Tacrolimus lipid microspheres, and preparation method and application thereof

Abstract

The application belongs to the technical field of medicines, and discloses a tacrolimus lipid microsphere, a preparation method and application thereof. Compared with the existing eye tacrolimus preparation (such as Talymus), the tacrolimus lipid microsphere has the advantages of no irritation, long retention time, better treatment effect on dry eye, can improve tear film stability, and can treat corneal damage caused by dry eye.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a tacrolimus lipid microsphere, its preparation method, and its application. Background Technology

[0002] Dry eye syndrome is a common, multifactorial ocular surface disease that can be accompanied by ocular surface inflammation, tissue damage, and neurological abnormalities. The appropriate use of eye drops is the best way to relieve eye strain and dry eye syndrome. However, traditional eye drop administration results in significant drug loss and reduced bioavailability due to physiological factors such as corneal barrier function and tear flushing.

[0003] Commercially available eye drops for treating dry eye include sodium hyaluronate eye drops, polyvinyl alcohol eye drops, recombinant bovine growth factor eye drops, and cyclosporine eye drops. Sodium hyaluronate and polyvinyl alcohol eye drops only relieve dry eye symptoms and require 5-6 applications daily, leading to poor patient compliance. Recombinant bovine growth factor eye drops can cause adverse reactions in some patients, such as itchy skin, sore throat, and difficulty breathing. Cyclosporine eye drops can cause blurred vision, gastrointestinal discomfort, and liver damage in patients who are intolerant to them.

[0004] Tacrolimus is a macrolide antibiotic with potent immunosuppressive properties. It is a highly lipophilic and hydrophobic compound, insoluble in water. When used for the prevention and treatment of dry eye, it is generally administered as 0.1% FK506 eye drops twice daily, gradually tapering the dosage. However, a side effect of ophthalmic tacrolimus preparations is mild and transient eye irritation; the incidence of side effects with 0.1% tacrolimus eye drops is 29%. Summary of the Invention

[0005] The first objective of this invention is to provide tacrolimus lipid microspheres.

[0006] The second objective of this invention is to provide a method for preparing tacrolimus lipid microspheres according to the first aspect of this invention.

[0007] A third aspect of the present invention is to provide a tacrolimus liposome microsphere gel.

[0008] The fourth aspect of this invention is to provide a method for preparing tacrolimus lipid microsphere gel according to the third aspect of this invention.

[0009] The fifth aspect of this invention is to provide a tacrolimus lipid microsphere suspension.

[0010] The sixth aspect of this invention aims to provide the use of tacrolimus lipid microspheres of the first aspect of this invention, tacrolimus lipid microsphere gel of the third aspect of this invention, or tacrolimus lipid microsphere suspension of the fifth aspect of this invention in the preparation of eye drops.

[0011] The seventh aspect of this invention is to provide an eye drop solution.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0013] In a first aspect, the present invention provides tacrolimus lipid microspheres, wherein the raw materials for preparing the tacrolimus lipid microspheres include: tacrolimus, a first emulsifier, an oil phase, a second emulsifier, and a lyophilization protectant.

[0014] Preferably, the first emulsifier comprises at least one of egg yolk lecithin, soy lecithin, gum arabic, astragalus gum, gelatin, apricot gum, polyoxyethylene fatty alcohol ether, and polyoxyethylene fatty acid ester; further comprises at least one of egg yolk lecithin and soy lecithin; and even more preferably, egg yolk lecithin.

[0015] Preferably, the oil phase comprises at least one of soybean oil, olive oil, castor oil, safflower oil, coconut oil, sunflower oil, almond oil, lanolin, medium-chain triglycerides, liquid paraffin, propylene glycol diallylate, silicone oil, and petrolatum; further comprises at least one of soybean oil, olive oil, castor oil, safflower oil, coconut oil, sunflower oil, and almond oil; and even more preferably, soybean oil.

[0016] Preferably, the second emulsifier comprises at least one of poloxamer (e.g., poloxamer 188, poloxamer 407), Tween, polyvinyl alcohol, sodium cholate, and sodium deoxycholate; further comprises at least one of poloxamer (e.g., poloxamer 188, poloxamer 407), Tween, and polyvinyl alcohol; and even more preferably, poloxamer 188.

[0017] Preferably, the freeze-drying protectant comprises at least one selected from mannitol, sucrose, trehalose, lactose, glucose, maltose, dextran, albumin, ethylene glycol, glycerol, L-serine, monosodium glutamate, alanine, glycine, sarcosine, phosphate, acetate, and citrate; further comprises at least one selected from mannitol, sucrose, trehalose, lactose, and glucose; and even more preferably, mannitol.

[0018] Preferably, the mass ratio of tacrolimus, the first emulsifier, the second emulsifier, the oil phase, and the lyophilization protectant is 12.5:(60-90):(60-90):(520-780):(280-420); further, it is 12.5:(67.5-82.5):(67.5-82.5):(585-715):(315-385); even further, it is 12.5:(71.25-78.75):(71.25-78.75):(617.5-682.5):(332.5-367.5); and still further, it is 12.5:(74-76):(74-76):(640-660):(340-360).

[0019] Preferably, the raw materials for preparation further include an aqueous phase.

[0020] Preferably, the aqueous phase comprises at least one of water and phosphate buffer; more preferably, it is water; and even more preferably, it is deionized water.

[0021] Preferably, the second emulsifier to the aqueous phase in a mass-volume ratio (g:mL) is 7.5:(100-600); further, 7.5:(304.5–565.5); even further, 7.5:(348-522); still further, 7.5:(391.5–478.5); and even further, 7.5:(420-450).

[0022] Preferably, the raw materials for preparation further include: an organic solvent.

[0023] Preferably, the organic solvent comprises at least one selected from ethanol, dichloromethane, chloroform, vinyl chloride, dimethyl sulfoxide, acetone, methyl acetate, and diethyl ether; further, it is ethanol; and even more preferably, it is anhydrous ethanol.

[0024] Preferably, the mixture of tacrolimus and the first emulsifier is mixed with the organic solvent in a mass-volume ratio (g:mL) of (0.7-1.1):8; further, (0.8-1.08):8; even further, (0.82-1.04):8; still further, (0.86-0.94):8; and even further, (0.87-0.88):8.

[0025] Preferably, the tacrolimus lipid microspheres have a particle size of 300-600 nm; more preferably 400-500 nm; and even more preferably 461.76 ± 15.13 nm.

[0026] Preferably, the PDI of the tacrolimus lipid microspheres is 0.05-0.15; more preferably, it is 0.10±0.02.

[0027] Preferably, the zeta potential of the tacrolimus lipid microspheres is -(15-25)mV; more preferably (-23.52±1.43)mV.

[0028] Preferably, the encapsulation efficiency of the tacrolimus lipid microspheres is 95%-99%; more preferably 97.18±1.80%.

[0029] A second aspect of the present invention provides a method for preparing tacrolimus lipid microspheres according to the first aspect of the present invention, comprising the following steps:

[0030] Tacrolimus, the first emulsifier, and an organic solvent are mixed to obtain a drug-containing organic solvent;

[0031] The drug-containing organic solvent is mixed with the oil phase to obtain an oil phase containing an emulsifier;

[0032] The second emulsifier is mixed with the aqueous phase to obtain an aqueous phase containing the emulsifier;

[0033] The oil phase containing emulsifier is mixed with the water phase containing emulsifier and emulsified to obtain an emulsion;

[0034] The emulsion was mixed with a lyophilization protectant and then freeze-dried to obtain tacrolimus lipid microspheres.

[0035] Preferably, the tacrolimus, the first emulsifier, and the organic solvent are mixed under the condition of stirring until dissolved and evenly dispersed.

[0036] Preferably, the stirring conditions are 200-600 rpm for 20-40 min; more preferably, magnetic stirring at 300-500 rpm for 25-35 min at room temperature.

[0037] Preferably, the oil phase containing emulsifier obtained after mixing the drug-containing organic solvent with the oil phase does not contain organic solvent.

[0038] Preferably, the conditions for mixing the drug-containing organic solvent with the oil phase are heating to 60-80°C; further, heating to 60-80°C and stirring; and even further, heating in a water bath to 65-75°C and stirring.

[0039] Preferably, the stirring conditions are stirring at 200-600 rpm; further, stirring at 300-500 rpm; and even further, magnetic stirring at 300-500 rpm.

[0040] Preferably, the second emulsifier is mixed with the aqueous phase under the condition of heating to 60-80°C; further, it is heated in a water bath to 65-75°C.

[0041] Preferably, the emulsification includes shearing and homogenization.

[0042] Preferably, the shearing conditions are 11,000-15,000 rpm for 5-15 min; more preferably, 12,000-14,000 rpm for 8-12 min.

[0043] Preferably, the shearing is performed using a homogenizer.

[0044] Preferably, the homogenization conditions are homogenization at 500-900 bar for 5-9 times; more preferably, homogenization at 600-800 bar for 6-8 times.

[0045] Preferably, homogenization should be performed for 2-4 minutes each time.

[0046] Preferably, the homogenization is performed using a high-pressure homogenizer.

[0047] Preferably, the freeze drying is performed using a freeze dryer.

[0048] A third aspect of the invention provides any one of a1)-a2) tacrolimus lipid microsphere gels:

[0049] a1) A tacrolimus lipid microsphere ordinary gel, wherein the raw materials for preparing the tacrolimus lipid microsphere ordinary gel include: an ordinary gel matrix and tacrolimus lipid microspheres of the first aspect of the present invention; wherein the ordinary gel matrix includes at least one of hydroxypropyl methylcellulose, ethylcellulose, and sodium carboxymethylcellulose;

[0050] a2) A tacrolimus liposome microsphere in situ gel, wherein the raw materials for preparing the tacrolimus liposome microsphere in situ gel include: an in situ gel matrix, a thickener, and tacrolimus liposome microspheres of the first aspect of the present invention; wherein the in situ gel matrix includes at least one of gellan gum, methylcellulose, sodium alginate, poloxamer, carbomer, and chitosan.

[0051] Preferably, the ordinary gel matrix described in a1) comprises at least one of hydroxypropyl methylcellulose and ethylcellulose; more preferably, it is hydroxypropyl methylcellulose or ethylcellulose.

[0052] Preferably, the in-situ gel matrix described in a2) is gellan gum.

[0053] Preferably, the thickener described in a2) comprises at least one of xanthan gum, carboxymethyl cellulose, methyl cellulose, sodium carboxymethyl cellulose, propylene glycol alginate, sodium starch phosphate, sodium alginate, casein, sodium polyacrylate, polyoxyethylene, and polyvinylpyrrolidone; further comprises at least one of xanthan gum, carboxymethyl cellulose, and methyl cellulose; and even more preferably xanthan gum.

[0054] Preferably, the mass ratio of the ordinary gel matrix and tacrolimus lipid microspheres in a1) is (15-40):100; further, it is (20-35):100; and even further, it is (25-30):100.

[0055] Preferably, the mass ratio of the in-situ gel matrix, thickener and tacrolimus lipid microspheres in a2) is (50-100):(15-25):1000; further, it is (60-90):(16-23):1000; and even further, it is (70-80):(17-22):1000.

[0056] Preferably, the raw materials for preparing the tacrolimus lipid microsphere ordinary gel described in a1) further include an osmotic pressure regulator.

[0057] Preferably, the raw materials for preparing the tacrolimus lipid microsphere in-situ gel described in a2) further include an osmotic pressure regulator.

[0058] Preferably, the osmotic pressure regulators described in a1) and a2) are each independently selected from at least one of mannitol, lactose, sucrose, and sodium chloride; more preferably, the osmotic pressure regulators described in a1) and a2) are mannitol.

[0059] Preferably, the mass ratio of the osmotic pressure regulator to the tacrolimus lipid microspheres in a1) is (25-70):100; further, (30-60):100; and even further, (45-51):100.

[0060] Preferably, the mass ratio of the osmotic pressure regulator to the tacrolimus lipid microspheres in a2) is (25-70):100; further, (30-60):100; and even further, (45-51):100.

[0061] Preferably, the raw materials for preparing the tacrolimus lipid microsphere ordinary gel described in a1) further include water; and more specifically, deionized water.

[0062] Preferably, the raw materials for preparing the tacrolimus lipid microsphere in-situ gel described in a2) further include water; and more specifically, deionized water.

[0063] Preferably, the mass ratio of water to tacrolimus lipid microspheres in a1) is (1-20):1; further, (5-16):1; and even further, (8-11):1.

[0064] Preferably, the mass ratio of water to tacrolimus lipid microspheres in a2) is (1-20):1; further, (5-16):1; and even further, (8-11):1.

[0065] Preferably, the pH of the tacrolimus lipid microsphere ordinary gel described in a1) is 6.0-8.0; further, it is 7.0-7.8; and even further, it is 7.12-7.70.

[0066] Preferably, the pH of the tacrolimus lipid microsphere in situ gel described in a2) is 6.0-8.0; further, it is 6.5-7.5; and even further, it is 7.3-7.5.

[0067] Preferably, the osmotic pressure of the tacrolimus lipid microsphere ordinary gel described in a1) is 248-370 mOsmol / kg; further, 270-320 mOsmol / kg; even further, 288-305 mOsmol / kg; and still further, 298-303 mOsmol / kg.

[0068] Preferably, the osmotic pressure of the tacrolimus lipid microsphere in-situ gel described in a2) is 248-370 mOsmol / kg; further, 270-320 mOsmol / kg; even further, 290-310 mOsmol / kg; and still further, 295-305 mOsmol / kg.

[0069] Preferably, the raw materials for preparing the tacrolimus lipid microsphere ordinary gel described in a1) further include a pH adjuster.

[0070] Preferably, the raw materials for preparing the tacrolimus lipid microsphere in situ gel described in a2) further include a pH adjuster.

[0071] Preferably, the pH adjuster described in a1) and a2) is independently selected from at least one of tris(hydroxymethyl)aminomethane, sodium hydroxide, triethylamine, boric acid, and hydrochloric acid; more preferably, it is tris(hydroxymethyl)aminomethane.

[0072] Preferably, the mass ratio of the pH adjuster to tacrolimus lipid microspheres in a1) is (0.01-0.3):1000; further, it is (0.08-0.25):1000; and even further, it is (0.1-0.15):1000.

[0073] Preferably, the mass ratio of the pH adjuster to tacrolimus lipid microspheres in a2) is (0.4-0.8):1000; further, (0.5-0.7):1000; and even further, (0.6-0.62):1000.

[0074] A fourth aspect of the present invention provides any one of the preparation methods b1)-b2):

[0075] b1) The method for preparing the tacrolimus lipid microsphere ordinary gel in the third aspect of the present invention includes the following steps:

[0076] A common gel matrix is ​​mixed with water to obtain a common gel matrix solution;

[0077] An osmotic pressure regulator, a pH regulator, and a common gel matrix solution are mixed to obtain a mixture;

[0078] The mixture was combined with tacrolimus lipid microspheres to obtain a tacrolimus lipid microsphere ordinary gel;

[0079] b2) The method for preparing the tacrolimus lipid microsphere in-situ gel according to the third aspect of the present invention includes the following steps:

[0080] The in-situ gel matrix, thickener, and water are mixed to obtain an in-situ gel matrix solution;

[0081] An osmotic pressure regulator, a pH regulator, and an in-situ gel matrix solution are mixed to obtain a mixture;

[0082] The mixture was combined with tacrolimus lipid microspheres to obtain tacrolimus lipid microsphere in situ gel.

[0083] Preferably, the ordinary gel matrix described in b1) is mixed with water under the condition of uniform dispersion at 300-700 rpm; more preferably, it is uniformly dispersed at 400-600 rpm.

[0084] Preferably, the ordinary gel matrix described in b1) is mixed with water at 60-80°C; further, it is mixed at 65-75°C.

[0085] Preferably, before the ordinary gel matrix solution described in b1) is mixed with the osmotic pressure regulator, the ordinary gel matrix solution is allowed to stand in an ice bath at 0-6°C until the gel matrix solution becomes clear and then returns to room temperature.

[0086] Preferably, before mixing the ordinary gel matrix solution described in b1) with the osmotic pressure regulator, the ordinary gel matrix solution is placed in an ice bath at 0-6°C for 18-30 hours to defoam, and after the gel matrix solution becomes clear, it is restored to room temperature.

[0087] Preferably, the osmotic pressure regulator, pH regulator, and ordinary gel matrix solution described in b1) are mixed under the condition of uniform dispersion at 300-700 rpm; more preferably, they are uniformly dispersed at 400-600 rpm.

[0088] Preferably, the mixture described in b1) is cooled to room temperature before being mixed with tacrolimus lipid microspheres.

[0089] Preferably, the mixture described in b1) is mixed with tacrolimus lipid microspheres under the condition of uniform dispersion at 600-1000 rpm; more preferably, it is uniformly dispersed at 700-900 rpm.

[0090] Preferably, the in-situ gel matrix, thickener, and water described in b2) are mixed under the following conditions: stirring until completely dissolved; further stirring at 50-90°C until completely dissolved; and even further stirring under a water bath at 60-80°C until completely dissolved.

[0091] Preferably, the stirring conditions are stirring at 300-700 rpm; more preferably, stirring at 400-600 rpm.

[0092] Preferably, before the in-situ gel matrix solution described in b2) is mixed with the osmotic pressure regulator, the in-situ gel matrix solution is allowed to stand in an ice bath at 0-6°C until the in-situ gel matrix solution becomes clear and then returns to room temperature.

[0093] Preferably, before mixing the in-situ gel matrix solution with the osmotic pressure regulator in b2), the in-situ gel matrix solution is placed in an ice bath at 0-6°C for 18-30 hours to defoam, and after the in-situ gel matrix solution becomes clear, it is restored to room temperature.

[0094] Preferably, the osmotic pressure regulator, pH regulator, and in-situ gel matrix solution described in b2) are mixed under the condition of uniform dispersion at 300-700 rpm; more preferably, they are uniformly dispersed at 400-600 rpm.

[0095] Preferably, the mixture described in b2) is cooled to room temperature before being mixed with tacrolimus lipid microspheres.

[0096] Preferably, the mixture described in b2) is mixed with tacrolimus lipid microspheres under the condition of uniform dispersion at 600-1000 rpm; more preferably, it is uniformly dispersed at 700-900 rpm.

[0097] A fifth aspect of the present invention provides a tacrolimus lipid microsphere suspension comprising an osmotic pressure regulator and tacrolimus lipid microspheres of the first aspect of the present invention.

[0098] Preferably, each of the osmotic pressure regulators is independently selected from at least one of mannitol, lactose, sucrose, and sodium chloride; more preferably, the osmotic pressure regulator is mannitol.

[0099] Preferably, the osmotic pressure regulator is present in a mass ratio of (1-2):1 with tacrolimus lipid microspheres.

[0100] Preferably, the tacrolimus lipid microsphere suspension further comprises water; more specifically, it comprises deionized water.

[0101] Preferably, the tacrolimus lipid microspheres and water are in a mass-to-volume ratio (g / mL) of 1:(5-15).

[0102] Preferably, the pH of the tacrolimus lipid microsphere suspension is 6.0-8.0; more preferably 6.5-7.5; and even more preferably 7.2-7.3.

[0103] Preferably, the osmotic pressure of the tacrolimus lipid microsphere suspension is 248-370 mOsmol / kg; further, 270-320 mOsmol / kg; even further, 288-305 mOsmol / kg; and still further, 298-303 mOsmol / kg.

[0104] Preferably, the tacrolimus lipid microsphere suspension is prepared by mixing an osmotic pressure regulator, tacrolimus lipid microspheres of the first aspect of the present invention, and water.

[0105] A sixth aspect of the present invention provides the use of tacrolimus lipid microspheres of the first aspect of the present invention, tacrolimus lipid microsphere gel of the third aspect of the present invention, or tacrolimus lipid microsphere suspension of the fifth aspect of the present invention in the preparation of eye drops.

[0106] A seventh aspect of the present invention provides an eye drop comprising: tacrolimus lipid microspheres of the first aspect of the present invention, tacrolimus lipid microsphere gel of the third aspect of the present invention, or tacrolimus lipid microsphere suspension of the fifth aspect of the present invention.

[0107] The beneficial effects of this invention are:

[0108] This invention provides a tacrolimus lipid microsphere, which, compared with existing ophthalmic tacrolimus formulations (such as Talymus), has advantages such as being non-irritating, having a long retention time, being more effective in treating dry eye, improving tear film stability, and treating corneal damage caused by dry eye.

[0109] This invention provides a tacrolimus lipid microsphere gel, which, compared with tacrolimus lipid microspheres, has advantages such as longer retention time, better therapeutic effect on dry eye syndrome, higher tear film stability, and better effect on treating corneal damage caused by dry eye syndrome, and has good prospects for clinical application. Attached Figure Description

[0110] Figure 1 The images show the appearance of tacrolimus lipid microspheres and the in-situ gel of tacrolimus lipid microspheres prepared in Example 2: (a) is an appearance of the tacrolimus lipid microsphere suspension prepared in Example 1; (b) is an appearance of the diluted tacrolimus lipid microsphere suspension prepared in the Effect Example after being irradiated with a laser pen; (c) is an appearance of the in-situ gel of tacrolimus lipid microspheres prepared in Example 2; and (d) is an appearance of the diluted tacrolimus lipid microsphere in-situ gel prepared in Example 2 after being irradiated with a laser pen.

[0111] Figure 2 The graph shows the in vitro release curves of the tacrolimus lipid microsphere suspension prepared in Example 1 and the commercially available formulation Talymus, where n = 3.

[0112] Figure 3 The images show the in vitro release curve and in situ gel erosion diagram of the tacrolimus lipid microspheres in situ gel prepared in Example 2, where n = 3.

[0113] Figure 4 The images show the in vitro release curve and in-situ gel erosion diagram of the tacrolimus lipid microsphere gel prepared in Example 3, where n = 3.

[0114] Figure 5 The images show the in vitro release curve and in-situ gel erosion diagram of the tacrolimus lipid microsphere gel prepared in Example 4, where n = 3.

[0115] Figure 6 The images show the bleeding, coagulation, and dissolution of blood vessels in chicken embryos in the saline group, positive control group, commercially available preparation Talymus, tacrolimus lipid microsphere suspension, and tacrolimus lipid microsphere in situ gel group prepared in Example 2. The camera lens magnification is 10x.

[0116] Figure 7 The images show the precorneal fluorescence retention of the commercially available formulation Talymus, tacrolimus lipid microsphere suspension, and the tacrolimus lipid microsphere in situ gel prepared in Example 2, with the camera lens magnification being 10x.

[0117] Figure 8 The tear film drug-time curves are those of the commercially available formulation Talymus, tacrolimus lipid microsphere suspension, and the tacrolimus lipid microsphere in situ gel prepared in Example 2, where n = 3.

[0118] Figure 9 This is a comparison chart of Schirmer I test results for the saline group, positive control group, Talymus, tacrolimus liposphere group, and tacrolimus liposphere in situ gel group, where n=3.

[0119] Figure 10 This is a comparative graph of tear film breakup time (TBUT) results for the saline group, positive control group, Talymus, tacrolimus lipid microsphere group, and tacrolimus lipid microsphere in situ gel group, where n=3.

[0120] Figure 11 This is a comparison chart of corneal fluorescence staining (FL) results among the saline group, positive control group, Talymus, tacrolimus lipid microsphere group, and tacrolimus lipid microsphere in situ gel group, where n=3.

[0121] In the above figures: * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001. Detailed Implementation

[0122] The present invention will be further described in detail below through specific embodiments.

[0123] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0124] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available.

[0125] In this invention, "room temperature" is 25±5℃.

[0126] The reagents used in the following examples and their manufacturers are shown in Table 1.

[0127] Table 1

[0128]

[0129]

[0130] Example 1: A method for preparing tacrolimus lipid microspheres

[0131] A method for preparing tacrolimus lipid microspheres includes the following steps:

[0132] (1) Accurately weigh 0.125g tacrolimus and 0.75g egg yolk lecithin into 8mL ethanol, and stir magnetically at 400rpm for 30min at room temperature until dissolved and evenly dispersed to obtain a drug-containing ethanol solution.

[0133] (2) Preparation of oil phase containing emulsifier: Accurately weigh 6.5g of soybean oil into a beaker, add the drug-containing ethanol solution obtained in step (1) above, heat in a water bath to 70°C, and magnetically stir at 400rpm until the ethanol is completely evaporated.

[0134] (3) Preparation of the aqueous phase containing emulsifier: Accurately weigh 0.75g of poloxamer 188 and measure 43.5mL of deionized water, mix them in a beaker, and heat in a water bath to 70℃.

[0135] (4) The oil phase containing emulsifier at 70°C obtained in step (2) was poured into the aqueous phase containing emulsifier at 70°C obtained in step (3) using a homogenizer (IKAT18, IKA GmbH, Germany) for emulsification. The shearing conditions were 13,000 rpm for 10 min.

[0136] (5) The emulsion prepared in step (4) was further emulsified in a high-pressure homogenizer (AH-2010, ATS Engineering Inc.) with a homogenization pressure of 700 bar, a homogenization number of 7 times, and a homogenization time of 3 min each time.

[0137] (6) Add 3.5g mannitol to the homogenized emulsion, mix well, and transfer to a freeze dryer (LGJ-10C, Beijing Sihuan Scientific Instruments Co., Ltd.) for freeze drying to obtain tacrolimus lipid microspheres. Figure 1 ).

[0138] 1 g of freeze-dried tacrolimus lipid microspheres and 1.5 g of mannitol were accurately weighed and added to 10 mL of deionized water. The mixture was stirred and dispersed evenly to obtain a tacrolimus lipid microsphere suspension. The osmotic pressure was measured to be 300 mOsmol / kg using an OSMOMAT 3000basic osmometer (Gonotec GmBHC, Germany) and the pH value was measured to be 7.23 using a pH meter (PHS-3C, Shanghai Leici Instrument Factory).

[0139] Example 2: A method for preparing tacrolimus lipid microsphere in situ gel

[0140] A method for preparing tacrolimus lipid microsphere in-situ gel, comprising the following steps:

[0141] (1) Accurately weigh 0.075 g gellan gum and 0.02 g xanthan gum into a beaker, add 10 mL of deionized water into the beaker, heat in a 70°C water bath and stir at 500 rpm until completely dissolved, and obtain an in-situ gel matrix. Place it in an ice bath at 4°C for 24 h to defoam, and wait for the solution to become clear. Then, restore the obtained clear in-situ gel matrix solution to room temperature.

[0142] (2) Accurately weigh 0.48 g of mannitol and measure 0.05 mL of tris(hydroxymethyl)aminomethane (12.2 mg / mL) and add it to the in-situ gel matrix obtained in step (1). Disperse it evenly at 500 rpm. The osmotic pressure of the mixture was measured to be 280 mOsmol / kg using an ice point osmometer (OSMOMAT3000basic, Gonotec GmBHC, Germany) and the pH value was measured to be 7.2 using a pH meter (PHS-3C, Shanghai Leici Instrument Factory).

[0143] (3) Accurately weigh 1g of the tacrolimus lipid microspheres obtained in step (6) of Example 1 and add them to the mixture cooled to room temperature in step (2). Stir at 800 rpm until evenly dispersed to obtain the tacrolimus lipid microsphere in-situ gel. Figure 1 Its osmotic pressure was measured to be 300 mOsmol / kg, and its pH value was measured to be 7.4.

[0144] Example 3: A method for preparing tacrolimus lipid microsphere gel

[0145] A method for preparing tacrolimus lipid microsphere gel includes the following steps:

[0146] (1) Accurately weigh 0.27g of hydroxypropyl methylcellulose into a beaker, add 10mL of deionized water into the beaker, heat in a 70℃ water bath at 500rpm to disperse evenly, and defoam the obtained gel matrix in an ice bath at 4℃ for 24h. After the solution becomes clear, the obtained clear gel matrix solution is restored to room temperature.

[0147] (2) Accurately weigh 0.48 g of mannitol and measure 0.01 mL of tris(hydroxymethyl)aminomethane (12.2 mg / mL) and add it to the gel matrix obtained in step (1). Disperse it evenly at 500 rpm. The osmotic pressure of the mixture was measured to be 278 mOsmol / kg using an OSMOMAT3000basic (Gonotec GmBHC, Germany) and the pH value was measured to be 7.58 using a pH meter (PHS-3C, Shanghai Leici Instrument Factory).

[0148] (3) Accurately weigh 1g of tacrolimus lipid microspheres after freeze-drying in step (6) of Example 1 and add them to the mixture obtained in step (2) that has been cooled to room temperature. Stir at 800rpm until evenly dispersed to obtain tacrolimus lipid microsphere gel. Its osmotic pressure is measured to be 303mOsmol / kg and its pH value is measured to be 7.70.

[0149] Example 4: A method for preparing tacrolimus lipid microsphere gel

[0150] A method for preparing tacrolimus lipid microsphere gel includes the following steps:

[0151] (1) Accurately weigh 0.27g of ethyl cellulose into a beaker, add 10mL of deionized water into the beaker, heat in a 70℃ water bath at 500rpm to disperse evenly, and defoam the resulting gel matrix in an ice bath at 4℃ for 24h. After the solution becomes clear, restore the resulting clear gel matrix solution to room temperature.

[0152] (2) Accurately weigh 0.48 g of mannitol and measure 0.01 mL of tris(hydroxymethyl)aminomethane (12.2 mg / mL) and add it to the gel matrix obtained in step (1). Disperse it evenly at 500 rpm. The osmotic pressure of the mixture was measured to be 275 mOsmol / kg using an ice point osmometer (OSMOMAT3000basic, Gonotec GmBHC, Germany) and the pH value was measured to be 7.00 using a pH meter (PHS-3C, Shanghai Leici Instrument Factory).

[0153] (3) Accurately weigh 1g of tacrolimus lipid microspheres after freeze-drying in step (6) of Example 1 and add them to the mixture obtained in step (2) and cooled to room temperature. Stir at 800rpm until evenly dispersed to obtain tacrolimus lipid microsphere gel. Its osmotic pressure was measured to be 298mOsmol / kg and its pH value was measured to be 7.12.

[0154] Effect Example

[0155] 1. Measure 1 mL of the tacrolimus lipid microsphere suspension prepared in Example 1 and the tacrolimus lipid microsphere in-situ gel prepared in Example 2, and dilute with 6 mL of deionized water. Place the diluted tacrolimus lipid microsphere suspension and tacrolimus lipid microsphere in-situ gel in a sample cell and analyze using a nano-laser particle size analyzer (Winner802, Jinan Micro-Nano Particle Instrument Co., Ltd.). Figure 1 The liposome particle size, polydispersity index (PDI), and Zeta potential of the liposome suspension and the liposome in situ gel were determined using a Zeta potentiometer (JS94J, Shanghai Zhongchen Digital Technology Equipment Co., Ltd.). 0.5 mL of the tacrolimus liposome suspension prepared in Example 1 and the tacrolimus liposome in situ gel prepared in Example 2 were respectively placed in centrifuge tubes, centrifuged at 3000 rpm for 15 min, and 0.4 mL of the filtrate was collected. Then, 0.5 mL of deionized water was added to the original centrifuge tube, and centrifugation was repeated three times. The filtrates were combined, filtered through a 0.22 μm microporous membrane, and the tacrolimus content was determined by HPLC (LC-20AT, Shimadzu Corporation, Japan). The encapsulation efficiency was calculated using the following formula, as shown in formula (1):

[0156] Wherein, W0 represents the free drug content in the tacrolimus liposome microsphere suspension / tacrolimus liposome in-situ gel; W1 represents the drug content added to the formulation.

[0157] The results are shown in Table 2 (n=3): The particle sizes of the tacrolimus lipid microsphere suspension prepared in Example 1 and the tacrolimus lipid microsphere in situ gel prepared in Example 2 were 461.76±15.13 nm and 441.64±19.96 nm, respectively; the PDI values ​​were 0.10±0.02 and 0.08±0.04, respectively, with no significant difference, and the particle size was relatively uniform; the Zeta potentials were -(23.52±1.43) mV and -(31.31±1.87) mV, respectively. This potential is beneficial to the dispersion of nanoparticles and makes the system more stable. The 2020 edition of the Chinese Pharmacopoeia requires that the encapsulation rate of microparticle formulations should generally not be less than 80%. The encapsulation rates of the tacrolimus lipid microsphere suspension prepared in Example 1 and the tacrolimus lipid microsphere in-situ gel prepared in Example 2 were 97.18±1.80% and 97.42±4.47%, respectively, both higher than 80%, which meets the requirements of the Pharmacopoeia.

[0158] Table 2 Physicochemical properties of tacrolimus liposomes and tacrolimus liposome in-situ gels (n=3)

[0159]

[0160] 2. Preparation of artificial tears: Accurately weigh 0.678g NaCl, 0.138g KCl, 0.218g NaHCO3 and 0.008g CaCl2·2H2O into a beaker, add 100mL deionized water, stir until completely dissolved, and then adjust the pH value to 7.4 with hydrochloric acid.

[0161] The in vitro release of tacrolimus lipid microspheres was investigated using dynamic dialysis, as detailed below:

[0162] (1) Measure 100 mL of freshly prepared release medium (acetonitrile:artificial tears volume ratio of 4:6) into a 250 mL stoppered conical flask and stopper it to prevent the release medium from evaporating during the test. Place the stoppered conical flask containing the release medium in a 34°C air bath constant temperature shaker for preheating.

[0163] (2) Cut an 8cm dialysis bag (MD25-8000-14000, molecular weight cutoff of 8000-14000D, purchased from Hunan Yibo Biotechnology Co., Ltd.), clamp one end with a dialysis clamp, add 4mL of commercially available preparation Talymus to one set of dialysis bags, and add 4mL of tacrolimus lipid microsphere suspension prepared in Example 1 to another set of dialysis bags. Then clamp the other end of the dialysis bag to ensure that the liquid inside the bag does not leak.

[0164] (3) Place the dialysis bag containing tacrolimus lipid microsphere suspension and the commercially available formulation Talymus into a preheated stoppered conical flask containing the release medium. Start timing at a shaking rate of 120 rpm. At the specified time points, take out 5 mL of the drug-containing release medium and add 5 mL of the drug-free release medium.

[0165] (4) After filtering the drug-containing release medium sample through a 0.22 μm microporous membrane, the tacrolimus content was determined by HPLC. The cumulative drug release rate (Q1) was calculated using the following formula (2), and a cumulative drug release curve was plotted based on the calculation results. The results are as follows: Figure 2 As shown: the in vitro release rate of the commercially available formulation Talymus was 35.06% at 0.5h and 92.52% at 3h; the in vitro release rate (cumulative release) of tacrolimus lipid microsphere suspension was 7.56% at 0.5h, 40.36% at 3h, and 76.77% at 14h.

[0166] Among them, C nC represents the drug concentration in the nth sample. i V represents the drug concentration of the sample taken in the i-th sampling; V represents the volume of the release medium in the stoppered conical flask; V0 represents the sample volume of each sampling; and W represents the total drug content of the preparation in the dialysis bag.

[0167] 3. The erosion and in vitro release behavior of the tacrolimus liposome in-situ gel were investigated using a membrane-free dissolution method. 1 mL of the tacrolimus liposome in-situ gel prepared in Example 2 was placed in a pre-weighed empty test tube. The tacrolimus liposome in-situ gel to artificial tears were added to the pre-weighed test tube at a volume ratio of 40:7. The tube was allowed to stand until complete gelation, and then weighed. Subsequently, 2.4 mL of artificial tears was slowly added along the test tube wall, taking care not to generate air bubbles. The tube was placed in a constant-temperature shaker at 34°C and 120 rpm. At specific time points, the entire medium was poured out, the test tube wall was wiped dry, and the tube was quickly weighed. Simultaneously, 2.4 mL of artificial tears was added, and the test tube was placed back in the constant-temperature shaker. The weight difference between adjacent time points represents the amount of gel erosion. Take 100 μL of the poured-out medium into a 1.5 mL centrifuge tube, add 100 μL of acetonitrile solution and mix well. Vortex for 5 min, sonicate for 10 min, centrifuge at 15000 rpm for 20 min, and determine the drug content by HPLC. The cumulative gel erosion rate R% is calculated by the following formula (3), and the cumulative drug release rate Q% is calculated by the following formula (4).

[0168]

[0169] Where M0 is the weight of the empty test tube, M i M represents the total weight of the tacrolimus lipid microsphere in-situ gel and the test tube in the i-th weighing. y W represents the total weight of the initial tacrolimus liposome microsphere in situ gel and the test tube. i denoted as , where is the measured drug content per milliliter of sample, and W is the total drug content.

[0170] The results are as follows Figure 3 As shown: The tacrolimus lipid microsphere in-situ gel prepared in Example 2 had a cumulative erosion rate of 87.17% after 8 hours and an in vitro release rate of 97.59% after 8 hours. Similar methods were used to conduct film-free dissolution and erosion experiments on the tacrolimus lipid microsphere gels prepared in Examples 3-4, and the results are as follows. Figure 4 , 5 As shown, the cumulative erosion rates of the gels after 8 hours were 91.58% and 78.67%, respectively; the in vitro release rates after 8 hours were 65.44% and 63.83%, respectively. The cumulative drug release rates were significantly lower than those of the tacrolimus liposphere in-situ gel prepared in Example 2. Because hydroxypropyl methylcellulose gel and ethylcellulose gel do not possess the characteristics of in-situ gels, they have strong adsorption to tacrolimus, and even if the gel matrix is ​​eroded, the drug cannot be effectively released.

[0171] 4. Twelve healthy New Zealand white rabbits without eye diseases were randomly divided into four groups. The rabbits were given 50 μL of physiological saline, the commercially available Talymus formulation, the tacrolimus lipid microsphere suspension prepared in Example 1, and the tacrolimus lipid microsphere in-situ gel prepared in Example 2, respectively. Immediately after administration, the rabbits' eyes were gently closed for 10 seconds, and the blinking frequency was recorded for 1 minute to assess the physical irritation of the formulations to the rabbit eyes. The results are shown in Table 3: The blinking frequency in the saline group was 1 ± 1, while the blinking frequencies in the tacrolimus lipid microsphere and tacrolimus lipid microsphere in-situ gel groups were 2 ± 1 and 2 ± 2, respectively, showing no significant difference from the saline group. No visible redness, swelling, or increased secretions were observed in the rabbit eyes. The blinking frequency in the Talymus formulation group was 4 ± 3, slightly higher than that in the saline group. This is attributed to the irritation caused by free tacrolimus drug molecules and the antibacterial agent benzalkonium chloride.

[0172] Table 3. Blinking frequency in rabbits in vivo with different formulations.

[0173]

[0174] 5. Place fertilized eggs (0 days old, Xinxing Dahua Poultry Co., Ltd.) in an incubator, add an appropriate amount of water to maintain the humidity in the incubator at 65-75%, set the incubator temperature to 37.8±0.2℃, and turn the eggs every 2 hours. Begin incubation, adding water as needed when humidity drops. Incubate under these conditions until day 10, when the complete allantoic membrane of the chicken embryo will have formed. Remove the eggs, candle them to locate the air cell, carefully tap the shell with tweezers, and peel off the shell portion containing the air cell. Apply a few drops of physiological saline injection to the surface of the shell membrane to thoroughly moisten it, then pour out the saline injection. Carefully peel off the shell membrane again, ensuring the exposed allantoic membrane of the chicken embryo is intact and undamaged. Observe the structure of the vascular system, assess its integrity and suitability for the experiment, and discard any incomplete vascular systems. Use physiological saline injection as a negative control group (physiological saline group), 0.1 mol·L⁻¹. -1 NaOH solution was used as the positive control group. The tacrolimus lipid microsphere suspension prepared in Example 1 and the tacrolimus lipid microsphere in situ gel prepared in Example 2 were used as the experimental groups to investigate the irritation of the three preparations. 300 μL of each of the above five solutions was directly applied to the chicken embryo allantoic membrane. After 5 min, the surface of the chicken embryo allantoic membrane was gently rinsed with pure water. The rinsing was completed within 30 s. The liquid was poured out, and the damage to the blood vessels of the chicken embryo allantoic membrane in each group was observed. The stimulation score was calculated using formula (5), and the irritation level of the preparation was determined according to the evaluation criteria of the stimulation score in Table 4. The results are shown in Table 5 (n=3) and Figure 6As shown: In the positive control group, when NaOH solution came into contact with the chicken embryo allantoic membrane, large-scale bleeding immediately occurred, demonstrating strong irritation. The stimulation score was 18.13±0.53, falling within the scoring range of 9-21, indicating severe irritation. In contrast, the saline group and other preparation groups did not exhibit vasoconstriction, bleeding, or coagulation within 5 minutes of contact with the chicken embryo allantoic membrane, with stimulation scores of 0. The tacrolimus lipid microspheres prepared in Example 1 and the tacrolimus lipid microsphere in-situ gel prepared in Example 2 both scored 0, indicating no irritation. This demonstrates that both the tacrolimus lipid microspheres prepared in Example 1 and the tacrolimus lipid microsphere in-situ gel prepared in Example 2 meet the safety requirements for ophthalmic preparations.

[0175]

[0176] In the formula, t1 is the initial bleeding time (s), t2 is the initial vasoconstriction time (s), and t3 is the initial coagulation time (s).

[0177] Table 4. Chicken Embryo Allantoic Membrane Experimental Stimulation Scoring Criteria

[0178]

[0179]

[0180] Table 5. Irritation scores of chicken embryo allantoic membrane test

[0181]

[0182] 6. Nine healthy New Zealand white rabbits without eye diseases were randomly divided into three groups. 0.008 g of sodium fluorescein was incubated with 4 mL of commercially available Talymus, the tacrolimus lipid microsphere suspension prepared in Example 1, and the tacrolimus lipid microsphere in situ gel prepared in Example 2 at room temperature in the dark for 4 hours. Before the experiment, the eye health of each group of healthy New Zealand white rabbits was examined under normal light using a slit lamp (YZ5S, Liuliu Vision Technology Co., Ltd.). Then, 50 μL of the commercially available Talymus, the tacrolimus lipid microsphere suspension, and the tacrolimus lipid microsphere in situ gel prepared in Example 2, which had been incubated with sodium fluorescein (recorded as 0 min at the time of instillation), were dripped into the conjunctival sac of the rabbits. The rabbit eyes were gently closed for 10 seconds, and the nasolacrimal duct was pressed simultaneously to reduce sample loss. At specific time points, photos were taken under cobalt blue light using a slit lamp to observe changes in fluorescence intensity. The results are shown in Table 6 (n=3). Figure 7As shown, the average retention times of the tacrolimus lipid microsphere suspension and the tacrolimus lipid microsphere in-situ gel group prepared in Example 2 were 17.67±2.08 min and 70.67±8.02 min, respectively, both longer than the commercially available formulation Talymus (8.00±1.73 min). The tacrolimus lipid microsphere in-situ gel group prepared in Example 2 had the longest average retention time, which was 8.83 times and 4.00 times that of the commercially available formulation Talymus group and the tacrolimus lipid microsphere suspension group, respectively, indicating that the tacrolimus lipid microsphere in-situ gel can rapidly gel in the conjunctival sac after contact with tear fluid.

[0183] Table 6. Corneal fluorescence retention time for different formulations

[0184]

[0185] 7. Take several filter paper strips (3×8cm) and place them in 1.5mL centrifuge tubes. Nine healthy New Zealand white rabbits without eye diseases were randomly selected and divided into three groups. 100μL of physiological saline was instilled into the left eye of each group of New Zealand white rabbits as a blank control. Equal volumes of commercially available Talymus, the tacrolimus lipid microsphere suspension prepared in Example 1, and the tacrolimus lipid microsphere in situ gel prepared in Example 2 were instilled into the right eye (recorded as 0min at the time of instillation). The nasolacrimal duct was gently pressed to prevent drug loss. At specific time points, pre-weighed filter paper strips were placed in the conjunctival sac, and the tear-containing filter paper strip samples were removed after 10s. The filter paper strips were dried with nitrogen and then reconstituted with 100μL of acetonitrile solution. After vortexing for 5min, sonication for 10min, and centrifugation at 15000rpm for 20min were performed. The supernatant was collected and the drug content in the tear fluid was determined by HPLC. Results Figure 8 As shown: The drug concentration of the tacrolimus lipid microsphere in situ gel prepared in Example 2 can be detected in tears for up to 60 min, and the drug concentration of the tacrolimus lipid microsphere suspension can be detected for up to 25 min, which is significantly longer than that of the commercially available formulation Talymus (15 min). This is consistent with the results of the fluorescence retention test, which further demonstrates that it can significantly prolong the retention time of the drug in tears.

[0186] 8. Twelve healthy New Zealand rabbits without eye diseases were randomly selected and divided into four groups. They were anesthetized intravenously with 3% pentobarbital and topically with 2% lidocaine eye drops. Then, a strip of filter paper approximately 10mm × 5mm in size was soaked in 1 mol·L⁻¹ eye drops… -1 A NaOH solution was placed on the bulbar conjunctiva approximately 2 mm above the limbus of the left rabbit's cornea. After 90 seconds, the conjunctival sac was immediately and repeatedly flushed with 100 mL of physiological saline (this day was recorded as day 0 of modeling). The right eye was in the physiological saline group. On days 0 and 7 of modeling, Schirmer I test, tear film breakup time, and corneal fluorescein staining were performed on the rabbit eyes. Figure 9 , Figure 10 , Figure 11As shown, on day 0, the Schirmer I test results of all groups were greater than 10 mm / 5 min, and the TBUT was greater than 7.5 s; on day 7, the Schirmer I test results of the experimental group were less than 5 mm / 5 min, the TBUT was less than 4 s, and the FL score was greater than that on day 0 (P<0.01). This indicates that the dry eye model created by burning the bulbar conjunctiva with NaOH was successful and can be used for subsequent experiments.

[0187] Starting from day 8 after modeling, 50 μL of physiological saline (positive control), commercially available formulation Talymus, tacrolimus lipid microsphere suspension prepared in Example 1, and tacrolimus lipid microsphere in situ gel prepared in Example 2 were instilled into the conjunctival sac of the left eye of each group of New Zealand rabbits. An equal volume of physiological saline was instilled into the right conjunctival sac as a control (physiological saline group). The nasolacrimal duct was gently pressed to prevent drug loss. Administration was twice daily. From day 11 to 14 after modeling, tear volume (Schirmer I test), tear film breakup time (TBUT), and corneal fluorescein staining (FL) were analyzed to evaluate the efficacy of the formulations in treating dry eye. All measurements were performed by the same operator under identical conditions.

[0188] Schirmer I test: After applying promecaine hydrochloride for topical anesthesia for 5 minutes, a 5×35mm strip of filter paper is folded at one end and placed in the conjunctival sac at the outer or middle third of the lower eyelid, with the remaining part suspended outside the palpebral fissure. The eye can be opened, and blinking can be performed freely without the filter paper strip falling out of the eye for 5 minutes. The grading criteria are shown in Table 7. Results are as follows: Figure 9 As shown: On day 11, the dry eye symptoms of the commercially available formulation Talymus, the tacrolimus lipid microsphere suspension, and the tacrolimus lipid microsphere in situ gel prepared in Example 2 were alleviated to some extent compared with those on day 7. The tear volume was 8.13±2.08mm / 5min, 9.57±0.59mm / 5min, and 12.71±2.17mm / 5min, respectively. The dry eye symptoms of the tacrolimus lipid microsphere in situ gel prepared in Example 2 were significantly improved. On day 14, the tear volume of the commercially available formulation Talymus, the tacrolimus lipid microsphere suspension, and the tacrolimus lipid microsphere in situ gel prepared in Example 2 were 10.37±1.58 mm / 5 min, 11.4±2.56 mm / 5 min, and 16.30±1.75 mm / 5 min, respectively. The tear volume of the tacrolimus lipid microsphere in situ gel prepared in Example 2 was similar to that of the saline group (15.03±1.85 mm / 5 min), indicating that the body had fully recovered to a normal physiological state.

[0189] Table 7 Schirmer I Test Grading Standards

[0190]

[0191] Tear film breakup time (TBUT) detection: Accurately weigh 0.008 g of sodium fluorescein and add it to 4 mL of deionized water, dissolving in the dark. Instill the sodium fluorescein solution into the lower conjunctival sac of the eye (record 0 s at instillation). Manually blink to distribute the fluorescein into the tear film. Then examine the tear film using a slit lamp; the time to detect the first dry spot on the cornea is defined as the TBUT. Grading criteria are shown in Table 8. Results are as follows: Figure 10 As shown, on day 11, the tear film breakup times of the commercially available formulation Talymus, the tacrolimus lipid microsphere suspension, and the tacrolimus lipid microsphere in situ gel prepared in Example 2 were 4.80±0.76s, 6.27±0.16s, and 11.39±2.54s, respectively. On day 14, the tear film breakup times of the tacrolimus lipid microsphere in situ gel prepared in Example 2 were 14.82±2.27s, and those of the tacrolimus lipid microsphere suspension were 6.73±1.15s, which were still better than those of the commercially available formulation Talymus (6.06±1.55s). Among them, the tacrolimus lipid microsphere in situ gel prepared in Example 2 was significantly better than that of the commercially available formulation Talymus, indicating that the tacrolimus lipid microspheres prepared in Example 1 and the tacrolimus lipid microsphere in situ gel prepared in Example 2 can improve the tear film stability of New Zealand rabbits, with the tacrolimus lipid microsphere in situ gel prepared in Example 2 being more significant.

[0192] Table 8 TBUT Grading Standards

[0193]

[0194] Corneal fluorescein examination: After fluorescein staining, the corneal surface was observed using a slit lamp. The appearance of punctate staining indicates the presence of dry patches on the ocular surface, which is considered a symptom of corneal dryness. The cornea was divided into four quadrants: superior, inferior, nasal, and temporal. The scoring criteria are shown in Table 9. The scores of the four quadrants were summed, and the total score ranged from 0 to 12 points. Results are as follows: Figure 11As shown: On day 11, the corneal fluorescence staining scores of the commercially available formulation Talymus, the tacrolimus lipid microsphere suspension, and the tacrolimus lipid microsphere in situ gel prepared in Example 2 were 6.33±2.31, 4.67±1.15, and 2.33±0.58, respectively. The corneal fluorescence staining score of the tacrolimus lipid microsphere in situ gel prepared in Example 2 was significantly lower than that of the positive control group (11.33±0.58). On day 14, the corneal fluorescence staining scores of the commercially available formulation Talymus, the tacrolimus lipid microsphere suspension, and the tacrolimus lipid microsphere in situ gel prepared in Example 2 were... The scores were 6.67±3.21, 3.67±1.53, and 1.00±1.00, respectively. The corneal fluorescence staining score of the commercially available formulation Talymus increased compared to day 11, while the fluorescence staining scores of the tacrolimus lipid microsphere suspension and the tacrolimus lipid microsphere in situ gel prepared in Example 2 decreased further compared to day 11. In particular, the fluorescence staining score of the tacrolimus lipid microsphere in situ gel prepared in Example 2 decreased significantly further compared to day 11, indicating that the tacrolimus lipid microsphere suspension and the tacrolimus lipid microsphere in situ gel can not only prevent and treat dry eye syndrome, but also treat corneal damage caused by dry eye syndrome.

[0195] The results above show that when the tacrolimus lipid microsphere in-situ gel prepared in Example 2 was used to treat dry eye in New Zealand rabbits, the dry eye symptoms significantly improved during the observation period. While the dry eye symptoms of New Zealand rabbits treated with the tacrolimus lipid microsphere suspension prepared in Example 1 and the commercially available formulation Talymus also improved, the effects were significantly worse than those of the tacrolimus lipid microsphere in-situ gel prepared in Example 2. This indicates that the tacrolimus lipid microsphere in-situ gel prepared in Example 2, based on the sustained-release properties of the tacrolimus lipid microspheres prepared in Example 1, has a longer corneal retention capacity, lower irritation, and better efficacy, demonstrating promising clinical application prospects.

[0196] Table 9 Corneal Fluorescein Staining Scoring Criteria

[0197]

[0198] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A tacrolimus lipid microsphere, wherein the raw materials for preparing the tacrolimus lipid microsphere comprise: tacrolimus, a first emulsifier, an oil phase, a second emulsifier, and a lyophilization protectant; wherein the mass ratio of tacrolimus, the first emulsifier, the second emulsifier, the oil phase, and the lyophilization protectant is 12.5: (60-90): (60-90): (520-780): (280-420), wherein the first emulsifier is egg yolk lecithin, the oil phase is soybean oil, the second emulsifier is poloxamer, and the lyophilization protectant is mannitol.

2. The tacrolimus lipid microspheres according to claim 1, characterized in that: The raw materials for preparation further include: an aqueous phase; the mass-volume ratio of the second emulsifier to the aqueous phase is 7.5:(100-600).

3. The tacrolimus lipid microspheres according to claim 1, characterized in that: The raw materials for preparation also include: an organic solvent; the mixture of the tacrolimus and the first emulsifier and the organic solvent are in a mass-volume ratio of (0.7 - 1.1):

8.

4. The tacrolimus lipid microspheres according to claim 1, characterized in that: The tacrolimus lipid microspheres have a particle size of 300-600 nm; or the zeta potential of the tacrolimus lipid microspheres is -(15-25) mV.

5. The method for preparing tacrolimus lipid microspheres according to claim 1, characterized in that: Includes the following steps: Tacrolimus, the first emulsifier, and an organic solvent are mixed to obtain a drug-containing organic solvent; The drug-containing organic solvent is mixed with the oil phase to obtain an oil phase containing an emulsifier; The second emulsifier is mixed with the aqueous phase to obtain an aqueous phase containing the emulsifier; The oil phase containing emulsifier is mixed with the water phase containing emulsifier and emulsified to obtain an emulsion; The emulsion was mixed with a lyophilization protectant and then freeze-dried to obtain tacrolimus lipid microspheres.

6. The preparation method according to claim 5, characterized in that: The oil phase containing emulsifier obtained by mixing the drug-containing organic solvent with the oil phase does not contain organic solvent; The conditions for mixing the drug-containing organic solvent with the oil phase are heating to 60-80°C; The second emulsifier is mixed with the aqueous phase under the condition of heating to 60-80°C.

7. A tacrolimus liposome microsphere gel comprising a conventional tacrolimus liposome microsphere gel or an in-situ tacrolimus liposome microsphere gel, characterized in that, The raw materials for preparing the tacrolimus lipid microsphere ordinary gel include: an ordinary gel matrix and the tacrolimus lipid microspheres according to any one of claims 1-4; the ordinary gel matrix includes at least one of hydroxypropyl methylcellulose, ethylcellulose, and sodium carboxymethylcellulose; or The raw materials for preparing the tacrolimus lipid microsphere in situ gel include: an in situ gel matrix, a thickener, and tacrolimus lipid microspheres as described in any one of claims 1-4; the in situ gel matrix includes at least one of gellan gum, methylcellulose, sodium alginate, poloxamer, carbomer, and chitosan.

8. The tacrolimus lipid microsphere gel according to claim 7, characterized in that: The mass ratio of the ordinary gel matrix to tacrolimus lipid microspheres is (15-40):100; or The mass ratio of the in-situ gel matrix, thickener, and tacrolimus lipid microspheres is (50 - 100): (15 - 25): 1000.

9. The tacrolimus lipid microsphere gel according to claim 7, characterized in that: The raw materials for preparing the gel also include osmotic pressure regulators; the mass ratio of the osmotic pressure regulators to tacrolimus lipid microspheres is (25-70):

100.

10. The tacrolimus lipid microsphere gel according to claim 7, characterized in that: The raw materials for preparing the gel also include water; the mass ratio of water to tacrolimus lipid microspheres is (1 - 20):

1.

11. The tacrolimus lipid microsphere gel according to claim 7, characterized in that: The raw materials for preparing the gel also include a pH adjuster; the mass ratio of the pH adjuster to tacrolimus lipid microspheres is (0.01-0.3):1000.

12. A method for preparing a conventional gel containing tacrolimus lipid microspheres as described in claim 7, characterized in that: Includes the following steps: A common gel matrix is ​​mixed with water to obtain a common gel matrix solution; An osmotic pressure regulator, a pH regulator, and a common gel matrix solution are mixed to obtain a mixture; The mixture was combined with tacrolimus lipid microspheres to obtain a tacrolimus lipid microsphere ordinary gel.

13. A method for preparing the tacrolimus lipid microsphere in-situ gel as described in claim 7, characterized in that, Includes the following steps: The in-situ gel matrix, thickener, and water are mixed to obtain an in-situ gel matrix solution; An osmotic pressure regulator, a pH regulator, and an in-situ gel matrix solution are mixed to obtain a mixture; The mixture was combined with tacrolimus lipid microspheres to obtain tacrolimus lipid microsphere in situ gel.

14. A tacrolimus lipid microsphere suspension comprising an osmotic pressure regulator and the tacrolimus lipid microspheres according to any one of claims 1-4.

15. An eye drop comprising: tacrolimus lipid microspheres according to any one of claims 1-4, tacrolimus lipid microsphere gel according to any one of claims 7-11, or tacrolimus lipid microsphere suspension according to claim 14.

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