Fusion polypeptide AJ007 and its use

Abstract

This application discloses the fusion polypeptide AJ007 and its use, and belongs to the art of ophthalmic drugs. This fusion polypeptide AJ007 is formed by linking a TAT membrane-permeable peptide to the tip of a polypeptide HM-1 molecule that inhibits neovascularization. The fusion polypeptide AJ007 was used in the preparation of polypeptide eye drops, and by selecting the formulation of this application, the fusion polypeptide AJ007 was prepared as an eye drop. In the prevention or treatment of wet age-related macular degeneration, the method of administering eye drops effectively avoids various serious side effects and low patient tolerance problems associated with intravitreal injection in clinical practice, and has the advantage of non-invasive treatment. Furthermore, in a laser-induced mouse wAMD model, it showed extremely good efficacy in improving abnormal hyperplasia of choroidal neovascularization, and possesses high clinical therapeutic potential.

Description

Technical Field

[0001] This application belongs to the technical field of ophthalmic drugs, specifically related to the fusion polypeptide AJ007 and its use, and more specifically related to the fusion polypeptide AJ007 and its use in the preparation of drugs for preventing or treating posterior segment neovascular diseases.

Background Art

[0002] With the progression of aging, the worsening of myopia, or the development of related metabolic diseases, when fundus diseases such as pathological myopia, age-related macular degeneration, and diabetic retinopathy occur, unnecessary neovascularization may form in the fundus. These neovascularizations invade the originally normal fundus tissues such as choroidal blood vessels and the photoreceptor cell layer of the retina, impair visual function, and may cause blindness in severe cases.

[0003] Age-related macular degeneration (AMD) is the most common cause of irreversible visual impairment, including dry (non-exudative or atrophic) and wet (neovascular or exudative) types. Among them, wet age-related macular degeneration (wAMD) is an important cause of low vision and blindness in humans, and its incidence is increasing year by year. Choroidal neovascularization (CNV) is the main pathological basis for impairing visual function and the most important cause. Therefore, there is a strong demand for the development of appropriate drugs to reduce the visual acuity decline of patients by improving CNV.

[0004] Vascular endothelial growth factor (VEGF) has already been shown to play a crucial role in the formation of CNVs (vascular vascular neovascularization). Therefore, currently, drugs used clinically for wet age-related macular degeneration (AMD) are all antibody drugs that target VEGF, such as ranibizumab, aflibercept, and convercept. While these drugs do improve visual acuity to some extent in patients with AMD, their large molecular weight prevents them from crossing the blood-retinal barrier and reaching the retina and choroid. Therefore, clinical treatment involves administering these drugs via intravitreal injection. However, intravitreal injections cause a variety of serious side effects, including intraocular hemorrhage, intraocular infection, retinal detachment, intraocular inflammation, cataracts, and elevated intraocular pressure, resulting in low patient adherence and tolerance. Therefore, there is a clinical need for more appropriate treatment regimens to avoid these serious side effects. Furthermore, while all of these antibody drugs directly neutralize VEGF, literature reports that VEGF plays a role in supplying nutrients to the retina and choroid. Long-term VEGF deficiency can lead to retinal and choroidal atrophy, potentially increasing the risk of vision impairment. Therefore, it is more appropriate to target VEGF receptors or other signaling pathways that suppress angiogenesis in the choroid.

[0005] In the applicant's prior Chinese invention patent (publication number CN104045718B), a multifunctional fusion polypeptide VI (HM-1) with the amino acid sequence Arg-Gly-Ala-Asp-Arg-Ala-Gly-Gly-Gly-Gly-Arg-Gly-Asp (SEQ ID NO: 1) is disclosed. This multifunctional fusion polypeptide VI has potent angiogenesis inhibitory activity, inhibiting angiogenesis mainly by suppressing the VEGFR2 and αvβ3 signaling pathways. Furthermore, it has been verified that this multifunctional fusion polypeptide VI can treat choroidal neovascularization in rats. However, since this multifunctional fusion polypeptide VI still needs to be administered by intravitreal injection, this method of administration is not considered to have good clinical adherence for patients and may cause other serious side effects.

[0006] Therefore, it is necessary to optimize this multifunctional fusion polypeptide VI by appropriate means to reduce side effects, improve patient adherence, and provide more appropriate clinical treatment regimens for patients with wet age-related macular degeneration. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] This application provides the fusion polypeptide AJ007 and its use to address problems such as the inability of conventional polypeptide drugs, such as multifunctional fusion polypeptide VI (HM-1), to cross the blood-retinal barrier, requiring administration by intravitreal injection, which is prone to causing serious side effects. This fusion polypeptide AJ007 is prepared by linking the cell membrane-permeable peptide TAT to the tip of the sequence of multifunctional fusion polypeptide VI (HM-1), which has potent angiogenesis inhibitory activity, thereby enhancing its ability to cross the blood-retinal barrier or sclera and reach the posterior segment of the eye. Furthermore, by using this fusion polypeptide AJ007 and its pharmaceutically acceptable salts and esters as polypeptide active pharmaceutical ingredients, and selecting appropriate auxiliary components, eye drops for the prevention and / or treatment of diseases such as wet age-related macular degeneration can be prepared, thereby not only enhancing the efficacy of fusion polypeptide AJ007 but also reducing side effects and facilitating clinical use. [Means for solving the problem]

[0008] To solve the above problems, the technical solutions adopted in this application are as follows:

[0009] This application provides a fusion polypeptide AJ007 comprising the amino acid sequence Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Arg-Gly-Ala-Asp-Arg-Ala-Gly-Gly-Gly-Gly-Arg-Gly-Asp (YGRKKRRQRRRRGADRAGGGGRGD, Sequence ID No. 2). By linking the cell membrane-permeable peptide TAT to the leading edge of the sequence of the multifunctional fusion polypeptide VI (HM-1), which the applicant previously disclosed has potent angiogenesis inhibitory activity, the ability to penetrate the blood-retinal barrier or sclera and reach the posterior segment of the eye is enhanced, and it becomes possible to penetrate the blood-retinal barrier or sclera and reach the choroid.

[0010] This application also provides the use of the above-mentioned fusion polypeptide AJ007 in the preparation of a drug for preventing or treating posterior segment neovascular disease, the drug being able to inhibit neovascularization.

[0011] Furthermore, the drugs for preventing or treating the above-mentioned posterior segment neovascular disease contain at least the fusion polypeptide AJ007 and / or a pharmaceutically acceptable carrier thereof.

[0012] Furthermore, the drugs for preventing or treating the above-mentioned posterior segment neovascular disease include eye drops containing the fusion polypeptide AJ007 and a pharmaceutically acceptable carrier thereof.

[0013] Furthermore, the posterior segment neovascular diseases mentioned above include one or more of the following: wet age-related macular degeneration (wAMD), retinal vein occlusion (RVO), diabetic retinopathy (DR), and retinopathy of prematurity (ROP). Diabetic retinopathy, retinal vein occlusion, and retinopathy of prematurity are all diseases that show a high correlation with abnormal neovascularization. Of these, Diabetic retinopathy (DR) is a chronic, progressive disease that worsens with the progression of diabetes and can manifest as retinal exudation, hemorrhage, macular edema, and retinal neovascularization. As the disease progresses to a certain extent, varying degrees of visual impairment occur, and in severe cases, it can lead to blindness. The main mechanism of DR is that a persistently high blood glucose environment impairs the blood-retinal barrier, increasing its permeability and causing leakage of microvessels. This leads to abnormal neovascularization and fibrous proliferation in the retina, ultimately altering the structure of retinal microvascular cells and eventually causing their complete disappearance. VEGF levels are closely related to the severity of DR, and anti-VEGF drug therapy can suppress or slow the progression of the disease and reduce visual loss.

[0014] Retinal vein occlusion (RVO) is a common retinal vascular disease caused by obstruction of blood flow in the retinal veins. The inability of the veins to return blood to the retina leads to exudation from the retinal vessels, resulting in macular edema and intraretinal hemorrhage, and ultimately, decreased vision. The pathogenesis of RVO is multifaceted, primarily involving changes in the vascular wall, blood rheology, and hemodynamics. There is no specific treatment for RVO, and current treatment methods cannot effectively reverse the pathological changes associated with retinal vein occlusion. On the other hand, treatment with anti-VEGF drugs can suppress macular edema and neovascularization caused by RVO, thus preserving the patient's vision.

[0015] Retinopathy of prematurity (ROP) is a disease of the retina of prematurity characterized by pathological neovascularization and associated fibrotic changes, and is a leading cause of childhood blindness worldwide. Its main pathological mechanism is abnormal hyperplasia of retinal blood vessels due to postnatal retinal vascular occlusion and hypoxia. Anti-VEGF therapy is a relatively good treatment option for ROP.

[0016] The pathological characteristics and mechanisms of onset of DR, RVO, and ROP share numerous similarities with wAMD, and it has already been demonstrated that anti-VEGF treatment can effectively control the disease progression of any of these three conditions. Therefore, AJ007 has relatively high potential to improve the progression of DR, RVO, and ROP by suppressing abnormal vascular formation in the retina.

[0017] Furthermore, the posterior segment neovascular diseases mentioned above include wet age-related macular degeneration.

[0018] This application also provides polypeptide eye drops for the prevention or treatment of posterior segment neovascular disease. These polypeptide eye drops comprise the above-mentioned fusion polypeptide AJ007 or a pharmaceutically acceptable salt form thereof, and auxiliary formulation components.

[0019] Furthermore, polypeptide eye drops for preventing or treating the above-mentioned posterior segment neovascular disease contain, per 100 mL: Fusion polypeptide AJ007 or its pharmaceutically acceptable salt form, 0-50 w / v%, 2-Hydroxypropyl-β-cyclodextrin 5-10 w / v%, Polysorbate 80 0.1~5 w / v%, Tyroxapole 0.05~5 w / v%, Poloxamer 407 0.05~10 w / v%, Sodium dihydrogen phosphate 0-1 w / v%, Preservatives 0-0.005 w / v%, Osmotic pressure adjusting agent 0-1 w / v%, which adjusts the osmotic pressure molar concentration to 250-350 mOsm / kg. Contains 0-1 w / v% of a pH adjuster to adjust the pH value to 6.0-8.0.

[0020] In this polypeptide eye drop solution, the fusion polypeptide AJ007 or a pharmaceutically acceptable salt form is used as the polypeptide active pharmaceutical ingredient, and the auxiliary compound is used as a carrier for the fusion polypeptide AJ007 or a pharmaceutically acceptable salt form. This effectively enhances the inclusion of the polypeptide active pharmaceutical ingredient, promotes the dissolution of the fusion polypeptide AJ007, improves the stability of the fusion polypeptide AJ007, and provides a higher efficacy in penetrating the blood-retinal barrier or sclera to reach the posterior segment of the eye. By linking the cell membrane-permeable peptide TAT to the leading edge of the sequence of the multifunctional fusion polypeptide VI (HM-1), which has potent angiogenesis inhibitory activity, and by optimizing two aspects of the auxiliary compound selected in this application, the fusion polypeptide AJ007, which has anti-neoangiogenic activity, can be delivered from the posterior segment retina to the choroid, thereby improving the CNV effect through the eye drop administration method.

[0021] Furthermore, polypeptide eye drops for preventing or treating the above-mentioned posterior segment neovascular disease contain, per 100 mL: Fusion polypeptide AJ007 or its pharmaceutically acceptable salt form 2 w / v%, 2-Hydroxypropyl-β-cyclodextrin 8.41 w / v%, Polysorbate 80 1 w / v%, Tyroxapole 0.1 w / v%, Poloxamer 407 2 w / v%, Sodium dihydrogen phosphate 0.142 w / v%, Preservative 0.005 w / v%, Osmotic pressure regulator (adjust the osmolarity to 276 mOsm / kg), pH regulator (adjust the pH value to 6.47).

[0022] Furthermore, the pharmaceutically acceptable salt forms of the above fusion polypeptide AJ007 include one or more of the forms of acetate, trifluoroacetate, hydrochloride, ammonium salt, sodium salt, pamoate, citrate, and salicylate. In addition, the pharmaceutically acceptable salt forms of the above fusion polypeptide AJ007 include the acetate form.

[0023] Furthermore, the above preservative includes one or more of benzalkonium chloride, benzalkonium bromide cationic surfactant, benzyl alcohol, trichloro t-butanol, and sorbic acid. In addition, the above preservative includes benzalkonium chloride at a ratio of 0.005 w / v%.

[0024] Furthermore, the above osmotic pressure regulator includes one or more of sodium chloride, boric acid, and glucose. In addition, the above osmotic pressure regulator includes sodium chloride at a ratio of 0.2 w / v%.

[0025] Furthermore, the above pH regulator includes one or more of phosphate buffer, borate buffer, hydrochloric acid, and sodium hydroxide. In addition, the above pH regulator includes sodium hydroxide at a ratio of 0.8 w / v%.

[0026] Furthermore, the above posterior segment neovascular diseases include one or more of diseases such as wet age-related macular degeneration, retinal vein occlusion, diabetic retinopathy, and retinopathy of prematurity.

[0027] Furthermore, the above posterior segment neovascular diseases include wet age-related macular degeneration.

[0028] This application also provides a method for preparing polypeptide eye drops for preventing or treating the above-mentioned posterior segment neovascular disease. This preparation method specifically (1) Preparation of auxiliary component solution: Add the prescribed amounts of sodium dihydrogen phosphate, benzalkonium chloride, polysorbate 80, poloxamer 407, and tyroxapole sequentially to ultrapure water, stir continuously until completely dissolved, filter through a 0.22 μm filter membrane, and prepare for use as solution A. (2) Preparation of 2-hydroxypropyl-β-cyclodextrin solution: Place solution A in a water bath stirring vessel and heat to 60°C, start stirring, add 2-hydroxypropyl-β-cyclodextrin in several batches, and after complete dispersion and dissolution, remove and cool to prepare for use as solution B. (3) Adjustment of pH and osmotic pressure: The pH value and osmotic pressure of solution B are detected, the pH and osmotic pressure are adjusted using a pH adjuster and an osmotic pressure adjuster, and the solution is filtered through a 0.22 μm filter membrane to prepare it for use as solution C. (4) Preparation of polypeptide eye drops: The preparation of polypeptide eye drops includes the steps of weighing the fusion polypeptide AJ007 or a pharmaceutically acceptable salt form thereof, adding it to solution C, and continuously shaking to completely disperse and dissolve the fusion polypeptide AJ007 or a pharmaceutically acceptable salt form thereof. [Effects of the Invention]

[0029] Compared to the prior art, the beneficial effects of this application are as follows:

[0030] (1) The fusion polypeptide AJ007 provided in this application enhances the ability to penetrate the blood-retinal barrier or sclera and reach the posterior segment of the eye by linking the cell membrane-permeable peptide TAT to the leading edge of the sequence of the multifunctional fusion polypeptide VI (HM-1), which has potent angiogenesis inhibitory activity and was previously disclosed by the applicant, and enables it to penetrate the blood-retinal barrier or sclera and reach the choroid. Furthermore, as can be seen from Example 3, after instilling the original multifunctional fusion polypeptide VI (HM-1), only active molecules of less than 10 ng / mL could reach the lesion target site of wAMD, i.e., the RPE / choroidal complex, whereas with fusion polypeptide AJ007, active molecules of 6291.38 ng / mL could reach the RPE / choroidal complex, thus significantly improving the ocular permeability of the original active molecules. In conventional technical solutions, polypeptide AT7 targeting VEGFR2 showed a 119-fold improvement in its ability to permeate endothelial cells after being linked to the transmembrane peptide TAT. In patent CN107129521A, bevacizumab, after being linked to the transmembrane peptide CPP, showed approximately a 10-fold improvement in its ability to penetrate the posterior segment of the eye after instillation. In patent CN114073774A, after being linked to the transmembrane peptide CC12, showed approximately an 8-fold improvement in its ability to penetrate the eye after instillation. In patent CN114073774A, after being linked to the transmembrane peptide 289WP, the concentration reaching the retina after instillation increased by approximately 100 times. In patent CN114668717A, after being linked to the transmembrane peptide PENE, the concentration reaching the retina after instillation increased by approximately 5 to 10 times. However, compared to HM-1, these conventional technological solutions showed approximately a 600-fold improvement in the posterior segment penetration of AJ007. The superiority of AJ007 is clear, and this superiority exceeds what was normally expected, demonstrating the specificity and creativity of this peptide.

[0031] (2) The polypeptide eye drops for preventing or treating posterior segment neovascular disease provided in this application are prepared by using fusion polypeptide AJ007 or a pharmaceutically acceptable salt form thereof as the polypeptide active pharmaceutical ingredient and selecting appropriate auxiliary components. By ligating the cell membrane permeable peptide TAT to the leading edge of the sequence of multifunctional fusion polypeptide VI (HM-1), which has potent angiogenesis inhibitory activity, and by optimizing two aspects of the auxiliary components selected in this application, a novel composition product is ultimately obtained, thereby differentiating this product from other peptide eye drops and giving it unique novelty. By delivering the fusion polypeptide AJ007, which has anti-neoangiogenic activity, between the posterior segment retina and choroid, the CNV effect can be improved by the eye drop administration method. Drug distribution to ocular tissue and efficacy experiments on laser-induced CNV in mice are compared with the original multifunctional fusion polypeptide VI (HM-1). Drug distribution experiments in ocular tissue demonstrate that the polypeptide ophthalmic solution of this application can deliver at least 100 times more active molecules to the lesion target site of wAMD, namely the RPE / choroidal complex, than multifunctional fusion polypeptide VI (HM-1). Efficacy experiments show that the polypeptide ophthalmic solution of this application has a far superior CNV leakage improvement effect compared to conventional multifunctional fusion polypeptide VI (HM-1), and is shown to be non-inferior to aflibercept, currently the best wAMD treatment drug in clinical practice. This is something that all other peptide ophthalmic solutions struggle to achieve. Technically, it demonstrates greater advancement and overcomes the shortcomings of conventional ophthalmic solutions in the treatment of fundus diseases where efficacy is limited. Therefore, it has relatively high clinical potential in the treatment of wAMD and is expected to change the current situation where wAMD patients must undergo treatment methods such as intravitreal injections, significantly improving patient adherence and providing wAMD patients with a treatment option that significantly improves adherence. [Brief explanation of the drawing]

[0032] [Figure 1] This figure shows the drug-time curve results for the original multifunctional fusion polypeptide VI (HM-1) in ophthalmic administration. [Figure 2] This figure shows the results of the drug-time curve when the polypeptide eye drop solution of this application is administered to the eye. [Figure 3] This is a schematic diagram of FFA imaging after administration of the polypeptide eye drop solution of this application to a mouse wAMD model. [Figure 4] This is a statistical graph of the CNV leakage area in FFA imaging after administration of the polypeptide eye drop solution of this application to a mouse wAMD model. [Figure 5] This is a schematic diagram of flat-mount staining of FITC-dextran after administration of the polypeptide eye drop solution of this application to a mouse wAMD model. [Figure 6] This is a statistical graph of CNV area obtained by flat-mount staining with FITC-dextran after administration of the polypeptide eye drop solution of this application to a mouse wAMD model. [Figure 7] This figure shows the drug-time curve results for the initial multifunctional fusion polypeptide VI (HM-1) in equimolar concentrations when administered as eye drops. [Figure 8] This figure shows the drug-time curve results for the initial multifunctional fusion polypeptide VI (HM-1) formulation at equimolar concentrations when administered as eye drops. [Figure 9] This figure shows the drug-time curve results for eye drops containing equimolar concentrations of the fusion polypeptide AJ007. [Figure 10] This figure shows the results of the drug-time curve when equimolar concentrations of the polypeptide eye drops of this application are administered to the eye. [Figure 11] This figure shows the drug-time curve results for intravitreal injection of the original multifunctional fusion polypeptide VI (HM-1) at equimolar concentrations. [Figure 12] This is a schematic diagram of FFA imaging after administering equimolar concentrations of various drug solutions to a mouse wAMD model. [Figure 13] This is a statistical graph of CNV leakage area in FFA imaging after administering equimolar concentrations of various drug solutions to a mouse wAMD model. [Figure 14]This is a schematic diagram of flat-mount staining of FITC-dextran after administration of equimolar concentrations of each drug solution to a mouse wAMD model. [Figure 15] This is a statistical graph of CNV area in flat-mount stained FITC-dextran samples after administration of equimolar concentrations of each drug solution to a mouse wAMD model. [Modes for carrying out the invention]

[0033] The present application will be further described below, along with specific examples.

[0034] Furthermore, terms such as "up," "down," "left," "right," and "center" used herein are used solely for the purpose of clarifying the explanation and are not intended to limit the scope of implementation. Any changes or adjustments to their relative relationships should be considered within the scope of implementation of this application, provided that they do not substantially alter the technical content.

[0035] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of this application. The term "and / or" as used herein includes any one or more and all combinations of the relevant enumerated items.

[0036] Unless otherwise specified in the examples, the procedures are carried out under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified by the manufacturer, the reagents and equipment used are all standard products available commercially.

[0037] As used herein, the term “about” is used to give flexibility and impreciseness to a given term, measure, or value. Those skilled in the art can readily determine the degree of flexibility to a particular variable. As used herein, the term "at least one of..." is synonymous with "one or more of...". For example, "at least one of A, B, and C" clearly includes A only, B only, C only, and any combination thereof.

[0038] In this specification, concentrations, quantities, and other numerical data may be presented in range format. Such range formatting is used solely for convenience and brevity, and should be interpreted flexibly to include not only the explicitly stated range limits, but also all individual numbers or subranges within the range, so that each number and subrange is clearly described. For example, a numerical range from approximately 1 to approximately 4.5 should be interpreted to include not only the explicitly stated limit values ​​from 1 to approximately 4.5, but also individual numbers (2, 3, 4, etc.) and subranges (1 to 3, 2 to 4, etc.). The same principle applies to describing only one numerical range, such as "less than approximately 4.5," which should be interpreted to include all the values ​​and ranges described above. Furthermore, such interpretation should apply regardless of the breadth of the described range or feature.

[0039] Example 1 In this example, the fusion polypeptide AJ007 is provided.

[0040] The amino acid sequence of fusion polypeptide AJ007 was Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Arg-Gly-Ala-Asp-Arg-Ala-Gly-Gly-Gly-Gly-Arg-Gly-Asp(YGRKKRRQRRRRGADRAGGGGRGD, Sequence ID No. 2). Of these, Arg-Gly-Ala-Asp-Arg-Ala-Gly-Gly-Gly-Gly-Arg-Gly-Asp(Sequence ID No. 1) was the multifunctional fusion polypeptide VI (HM-1) disclosed in the applicant's Chinese invention patent (publication number CN104045718B). This multifunctional fusion polypeptide VI has potent angiogenesis inhibitory activity and inhibited neovascularization mainly by suppressing the VEGFR2 and αvβ3 signaling pathways. Therefore, we were able to improve the formation and leakage of laser-induced choroidal neovascularization in mice.

[0041] Example 2 This embodiment provides the use of the fusion polypeptide AJ007 described in Example 1 in the preparation of a drug for preventing or treating posterior segment neovascular disease. This drug was a polypeptide eye drop for preventing or treating posterior segment neovascular disease. This embodiment also provides a polypeptide eye drop for preventing or treating posterior segment neovascular disease and a method for preparing the same.

[0042] The polypeptide eye drops for preventing or treating posterior segment neovascular disease provided in this embodiment contain, per 100 mL: Acetate form of fusion polypeptide AJ007, 2 w / v%, 2-Hydroxypropyl-β-cyclodextrin 8.41 w / v%, Polysorbate 80 1 w / v%, Tyroxapole 0.1 w / v%, Poloxamer 407 2w / v%, Sodium dihydrogen phosphate 0.142 w / v%, Preservative (benzalkonium chloride) 0.005 w / v%, Osmotic pressure adjusting agent (sodium chloride) 0.2 w / v%, adjusting the osmotic pressure molar concentration to 276 mOsm / kg. It contained 8% w / v of a pH adjuster (sodium hydroxide) to adjust the pH value to 6.47.

[0043] The method for preparing polypeptide eye drops for preventing or treating posterior segment neovascular disease provided in this embodiment specifically included the following steps:

[0044] (1) Preparation of auxiliary component solution: Sodium dihydrogen phosphate, benzalkonium chloride, polysorbate 80, poloxamer 407, and tyroxapol in the prescribed amounts were added sequentially to 100 mL of ultrapure water, and the mixture was continuously stirred until completely dissolved. The mixture was then filtered through a 0.22 μm filter membrane to obtain solution A, which would be used later.

[0045] (2) Preparation of 2-hydroxypropyl-β-cyclodextrin solution: 100 mL of solution A was placed in a water bath stirring vessel and heated to 60°C. Stirring was started, and 2-hydroxypropyl-β-cyclodextrin was added in several batches. After complete dispersion and dissolution, it was removed and cooled to obtain solution B, which would be used later.

[0046] (3) pH and osmotic pressure adjustment: The pH and osmotic pressure of solution B were detected, and the pH was adjusted to 7.5 and the osmotic pressure to 250 using a pH adjuster and an osmotic pressure adjuster. The solution was then filtered through a 0.22 μm filter membrane to obtain solution C, which would be used later.

[0047] (4) Preparation of polypeptide ophthalmic solution: 2000 mg of the acetate form of fusion polypeptide AJ007 was weighed and added to 100 mL of solution C. The solution was continuously shaken to completely disperse and dissolve the fusion polypeptide AJ007 or its pharmaceutically acceptable salt form, thereby obtaining polypeptide ophthalmic solution with a concentration of 20 mg / mL. The results showed an osmotic molar concentration of 276 mOsm / kg and a pH of 6.47.

[0048] Example 3 This example provides a comparative experiment on the distribution of polypeptide eye drops (AJ007 eye drops) from Example 2 and multifunctional fusion polypeptide VI (HM-1) in ocular tissue. Specifically, the results were as follows:

[0049] Drug solution: Polypeptide ophthalmic solution containing 20 mg / mL of fusion polypeptide AJ007 (AJ007 ophthalmic solution), and multifunctional fusion polypeptide VI (HM-1) solution containing 20 mg / mL of multifunctional fusion polypeptide VI (HM-1).

[0050] Animal grouping: 24 female C57BL / 6J mice were randomly divided into six time point groups of two mice each at 0h, 1h, 3h, 7h, 12h, and 24h.

[0051] Experimental Procedure: In all eye drop groups, 5 μL of the corresponding drug solution was administered to each eye by instilling it once into both eyes of the mice. At six time points (0h, 1h, 3h, 7h, 12h, and 24h) after administration, the eyeballs of the mice in the corresponding groups were extracted and blood was collected. Immediately after obtaining the whole blood, it was centrifuged at 6000 rpm for 5 minutes, and the supernatant was aspirated and stored as the test sample. In addition, three tissue regions—vitreous humor, retina, and RPE / choroidal complex—were separated from the eyeball under a stereomicroscope. After dilution with PBS at a ratio of 1:10 and polishing, the tissues were centrifuged at 12000 rpm for 5 minutes, and the supernatant was aspirated as the test sample. Subsequently, the concentration of fusion polypeptide AJ007 or multifunctional fusion polypeptide VI (HM-1) in each tissue was measured by ELISA. The ELISA method was as follows.

[0052] (1) Coating: HM-1 was diluted to the specified concentration of 1 μg / mL with coating buffer (CBS), 100 μL was added to each well of the ELISA plate, and left overnight at 4°C.

[0053] (2) Plate washing: Remove the coating buffer, add 265 μL of PBST washing solution to each well, wash for 3 minutes three times, and then tap to dry.

[0054] (3) Blocking: 200 μL of 1% BSA blocking solution was added to each well, and the plates were left in a 37°C water bath for 1.5 hours. The plate washing process was repeated, and the plates were tapped to dry.

[0055] (4) Addition of test sample and antibody: 100 μL of the test sample was added to the ELISA plate, followed by 100 μL of HM-1 monoclonal antibody dilution at the specified concentration. Two to three repeat wells were created, and the plate was shaken uniformly for 2 minutes in a 37°C shaker. Then, it was transferred to a 37°C water bath and left for 2 hours. After sufficient reaction, the plate was removed, and the plate washing process was repeated.

[0056] (5) Reaction with enzyme-labeled secondary antibody: 100 μL of HRP enzyme-labeled goat anti-mouse IgG at a predetermined concentration was added to each well and incubated at 37°C for 1.5 hours. The plate washing process was repeated and the plates were tapped to dry.

[0057] (6) Addition of substrate: 100 μL of TMB substrate colorant was added to each well and allowed to develop color in the dark at room temperature for the specified time.

[0058] (7) Termination: The reaction was stopped by adding 50 μL of stop solution to each well.

[0059] (8) Detection: The OD450nm value of each well was read using an ELISA reader.

[0060] Experimental results and conclusions: Comparing the drug-time curves (Figures 1 and 2) for HM-1 eye drops and AJ007 eye drops, it was found that after instilling HM-1, only a small number of active molecules could reach the lesion target site of wAMD, i.e., the RPE / choroidal complex, and the maintenance time was relatively short. However, when the same concentration of AJ007 eye drops was instilled, at least 100 times more active molecules could reach the RPE / choroidal complex, and a considerable number of active molecules were still present at this site after 24 hours, significantly improving bioavailability. When multifunctional fusion polypeptide VI (HM-1) was instilled, it could not cross the blood-retinal barrier and reach the RPE / choroidal complex, and therefore could not achieve the therapeutic effect of improving CNV leakage. On the other hand, by adding TAT peptide, it was possible to promote the arrival of more active molecules at the RPE / choroidal complex. While the improvement in reaching the posterior orbit of typical membrane-permeable peptides when linked to low or high molecular weight molecules is only a few times (CN107129521A, CN114073774A, CN114668717A), this study showed an extremely significant improvement.

[0061] Example 4 This example provides a comparative experiment on the efficacy of the polypeptide eye drop solution (AJ007 eye drop solution) from Example 2 and the multifunctional fusion polypeptide VI (HM-1). Specifically, the results were as follows:

[0062] Animal grouping: 56 female C57BL / 6J mice were randomly divided into four groups: a complete blank group, a control group consisting of a model + solvent (a control solution containing equal amounts of all auxiliary components but without the fusion polypeptide AJ007), a model + intravitreal injection of the positive drug aflibercept (10 mg / mL), a model + low-dose HM-1 (20 mg / mL), a model + high-dose HM-1 (80 mg / mL), a model + low-dose AJ007 eye drops (20 mg / mL), a model + medium-dose AJ007 eye drops (40 mg / mL), and a model + high-dose AJ007 eye drops (80 mg / mL), with 7 mice in each group.

[0063] Experimental Procedure: First, the pupils of the mice were dilated using a complex tropicamide eye drop solution, and then anesthetized by intraperitoneal injection of a 4% chloral hydrate solution (0.2 mL per 20 g of mouse body weight). After anesthesia, sodium hyaluronate gel was applied to protect the eye, and a coverslip was attached. A left eye laser model was created using a 532 nm argon ion laser with an output of 120 mW, a burst time of 0.01 s, and a spot size of 100 μm. Medication was started on the day the model was created. Each group received medication only in the left eye using a different administration method. In the eye drop group, mice received eye drops five times daily at one-hour intervals, with 5 μL of the corresponding drug solution administered each time, for seven days. In the intravitreal injection group, mice were anesthetized on the day the model was created and then administered by intravitreal injection, with 1 μL of the corresponding drug solution injected into each mouse using a micro-syringe. After creating the model, a fundus fluorescein angiography (FFA) examination was performed once on day 3 after model creation to evaluate the level of CNV leakage. On day 7 after model creation, the mice were euthanized, and FITC-dextran was injected into the ventricles. Changes in CNV area were then measured by flat-mount fluorescence staining of the RPE-choroidal / scleral complex.

[0064] Experimental Results and Conclusions: As shown by the FFA test results, neither low-dose nor high-dose administration of HM-1 significantly improved laser-induced CNV leakage. On the other hand, low-dose administration of AJ007 eye drops significantly improved CNV leakage and was clearly superior to both low-dose and high-dose HM-1. Furthermore, its effect was superior to that of medium-dose and high-dose AJ007 eye drops, achieving efficacy equivalent to that of the positive drug aflibercept administered by intravitreous injection (Figures 3 and 4). As shown by the flat-mount staining results after FITC-dextran injection, low-dose HM-1 could not reduce the laser-induced CNV area, while high-dose HM-1 could partially reduce the CNV area. However, low-dose AJ007 eye drops significantly reduced the CNV area and was significantly superior to high-dose HM-1. Furthermore, its efficacy was better than that of medium- and high-dose AJ007 ophthalmic solutions, and its effectiveness was even slightly superior to that of aflibercept administered by intravitreous injection (Figures 5 and 6). Combined with the results of FFA testing and flat-mount fluorescence staining after FITC-dextran injection, 20 mg / mL of AJ007 ophthalmic solution significantly improved neovascularization in the mouse wAMD model, far surpassing the efficacy of low- and high-dose HM-1. Its efficacy in this typical wAMD model was found to be equivalent to, or slightly superior to, that of aflibercept administered by intravitreous injection, which is currently the best treatment method for wAMD in clinical practice.

[0065] Example 5 This example provides comparative experiments on the distribution of ocular tissue after instillation of the polypeptide ophthalmic solution of Example 2 (AJ007 ophthalmic solution), equimolar concentration of multifunctional fusion polypeptide VI (HM-1) ophthalmic solution, multifunctional fusion polypeptide VI preparation (HM-1 preparation, polypeptide is HM-1, other auxiliary components are the same as in AJ007 ophthalmic solution), and fusion polypeptide AJ007 preparation (TAT-HM-1, polypeptide is AJ007, solvent is pure water), as well as after intravitreal injection of multifunctional fusion polypeptide VI (HM-1). Specifically, the results were as follows.

[0066] Animal grouping: 70 female C57BL / 6J mice were randomly divided into seven time point groups of two mice each at 0h, 10min, 1h, 3h, 7h, 12h, and 24h.

[0067] Experimental Procedure: In the eye drop group, all mice received a single eye drop instillation, administering 5 μL of the corresponding drug solution to each eye. In contrast, in the HM-1 intravitreal injection group, 5 μL of the corresponding drug solution was injected into each eye. At seven time points (0h, 10min, 1h, 3h, 7h, 12h, and 24h) after administration, eyeballs were extracted from the corresponding group of mice, and blood was collected. Immediately after obtaining whole blood, the cells were centrifuged at 6000 rpm for 5 minutes, and the supernatant was aspirated and stored as the test sample. Three tissue regions—vitreous humor, retina, and RPE / choroidal complex—were separated from the eyeball under a stereomicroscope. After dilution with PBS at a 1:10 ratio, the cells were polished, centrifuged at 12000 rpm for 5 minutes, and the supernatant was aspirated as the test sample. Subsequently, the concentration of HM-1 or fusion polypeptide AJ007 in each tissue was measured by ELISA.

[0068] Experimental results and conclusions: As can be seen from the drug-time curves after instillation of AJ007 eye drops, equimolar concentrations of HM-1, HM-1 preparations, TAT-HM-1, and HM-1 after intravitreal injection (Figures 7, 8, 9, 10, and 11), whether it was HM-1 or an HM-1 preparation, only active molecules at concentrations of less than 10 ng / mL could reach the lesion target site of wAMD, i.e., the RPE / choroidal complex, after instillation, and the maintenance time was very short. However, when TAT was linked to HM-1, 843.28 ng / mL of active molecules were able to reach the RPE / choroidal complex, and although the concentration was much higher than that of the HM-1 group and the HM-1 preparation group, the maintenance time was also relatively short. When HM-1 was injected into the vitreous humor, approximately 5729.7 ng / mL of active molecules reached the RPE / choroidal complex. Although the concentration was higher than in the TAT-HM-1 group, the maintenance time was also relatively short. In contrast, when AJ007 eye drops were instilled, approximately 6291.38 ng / mL of active molecules reached the RPE / choroidal complex. This was significantly higher than in the HM-1 group, the HM-1 formulation group, and the TAT-HM-1 eye drop group, and slightly higher than the HM-1 intravitreal injection group. Furthermore, the maintenance time was longer than in the HM-1 intravitreal injection group. This indicates that the optimized and improved AJ007 eye drops allowed a large amount of active molecules to reach the RPE / choroidal complex, providing pharmacokinetic data that supports its effectiveness in effectively improving angiogenesis abnormalities in the choroid.

[0069] Example 6 This example provides a comparative experiment on the efficacy of the polypeptide eye drops of Example 2 (AJ007 eye drops), equimolar concentrations of multifunctional fusion polypeptide VI (HM-1), multifunctional fusion polypeptide VI formulation (HM-1 formulation), fusion polypeptide AJ007 (TAT-HM-1) after eye drops, and multifunctional fusion polypeptide VI (HM-1) after intravitreal injection. Specifically, the results were as follows.

[0070] Animal grouping: 42 female C57BL / 6J mice were randomly divided into four groups: model + solvent control group, model + positive drug aflibercept intravitreal injection (10 mg / mL) group, model + HM-1 (8.76 mg / mL) group, model + HM-1 preparation (8.76 mg / mL) group, model + TAT-HM-1 (20 mg / mL) group, model + AJ007 eye drops (20 mg / mL) group, and model + HM-1 intravitreal injection (8.76 mg / mL), with 6 mice in each group.

[0071] Experimental procedure: First, the pupils of the mice were dilated using a complex tropicamide eye drop solution, and then anesthetized by intraperitoneal injection of a 4% chloral hydrate solution (0.2 mL per 20 g of mouse body weight). After anesthesia, sodium hyaluronate gel was applied to protect the eye, and a coverslip was attached. A left eye laser model was created using a 532 nm argon ion laser with an output of 120 mW, a burst time of 0.01 s, and a spot size of 100 μm. Medication was started on the day the model was created. Each group received medication only in the left eye using a different administration method. In the eye drop group, 5 μL of the corresponding drug solution was dropped into the eye five times daily at one-hour intervals, and this continued for seven days. In the intravitreal injection group, mice were anesthetized on the day the model was created and administered the drug by intravitreal injection. Each mouse received the corresponding drug solution using a micro-syringe (1 μL injected into each eye in the aflibercept group, and 5 μL injected into each eye in the HM-1 group). After the model was created, a fundus fluorescein angiography (FFA) examination was performed once on the third day after the model was created using a fundus fluorescein system to evaluate the CNV leakage level. On the seventh day after the model was created, the mice were euthanized, and FITC-dextran was injected into the ventricle. Changes in CNV area were then measured by flat-mount fluorescence staining of the RPE-choroidal / scleral complex.

[0072] Experimental Results and Conclusions: As shown by the FFA test results, none of the eye drops of HM-1, HM-1 preparations, or TAT-HM-1 significantly improved laser-induced CNV leakage. However, both intravitreal injection of HM-1 and eye drops of AJ007 showed significant improvement in CNV leakage, achieving efficacy equivalent to that of the positive drug aflibercept administered by intravitreal injection (Figures 12 and 13). Of these, the efficacy of AJ007 eye drops after administration was slightly superior to that of the positive drug aflibercept administered by intravitreal injection. Flat-mount staining results after FITC-dextran injection showed that HM-1, HM-1 preparations, and TAT-HM-1 eye drops were unable to reduce laser-induced CNV area. However, both intravitreal injection of HM-1 and instillation of AJ007 eye drops significantly reduced CNV area, and their efficacy was slightly superior to that of the positive drug aflibercept administered intravitreally (Figures 14, 15). As can be seen in conjunction with the results of FFA testing and flat-mount fluorescence staining after FITC-dextran injection, HM-1, HM-1 formulations, or HM-1 linked to TAT did not improve angiogenesis abnormalities in the mouse wAMD model. However, when AJ007 ophthalmic solution, obtained by linking HM-1 to TAT and then formulation, was administered as eye drops, its efficacy against this typical wAMD model was equivalent to, or slightly better than, equimolar HM-1 administered by intravitreous injection. It was also equivalent to, or slightly better than, aflibercept administered by intravitreous injection, which is currently the best clinical treatment method for wAMD. Considering that patient adherence to the eye drop method was significantly better than that of the intravitreous injection method, the polypeptide eye drops of the present invention have relatively good clinical potential, providing a better treatment option for wAMD patients and generating tangible economic benefits.

[0073] Based on the results of the above examples, this application successfully optimized and improved the molecule by ligating the cell membrane-permeable peptide TAT to the tip of the sequence of multifunctional fusion polypeptide VI (HM-1), which has relatively potent angiogenesis inhibitory activity, and preparing an ophthalmic solution by selecting auxiliary components. As shown by the ophthalmic pharmacokinetic and efficacy results in a mouse laser-induced AMD model, AJ007 ophthalmic solution significantly improved tissue concentration and efficacy in the retina and choroid compared to the original HM-1 molecule. Its efficacy was slightly better than aflibercept, currently the best drug for wAMD in clinical practice, achieving an overall effect of 1+1>2. The efficacy of this ophthalmic solution was significantly better than the efficacy of ophthalmic solutions with membrane-permeable peptides ligated in other references. Furthermore, considering that aflibercept is administered via the invasive method of intravitreal injection, the non-invasive method of administering this polypeptide eye drop avoids the various serious side effects associated with intravitreal injection and offers greater clinical therapeutic potential, making it safer and more convenient for patients.

Claims

1. Fusion polypeptide AJ007, characterized in that the amino acid sequence of the fusion polypeptide is shown in SEQ ID NO:

2.

2. Use of the fusion polypeptide AJ007 according to claim 1 in the preparation of a drug for preventing or treating posterior segment neovascular disease.

3. The use according to claim 2, characterized in that the posterior segment neovascular disease includes one or more of the following diseases: wet age-related macular degeneration, retinal vein occlusion, diabetic retinopathy, and retinopathy of prematurity.

4. The use according to claim 3, characterized in that the aforementioned posterior segment neovascular disease includes wet age-related macular degeneration.

5. The use according to any one of claims 2 to 4, characterized in that the drug for preventing or treating posterior segment neovascular disease contains at least fusion polypeptide AJ007 and / or a pharmaceutically acceptable carrier thereof.

6. A polypeptide eye drop for preventing or treating posterior segment neovascular disease, characterized by comprising the fusion polypeptide AJ007 described in claim 1 or a pharmaceutically acceptable salt form thereof, and auxiliary formulation components.

7. The polypeptide eye drops contain, per 100 mL, Fusion polypeptide AJ007 or its pharmaceutically acceptable salt form, 0-50 w / v%, 2-hydroxypropyl-β-cyclodextrin 5-10 w / v%, Polysorbate 80 0.1-5 w / v%, Tyroxapole 0.05-5 w / v%, Poloxamer 407 0.05-10 w / v%, Sodium dihydrogen phosphate 0-1 w / v%, Preservatives 0-0.005 w / v%, Osmotic pressure adjusting agent 0-1 w / v%, which adjusts the osmotic pressure molar concentration to 250-350 mOsm / kg. A polypeptide eye drop for preventing or treating posterior segment neovascular disease according to claim 6, characterized by containing 0 to 1 w / v% of a pH adjusting agent that adjusts the pH value to 6.0 to 8.

0.

8. The pharmaceutically acceptable salt forms of the fusion polypeptide AJ007 include one or more of the forms of acetate, trifluoroacetate, hydrochloride, ammonium salt, sodium salt, pamoate, citrate, and salicylate, and / or The preservatives include one or more of benzalkonium chloride, benzalkonium bromide cationic surfactant, benzyl alcohol, trichloro-t-butanol, and / or sorbic acid, and / or The osmotic pressure adjusting agent includes one or more of sodium chloride, boric acid, and / or glucose. The polypeptide eye drop for preventing or treating posterior segment neovascular disease according to claim 7, characterized in that the pH adjusting agent includes one or more of phosphate buffer, borate buffer, hydrochloric acid, and sodium hydroxide.

9. The polypeptide eye drop for preventing or treating posterior segment neovascular disease according to claim 8, characterized in that the pharmaceutically acceptable salt form of the fusion polypeptide AJ007 includes an acetate form, the preservative includes benzalkonium chloride, the osmotic pressure regulator includes sodium chloride, and the pH regulator includes sodium hydroxide.

10. A method for preparing polypeptide eye drops for preventing or treating posterior segment neovascular disease according to any one of claims 6 to 9, (1) Preparation of auxiliary component solution: Add the prescribed amounts of sodium dihydrogen phosphate, benzalkonium chloride, polysorbate 80, poloxamer 407, and tyroxapole sequentially to ultrapure water, stir continuously until completely dissolved, filter through a 0.22 μm filter membrane, and prepare for use as solution A. (2) Preparation of 2-hydroxypropyl-β-cyclodextrin solution: Place solution A in a water bath stirring vessel and heat to 60°C, start stirring, add 2-hydroxypropyl-β-cyclodextrin in several batches, and after it is completely dispersed and dissolved, remove and cool to prepare for use as solution B, (3) Adjustment of pH and osmotic pressure: The pH value and osmotic pressure of solution B are detected, the pH and osmotic pressure are adjusted using a pH adjuster and an osmotic pressure adjuster, and the solution is filtered through a 0.22 μm filter membrane to prepare it for use as solution C. (4) Preparation of polypeptide eye drops: A method comprising the steps of weighing fusion polypeptide AJ007 or a pharmaceutically acceptable salt form thereof, adding it to solution C, and continuously shaking to completely disperse and dissolve the fusion polypeptide AJ007 or a pharmaceutically acceptable salt form thereof, thereby obtaining polypeptide eye drops.

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