Tacrolimus lipid microspheres as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and discloses tacrolimus lipid microspheres as well as a preparation method and application thereof. Compared with the existing ophthalmic tacrolimus preparation (such as Talymus), the tacrolimus lipid microsphere has the advantages of no stimulation, long residence time, better xerophthalmia treatment effect, improvement of tear film stability, treatment of corneal injury caused by xerophthalmia and the like.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical technology, and specifically relates to tacrolimus lipid microspheres and a preparation method and application thereof. Background Art

[0002] Dry eye is a common multifactorial ocular surface disease, which may be accompanied by ocular surface inflammation, tissue damage and neurological abnormalities. The rational use of eye drops is the best way to relieve visual fatigue and dry eye, but traditional eye drops cause a large amount of drug loss due to physiological reasons such as corneal barrier and tear washout, resulting in reduced bioavailability.

[0003] The eye drops for treating dry eyes on the market include sodium hyaluronate eye drops, polyvinyl alcohol eye drops, recombinant bovine growth factor eye drops and cyclosporine eye drops. Among them, sodium hyaluronate eye drops and polyvinyl alcohol eye drops can only relieve the symptoms of dry eyes, and need to be dropped into the eyes 5-6 times a day, and the patient compliance is poor. Recombinant bovine growth factor eye drops can cause adverse reactions such as skin itching, throat swelling, and difficulty breathing in some patients. Cyclosporine eye drops cause patients who are intolerant to them to experience symptoms such as blurred vision, gastrointestinal discomfort, and impaired liver function.

[0004] Tacrolimus is a macrolide drug with strong immunosuppressive properties. It is a highly lipophilic and hydrophobic compound that is insoluble in water. When used for the prevention and treatment of dry eye, 0.1% FK506 eye drops are generally used twice a day, and then the dosage is gradually reduced. However, the side effect of ophthalmic tacrolimus preparations is mild and transient eye irritation. The incidence of side effects of 0.1% tacrolimus eye drops is 29%. Summary of the invention

[0005] The purpose of the first aspect of the present invention is to provide a tacrolimus lipid microsphere.

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

[0007] The third aspect of the present invention aims to provide a tacrolimus lipid microsphere gel.

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

[0009] The fifth aspect of the present invention aims to provide a tacrolimus lipid microsphere suspension.

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

[0011] The seventh aspect of the present invention aims to provide an eye drop.

[0012] In order to achieve the above object, the technical solution adopted by the present invention is:

[0013] The first aspect of the present invention provides a tacrolimus lipid microsphere, wherein the raw materials for preparing the tacrolimus lipid microsphere 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, soybean lecithin, gum arabic, tragacanth gum, gelatin, apricot gum, polyoxyethylene fatty alcohol ether, and polyoxyethylene fatty acid ester; further comprises at least one of egg yolk lecithin and soybean lecithin; and further is 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 dienoate, silicone oil, and vaseline; further comprises at least one of soybean oil, olive oil, castor oil, safflower oil, coconut oil, sunflower oil, and almond oil; and further comprises 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 further comprises poloxamer 188.

[0017] Preferably, the lyoprotectant comprises at least one of mannitol, sucrose, trehalose, lactose, glucose, maltose, dextran, albumin, ethylene glycol, glycerol, L-serine, sodium glutamate, alanine, glycine, sarcosine, phosphate, acetate, and citrate; further comprises at least one of mannitol, sucrose, trehalose, lactose, and glucose; and further comprises mannitol.

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

[0019] Preferably, the preparation raw material further comprises: an aqueous phase.

[0020] Preferably, the aqueous phase comprises at least one of water and phosphate buffer; further comprises water; and further comprises deionized water.

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

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

[0023] Preferably, the organic solvent comprises at least one of ethanol, dichloromethane, chloroform, vinyl chloride, dimethyl sulfoxide, acetone, methyl acetate, and ether; further comprises ethanol; and further comprises anhydrous ethanol.

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

[0025] Preferably, the particle size of the tacrolimus lipid microspheres is 300-600 nm; further 400-500 nm; further 461.76±15.13 nm.

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

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

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

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

[0030] mixing tacrolimus, a first emulsifier, and an organic solvent to obtain a drug-containing organic solvent;

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

[0032] mixing the second emulsifier with the water phase to obtain an emulsifier-containing water phase;

[0033] mixing an oil phase containing an emulsifier with an aqueous phase containing an emulsifier, emulsifying, and obtaining an emulsion;

[0034] The emulsion is mixed with a freeze-drying protectant, and freeze-dried to obtain tacrolimus lipid microspheres.

[0035] Preferably, the tacrolimus, the first emulsifier and the organic solvent are mixed by stirring until they are dissolved and uniformly dispersed.

[0036] Preferably, the stirring condition is 200-600 rpm stirring for 20-40 min; further, 300-500 rpm magnetic stirring for 25-35 min at room temperature.

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

[0038] Preferably, the drug-containing organic solvent is mixed with the oil phase by heating to 60-80°C; further heating to 60-80°C and stirring; further heating to 65-75°C in a water bath and stirring.

[0039] Preferably, the stirring condition is stirring at 200-600 rpm; further stirring at 300-500 rpm; further magnetic stirring at 300-500 rpm.

[0040] Preferably, the second emulsifier is mixed with water by heating to 60-80°C; further by heating in a water bath to 65-75°C.

[0041] Preferably, the emulsifying comprises shearing, and homogenizing.

[0042] Preferably, the shearing conditions are 11000-15000 rpm, 5-15 min; further 12000-14000 rpm, 8-12 min.

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

[0044] Preferably, the homogenization condition is 500-900 bar for 5-9 times; further, 600-800 bar for 6-8 times.

[0045] Preferably, homogenize for 2-4 minutes each time.

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

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

[0048] The third aspect of the present invention provides any one of a1) to a2) tacrolimus lipid microsphere gels:

[0049] a1) a tacrolimus lipid microsphere common gel, wherein the raw materials for preparing the tacrolimus lipid microsphere common gel include: a common gel matrix, and the tacrolimus lipid microsphere of the first aspect of the present invention; the common gel matrix includes at least one of hydroxypropyl methylcellulose, ethyl cellulose, and sodium carboxymethyl cellulose;

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

[0051] Preferably, the common gel matrix in a1) comprises at least one of hypromellose and ethyl cellulose; further comprises hypromellose or ethyl cellulose.

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

[0053] Preferably, the thickener 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 polyvinyl pyrrolidone; further comprises at least one of xanthan gum, carboxymethyl cellulose, and methyl cellulose; and further comprises xanthan gum.

[0054] Preferably, the mass ratio of the common gel matrix and the tacrolimus lipid microspheres in a1) is (15-40):100; further (20-35):100; further (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 (60-90):(16-23):1000; further (70-80):(17-22):1000.

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

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

[0058] Preferably, the osmotic pressure regulators in a1) and a2) are each independently selected from at least one of mannitol, lactose, sucrose and sodium chloride; further preferably, the osmotic pressure regulators 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; 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; further (45-51):100.

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

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

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

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

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

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

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

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

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

[0071] Preferably, the pH adjusters described in a1) and a2) are each independently selected from at least one of tris(hydroxymethyl)aminomethane, sodium hydroxide, triethylamine, boric acid, and hydrochloric acid; further selected from tris(hydroxymethyl)aminomethane.

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

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

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

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

[0076] mixing a common gel base with water to obtain a common gel base solution;

[0077] mixing an osmotic pressure regulator, a pH regulator, and a common gel matrix solution to obtain a mixture;

[0078] The mixture is mixed with tacrolimus lipid microspheres to obtain tacrolimus lipid microspheres ordinary gel;

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

[0080] Mixing the in situ gel matrix, the thickener, and water to obtain an in situ gel matrix solution;

[0081] mixing an osmotic pressure regulator, a pH regulator, and the in situ gel matrix solution to obtain a mixture;

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

[0083] Preferably, the common gel matrix and water in b1) are mixed at 300-700 rpm to achieve uniform dispersion; further, at 400-600 rpm to achieve uniform dispersion.

[0084] Preferably, the common gel matrix and water in b1) are mixed at 60-80°C; further at 65-75°C.

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

[0086] Preferably, before the common gel matrix solution in b1) is mixed with the osmotic pressure regulator, the common gel matrix solution is placed in an ice bath at 0-6° C. for 18-30 h to defoam, and the gel matrix solution is clarified and returned to room temperature.

[0087] Preferably, the osmotic pressure regulator, pH regulator and common gel matrix solution in b1) are mixed at 300-700 rpm to achieve uniform dispersion; further, at 400-600 rpm to achieve uniform dispersion.

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

[0089] Preferably, the mixture in b1) is mixed with tacrolimus lipid microspheres to be uniformly dispersed at 600-1000 rpm; further, uniformly dispersed at 700-900 rpm.

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

[0091] Preferably, the stirring condition is stirring at 300-700 rpm; further stirring at 400-600 rpm.

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

[0093] Preferably, before the in situ gel matrix solution is mixed 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 h to defoam, and the in situ gel matrix solution is clarified and returned to room temperature.

[0094] Preferably, the osmotic pressure regulator, pH regulator, and in situ gel matrix solution are mixed at 300-700 rpm to achieve uniform dispersion; further, at 400-600 rpm to achieve uniform dispersion.

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

[0096] Preferably, the mixture in b2) is mixed with tacrolimus lipid microspheres to be uniformly dispersed at 600-1000 rpm; further, uniformly dispersed at 700-900 rpm.

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

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

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

[0100] Preferably, the tacrolimus lipid microsphere suspension also contains water; further contains deionized water.

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

[0102] Preferably, the pH of the tacrolimus lipid microsphere suspension is 6.0-8.0; further 6.5-7.5; further 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; further 288-305 mOsmol / kg; further 298-303 mOsmol / kg.

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

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

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

[0107] The beneficial effects of the present invention are:

[0108] The present invention provides a tacrolimus lipid microsphere, which, compared with existing tacrolimus preparations for eyes (such as Talymus), has the advantages of being non-irritating, having a long retention time, having a better effect in treating dry eye, being able to improve the stability of the tear film, and treating corneal damage caused by dry eye.

[0109] The present invention provides a tacrolimus lipid microsphere gel, which, compared with tacrolimus lipid microspheres, has the advantages of long retention time, better effect in treating dry eye, higher tear film stability, better effect in treating corneal damage caused by dry eye, etc., and has good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] Figure 1 The figures are the appearance pictures of tacrolimus lipid microspheres and the tacrolimus lipid microsphere in situ gel prepared in Example 2: wherein, (a) is the appearance picture of the tacrolimus lipid microsphere suspension prepared in Example 1; (b) is the appearance picture of the diluted solution of the tacrolimus lipid microsphere suspension prepared in Example 1 irradiated by a laser pen; (c) is the appearance picture of the tacrolimus lipid microsphere in situ gel prepared in Example 2; (d) is the appearance picture of the diluted solution of the tacrolimus lipid microsphere in situ gel prepared in Example 2 irradiated by a laser pen.

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

[0112] Figure 3 The in vitro release curve and in situ gel dissolution diagram of the tacrolimus lipid microsphere in situ gel prepared in Example 2, wherein n=3.

[0113] Figure 4 The in vitro release curve and in situ gel dissolution diagram of the tacrolimus lipid microsphere gel prepared in Example 3, wherein n=3.

[0114] Figure 5 The in vitro release curve and in situ gel dissolution diagram of the tacrolimus lipid microsphere gel prepared in Example 4, wherein n=3.

[0115] Figure 6 The diagrams are of chicken embryo vascular bleeding, coagulation and melting in the normal saline group, the positive control group, the commercially available preparation Talymus, the tacrolimus lipid microsphere suspension and the tacrolimus lipid microsphere in situ gel group prepared in Example 2, wherein the camera lens magnification is 10 times.

[0116] Figure 7 The precorneal fluorescence retention of the commercially available preparation Talymus, the tacrolimus lipid microsphere suspension and the tacrolimus lipid microsphere in situ gel prepared in Example 2, wherein the camera lens magnification is 10 times.

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

[0118] Fig. 9 It is a comparison chart of Schirmer I test results of normal saline group, positive control group, Talymus, tacrolimus lipid microsphere group, and tacrolimus lipid microsphere in situ gel group, wherein n=3.

[0119] Fig.10 This is a comparison chart of the tear breakup time (TBUT) results of the normal saline group, positive control group, Talymus, tacrolimus lipid microsphere group, and tacrolimus lipid microsphere in situ gel group, wherein n=3.

[0120] Fig.11 It is a comparison chart of corneal fluorescence staining (FL) results of normal saline group, positive control group, Talymus, tacrolimus lipid microsphere group, and tacrolimus lipid microsphere in situ gel group, wherein 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 DESCRIPTION

[0122] The present invention is further described in detail below through specific examples.

[0123] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0124] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The materials and reagents used in the examples are commercially available unless otherwise specified.

[0125] In the present invention, "room temperature" is 25±5°C.

[0126] The reagents and their manufacturers used in the following examples 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 comprises the following steps:

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

[0133] (2) Preparation of the oil phase containing the emulsifier: Accurately weigh 6.5 g 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 stir with a magnetic stirrer at 400 rpm until the ethanol is completely evaporated.

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

[0135] (4) The 70° C. emulsifier-containing oil phase obtained in step (2) was poured into the 70° C. emulsifier-containing water phase obtained in step (3) using a homogenizer (IKAT18, IKA, Germany) for emulsification under shear conditions of 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.) at a homogenization pressure of 700 bar, 7 homogenization times, and each homogenization time of 3 min.

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

[0138] Accurately weigh 1 g of freeze-dried tacrolimus lipid microspheres and 1.5 g of mannitol, add 10 mL of deionized water and stir to disperse evenly to obtain a tacrolimus lipid microsphere suspension. The osmotic pressure was measured by an ice point osmometer (OSMOMAT 3000basic, Gonotec GmBHC, Germany) to be 300 mOsmol / kg, and the pH value was measured by a pH meter (PHS-3C, Shanghai Leici Instrument Factory) to be 7.23.

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

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

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

[0142] (2) Accurately weigh 0.48 g of mannitol and 0.05 mL of tris(hydroxymethyl)aminomethane (12.2 mg / mL) were added to the in situ gel matrix obtained in step (1), and dispersed evenly at 500 rpm. The osmotic pressure of the resulting mixture was measured to be 280 mOsmol / kg using an 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 1 g of the freeze-dried tacrolimus lipid microspheres obtained in step (6) of Example 1 and add them to the mixture cooled to room temperature in step (2), and stir at 800 rpm until the mixture is evenly dispersed to obtain the tacrolimus lipid microsphere in situ gel ( Figure 1 ), the osmotic pressure was measured to be 300mOsmol / kg, and the 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 comprises the following steps:

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

[0147] (2) Accurately weigh 0.48 g of mannitol and 0.01 mL of tris(hydroxymethyl)aminomethane (12.2 mg / mL) were added to the gel matrix obtained in step (1) and dispersed evenly at 500 rpm. The osmotic pressure of the resulting mixture was measured to be 278 mOsmol / kg using an osmometer (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 1 g of the freeze-dried tacrolimus lipid microspheres obtained in step (6) of Example 1 and add them to the mixture cooled to room temperature obtained in step (2), and stir at 800 rpm until the mixture is evenly dispersed to obtain tacrolimus lipid microsphere gel. The osmotic pressure of the tacrolimus lipid microsphere gel was measured to be 303 mOsmol / kg, and the pH value was 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 comprises the following steps:

[0151] (1) Accurately weigh 0.27 g of ethyl cellulose into a beaker, measure 10 mL of deionized water and add it to the beaker, heat in a 70°C water bath at 500 rpm to disperse evenly, and place the obtained gel matrix in an ice bath at 4°C for 24 h to defoam. When the solution is clarified, the obtained clear gel matrix solution is returned to room temperature.

[0152] (2) Accurately weigh 0.48 g of mannitol and 0.01 mL of tris(hydroxymethyl)aminomethane (12.2 mg / mL) were added to the gel matrix obtained in step (1) and dispersed evenly at 500 rpm. The osmotic pressure of the resulting mixture was measured to be 275 mOsmol / kg using an 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 1 g of the freeze-dried tacrolimus lipid microspheres obtained in step (6) of Example 1 and add them to the mixture cooled to room temperature obtained in step (2), and stir at 800 rpm until uniformly dispersed to obtain tacrolimus lipid microsphere gel. The osmotic pressure was measured to be 298 mOsmol / kg, and the pH value was measured to be 7.12.

[0154] Effect Example

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

[0156] Wherein, W0 is the free drug content in the tacrolimus lipid microsphere suspension / tacrolimus lipid microsphere in situ gel; W1 is the drug content added in the prescription.

[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 sizes were relatively uniform; the Zeta potentials were -(23.52±1.43) mV and -(31.31±1.87) mV, respectively, which was conducive to the dispersion of nanoparticles and made the system more stable. The requirement for the encapsulation rate of microparticle preparations in the 2020 edition of the Chinese Pharmacopoeia is generally not 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 are 97.18±1.80% and 97.42±4.47%, respectively, both higher than 80%, which are in compliance with the provisions of the Pharmacopoeia.

[0158] Table 2 Physicochemical properties of tacrolimus lipid microspheres and tacrolimus lipid microsphere in situ gel (n=3)

[0159]

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

[0161] The in vitro release of tacrolimus lipid microspheres was investigated by dynamic dialysis method, as follows:

[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 plug it to prevent the release medium from evaporating during the experiment. Place the stoppered conical flask containing the release medium in a 34°C air bath constant temperature oscillator for preheating.

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

[0164] (3) Place the dialysis bag containing tacrolimus lipid microsphere suspension and the commercially available preparation Talymus into a preheated stoppered conical flask containing the release medium. Start the timing at an oscillation 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 filter membrane, the content of tacrolimus was determined by HPLC. The drug cumulative release rate (Q1) was calculated by the following formula (2), and the drug cumulative release curve was drawn based on the calculation results. Figure 2 As shown: the in vitro release rate of the commercially available preparation Talymus is 35.06% at 0.5h and 92.52% at 3h; the in vitro release rate (cumulative release amount) of tacrolimus lipid microsphere suspension is 7.56% at 0.5h, 40.36% at 3h and 76.77% at 14h.

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

[0167] 3. The dissolution and in vitro release behavior of the tacrolimus lipid microsphere in situ gel were investigated using a membraneless dissolution method. Take 1 mL of the tacrolimus lipid microsphere in situ gel prepared in Example 2 in a weighed empty test tube, add it to the weighed test tube with a volume ratio of 40:7 of tacrolimus lipid microsphere in situ gel to artificial tears, let it stand until it is completely gelled, and weigh it. Then slowly add 2.4 mL of artificial tears along the wall of the test tube, being careful not to produce bubbles, place it in an air bath constant temperature oscillator at 34 ° C and 120 rpm, pour out all the medium at a specific time point, wipe off the water droplets on the wall of the test tube and weigh it quickly, add 2.4 mL of artificial tears at the same time, and place the test tube in the constant temperature oscillator again. The weight difference between adjacent time points is the amount of gel dissolution. Take 100 μL of the poured medium into a 1.5 mL centrifuge tube, add 100 μL of acetonitrile solution and mix well, vortex for 5 minutes, ultrasonicate for 10 minutes, centrifuge at 15000 rpm for 20 minutes, and determine the drug content by HPLC. The cumulative gel dissolution 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] Among them, M0 is the weight of the empty test tube, M i is the total weight of the tacrolimus lipid microsphere in situ gel and the test tube weighed for the i-th time, M y is the total weight of the initial tacrolimus lipid microsphere in situ gel and the test tube, W i is the measured drug content per ml of sample, and W is the total drug content.

[0170] The results are as follows Figure 3 As shown in the figure, the cumulative erosion rate of the tacrolimus lipid microsphere in situ gel prepared in Example 2 was 87.17% in 8 hours, and the in vitro release rate in 8 hours was 97.59%. The membraneless dissolution and erosion test of the tacrolimus lipid microsphere gel prepared in Examples 3-4 was carried out by a similar method. The results are shown in the figure. Figure 4 , 5As shown in the figure, the cumulative dissolution rates of the gels at 8 h were 91.58% and 78.67% respectively; the in vitro release rates at 8 h were 65.44% and 63.83% respectively, and the cumulative release rates of the drugs were much lower than those of the tacrolimus lipid microsphere in situ gel prepared in Example 2. Since the hydroxypropyl methylcellulose gel and the ethylcellulose gel do not have the characteristics of in situ gel, they have strong adsorption capacity for tacrolimus drugs, and even if the gel matrix is ​​dissolved, the drug cannot be effectively released.

[0171] 4. 12 healthy New Zealand white rabbits without eye diseases were selected and randomly divided into 4 groups. 50 μL of saline, commercial preparation Talymus, tacrolimus lipid microsphere suspension prepared in Example 1 and tacrolimus lipid microsphere in situ gel prepared in Example 2 were respectively given to the rabbit eyes. Immediately after administration, the rabbit eyes were gently closed for 10 seconds, and the timing was started for 1 minute. The number of rabbit blinks in each group within 1 minute was recorded to investigate the physical irritation of the preparation to the rabbit eyes. The results are shown in Table 3: The blinking frequency of the saline group was 1±1, and the tacrolimus lipid microsphere and tacrolimus lipid microsphere in situ gel groups were 2±1 times and 2±2 times, respectively, which were not significantly different from the saline group. The rabbit eyes did not show visible redness, swelling, increased secretions, etc. The blinking frequency of the commercial preparation Talymus group was 4±3 times, slightly higher than that of the saline group. This is the irritation caused by free tacrolimus drug molecules and antibacterial agent benzalkonium chloride.

[0172] Table 3 Blinking frequency of rabbits in vivo with different preparations

[0173]

[0174] 5. Place the fertilized eggs (0 days old, Xinxing Dahua Agricultural and Poultry Co., Ltd.) in an incubator, inject an appropriate amount of water into the water tank to maintain the humidity in the incubator at 65-75%, set the temperature in the incubator to 37.8±0.2℃, and turn over once every 2 hours. Start incubating the eggs, and replenish water in time when the humidity drops. When incubated under this condition to the 10th day, a complete chicken embryo chorioallantoic membrane is formed. Take out the eggs, illuminate the eggs to find the location of the air chamber, carefully tap the eggshell with tweezers, and peel off the eggshell part of the air chamber. Drop a few drops of saline injection on the surface of the eggshell membrane to fully moisten it, then pour out the saline injection, and then carefully peel off the eggshell membrane to ensure that the exposed chicken embryo chorioallantoic membrane is intact and not damaged. Observe the structure of the vascular system, judge its integrity and whether it is suitable for the experiment, and discard it if it is incomplete. The saline injection was used as the negative control group (saline group), 0.1mol·L -1The 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 group, and the irritation of the three preparations was investigated. 300 μL of the above five solutions were taken to act directly on the chicken embryo chorioallantoic membrane. After 5 minutes, the surface of the chicken embryo chorioallantoic membrane was gently rinsed with pure water. The rinsing was completed within 30 seconds, and the liquid was poured out. The damage to the blood vessels of the chicken embryo chorioallantoic membrane of each group was observed, and the irritation score was calculated by formula (5). The irritation of the preparation was determined according to the evaluation criteria of the irritation score in Table 4. The results are shown in Table 5 (n=3) and Figure 6 As shown: In the positive control group, when the NaOH solution came into contact with the chicken embryo chorioallantoic membrane, large-scale bleeding occurred immediately, showing strong irritation, with an irritation score of 18.13±0.53, which belongs to the score range of 9 to 21, and is a severe irritation. In the physiological saline group and other preparation groups, no vasoconstriction, bleeding, coagulation and other phenomena occurred within 5 minutes after contacting the chicken embryo chorioallantoic membrane, and the irritation score was 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. It shows that the tacrolimus lipid microspheres prepared in Example 1 and the tacrolimus lipid microsphere in situ gel prepared in Example 2 both meet the safety requirements of ophthalmic preparations.

[0175]

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

[0177] Table 4 Chicken embryo chorioallantoic membrane test stimulation scoring standard

[0178]

[0179]

[0180] Table 5 Chicken embryo allantoic membrane test irritation scoring results

[0181]

[0182] 6. Select 9 healthy New Zealand white rabbits without eye diseases and randomly divide them into 3 groups. Take 0.008g of sodium fluorescein and incubate with 4mL of the commercially available preparation Talymus, the tacrolimus lipid microsphere suspension prepared in Example 1, and the tacrolimus lipid microsphere in situ gel prepared in Example 2 in the dark at room temperature for 4h. Before the experiment, use a slit lamp (YZ5S, Liuliu Vision Technology Co., Ltd.) to check the health of the rabbit eyes of each group of healthy New Zealand rabbits under normal light. Then, 50μL of the commercially available preparation Talymus, tacrolimus lipid microsphere suspension, and the tacrolimus lipid microsphere in situ gel prepared in Example 2 that have been incubated with sodium fluorescein are instilled into the conjunctival sac of the rabbits (the time of instillation is marked as 0min), and gently close the rabbit eyes for 10s, while pressing the nasolacrimal duct to reduce sample loss. Take pictures under cobalt blue light with a slit lamp at specific time points to observe changes in fluorescence intensity. The results are shown in Table 6 (n=3) and Figure 7 As shown in the figure: the average retention time of the tacrolimus lipid microsphere suspension and the tacrolimus lipid microsphere in situ gel group prepared in Example 2 were 17.67±2.08min and 70.67±8.02min, respectively, both longer than the commercially available preparation Talymus (8.00±1.73min). The average retention time of the tacrolimus lipid microsphere in situ gel group prepared in Example 2 was the longest, which was 8.83 times and 4.00 times that of the commercially available preparation Talymus group and the tacrolimus lipid microsphere suspension group, respectively, indicating that the tacrolimus lipid microsphere in situ gel can be quickly gelled in the conjunctival sac after contacting with tears.

[0183] Table 6 Corneal fluorescence retention time of different preparations

[0184]

[0185] 7. Take several filter paper strips (specifications are 3×8 cm) and put them in a 1.5 mL centrifuge tube. 9 healthy New Zealand white rabbits without eye diseases were randomly selected and divided into 3 groups. 100 μL of normal saline was dropped into the left eye of each group of New Zealand white rabbits as a blank control, and equal volumes of the commercially available preparation Talymus, the tacrolimus lipid microsphere suspension prepared in Example 1, and the tacrolimus lipid microsphere in situ gel prepared in Example 2 were dropped into the right eye (the time of instillation was marked as 0 min), and the nasolacrimal duct was gently pressed to prevent drug loss. At a specific time point, the pre-weighed filter paper strips were placed in the conjunctival sac, and the filter paper strip samples containing tears were taken out after 10 seconds. After the filter paper strips were dried with a nitrogen blower, 100 μL of acetonitrile solution was added to dissolve them, vortexed for 5 minutes, and ultrasonicated for 10 minutes. Centrifuged at 15000 rpm for 20 minutes, the supernatant was taken and the drug content in the tears was determined by HPLC. Results Figure 8As shown: the drug concentration of the tacrolimus lipid microsphere in situ gel prepared in Example 2 in tears can be continuously detected for 60 minutes, and the tacrolimus lipid microsphere suspension can be continuously detected for 25 minutes, which is significantly longer than the commercially available preparation Talymus (15 minutes), which is consistent with the results of the fluorescence retention test, once again indicating that it can significantly prolong the retention time of the drug in tears.

[0186] 8. Randomly select 12 healthy New Zealand rabbits without eye diseases and divide them into 4 groups. Use 3% pentobarbital intravenous anesthesia and 2% lidocaine eye drops for surface anesthesia. Then use a filter paper strip of about 10mm×5mm size to dip 1mol·L -1 The NaOH solution was placed on the bulbar conjunctiva about 2 mm above the corneal margin of the left eye of the rabbit. After 90 seconds, the conjunctival sac was repeatedly rinsed with 100 mL of normal saline (the day was recorded as the 0th day of modeling). The right eye was in the normal saline group. On the 0th and 7th days of modeling, the rabbit eyes were examined by Schirmer I test, tear film breakup time and corneal fluorescence staining. Fig. 9 , Fig.10 , Fig.11 As shown in the data, on day 0, the Schirmer I test results of each group 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 scores were greater than those on day 0 (P<0.01), indicating that the dry eye model was successfully created by burning the bulbar conjunctiva with NaOH and could be used for subsequent experiments.

[0187] From the 8th day of modeling, 50 μL of normal saline (positive control group), commercially available preparation Talymus (Talymus), tacrolimus lipid microsphere suspension prepared in Example 1 (tacrolimus lipid microsphere) and tacrolimus lipid microsphere in situ gel prepared in Example 2 (tacrolimus lipid microsphere in situ gel) were respectively dripped into the conjunctival sac of the left eye of each group of New Zealand rabbits, and an equal volume of normal saline was dripped into the conjunctival sac of the right eye as a control (normal saline group), and the nasolacrimal duct was gently pressed to prevent drug loss, and the drug was administered twice a day. On the 11th to 14th day after modeling, the amount of rabbit tears (Schirmer I test), tear film breakup time (TBUT) and corneal fluorescence staining examination (FL) were analyzed to evaluate the effect of the preparation in treating dry eye. All measurements were performed by the same operator under the same environment.

[0188] Schirmer I test: After dripping proparacaine hydrochloride as a surface 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 1 / 3 of the lower eyelid, and the rest is hung outside the eyelid from the palpebral fissure. The eye can be opened and blinking can be done at will without causing the filter paper to fall out of the eye. This lasts for 5 minutes. The grading standard is shown in Table 7. Fig. 9 As shown: On the 11th day, the commercially available preparation Talymus, tacrolimus lipid microsphere suspension and the tacrolimus lipid microsphere in situ gel group prepared in Example 2 had a certain degree of relief of dry eye symptoms compared to the 7th day, and 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 group prepared in Example 2 were significantly improved. On the 14th day, the tear volumes of the commercially available preparation Talymus, the tacrolimus lipid microsphere suspension, and the tacrolimus lipid microsphere in situ gel group prepared in Example 2 were 10.37±1.58mm / 5min, 11.4±2.56mm / 5min, and 16.30±1.75mm / 5min, respectively. The tacrolimus lipid microsphere in situ gel group prepared in Example 2 was similar to the normal saline group (15.03±1.85mm / 5min), indicating that the mice had fully recovered to a normal physiological state.

[0189] Table 7 Schirmer I test grading standards

[0190]

[0191] Tear breakup time (TBUT) test: accurately weigh 0.008g of sodium fluorescein and add it to 4mL of deionized water, dissolve it in the dark, drip the sodium fluorescein solution into the lower conjunctival sac of the eye (0s when dripping), and manually blink to distribute the fluorescein in the tear film. Then check the tear film with a slit lamp to find the time when the first dry spot on the cornea appears as TBUT. The grading standard is shown in Table 8. The results are as follows Fig.10 As shown, on the 11th day, the tear film breakup times of the commercially available preparation Talymus, the tacrolimus lipid microsphere suspension and the tacrolimus lipid microsphere in situ gel group prepared in Example 2 were 4.80±0.76s, 6.27±0.16s and 11.39±2.54s, respectively. On the 14th day, the tacrolimus lipid microsphere in situ gel group prepared in Example 2 was 14.82±2.27s, and the tacrolimus lipid microsphere suspension was 6.73±1.15s, which were still better than the commercially available preparation Talymus (6.06±1.55s). Among them, the tacrolimus lipid microsphere in situ gel group prepared in Example 2 was significantly better than the commercially available preparation 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, among which the tacrolimus lipid microsphere in situ gel prepared in Example 2 is more significant.

[0192] Table 8 TBUT grading standards

[0193]

[0194] Corneal fluorescence examination: After fluorescein staining, the corneal surface is observed with a slit lamp. The appearance of punctate staining indicates the presence of dry spots on the ocular surface, which is considered a symptom of corneal dryness. The cornea is divided into four quadrants: superior, inferior, nasal, and temporal. The scoring criteria are shown in Table 9. The scores of the four quadrants are added together, with a total score of 0 to 12 points. The results are as follows Fig.11 As shown in the figure: on the 11th day, the corneal fluorescence staining scores of the commercially available Talymus, tacrolimus lipid microsphere suspension and the tacrolimus lipid microsphere in situ gel group 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 group prepared in Example 2 was significantly lower than that of the positive control group (11.33±0.58). On the 14th day, the corneal fluorescence staining scores of the commercially available Talymus, tacrolimus lipid microsphere suspension and the tacrolimus lipid microsphere in situ gel group 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 group prepared in Example 2 was significantly lower than that of the positive control group (11.33±0.58). The corneal fluorescence staining score of the commercially available preparation Talymus increased compared with that on the 11th day, while the fluorescence staining score of the tacrolimus lipid microsphere suspension and the tacrolimus lipid microsphere in situ gel prepared in Example 2 was further reduced compared with that on the 11th day. In particular, the fluorescence staining score of the tacrolimus lipid microsphere in situ gel prepared in Example 2 was further significantly reduced compared with that on the 11th day, indicating that the tacrolimus lipid microsphere suspension and the tacrolimus lipid microsphere in situ gel can not only prevent and treat dry eye, but also treat corneal damage caused by dry eye.

[0195] From the above results, it can be seen that when the tacrolimus lipid microsphere in situ gel prepared in Example 2 is used for New Zealand rabbits with dry eye, the symptoms of dry eye are significantly restored 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 preparation Talymus are restored, but the effect is significantly worse than that of the tacrolimus lipid microsphere in situ gel prepared in Example 2. It shows that the tacrolimus lipid microsphere in situ gel prepared in Example 2 can have a longer corneal retention ability, lower irritation, better efficacy, and good clinical application prospects on the basis of the sustained release of the tacrolimus lipid microsphere prepared in Example 1, and has a good clinical application prospect.

[0196] Table 9 Corneal fluorescence staining scoring criteria

[0197]

[0198] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A tacrolimus lipid microsphere, wherein the raw materials for preparing the tacrolimus lipid microsphere include: tacrolimus, a first emulsifier, an oil phase, a second emulsifier, and a lyophilization protective agent.

2. The tacrolimus lipid microsphere according to claim 1, characterized in that: The first emulsifier comprises at least one of egg yolk lecithin, soybean lecithin, gum arabic, gum tragacanth, gelatin, apricot gum, polyoxyethylene fatty alcohol ether, and polyoxyethylene fatty acid ester; 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 dienoate, silicone oil, and vaseline; Preferably, the second emulsifier comprises at least one of poloxamer, Tween, polyvinyl alcohol, sodium cholate, and sodium deoxycholate; Preferably, the lyoprotectant comprises at least one of mannitol, sucrose, trehalose, lactose, glucose, maltose, dextran, albumin, ethylene glycol, glycerol, L-serine, sodium glutamate, alanine, glycine, sarcosine, phosphate, acetate, and citrate.

3. The tacrolimus lipid microsphere according to claim 1 or 2, characterized in that: The mass ratio of tacrolimus, the first emulsifier, the second emulsifier, the oil phase, and the lyophilization protective agent is 12.5:(60-90):(60-90):(520-780):(280-420); Preferably, the preparation raw material further comprises: an aqueous phase; the mass volume ratio of the second emulsifier to the aqueous phase is 7.5:(100-600); Preferably, the preparation raw material further comprises: an organic solvent; the mass volume ratio of the mixture of tacrolimus and the first emulsifier to the organic solvent is (0.7-1.1):8; Preferably, the particle size of the tacrolimus lipid microspheres is 300-600 nm; Preferably, the Zeta potential of the tacrolimus lipid microspheres is -(15-25) mV.

4. The method for preparing tacrolimus lipid microspheres according to claim 3, characterized in that: The steps include: Mixing tacrolimus, a first emulsifier, and an organic solvent to obtain a drug-containing organic solvent; mixing the drug-containing organic solvent with the oil phase to obtain an emulsifier-containing oil phase; mixing the second emulsifier with the water phase to obtain an emulsifier-containing water phase; mixing an oil phase containing an emulsifier with an aqueous phase containing an emulsifier, emulsifying, and obtaining an emulsion; The emulsion is mixed with a freeze-drying protectant, and freeze-dried to obtain tacrolimus lipid microspheres.

5. The preparation method according to claim 4, characterized in that: The emulsifier-containing oil phase obtained by mixing the drug-containing organic solvent with the oil phase does not contain an organic solvent; Preferably, the drug-containing organic solvent is mixed with the oil phase under the condition of heating to 60-80°C; Preferably, the second emulsifier is mixed with water under the condition of heating to 60-80°C.

6. Any one of a1)-a2) of tacrolimus lipid microsphere gel: a1) a tacrolimus lipid microsphere common gel, wherein the raw materials for preparing the tacrolimus lipid microsphere common gel include: a common gel matrix, and the tacrolimus lipid microsphere according to any one of claims 1 to 3; the common gel matrix includes at least one of hydroxypropyl methylcellulose, ethyl cellulose, and sodium carboxymethyl cellulose; a2) A tacrolimus lipid microsphere in situ gel, wherein the raw materials for preparing the tacrolimus lipid microsphere in situ gel comprise: an in situ gel matrix, a thickener and the tacrolimus lipid microsphere according to any one of claims 1 to 3; the in situ gel matrix comprises at least one of gellan gum, methylcellulose, sodium alginate, poloxamer, carbomer and chitosan.

7. The tacrolimus lipid microsphere gel according to claim 6, characterized in that: The mass ratio of the common gel matrix and the tacrolimus lipid microspheres in a1) is (15-40):100; Preferably, the mass ratio of the in situ gel matrix, thickener and tacrolimus lipid microspheres in a2) is (50-100):(15-25):1000; Preferably, the raw materials for preparing the gel in a1) and a2) further include an osmotic pressure regulator, respectively; the mass ratio of the osmotic pressure regulator to the tacrolimus lipid microspheres is (25-70):100; Preferably, the raw materials for preparing the gel in a1) and a2) further comprise water respectively; the mass ratio of water to tacrolimus lipid microspheres is (1-20):1; Preferably, the raw materials for preparing the gel in a1) and a2) further comprise a pH regulator, respectively; the mass ratio of the pH regulator to the tacrolimus lipid microspheres is (0.01-0.3):1000.

8. Any one of b1) to b2) preparation methods: b1) The method for preparing the tacrolimus lipid microsphere ordinary gel as claimed in claim 7 comprises the following steps: mixing a common gel base with water to obtain a common gel base solution; mixing an osmotic pressure regulator, a pH regulator, and a common gel matrix solution to obtain a mixture; The mixture is mixed with tacrolimus lipid microspheres to obtain tacrolimus lipid microspheres ordinary gel; b2) The method for preparing the tacrolimus lipid microsphere in situ gel as claimed in claim 7 comprises the following steps: mixing the in situ gel matrix, the thickener, and water to obtain an in situ gel matrix solution; mixing an osmotic pressure regulator, a pH regulator, and the in situ gel matrix solution to obtain a mixture; The mixture is mixed with tacrolimus lipid microspheres to obtain tacrolimus lipid microsphere in situ gel.

9. A tacrolimus lipid microsphere suspension comprising an osmotic pressure regulator and the tacrolimus lipid microsphere according to any one of claims 1 to 3.

10. An eye drop comprising: the tacrolimus lipid microspheres according to any one of claims 1 to 3, the tacrolimus lipid microsphere gel according to any one of claims 6 to 7, or the tacrolimus lipid microsphere suspension according to claim 9.

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