the rubber divider inside the bottle and the squeeze pad on the bottle body are perpendicular to each other.

[0080] There are two outlets, a and b, at the bottle opening, which correspond to the administration portions of the two therapeutic agents, respectively.

[0081] The bottle cap has raised rubber caps corresponding to the two liquid outlets (a and b) at the bottle opening, ensuring a tight seal at outlets a and b. These raised caps are independent of the bottle cap and do not rotate with it. To use, align the raised caps (a and b) on the bottle cap with the corresponding outlets (a and b) at the bottle opening, then tighten the cap.

[0082] When using: Place the metronidazole ion-sensitive in-situ gel preparation in one cavity and the 5% tea tree oil in the other cavity. The bottle has a separator in the middle to ensure that the two treatment agents are independent of each other. When using, point the bottle mouth at the eye and press the squeeze pad to make the two treatment solutions flow out from outlets a and b respectively and act on the eye. Because there is a separator at the bottle mouth, the treatment agents will converge in the eye and will not contaminate each other.

[0083] Under the action of the ion-sensitive gel matrix, a liquid-to-solid transition is observed in a very short time, while retaining both active ingredients. Then, align the protrusion in the cap with outlets a and b. Due to the presence of the retainer, outlets a and b are independent of the cap. Afterward, tighten the cap for future use.

[0084] Example 5: In vitro release comparison test between metronidazole solution without ion-sensitive gel matrix and metronidazole ion-sensitive in-situ gelling agent.

[0085] The formulations without ion-sensitive gel matrices are shown in the table below.

[0086] Table 6 Metronidazole Solution Prescription

[0087] Element Dosage Metronidazole 2% Mannitol 2.5% Tris solution Appropriate amount (adjust pH to 7.3)

[0088] Add water for injection to 100 ml and prepare metronidazole solution using standard methods.

[0089] Release tests were conducted on two solution formulations using dialysis bags.

[0090] First, cut the dialysis bags into 10cm segments. Boil the dialysis bags in a large volume (500mL) of 2% (w / v) sodium bicarbonate and 1mmol / L EDTA·2Na (pH=8.0) solution for 10 minutes. Then thoroughly rinse the dialysis bags with deionized water. Next, boil them in 500mL of 1mmol / L EDTA·2Na (pH=8.0) solution for 10 minutes. After cooling, store them at 4°C for later use.

[0091] Using 500 mL of artificial tears as the release medium, 5 g of the formulation was added to a dialysis bag. The dissolution apparatus was set at 50 rpm and 34 °C. Samples were taken at 0.5, 1, 2, 4, 6, 8, 10, and 12 h, with 5 mL of sample taken each time and 5 mL of LTS added simultaneously. The samples were treated with sulfuric acid colorimetric method, and absorbance was measured according to the dissolution assay conditions. The cumulative release percentage was calculated, and a drug release curve was plotted. A comparison of results between ordinary ophthalmic formulations and ion-sensitive in-situ gel formulations is provided. Figure 9 As shown.

[0092] Experimental results: From Figure 9The drug release curves show that the self-made ordinary metronidazole solution was completely released after 4 hours, while the metronidazole ion-sensitive in-situ gel formulation was nearly completely released after 12 hours, with a cumulative release rate of 90% after 10 hours. This comparison demonstrates that the metronidazole ion-sensitive in-situ gel formulation has a better sustained-release effect compared to ordinary ophthalmic formulations.

[0093] The above results, combined with the accidental discovery made by the inventors during the experiment, demonstrate that by using a dual-chamber administration bottle to simultaneously apply metronidazole ion-sensitive in-situ gel matrix and 5% tea tree oil, a gel can rapidly form within 3 seconds upon contact with the corneal anterior tear film, simultaneously retaining both the tea tree oil and metronidazole therapeutic components. This indicates that the dual-chamber administration method allows the composite formulation of this application to achieve the same sustained-release effect as the metronidazole ion-sensitive in-situ gel.

[0094] Example 6: Quality assessment test of metronidazole ion-sensitive in-situ gel

[0095] Appearance and visible foreign matter detection test: This product is light yellow. Three batches of samples were prepared and observed at eye level in front of a black background. The samples were then placed in a clarity tester with the illuminance adjusted to 2000-3000 LX. Under white and black backgrounds, the sample vials were held in hand to observe for any visible foreign matter in the solution. The results are shown in the table below.

[0096] Table 7. Properties and Visible Foreign Matter Examination of Metronidazole Ion-Sensitive In-Situ Gel

[0097] serial number 1 2 3 Properties light yellow transparent liquid light yellow transparent liquid light yellow transparent liquid Visible foreign objects none none none

[0098] The results showed that the properties and visible foreign matter content of the metronidazole ion-sensitive in-situ gel met the standard requirements.

[0099] Viscosity testing: Three batches of ion-sensitive in-situ gel formulations were prepared in parallel. The viscosity of the formulations was measured using a rotational viscometer at 25°C. The viscosity of the gel after mixing the formulation with simulated tear fluid was measured at the physiological ocular temperature (34°C). The samples were measured three times in parallel, and the average value was taken. The results are shown in the table below. The results show that the viscosity of all three batches of formulation samples met the requirements.

[0100] Table 8. Viscosity test results of metronidazole ion-sensitive in-situ gel (Mean±SD, n=3)

[0101] serial number Formulation viscosity (mPa.s) Gel viscosity (mPa.s) A 1004.6±7.1 2313.4±13.6 B 980.5±11.6 2287.4±13.1 C 986.8±6.8 2295.7±17.3

[0102] The results showed that the viscosity of all three batches of metronidazole ion-sensitive in-situ gel formulation samples met the requirements.

[0103] pH testing: Three batches of ion-sensitive in-situ gel formulation were prepared in parallel, 200 mL per batch. The pH value of each sample was measured three times in parallel under the same conditions, and the average value was taken. The pH meter should be calibrated before measurement to ensure the accuracy of the results.

[0104] Table 9. Results of pH determination using metronidazole ion-sensitive in-situ gel (Mean ± SD, n = 3)

[0105] serial number First Second C pH value 7.29±0.01 7.31±0.01 7.30±0.01

[0106] The results showed that the pH values ​​of all three batches of metronidazole ion-sensitive in-situ gel formulation samples met the requirements.

[0107] Rheological testing: The effects of temperature and rotation speed on the viscosity of the ion-sensitive in-situ gel formulation were investigated using rotor No. 3. Temperature conditions ranged from 10-40℃, and rotation speeds were 5, 10, 15, 20, 25, and 30 rpm. Based on the physiological condition of the eye, the normal eyeball rotation frequency should be between 10-30 times per minute. By observing the formulation state at the corresponding rotation speeds, the viscosity of the ion-sensitive in-situ gel formulation at 25-35℃ was 500-850 mPa·s. This indicates that the formulation itself has a certain viscosity before gel formation and before it acts on the eye. Compared to ordinary eye drops, it is not easily lost; its low viscosity makes it easy to distribute on the surface of the eyeball without causing eye discomfort.

[0108] In summary, the results of measurements on the appearance, visible foreign matter, viscosity, pH, and rheology of the ion-sensitive in-situ gel formulation show that it is a light yellow-green transparent liquid with no visible foreign matter, a pH of approximately 7.3 (within the physiological pH range of the eye), and rheological results indicating that it is a non-Newtonian fluid. Upon contact with the eye, it can rapidly disperse on the eye surface, forming a gel of suitable viscosity, thereby increasing its retention effect. Therefore, the ion-sensitive in-situ gel formulation meets the quality requirements for ophthalmic preparations.

[0109] Example 7: Ocular irritation test of compound ophthalmic formulation

[0110] The Draize ocular irritation experiment was designed to evaluate the irritation of a compound preparation (metronidazole ion-sensitive in-situ gel and tea tree oil) administered via a dual-chamber vial to the eyes of rabbits. Ten healthy rabbits (five males and five females) were selected. The ocular physiological condition of each rabbit was observed and recorded before administration to confirm that the cornea, iris, and other parts of the eye were in good condition. The rabbits were provided with normal food and water before and during the experiment. A self-controlled method was used, with the left eye of each animal serving as the control group (50 µL of physiological saline) and the right eye as the experimental group (50 µL of the compound ocular preparation).

[0111] Single-dose test: Administer a single dose of saline or gel preparation, gently close the eyelids for about 1 second to ensure sufficient contact between the solution and the local area, and record the damage observed in the cornea, iris, conjunctiva and other tissues at 1, 6, 12, 24 and 48 hours after administration, expressed as the Draize eye irritation test score.

[0112] Multiple-dose trial: After continuous administration for more than one week, continue to observe for 7-14 days, and record the results using the Draize eye irritation test score.

[0113] Evaluation criteria and conclusions: Table 10 is the evaluation criteria table, and Table 11 is the evaluation results table.

[0114] Table 10 Drauze Ocular Irritation Test Scoring Sheet

[0115]

[0116] Table 11 Results of Ocular Irritation Scores for Compound Ophthalmic Preparations

[0117]

[0118] As shown in Table 11, no irritation symptoms were detected in the saline control group in both the single-dose and multiple-dose experiments. In the single-dose experimental group, mild pericorneal hyperemia was observed in rabbits 1, 2, and 8, while other rabbits showed no obvious irritation symptoms. In the multiple-dose experiment, five rabbits showed mild hyperemia, and the result was recorded as 1 point according to the eye irritation evaluation criteria. The average scores for eye irritation of the test substance were calculated based on the results of the single-dose and multiple-dose experiments, and the results were 0.3 and 0.5, respectively, both less than 3. Therefore, the compound preparation (metronidazole ion-sensitive in-situ gel and tea tree oil) administered via a dual-chamber administration bottle is non-irritating to rabbit eyes and has good biocompatibility.

[0119] Example 8: Stability test of metronidazole ion-sensitive in-situ gel

[0120] Centrifugation test: Take an appropriate amount of metronidazole ion-sensitive in situ gel and place it in a centrifuge tube. Centrifuge it at 4000 rpm for 10 min, 20 min and 30 min and observe the changes in appearance. The results are shown in the table below.

[0121] Table 12 Results of Ion-Sensitive In-Situ Gel Centrifugation Test

[0122] Centrifugation time (min) color Is it layered? 15min light yellow no 30min light yellow no 45min light yellow no

[0123] High-temperature test: Take an appropriate amount of metronidazole ion-sensitive in-situ gel and place it in a sterile colorless and transparent PET eye drop bottle. Place it at 60℃ and take samples at 0, 5, and 10 days respectively. Observe the appearance of the samples and determine the changes in pH, content, and total impurities. If the content of the test sample is lower than the specified limit, the test should be carried out at 40℃ using the same method. If there is no significant change at 60℃, the 40℃ test should not be carried out. The results are shown in the table below.

[0124] Table 13 Metronidazole ion-sensitive in situ gels at 60℃

[0125] Time (days) pH content General Miscellaneous 0 days 7.35 100.12 0.70% 5 days 7.29 99.63 1.25% 10 days 7.21 98.91 2.48%

[0126] Table 14 Metronidazole ion-sensitive in situ gels at 40℃

[0127] Time (days) pH content General Miscellaneous 0 days 7.33 100.12 0.70% 5 days 7.29 99.72 1.02% 10 days 7.28 99.46 1.11%

[0128] The results show that when the metronidazole in situ gel formulation is placed at 60℃ for 10 days, the pH value and content both decrease, while the total amount of impurities increases. When the formulation is placed at 40℃ for 10 days, the pH value and content both decrease, while the total amount of impurities increases slightly, but both are within the limits. The results indicate that ion-sensitive in situ gels are not suitable for long-term storage in high-temperature environments.

[0129] Low temperature test: Take an appropriate amount of metronidazole ion-sensitive in situ gel and put it into a sterile colorless transparent PET eye drop bottle. Place it at 4℃ and -20℃ for 10 days. Observe the appearance of the sample and measure the changes in pH, content and total impurities. The results are shown in the table below.

[0130] Table 15. Test results of metronidazole ion-sensitive in-situ gel under low temperature conditions.

[0131] Temperature (°C) pH content General Miscellaneous 4 7.32 99.98 0.83% -20 7.29 99.89 0.77%

[0132] The results show that metronidazole ion-sensitive in-situ gel formulation is relatively stable at low temperatures, with no significant changes in pH, content, or impurities. Formulation stored at -20℃ will freeze and thaw at room temperature, but can be restored to a liquid state without significant changes in viscosity.

[0133] Light exposure test: Take an appropriate amount of metronidazole ion-sensitive in situ gel and put it into a sterile colorless neutral ampoule. Place it under 4500±500 LX conditions for 10 days. Take samples at 0, 5 and 10 days respectively, observe the appearance of the samples, and measure the changes in pH, content and total impurities. The results are shown in the table below.

[0134] Table 16 Results of low-temperature test of metronidazole ion-sensitive in situ gel

[0135] Time (days) pH content General Miscellaneous 0 days 7.31 99.12 0.80% 5 days 7.15 97.61 2.12% 10 days 7.07 96.16 2.98%

[0136] The results show that metronidazole ion-sensitive in-situ gel is unstable under light conditions, with a decrease in pH value and content, and a significant increase in total impurities. Therefore, metronidazole ion-sensitive in-situ gel needs to be stored away from light. Furthermore, due to the inherent properties of tea tree oil, it also requires storage in a cool, dark place away from light. Therefore, the double-chamber ophthalmic drug delivery bottle used in this application needs to have a dark-colored, light-proof outer wall, or an additional light-proof protective sleeve added to the outside of the bottle.

[0137] Thus far, the inventors have clearly described the in-situ gel matrix screening, specific drug delivery devices, therapeutic effects of the compound preparation, ocular irritation test of the compound ophthalmic preparation, and the properties and evaluation process of the compound preparation in the technical solution.

[0138] This invention aims to provide a novel compound preparation for improving the symptoms of blepharitis caused by Demodex mites. It breaks through traditional technical biases, optimizes existing treatment methods, and utilizes a dual-chamber ophthalmic administration bottle provided by the inventor to simultaneously apply metronidazole and tea tree oil to the affected area. Furthermore, during the inventor's research and development process, continuous experimentation revealed unexpected effects from the use of an ion-sensitive matrix, overcoming existing technologies and improving upon the conventional thermosensitive gel approach using poloxamer. The invention also explored and broke through the issue of gelation time. After comprehensively considering the drug properties and administration method, this invention was finally obtained: a compound preparation for improving the symptoms of blepharitis caused by Demodex mites, consisting of metronidazole ion-sensitive in-situ gel and tea tree oil combined through a dual-chamber administration bottle.

[0139] It should be noted that the description of the embodiments in this application is merely an illustration of the principles and applications of the technical solutions of the present invention. References to details of the illustrated embodiments in this application are not intended to limit the scope of the claims, which themselves contain features considered essential to the present invention.

Claims

1. A long-acting ophthalmic compound formulation for improving symptoms of blepharitis caused by Demodex mites, comprising a combination of two active ingredients in therapeutically effective amounts with an ion-sensitive gel matrix or a compound system thereof, characterized in that: A. The two active ingredients in the long-acting ophthalmic compound preparation are metronidazole and tea tree oil, wherein metronidazole exists in the preparation at an amount of 1-2% and tea tree oil exists in the preparation at an amount of 5%. B. The two active ingredients in the long-acting ophthalmic compound preparation are stored in different cavities of the dual-cavity administration bottle, with 5% tea tree oil stored separately in one cavity and 1-2% metronidazole stored in the other cavity in the form of an ion-sensitive in-situ gel preparation. C. The ion-sensitive gel matrix is ​​a deacetylated gellan gum-xanthan gum complex, wherein the deacetylated gellan gum is present in the formulation at an amount of 0.50-0.60% and the xanthan gum is present in the formulation at an amount of 0.20-0.25%; D. The long-acting ophthalmic compound formulation also includes an osmotic pressure regulator, specifically mannitol, which is present in the formulation at a concentration of 2.5%. E. The long-acting ophthalmic compound preparation does not contain sodium chloride; F. Preparation process of the metronidazole ion-sensitive in-situ gel formulation in the long-acting ophthalmic compound preparation: (1) Weigh an appropriate amount of metronidazole, add an appropriate amount of sterile deionized water, sonicate to dissolve, then weigh an appropriate amount of mannitol and add it to the metronidazole solution, mix it evenly, and adjust the pH value with 2% Tris solution. (2) Weigh out an appropriate amount of deacetylated gellan gum and xanthan gum, add sterile deionized water, stir in a water bath at 90°C for 30 minutes, and cool to room temperature for later use. (3) Under sterile conditions, combine the products of steps (1) and (2), adjust the pH of the mixture with 2% Tris solution, add the remaining water for injection, sonicate to mix evenly, dispense, sterilize, and obtain the final product.

2. The long-acting ophthalmic compound preparation for improving blepharitis symptoms caused by Demodex mites according to claim 1, characterized in that, The pH value of the metronidazole ion-sensitive in-situ gel formulation is between 7.0 and 7.

5.

3. The long-acting ophthalmic compound preparation for improving blepharitis symptoms caused by Demodex mites according to claim 1, characterized in that, The metronidazole ion-sensitive in-situ gel formulation has a viscosity of 500-850 mPa·s between 25-35°C.

4. A long-acting ophthalmic compound preparation for improving blepharitis symptoms caused by Demodex mites according to claim 1, characterized in that, The portion of the dual-chamber drug delivery bottle where the drug is stored is dark brown or has a light-proof protective cover.

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