Tacrolimus microcrystal and preparation method and application thereof

Abstract

The application belongs to the technical field of medicines, and discloses a tacrolimus microcrystal and a preparation method and application thereof. Compared with the raw medicine, the tacrolimus microcrystal has a significantly improved dissolution rate, cumulative dissolution rate, hydrophobicity and safety (non-irritation). Compared with the existing medicine preparation, the tacrolimus microcrystal has the advantages of long retention time and better treatment effect.

Description

Technical Field

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

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

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

[0004] Very few tacrolimus eye drops are available on the market, both domestically and internationally. Currently, the only one available is the tacrolimus eye drops from SENJU Pharmaceutical Co., Ltd. in Japan. (5mg:5mL) Administered once daily. Due to its extremely low water solubility and high molecular weight, tacrolimus is hindered from being transported through the tear film, cornea, and transcorneal region into the eye. As a result, its bioavailability is extremely low and it requires multiple daily eye drops, leading to poor patient compliance.

[0005] The sodium borohydride used in the preparation of layer-by-layer self-assembled tacrolimus microcrystals (TAM MCs@(PEI / HA)3) 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.) is a potentially explosive substance. It reacts violently with water, humid air, acids, oxidants, high heat and open flame. It is corrosive to human eyes and skin, and is a highly toxic substance. Its use is dangerous and does not meet the requirements for related reagents in pharmaceutical preparations. Summary of the Invention

[0006] The first aspect of the present invention is to provide a tacrolimus microcrystal.

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

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

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

[0010] The fifth aspect of the present application aims to provide the use of the tacrolimus microcrystal of the first aspect of the present application or the long-acting tacrolimus microcrystal of the third aspect of the present application in the preparation of eye drops.

[0011] The sixth aspect of the present application aims to provide an eye drop.

[0012] To achieve the above-mentioned purposes, the technical solutions adopted by the present application are as follows:

[0013] The first aspect of the present application provides a tacrolimus microcrystal, wherein the raw material for preparing the tacrolimus microcrystal comprises: tacrolimus, an oil-phase solvent, a stabilizer, a surfactant, and an aqueous-phase solvent; and the oil-phase solvent comprises acetonitrile.

[0014] Preferably, the stabilizer comprises at least one of polylactic acid, polyhydroxybutyric acid, polyhydroxyvaleric acid, polyvinyl alcohol, polycaprolactone, and polybutylene succinate; further comprises at least one of polylactic acid, polyvinyl alcohol, and polycaprolactone; and more 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 more 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 more further comprises deionized water.

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

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

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

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

[0021] In a second aspect, the present application provides a method for preparing the tacrolimus microcrystal of the first aspect of the present application,

[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] mixing the oil phase and the aqueous phase to obtain a tacrolimus microcrystal suspension;

[0025] volatilizing the oil phase solvent in the tacrolimus microcrystal suspension, and drying to obtain the tacrolimus microcrystal.

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

[0027] Preferably, the mixing of the stabilizer, the surfactant and the aqueous phase solvent is swelling overnight.

[0028] Preferably, the mixing of the oil phase and the aqueous phase is adding the oil phase to the aqueous phase under shearing; further, adding the oil phase to the aqueous phase under shearing at a constant speed.

[0029] Preferably, the speed of adding the oil phase is 0.5-1.5 mL / min.

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

[0031] Preferably, after adding the oil phase to the aqueous phase under shearing, the method further comprises the step of shearing homogenization.

[0032] Preferably, the conditions of shearing homogenization are shearing at 10000-15000 rpm for 2-4 min.

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

[0034] Preferably, after volatilizing the oil phase solvent in the tacrolimus microcrystal suspension, the method further comprises the step of solid-liquid separation and washing the tacrolimus microcrystal before drying.

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

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

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

[0038] Preferably, the drying is freeze drying.

[0039] A 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; more preferably, at least one of chitosan and polyethyleneimine; and even more preferably, 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; more preferably, it is hyaluronic acid.

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

[0043] Preferably, the number of positively charged material layers 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 solution of a positively charged material; more preferably, it is an aqueous solution of a positively charged material.

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

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

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

[0050] Preferably, the long-retained tacrolimus microcrystal has an average particle size of 0.1-8 μm; further 1-3 μm; and more further 1.41±0.05 μm.

[0051] Preferably, the long-retained tacrolimus microcrystal has a PDI of 0.1-0.4; further 0.25-0.3; and more further 0.27±0.08.

[0052] Preferably, the long-retained tacrolimus microcrystal has a Zeta potential of -(3-7) mV; further -(5.45±0.29) mV.

[0053] In a fourth aspect, the present application provides a preparation method of the long-retained tacrolimus microcrystal of the third aspect of the present application,

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

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

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

[0057] Preferably, the method further comprises the following step after S2 and before S3: repeating S1 and S2.

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

[0059] Preferably, the method of depositing the positively charged material on the tacrolimus microcrystal is placing the tacrolimus microcrystal in the positively charged material solution, solid-liquid separation, and washing the tacrolimus microcrystal with deposited positively charged material.

[0060] Preferably, the placing of the tacrolimus microcrystal in the positively charged material solution is vortexing the tacrolimus microcrystal in the positively charged material solution; and further vortexing the tacrolimus microcrystal in the positively charged material solution for 15-25 min.

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

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

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

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

[0065] Preferably, the method for placing the deposited positively charged material tacrolimus microcrystal in the negatively charged material solution is vortexing the deposited positively charged material tacrolimus microcrystal in the negatively charged material solution; further is vortexing the deposited positively charged material tacrolimus microcrystal in the negatively charged material solution for 15-25 min.

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

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

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

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

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

[0071] In the fifth aspect of the present application, the use of the tacrolimus microcrystal of the first aspect of the present application, or the long-retention tacrolimus microcrystal of the third aspect of the present application in the preparation of eye drops is provided.

[0072] In the sixth aspect of the present application, an eye drop is provided, which comprises: the tacrolimus microcrystal of the first aspect of the present application, or the long-retention tacrolimus microcrystal of the third aspect of the present application.

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

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

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

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

[0077] Preferably, the buffer comprises at least one of boric acid-borax buffer, sodium phosphate dibasic-sodium phosphate monobasic buffer, carbonic acid-sodium bicarbonate buffer; further is sodium phosphate dibasic-sodium phosphate monobasic buffer.

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

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

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

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

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

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

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

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

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

[0087] The present application provides a tacrolimus microcrystal, which has significantly improved dissolution rate, cumulative dissolution rate, hydrophobicity, safety (non-irritation) compared with the raw material drug; and has the advantages of long retention time, better treatment effect and the like compared with the existing drug preparation.

[0088] The present application provides a long retention tacrolimus microcrystal, which has the advantages of good sustained release effect, long retention time, better treatment effect and the like compared with the tacrolimus microcrystal, and has good clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0089] Figure 1 are the appearance and particle size distribution diagrams of tacrolimus raw material drug, tacrolimus microcrystal prepared in Example 1, long retention tacrolimus microcrystal prepared in Example 5 and tacrolimus commercial preparation , wherein (a) is the scanning electron microscope diagram of tacrolimus raw material drug (the scale is 200 μm); (b) is the scanning electron microscope diagram of tacrolimus microcrystal prepared in Example 1 (the scale is 2 μm); (c) is the scanning electron microscope diagram of long retention tacrolimus microcrystal prepared in Example 5 (the scale is 2 μm); (d) is the scanning electron microscope diagram of tacrolimus commercial preparation (the scale is 10 μm); (e) is the particle size distribution diagram of tacrolimus microcrystal prepared in Example 1; (f) is the particle size distribution diagram of long retention tacrolimus microcrystal prepared in Example 5.

[0090] Figure 2 is the potential flip diagram of the surface of the microparticle after each layer of high molecular material is wrapped in the preparation of long retention tacrolimus microcrystal in Example 5, wherein n = 3.

[0091] Figure 3 is the dissolution curve diagram of tacrolimus raw material drug and tacrolimus microcrystal prepared in Example 1, wherein n = 3.

[0092] Figure 4 is the hydrophobic binding diagram of tacrolimus raw material drug, tacrolimus microcrystal prepared in Example 1 and long retention tacrolimus microcrystal prepared in Example 5.

[0093] Figure 5 is the in-vitro release curve diagram of tacrolimus raw material drug eye drop prepared in Comparative Example 2, tacrolimus microcrystal eye drop prepared in Example 6, long retention tacrolimus microcrystal eye drop prepared in Example 7 and tacrolimus commercial preparation , wherein n = 3.

[0094] Figure 6The groups included a positive control group, a saline group, tacrolimus raw material eye drops prepared in Comparative Example 2, tacrolimus microcrystalline eye drops prepared in Example 6, long-retention tacrolimus microcrystalline eye drops prepared in Example 7, and commercially available tacrolimus formulations. The image shows the bleeding, coagulation, and dissolution of blood vessels in a chicken embryo, with the camera lens magnified 10 times.

[0095] Figure 7 The examples are: tacrolimus active pharmaceutical ingredient eye drops prepared in Comparative Example 2, tacrolimus microcrystalline eye drops prepared in Example 6, long-retention tacrolimus microcrystalline eye drops prepared in Example 7, and commercially available tacrolimus formulations. The image shows the pre-corneal fluorescence retention, with the camera lens magnification being 10x.

[0096] Figure 8 The examples are: tacrolimus active pharmaceutical ingredient eye drops prepared in Comparative Example 2, tacrolimus microcrystalline eye drops prepared in Example 6, long-retention tacrolimus microcrystalline eye drops prepared in Example 7, and commercially available tacrolimus formulations. The tear drug-time curve, where n = 3.

[0097] Figure 9 The groups included a negative control group (Blank control), a saline group (positive control group), tacrolimus raw material eye drops prepared in Comparative Example 2, tacrolimus microcrystalline eye drops prepared in Example 6, long-retention tacrolimus microcrystalline eye drops prepared in Example 7, and commercially available tacrolimus formulations. A comparison chart of Schirmer I test results, where n = 3.

[0098] Figure 10 The groups included a negative control group (Blank control), a saline group (positive control group), tacrolimus raw material eye drops prepared in Comparative Example 2, tacrolimus microcrystalline eye drops prepared in Example 6, long-retention tacrolimus microcrystalline eye drops prepared in Example 7, and commercially available tacrolimus formulations. A comparison of tear film breakup time (TBUT) results, where n=3.

[0099] Figure 11 This is an optical microscope image (scale bar 50 μm) of the tacrolimus formulation prepared in Comparative Example 1.

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

[0101] Figure 13Figure 4 is a graph of the in vitro release profile of the tacrolimus microcrystal (TAC MCs) eye drops prepared in Comparative Examples 5 and 6 and the layer-by-layer self-assembled tacrolimus microcrystal (TAC-(PAH / CMC)3 eye drops, 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 application will be further described in detail by specific examples.

[0104] It should be understood that the examples are only used for illustrating the present application but not for limiting the scope of the present application.

[0105] The experimental methods not specified in the following examples are generally carried out according to the conventional conditions or the conditions suggested by the manufacturers. The materials, reagents, etc. used in the present examples are commercially available reagents and materials unless otherwise specified.

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

[0107] The reagents used in the following examples and their manufacturers 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, comprising the following steps:

[0113] (1) Preparation of the oil phase: 10 mg of tacrolimus raw material was precisely weighed into a beaker, 1 mL of acetonitrile was added, and the beaker was tightly sealed. The tacrolimus was fully dispersed and dissolved by gently shaking to obtain a tacrolimus acetonitrile solution.

[0114] (2) Preparation of the water phase: 10 mg of polyvinyl alcohol and 10 mg of poloxamer 188 were precisely weighed into a beaker, and 10 mL of deionized water was added for swelling overnight.

[0115] (3) The water phase was placed in a homogenizer (IKAT18, IKA, Germany), and the shearing speed was 13000 rpm. The oil phase was completely sucked into a 1 mL syringe, and then the oil phase was uniformly injected into the water phase at a speed of 1 mL / min. After the end, the shearing homogenization was continued for 3 min to obtain a tacrolimus microcrystal suspension.

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

[0117] (5) The Tacrolimus microcrystals suspension obtained in step (4) in which the acetonitrile was completely volatilized was placed in a 10 mL centrifuge tube and centrifuged at 12000 rpm for 8 min, the supernatant was removed, and deionized water was added to wash away the excess water phase on the outer surface of the Tacrolimus microcrystals. This water washing was repeated three times.

[0118] (6) The washed Tacrolimus microcrystals suspension obtained in step (5) was placed in a freeze dryer (LGJ-10C, Beijing Sihuan Scientific Instrument Co., Ltd.) for freeze drying, thereby obtaining 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 the oil phase: 10 mg of Tacrolimus raw material was accurately weighed into a beaker, 1 mL of acetonitrile was added, and the beaker was tightly sealed. The Tacrolimus was fully dispersed and dissolved by gently shaking, thereby obtaining a Tacrolimus acetonitrile solution.

[0122] (2) Preparation of the water phase: 10 mg of polyvinyl alcohol and 10 mg of Tween 80 were accurately weighed into a beaker, and 10 mL of deionized water was added for swelling overnight.

[0123] (3) The water phase was placed in a homogenizer (IKAT18, IKA, Germany) with a shear speed of 13000 rpm. The oil phase was completely sucked into a 1 mL syringe, and then the oil phase was uniformly injected into the water phase at a speed of 1 mL / min. After the end, the homogenization was continued for 3 min, thereby obtaining a Tacrolimus microcrystals suspension.

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

[0125] (5) The Tacrolimus microcrystals suspension obtained in step (4) in which the acetonitrile was completely volatilized was placed in a 10 mL centrifuge tube and centrifuged at 12000 rpm for 8 min, the supernatant was removed, and deionized water was added to wash away the excess water phase on the outer surface of the Tacrolimus microcrystals. This water washing was repeated three times.

[0126] (6) The washed Tacrolimus microcrystals suspension obtained in step (5) was placed in a freeze dryer (LGJ-10C, Beijing Sihuan Scientific Instrument Co., Ltd.) for freeze drying, thereby obtaining Tacrolimus microcrystals.

[0127] Example 3 A method for preparing tacrolimus microcrystal

[0128] A method for preparing tacrolimus microcrystal, comprising the following steps:

[0129] (1) Preparation of oil phase: precisely weigh 10 mg of tacrolimus raw material into a beaker, add 1 mL of acetonitrile, then seal the beaker opening, shake gently to disperse and dissolve tacrolimus, and obtain tacrolimus acetonitrile solution.

[0130] (2) Preparation of water phase: precisely 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) Place the water phase in a homogenizer (IKAT18, IKA, Germany), with a shear speed of 13000 rpm, and then inject the oil phase into the water phase at a speed of 1 mL / min, and continue to shear and homogenize for 3 min after the end, to obtain tacrolimus microcrystal suspension.

[0132] (4) Place the tacrolimus microcrystal suspension obtained in step (3) in a dark room and magnetically stir at 300 rpm overnight until the acetonitrile is completely volatilized.

[0133] (5) Place the tacrolimus microcrystal suspension obtained in step (4) in a 10 mL centrifuge tube, centrifuge at 12000 rpm for 8 min, remove the supernatant, and wash the tacrolimus microcrystal with deionized water to remove the excess water phase on the outside, and repeat this washing three times.

[0134] (6) Place the washed tacrolimus microcrystal suspension obtained in step (5) in a freeze dryer (LGJ-10C, Beijing Sihuan Scientific Instrument Co., Ltd.), and freeze dry to obtain tacrolimus microcrystal.

[0135] Example 4 A method for preparing tacrolimus microcrystal

[0136] A method for preparing tacrolimus microcrystal, comprising the following steps:

[0137] (1) Preparation of oil phase: precisely weigh 10 mg of tacrolimus raw material into a beaker, add 1 mL of acetonitrile, then seal the beaker opening, shake gently to disperse and dissolve tacrolimus, and obtain tacrolimus acetonitrile solution.

[0138] (2) Preparation of water phase: precisely weigh 15 mg of polyvinyl alcohol and 5 mg of poloxamer 188 into a beaker, add 10 mL of deionized water and swell overnight.

[0139] (3) The water phase was placed in a homogenizer (IKAT18, IKA, Germany) with a shear speed of 13000 rpm. The oil phase was completely absorbed into a 1 mL syringe, and then injected into the water phase at a speed of 1 mL / min. After the end of the injection, the homogenization was continued for 3 min. Thus, the tacrolimus microcrystal suspension was obtained.

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

[0141] (5) The tacrolimus microcrystal suspension obtained in step (4) was placed in a 10 mL centrifuge tube and centrifuged at 12000 rpm for 8 min. The supernatant was removed, and the excess water phase on the surface of the tacrolimus microcrystals was washed away with deionized water. This water washing was repeated three times.

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

[0143] Example 5: A preparation method of long-retained tacrolimus microcrystals

[0144] A preparation method of long-retained tacrolimus microcrystals, comprising the following steps:

[0145] (1) 50 mg of the tacrolimus microcrystals prepared in Example 1 were dispersed in 5 mL of a polyethyleneimine (PEI) aqueous solution with a concentration of 2 mg / mL and vortexed for 20 min. Then, the mixture was centrifuged at 12000 rpm for 15 min. The supernatant was removed, and the excess PEI was washed away with deionized water. This water washing was repeated twice.

[0146] (2) The sediment obtained in step (1) was dispersed in 5 mL of a hyaluronic acid (HA) aqueous solution with a concentration of 1 mg / mL and vortexed for 20 min. Then, the mixture was centrifuged at 12000 rpm for 15 min. The supernatant was removed, and the excess HA was washed away with deionized water. This water washing was repeated twice.

[0147] (3) Steps (1) and (2) were repeated twice.

[0148] (4) The final sediment was dispersed in an appropriate amount of deionized water and placed in a freeze dryer for freeze-drying. Thus, the long-retained tacrolimus microcrystals (TAM MCs@(PEI / HA)3) were obtained.

[0149] Example 6: A preparation method of an eye drop

[0150] A method for preparing an eye drop: 10 mg of tacrolimus microcrystals (TAM MCs) of Example 1 were dispersed in 10 mL of a sodium dihydrogen phosphate-sodium hydrogen phosphate buffer in which 15 mg of carbomer 934 had been swelled, 0.31 g of mannitol, 0.001 g of Tween 80, and 0.002 g of benzalkonium chloride were added, and a TAM MCs eye drop having a concentration of 1 mg / mL, a pH of 6.62, and an osmotic pressure of 267 mOsmol / kg was prepared.

[0151] Example 7 A method for preparing an eye drop

[0152] A method for preparing an eye drop: 10 mg of long-retained tacrolimus microcrystals (TAM MCs@(PEI / HA)3) of Example 5 were dispersed in 10 mL of a sodium dihydrogen phosphate-sodium hydrogen phosphate buffer in which 15 mg of carbomer 934 had been swelled, 0.31 g of mannitol, 0.001 g of Tween 80, and 0.002 g of benzalkonium chloride were added, and a TAM MCs@(PEI / HA)3 eye drop having a concentration of 1 mg / mL, a pH of 6.63, and an osmotic pressure of 271 mOsmol / kg was prepared.

[0153] Comparative Example 1 A method for preparing a tacrolimus formulation

[0154] A method for preparing a tacrolimus formulation, comprising the following steps:

[0155] (1) Preparation of an oil phase: 10 mg of tacrolimus raw material was precisely weighed into a beaker, 1 mL of ethanol was added, the beaker was tightly closed, and the tacrolimus was fully dispersed and dissolved by gentle shaking to obtain a tacrolimus ethanol solution.

[0156] (2) Preparation of an aqueous phase: 10 mg of polyvinyl alcohol and 10 mg of poloxamer 188 were precisely weighed into a beaker, and 10 mL of deionized water was added to swell overnight.

[0157] (3) The aqueous phase was placed in a homogenizer (IKAT18, IKA, Germany), the shear speed was 13000 rpm, the oil phase was completely sucked into a 1 mL syringe, and the oil phase was uniformly injected into the aqueous phase at a speed of 1 mL / min, and after the end, the homogenization was continued for 3 min, and a tacrolimus formulation suspension was obtained.

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

[0159] (5) The heparin preparation suspension obtained in step (4) was placed in a 10 mL centrifuge tube, centrifuged at 12000 rpm for 8 min, the supernatant was removed, and the excess water phase on the outside of the heparin preparation was washed with deionized water. This water washing was repeated three times.

[0160] (6) The washed heparin preparation suspension obtained in step (5) was placed in a freeze dryer (LGJ-10C, Beijing Sihuan Scientific Instrument Co., Ltd.) for freeze drying, and heparin preparation was obtained.

[0161] Preparation method of a kind of eye drops

[0162] Preparation method of a kind of eye drops: 10 mg tacrolimus raw material drug (Tacrolimus, TAM) was dispersed in 10 mL sodium dihydrogen phosphate-sodium hydrogen phosphate buffer solution in which 15 mg carbomer 934 was swelled, 0.31 g mannitol, 0.001 g Tween 80 and 0.002 g benzalkonium chloride were added, and a TAM eye drop with a concentration of 1 mg / mL, a pH of 6.59 and an osmotic pressure of 269 mOsmol / kg was prepared.

[0163] Comparative Example 3

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

[0165] Comparative Example 4

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

[0167] Comparative Example 5

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

[0169] Comparative Example 6

[0170] TAC-(PAH / CMC)3 eye drops were prepared according to the preparation method in the literature (Zhang C J. Carboxymethyl cellulose modified tacrolimus non-spherical microcrystals for the treatment of dry eye in mice [D]. Tianjin Medical University, 2020. DOI: 10.27366 / d.cnki.gtyku.2020.000348.) (page 17).

[0171] Effect examples

[0172] 1. Tacrolimus raw material (TAM), TAM MCs prepared in Example 1, TAM MCs@ (PEI / HA) 3 prepared in Example 5, and a commercially available preparation After drying, gold was sprayed, and the morphology was observed under a scanning electron microscope (Scanning electron microscope, SEM, Sigma 300, Carl Zeiss, Germany) to capture SEM images. The particle size of tacrolimus raw material, TAM MCs prepared in Example 1, and TAM MCs@ (PEI / HA) 3 prepared in Example 5 was measured and counted under an optical microscope, and the particle size distribution of TAM MCs prepared in Example 1 and TAM MCs@ (PEI / HA) 3 prepared in Example 5 was plotted. The potential of tacrolimus raw material, TAM MCs prepared in Example 1, and TAM MCs@ (PEI / HA) 3 prepared in Example 5 was measured by a Zeta potential instrument (JS94J, Shanghai Zhongchen Digital Technology Equipment Co., Ltd.).

[0173] The results are shown in Figure 1 , Table 2. The average particle size of TAM MCs and TAM MCs@ (PEI / HA) 3 prepared in Examples 1 and 5 was less than 10 μm, which met the requirements of ophthalmic preparations and could avoid the loss of drugs on the ocular surface due to the blinking reflex caused by large particle size. No microcrystals were generated in Comparative Example 1 Figure 11 ), and the microcrystal morphology in Examples 2-4 was not clear, and there were large lumps (microcrystal lumps).

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

[0175]

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

[0177] 3. Dissolution determination of tacrolimus active pharmaceutical ingredient (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 of deionized water, and adjusting the pH to 7.4 with HCl. Using artificial tears containing 30% v / v acetonitrile as the dissolution medium for TAM and TAM MCs, 100 mL of each dissolution medium was accurately measured into 250 mL stoppered conical flasks and preheated in a gas bath constant-temperature shaker (THZ-82, Shanghai Xiaohan Industrial Development Co., Ltd.) at 34 °C and a shaking speed of 100 rpm / min. Accurately weigh 10 mg of TAM and TAM MCs into 500-mesh filter bags, respectively, and add them to the corresponding dissolution medium. Start timing. At different time points, accurately transfer 5 mL of the dissolution medium outside the filter bag, i.e., the sample, and then immediately add an equal volume of blank dissolution medium. Analyze the obtained samples using a high-performance liquid chromatograph (HPLC, LC-20AT, Shimadzu Corporation, Japan), and calculate the cumulative dissolution rate using the following formula:

[0178]

[0179] Among them, Q a For cumulative dissolution rate, m t Let m be the total mass of substances dissolved at time t, and m be the amount added.

[0180] The results are as follows Figure 3 As shown, after 45 minutes of dissolution, the cumulative dissolution rate of TAM MCs reached as high as 91.11%, while the cumulative dissolution rate of TAM was only 45.54%, and dissolution continued for 8.5 hours before it was basically completely dissolved. It is evident that the dissolution rate of TAM MCs is much greater than that of TAM. Figure 12 The cumulative dissolution rate of TAC MCs in Comparative Example 3 was only 56% at 45 min, and it took 6.5 h for dissolution to reach 90.91%.

[0181] 4. Rose Bengal (RB) experiment was used to explore the hydrophobicity of the surface of the drug particles. 2 mg of tacrolimus raw material (TAM), TAM MCs prepared in Example 1, and TAM MCs@ (PEI / HA) 3 powder prepared in Example 5 were respectively taken into centrifuge tubes, and 1.5 mL of RB solution with a concentration of 0.020, 0.025, 0.030, 0.035, 0.040, 0.045 mg / mL was added to each tube. The mixture was incubated at 4°C in a dark room for 3 h. Then the mixture was centrifuged at 15000 rpm for 20 min. The supernatant was filtered through a 0.22 μm organic microporous filter, and the filtrate was measured for absorbance by an ultraviolet spectrophotometer (Ultraviolet spectrophotometer, UV, UV-1800PC, Shanghai Meishidun Instrument Co., Ltd.). The unbound free RB was calculated. The binding constant K was obtained according to the Scatchard-plot formula:

[0182]

[0183] wherein 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 surface of the particles, and N is the maximum binding amount of RB.

[0184] The results are shown in Table 3. Figure 4 , and Table 3: TAM MCs@ (PEI / HA) 3、 The K value of TAM MCs is greater than that of TAM (wherein the K value of TAM MCs@ (PEI / HA) 3 is greater than that of TAM MCs), which has strong hydrophobicity and affinity with the hydrophobic cornea, providing conditions for prolonging the residence time of the drug in the cornea. The TAC-(PAH / CMC) 3 has too strong hydrophobicity, which affects the 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. STF solution containing 40% v / v acetonitrile was prepared 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 Commercially available eye drops, tacrolimus microcrystals (TAC MCs) eye drops prepared in Comparative Examples 5 and 6, and layer-by-layer self-assembled microcrystalline tacrolimus microcrystals (TAC-(PAH / CMC)3 eye drops) were all placed in 50 mL of STF solution containing 40% v / v acetonitrile in an air bath at 34 °C and 120 rpm / min for in vitro release. 1 mL samples were taken at different time points, and an equal volume of dialysis medium was immediately added. The filtrate after passing through a 0.22 μm microporous membrane was used to determine the peak area of ​​the drug using HPLC, calculate the drug concentration, and calculate the cumulative release (Q) according to the following formula. Release curves were plotted. Each formulation was tested in triplicate.

[0188]

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

[0190] The results are as follows Figure 5 As shown: The cumulative drug release in the first 0.5 hours was 29.65%, which is 1.65 times that of TAM MCs@(PEI / HA)3 (18.00%). The release plateau was reached after 6 hours, indicating weak sustained-release properties. TAM MCs@(PEI / HA)3, however, maintained drug release for 24 hours, ensuring stable and continuous drug release in the eye, maintaining long-term effective therapeutic concentrations at the target site, and achieving higher bioavailability. Figure 13 The cumulative in vitro release rates of tacrolimus microcrystals (TAC MCs) eye drops and layer-by-layer self-assembled microcrystalline tacrolimus microcrystals (TAC-(PAH / CMC)3 eye drops prepared in Comparative Examples 5 and 6 were both less than 4%, indicating almost no drug release.

[0191] 6. Fertilized eggs were placed in an incubator at a temperature of 37.8±0.2℃ and a humidity of 65%-75% for 9 consecutive days. At 9 days old, the chicken embryo allantoic membrane (CAM) had formed. After determining the air cell location by light, the outer shell at the top of the air cell was removed. The shell membrane was thoroughly moistened with physiological saline and carefully removed to obtain a complete and undamaged CAM. If blood vessel rupture and bleeding occurred, the egg was discarded and not used for further experiments. A 0.1 mol / L NaOH solution was used as the positive control group, and physiological saline as the negative control group. Comparative control groups included the TAM eye drops prepared in Example 2, the TAM MCs eye drops prepared in Example 6, and the TAM MCs@(PEI / HA)3 eye drops prepared in Example 7. The commercially available eye drops were used as the experimental group for the irritation experiment. 0.3 mL of each group of preparation was taken on the CAM, and then the surface of the CAM was washed with normal saline to remove the excess liquid, and then photographed. The irritation score (IS) was calculated according to the following formula, and the irritation was evaluated according to the scoring standard shown in Table 4.

[0192] The calculation formula of IS is:

[0193]

[0194] In the formula, t1 is the bleeding time (s), t2 is the initial time of blood vessel contraction (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 shown in Table 5: Figure 6 TAM MCs@(PEI / HA)3, TAM MCs eye drops have no irritation to CAM.

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

[0199]

[0200] 7. In 1 mL of commercially available preparation TAM eye drops prepared in Comparative Example 2, TAM MCs eye drops prepared in Example 6 and TAM MCs@(PEI / HA)3 eye drops prepared in Example 7, 0.002 g of fluorescein sodium was added. New Zealand white rabbits of both sexes and healthy eye conditions were selected and randomly divided into 4 groups, 3 in each group. 50 μL of each fluorescein sodium-containing preparation was dropped into the conjunctival sac of each rabbit eye, and after closing the rabbit eye for 10 s, the retention of the preparation on the ocular surface of the rabbit was observed using the cobalt blue lamp of the slit lamp (YZ5S, Liu Liu Vision Technology Co., Ltd.).

[0201] The results are shown in Table 6: Figure 7 The retention time of TAM MCs@(PEI / HA)3 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 ± 3.69 min (Table 6). Compared with the raw material drug, TAM MCs@(PEI / HA)3 has a more uniform and easy-to-disperse basis, and also has hydrophobicity, which is more compatible with the hydrophobic corneal epithelium; secondly, TAM MCs@(PEI / HA)3 specifically targets micro-interaction with CD44 receptors.

[0202] Table 6: Corneal pre-fluorescent retention time of different preparations (n = 3)

[0203]

[0204] 8. Conduct pharmacokinetic studies of tears. New Zealand white rabbits of any sex and with healthy eyes were randomly divided into 4 groups of 3 rabbits each. The left eye of each rabbit was designated as the experimental group, and the following eye drops were instilled into the conjunctival sac: TAM eye drops prepared in Comparative Example 2, TAM MCs eye drops prepared in Example 6, and TAM MCs@(PEI / HA)3 eye drops prepared in Example 7. Commercially available eye drops were used, with an equal volume of physiological saline instilled in the right rabbit eye as a blank control. Two strips of filter paper (3×8mm) were cut and placed in a 1.5mL centrifuge tube, and their weight was accurately recorded as m0. 100μL of each of the above preparations were added to the rabbit eye conjunctival sac, and the nasolacrimal duct was gently pressed to reduce preparation loss. At a specific time point, the filter paper strip was placed into the rabbit eye conjunctival sac and timed for 1 minute. The filter paper strip was then removed and placed at the bottom of the original centrifuge tube, and its weight was accurately recorded as m1. The filter paper strip was dried using a nitrogen blower (MD200-1, Hangzhou Aosheng Instrument Co., Ltd.), and 100μL of acetonitrile was added to reconstitute the solution. The mixture was vortexed for 5 minutes, sonicated for 5 minutes, and then centrifuged at 15000rpm for 20 minutes. The supernatant was collected for HPCL analysis to determine the drug content. The drug concentration in the tear fluid was calculated using the following formula:

[0205]

[0206] Where C is the drug content in the tear fluid, C1 is the drug concentration in the sample, V is the volume of acetonitrile used for reconstitution, and ρ is the tear fluid density, calculated at 1.005 g / mL.

[0207] The results are as follows Figure 8 Table 7 shows the AUC of the TAM MCs@(PEI / HA) 3 group compared to the other three groups. 0-t and MRT 0-t All are of the highest quality. In particular, TAM MCs@(PEI / HA)3 eye drops have a much greater retention capacity on the ocular surface than commercially available formulations that are completely eliminated within 15 minutes of administration. It should be noted that the drug concentration of this commercially available formulation was only 674.31 ± 19.05 μg / mL 5 minutes after administration. The AUC of TAM MCs@(PEI / HA)3 eye drops... 0-t yes 5.67 times that of MRT 0-t yes 1.97 times. Furthermore, Table 7 shows the MRT for each formulation. 0-t The pattern is consistent with the pattern of corneal pre-fluorescence retention time of the above-mentioned formulations (Table 6).

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

[0209]

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

[0211] 9. Dry eye model was established by alkali burn method. New Zealand white rabbits with healthy eye condition, male or female, were selected. Intravenous general anesthesia was performed using 3% pentobarbital, and topical anesthesia of the ocular surface was performed using 2% lidocaine eye drops. A filter paper strip (5x10mm) was dipped in a 1 mol / L NaOH solution and then directly contacted with the bulbar conjunctiva 2-3mm away from the corneal edge for 90s. Subsequently, a large amount of normal saline was used for flushing 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 break-up time (TBUT) was greater than 10s. After modeling for 7 days (Modeling 7), the Schirmer I test results of the rabbit eyes were less than 7mm / 5min, and the TBUT was less than 4s. This indicated that the dry eye model was successfully created. The successfully modeled New Zealand rabbits were randomly divided into 6 groups, namely the positive control group, the commercial preparation group, the TAM eye drop group, the TAM MCs eye drop group, the TAM MCs@(PEI / HA)3 eye drop (twice a day) group and the TAM MCs@(PEI / HA)3 eye drop (once a day) group. The unmodeled healthy eyes of New Zealand rabbits were used as the Blank control group (negative control group).

[0212] From the 8th day of modeling, 50μL of normal saline was respectively added to the conjunctival sac of the left eye of each group of New Zealand rabbits (added twice a day in the positive control group and the negative control group), the commercial preparation (twice a day, TAM eye drops prepared in Example 6 (TAM MCs eye drops bid. group, administered twice a day), TAM MCs@ (PEI / HA)3eye drops prepared in Example 7 (TAM MCs@ (PEI / HA)3bid. group, administered twice a day), TAM MCs@ (PEI / HA)3eye drops prepared in Example 7 (TAM MCs@ (PEI / HA)3qd. group (TAM MCs@ (PEI / HA)3pd.)), and an equal volume of normal saline was instilled into the right conjunctival sac of the rabbits as a control (normal saline group), and the nasolacrimal duct was gently pressed to prevent drug loss. The tear volume (Schirmer I test), tear film break-up time (TBUT), and corneal fluorescein staining (FL) of the rabbits were analyzed on the 11th to 14th day after modeling to evaluate the effect of the preparations on dry eye. All measurements were performed by the same operator in the same environment.

[0213] Schirmer I test: Two drops of propicacaine hydrochloride eye drops were instilled into the conjunctival sac of the rabbits for 5 min to anesthetize the ocular surface. Then, a Schirmer I test paper (5 x 35 mm) was folded at 5 mm, and the folded part was inserted into the conjunctival sac of the lower 1 / 3 of the lower eyelid of the rabbits. The test paper was kept stable, and the rabbits could normally blink. The lowest value of the test paper wetted by the tears was read immediately after 5 min of timing, and the degree of dry eye was classified according to Table 8. The results are shown in Table 9. Figure 9 The tear volumes of the TAM MCs@ (PEI / HA)3bid. group and the TAM MCs@ (PEI / HA)3qd. group were 2.90 and 2.46 times that of the TAM MCs bid. group, respectively, on the 4th day of administration, and the difference was very significant (P < 0.001) compared with the positive control group. On the 7th day of administration, the tear volume of each group of rabbits increased significantly, and the tear secretion of the TAM MCs@ (PEI / HA)3bid. group and the TAM MCs@ (PEI / HA)3qd. group was slightly better than that of the negative control group.

[0214] ​​TBUT: 50 μL of 0.2% (w / v) sodium fluorescein solution was added to the conjunctival sac of the rabbit eye, and the rabbit eye was closed gently to make the fluorescein distribute evenly in the tear film. The rabbit eye was opened under the cobalt blue light of the slit lamp, and the time was started. The state of the tear film was observed, and the time when the first dry spot or crack appeared in the tear film was recorded as the tear film break-up time. The dry eye degree was graded according to Table 8. The results are shown in Table 9. Figure 10 As shown in Table 9, the TBUT of the TAM MCs@ (PEI / HA)3 bid. group was 5.55 ± 0.44 s, which was higher than that of the positive control group (3.72 ± 0.18 s), but there was no significant difference. The TBUT of the TAM MCs@ (PEI / HA)3 bid. group was significantly different from that of the positive control group (P < 0.001). On the seventh day of administration, the dry eye degree of each experimental group was improved, especially the TAM MCs@ (PEI / HA)3 bid. group and the TAM MCs@ (PEI / HA)3 qd. group.

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

[0216]

[0217] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples. Any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

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; wherein the oil phase solvent is acetonitrile; The stabilizer is polyvinyl alcohol; The surfactant is poloxamer 188; The tacrolimus, oil phase solvent, stabilizer, surfactant and aqueous phase solvent are present in a mass-volume ratio of (8-12) mg: 1 mL: (8-12) mg: (8-12) mg: (8-12) mL; The method for preparing the tacrolimus microcrystals includes the following steps: Tacrolimus was mixed with an oil phase solvent to obtain an oil phase; The stabilizer, surfactant, and aqueous solvent are mixed to obtain an aqueous phase; The oil phase and the aqueous phase were mixed to obtain a tacrolimus microcrystalline suspension; The oil phase solvent in the tacrolimus microcrystal suspension was evaporated, and the suspension was dried to obtain tacrolimus microcrystals.

2. The tacrolimus microcrystals according to claim 1, characterized in that: The aqueous solvent comprises at least one of water, phosphate buffer, and aqueous sodium chloride solution.

3. The tacrolimus microcrystals according to any one of claims 1-2, characterized in that: The aqueous solvent is water; and / or The average particle size of the tacrolimus microcrystals is 0.1-10 μm; and / or The PDI of the tacrolimus microcrystals is 0.05 - 0.2; and / or The zeta potential of the tacrolimus microcrystals is -(2 - 5) mV.

4. The method for preparing tacrolimus microcrystals according to any one of claims 1-3, Tacrolimus was mixed with an oil phase solvent to obtain an oil phase; The stabilizer, surfactant, and aqueous solvent are mixed to obtain an aqueous phase; The oil phase and the aqueous phase were mixed to obtain a tacrolimus microcrystalline suspension; The oil phase solvent in the tacrolimus microcrystal suspension was evaporated, and the suspension was dried to obtain tacrolimus microcrystals.

5. 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 as described in any one of claims 1-3; The positively charged material is polyethyleneimine; The negatively charged material is hyaluronic acid; The method for preparing the long-retention tacrolimus microcrystals includes the following steps: S1: Deposit the positively charged material onto the tacrolimus microcrystals to obtain tacrolimus microcrystals with deposited positively charged material; S2: Deposit the negatively charged material onto the tacrolimus microcrystals of the positively charged material to obtain the positively charged material and the negatively charged material tacrolimus microcrystals. S3: Dry the deposited positively charged and negatively charged tacrolimus microcrystals to obtain long-retention tacrolimus microcrystals.

6. The long-retention tacrolimus microcrystals according to claim 5, characterized in that: The positively charged material is a positively charged material solution; and / or The negatively charged material is a solution of negatively charged material.

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

8. The long-retention tacrolimus microcrystals according to any one of claims 5-7, characterized in that: The steps following S2 and before S3 include: repeating S1 and S2; and / or The method for depositing the positively charged material on the tacrolimus microcrystals is as follows: placing the tacrolimus microcrystals in the solution of the positively charged material, performing solid-liquid separation, and cleaning the tacrolimus microcrystals on which the positively charged material was deposited; and / or The method for depositing the negatively charged material onto the tacrolimus microcrystals of the positively charged material is as follows: placing the tacrolimus microcrystals of the positively charged material into the solution of the negatively charged material, performing solid-liquid separation, and cleaning the positively charged material and the tacrolimus microcrystals of the negatively charged material.

9. The long-retention tacrolimus microcrystals according to claim 8, characterized in that: The repetition is performed 2-4 times.

10. The method for preparing long-retention tacrolimus microcrystals according to any one of claims 5-9, S1: Deposit the positively charged material onto the tacrolimus microcrystals to obtain tacrolimus microcrystals with deposited positively charged material; S2: Deposit the negatively charged material onto the tacrolimus microcrystals of the positively charged material to obtain the positively charged material and the negatively charged material tacrolimus microcrystals. S3: Dry the deposited positively charged and negatively charged tacrolimus microcrystals to obtain long-retention tacrolimus microcrystals.

11. The preparation method according to claim 10, characterized in that: The steps following S2 and before S3 include: repeating S1 and S2; and / or The method for depositing the positively charged material on the tacrolimus microcrystals is as follows: placing the tacrolimus microcrystals in the solution of the positively charged material, performing solid-liquid separation, and cleaning the tacrolimus microcrystals on which the positively charged material was deposited; and / or The method for depositing the negatively charged material onto the tacrolimus microcrystals of the positively charged material is as follows: placing the tacrolimus microcrystals of the positively charged material into the solution of the negatively charged material, performing solid-liquid separation, and cleaning the positively charged material and the tacrolimus microcrystals of the negatively charged material.

12. The preparation method according to claim 11, characterized in that: The repetition is performed 2-4 times.

13. The use of the tacrolimus microcrystals according to any one of claims 1-3, or the long-retention tacrolimus microcrystals according to any one of claims 5-9, in the preparation of eye drops.

14. An eye drop comprising: tacrolimus microcrystals as described in any one of claims 1-3, or long-retention tacrolimus microcrystals as described in any one of claims 5-9.

15. The eye drops according to claim 14, characterized in that: The eye drops also contain at least one of the following: stabilizer, wetting agent, osmotic pressure regulator, buffer, and antibacterial agent.

16. The eye drops according to claim 15, characterized in that: The eye drops also contain stabilizers, wetting agents, osmotic pressure regulators, buffer solutions, and antibacterial agents.

17. The eye drops according to any one of claims 15-16, characterized in that: The stabilizer is at least one of carbomer 934, carbomer 940, and carbomer 941; and / or The wetting agent is at least one of Tween 60, Tween 80, and Span 80; and / or The osmotic pressure regulator comprises at least one of sodium chloride, glucose, phosphate, citrate, mannitol, and sorbitol; and / or The buffer solution comprises at least one of borate-borax buffer, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, and carbonate-sodium bicarbonate buffer; and / or The antibacterial agent comprises at least one of benzalkonium chloride, benzalkonium bromide, chlorobutanol, methylparaben, ethylparaben, sodium perborate, and sorbic acid; and / or The concentration of the tacrolimus microcrystals, or long-retention tacrolimus microcrystals, in the eye drops is 0.5-1.5 mg / mL; and / or The stabilizer is present at a concentration of 1-3 mg / mL in the eye drops; and / or The concentration of the wetting agent in the eye drops is 0.05-0.15 mg / mL; and / or The concentration of the osmotic pressure regulator in the eye drops is 250-350 mg / mL; and / or The concentration of the antibacterial agent in the eye drops is 0.1-0.3 mg / mL.

18. The eye drops according to any one of claims 15-16, characterized in that: The osmotic pressure of the eye drops is 250-300 mOsmol / kg; and / or The pH of the eye drops is 6.0-8.0.

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