Tacrolimus microcrystal as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and discloses a tacrolimus microcrystal as well as a preparation method and application thereof. Compared with a raw material medicine, the tacrolimus microcrystal has the advantages that the dissolution rate, the cumulative dissolution rate, the hydrophobicity and the safety (without irritation) are obviously improved; compared with the existing pharmaceutical preparation, the pharmaceutical composition has the advantages of long residence time, better treatment effect and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and specifically relates to a tacrolimus microcrystal and a preparation method and application thereof. Background Art

[0002] Dry eye disease, with an incidence of 21-30% in China, is a common and complex ophthalmic disease caused by insufficient tear secretion, excessive evaporation or abnormal tear composition, which can cause a variety of eye discomfort symptoms and visual dysfunction. The first choice for dry eye disease treatment is drug therapy, but the bioavailability of traditional eye drops is usually less than 5%.

[0003] In the field of ophthalmology, tacrolimus is an effective drug for the treatment of inflammatory eye diseases such as recurrent refractory uveitis, vernal catarrhal conjunctivitis or dry eye. Tacrolimus is a macrolide immunosuppressant that is almost insoluble in water, which limits its application in the eye.

[0004] There are very few tacrolimus eye drops on the market at home and abroad. Currently on the market are tacrolimus eye drops from Japan's SENJU Pharmaceutical Co., Ltd. (Specification: 5mg:5mL), administered once a day. Due to the extremely low water solubility and high molecular weight of tacrolimus, which hinder the transport of tacrolimus in the tear film, cornea and through the cornea into the eye, its bioavailability is extremely low and multiple eye drops are required per day, resulting in poor patient compliance.

[0005] Literature (Zhang Caijie. Treatment of dry eye in mice with non-spherical tacrolimus microcrystals modified with carboxymethyl cellulose [D]. Tianjin Medical University, 2020. DOI: 10.27366 / d.cnki.gtyku.2020.000348.) Sodium borohydride used in the preparation of layer-by-layer self-assembled tacrolimus microcrystals (TAM MCs@(PEI / HA)3) is an explosive product that can react violently with water, humid air, acids, oxidants, high heat and open flames. It is corrosive to human eyes and skin and is a highly toxic substance. It is dangerous to use and does not meet the requirements for relevant reagents in pharmaceutical preparations. Summary of the invention

[0006] The first aspect of the present invention aims to provide tacrolimus microcrystals.

[0007] The object of the second aspect of the present invention is to provide a method for preparing the tacrolimus microcrystals of the first aspect of the present invention.

[0008] The third aspect of the present invention aims to provide a long-retention tacrolimus microcrystal.

[0009] The fourth aspect of the present invention aims to provide a method for preparing the long-retention tacrolimus microcrystals according to the third aspect of the present invention.

[0010] The purpose of the fifth aspect of the present invention is to provide use of the tacrolimus microcrystals of the first aspect of the present invention or the long-retention tacrolimus microcrystals of the third aspect of the present invention in the preparation of eye drops.

[0011] The sixth 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 microcrystal, wherein the raw materials for preparing the tacrolimus microcrystal include: tacrolimus, an oil phase solvent, a stabilizer, a surfactant and an aqueous phase solvent; and the oil phase solvent includes acetonitrile.

[0014] Preferably, the stabilizer comprises at least one of polylactic acid, polyhydroxybutyrate, polyhydroxyvalerate, polyvinyl alcohol, polycaprolactone, and polybutylene succinate; further comprises at least one of polylactic acid, polyvinyl alcohol, and polycaprolactone; and further comprises polyvinyl alcohol.

[0015] Preferably, the surfactant comprises at least one of Tween, Span, sodium dodecyl sulfate, sodium cholate, sodium deoxycholate, poloxamer 188, and poloxamer 407; further comprises at least one of Tween 80, poloxamer 188, and poloxamer 407; and further comprises poloxamer 188.

[0016] Preferably, the aqueous phase solvent comprises at least one of water, phosphate buffer, and sodium chloride aqueous solution; further comprises water; and further comprises deionized water.

[0017] Preferably, the mass volume ratio of tacrolimus, oil phase solvent, stabilizer, surfactant and water phase solvent (mg:mL:mg:mg:mL) is (5-15):1:(5-15):(5-15):(5-15); further (8-12):1:(8-15):(5-12):(8-12); further (8-12):1:(8-12):(8-12):(8-12).

[0018] Preferably, the average particle size of the tacrolimus microcrystals is 0.1-10 μm; further 2-5 μm; further 3.38±0.11 μm.

[0019] Preferably, the PDI of the tacrolimus microcrystals is 0.05-0.2; further 0.17-0.19; further 0.18±0.04.

[0020] Preferably, the Zeta potential of the tacrolimus microcrystals is -(2-5) mV; further -(3.44±0.29) mV.

[0021] The second aspect of the present invention provides a method for preparing tacrolimus microcrystals according to the first aspect of the present invention.

[0022] mixing tacrolimus with an oil phase solvent to obtain an oil phase;

[0023] Mixing a stabilizer, a surfactant and an aqueous phase solvent to obtain an aqueous phase;

[0024] The oil phase is mixed with the water phase to obtain a tacrolimus microcrystalline suspension;

[0025] The oil phase solvent in the tacrolimus microcrystal suspension is evaporated and dried to obtain tacrolimus microcrystals.

[0026] Preferably, the mixing of tacrolimus and the oil phase solvent comprises: dispersing and dissolving tacrolimus in the oil phase.

[0027] Preferably, the stabilizer, surfactant and aqueous solvent are mixed to swell overnight.

[0028] Preferably, the mixing of the oil phase and the water phase comprises: adding the oil phase to the water phase under shearing; further comprising adding the oil phase to the water phase under shearing at a uniform speed.

[0029] Preferably, the oil phase is added at a rate of 0.5-1.5 mL / min.

[0030] Preferably, the shearing speed is 10000-15000 rpm.

[0031] Preferably, after the oil phase is added to the water phase in shear, the method further comprises the following step: shear homogenization.

[0032] Preferably, the shear homogenization condition is shearing at 10000-15000 rpm for 2-4 min.

[0033] Preferably, the method for volatilizing the oil phase solvent in the tacrolimus microcrystalline suspension is stirring; further, placing it in a dark room with magnetic stirring at 200-400 rpm overnight.

[0034] Preferably, after the oil phase solvent in the tacrolimus microcrystal suspension is volatilized and before drying, the method further comprises the following steps: solid-liquid separation and washing the tacrolimus microcrystals.

[0035] Preferably, the solid-liquid separation method is centrifugation; further, centrifugation is performed at 10000-15000 rpm for 6-10 min.

[0036] Preferably, the washing is performed with water (preferably deionized water).

[0037] Preferably, the washing is repeated multiple times; further repeated 2-4 times.

[0038] Preferably, the drying is freeze-drying.

[0039] The third aspect of the present invention provides a long-retention tacrolimus microcrystal, wherein the raw materials for preparing the long-retention tacrolimus microcrystal include: a positively charged material, a negatively charged material, and the tacrolimus microcrystal of the first aspect of the present invention.

[0040] Preferably, the positively charged material comprises at least one of chitosan, polyethyleneimine, hydroxymethyl cellulose, sodium hydroxymethyl cellulose, and hydroxypropyl methyl cellulose; further comprises at least one of chitosan and polyethyleneimine; and further comprises polyethyleneimine (preferably polyethyleneimine with a Mw of 20,000-30,000).

[0041] Preferably, the negatively charged material comprises at least one of polyacrylic acid, polyacrylamide and hyaluronic acid; further comprises hyaluronic acid.

[0042] Preferably, the positively charged material and the negatively charged material are sequentially modified on the tacrolimus microcrystals.

[0043] Preferably, the number of layers of the positively charged material in the long-retention tacrolimus microcrystals is 2-4.

[0044] Preferably, the number of layers of the negatively charged material in the long-retention tacrolimus microcrystals is 2-4.

[0045] Preferably, the number of layers of the positively charged material in the long-retention tacrolimus microcrystal is the same as the number of layers of the negatively charged material in the long-retention tacrolimus microcrystal.

[0046] Preferably, the positively charged material is a positively charged material solution; further, an aqueous solution of the positively charged material.

[0047] Preferably, the concentration of the aqueous solution of the positively charged material is 0.1-4 mg / mL; further 1-3.5 mg / mL; further 1.5-2.5 mg / mL.

[0048] Preferably, the negatively charged material is a negatively charged material solution; further, an aqueous solution of the negatively charged material.

[0049] Preferably, the concentration of the aqueous solution of the negatively charged material is 0.03-1.3 mg / mL; further 0.5-1.1 mg / mL; further 0.9-1.1 mg / mL.

[0050] Preferably, the average particle size of the long-retention tacrolimus microcrystals is 0.1-8 μm; further 1-3 μm; further 1.41±0.05 μm.

[0051] Preferably, the PDI of the long-retention tacrolimus microcrystals is 0.1-0.4; further 0.25-0.3; further 0.27±0.08.

[0052] Preferably, the Zeta potential of the long-retention tacrolimus microcrystals is -(3-7) mV; further -(5.45±0.29) mV.

[0053] The fourth aspect of the present invention provides a method for preparing the long-retention tacrolimus microcrystals of the third aspect of the present invention.

[0054] S1: depositing the positively charged material on the tacrolimus microcrystals to obtain tacrolimus microcrystals with deposited positively charged material;

[0055] S2: depositing the negatively charged material on the tacrolimus microcrystals deposited with the positively charged material, to obtain tacrolimus microcrystals deposited with the positively charged material and the negatively charged material;

[0056] S3: drying the tacrolimus microcrystals deposited with the positively charged material and the negatively charged material to obtain long-retention tacrolimus microcrystals.

[0057] Preferably, after S2 and before S3, the following steps are further included: repeating S1 and S2.

[0058] Preferably, the repetition number is 2-4 times.

[0059] Preferably, the method for depositing the positively charged material on the tacrolimus microcrystals is: placing the tacrolimus microcrystals in a solution of the positively charged material, performing solid-liquid separation, and washing the tacrolimus microcrystals on which the positively charged material is deposited.

[0060] Preferably, placing the tacrolimus microcrystals in the positively charged material solution specifically includes placing the tacrolimus microcrystals in the positively charged material solution and vortexing, and further includes placing the tacrolimus microcrystals in the positively charged material solution and vortexing for 15-25 minutes.

[0061] Preferably, the solid-liquid separation method is centrifugation; further, centrifugation is performed at 10000-15000 rpm for 10-20 min.

[0062] Preferably, the washing is performed with water (preferably deionized water).

[0063] Preferably, the washing is repeated multiple times; further repeated 2-4 times.

[0064] Preferably, the method for depositing the negatively charged material on the tacrolimus microcrystals deposited with the positively charged material is: placing the tacrolimus microcrystals deposited with the positively charged material in a solution of the negatively charged material, performing solid-liquid separation, and washing the deposited positively charged material and the tacrolimus microcrystals deposited with the negatively charged material.

[0065] Preferably, placing the tacrolimus microcrystals deposited with the positively charged material in the negatively charged material solution specifically comprises placing the tacrolimus microcrystals deposited with the positively charged material in the negatively charged material solution and vortexing; further comprising placing the tacrolimus microcrystals deposited with the positively charged material in the negatively charged material solution and vortexing for 15-25 minutes.

[0066] Preferably, the solid-liquid separation method is centrifugation; further, centrifugation is performed at 10000-15000 rpm for 10-20 min.

[0067] Preferably, the washing is performed with water (preferably deionized water).

[0068] Preferably, the washing is repeated multiple times; further repeated 2-4 times.

[0069] Preferably, before drying the tacrolimus microcrystals deposited with the positively charged material and the negatively charged material, the tacrolimus microcrystals deposited with the positively charged material and the negatively charged material are dispersed in water (preferably deionized water).

[0070] Preferably, the drying is freeze-drying.

[0071] The fifth aspect of the present invention provides use of the tacrolimus microcrystals according to the first aspect of the present invention or the long-retention tacrolimus microcrystals according to the third aspect of the present invention in the preparation of eye drops.

[0072] The sixth aspect of the present invention provides an eye drop comprising: the tacrolimus microcrystals according to the first aspect of the present invention, or the long-retention tacrolimus microcrystals according to the third aspect of the present invention.

[0073] Preferably, the eye drops further comprise: at least one of a stabilizer, a wetting agent, an osmotic pressure regulator, a buffer, and an antibacterial agent; and further comprise a stabilizer, a wetting agent, an osmotic pressure regulator, a buffer, and an antibacterial agent.

[0074] Preferably, the stabilizer is at least one of Carbomer 934, Carbomer 940, and Carbomer 941; further Carbomer 934.

[0075] Preferably, the wetting agent is at least one of Tween 60, Tween 80 and Span 80; further Tween 80.

[0076] Preferably, the osmotic pressure regulator comprises at least one of sodium chloride, glucose, phosphate, citrate, mannitol and sorbitol; further comprises at least one of glucose, mannitol and sorbitol; further comprises mannitol.

[0077] Preferably, the buffer comprises at least one of boric acid-borax buffer, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, and carbonic acid-sodium bicarbonate buffer; further comprising sodium dihydrogen phosphate-disodium hydrogen phosphate buffer.

[0078] Preferably, the antibacterial agent comprises at least one of benzalkonium chloride, benzalkonium bromide, chlorobutanol, methylparaben, ethylparaben, sodium perborate, and sorbic acid; further comprises at least one of benzalkonium chloride and benzalkonium bromide; and further comprises benzalkonium chloride.

[0079] Preferably, the concentration of the tacrolimus microcrystals or long-retention tacrolimus microcrystals in the eye drops is 0.5-1.5 mg / mL; further 0.9-1.1 mg / mL.

[0080] Preferably, the concentration of the stabilizer in the eye drops is 1-3 mg / mL; further 1.4-1.6 mg / mL.

[0081] Preferably, the concentration of the wetting agent in the eye drops is 0.05-0.15 mg / mL; further 0.09-0.11 mg / mL.

[0082] Preferably, the concentration of the osmotic pressure regulator in the eye drops is 250-350 mg / mL; further 300-320 mg / mL.

[0083] Preferably, the concentration of the antibacterial agent in the eye drops is 0.1-0.3 mg / mL; further 0.19-0.21 mg / mL.

[0084] Preferably, the osmotic pressure of the eye drops is 250-300 mOsmol / kg; further 260-280 mOsmol / kg; further 267-271 mOsmol / kg.

[0085] Preferably, the pH of the eye drops is 6.0-8.0; further 6.5-7.5; further 6.5-6.7.

[0086] The beneficial effects of the present invention are:

[0087] The present invention provides a tacrolimus microcrystal. Compared with a raw drug, the tacrolimus microcrystal has significantly improved dissolution rate, cumulative dissolution rate, hydrophobicity, and safety (non-irritating). Compared with existing drug preparations, the tacrolimus microcrystal has the advantages of long retention time, better therapeutic effect, and the like.

[0088] The present invention provides a long-retention tacrolimus microcrystal, which, compared with tacrolimus microcrystals, has the advantages of good sustained-release effect, long retention time, better therapeutic effect, etc., and has good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 The tacrolimus bulk drug, the tacrolimus microcrystals prepared in Example 1, the long-retention tacrolimus microcrystals prepared in Example 5, and the commercially available tacrolimus preparation The appearance morphology and particle size distribution diagram of: wherein, (a) is a scanning electron micrograph of tacrolimus raw material (scale is 200 μm); (b) is a scanning electron micrograph of tacrolimus microcrystals prepared in Example 1 (scale is 2 μm); (c) is a scanning electron micrograph of long-retention tacrolimus microcrystals prepared in Example 5 (scale is 2 μm); (d) is a scanning electron micrograph of tacrolimus commercially available preparation (e) is the particle size distribution diagram of the tacrolimus microcrystals prepared in Example 1; (f) is the particle size distribution diagram of the long-retention tacrolimus microcrystals prepared in Example 5.

[0090] Figure 2 This is a potential reversal diagram of the surface of the microparticles after each layer of polymer material is wrapped during the preparation of long-retention tacrolimus microcrystals in Example 5, wherein n=3.

[0091] Figure 3 It is the dissolution curve diagram of tacrolimus bulk drug and tacrolimus microcrystals prepared in Example 1, wherein n=3.

[0092] Figure 4 It is a diagram showing the hydrophobic binding of Rose Bengal to tacrolimus bulk drug, tacrolimus microcrystals prepared in Example 1, and long-retention tacrolimus microcrystals prepared in Example 5.

[0093] Figure 5 The tacrolimus raw material eye drops prepared in comparative example 2, the tacrolimus microcrystalline eye drops prepared in example 6, the long-retention tacrolimus microcrystalline eye drops prepared in example 7 and the commercially available tacrolimus preparations are In vitro release curve of , wherein n=3.

[0094] Figure 6The positive control group, the normal saline group, the tacrolimus raw material eye drops prepared in comparative example 2, the tacrolimus microcrystalline eye drops prepared in example 6, the long-retention tacrolimus microcrystalline eye drops prepared in example 7, and the commercially available tacrolimus preparation A diagram of the bleeding, coagulation and melting of chicken embryo blood vessels. The camera lens has a magnification of 10 times.

[0095] Figure 7 The tacrolimus raw material eye drops prepared in comparative example 2, the tacrolimus microcrystalline eye drops prepared in example 6, the long-retention tacrolimus microcrystalline eye drops prepared in example 7 and the commercially available tacrolimus preparations are Diagram of anterior corneal fluorescence retention, where the camera lens magnification is 10 times.

[0096] Figure 8 The tacrolimus raw material eye drops prepared in comparative example 2, the tacrolimus microcrystalline eye drops prepared in example 6, the long-retention tacrolimus microcrystalline eye drops prepared in example 7 and the commercially available tacrolimus preparations are Tear drug-time curve, where n=3.

[0097] Fig. 9 The negative control group (Blank control), the normal saline group (positive control group), the tacrolimus raw material eye drops prepared in comparative example 2, the tacrolimus microcrystalline eye drops prepared in example 6, the long-retention tacrolimus microcrystalline eye drops prepared in example 7, and the commercially available tacrolimus preparation Comparison of Schirmer I test results, where n=3.

[0098] Fig.10 The negative control group (Blank control), the normal saline group (positive control group), the tacrolimus raw material eye drops prepared in comparative example 2, the tacrolimus microcrystalline eye drops prepared in example 6, the long-retention tacrolimus microcrystalline eye drops prepared in example 7, and the commercially available tacrolimus preparation Comparison of tear breakup time (TBUT) results, where n=3.

[0099] Fig.11 It is an optical microscope image of the tacrolimus preparation prepared in Comparative Example 1 (scale bar: 50 μm).

[0100] Fig.12 It is the dissolution curve of tacrolimus microcrystals (TAC MCs) prepared in Comparative Example 3, wherein n=3.

[0101] Fig.13The in vitro release curves of the tacrolimus microcrystals (TAC MCs) eye drops and the layer-by-layer self-assembled microcrystals tacrolimus microcrystals (TAC-(PAH / CMC) 3) eye drops prepared in Examples 5 and 6 are shown, wherein n=3.

[0102] In the above figures: * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. DETAILED DESCRIPTION

[0103] The present invention is further described in detail below through specific examples.

[0104] 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.

[0105] 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.

[0106] In the present invention, "room temperature" is 25±5°C.

[0107] The reagents and their manufacturers used in the following examples are shown in Table 1.

[0108] Table 1

[0109]

[0110]

[0111] Example 1 A method for preparing tacrolimus microcrystals

[0112] A method for preparing tacrolimus microcrystals comprises the following steps:

[0113] (1) Preparation of oil phase: Accurately weigh 10 mg of tacrolimus API and place it in a beaker. Add 1 mL of acetonitrile and seal the beaker tightly. Gently shake the beaker to fully disperse and dissolve the tacrolimus to obtain a tacrolimus acetonitrile solution.

[0114] (2) Preparation of aqueous phase: Accurately weigh 10 mg of polyvinyl alcohol and 10 mg of poloxamer 188 into a beaker, add 10 mL of deionized water and swell overnight.

[0115] (3) The aqueous phase was placed in a homogenizer (IKAT18, IKA, Germany) at a shear rate of 13,000 rpm. The oil phase was completely drawn into a 1 mL syringe and then uniformly injected into the aqueous phase at a rate of 1 mL / min. After completion, shear homogenization was continued for 3 min to obtain a tacrolimus microcrystalline suspension.

[0116] (4) The tacrolimus microcrystalline suspension obtained in step (3) was placed in a dark room and magnetically stirred at 300 rpm overnight until the acetonitrile was completely evaporated.

[0117] (5) The tacrolimus microcrystal suspension from which the acetonitrile obtained in step (4) has been completely evaporated is placed in a 10 mL centrifuge tube, centrifuged at 12,000 rpm for 8 min, the supernatant is removed, and deionized water is added to wash away the excess water phase on the surface of the tacrolimus microcrystals. The water washing is repeated three times.

[0118] (6) The washed tacrolimus microcrystal suspension obtained in step (5) is placed in a freeze dryer (LGJ-10C, Beijing Sihuan Scientific Instrument Co., Ltd.) for freeze drying to obtain tacrolimus microcrystals (TAM MCs).

[0119] Example 2 A method for preparing tacrolimus microcrystals

[0120] A method for preparing tacrolimus microcrystals comprises the following steps:

[0121] (1) Preparation of oil phase: Accurately weigh 10 mg of tacrolimus API and place it in a beaker. Add 1 mL of acetonitrile and seal the beaker tightly. Gently shake the beaker to fully disperse and dissolve the tacrolimus to obtain a tacrolimus acetonitrile solution.

[0122] (2) Preparation of aqueous phase: Accurately weigh 10 mg of polyvinyl alcohol and 10 mg of Tween 80 into a beaker, add 10 mL of deionized water and swell overnight.

[0123] (3) The aqueous phase was placed in a homogenizer (IKAT18, IKA, Germany) at a shear rate of 13,000 rpm. The oil phase was completely drawn into a 1 mL syringe and then uniformly injected into the aqueous phase at a rate of 1 mL / min. After completion, shear homogenization was continued for 3 min to obtain a tacrolimus microcrystalline suspension.

[0124] (4) The tacrolimus microcrystalline suspension obtained in step (3) was placed in a dark room and magnetically stirred at 300 rpm overnight until the acetonitrile was completely evaporated.

[0125] (5) The tacrolimus microcrystal suspension from which the acetonitrile obtained in step (4) has been completely evaporated is placed in a 10 mL centrifuge tube, centrifuged at 12,000 rpm for 8 min, the supernatant is removed, and deionized water is added to wash away the excess water phase on the surface of the tacrolimus microcrystals. The water washing is repeated three times.

[0126] (6) The washed tacrolimus microcrystal suspension obtained in step (5) is placed in a freeze dryer (LGJ-10C, Beijing Sihuan Scientific Instrument Co., Ltd.) for freeze drying to obtain tacrolimus microcrystals.

[0127] Example 3 A method for preparing tacrolimus microcrystals

[0128] A method for preparing tacrolimus microcrystals comprises the following steps:

[0129] (1) Preparation of oil phase: Accurately weigh 10 mg of tacrolimus API and place it in a beaker. Add 1 mL of acetonitrile and seal the beaker tightly. Gently shake the beaker to fully disperse and dissolve the tacrolimus to obtain a tacrolimus acetonitrile solution.

[0130] (2) Preparation of aqueous phase: Accurately weigh 15 mg of polyvinyl alcohol and 5 mg of poloxamer 188 into a beaker, add 10 mL of deionized water and swell overnight.

[0131] (3) The aqueous phase was placed in a homogenizer (IKAT18, IKA, Germany) at a shear rate of 13,000 rpm. The oil phase was completely drawn into a 1 mL syringe and then uniformly injected into the aqueous phase at a rate of 1 mL / min. After completion, shear homogenization was continued for 3 min to obtain a tacrolimus microcrystalline suspension.

[0132] (4) The tacrolimus microcrystalline suspension obtained in step (3) was placed in a dark room and magnetically stirred at 300 rpm overnight until the acetonitrile was completely evaporated.

[0133] (5) The tacrolimus microcrystal suspension from which the acetonitrile obtained in step (4) has been completely evaporated is placed in a 10 mL centrifuge tube, centrifuged at 12,000 rpm for 8 min, the supernatant is removed, and deionized water is added to wash away the excess water phase on the surface of the tacrolimus microcrystals. The water washing is repeated three times.

[0134] (6) The washed tacrolimus microcrystal suspension obtained in step (5) is placed in a freeze dryer (LGJ-10C, Beijing Sihuan Scientific Instrument Co., Ltd.) for freeze drying to obtain tacrolimus microcrystals.

[0135] Example 4 A method for preparing tacrolimus microcrystals

[0136] A method for preparing tacrolimus microcrystals comprises the following steps:

[0137] (1) Preparation of oil phase: Accurately weigh 10 mg of tacrolimus API and place it in a beaker. Add 1 mL of acetonitrile and seal the beaker tightly. Gently shake the beaker to fully disperse and dissolve the tacrolimus to obtain a tacrolimus acetonitrile solution.

[0138] (2) Preparation of aqueous phase: Accurately weigh 15 mg of polyvinyl alcohol and 5 mg of Tween 80 into a beaker, add 10 mL of deionized water and swell overnight.

[0139] (3) The aqueous phase was placed in a homogenizer (IKAT18, IKA, Germany) at a shear rate of 13,000 rpm. The oil phase was completely drawn into a 1 mL syringe and then uniformly injected into the aqueous phase at a rate of 1 mL / min. After completion, shear homogenization was continued for 3 min to obtain a tacrolimus microcrystalline suspension.

[0140] (4) The tacrolimus microcrystalline suspension obtained in step (3) was placed in a dark room and magnetically stirred at 300 rpm overnight until the acetonitrile was completely evaporated.

[0141] (5) The tacrolimus microcrystal suspension from which the acetonitrile obtained in step (4) has been completely evaporated is placed in a 10 mL centrifuge tube, centrifuged at 12,000 rpm for 8 min, the supernatant is removed, and deionized water is added to wash away the excess water phase on the surface of the tacrolimus microcrystals. The water washing is repeated three times.

[0142] (6) The washed tacrolimus microcrystal suspension obtained in step (5) is placed in a freeze dryer (LGJ-10C, Beijing Sihuan Scientific Instrument Co., Ltd.) for freeze drying to obtain tacrolimus microcrystals.

[0143] Example 5 A method for preparing long-retention tacrolimus microcrystals

[0144] A method for preparing long-retention tacrolimus microcrystals comprises the following steps:

[0145] (1) 50 mg of tacrolimus microcrystals prepared in Example 1 were dispersed in 5 mL of 2 mg / mL polyethyleneimine (PEI) aqueous solution and vortexed for 20 min. The mixture was then centrifuged at 12,000 rpm for 15 min. The supernatant was removed and the excess PEI was washed with deionized water. The washing was repeated twice.

[0146] (2) The sediment obtained in step (1) was dispersed in 5 mL of 1 mg / mL hyaluronic acid (HA) aqueous solution and vortexed for 20 min. The mixture was then centrifuged at 12,000 rpm for 15 min. The supernatant was removed and the excess HA was washed with deionized water. The washing was repeated twice.

[0147] (3) Repeat steps (1) and (2) twice.

[0148] (4) The final sediment was dispersed in an appropriate amount of deionized water and freeze-dried in a freeze dryer to obtain long-retention tacrolimus microcrystals (TAM MCs@(PEI / HA)3).

[0149] Example 6 A method for preparing eye drops

[0150] A method for preparing eye drops: 10 mg of tacrolimus microcrystals (TAM MCs) of Example 1 is dispersed in 10 mL of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer swollen with 15 mg of carbomer 934, and 0.31 g of mannitol, 0.001 g of Tween 80 and 0.002 g of benzalkonium chloride are added to prepare TAM MCs eye drops with a concentration of 1 mg / mL, a pH of 6.62 and an osmotic pressure of 267 mOsmol / kg.

[0151] Example 7 A method for preparing eye drops

[0152] A method for preparing eye drops: 10 mg of long-retention tacrolimus microcrystals (TAM MCs@(PEI / HA)3) of Example 5 is dispersed in 10 mL of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer swollen with 15 mg of carbomer 934, and 0.31 g of mannitol, 0.001 g of Tween 80 and 0.002 g of benzalkonium chloride are added to prepare TAM MCs@(PEI / HA)3 eye drops with a concentration of 1 mg / mL, a pH of 6.63 and an osmotic pressure of 271 mOsmol / kg.

[0153] Comparative Example 1: Preparation method of a tacrolimus preparation

[0154] A method for preparing a tacrolimus preparation comprises the following steps:

[0155] (1) Preparation of oil phase: Accurately weigh 10 mg of tacrolimus API and place it in a beaker. Add 1 mL of ethanol and seal the beaker tightly. Gently shake the beaker to fully disperse and dissolve the tacrolimus to obtain a tacrolimus ethanol solution.

[0156] (2) Preparation of aqueous phase: Accurately weigh 10 mg of polyvinyl alcohol and 10 mg of poloxamer 188 into a beaker, add 10 mL of deionized water and swell overnight.

[0157] (3) The aqueous phase was placed in a homogenizer (IKAT18, IKA, Germany) at a shear rate of 13,000 rpm. The oil phase was completely drawn into a 1 mL syringe and then uniformly injected into the aqueous phase at a rate of 1 mL / min. After completion, shear homogenization was continued for 3 min to obtain a tacrolimus preparation suspension.

[0158] (4) The tacrolimus preparation suspension obtained in step (3) was placed in a dark room and magnetically stirred at 300 rpm overnight until the ethanol was completely evaporated.

[0159] (5) The tacrolimus preparation suspension from which the ethanol obtained in step (4) has been completely evaporated is placed in a 10 mL centrifuge tube, centrifuged at 12,000 rpm for 8 min, the supernatant is removed, and deionized water is added to wash away the excess water phase on the surface of the tacrolimus preparation. The water washing is repeated three times.

[0160] (6) The washed tacrolimus preparation suspension obtained in step (5) is placed in a freeze dryer (LGJ-10C, Beijing Sihuan Scientific Instrument Co., Ltd.) and freeze-dried to obtain a tacrolimus preparation.

[0161] Comparative Example 2: A method for preparing eye drops

[0162] A method for preparing eye drops: 10 mg of tacrolimus raw material (TAM) is dispersed in 10 mL of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer in which 15 mg of carbomer 934 has been dissolved, and 0.31 g of mannitol, 0.001 g of Tween 80 and 0.002 g of benzalkonium chloride are added to prepare TAM eye drops with a concentration of 1 mg / mL, a pH of 6.59 and an osmotic pressure of 269 mOsmol / kg.

[0163] Comparative Example 3

[0164] Tacrolimus microcrystals (TAC MCs) were prepared according to the preparation method in the literature (Zhang Caijie. Treatment of dry eye in mice with carboxymethyl cellulose-modified tacrolimus non-spherical microcrystals [D]. Tianjin Medical University, 2020. DOI: 10.27366 / d.cnki.gtyku.2020.000348.) (page 8).

[0165] Comparative Example 4

[0166] According to the preparation method in the literature (Zhang Caijie. Treatment of dry eye in mice with non-spherical tacrolimus microcrystals modified with carboxymethyl cellulose [D]. Tianjin Medical University, 2020. DOI: 10.27366 / d.cnki.gtyku.2020.000348.) (pages 8-9), layer-by-layer self-assembled microcrystals (TAC-(PAH / CMC)3 were prepared.

[0167] Comparative Example 5

[0168] Tacrolimus microcrystals (TAC MCs) eye drops were prepared according to the preparation method in the literature (Zhang Caijie. Treatment of dry eye in mice with carboxymethyl cellulose-modified tacrolimus non-spherical microcrystals [D]. Tianjin Medical University, 2020. DOI: 10.27366 / d.cnki.gtyku.2020.000348.) (page 17).

[0169] Comparative Example 6

[0170] According to the preparation method in the literature (Zhang Caijie. Treatment of dry eye in mice with non-spherical tacrolimus microcrystals modified with carboxymethyl cellulose [D]. Tianjin Medical University, 2020. DOI: 10.27366 / d.cnki.gtyku.2020.000348.) (page 17), layer-by-layer self-assembled microcrystal tacrolimus microcrystals (TAC-(PAH / CMC)3 eye drops) were prepared.

[0171] Effect Example

[0172] 1. Tacrolimus API (TAM), TAM MCs prepared in Example 1, TAMMCs@(PEI / HA)3 prepared in Example 5 and commercially available preparations were mixed. After drying, the particles were sprayed with gold, placed under a scanning electron microscope (SEM, Sigma 300, Zeiss, Germany) to observe the morphology, and captured SEM images. The particle sizes of the tacrolimus API, the TAM MCs prepared in Example 1, and the TAM MCs@(PEI / HA)3 prepared in Example 5 were measured and counted under an optical microscope, and the particle size distribution diagrams of the TAM MCs prepared in Example 1 and the TAM MCs@(PEI / HA)3 prepared in Example 5 were drawn. The potentials of the tacrolimus API, the TAM MCs prepared in Example 1, and the TAM MCs@(PEI / HA)3 prepared in Example 5 were measured using a Zeta potential meter (JS94J, Shanghai Zhongchen Digital Technology Equipment Co., Ltd.).

[0173] The results are as follows Figure 1 , as shown in Table 2. The average particle size of TAM MCs and TAM MCs@(PEI / HA)3 prepared in Example 1 and Example 5 is less than 10 μm, which meets the requirements of ophthalmic preparations and can avoid the loss of drugs on the ocular surface due to blinking reflex caused by excessive particle size. Fig.11 ), the microcrystalline morphology of Example 2-4 is not clear, and large agglomerates (microcrystalline agglomerates) exist.

[0174] Table 2 Physicochemical properties of tacrolimus series preparations (n=3)

[0175]

[0176] 2. In the process of preparing TAM MCs@(PEI / HA)3 in Example 5, after each layer of PEI or HA was assembled, an appropriate amount of sample was taken out to measure the Zeta potential. The results are shown in Figure 2 shown.

[0177] 3. Determination of the dissolution rate of tacrolimus bulk drug (TAM), TAM MCs prepared in Example 1, and tacrolimus microcrystals (TAC MCs) prepared in Comparative Example 3. Artificial tears (STF) were prepared by dissolving 2.18 g NaHCO3, 1.38 g KCl, 6.78 g NaCl, and 0.084 g CaCl2·2H2O in 1000 mL deionized water, and adjusting the pH to 7.4 with HCl. Artificial tears containing 30% v / v acetonitrile were used as the dissolution medium for TAM and TAM MCs. 100 mL of each dissolution medium was accurately measured in a 250 mL stoppered conical flask, and preheated in an air bath constant temperature oscillator (THZ-82, Shanghai Xiaohan Industrial Development Co., Ltd.) at a temperature of 34°C and an oscillation speed of 100 rpm / min. Weigh 10 mg of TAM and TAM MCs respectively and place them in a 500-mesh filter bag, add the corresponding dissolution medium, and start timing. At different time points, accurately pipette 5 mL of the dissolution medium outside the filter bag, i.e., the sample, and then immediately add an equal amount of blank dissolution medium. The obtained samples were measured using a high-performance liquid chromatograph (HPLC, LC-20AT, Shimadzu Corporation, Japan), and the cumulative dissolution rate was calculated using the following formula:

[0178]

[0179] Among them, Q a is the cumulative dissolution rate, m t is the total mass of substance dissolved at time t, and m is the input amount.

[0180] The results are as follows Figure 3 As shown in the figure, after 45 minutes of dissolution, the cumulative dissolution rate of TAM MCs was as high as 91.11%, while the cumulative dissolution rate of TAM was only 45.54% and the dissolution lasted until 8.5 hours before the dissolution was basically complete. It can be seen that the dissolution rate of TAM MCs is much greater than that of TAM. Fig.12 It shows that the cumulative dissolution rate of TAC MCs in Comparative Example 3 is only 56% at the 45th minute and the dissolution lasts for 6.5 hours to reach 90.91%.

[0181] 4. The Rose Bengal (RB) experiment was used to investigate whether the surface of the drug particles has hydrophobicity. Take 2 mg of each of tacrolimus bulk drug (TAM), TAM MCs prepared in Example 1, and TAM MCs@(PEI / HA)3 powder prepared in Example 5, respectively, and incubate them in a centrifuge tube with 1.5 mL of RB solution with concentrations of 0.020, 0.025, 0.030, 0.035, 0.040, and 0.045 mg / mL in a dark room at 4°C for 3 hours. Then centrifuge at 15000 rpm for 20 minutes. The filtrate after the upper clear liquid passes through a 0.22 μm organic microporous filter membrane is measured for absorbance using an ultraviolet spectrophotometer (Ultraviolet spectrophotometer, UV, UV-1800PC, Shanghai Meipu Instrument Co., Ltd.) to calculate the unbound free RB. The binding constant K is obtained according to the Scatchard-plot formula:

[0182]

[0183] Where r is the binding concentration of RB; a is the RB concentration at equilibrium; K is the binding constant for evaluating the hydrophobicity of the particle surface, and N is the maximum binding amount of RB.

[0184] The results are as follows Figure 4 , as shown in Table 3: TAM MCs@(PEI / HA) 3、 The K value of TAM MCs is greater than that of TAM (among which, the K value of TAMMCs@(PEI / HA)3 is greater than that of TAM MCs), and they are highly hydrophobic and have affinity with the hydrophobic cornea, providing conditions for prolonging the retention time of drugs in front of the cornea. The excessive hydrophobicity of TAC-(PAH / CMC)3 affects its cumulative drug release rate in the in vitro release experiment.

[0185] Table 3 Scatchard-plot equation and rose bengal binding constant of tacrolimus series preparations

[0186]

[0187] 5. Prepare STF solution containing 40% v / v acetonitrile as the dialysis medium for in vitro release study of tacrolimus series preparations. 2 mL of TAM eye drops prepared in Comparative Example 2, TAM MCs eye drops prepared in Example 6, TAM MCs@(PEI / HA)3 eye drops prepared in Example 7 and The dialysis bags (molecular cutoff of 8-14 KDa) of commercially available eye drops, tacrolimus microcrystals (TAC MCs) eye drops prepared in comparative examples 5 and 6, and layer-by-layer self-assembled microcrystals tacrolimus microcrystals (TAC-(PAH / CMC)3 eye drops) were sealed at both ends and placed in 50 mL of STF solution containing 40% v / v acetonitrile, and released in vitro in an air bath constant temperature oscillator at 34°C and 120 rpm / min. 1 mL of samples were taken at different time points, and an equal amount of dialysis medium was immediately supplemented. The filtrate of each sample after passing through a 0.22 μm microporous filter membrane was taken, and the drug peak area was determined by HPLC, the drug concentration was calculated, the cumulative release amount (Q) was calculated according to the following formula, and the release curve was drawn. Three groups of experiments were conducted in parallel for each preparation.

[0188]

[0189] Where W is the total amount of drug contained in the dialysis bag; V is the volume of the dialysis medium; V0 is the sample volume for each sampling; C n is the drug concentration in the sample taken for the nth time.

[0190] The results are as follows Figure 5 As shown: The cumulative drug release in the first 0.5h was 29.65%, which was 1.65 times that of TAM MCs@(PEI / HA)3 (18.00%), and the drug release platform was basically reached at 6h, with weak sustained release performance. The drug release of TAM MCs@(PEI / HA)3 can last for 24h, which means that the drug can be stably and continuously released in the eye, maintain the long-term effective therapeutic concentration of the target and obtain higher bioavailability. Fig.13 The results show that the cumulative in vitro release rates of tacrolimus microcrystals (TAC MCs) eye drops prepared in Comparative Examples 5 and 6 and tacrolimus microcrystals (TAC-(PAH / CMC) 3) prepared in Comparative Examples 5 and 6 were both lower than 4%, indicating almost no drug release.

[0191] 6. Place the fertilized eggs in an egg incubator at a temperature of 37.8±0.2℃ and a humidity of 65%-75% for continuous incubation for 9 days. The 9-day-old breeding eggs have formed a complete chicken embryo chorioallantoic membrane (CAM). After the position of the air chamber is determined by illumination, the outer eggshell on the top of the air chamber is removed. The eggshell membrane is fully moistened with physiological saline and the membrane is carefully removed to obtain a complete and undamaged CAM. If blood vessels are damaged and bleeding occurs, the egg is discarded and no subsequent experiments are performed. A 0.1mol / L NaOH solution was used as the positive control group, and physiological saline was used as the negative control group. The TAM eye drops prepared in Comparative Example 2, the TAM MCs eye drops prepared in Example 6, the TAM MCs@(PEI / HA)3 eye drops prepared in Example 7, and The commercially available eye drops were used as the experimental group for the irritation test. 0.3 mL of each group of preparations was taken on the CAM, and then the surface of the CAM was rinsed with normal saline. After removing the excess liquid, photos were taken and recorded. The irritation score (IS) was calculated according to the following formula, and the irritation was evaluated according to the scoring criteria shown in Table 4.

[0192] The calculation formula of IS is:

[0193]

[0194] Where t1 is the bleeding time (s), t2 is the initial time of vasoconstriction (s), and t3 is the initial time of coagulation (s).

[0195] Table 4 Chicken embryo chorioallantoic membrane irritation scoring standard

[0196]

[0197] The results are as follows Figure 6 , As shown in Table 5: TAM MCs@(PEI / HA)3. TAM MCs eye drops are non-irritating to CAM.

[0198] Table 5 Chicken embryo chorioallantoic membrane test irritation scoring results (n = 3)

[0199]

[0200] 7. In 1mL commercial preparation 0.002 g of sodium fluorescein was added to the TAM eye drops prepared in Comparative Example 2, the TAMMCs eye drops prepared in Example 6, and the TAM MCs@(PEI / HA)3 eye drops prepared in Example 7. New Zealand white rabbits of either sex with healthy eyes were selected and randomly divided into 4 groups, 3 in each group. 50 μL of each preparation containing sodium fluorescein was dripped into the conjunctival sac of each group of rabbit eyes, and the retention of the preparation on the ocular surface of the rabbit eyes was observed using the cobalt blue light of a slit lamp (YZ5S, Six Six Vision Technology Co., Ltd.) after closing the rabbit eyes for 10 seconds.

[0201] The results are as follows Figure 7 As shown in Table 6: The retention time of TAM MCs@(PEI / HA)3 group in the tear film was longer than that of the other three groups, and the longest retention time on the ocular surface was 40.83±3.69min (Table 6). Compared with the API, TAM MCs@(PEI / HA)3 is more uniform and easy to disperse, and it is also hydrophobic and more compatible with the hydrophobic corneal epithelium; secondly, TAM MCs@(PEI / HA)3 specifically interacts with the CD44 receptor at the microscopic level.

[0202] Table 6 Precorneal fluorescence retention time of different preparations (n=3)

[0203]

[0204] 8. Conduct tear pharmacokinetic studies. New Zealand white rabbits of either sex with healthy eyes were randomly divided into 4 groups, 3 rabbits in each group. The left eye of each rabbit was the experimental group, and the TAM eye drops prepared in Comparative Example 2, the TAM MCs eye drops prepared in Example 6, and the TAM MCs@(PEI / HA)3 eye drops prepared in Example 7 were dripped into the conjunctival sac of the rabbits. Commercially available eye drops were used, and an equal amount of normal saline was dripped into the right eye of the rabbit as a blank control. Two filter paper strips (3×8mm) were cut and placed in a 1.5mL centrifuge tube, and their weight was accurately weighed as m0. 100μL of each of the above preparations was added to the conjunctival sac of the rabbit eye, and the nasolacrimal duct was gently pressed to reduce the loss of the preparation. At a specific time point, the filter paper strip was placed in the conjunctival sac of the rabbit eye, and the time was 1min. Then the filter paper strip was taken out and placed at the bottom of the original centrifuge tube, and the weight was accurately weighed as m1. Use a nitrogen blower (MD200-1, Hangzhou Aosheng Instrument Co., Ltd.) to blow dry the filter paper strip, add 100μL of acetonitrile to dissolve, vortex for 5min, sonicate for 5min, and then place it in a centrifuge at 15000rpm for 20min. The supernatant was taken for HPCL detection and the drug content was analyzed. The drug concentration in tears was calculated according to the following formula:

[0205]

[0206] Wherein, C is the drug content in tears, C1 is the drug concentration in the sample, V is the volume of acetonitrile used for reconstitution, and ρ is the density of tears, which is calculated as 1.005 g / mL.

[0207] The results are as follows Figure 8 , as shown in Table 7. AUC of TAM MCs@(PEI / HA)3 group compared with the other three groups 0-t and MRT 0-t In particular, the retention capacity of TAM MCs@(PEI / HA)3 eye drops on the ocular surface is much greater than that of commercial preparations, which are completely eliminated 15 minutes after administration. It should be noted that the drug concentration of the commercially available preparation was only 674.31±19.05 μg / mL 5 minutes after administration. 0-t yes 5.67 times of MRT 0-t yes In addition, the MRT of each preparation shown in Table 7 0-t This is consistent with the pattern of the precorneal fluorescence retention time of the above-mentioned preparations (Table 6).

[0208] Table 7 Tear elimination pharmacokinetic parameters of each preparation (n=3)

[0209]

[0210] Note: AUC 0-t express Figure 8 Area under the drug concentration-time curve; MRT 0-t It indicates the time required for 63.2% of the drug dose or concentration to be eliminated.

[0211] 9. Alkali burn method was used to model dry eye in rabbit eyes. New Zealand white rabbits with healthy eyes and of any gender were selected. 3% pentobarbital was used for intravenous general anesthesia, and 2% lidocaine was used for local anesthesia of the ocular surface. Filter paper strips (5×10mm) were clamped with tweezers and dipped in 1mol / L NaOH solution. The strips were directly contacted with the bulbar conjunctiva 2-3mm away from the corneal edge of the rabbit eyes for 90s, and then immediately rinsed with a large amount of saline to ensure that there was no NaOH residue. Before modeling, the Schirmer I test results of all rabbit eyes were greater than 11mm / 5min, and the tear film break-up time (TBUT) was greater than 10s; after modeling 7 days (Modeling 7), the Schirmer I test results of all rabbit eyes were less than 7mm / 5min, and the TBUT was less than 4s. This shows that the dry eye model was successfully established. The New Zealand rabbits with successful modeling were randomly divided into 6 groups, namely, positive control group, commercial preparation group, TAM eye drops group, TAM MCs eye drops group, TAM MCs@(PEI / HA)3 eye drops (twice a day) group and TAM MCs@(PEI / HA)3 eye drops (once a day) group; New Zealand rabbits with healthy eyes without modeling were used as the Blank control group (negative control group).

[0212] From the 8th day of modeling, 50 μL of normal saline (added to the positive control group and the negative control group, twice a day), commercially available preparations, and 10 μL of ... (Twice daily, bid. group), TAM eye drops prepared in Comparative Example 2 (administered twice a day, TAM eye drops bid. group), TAM MCs eye drops prepared in Example 6 (administered twice a day, TAM MCs eye drops bid. group), TAM MCs@(PEI / HA)3 eye drops prepared in Example 7 (administered twice a day, TAM MCs@(PEI / HA)3bid. group), and TAM MCs@(PEI / HA)3 eye drops prepared in Example 7 (administered once a day, TAM MCs@(PEI / HA)3qd. group (TAM MCs@(PEI / HA)3pd.)). An equal volume of normal saline was instilled 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. On days 11-14 after modeling, the rabbit tear volume (Schirmer I test), tear film breakup time (TBUT) and corneal fluorescence staining (FL) were analyzed to evaluate the efficacy of the preparation in treating dry eye. All measurements were performed by the same operator under the same environment.

[0213] Schirmer I test: Add two drops of proparacaine hydrochloride eye drops into the conjunctival sac of the rabbit eye for 5 minutes as an ocular surface anesthesia. Then take a Schirmer I test paper (5×35mm) and fold it at 5mm. Pull open the rabbit's lower eyelid and insert the folded part of the test paper into the conjunctival sac at the back 1 / 3 of the lower eyelid. Keep the test paper hanging stably. The rabbit eye can blink normally. After timing for 5 minutes, immediately read the lowest point value of the test paper wetted by tears, and grade the degree of dry eyes according to Table 8. The results are as follows Fig. 9 As shown: On the 4th day of administration (Administration 4d), The tear volumes of the bid. group, TAM eye drops bid. group, and TAM MCs eye drops bid. group were 8.13±2.08mm / 5min, 8.77±4.53mm / 5min, and 10.63±2.05mm / 5min, respectively. Although there was no significant difference compared with the positive control group (5.10±3.80mm / 5min), the tear volumes were higher than those of the positive control group. The tear volumes of the TAM MCs@(PEI / HA)3bid. group and the TAM MCs@(PEI / HA)3qd. group on the 4th day of administration were significantly different from those of the positive control group (P<0.001). The tear volumes of the two groups were bid. group, 2.90 times and 2.46 times. On the 7th day of administration, the tear volume of rabbits in each group recovered significantly, and the tear secretion of TAM MCs@(PEI / HA)3bid. group and TAMMCs@(PEI / HA)3qd. group was slightly better than that of the negative control group.

[0214] TBUT: Add 50 μL of 0.2% (w / v) sodium fluorescein solution to the conjunctival sac of the rabbit eye, close the rabbit eye and gently distribute the fluorescein evenly in the tear film. Open the rabbit eye under the cobalt blue light of the slit lamp and start timing to observe the state of the tear film. The time when the first dry spot or crack appears in the tear film is the tear film breakup time, and the degree of dry eye is graded according to Table 8. The results are shown in Table 8. Fig.10 As shown: On the 4th day of administration, The TBUT of the bid. group, TAM bid. group, and TAM MCs bid. group were 4.47±0.79s, 3.78±0.86s, and 5.55±0.44s, respectively. Although there was no significant difference compared with the positive control group (3.72±0.18s), the tear film breakup time was higher than that of the positive control group. The TBUT of the TAM MCs@(PEI / HA)3bid. group and the TAM MCs@(PEI / HA)3qd. group was significantly different from that of the positive control group (P<0.001). On the seventh day of administration, all experimental groups showed improvement, especially the TAM MCs@(PEI / HA)3bid. group and the TAM MCs@(PEI / HA)3qd. group.

[0215] Table 8 Schirmer I test and TBUT grading standards

[0216]

[0217] 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 microcrystal, wherein the raw materials for preparing the tacrolimus microcrystal comprise: tacrolimus, an oil phase solvent, a stabilizer, a surfactant and an aqueous phase solvent; the oil phase solvent comprises acetonitrile.

2. The tacrolimus microcrystal according to claim 1, characterized in that: The stabilizer comprises at least one of polylactic acid, polyhydroxybutyrate, polyhydroxyvalerate, polyvinyl alcohol, polycaprolactone, and polybutylene succinate; Preferably, the surfactant comprises at least one of Tween, Span, sodium dodecyl sulfate, sodium cholate, sodium deoxycholate, poloxamer 188, and poloxamer 407; Preferably, the aqueous phase solvent comprises at least one of water, phosphate buffer, and sodium chloride aqueous solution; Preferably, the mass volume ratio of tacrolimus, oil phase solvent, stabilizer, surfactant and water phase solvent (mg:mL:mg:mg:mL) is (5-15):1:(5-15):(5-15):(5-15); Preferably, the average particle size of the tacrolimus microcrystals is 0.1-10 μm; Preferably, the PDI of the tacrolimus microcrystals is 0.05-0.2; Preferably, the Zeta potential of the tacrolimus microcrystals is -(2-5) mV.

3. The method for preparing tacrolimus microcrystals according to claim 1 or 2, mixing tacrolimus with an oil phase solvent to obtain an oil phase; Mixing a stabilizer, a surfactant and an aqueous phase solvent to obtain an aqueous phase; The oil phase is mixed with the water phase to obtain a tacrolimus microcrystalline suspension; The oil phase solvent in the tacrolimus microcrystal suspension is evaporated and dried to obtain tacrolimus microcrystals.

4. A long-retention tacrolimus microcrystal, wherein the raw materials for preparing the long-retention tacrolimus microcrystal comprise: a positively charged material, a negatively charged material, and the tacrolimus microcrystal according to claim 1 or 2.

5. The long-retention tacrolimus microcrystal according to claim 4, characterized in that: The positively charged material comprises at least one of chitosan, polyethyleneimine, hydroxymethyl cellulose, sodium hydroxymethyl cellulose, and hydroxypropyl methyl cellulose; Preferably, the negatively charged material comprises at least one of polyacrylic acid, polyacrylamide, and hyaluronic acid; Preferably, the positively charged material is a positively charged material solution; Preferably, the negatively charged material is a negatively charged material solution.

6. The long-retention tacrolimus microcrystal according to claim 4 or 5, characterized in that: The average particle size of the long-retention tacrolimus microcrystals is 0.1-8 μm; Preferably, the PDI of the long-retention tacrolimus microcrystals is 0.1-0.4; Preferably, the Zeta potential of the long-retention tacrolimus microcrystals is -(3-7) mV.

7. A method for preparing the long-retention tacrolimus microcrystals according to any one of claims 4 to 6, S1: depositing the positively charged material on the tacrolimus microcrystals to obtain tacrolimus microcrystals with deposited positively charged material; S2: depositing the negatively charged material on the tacrolimus microcrystals deposited with the positively charged material, to obtain tacrolimus microcrystals deposited with the positively charged material and the negatively charged material; S3: drying the deposited positively charged material and the negatively charged material tacrolimus microcrystals to obtain long-retention tacrolimus microcrystals; Preferably, after S2 and before S3, the method further comprises the following steps: repeating S1 and S2; Preferably, the number of repetitions is 2-4 times; Preferably, the method for depositing the positively charged material on the tacrolimus microcrystals is: placing the tacrolimus microcrystals in a solution of the positively charged material, performing solid-liquid separation, and washing the tacrolimus microcrystals on which the positively charged material is deposited; Preferably, the method for depositing the negatively charged material on the tacrolimus microcrystals deposited with the positively charged material is: placing the tacrolimus microcrystals deposited with the positively charged material in a solution of the negatively charged material, performing solid-liquid separation, and washing the deposited positively charged material and the tacrolimus microcrystals deposited with the negatively charged material.

8. Use of the tacrolimus microcrystals according to any one of claims 1 to 2 or the long-retention tacrolimus microcrystals according to any one of claims 4 to 6 in the preparation of eye drops.

9. An eye drop comprising: the tacrolimus microcrystals according to any one of claims 1 to 2, or the long-retention tacrolimus microcrystals according to any one of claims 4 to 6.

10. The eye drops according to claim 9, characterized in that: The eye drops further comprise: at least one of a stabilizer, a wetting agent, an osmotic pressure regulator, a buffer, and an antibacterial agent; further comprising a stabilizer, a wetting agent, an osmotic pressure regulator, a buffer, and an antibacterial agent; Preferably, the stabilizer is at least one of Carbomer 934, Carbomer 940, and Carbomer 941; Preferably, the wetting agent is at least one of Tween 60, Tween 80 and Span 80; Preferably, the osmotic pressure regulator comprises at least one of sodium chloride, glucose, phosphate, citrate, mannitol, and sorbitol; Preferably, the buffer comprises at least one of boric acid-borax buffer, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, and carbonic acid-sodium bicarbonate buffer; Preferably, the antibacterial agent comprises at least one of benzalkonium chloride, benzalkonium bromide, chlorobutanol, methylparaben, ethylparaben, sodium perborate, and sorbic acid; Preferably, the concentration of the tacrolimus microcrystals or long-retention tacrolimus microcrystals in the eye drops is 0.5-1.5 mg / mL; Preferably, the concentration of the stabilizer in the eye drops is 1-3 mg / mL; Preferably, the concentration of the wetting agent in the eye drops is 0.05-0.15 mg / mL; Preferably, the concentration of the osmotic pressure regulator in the eye drops is 250-350 mg / mL; Preferably, the concentration of the antibacterial agent in the eye drops is 0.1-0.3 mg / mL; Preferably, the osmotic pressure of the eye drops is 250-300 mOsmol / kg; Preferably, the pH of the eye drops is 6.0-8.0.

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