Oily tacrolimus preparation for treating dry eye syndrome and method for preparing same

Abstract

Provided is a tacrolimus-containing ophthalmic composition, comprising tacrolimus, medium-chain triglyceride, white petrolatum, and a matrix. On the basis of the total weight of the tacrolimus-containing ophthalmic composition, the content of the medium-chain triglyceride is greater than or equal to 3 wt % and less than or equal to 30 wt %, and the content of white petrolatum is greater than or equal to 20 wt % and less than or equal to 25 wt %. In the present invention, tacrolimus is prepared into an oily eye drop with good content uniformity, ease-to-use, good dispersion properties in the eyes, and sufficient retention time in the eyes. The preparation features good tacrolimus solubility, fluidity, and stability.

Description

Tacrolimus oily preparation for treating dry eye and preparation method thereof

[0001] Cross-references

[0002] This application claims priority to Chinese Patent Application No. 2023117731075, filed on December 21, 2023, entitled “A Tacrolimus Oily Preparation for Treating Dry Eye and Its Preparation Method,” the entire disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to an ophthalmic composition, in particular to an ophthalmic composition containing tacrolimus. Background Art

[0004] The present invention relates to an ophthalmic composition, and more particularly to an ophthalmic composition containing tacrolimus.

[0005] Tacrolimus (also known as FK506) is a macrolide antibiotic that possesses antimicrobial activity as well as immunosuppressive and anti-inflammatory effects. Tacrolimus's immunosuppressive and anti-inflammatory effects are 10 to 100 times greater than those of cyclosporine, another immunosuppressant. Therefore, it is a potent new immunosuppressant. Its chemical structure is shown below:

[0006] Tacrolimus's immunosuppressive effects are primarily achieved by binding to FK-binding protein (FKBPS) in cells, inhibiting the activation of calcineurin (CaN). This, in turn, blocks the calcium-induced signaling pathway in T cells, further inhibiting the production of interleukin-2 (IL-2), resulting in immunosuppressive and anti-inflammatory effects. Therefore, tacrolimus is widely used to prevent and treat rejection reactions following organ transplantation, such as heart and kidney transplants, as well as autoimmune diseases such as atopic dermatitis (AD) and systemic lupus erythematosus (SLE). Furthermore, studies have demonstrated that tacrolimus is also significantly effective in improving dry eye syndrome (DES).

[0007] Dry eye, also known as keratoconjunctivitis sicca (KCS), is a disease of the ocular surface that typically results from insufficient tear production, uneven distribution, or excessive tear evaporation. This prevents tears from properly moistening the ocular surface, leading to increased tear osmotic pressure and inflammation. This in turn causes symptoms such as dryness or burning eyes, photophobia, eye fatigue, or temporary blurred vision. Dry eye can be caused by a variety of factors, including aging, immune disorders (such as xerosis and atopic dermatitis), skin diseases (such as seborrheic dermatitis or chronic blepharitis), medication, infection, or prolonged contact lens wear.

[0008] Literature shows that currently, ophthalmic products with tacrolimus as the main active ingredient include eye drops, eye gels, and eye ointments, which can be used to treat symptoms such as dry eyes, conjunctivitis, or keratitis. However, due to the hydrophobic properties of tacrolimus, the commercially available tacrolimus eye drops are Because suspensions contain a large number of insoluble particles, application can be accompanied by severe eye irritation and a foreign body sensation, often leading to more severe eye discomfort and significantly reducing user comfort. Tacrolimus-containing ophthalmic gels or ointments, due to their inherent low fluidity, can cause side effects such as temporary blurred vision, a foreign body sensation, and eye irritation, similarly reducing user comfort.

[0009] Therefore, there is an urgent need to develop new technical means to enable ophthalmic compositions containing tacrolimus to avoid eye irritation, have good solubility for tacrolimus, and meet the requirements of the United States Pharmacopoeia for insoluble particulate matter in ophthalmic solutions. At the same time, they also have good flowability and stability, thereby effectively reducing discomfort caused during application and improving compliance during treatment, thereby providing users with an effective and comfortable treatment option.

[0010] Summary of the Invention

[0011] In view of the problems faced by the prior art, the object of the present invention is to provide an ophthalmic composition containing tacrolimus, which is an oily preparation. Compared with commercially available ophthalmic preparations containing tacrolimus, it can avoid eye irritation, and has good solubility for tacrolimus, which can meet the requirements of the United States Pharmacopoeia regarding the presence of insoluble particulates in ophthalmic drugs. It also has good fluidity and stability, and can therefore effectively reduce the discomfort caused by eye irritation, foreign body sensation and temporary visual blurring during application, thereby significantly improving the comfort and compliance of users.

[0012] To achieve the aforementioned objectives, the present invention provides a tacrolimus-containing ophthalmic composition, comprising tacrolimus, medium-chain triglycerides (MCT), white vaselin, and a base component; the base component comprises light liquid paraffin, isopropyl myristate, vegetable oil, animal oil, or a combination thereof; based on the total weight of the tacrolimus-containing ophthalmic composition, the content of medium-chain triglycerides is greater than or equal to 3 weight percent (wt%) and less than or equal to 30 weight percent, and the content of white vaselin is greater than or equal to 20 weight percent and less than or equal to 25 weight percent.

[0013] By adopting the technical means of simultaneously containing tacrolimus, a specific amount of medium-chain triglycerides, a specific amount of white petrolatum, and a specific type of matrix component in an ophthalmic composition, the tacrolimus-containing ophthalmic composition of the present invention can avoid eye irritation compared to commercially available tacrolimus eye drops, and has good solubility for tacrolimus and can meet the requirements of the United States Pharmacopoeia regarding the presence of insoluble particles in ophthalmic drugs. It also has good fluidity and stability, thereby effectively reducing the discomfort caused to the user during the application process and improving the user's comfort and compliance.

[0014] In some embodiments of the present invention, the total proportion of carbon chains with 8 carbon atoms and carbon chains with 10 carbon atoms in the medium-chain triglycerides may be greater than or equal to 95%.

[0015] According to the present invention, the melting point of the white vaseline can be 38°C to 60°C; the dropping point of the white vaseline can be 35°C to 70°C; and the puncture needle penetration distance of the white vaseline measured after a cone penetration test (CPT) at a temperature of approximately 25°C can be greater than or equal to 6 mm and less than or equal to 30 mm.

[0016] Preferably, the vegetable oil may be peanut oil, sesame oil, castor oil, soybean oil, olive oil, corn oil, canola oil, almond oil, or a combination thereof, but is not limited thereto. The animal oil may be lanolin oil, lanolin, or a combination thereof, but is not limited thereto.

[0017] In some embodiments of the present invention, the matrix component comprises light liquid paraffin. The relative density of the light liquid paraffin may be 0.810 to 0.880, wherein the relative density is obtained by comparing the relative density with the same volume of pure water at a temperature of about 20°C; the kinematic viscosity of the light liquid paraffin measured by a capillary viscometer at a temperature of 40°C shall not be less than 3 mm. 2 / s.

[0018] Preferably, based on the total weight of the tacrolimus-containing ophthalmic composition, the content of tacrolimus is greater than or equal to 0.001 wt % and less than or equal to 0.1 wt %.

[0019] In some embodiments of the present invention, the amount of tacrolimus is greater than or equal to 0.005 wt % and less than or equal to 0.1 wt %, based on the total weight of the tacrolimus-containing ophthalmic composition. In other embodiments of the present invention, the amount of tacrolimus is greater than or equal to 0.01 wt % and less than or equal to 0.1 wt %, based on the total weight of the tacrolimus-containing ophthalmic composition.

[0020] In some embodiments of the present invention, the weight ratio of the solid oil phase to the liquid oil phase in the tacrolimus-containing ophthalmic composition may be 1:1 to 1:4. For example, if the tacrolimus-containing ophthalmic composition comprises tacrolimus, medium-chain triglycerides, white petrolatum, and light liquid paraffin, the weight ratio of the solid oil phase (i.e., white petrolatum) to the liquid oil phase (i.e., medium-chain triglycerides and light liquid paraffin) may be 1:1 to 1:4. Specifically, the solid oil phase and liquid oil phase are determined by the states of the components at room temperature and pressure. For example, white petrolatum or lanolin constitutes the solid oil phase, while medium-chain triglycerides, light liquid paraffin, isopropyl myristate, peanut oil, sesame oil, castor oil, soybean oil, olive oil, corn oil, canola oil, almond oil, or sheep oil constitute the liquid oil phase.

[0021] In some embodiments of the present invention, the ophthalmic composition containing tacrolimus may further comprise any ingredient that is suitable for ophthalmic medication but does not affect the therapeutic effect of tacrolimus. For example, the ophthalmic composition containing tacrolimus may further comprise a preservative, an antioxidant, or a combination thereof, but is not limited thereto. The preservative may be benzalkonium chloride, benzethonium chloride, benzyl alcohol, chlorobutanol, parabens, or a combination thereof, but is not limited thereto. The antioxidant may be tocopherol, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), or a combination thereof, but is not limited thereto.

[0022] In addition, the present invention further provides a method for preparing the aforementioned tacrolimus-containing ophthalmic composition, comprising the following steps: step (A): uniformly mixing tacrolimus, medium-chain triglycerides, white petrolatum, and the base component to obtain a mixed solution, wherein the base component comprises light liquid paraffin, isopropyl myristate, the vegetable oil, the animal oil, or a combination thereof, and the content of medium-chain triglycerides is greater than or equal to 3 wt % and less than or equal to 30 wt %, and the content of white petrolatum is greater than or equal to 20 wt % and less than or equal to 25 wt %, based on the total weight of the mixed solution; and step (B): heating the mixed solution to about 60° C. to 90° C. to completely dissolve the components, and then cooling the mixed solution to obtain the tacrolimus-containing ophthalmic composition.

[0023] By uniformly mixing tacrolimus, a specific amount of medium-chain triglycerides, a specific amount of white petrolatum, and a specific type of matrix component, and then heating and cooling the mixture, the ophthalmic composition containing tacrolimus can avoid eye irritation compared to commercially available tacrolimus eye drops. The ophthalmic composition has good solubility for tacrolimus and meets the requirements of the United States Pharmacopoeia regarding insoluble particles in ophthalmic drugs. It also has good fluidity and stability, thereby effectively reducing the discomfort caused to the user during application and improving the user's comfort and compliance.

[0024] Preferably, based on the total weight of the mixed solution, the content of tacrolimus is greater than or equal to 0.001 wt % and less than or equal to 0.1 wt %.

[0025] In some embodiments of the present invention, the content of tacrolimus is greater than or equal to 0.005 wt % and less than or equal to 0.1 wt %, based on the total weight of the mixed solution. In other embodiments of the present invention, the content of tacrolimus is greater than or equal to 0.01 wt % and less than or equal to 0.1 wt %, based on the total weight of the mixed solution.

[0026] In addition, the present invention further provides a use of the aforementioned tacrolimus-containing ophthalmic composition for preparing a medicament for treating dry eye.

[0027] According to the present invention, the drug for treating dry eye contains a therapeutically effective amount of tacrolimus. For example, based on the total weight of the drug for treating dry eye, the content of tacrolimus may be 0.001 wt% to 0.1 wt%, but is not limited thereto.

[0028] In this specification, a range expressed as "from a smaller value to a larger value" unless otherwise specified indicates that the range is greater than or equal to the smaller value and less than or equal to the larger value. For example, a melting point of 38°C to 60°C would be considered "greater than or equal to 38°C and less than or equal to 60°C." BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG1 is a photograph showing the appearance of the tacrolimus-containing ophthalmic composition of Example 5.

[0030] FIG2 is a photograph of the appearance of the tacrolimus-containing ophthalmic composition of Comparative Example 1.

[0031] FIG3 shows the corneal fluorescence staining scoring results on days 6, 13, and 17 of the adaptation and model periods in Experimental Example 5.

[0032] FIG4 shows the corneal fluorescence staining scoring results on days 2, 6, 10, and 14 of the administration period in Experimental Example 5.

[0033] FIG5A is a photograph showing the results of optical coherence tomography (OCT) examination of the cornea of ​​the experimental animal before modeling in Experimental Example 5. ...

[0034] FIG5B is an OCT image showing the cornea of ​​the experimental animals in Experimental Example 5 on the 7th day of the model period.

[0035] FIG5C is an OCT image showing the cornea of ​​the experimental animals in Experimental Example 5 on the 14th day of the model period.

[0036] FIG5D is an OCT image showing the cornea of ​​the experimental animals in Experimental Example 5 on the 17th day of the model period.

[0037] FIG5E is an OCT image showing the cornea of ​​the experimental animals on the third day of the administration period in Experimental Example 5. ...

[0038] FIG5F is an OCT image showing the cornea of ​​the experimental animals in Experimental Example 5 on the 7th day of the administration period.

[0039] FIG5G is an OCT image showing the cornea of ​​the experimental animals in Experimental Example 5 on the 11th day of the administration period.

[0040] FIG5H is an OCT image showing the cornea of ​​the experimental animals in Experimental Example 5 on the 15th day of the administration period.

[0041] FIG6 shows the tear secretion results during the adaptation period, model period, and drug administration period in Experimental Example 5. DETAILED DESCRIPTION

[0042] Several embodiments are listed below to illustrate the implementation of the present invention. Those skilled in the art can easily understand the advantages and effects that can be achieved by the present invention through the contents of this specification, and make various modifications and changes without departing from the spirit of the present invention to implement or apply the contents of the present invention.

[0043] Examples 1 to 10: Ophthalmic compositions containing tacrolimus

[0044] According to the ingredients and amounts of each ingredient listed in Table 1 below, appropriate amounts of tacrolimus, medium-chain triglycerides, white petrolatum, and a base component were placed in a glass beaker. The beaker was then heated to approximately 90° C. and magnetically stirred until the ingredients were completely dissolved to obtain a uniformly mixed solution. Stirring was then stopped and the mixed solution was cooled to obtain the tacrolimus-containing ophthalmic compositions of Examples 1 to 10.

[0045] Comparative Example 1: Commercially available tacrolimus eye drops

[0046] Comparative Example 1 uses commercially available tacrolimus eye drops, which are manufactured by Senju Pharmaceutical Co., Ltd. Fukusaki Plant, Japan, under the trade name The invention also contains 0.1 wt% of tacrolimus.

[0047] Comparative Examples 2 to 7: Ophthalmic compositions containing tacrolimus

[0048] The preparation process for Comparative Examples 2 to 7 was similar to that of the Examples. Specifically, according to the ingredients and their contents listed in Table 1 below, appropriate amounts of the ingredients were placed in a glass beaker. The beaker was then heated to approximately 90°C and magnetically stirred until the ingredients were completely dissolved to obtain a uniformly mixed solution. Stirring was then stopped and the mixed solution was cooled to obtain the tacrolimus-containing ophthalmic compositions of Comparative Examples 2 to 7. The main difference between Comparative Examples 2 to 7 and Examples 1 to 10 is that the tacrolimus-containing ophthalmic compositions of Comparative Examples 2 to 7 did not contain both medium-chain triglycerides and white petrolatum.

[0049] Table 1: Compositions of the tacrolimus-containing ophthalmic compositions of Examples 1 to 10 and Comparative Examples 2 to 7.

[0050] Test Example 1: Eye irritation evaluation

[0051] (1) In vitro test

[0052] This test used Examples 1 to 10 and Comparative Examples 1 to 7. The test followed the in vitro ocular irritation testing method described in the Guideline for the Testing of Chemicals (Test Guideline No. 496) published by the Organization for Economic Cooperation and Development (OECD). Each group underwent an in vitro chemical reaction to simulate human behavior. This reaction primarily involved protein denaturation, which results in turbidity in the solution. The light transmittance of each group at a wavelength of approximately 405 nm was then measured using a UV-Vis spectrophotometer (Manufacturer: Hitachi; Model: U-2900) in accordance with the transmittance requirements of the United States Pharmacopeia (USP). The results are shown in Table 2 below. The light transmittance can be used to assess the degree of eye irritation, with higher light transmittance indicating lower eye irritation and lower light transmittance indicating higher eye irritation. Specifically, when the light transmittance is greater than 90%, it is considered to be non-eye irritating, when the light transmittance is greater than 80% and less than or equal to 90%, it is considered to be low eye irritating, when the light transmittance is greater than 70% and less than or equal to 80%, it is considered to be moderate eye irritating, and when the light transmittance is less than or equal to 70%, it is considered to be high eye irritating.

[0053] Table 2: Light transmittance and corresponding eye irritation of Examples 1 to 10 and Comparative Examples 1 to 7 measured after conducting in vitro chemical reactions simulating the human body.

[0054] As shown in Table 2, the light transmittances of the tacrolimus-containing ophthalmic compositions of Examples 1 to 10 all exceed 95%, and therefore are non-irritating to the eyes. Specifically, the light transmittances of the tacrolimus-containing ophthalmic compositions of Examples 1 to 5 and 7 to 10 all exceed 98%. In contrast, the light transmittance of the commercially available tacrolimus eye drops of Comparative Example 1 is only 58.2%, clearly highly irritating to the eyes. The light transmittances of the tacrolimus-containing ophthalmic compositions of Comparative Examples 2, 4, 6, and 7 are all less than 90%, indicating that they still present eye irritation. The light transmittances of the tacrolimus-containing ophthalmic compositions of Comparative Examples 3 and 5 are 91.3% and 91.0%, respectively, which are less than 95%, and therefore still present higher eye irritation than the tacrolimus-containing ophthalmic compositions of Examples 1 to 10.

[0055] This demonstrates that, compared to the commercially available tacrolimus eye drops with high eye irritation, the tacrolimus-containing ophthalmic composition of the present invention indeed has a better effect in avoiding eye irritation.

[0056] (2) Animal testing

[0057] In this study, the tacrolimus-containing ophthalmic compositions of Examples 4 and 5 were used as test samples, and six male New Zealand white rabbits weighing approximately 2 kg to 3 kg were used as test subjects. The tacrolimus-containing ophthalmic compositions of Examples 4 and 5 were administered to the left and right eyes, respectively, by eye drops. The frequency of administration was 3 times a day, 4 hours apart, for 14 days, and the volume of each administration was approximately 50 microliters (μL).

[0058] The ocular irritation assessment included examinations of corneal opacity, corneal affected area, iris abnormalities, conjunctival congestion, chemosis, ocular discharge, corneal fluorescein staining, and the area affected by corneal fluorescein staining. Scores were assigned to these various ocular irritation assessment items at different time points to comprehensively evaluate the ocular irritation of the test samples. The assessment time points were: before test sample administration on Day 0, one hour after the first test sample administration on Day 0, and one hour after the first test sample administration on Days 1, 2, 3, 7, and 14. The specific assessment items, scoring indicators, and their corresponding scores are shown in Table 3 below. Corneal fluorescein staining and the area affected by corneal fluorescein staining were assessed by first instilling approximately 20 μL of 0.5% sodium fluorescein into the superior conjunctival sac. The eyelids were then gently closed for approximately 30 seconds. The cornea was then rinsed with saline and the corneal staining was observed and scored using a slit lamp. The degree of eye irritation of the test sample can be evaluated based on the total score obtained from the above-mentioned different evaluation items, where a total score of 0 to 5 points represents no eye irritation, a total score of 6 to 14 points represents mild eye irritation, a total score of 15 to 23 points represents moderate eye irritation, and a total score of 24 to 28 points represents severe eye irritation.

[0059] The tacrolimus-containing ophthalmic compositions of Examples 4 and 5 were subjected to the aforementioned tests, and the scores obtained after performing the aforementioned evaluation items at the aforementioned different time points and the total scores for each item are respectively listed in Tables 4 and 5 below, wherein "Before Administration" represents the test results before administration of the test sample on Day 0, "Day 0" represents the test results one hour after administration of the first test sample on Day 0, "Day 1" represents the test results one hour after administration of the first test sample on Day 1, "Day 2" represents the test results one hour after administration of the first test sample on Day 2, "Day 3" represents the test results one hour after administration of the first test sample on Day 3, "Day 7" represents the test results one hour after administration of the first test sample on Day 7, and "Day 14" represents the test results one hour after administration of the first test sample on Day 14.

[0060] Table 3: Eye irritation assessment items, scoring indicators and their corresponding scores.

[0061] Table 4: The scores of the tacrolimus-containing ophthalmic composition of Example 4 obtained after different evaluation items were performed at different time points and the sum of the scores of each item.

[0062] Table 5: The scores of the tacrolimus-containing ophthalmic composition of Example 5 obtained after different evaluation items were performed at different time points and the sum of the scores of each item.

[0063] As shown in Tables 4 and 5 above, the sum of the scores for each of the aforementioned ocular irritation assessment items measured at different time points for the tacrolimus-containing ophthalmic compositions of Examples 4 and 5 ranged from 0 to 5, indicating that the tacrolimus-containing ophthalmic compositions of Examples 4 and 5 were indeed non-ocular irritants. This demonstrates that the tacrolimus-containing ophthalmic compositions of the present invention are indeed non-ocular irritants, as confirmed by animal experiments.

[0064] Test Example 2: Insoluble Particle Detection

[0065] This test selected Examples 1 to 10 and Comparative Examples 1 to 7, and followed the USP <789> Particulate matter in eye drops (USP <789> The number of particulate matter with a diameter greater than or equal to 25 microns (hereinafter referred to as insoluble particles) in each group of solutions is determined by microscopic examination according to the requirements of USP Particulate Matter In Ophthalmic Solutions. The number of particulate matter with a diameter greater than or equal to 25 microns (hereinafter referred to as insoluble particles) in each group of solutions must be less than 5 particles (based on a volume of 1 ml) to be considered in compliance with USP. <789> The amount of insoluble particles in the solutions measured in Examples 1 to 10 and Comparative Examples 1 to 7 is shown in Table 6 below, wherein the volume of each test sample is 1 ml.

[0066] Table 6: The amount of insoluble particles contained in the solutions measured in Examples 1 to 10 and Comparative Examples 1 to 7.

[0067] As can be seen from the results in Table 6 above, the number of insoluble particles detected in the tacrolimus-containing ophthalmic compositions of Examples 1 to 10 at a volume of 1 ml was all 0, i.e., no insoluble particles were detected, indicating that the tacrolimus-containing ophthalmic compositions of Examples 1 to 10 all complied with the USP <789> In contrast, the commercially available tacrolimus eye drops in Comparative Example 1 are suspensions and contain a large number of insoluble particles, making it difficult to calculate the total number of insoluble particles. Therefore, the commercially available tacrolimus eye drops in Comparative Example 1 do not comply with the USP <789> The ophthalmic composition containing tacrolimus in Comparative Example 2 had a volume of 1 ml and the number of insoluble particles measured was 5, which also did not meet the requirements of USP. <789> The ophthalmic compositions containing tacrolimus of Comparative Examples 3 to 7, although the number of insoluble particles measured in these compositions at a volume of 1 ml was less than 5, still contained insoluble particles, indicating that their solubility in tacrolimus was inferior to that of the ophthalmic compositions containing tacrolimus of Examples 1 to 10.

[0068] In addition, to more intuitively highlight the difference in the amount of insoluble particulates between commercially available tacrolimus eye drops and the tacrolimus-containing ophthalmic composition of this invention, the tacrolimus-containing ophthalmic composition of Example 5 and the commercially available tacrolimus eye drops of Comparative Example 1 were further selected. After these compositions were allowed to stand at room temperature for approximately 2 hours, photographs of the appearance of these compositions were taken using a camera under the same conditions, as shown in Figures 1 and 2, respectively.

[0069] As shown in Figure 1 , the tacrolimus-containing ophthalmic composition of Example 5 is a light white oily solution, and no insoluble particles are observed to settle even after 2 hours of standing. In contrast, as shown in Figure 2 , after 2 hours of standing, a large amount of white insoluble particles are clearly observed to settle at the bottom of the commercially available tacrolimus eye drops of Comparative Example 1, forming a distinct stratification from the clear solution above.

[0070] As can be seen, compared to commercially available tacrolimus eye drops, which are suspensions and contain a large amount of insoluble particulates, thus failing to meet the USP regulations for insoluble particulates in eye drops, the tacrolimus-containing ophthalmic composition of the present invention, as no insoluble particulates were detected, does meet the USP requirements and also demonstrates good solubility of tacrolimus. Therefore, the tacrolimus-containing ophthalmic composition of the present invention has excellent patient compatibility.

[0071] Test Example 3: Stability Evaluation

[0072] This study used Examples 1 to 10 and Comparative Example 1 for stability testing. Specifically, the tacrolimus-containing ophthalmic compositions of Examples 1 to 10 and the commercially available tacrolimus eye drops of Comparative Example 1 were placed in a 25°C environment for one month and then in a 70°C environment for three days. The tacrolimus content of each group was determined by high-performance liquid chromatography (HPLC-UV) using octadecylsilane-bonded silica gel as a filler (4.6×150 mm, 3 μm) according to the USP method for tacrolimus raw material testing. The flow rate of the HPLC mobile phase was approximately 1.5 ml / min. The composition of the mobile phase is shown in Table 7 below, and the composition ratios of the mobile phase at different times are shown in Table 8 below. The tacrolimus content determination results for Examples 1 to 10 and Comparative Example 1 are listed in Table 9 below.

[0073] Table 7: Preparation of mobile phase

[0074] Table 8: Composition ratio of mobile phase.

[0075] Table 9: Tacrolimus content determination results in Examples 1 to 10 and Comparative Example 1.

[0076] As shown in the results of Table 9 above, after stability testing, the tacrolimus content in the tacrolimus-containing ophthalmic compositions of Examples 1 to 10 remained above 85%. Specifically, the tacrolimus content in the tacrolimus-containing ophthalmic compositions of Examples 1 to 7 and 9 was above 90%. In contrast, the commercially available tacrolimus eye drops of Comparative Example 1, after undergoing stability testing under the same conditions as the Examples, contained only 71.8% tacrolimus, significantly lower than the results in the Examples. This demonstrates that the tacrolimus-containing ophthalmic compositions of the present invention exhibit superior stability.

[0077] Test Example 4: Fluidity Evaluation

[0078] In this test, the composition of the tacrolimus-containing ophthalmic composition of the present invention was prepared by uniformly mixing different amounts of medium-chain triglycerides, white petrolatum, and light liquid paraffin as described in Table 10 below to produce Test Samples 1 to 3. Furthermore, Test Samples 4 and 5 were also uniformly mixed with different amounts of white petrolatum and light liquid paraffin as described in Table 10 below. The appearance of Test Samples 1 to 5 was then visually observed to determine whether stratification occurred and whether the compositions had good flowability. The results are also listed in Table 10 below.

[0079] Table 10: Composition and appearance of test samples 1 to 5.

[0080] As can be seen from the results in Table 10 above, Test Samples 4 and 5 do not contain medium-chain triglycerides, and the white petrolatum contents are 10 wt % and 94 wt %, respectively. These contents do not meet the requirements of the present invention for a white petrolatum content of greater than or equal to 20 wt % and less than or equal to 25 wt %. As a result, Test Sample 4 exhibited delamination, while Test Sample 5 was solid and lacked fluidity.

[0081] Looking at Test Samples 1 to 3 again, except for the absence of tacrolimus, the remaining compositions and contents of Test Samples 1 to 3 correspond to the tacrolimus-containing ophthalmic composition of the present invention. That is, Test Samples 1 to 3 should have the same physical properties as the tacrolimus-containing ophthalmic composition of the present invention. Furthermore, Test Samples 1 to 3 showed no delamination and had good fluidity. Therefore, it can be seen that the tacrolimus-containing ophthalmic composition of the present invention should also show no delamination and have good fluidity, thereby avoiding the problems of temporary blurred vision and foreign body sensation during application to the eye.

[0082] In addition, the viscosities of test samples 1 to 3, each with a volume of approximately 200 ml, were further measured using a viscometer at a temperature of approximately 28°C, using spindle No. 2, and a rotation speed of approximately 50 revolutions per minute (RPM). The results showed that the viscosities of these groups ranged from approximately 200 centipoise (Cps) to 1000 centipoise (Cps). This indicates that the viscosities of the tacrolimus-containing ophthalmic compositions of the present invention also ranged from approximately 200 Cps to 1000 Cps, demonstrating good flowability.

[0083] Test Example 5: Pharmacological efficacy of an ophthalmic composition containing tacrolimus

[0084] This test example tests the efficacy of the ophthalmic composition containing tacrolimus of the present invention. The ophthalmic composition containing tacrolimus used in this test example is referred to as RX-101, and the test group is referred to as the RX-101 group.

[0085] In this study, a New Zealand rabbit dry eye model was established using nictitating membrane and lacrimal gland removal combined with benzalkonium chloride (BAC) eye drops. Eighteen all-female rabbits were divided into six groups: model control, vehicle control, 0.09% cyclosporine, 0.005% RX-101, 0.01% RX-101, and 0.03% RX-101, with three animals in each group. All animals underwent nictitating membrane and lacrimal gland removal on day 1 of the model phase. From days 8 to 14 of the model phase, 40 μL / eye / time of 0.1% BAC solution was administered to both eyes twice daily. The frequency of BAC eye drops was increased to three times daily from days 15 to 17 of the model phase. Animals were then grouped on day 17 of the model phase. Starting from day 18 of the model period (i.e., day 1 of the dosing period), animals in the vehicle control group, 0.09% cyclosporine group, 0.005% RX-101 group, 0.01% RX-101 group, and 0.03% RX-101 group were given eye drops of the control article or the test article at 30 μL / eye / time, twice a day for 14 consecutive days to evaluate the efficacy of the test article.

[0086] The components and contents of each group used in the experiment are shown in Table 11 below:

[0087] Table 11: Components and contents of each group

[0088] The 0.09% cyclosporine group was prepared with reference to the prescription I in Table 1, paragraph 0036 of patent CN101897949B, except that the content of cyclosporine in the 0.09% cyclosporine group was 0.09%.

[0089] The general condition of the rabbits in each group was observed every day after administration. Body weight was measured during the adaptation period, on days 1, 8, 14, 15, 16, 17, and 18 of the model period, and on days 2, 3, 4, 5, 6, 7, 8, and 9 of the administration period. Corneal fluorescence staining was observed and scored before modeling (same as the adaptation period), on days 6, 13, and 17 of the model period, and on days 2, 6, 10, and 14 of the administration period. Tear secretion and corneal optical coherence tomography (OCT) examinations were performed before modeling (same as the adaptation period), on days 7, 14, and 17 of the model period, and on days 3, 7, 11, and 15 of the administration period.

[0090] Corneal fluorescence staining scores and tear secretion volume were expressed as mean ± standard deviation. Corneal fluorescence staining scores were statistically analyzed using Stata / IC 15.0 for Windows, and differences between groups were compared using one-way analysis of variance (ANOVA) or Kruskal-Wallish rank sum test (KW method).

[0091] The main results are as follows:

[0092] 1. General condition

[0093] During the trial, some rabbits exhibited some or all of the following symptoms, including unformed discharge, eyelid swelling, and swelling of the head skin, which were related to the BAC eye drops and surgical procedure. All rabbits were in good spirits, exhibited normal spontaneous activity, had clean fur, and showed no abnormal reactions to feeding, feces, or urine, nor any abnormal symptoms such as eye congestion.

[0094] 2. Weight

[0095] Before modeling, during the model period and during the drug administration period, there was no obvious abnormality in the body weight of all rabbits.

[0096] 3. Dry eye model

[0097] On day 1 of the modeling phase, the nictitating membranes, supraorbital lacrimal glands, inferior lacrimal glands, and superior lacrimal glands of both eyes were removed. From days 8 to 17 of the modeling phase, 0.1% BAC was administered topically to both eyes. On day 17 of the modeling phase, corneal fluorescence staining revealed diffuse punctate staining or large patches of fluorescence in all animals. Corneal optical coherence tomography (OCT) revealed changes such as poor corneal epithelial continuity, corneal thickening, and increased reflectivity at various layers. These changes indicate successful modeling.

[0098] 4. Corneal fluorescence staining scoring

[0099] According to the National Eye Institute scoring system, the cornea is divided into five regions: superior, inferior, temporal, nasal, and central.

[0100] Before modeling, all animals showed no corneal staining or only a small amount of punctate staining. Starting on day 6 of the model period, some animals showed an increase in the number and coverage of corneal punctate staining. By day 17 of the model period, all animals showed diffuse large punctate staining or large sheet-like fluorescence in both eyes, covering the five aforementioned corneal regions. From day 2 to day 14 of the dosing period, diffuse large punctate staining or flake fluorescence was observed in both eyes of the corneas of the animals in the model control and vehicle control groups. The range of the flake fluorescence in the corneas of some animals decreased, and corneal neovascularization of varying degrees occurred. Punctate staining or flake fluorescence was observed in both eyes of the corneas of the animals in the 0.09% cyclosporine, 0.005% RX-101, 0.01% RX-101, and 0.03% RX-101 groups. The range of staining decreased to varying degrees. In addition, from day 6 of the dosing period, corneal neovascularization of varying degrees was observed in one or both eyes of some animals in the 0.09% cyclosporine, 0.005% RX-101, 0.01% RX-101, and 0.03% RX-101 groups.

[0101] The statistical data of corneal fluorescence staining scores and statistical test results of the experimental animals are shown in Table 12 below.

[0102] Table 12: Statistical data and statistical test results of corneal fluorescence staining scores of experimental animals

[0103] Note: 1) * Compared with the model control group, the difference in mean values ​​was statistically significant (P≤0.05).

[0104] 2)▲Compared with the vehicle control group, the difference in mean values ​​was statistically significant (P≤0.05).

[0105] 3)●Compared with the 0.09% cyclosporine group, the difference in mean values ​​was statistically significant (P≤0.05).

[0106] 4)★Compared with the 0.005% RX-101 group, the mean difference was statistically significant (P≤0.05).

[0107] Specifically, the corneal fluorescence staining scores of the model control group, vehicle control group, 0.09% cyclosporine group, 0.005% RX-101 group, 0.01% RX-101 group, and 0.03% RX-101 group during the adaptation period were 0±0.5, 0±0.4, 0±0.5, 0±0.0, 0±0.5, and 1±0.8, respectively; on the 6th day of the model period, they were 1±1.8, 5±7.7, 1±1.6, 2±1.9, 4±2.0, and 0±0.5, respectively; on the 13th day of the model period, they were 17±1.4, 16±3.4, 17±3.5, 9±6.7, 13±3.8, and 16±1.9, respectively; on the 17th day of the model period, they were 19±1.0, 20±0.0, 20±0.0, 18±2.2, 19±1.7, and 20±0.4, respectively; On the second day of the dosing period, the respective rates were 18±2.4, 20±0.5, 20±0.8, 15±1.9, 19±2.0, and 18±1.3; on the sixth day of the dosing period, the respective rates were 19±1.6, 19±1.0, 16±1.3, 13±3.0, 17±2.1, and 15±2.2; on the tenth day of the dosing period, the respective rates were 19±1.5, 19±2.0, 14±2.6, 11±2.9, 13±2.9, and 16±2.3; on the fourteenth day of the dosing period, the respective rates were 18±1.9, 19±1.3, 15±3.3, 12±3.7, 13±2.7, and 13±2.6.

[0108] Corneal fluorescence staining scores gradually increased in all groups from days 6 to 17 of the model period. By day 17, all scores were significantly higher than those in the adaptation period, indicating successful model establishment. On day 17 of the model period, the corneal fluorescence staining score in the 0.005% RX-101 group was slightly lower than that in the vehicle control group, the 0.09% cyclosporine group, and the 0.03% RX-101 group, with statistically significant differences (P ≤ 0.05).

[0109] There were no significant changes in corneal fluorescence staining scores between days 2 and 14 of the dosing period compared with day 17 of the model period in the model and vehicle control groups. On day 2 of the dosing period, the corneal fluorescence staining score in the 0.005% RX-101 group was significantly lower than that in the model, vehicle, 0.09% cyclosporine, 0.01% RX-101, and 0.03% RX-101 groups, with statistically significant differences (P ≤ 0.05). From days 6 to 14 of the dosing period, the corneal fluorescence staining scores in the 0.09% cyclosporine, 0.005% RX-101, 0.01% RX-101, and 0.03% RX-101 groups showed a downward trend and were significantly lower than those in the model and vehicle control groups, with statistically significant differences compared with the model and / or vehicle control groups (P ≤ 0.05). On days 6 and 10 of the administration period, the corneal fluorescence staining score in the 0.005% RX-101 group was significantly lower than that in the 0.09% cyclosporine group, 0.01% RX-101 group, and 0.03% RX-101 group, and the difference was statistically significant compared with the 0.09% cyclosporine group (P≤0.05). On day 6 of the administration period, the difference between the 0.005% RX-101 group and the 0.01% RX-101 group was statistically significant (P≤0.05). On day 10 of the administration period, the difference between the 0.005% RX-101 group and the 0.03% RX-101 group was statistically significant (P≤0.05). On the 14th day of the administration period, the corneal fluorescence staining scores of the 0.05% RX-101 group, 0.01% RX-101 group, and 0.03% RX-101 group were all lower than those of the 0.09% cyclosporine group, and the difference between the 0.05% RX-101 group and the 0.09% cyclosporine group was statistically significant (P≤0.05).

[0110] Figure 3 shows the corneal fluorescence staining scores on days 6, 13, and 17 of the adaptation and model periods. The differences between the groups were statistically analyzed using the KW method or ANOVA method. The data are described in the form of a table. Sample size: N = 6. *Compared with the model control group, the mean difference was statistically significant (P ≤ 0.05); ▲Compared with the vehicle control group, the mean difference was statistically significant (P ≤ 0.05); ●Compared with the 0.09% cyclosporine group, the mean difference was statistically significant (P ≤ 0.05); ★Compared with the 0.005% RX-101 group, the mean difference was statistically significant (P ≤ 0.05).

[0111] Figure 4 shows the corneal fluorescence staining scores on days 2, 6, 10, and 14 of the administration period. The differences between the groups were statistically analyzed using the KW method or ANOVA method. The data are described in the form of a table. Sample size: N = 6. *Compared with the model control group, the mean difference was statistically significant (P ≤ 0.05); ▲Compared with the vehicle control group, the mean difference was statistically significant (P ≤ 0.05); ●Compared with the 0.09% cyclosporine group, the mean difference was statistically significant (P ≤ 0.05); ★Compared with the 0.005% RX-101 group, the mean difference was statistically significant (P ≤ 0.05).

[0112] 5Corneal optical coherence tomography (OCT) examination

[0113] Before modeling, the structures of the corneal epithelial cell layer, anterior Descemet's layer, stromal layer, posterior Descemet's layer, endothelial cell layer, etc. in both eyes of all animals were complete, continuous, and of uniform thickness. The reflections of each layer were normal, and no obvious abnormal changes were observed.

[0114] On day 7 of the model period, some animals showed a loss of continuity in the corneal epithelium, and some showed thickening of the corneal stroma and increased reflexes. From day 14 to 17 of the model period, all animals gradually showed an incomplete structure and loss of continuity in the corneal epithelium. In some animals, the epithelium and Descemet's membrane were destroyed, the stroma thickened, the reflexes increased, and the structure of each layer was unclear.

[0115] From day 3 to day 15 of the drug administration period, the corneas of the animals in the model control group and the vehicle control group showed persistent destruction and discontinuity of the epithelial cell layer structure, which may be accompanied by destruction of the anterior Descemet's membrane structure, unclear structures of the various layers, thickening of the stromal layer with enhanced reflections, and bulging of the corneal surface, which was considered to be corneal scarring. In the 0.09% cyclosporine group, two animals (2 / 3) showed persistent destruction and discontinuity of the epithelial cell layer structure, thickening of the stromal layer with enhanced reflections, and unclear structures of the various layers of the cornea. The thickness of the corneal stromal layer of the right eye of one animal (1 / 3) decreased. In all animals in the 0.005% RX-101 group and in two animals (2 / 3) in the 0.01% RX-101 group, the corneal epithelial cell layer structure recovered to varying degrees in both eyes, and the stromal layer thickness also decreased to varying degrees. In the 0.01% RX-101 group, one animal (1 / 3) showed thickening of the corneal stromal layer in both eyes, enhanced reflections, and unclear structures of the various layers of the cornea. All animals in the RX-101 group had varying degrees of decreased corneal stroma thickness in the right eye or both eyes. In one animal (1 / 3), a portion of the stroma thickened and bulged toward the corneal surface, suggesting corneal scarring. Figures 5A-5H show the corneal optical coherence tomography (OCT) examination results for one animal in Experimental Example 5 before modeling, on model day 7, model day 14, model day 17, and on dosing day 3, dosing day 7, dosing day 11, and dosing day 15, respectively.

[0116] 6. Tear secretion measurement

[0117] Before modeling, the tear secretion volumes of the model control group, vehicle control group, cyclosporine group, 0.005% RX-101 group, 0.01% RX-101 group, and 0.03% RX-101 group were 19.4±6.50mm, 18.1±1.93mm, 20.9±5.89mm, 15.6±5.09mm, 18.4±5.65mm, and 20.1±3.29mm, respectively; on the 7th day of the model period, they were 12.2±5.10mm, 9.8±3.54mm, 12.4±4.72mm, 11.0±6.71mm, and 10.2±3.76mm, respectively. m, 7.9±2.30mm; on the 14th day of the model period, they were 15.7±2.59mm, 8.8±2.61mm, 11.0±2.69mm, 9.9±4.06mm, 14.5±5.45mm, 11.9±4.28mm; on the 17th day of the model period, they were 21.7±8.37mm, 10.4±1.91mm, 12.3±2.13mm, 11.2±6.20mm, 15.2±5.24mm, 10. 2±3.73mm; on the third day of the administration period, they were 15.7±7.86mm, 11.7±5.63mm, 17.5±5.74mm, 12.6±6.38mm, 12.6±6.09mm, and 8.0±3.08mm; on the seventh day of the administration period, they were 15.3±7.92mm, 9.6±2.86mm, 18.9±4.62mm, 14.2±8.46mm, 11.8±6.91mm, and 12.9±2.61mm. mm; on the 11th day of the administration period, they were 15.9±5.47mm, 12.2±4.70mm, 15.4±5.86mm, 14.5±3.07mm, 14.8±4.86mm, and 12.7±3.83mm; on the 15th day of the administration period, they were 16.4±8.93mm, 12.5±6.32mm, 15.8±4.20mm, 14.4±8.86mm, 12.6±4.65mm, and 11.3±2.84mm. Specifically, the statistical data of the tear secretion volume of the test animals are shown in Table 13 below.

[0118] Table 13: Statistical data of tear secretion of experimental animals (Mm)

[0119] Compared with the pre-modeling period, the tear secretion of the animals in the above groups decreased significantly on the 7th day of the model period. From the 14th day of the model period to the 15th day of the drug administration period, it can be observed that the tear secretion has a certain recovery trend, but it is still lower than before the modeling. It is speculated that this may be related to the compensatory secretion of fluid by conjunctival cells after lacrimal gland resection. Figure 6 shows the tear secretion results during the adaptation period, model period and drug administration period. The sample size is N=6.

[0120] In conclusion, under the conditions of this study, topical administration of 0.09% cyclosporine, 0.005% RX-101, 0.01% RX-101, and 0.03% RX-101 eye drops had a certain effect on inhibiting corneal epithelial damage in the New Zealand rabbit dry eye model induced by nictitating membrane and lacrimal gland removal combined with benzalkonium chloride eye drops. The effect of RX-101 was better than that of cyclosporine. However, no obvious dose-related effect was observed in the results of RX-101 in inhibiting corneal epithelial damage.

[0121] In summary, compared to commercially available tacrolimus eye drops, which are highly irritating to the eyes, the tacrolimus-containing ophthalmic composition of the present invention can avoid eye irritation by controlling the types and content ranges of its ingredients. Furthermore, the composition has good solubility for tacrolimus and meets the requirements of the United States Pharmacopoeia regarding the presence of insoluble particulates in ophthalmic drugs. It also has good stability and flowability, thereby effectively reducing user discomfort during application and improving user comfort. It can also enhance compliance during treatment, and thus has high commercial development potential. Industrial Applicability

[0122] The present invention provides an ophthalmic composition containing tacrolimus, comprising tacrolimus, medium-chain triglycerides, white vaseline, and a matrix component; based on the total weight of the ophthalmic composition containing tacrolimus, the content of medium-chain triglycerides is greater than or equal to 3wt% and less than or equal to 30wt%, and the content of white vaseline is greater than or equal to 20wt% and less than or equal to 25wt%. The present invention is the first to prepare tacrolimus into an oily eye drop. The prepared ophthalmic preparation has good content uniformity, is easy to use, spreads evenly after instillation into the eye, and has a longer residence time in the eye. Compared with commercially available tacrolimus eye drops, it can avoid eye irritation. In addition, the ophthalmic composition has good solubility for tacrolimus, thus meeting the requirements for insoluble particulates of ophthalmic drugs in the United States Pharmacopoeia. It also has good fluidity and stability, thereby significantly improving user comfort. It can be used clinically and has good economic value and application prospects.

Claims

1. An ophthalmic composition containing tacrolimus, comprising tacrolimus, medium-chain triglycerides, white vaseline and a matrix component; the matrix component comprises light liquid paraffin, isopropyl myristate, vegetable oil, animal oil or a combination thereof; based on the total weight of the ophthalmic composition containing tacrolimus, the content of the medium-chain triglycerides is greater than or equal to 3 weight percent and less than or equal to 30 weight percent, and the content of the white vaseline is greater than or equal to 20 weight percent and less than or equal to 25 weight percent.

2. The ophthalmic composition containing tacrolimus according to claim 1, wherein The vegetable oil comprises peanut oil, sesame oil, castor oil, soybean oil, olive oil, corn oil, canola oil, almond oil or a combination thereof, and the animal oil comprises sheep oil, lanolin or a combination thereof.

3. The ophthalmic composition containing tacrolimus according to claim 1, wherein The matrix component is light liquid paraffin, and the relative density of the light liquid paraffin is 0.810 to 0.

880.

4. The tacrolimus-containing ophthalmic composition according to any one of claims 1 to 3, wherein Based on the total weight of the tacrolimus-containing ophthalmic composition, the tacrolimus content is greater than or equal to 0.001 weight percent and less than or equal to 0.1 weight percent.

5. The tacrolimus-containing ophthalmic composition according to any one of claims 1 to 3, wherein The ophthalmic composition containing tacrolimus further comprises an antibacterial agent, an antioxidant or a combination thereof.

6. A method for preparing an ophthalmic composition containing tacrolimus according to any one of claims 1 to 5, comprising the following steps: Step (A): uniformly mixing tacrolimus, medium chain triglycerides, white vaseline and a matrix component to obtain a mixed solution, wherein: The matrix component comprises light liquid paraffin, isopropyl myristate, vegetable oil, animal oil or a combination thereof, and based on the total weight of the mixed solution, the content of medium chain triglycerides is greater than or equal to 3 weight percent and less than or equal to 30 weight percent, and the content of white vaseline is greater than or equal to 20 weight percent and less than or equal to 25 weight percent; and Step (B): heating the mixed solution to about 60° C. to 90° C. to completely dissolve the components, and then cooling it to obtain the tacrolimus-containing ophthalmic composition.

7. The method according to claim 6, wherein: Based on the total weight of the mixed solution, the content of tacrolimus is greater than or equal to 0.001 weight percent and less than or equal to 0.1 weight percent.

8. Use of the tacrolimus-containing ophthalmic composition according to any one of claims 1 to 5 for preparing a medicament for treating dry eye.

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