Cyclosporin A-entrapped albumin-bound drug as well as preparation method and application of cyclosporin A-entrapped albumin-bound drug

Abstract

The invention relates to the field of albumin binding type drugs, in particular to a cyclosporin A entrapped albumin binding type drug as well as a preparation method and application thereof. The albumin binding type medicine comprises cyclosporin A or a derivative thereof and albumin, the mass ratio of the cyclosporin A or the derivative thereof to the albumin is 1: (1-10), and albumin molecules are connected through disulfide bonds formed by self-crosslinking of free sulfydryl of the albumin. The albumin combined medicine disclosed by the invention shows a remarkable curative effect in the aspect of treating xerophthalmia, and can be used for remarkably improving tear secretion and quality, effectively repairing corneal injury, relieving eye inflammation and promoting overall recovery of eye tissues. According to the albumin binding type medicine, the retention time of the medicine on the ocular surface can be remarkably prolonged, the local medicine concentration is improved, the eye tissue recovery is comprehensively promoted while symptoms are rapidly relieved, and good safety and potential clinical application value are shown.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of albumin-binding drugs, in particular to an albumin-binding drug for loading cyclosporine A and a preparation method and application thereof. BACKGROUND

[0002] Dry eye disease (DED) is a multifactorial ocular surface disease caused by dysfunction of tear film homeostasis. Hyperosmolarity, tear film instability, neurosensory abnormalities, ocular surface inflammation, and tissue injury are common causes of DED symptoms. Among them, ocular surface inflammation is mainly mediated by CD4+ T cells, and the concentration of inflammatory cytokines related to conjunctival T cells is often found to be elevated in the tear film of DED patients.

[0003] Cyclosporine A (CyA) has become the first choice anti-inflammatory drug for DED treatment due to its long-term use without side effects such as corticosteroids. However, the poor water solubility and large molecular weight of CyA limit its possibility of using traditional local ophthalmic delivery systems. Existing drug delivery strategies, including hydrogels, nanoparticles, etc., have been improved, but it is usually difficult to completely overcome the challenge of poor solubility.

[0004] The most commonly used CyA ophthalmic preparation on the market is 0.05% cyclosporine emulsion (trade name: Restasis), which uses a complex emulsion technology to increase the stability and sustained-release effect of the drug. However, Restasis still has obvious deficiencies in clinical application. First of all, there is a problem of bioavailability. Although the emulsion form can prolong the residence time of the drug on the ocular surface to some extent, it also limits the release and absorption efficiency of the active ingredient of the drug, resulting in low overall bioavailability. This inefficient limitation often forces patients to take medication frequently to achieve the desired therapeutic effect. Secondly, the complexity of the preparation structure is a key problem. The formula design of Restasis is relatively complex, and strict control must be carried out during the production process to ensure the stability of the emulsion. This complexity not only increases the production and storage costs, but also may cause certain physical instability, thereby negatively affecting the drug efficacy. In addition, patients report a high incidence of adverse reactions during use. Common symptoms include blurred vision, eye burning and stinging, which significantly affect the quality of life and compliance of patients. Furthermore, the risk of local irritation cannot be ignored. The interfacial active agent and other additives in the emulsion may cause ocular surface irritation and exacerbate existing inflammatory reactions when used for a long time, which is not conducive to the recovery of eye health.

[0005] Human Serum Albumin (HSA) is the most abundant protein in human plasma. According to the analysis of tear proteome, HSA is also distributed in tears, with a proportion of about 1%. The excellent physical properties (water storage, viscosity, and gelatinous nature) of HSA can maintain the corneal and conjunctival epithelium moist for a long time, preventing the ocular surface from drying. Studies have shown that natural bovine serum albumin, human serum albumin, and recombinant human serum albumin can all inhibit the apoptosis of umbilical vein and microvascular cells at physiological concentrations, with the same dose-response curve. In addition, HSA has a certain therapeutic effect on dry eye, and its efficacy may be related to its inhibition of cell apoptosis. HSA can also increase the secretion of mucin, which may improve the stability of the tear film. Due to its chemical stability, biocompatibility, and high affinity for small molecule drugs, HSA is considered an ideal drug carrier.

[0006] CN111163757A discloses an albumin eye preparation that reversibly binds active compound PP-001 with albumin to reduce the side effect of cyclosporine's burning sensation on the eye. CN104434808A discloses a nano-preparation containing albumin and cyclosporine, but the drug has poor stability and is prone to burst release, which is not conducive to the exertion of its efficacy.

[0007] Although existing albumin nano-preparation technologies have improved the problems of poor drug delivery or stability to some extent, these methods often involve a large amount of organic solvent or are limited to specific drug types. SUMMARY

[0008] In view of the deficiencies of the prior art, the present application relates to a combination drug composition based on albumin as a carrier for loading cyclosporin A and a preparation method thereof, which is particularly suitable for the treatment of inflammatory and / or autoimmune ophthalmic diseases and eyelid diseases. The composition has a high drug loading capacity, a stable particle size distribution, and good ophthalmic drug adaptability, and can significantly improve the bioavailability and efficacy of cyclosporin A.

[0009] The present application provides an albumin combination drug for loading cyclosporin A, which comprises cyclosporin A and albumin, and the mass ratio of active ingredients to albumin is 1:1-10, wherein the albumin molecules are connected by disulfide bonds formed by self-crosslinking of free sulfhydryl groups of albumin.

[0010] Further, the cyclosporin A includes one or more of cyclosporin A drugs themselves, pharmaceutically acceptable salts thereof, or basic complexes thereof.

[0011] Further, the albumin includes animal albumin; preferably, the albumin includes at least one of natural albumin and recombinant albumin, more preferably, the albumin includes at least one of human serum albumin, recombinant human serum albumin, bovine serum albumin, ovalbumin, lactalbumin.

[0012] Further, the particle size of the albumin-bound drug is in the range of 5 nm to 500 nm, preferably 100 nm to 200 nm, more preferably, the particle size is 130-190 nm.

[0013] In some embodiments, the present application surprisingly found that the particle size control of the albumin drug composition of cyclosporine A significantly affects the stability of the drug composition (e.g. aggregation, precipitation, particle size change, etc.). When the average particle size of the drug composition is controlled to be 130-190 nm, the drug composition can maintain its physical state after storage (e.g. maintain its suspension state after storage at room temperature or 2-8℃ for 7 days, no precipitation or aggregation occurs, and no significant increase in particle size occurs).

[0014] Further, the drug loading of cyclosporine A in albumin is in the range of 5% to 20%. Preferably 10% to 15%, most preferably 12%.

[0015] In a second aspect of the present application, a method for preparing an albumin-bound drug loaded with cyclosporine A is provided, the method comprising at least the following steps: S1. Dissolve albumin in water to prepare an aqueous phase, preferably the concentration of the albumin is in the range of 0.1% to 1%; S2. Dissolve cyclosporine A or its derivative in an organic solvent to prepare an organic phase, preferably the concentration of cyclosporine A or its derivative is in the range of 5 mg / mL to 40 mg / mL; S3. Slowly add the organic phase to the aqueous phase using a probe for ultrasonic treatment to form a primary emulsion solution, preferably the mass ratio of active ingredient to albumin is 1:1~10; S4. Transfer the primary emulsion to a high-pressure homogenizer and perform cycle homogenization at a fixed pressure of 800~1200 bar until a translucent emulsion liquid is obtained; S5. Remove the organic solvent to obtain an albumin-bound drug solution loaded with cyclosporine A.

[0016] Further, the concentration of albumin in step S1 is in the range of 0.1% to 0.5%, and in a specific embodiment of the present application, the concentration is 0.25%.

[0017] Further, the organic solvent in step S2 comprises at least one of ethanol, methanol, chloroform. Further, the concentration of the cyclosporin A or its derivative ranges from 5 mg / mL to 25 mg / mL.

[0018] Further, the mass ratio of the cyclosporin A or its derivative to the albumin in step S3 is 1:1~5.

[0019] Further, the high pressure homogenization in step S4 is performed under a fixed pressure of 800~1200 bar for 15-20 cycles.

[0020] Further, the method for removing the organic solvent in step S5 comprises at least one of reduced pressure distillation, dialysis, ultrafiltration, freeze-drying, and air flow evaporation.

[0021] Further, the method further comprises a step of pre-freezing or freeze-drying the obtained albumin-bound drug solution for loading cyclosporin A.

[0022] In a third aspect of the present application, a composition is provided, which comprises the albumin-bound drug and an additive acceptable in the pharmaceutical field. Preferably, the additive comprises at least a freeze-drying protective agent.

[0023] Further, the freeze-drying protective agent comprises at least one or more of mannitol, trehalose, sucrose, lactose, glucose, and sodium caprylate. Preferably, the composition nanoparticles of the present disclosure are suspended in 1% to 5% of the freeze-drying protective agent, and then freeze-dried to prepare a pharmaceutical composition in the form of a freeze-dried powder. In a specific embodiment of the present application, the freeze-drying protective agent comprises mannitol with a mass fraction of 0.5%~2% and trehalose with a mass fraction of 0.3%~1%.

[0024] In certain embodiments, the pharmaceutical composition is a nanosuspension or a nanofreeze-dried preparation, and the average particle size of the particles or crystals before and after reconstitution can be 50-400 nm. In certain embodiments, the average particle size of the nanosuspension or freeze-dried preparation is 130-190 nm.

[0025] In a fourth aspect of the present application, the albumin-bound drug or the composition is applied in any of the following aspects: A1. application in the preparation of a product for promoting tear secretion; A2. application in the preparation of a product for repairing corneal damage; A3. application in the preparation of a product for improving the structure of corneal epithelial cells; A4. application in the preparation of an anti-inflammatory product.

[0026] In a fifth aspect, the present application provides the use of the albumin-binding drug or the composition in the preparation of a medicament for treating and / or inflammatory and / or autoimmune ophthalmic diseases and eyelid margin diseases, preferably at least one of dry eye, vernal keratoconjunctivitis, atopic keratoconjunctivitis, allergic conjunctivitis, ocular rosacea, uveitis, ocular cicatricial pemphigoid, ocular graft versus host disease and immune corneal ulcer disease.

[0027] The beneficial effects of the present application include, but are not limited to: The albumin-binding drug for loading cyclosporin A of the present application has a drug loading ratio of cyclosporin A in albumin of 10% to 15%. The drug composition can be in a lyophilized powder state, thereby improving its stability and storage convenience. Before use, the drug composition is reconstituted with an aqueous solution to form an eye drop, an eye gel or other suitable local delivery system. The composition is mainly used for preventing or treating inflammatory and autoimmune ophthalmic diseases such as dry eye, uveitis, keratitis, etc. The reconstitution process is simple and only needs sterile normal saline or other suitable buffer solution to be completely dissolved within about 10 seconds at room temperature.

[0028] The albumin-binding drug for loading cyclosporin A of the present application provides a treatment regimen that can enhance drug efficacy, high bioavailability, excellent biocompatibility, no irritation to the eye, and no obvious toxic side effects with long-term use through non-covalent binding of cyclosporin A and albumin. Its lyophilized powder form significantly improves the stability and shelf life of the drug, and after reconstitution, it can form an eye drop or a gel suitable for the eye, making it convenient for patients to use.

[0029] The albumin-binding drug for loading cyclosporin A of the present application exhibits significant therapeutic effect in treating dry eye, can significantly improve tear secretion and quality, effectively repair corneal damage, relieve eye inflammation, and promote overall recovery of eye tissue. Compared with traditional 0.05% CyA and 0.39% HSA-CyA Nps (0.05% CyA), the drug combines the immunomodulatory effect of CyA and the moisturizing and repair properties of rHA, improves the bioavailability and targeting of the drug through a nanoparticle delivery system, allows lower doses to achieve stronger efficacy, and reduces the risk of side effects. It prolongs the residence time of the drug on the ocular surface, improves the local drug concentration, rapidly relieves symptoms while comprehensively promoting the recovery of eye tissue, and exhibits good safety and potential clinical application value, especially for dry eye patients who need rapid and comprehensive repair. BRIEF DESCRIPTION OF DRAWINGS

[0030] The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings: Figure 1 Figure 1 is a graph showing the particle size distribution (A) and Zeta potential (B) of the albumin-bound drug of Example 1 of the present application, wherein the recombinant human serum albumin encapsulates cyclosporin A.

[0031] Figure 2 Figure 2 is a graph showing the particle size distribution (A) and Zeta potential (B) of the albumin-bound drug of Example 2 of the present application, wherein the human serum albumin encapsulates cyclosporin A.

[0032] Figure 3 Figure 3 is a transmission electron micrograph of the albumin-bound drug of Example 3 of the present application, wherein the recombinant human serum encapsulates cyclosporin A.

[0033] Figure 4 Figure 4 is a transmission electron micrograph of the albumin-bound drug of Example 4 of the present application, wherein the human serum albumin encapsulates cyclosporin A.

[0034] Figure 5 Figure 5 is a graph showing the results of the stability test of the albumin-bound drug of Example 5 of the present application, wherein the recombinant human serum albumin encapsulates cyclosporin A.

[0035] Figure 6 Figure 6 is a graph showing the results of the stability test of the albumin-bound drug of Example 6 of the present application, wherein the human serum albumin encapsulates cyclosporin A.

[0036] Figure 7 Figure 7 is a graph showing the appearance of the lyophilized preparation and the reconstituted product of the albumin-bound drug of Example 8 of the present application, wherein the recombinant human serum albumin encapsulates cyclosporin A.

[0037] Figure 8 Figure 8 is a graph showing the results of the tear test of mice treated with the albumin solution of Test Example 1 of the present application, wherein p<0.05 indicates a significant difference, usually marked with *, p<0.01 indicates a highly significant difference, usually marked with **, p<0.001 indicates an extremely significant difference, usually marked with ***, and p<0.0001 indicates a very significant difference, usually marked with ****.

[0038] Figure 9Figure 1 is a diagram of the results of the evaluation of the damaged area of the cornea of the mouse treated with the albumin solution of the present application in Example 1, wherein p<0.05 indicates a significant difference, usually marked with *, p<0.01 indicates a highly significant difference, usually marked with **, p<0.001 indicates an extremely significant difference, usually marked with ***, and p<0.0001 indicates a very significant difference, usually marked with ****.

[0039] Figure 10 Figure 2 is a diagram of the results of the detection of fern-like changes in the tear of the mouse treated with the albumin solution of the present application in Example 1, wherein p<0.05 indicates a significant difference, usually marked with *, p<0.01 indicates a highly significant difference, usually marked with **, p<0.001 indicates an extremely significant difference, usually marked with ***, and p<0.0001 indicates a very significant difference, usually marked with ****.

[0040] Figure 11 Figure 3 is a HE staining diagram of the cornea treated with the albumin solution of the present application in Example 1.

[0041] Figure 12 Figure 4 is a diagram of the results of the detection of the tear of the mouse in Example 2 of the present application, wherein p<0.05 indicates a significant difference, usually marked with *, p<0.01 indicates a highly significant difference, usually marked with **, p<0.001 indicates an extremely significant difference, usually marked with ***, and p<0.0001 indicates a very significant difference, usually marked with ****.

[0042] Figure 13 Figure 5 is a diagram of the results of the state of the ocular surface of the mouse without fluorescein sodium staining in Example 2 of the present application.

[0043] Figure 14 Figure 6 is a diagram of the results of the damaged area of the cornea of the mouse with fluorescein sodium staining in Example 2 of the present application.

[0044] Figure 15 Figure 7 is a diagram of the results of the evaluation of the damaged area of the cornea of the mouse in Example 2 of the present application, wherein p<0.05 indicates a significant difference, usually marked with *, p<0.01 indicates a highly significant difference, usually marked with **, p<0.001 indicates an extremely significant difference, usually marked with ***, and p<0.0001 indicates a very significant difference, usually marked with ****.

[0045] Figure 16 Figure 8 is a diagram of the results of the fern-like shape of the tear in Example 2 of the present application.

[0046] Figure 17Figure 2 is a schematic diagram of the results of the detection of fern-like changes in the tear fluid of mice in the test example 2 of the present application, wherein p<0.05 indicates a significant difference, usually marked with *, p<0.01 indicates a highly significant difference, usually marked with **, p<0.001 indicates an extremely significant difference, usually marked with ***, p<0.0001 indicates a very significant difference, usually marked with ****.

[0047] Figure 18 Figure 3 is a corneal HE staining diagram in the test example 2 of the present application.

[0048] Figure 19 Figure 5 is a schematic diagram of the results of the detection of fern-like changes in the tear fluid of mice in the test example 3 of the present application, wherein p<0.05 indicates a significant difference, usually marked with *, p<0.01 indicates a highly significant difference, usually marked with **, p<0.001 indicates an extremely significant difference, usually marked with ***, p<0.0001 indicates a very significant difference, usually marked with ****.

[0049] Figure 20 Figure 6 is a schematic diagram of the results of the scoring of the damaged areas of the corneas of mice in the test example 3 of the present application, wherein p<0.05 indicates a significant difference, usually marked with *, p<0.01 indicates a highly significant difference, usually marked with **, p<0.001 indicates an extremely significant difference, usually marked with ***, p<0.0001 indicates a very significant difference, usually marked with ****.

[0050] Figure 21 Figure 7 is a schematic diagram of the results of the detection of fern-like changes in the tear fluid of mice in the test example 3 of the present application, wherein p<0.05 indicates a significant difference, usually marked with *, p<0.01 indicates a highly significant difference, usually marked with **, p<0.001 indicates an extremely significant difference, usually marked with ***, p<0.0001 indicates a very significant difference, usually marked with ****.

[0051] Figure 22 Figure 8 is an ocular pharmacokinetic experiment of the albumin-bound drug for encapsulating cyclosporin A in the test example 4 of the present application, wherein Figure A is a diagram of the distribution of the albumin-bound drug in the cornea, Figure B is a diagram of the distribution of the albumin-bound drug in the conjunctiva, and Figure C is a diagram of the distribution of the albumin-bound drug in the eyelid. DETAILED DESCRIPTION

[0052] The present application will be described in detail below with reference to the examples, but the present application is not limited to these examples. Unless otherwise specified, the raw materials and catalysts in the examples of the present application are all purchased through commercial channels.

[0053] BAC (Benzalkonium chloride) was purchased from Sigma-Aldrich, item number 63449-41-2, diluted with PBS solution to a final concentration of 0.2%.

[0054] Human serum albumin (HSA): self-made by Tonghua Anruit Biological Pharmaceutical Co., Ltd.

[0055] Recombinant human serum albumin (rHA): self-made by Tonghua Anruit Biological Pharmaceutical Co., Ltd.

[0056] Recombinant human serum albumin encapsulated cyclosporin A albumin binding drug: rHA-CyA Nps.

[0057] Human serum albumin encapsulated cyclosporin A albumin binding drug: HSA-CyA Nps.

[0058] Example 1 Preparation of recombinant human serum albumin encapsulated cyclosporin A albumin binding drug a) A 20% recombinant human serum albumin (rHA) solution was diluted 40 times with pure water to prepare a rHA aqueous solution with a concentration of 5 mg / mL as the water phase.

[0059] b) An appropriate amount of cyclosporin A was weighed and prepared into a cyclosporin A chloroform solution with a concentration of 25 mg / mL as the organic phase.

[0060] c) The organic phase was slowly added to the water phase while the water phase was being ultrasonically treated using a probe. The ultrasonic parameters were 200 W, on for 2 s and off for 1 s, and the ultrasonic treatment lasted for 3 min to form a uniform initial emulsion with a mass ratio of cyclosporin A to recombinant human serum albumin of 1:1-10.

[0061] d) The initial emulsion was transferred to a high-pressure homogenizer and subjected to cycle homogenization at a fixed pressure of 1000 bar for a total of 15 times to obtain a translucent emulsion liquid.

[0062] e) The obtained emulsion liquid was transferred to a flask and subjected to rotary evaporation at 45°C for 10 min to remove chloroform, and finally a recombinant human serum albumin encapsulated cyclosporin A albumin binding drug (rHA-CyA Nps) solution was obtained.

[0063] f) The nanomedicine solution was diluted by an appropriate multiple, and its particle size and dispersity were determined using a Malvern laser particle size analyzer to evaluate the uniformity and stability of the nanoparticles, and the particle size distribution is shown in Figure 1 The particle size of rHA-CyA Nps was 179.00±4.64 nm, and the Zeta potential was -20 mV.

[0064] g) The nanoparticle solution was dialyzed for 24 h to remove unbound drug. After dialysis, the solution was stored in a freezer at -80°C for 12 h.

[0065] h) The frozen sample was lyophilized for 24 h to obtain the white solid of the recombinant human serum albumin encapsulated cyclosporin A albumin-bound drug (rHA-CyA Nps).

[0066] Example 2 Preparation of human serum albumin encapsulated cyclosporin A albumin-bound drug Example 1 was repeated using human serum albumin (HSA) instead of recombinant human serum albumin (rHA) in step a) to obtain the human serum albumin encapsulated cyclosporin A albumin-bound drug (HSA-CyA Nps) as a white solid. The particle size distribution is shown in Figure 2 The particle size of HSA-CyA Nps was determined to be 173.72 ± 5.34 nm and the zeta potential was -22 mV.

[0067] Example 3 The average particle size of the recombinant human serum albumin encapsulated cyclosporin A albumin-bound drug prepared in Example 1 was 160 nm and the suspension was translucent with blue opalescence. The drug composition solution was stored at room temperature for 48 h without precipitation. 10 μL of the albumin cyclosporin nanoparticle solution was dropped onto a copper grid covered with carbon film, and allowed to stand for 6 min. An appropriate amount of 2% sodium tungstate phosphorus negative staining solution was added, and allowed to stand for 5 min. The sample was re-stained once and dried for 1 min. The morphology and distribution of the nanoparticles were observed under a transmission electron microscope. The experimental results are shown in Figure 3 .

[0068] Example 4 The average particle size of the human serum albumin encapsulated cyclosporin A albumin-bound drug prepared in Example 2 was 168 nm and the suspension was translucent with blue opalescence. The drug composition solution was stored at room temperature for 48 h without precipitation. The procedure of Example 5 was repeated, and the experimental results are shown in Figure 4 .

[0069] Example 5 Stability Investigation The same batch of nanoparticle suspension prepared in Examples 1-2 was stored at room temperature, and samples were taken at 0, 1, 3, 5, 7, and 9 days to determine the particle size, PDI, and record the data to investigate the stability. The experimental results are shown in Figure 5 , 6The results show that the particle size and PDI of the nanoparticles do not change significantly with the extension of the storage time, and no precipitation or turbidity is observed, and the solution is still translucent with blue opalescence. It is preliminarily indicated that the albumin-bound drug solution loaded with cyclosporin A is relatively stable in short-term storage at room temperature.

[0070] Example 6 Investigation of Freeze-drying Protectants The albumin-bound drug loaded with cyclosporin A obtained in Example 1 was added into mannitol, trehalose, sucrose, lactose, glucose and sodium octanoate at a proportion of 1% and 5% (w / v). The solution was divided into a vial and quickly placed in a refrigerator at -80°C for pre-freezing for more than 24 hours, and then quickly taken out and placed on a shelf of a freeze dryer whose temperature had been reduced to -45°C. The switch of the vacuum pump was turned on, and the freeze-drying was performed for 48 hours to obtain the freeze-dried preparation of the albumin-bound drug loaded with cyclosporin A, which was stored after capping.

[0071] Example 7 The albumin-bound drug loaded with cyclosporin A obtained in Example 2 was added into mannitol and trehalose as freeze-drying protectants at a proportion of 0.5%-2% and 0.3%-1% (w / v) respectively. The solution was divided into a vial and quickly placed in a refrigerator at -80°C for pre-freezing for more than 24 hours, and then quickly taken out and placed on a shelf of a freeze dryer whose temperature had been reduced to -45°C. The switch of the vacuum pump was turned on, and the freeze-drying was performed for 48 hours to obtain the freeze-dried preparation of the albumin-bound drug loaded with cyclosporin A, which was stored after capping.

[0072] Example 8 The freeze-dried products obtained in Example 6 and Example 7 were white loose solids. After reconstitution with ultrapure water, the particle size of the reconstituted product was determined by a laser particle size analyzer, and each sample was repeatedly determined for three times. The experimental results are shown in Table 1 and Table 2 and Figure 7 It is shown that the addition of 1% mannitol and 0.3% trehalose has a better protective effect on the freeze-dried preparation.

[0073] Table 1 Investigation of the freeze-drying protective effect of different concentrations of mannitol, trehalose, sucrose, lactose, glucose and sodium octanoate on rHA-CyA Nps.

[0074]

[0075] Table 2 Investigation of the protective effect of different concentrations of mannitol and trehalose mixed freeze-drying protectants on rHA-CyA Nps.

[0076]

[0077] Note: Appearance: flat, smooth, little change in volume before and after freeze-drying, no collapse, no shrinkage is good. Redispersibility: take the freeze-dried powder, add the original volume of pure water, shake to quickly restore to the original state is good, the shorter the dispersion time the better. “++++” represents “excellent”, “+++” represents “good”, “++” represents “medium”, “+” represents “poor”.

[0078] Comparative Example The marketed drug 0.05% cyclosporine A eye emulsion (trade name: Ziru, purchased from Shenyang Xingqi Pharmaceutical) was purchased as a control drug 1 of the pharmaceutical composition of the present application.

[0079] Referring to the published data (CN111163757A), a human serum albumin non-covalent binding type eye composition preparation of compound 3-(2, 3, 5, 6-tetrafluoro-3'-trifluoromethoxy-biphenyl-4-yl carbamoyl)-thiophene-2-carboxylic acid (PP-001) was prepared, wherein the content of PP-001 was 3 mg / mL, and the content of albumin was 50 mg / mL, as a control drug 2.

[0080] Referring to the published data (CN104434808A), paclitaxel and cyclosporine albumin nanoparticle suspensions were prepared, wherein the content of albumin was 0.1-2 mg / mL, and the ratio of paclitaxel and cyclosporine to albumin was 1:0.14 and 1:0.26, respectively, and the average particle size was in the range of 70-150 nm, which were control drug 3 (paclitaxel albumin nanoparticle suspension) and control drug 4 (cyclosporine albumin nanoparticle suspension), respectively.

[0081] The following specific test examples demonstrate the beneficial effects of the present application compared with the existing commercially available preparations.

[0082] Test Example 1 Effect of Albumin Solution on Treatment of Dry Eye 1. Research Method 1.1 Research Animals 24 female C57BL / 6 mice aged 6-8 weeks (general grade, body weight 18-20 g, purchased from Zhejiang Weitong Lihua Experimental Technology Co., Ltd.) were used in the experiment and were raised in a SPF level environment in accordance with the national standard GB14925-2010 (license number: SYXK (Su) 2023-0018). All animal operations were in accordance with the experimental protocol approved by the institutional animal ethics committee.

[0083] 1.2 Animal Grouping Group 1 design: 6 in the modeling group, 6 in the 0.25% rHA solution group, 6 in the 0.25% HSA solution group, and 6 in the 5% rHA solution group.

[0084] Grouping method: Random grouping.

[0085] 1.3 Modeling method 1) 0.2% BAC solution eye drops Modeling animals: modeling group, 0.25% rHA solution group, 0.25% HSA solution group, 5% rHA solution group; Modeling method: 5 μL of 0.2% BAC solution was dropped onto the ocular surface of the mouse, 2 times / day (8:30 am, 6:30 pm), and the first day of modeling was recorded as D1, a total of 7 days of modeling, 7 days of modeling, a total of 14 days.

[0086] 1.4 Administration method Administration method: 5 μL of drug (0.25% rHA solution, 0.25% HSA solution, 5% rHA solution) solution was dropped onto the ocular surface of the mouse, avoiding drug overflow, and the administration of each group is shown in Table 3; Administration frequency: 2 times / day (9:30 am, 7:30 pm); Administration time: D8-D14 (8th-14th day of the experiment, D8 is the first day after modeling).

[0087] Table 3. Administration table of each group

[0088] 1.5 Phenol red cotton tear test Detection time: D7, D10, D14; Detection method: After the mouse was anesthetized with isoflurane, phenol red cotton thread (Tianjin Jingming New Technology Development Co., Ltd.) was used to detect the tear fluid, the phenol red cotton thread was inserted into the outer corner of the mouse eye, and the mouse cornea was avoided during the detection process. After 30 s, the phenol red cotton thread was removed and the total length of the wet part of the cotton thread was measured.

[0089] 1.6 Corneal fluorescein sodium staining and scoring Detection time: D7, D10, D14; Detection method: After the mouse was anesthetized with isoflurane, 1 μL of 0.2% fluorescein sodium solution was added to the ocular surface, and the eyelids were gently blinked 3 times with the hand. Under the blue cobalt light, the corneal fluorescence staining was observed. The scoring standard is: 0 level indicates no staining, 1 level indicates staining area not more than 1 / 8, 2 level indicates not more than 1 / 4, 3 level indicates not more than 1 / 2, 4 level indicates staining area reaches or exceeds 1 / 2.

[0090] 1.7 Tear fern test and scoring Detection time: D7, D10, D14; Detection method: After the mice were anesthetized with isoflurane, 1 μL of 0.9% saline was added to the ocular surface, and the sample capillary glass tube was placed at the lacrimal fornix of the lower eyelid conjunctival sac. The tear fluid sample was collected by capillary action while avoiding contact with the ocular surface. Then, the tear fluid sample was evenly coated on a clean glass slide and dried for 10 to 20 minutes in a 25°C air-conditioned environment. Finally, the morphology of the fern-like crystals in the tear fluid was observed under an optical microscope and photographed. The scoring criteria were as follows: the crystal morphology was divided into four levels. Level I crystal pattern was uniform fern-like branching with relatively narrow spacing; level II crystal pattern was small in size, with few branches and increased spacing; level III crystal pattern had significantly reduced branches, with spacing increased to the size of the crystals; and level IV had no fern-like crystal pattern, with a small amount of amorphous crystals. The general results of the tear fern test in dry eye animal models were levels III and IV crystals.

[0091] 1.8 Corneal HE staining Detection time: D14; Detection method: After the mice were sacrificed by cervical dislocation, the corneal tissue was taken and fixed in 4% paraformaldehyde for 48 hours. The corneal tissue sections were prepared by routine paraffin embedding and continuous sectioning. After HE staining, the pathological damage of the mouse corneal tissue was observed under a microscope and photographed.

[0092] 1.9 Statistics All data were analyzed using GraphPad Prism 8 software. For comparison between multiple groups, two-way ANOVA analysis method was used, combined with Tukey's test for pairwise comparison between groups to determine the significant differences between different groups. This statistical method is suitable for handling complex data involving multiple independent variables and can provide more accurate difference analysis results. For all statistical analyses, the significance level was set at p<0.05 to ensure the reliability and scientificity of the results.

[0093] 2. Results 2.1 Phenol red cotton thread tear test In the experiment, phenol red cotton thread was used to measure the tear secretion of mice. As Figure 8 shown in the color change length statistics of the phenol red cotton thread. On the 3rd day after administration, the tear secretion of mice in the 0.25% rHA group and the 0.25% HSA group was significantly higher than that in the model control group. In particular, the 0.25% HSA group not only showed a significant difference compared with the model group (P<0.01), but also showed a more excellent effect compared with the 5% rHA group (P<0.01), with an increase of about 40-50% in tear secretion, indicating that 0.25% HSA has a significant improvement effect on promoting tear secretion.

[0094] 2.3 Corneal fluorescein sodium staining score results In this experiment, the therapeutic effect was observed by evaluating the grading of the damaged area of the mouse cornea. As shown in Figure 9 The grading of the damaged area of the cornea of each group of mice is shown. On the 3rd day after administration, the corneal damage of the mice in the 0.25% rHA group and the 0.25% HSA group was significantly repaired, and the effect of corneal repair was significantly better than that of the model control group (P<0.001), with an improvement of about 60% in the degree of corneal damage, indicating that 0.25% rHA and 0.25% HSA effectively promoted the repair of the cornea. In addition, on the 7th day after treatment, the score of the model group also decreased, and the degree of corneal damage improved by about 50-60%, which may be due to the influence of environmental factors.

[0095] 2.4 Results of tear fern examination and scoring This study evaluated the effect of 0.25% rHA and 0.25% HSA on the recovery of corneal damage in mice. As shown in Figure 10 The crystal morphology score is shown. On the 3rd day after administration, the mice in the 0.25% rHA group and the 0.25% HSA group had a significant improvement in tear fern grading compared to the model control group, with a decrease in tear fern score of about 30-40%. In particular, the 0.25% HSA group showed a significant improvement, with a statistical significance of p<0.01. By the 7th day, the tear fern grading of the mice in these two groups further improved, indicating that both 0.25% rHA and 0.25% HSA effectively promoted the repair of corneal damage.

[0096] 2.5 Results of corneal HE staining This study observed the morphological changes of the mouse corneal epithelial cells by HE staining method. As shown in Figure 11 In the model control group, the corneal epithelial cells showed severe structural damage, with excessive proliferation of goblet cells and extensive infiltration of inflammatory factors, as well as obvious formation of new blood vessels. After 7 days of treatment, the corneal epithelial cells of the mice in the 0.25% rHA treatment group showed significant improvement in structure, with more orderly arrangement of cells, clear cell stratification, and significant reduction in inflammatory response. These observations indicate that 0.25% rHA has potential effects in promoting the repair of corneal damage and reducing inflammation.

[0097] 2.6 Summary In this study, 0.25% rHA and 0.25% HSA showed significant therapeutic effects in treating corneal damage in mice. In particular, 0.25% HSA showed better effects than 5% rHA in multiple evaluation indicators. This highlights the importance of selecting appropriate concentrations for therapeutic effects, and the concentration of 0.25% provides evidence for achieving the best balance between efficacy and safety.

[0098] Specifically, 0.25% rHA showed excellent performance in promoting the repair of the damaged corneal area, improving the epithelial cell structure, reducing the infiltration of inflammatory factors, and controlling the formation of new blood vessels, showing its potential clinical value in promoting the repair of corneal damage and relieving inflammation. These therapeutic effects of rHA are particularly superior in promoting the orderly arrangement of epithelial cell structure and reducing inflammatory response, which may be attributed to its unique biological activity and excellent cell compatibility.

[0099] In summary, 0.25% rHA and HSA not only can significantly improve tear secretion, but also can accelerate the repair process of the cornea, making these two concentrations of drugs strong candidates for the development of new ophthalmic therapeutic drugs. Future studies should further explore the optimal application scheme and long-term efficacy of these drugs to provide more effective treatment strategies for clinical practice and provide important scientific basis for determining the optimal concentration and composition of drugs.

[0100] Test Example 2 Effect of Albumin-Bound Drug Containing Cyclosporin A of the Invention on Dry Eye Research Method 1.1 Research Animals The experiment used 15 research animals in Test Example 1.

[0101] 1.2 Animal Grouping Group Design: 3 in the control group, 3 in the modeling group, 3 in the cyclosporin eye drop (II) (trade name Zirun, specification 0.05%, Shenyang Xingqi Pharmaceutical) group, 3 in the rHA-CyA Nps (0.05% CyA) group, and 3 in the HSA-CyA Nps (0.05% CyA) group.

[0102] Grouping Method: Random grouping.

[0103] 1.3 Modeling Method The experimental steps were the same as the modeling method in Test Example 1.

[0104] 1.4 Administration Method Drug Nanoparticle Prescription: The concentration of cyclosporin A was 25 mg / mL; the concentration of albumin was 0.25%; the mass ratio of cyclosporin A to albumin was 5:1; Administration Method: 5 μL of drug (cyclosporin eye drop (II), rHA-CyA Nps (0.05% CyA), and HSA-CyA Nps (0.05% CyA)) solution was dropped onto the mouse ocular surface, avoiding drug overflow, and the drugs for each group are shown in Table 4; Administration Frequency: 3 times / day (9:30 am, 19:30 pm); Administration Time: D8-D14 (8th-14th day of the experiment, D8 being the first day after modeling).

[0105] Table 4. Dosage schedule for each group

[0106] 1.5 Phenol red cotton tear test The experimental steps are the same as 1.5 of Test Example 1.

[0107] 1.6 Periocular Examination Detection time: D7, D10, D14; Detection method: After anesthetizing mice with isoflurane, observe the degree of keratinization of the corneal epithelium of the mice under normal light to see whether there are abnormalities such as blisters, ulcers, and pannus; whether there is debris on the surface of the cornea and in the lower fornix; whether the conjunctiva is congested or has tissue hyperplasia; whether the conjunctival sac and conjunctiva are loose and wrinkled, whether the eyelid margin is congested, irregular, thickened, or everted; whether the glandular opening is blocked by clear or yellow serous fluid, and whether the glandular duct is blurred.

[0108] The experimental steps from 1.7 to 1.10 are the same as those from 1.6 to 1.9 in Experimental Example 1.

[0109] 2. Results 2.1 Phenol red cotton tear examination like Figure 12 Shown are statistics on the discoloration length of the phenol red cotton thread. On day 3 after administration, tear secretion in mice generally increased by approximately 40-50%. Tear secretion in the 0.05% CyA and 0.41% rHA-CyA Nps (0.05% CyA) groups was significantly higher than that in the 0.39% HSA-CyA Nps (0.05% CyA) group (p < 0.05). By day 7, tear secretion in mice remained similar to that on day 3, presumably indicating that tear production had returned to normal levels.

[0110] 2.2 Periocular Examination Results like Figure 13 As shown, on day 3 after administration, periocular congestion was significantly improved in the mice in the 0.05% CyA group and the 0.41% rHA-CyA Nps (0.05% CyA) group. By day 7, periocular congestion in the mice in the 0.41% rHA-CyA Nps (0.05% CyA) group had almost completely resolved.

[0111] Meanwhile, on day 3, mice in the 0.05% CyA group (1L and 1R) and mice in the 1R group receiving 0.39% HSA-CyA Nps (0.05% CyA) developed blisters in the corners of their eyes. On day 7, the blisters in the 0.05% CyA group not only did not subside, but actually worsened. However, the 0.39% HSA-CyA Nps (0.05% CyA) group showed improvement.

[0112] 2.3 Corneal fluorescein sodium staining and scoring results like Figure 14 Shown are stained images of the damaged corneas in each group of mice. Three days after administration, the damaged area of ​​the corneas in mice treated with 0.05% CyA and 0.41% rHA-CyA Nps (0.05% CyA) decreased by approximately 50-70%. Seven days after administration, the damaged area of ​​the corneas in mice treated with 0.05% CyA, 0.41% rHA-CyA Nps (0.05% CyA), and 0.39% HSA-CyANps (0.05% CyA) all showed significant reductions, by approximately 70-80%. In particular, the damaged area in mice (3L and 3R) treated with 0.41% rHA-CyA Nps (0.05% CyA) was almost completely healed.

[0113] like Figure 15 Shown are the corneal damage area ratings for each group of mice. Seven days after administration, mice in the 0.05% CyA and 0.41% rHA-CyA Nps (0.05% CyA) groups showed significant differences compared to the model group (p < 0.05), indicating that both drugs are able to effectively repair corneal damage.

[0114] 2.4 Tear Fern Examination and Scoring Results like Figure 16 The morphology of tear ferns is shown. Figure 17 Crystal morphology grade classification was performed. On day 3 after administration, the tear fern grade of mice in the 0.05% CyA, 0.41% rHA-CyA Nps (0.05% CyA), and 0.39% HSA-CyA Nps (0.05% CyA) groups showed significant improvement compared to the model group. The improvement in the 0.41% rHA-CyA Nps (0.05% CyA) group was significantly greater than that in the 0.05% CyA group (p < 0.05), with an approximately 50% improvement in tear fern grade. On day 7 after administration, the tear fern grade of mice in the 0.05% CyA and 0.41% rHA-CyA Nps (0.05% CyA) groups showed no significant difference from that in the control group, indicating that the grade had returned to normal.

[0115] 2.5 Corneal HE staining results like Figure 18HE staining of cornea of each group of mice is shown. Compared with the control group, the corneal epithelial cell layer of the DED group is rough and broken, with vacuole-like cells, and inflammatory cell infiltration appears in the corneal stroma. After 7 days of administration, the corneal epithelial cells of the 0.05% CyA and 0.41% rHA-CyA Nps (0.05% CyA) and 0.39% HSA-CyA Nps (0.05% CyA) groups are re-arranged in an orderly manner, and the stratification is clear.

[0116] 2.7 Summary 0.41% rHA-CyA Nps (0.05% CyA) showed significant efficacy in treating dry eye, which can significantly improve tear secretion and quality, effectively repair corneal damage, relieve eye inflammation, and promote the overall recovery of eye tissues. Compared with traditional 0.05% CyA and 0.39% HSA-CyA Nps (0.05% CyA), this drug combines the immunomodulatory effect of CyA and the moisturizing and repair properties of rHA, improves the bioavailability and targeting of the drug through the nanoparticle delivery system, so that a lower dose can achieve stronger efficacy, while reducing the risk of side effects. It prolongs the residence time of the drug on the ocular surface, increases the local drug concentration, rapidly relieves symptoms, and comprehensively promotes the recovery of eye tissues, showing good safety and potential clinical application value, especially for dry eye patients who need rapid and comprehensive repair.

[0117] Comparison of the effects of test example 3 control drugs 2-4 on treating dry eye Referring to the test method (test animals, modeling method, administration method and frequency) and evaluation method (evaluation time is D7, D10, D14) of test example 2, the anti-dry eye effects of the albumin-bound drug loaded with cyclosporin A of the application and control drugs 2-4 are compared.

[0118] The results are summarized as follows: As Figure 19 The color change length of the phenol red cotton thread is shown. On the 3rd day after administration, the tear secretion of the mice generally increased, and the tear secretion of the mice in the preferred rHA-CyA Nps group of the application was significantly higher than that in the 0.2% BAC model group (p<0.0001), the control drug 2 group (p<0.01), the control drug 3 group (p<0.0001), and the control drug 4 group (p<0.0001). On the 7th day, the tear secretion of the mice in the rHA-CyA Nps group was also significantly higher than that in the 0.2% BAC model group (p<0.001), the control drug 2 group (p<0.05), the control drug 3 group (p<0.001), and the control drug 4 group (p<0.05). This indicates that the drug in the rHA-CyA Nps group has an advantage in promoting tear secretion in dry eye mice.

[0119] AsFigure 20 The corneal lesion area of each group of mice was rated as shown. By the 7th day after administration, the corneal lesion area of the mice in the rHA-CyA Nps group was significantly lower than that of the 0.2% BAC model group (p<0.0001), the control drug 2 group (p<0.001), the control drug 3 group (p<0.01), and the control drug 4 group (p<0.0001). This indicates that the drug in the rHA-CyA Nps group can effectively repair corneal damage.

[0120] As shown in Table 3, the tear fluid volume of the mice in the rHA-CyA Nps group was significantly improved on the 3rd day after administration, significantly better than that of the 0.2% BAC model group (p<0.0001), the control drug 2 group (p<0.001), the control drug 3 group (p<0.001), and the control drug 4 group (p<0.01). By the 7th day, the improvement effect was further improved, and was better than that of the other groups (all p<0.0001). This indicates that the drug in the rHA-CyA Nps group can effectively improve the tear fluid volume. Figure 21 The tear fluid morphology of each group of mice was rated as shown in Table 4. On the 3rd day after administration, the mice in the preferred rHA-CyA Nps group of the present application had significant improvement in the tear fern rating, significantly better than the 0.2% BAC model group (p<0.0001), the control drug 2 group (p<0.001), the control drug 3 group (p<0.001), and the control drug 4 group (p<0.01). By the 7th day, the improvement effect was further improved, and was better than that of the other groups (all p<0.0001). This indicates that the drug in the rHA-CyA Nps group can effectively improve the tear fluid morphology.

[0121] In summary, the preferred rHA-CyA Nps group of the present application is significantly better than the marketed drugs and some of the disclosed control groups, and has significant development potential.

[0122] Test Example 4: Rabbit pharmacokinetic investigation Forty male New Zealand rabbits were divided into 8 groups and received a one-time eye drop of rHA-CyA or cyclosporine A (CyA) eye drop emulsion 0.05% (Ziru, Xingqi Pharmaceutical) as a control, 50 μL per eye. Samples were collected at 6, 12, 24, and 48 hours, including tear fluid and eye tissues / fluids (aqueous humor, choroid-retina, conjunctiva, cornea, upper eyelid, third eyelid, iris / ciliary body, lacrimal gland, lens, sclera, and vitreous body). The CyA concentration was analyzed using liquid chromatography-tandem mass spectrometry. The results are shown in Table 5. Figure 22 The albumin-bound drug release effect of cyclosporine A was obvious, and there was still obvious distribution in the cornea at 48 h. The pharmacokinetic experiment groups are shown in Table 5.

[0123] Table 5: Pharmacokinetic experiment groups

[0124] Test Example 5: Screening of process prescriptions Table 6: Process prescription table

[0125] The experimental steps of Example 1 were repeated by replacing the corresponding ingredients in Example 1 with the data in each of the process prescription table in Table 6.

[0126] For the above test example, the albumin cyclosporin A nanoparticle solution obtained in step e was appropriately diluted, and the particle size and dispersibility thereof were measured using a Malvern laser particle size analyzer to evaluate the uniformity and stability of the nanoparticles. The results are shown in Table 7.

[0127] Table 7 Evaluation of albumin cyclosporin A nanoparticle solution

[0128] Table 7 shows that, since Test Example 2 has a lower average particle size, a higher absolute value of Zeta potential, and a lower PDI value, the preparation method for preparing the albumin cyclosporin A nanoparticle solution is preferably a concentration ratio of 0.5% of albumin in the aqueous phase, a concentration of 25 mg / mL of cyclosporin A in the organic phase, and a mass ratio of 1:5 of the mass ratio of cyclosporin A to albumin, considering the actual use and production convenience of the product.

[0129] The above description is merely illustrative of the embodiments of the present application and the scope of the present application is not limited to these specific embodiments. The present application can be variously changed and modified by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the technical idea and principles of the present application should be included in the scope of the present application.

Claims

1. An albumin-bound drug containing cyclosporin A, characterized in that: The albumin-binding drug comprises cyclosporin A or its derivatives and albumin, wherein the mass ratio of cyclosporin A or its derivatives to albumin is 1:1-10, wherein the albumin molecules are connected through disulfide bonds formed by self-crosslinking of free thiol groups of the albumin.

2. The albumin-binding drug according to claim 1, characterized in that The cyclosporin A derivatives include one or more of its pharmaceutically acceptable salts or basic complexes.

3. The albumin-binding drug according to claim 1, characterized in that The albumin includes animal albumin.

4. A method for preparing an albumin-bound drug containing cyclosporin A, characterized in that: The method comprises at least the following steps: S1. dissolving albumin in water to prepare an aqueous phase, wherein the albumin concentration ranges from 0.1% to 1%; S2. dissolving cyclosporin A or a derivative thereof in an organic solvent to prepare an organic phase, wherein the concentration of cyclosporin A or a derivative thereof ranges from 5 mg / mL to 40 mg / mL; S3. while the aqueous phase is ultrasonically treated using a probe, the organic phase is slowly added dropwise to form a colostrum solution, wherein the mass ratio of sporin A or its derivative to albumin is 1:1 to 10; S4. Transfer the colostrum to a high-pressure homogenizer and homogenize it under a constant pressure of 800-1200 bar until a translucent milky liquid is obtained. S5. The organic solvent is removed to obtain an albumin-bound drug solution containing cyclosporin A.

5. The method according to claim 4, characterized in that The organic solvent in step S2 includes at least one of ethanol, methanol, and chloroform.

6. The method according to claim 4, characterized in that The method for removing the organic solvent in step S5 includes at least one of reduced pressure distillation, dialysis, ultrafiltration, freeze drying, and air evaporation.

7. The method according to claim 4, characterized in that The method further comprises the step of pre-freezing or freeze-drying the obtained albumin-bound drug solution containing cyclosporin A for storage.

8. A composition, characterized in that The composition comprises the albumin-binding drug according to any one of claims 1 to 3 and additives acceptable in the medical field.

9. Use of the albumin-binding drug according to any one of claims 1 to 3 or the composition according to claim 8 in any of the following aspects: A1. Application in the preparation of products that promote tear secretion; A2. Application in the preparation of products for repairing corneal damage; A3. Application in the preparation of products for improving corneal epithelial cell structure; A4. Application in the preparation of anti-inflammatory products.

10. Use of the albumin-binding drug according to any one of claims 1 to 3 or the composition according to claim 8 in the preparation of a medicament for treating and / or treating inflammatory and / or autoimmune ophthalmic diseases and eyelid margin diseases.

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