Fusion polypeptide AJ007 and use thereof

Abstract

A fusion polypeptide AJ007 and a use thereof. The fusion polypeptide AJ007 is formed by linking a TAT cell-penetrating peptide to the front end of a polypeptide HM-1 molecule inhibiting neovascularization. The fusion polypeptide AJ007 is prepared into eye drops by means of a specific prescription, and when wet age-related macular degeneration is prevented or treated, the eye drop administration manner can effectively avoid various serious side effects caused by intravitreal injection in clinic and poor patient tolerance, has the advantage of non-invasive treatment, shows an extremely good efficacy in improving abnormal hyperplasia of choroid neovascularization in a laser-induced mouse wAMD model, and has good clinical treatment potential.

Description

A fusion polypeptide AJ007 and its application Technical Field

[0001] The present application belongs to the field of ophthalmic drug technology, and specifically relates to a fusion polypeptide AJ007 and its application, and more specifically to a fusion polypeptide AJ007 and its application in the preparation of a drug for preventing or treating posterior ocular neovascular diseases. Background Art

[0002] With 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 blood vessels will form in the fundus. These new blood vessels can invade the choroidal blood vessels, retinal photoreceptor cells, and other normal fundus tissues, impairing visual function and even leading to blindness in severe cases.

[0003] Age-related macular degeneration (AMD) is the most common cause of irreversible vision loss. It includes both dry (non-exudative or atrophic) and wet (neovascular or exudative) forms. Wet AMD (wAMD) is a major cause of low vision and blindness, and its incidence is increasing annually. Choroidal neovascularization (CNV) is the primary pathological basis and primary cause of AMD's visual impairment. Therefore, it is crucial to develop appropriate drugs to alleviate vision loss by ameliorating CNV.

[0004] Vascular endothelial growth factor (VEGF) has been shown to play a key role in CNV formation. Therefore, current clinical treatments for wet age-related macular degeneration are antibodies targeting VEGF, such as ranibizumab, aflibercept, and conbercept. These drugs have some potential to improve vision in patients with wet age-related macular degeneration. However, these antibodies are large in molecular weight and cannot penetrate the blood-ocular barrier to reach the retina and choroid. Therefore, they are typically administered via intravitreal injection. However, intravitreal injections can cause multiple serious side effects, including intraocular hemorrhage, infection, retinal detachment, inflammation, cataracts, and increased intraocular pressure. Patient compliance and tolerance are poor, leading to the need for more appropriate treatment options to avoid these serious side effects. In addition, these antibody drugs directly neutralize VEGF. However, there are literature reports that VEGF has a nutritional effect on the retina and choroid. Long-term VEGF deficiency can easily lead to atrophy of the retina and choroid, which in turn increases the risk of vision impairment. Therefore, it is more appropriate to target VEGF receptors or other signaling pathways that inhibit choroidal angiogenesis for treatment.

[0005] The applicant's prior Chinese invention patent, with authorization publication number CN104045718B, discloses 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). This multifunctional fusion polypeptide VI has a strong ability to inhibit neovascularization, primarily by inhibiting VEGFR2 and αvβ3 signaling pathways, thereby inhibiting angiogenesis. The results further validated that this multifunctional fusion polypeptide VI can treat choroidal neovascularization in rats. However, this multifunctional fusion polypeptide VI still requires intravitreal injection, a method of administration that is not clinically effective for patient compliance and can also lead to other serious side effects.

[0006] Therefore, it is necessary to optimize the multifunctional fusion polypeptide VI through appropriate means, reduce side effects, improve patient compliance, and provide a more suitable clinical treatment plan for patients with wet age-related macular degeneration.

[0007] Summary of the Invention

[0008] 1. Problem to be solved

[0009] The present application is directed to the problem that polypeptide drugs in the prior art, such as multifunctional fusion polypeptide VI (HM-1), cannot penetrate the blood-ocular barrier and must be administered through intravitreal injection, which is prone to cause serious side effects. A fusion polypeptide AJ007 and its application are provided. The fusion polypeptide AJ007 is prepared by connecting the cell-penetrating peptide TAT to the front end of the multifunctional fusion polypeptide VI (HM-1) sequence with strong neovascularization inhibitory ability, thereby enhancing its ability to penetrate the blood-ocular barrier or sclera to reach the posterior segment of the eye. At the same time, the fusion polypeptide AJ007 and its pharmaceutically acceptable salts, esters, etc. are used as polypeptide raw materials, and suitable excipients are selected to prepare eye drops for the prevention and / or treatment of diseases such as wet age-related macular degeneration, which not only increases the efficacy of the fusion polypeptide AJ007, but also reduces side effects and is convenient for clinical use.

[0010] 2. Technical solution

[0011] In order to solve the above problems, the technical solutions adopted in this application are as follows:

[0012] The present application provides a fusion polypeptide AJ007, which comprises the following amino acid sequence: Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Arg-Gly-Ala-Asp-Arg-Ala-Gly-Gly-Gly-Gly-Gly-Arg-Gly-Asp (YGRKKRRQRRRRGADRAGGGGRGD, SEQ ID NO: 2). By connecting the cell-penetrating peptide TAT to the front end of the multifunctional fusion polypeptide VI (HM-1) sequence previously disclosed by the applicant with strong neovascularization inhibitory ability, the fusion polypeptide AJ007 has enhanced its ability to penetrate the blood-ocular barrier or the sclera to reach the posterior segment of the eye, and can penetrate the blood-ocular barrier or the sclera to reach the choroid.

[0013] The present application also provides the use of the above-mentioned fusion polypeptide AJ007 in the preparation of a drug for preventing or treating posterior ocular neovascular diseases, which can inhibit neovascularization.

[0014] Furthermore, the above-mentioned drug for preventing or treating posterior ocular neovascular diseases contains at least the fusion polypeptide AJ007 and / or a pharmaceutically acceptable carrier thereof.

[0015] Furthermore, the above-mentioned drug for preventing or treating posterior ocular neovascular diseases includes: eye drops containing the fusion polypeptide AJ007 and a pharmaceutically acceptable carrier thereof.

[0016] Furthermore, the above-mentioned posterior segment neovascular diseases include one or more of wet age-related macular degeneration (wAMD), retinal vein occlusion (RVO), diabetic retinopathy (DR), retinopathy of prematurity (ROP), etc. Diabetic retinopathy, retinal vein occlusion, and retinopathy of prematurity are all diseases that are highly correlated with abnormal angiogenesis, among which:

[0017] Diabetic retinopathy (DR) is a chronic, progressive disease that worsens with the progression of diabetes. It can manifest as retinal exudates, hemorrhages, macular edema, and retinal neovascularization. When the disease progresses to a certain extent, varying degrees of vision loss begin to occur, and in severe cases, blindness can occur. The main pathogenesis of DR is that persistent hyperglycemia damages the blood-retinal barrier, increasing permeability and microvascular permeability, leading to abnormal retinal neovascularization and fibrosis, ultimately altering or even completely losing the structure of retinal microvascular cells. VEGF levels are closely related to the severity of DR, and anti-VEGF drug therapy can control or delay disease progression and reduce vision loss.

[0018] Retinal vein occlusion (RVO) is a common retinal vascular disease caused by blockage of retinal venous blood flow. Its primary manifestation is the inability of the veins to drain retinal blood, leading to retinal blood vessel exudation, macular edema, intraretinal hemorrhage, and decreased vision. RVO has multiple pathogenesis mechanisms, primarily alterations in vascular walls, blood rheology, and hemodynamics. There is no specific treatment for RVO, and current treatments are ineffective in reversing the pathological changes of RVO. However, anti-VEGF drugs can inhibit RVO-induced macular edema and neovascularization, thereby maintaining the patient's vision.

[0019] Retinopathy of prematurity (ROP) is a fundus disease of premature infants characterized by pathological neovascularization and fibrotic changes. It is the leading cause of death in children worldwide. The main pathological mechanism is abnormal retinal vascular proliferation caused by postnatal retinal vascular occlusion and hypoxia. Anti-VEGF therapy is a promising treatment for ROP.

[0020] The pathological manifestations and pathogenesis of DR, RVO, and ROP share many similarities with wAMD, and anti-VEGF therapy has been shown to effectively control the disease in all of these conditions. Therefore, AJ007 has great potential to improve the course of DR, RVO, and ROP by inhibiting abnormal retinal angiogenesis.

[0021] Furthermore, the above-mentioned posterior segment neovascular disease includes: wet age-related macular degeneration.

[0022] The present application also provides a polypeptide eye drop for preventing or treating posterior ocular neovascular diseases, which comprises the above-mentioned fusion polypeptide AJ007 or a pharmaceutically acceptable salt form thereof, and excipients.

[0023] Furthermore, the polypeptide eye drops for preventing or treating posterior ocular neovascular diseases comprise the following components per 100 mL:

[0024] Fusion polypeptide AJ007 or a pharmaceutically acceptable salt thereof, 0-50% w / v;

[0025] 2-Hydroxypropyl-β-cyclodextrin, 5-10% w / v;

[0026] Polysorbate 80, 0.1-5% w / v;

[0027] Tyloxapol, 0.05-5% w / v;

[0028] Poloxamer 407, 0.05-10% w / v;

[0029] Sodium dihydrogen phosphate, 0-1% w / v;

[0030] Preservatives, 0-0.005% w / v;

[0031] Osmotic pressure regulator, 0-1% w / v, adjusts the osmotic pressure molar concentration to 250-350mOsm / kg;

[0032] pH adjuster, 0-1% w / v, adjusts pH to 6.0-8.0;

[0033] In the polypeptide eye drops, the fusion polypeptide AJ007 or a pharmaceutically acceptable salt thereof is used as the polypeptide API, and the excipient combination serves as a carrier of the fusion polypeptide AJ007 or a pharmaceutically acceptable salt thereof, which can effectively increase the inclusion of the polypeptide API, promote the dissolution of the fusion polypeptide AJ007, enhance the stability of the fusion polypeptide AJ007, and enable it to better penetrate the blood-ocular barrier or sclera to reach the posterior segment of the eye. By optimizing the two aspects of connecting the cell-penetrating peptide TAT to the front end of the multifunctional fusion polypeptide VI (HM-1) sequence with strong neovascularization inhibitory ability and the excipient combination selected in this application, the fusion polypeptide AJ007 with anti-angiogenesis function is delivered to the retina to the choroid in the posterior segment of the eye, thereby achieving the effect of improving CNV by eye drop administration.

[0034] Furthermore, the polypeptide eye drops for preventing or treating posterior ocular neovascular diseases comprise the following components per 100 mL:

[0035] Fusion polypeptide AJ007 or a pharmaceutically acceptable salt thereof, 2% w / v;

[0036] 2-Hydroxypropyl-β-cyclodextrin, 8.41% w / v;

[0037] Polysorbate 80, 1% w / v;

[0038] Tyloxapol, 0.1% w / v;

[0039] Poloxamer 407, 2% w / v;

[0040] Sodium dihydrogen phosphate, 0.142% w / v;

[0041] Preservative, 0.005% w / v;

[0042] Osmotic pressure regulator, adjust the osmotic pressure molar concentration to 276mOsm / kg;

[0043] pH regulator, adjust the pH to 6.47.

[0044] Furthermore, pharmaceutically acceptable salt forms of the fusion polypeptide AJ007 include one or more of acetate, trifluoroacetate, hydrochloride, ammonium salt, sodium salt, pamoate, citrate, and salicylate. Furthermore, pharmaceutically acceptable salt forms of the fusion polypeptide AJ007 include acetate.

[0045] Furthermore, the preservative includes one or more of benzalkonium chloride, benzalkonium bromide cationic surfactant, benzyl alcohol, chlorobutanol, and sorbic acid. Furthermore, the preservative includes benzalkonium chloride in an amount of 0.005% w / v.

[0046] Furthermore, the osmotic pressure regulator includes one or more of sodium chloride, boric acid, and glucose. Still further, the osmotic pressure regulator includes sodium chloride, accounting for 0.2% w / v.

[0047] Furthermore, the pH adjuster includes one or more of phosphate buffer, borate buffer, hydrochloric acid, and sodium hydroxide. Still further, the pH adjuster includes sodium hydroxide, accounting for 0.8% w / v.

[0048] Furthermore, the above-mentioned posterior segment neovascular diseases include: one or more of wet age-related macular degeneration, retinal vein occlusion, diabetic retinopathy, retinopathy of prematurity and the like.

[0049] Furthermore, the above-mentioned posterior segment neovascular disease includes: wet age-related macular degeneration.

[0050] The present application also provides a method for preparing the polypeptide eye drops for preventing or treating posterior ocular neovascular diseases, which specifically comprises the following steps:

[0051] (1) Preparation of excipient solution: Take ultrapure water and add the prescribed amount of sodium dihydrogen phosphate, benzalkonium chloride, polysorbate 80, poloxamer 407, and tyloxapol in sequence, stirring continuously until completely dissolved; filter with a 0.22 μm filter membrane and prepare solution A for later use;

[0052] (2) Preparation of 2-hydroxypropyl β-cyclodextrin solution: Take solution A and place it in a water bath stirring pot and heat it to 60°C. Start stirring and add 2-hydroxypropyl β-cyclodextrin in several times. After it is completely dispersed and dissolved, take it out and cool it down. It is used as solution B for later use.

[0053] (3) Adjusting pH and osmotic pressure: Detect the pH value and osmotic pressure of Solution B, adjust the pH and osmotic pressure using a pH regulator and an osmotic pressure regulator, filter with a 0.22 μm filter membrane, and prepare Solution C for later use;

[0054] (4) Preparation of polypeptide eye drops: Weigh the fusion polypeptide AJ007 or its pharmaceutically acceptable salt form and add it to solution C. Shake continuously to allow the fusion polypeptide AJ007 or its pharmaceutically acceptable salt form to be dispersed and dissolved completely. This is the polypeptide eye drops.

[0055] 3. Beneficial effects

[0056] Compared with the prior art, the present application has the following advantages:

[0057] (1) The present application provides a fusion polypeptide AJ007, which enhances its ability to penetrate the blood-ocular barrier or sclera to reach the posterior segment of the eye by connecting the cell-penetrating peptide TAT to the front end of the multifunctional fusion polypeptide VI (HM-1) sequence previously disclosed by the applicant, which has strong neovascularization inhibitory ability. It can penetrate the blood-ocular barrier or sclera to reach the choroid. Moreover, it can be seen from Example 3 that after the original multifunctional fusion polypeptide VI (HM-1) is dropped into the eye, only less than 10 ng / mL of active molecules can reach the target location of the wAMD lesion - the RPE / choroid complex, while the fusion polypeptide AJ007 has 6291.38 ng / mL of active molecules that can reach the RPE / choroid complex, greatly improving the eye penetration ability of the original active molecules. In the existing technical solutions, after a peptide AT7 targeting VEGFR2 is linked to the cell-penetrating peptide TAT, its ability to penetrate into endothelial cells increases by 119 times; after bevacizumab is linked to the cell-penetrating peptide CPP, its ability to penetrate into the posterior segment of the eye after eye drops increases by about 10 times; in patent CN107129521A, after the cell-penetrating peptide CC12 is linked to KV12, its ability to penetrate into the eye after eye drops increases by about 8 times; in patent CN114073774A, after the cell-penetrating peptide 289WP is linked to ranibizumab, its concentration reaching the retina after eye drops increases by about 100 times; in patent CN114668717A, after the cell-penetrating peptide PENE is linked to axitinib, its concentration reaching the retina after eye drops increases by about 5 to 10 times. However, compared with the above-mentioned existing technical solutions, AJ007 has an approximately 600-fold improvement in its ability to penetrate the posterior segment of the eye compared to HM-1. The superior efficacy of AJ007 is still obvious, and this superior efficacy exceeds normal expectations, indicating the uniqueness and creativity of this peptide.

[0058] (2) The present application provides a polypeptide eye drop for preventing or treating neovascular diseases in the posterior segment of the eye, which is prepared by using the fusion polypeptide AJ007 or its pharmaceutically acceptable salt form as the polypeptide raw material and selecting suitable excipients. By optimizing the two aspects of connecting the cell-penetrating peptide TAT to the front end of the multifunctional fusion polypeptide VI (HM-1) sequence with strong neovascularization inhibitory ability and the excipient compatibility selected in this application, a new composition product is finally obtained, which distinguishes the product from other peptide eye drops and has unique novelty. It can deliver the fusion polypeptide AJ007 with anti-angiogenesis function to the retina of the posterior segment of the eye to the choroid, and can achieve the effect of improving CNV by eye drop administration. Comparisons of ocular tissue drug distribution and laser-induced CNV efficacy in mice with the original multifunctional fusion peptide VI (HM-1) revealed that the peptide eye drops of the present application have at least 100 times more active molecules reaching the target site of wAMD lesions—the RPE / choroid complex—than the multifunctional fusion peptide VI (HM-1). Efficacy studies also demonstrated that the peptide eye drops of the present application have significantly superior CNV leakage improvement compared to the original multifunctional fusion peptide VI (HM-1), demonstrating non-inferiority even compared to aflibercept, the currently most effective wAMD treatment. This is a feat unattainable by any other peptide eye drops. This technological advancement overcomes the limited efficacy of previous eye drops for treating fundus diseases, and therefore possesses strong clinical potential for the treatment of wAMD. It is expected to change the current situation in which wAMD patients must undergo intravitreal injections for treatment, significantly increase patient compliance, and provide a treatment option with significantly improved compliance for wAMD patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] FIG1 is a drug-time curve result of the original multifunctional fusion polypeptide VI (HM-1) eye drops.

[0060] FIG2 is a drug-time curve result of the polypeptide eye drops of the present application.

[0061] FIG3 is a schematic diagram of FFA angiography after administration of the polypeptide eye drops of the present application to a wAMD model mouse.

[0062] FIG4 is a statistical graph of CNV leakage area obtained by FFA angiography after administration of the polypeptide eye drops of the present application to a wAMD model mouse.

[0063] FIG5 is a schematic diagram of FITC-dextran staining after administration of the polypeptide eye drops of the present application to a wAMD model mouse.

[0064] FIG6 is a statistical graph of CNV area of ​​FITC-dextran staining after administration of the polypeptide eye drops of the present application to a wAMD model mouse.

[0065] FIG7 is a drug-time curve result of eye drops of original multifunctional fusion polypeptide VI (HM-1) at equimolar concentrations.

[0066] FIG8 is a drug-time curve of the original multifunctional fusion polypeptide VI (HM-1) preparation of equimolar concentrations for eye drops.

[0067] FIG9 is a drug-time curve of equimolar concentrations of the fusion polypeptide AJ007 eye drops.

[0068] FIG10 is a drug-time curve of the polypeptide eye drops of the present application at equimolar concentrations.

[0069] FIG11 shows the drug-time curve of the original multifunctional fusion polypeptide VI (HM-1) injected into the vitreous cavity at equimolar concentrations.

[0070] FIG12 is a schematic diagram of FFA angiography of wAMD model mice after administration of equimolar concentrations of various drug solutions.

[0071] FIG13 is a statistical graph showing CNV leakage area obtained by FFA angiography after administration of equimolar concentrations of various drug solutions to a wAMD mouse model.

[0072] FIG14 is a schematic diagram of FITC-dextran staining of equimolar concentrations of various drug solutions administered to a wAMD mouse model.

[0073] FIG15 is a statistical graph showing the CNV area of ​​FITC-dextran staining of wAMD model mice administered with equimolar concentrations of various drug solutions. DETAILED DESCRIPTION

[0074] The present application is further described below with reference to specific embodiments.

[0075] It should be noted that the terms such as "upper", "lower", "left", "right", and "middle" cited in this specification are only for the convenience of description and are not used to limit the scope of implementation. Changes or adjustments to their relative relationships should be regarded as the scope of implementation of this application without substantially changing the technical content.

[0076] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0077] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0078] As used herein, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement, or value. One skilled in the art can readily determine the degree of flexibility for a particular variable.

[0079] As used herein, the term "at least one of" is intended to be synonymous with "one or more of." For example, "at least one of A, B, and C" explicitly includes only A, only B, only C, and combinations of each thereof.

[0080] Concentration, amount and other numerical data can be presented in range format in this article.Should be understood that such range format is only used for convenience and brevity, and should be flexibly interpreted as not only including the numerical value clearly described as range limit, but also including all independent numerical values ​​or subranges encompassed within the scope, just as each numerical value and subrange are clearly described.For example, the numerical range of about 1 to about 4.5 should be interpreted as not only including the limit value of 1 to about 4.5 clearly described, but also including independent numerals (such as 2,3,4) and subranges (such as 1 to 3,2 to 4 etc.).The same principle is applicable to the scope of only narrating a numerical value, such as "less than about 4.5", which should be interpreted as including all above-mentioned values ​​and scopes.In addition, no matter how the breadth of described scope or feature is, this explanation should be applicable.

[0081] Example 1

[0082] This example provides a fusion polypeptide AJ007.

[0083] The amino acid sequence of the fusion polypeptide AJ007 is: 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, SEQ ID NO: 2). Among them, Arg-Gly-Ala-Asp-Arg-Ala-Gly-Gly-Gly-Gly-Arg-Gly-Asp (SEQ ID NO: 1) is a multifunctional fusion polypeptide VI (HM-1) disclosed in the applicant's prior Chinese invention patent with authorization publication number CN104045718B. The multifunctional fusion polypeptide VI has a strong ability to inhibit neovascularization, which mainly inhibits the formation of neovascularization by inhibiting VEGFR2 and αvβ3 signaling pathways. Therefore, it can improve laser-induced choroidal neovascularization and leakage in mice.

[0084] Example 2

[0085] This example provides the use of the fusion polypeptide AJ007 described in Example 1 in the preparation of a medicament for preventing or treating posterior ocular neovascular disease. The medicament is a polypeptide eye drop for preventing or treating posterior ocular neovascular disease. This example also provides polypeptide eye drops for preventing or treating posterior ocular neovascular disease and a method for preparing the same.

[0086] This embodiment provides a polypeptide eye drop for preventing or treating posterior ocular neovascular disease, each 100 mL of which comprises the following components:

[0087] acetate form of fusion peptide AJ007, 2% w / v;

[0088] 2-Hydroxypropyl-β-cyclodextrin, 8.41% w / v;

[0089] Polysorbate 80, 1% w / v;

[0090] Tyloxapol, 0.1% w / v;

[0091] Poloxamer 407, 2% w / v;

[0092] Sodium dihydrogen phosphate, 0.142% w / v;

[0093] Preservative (benzalkonium chloride), 0.005% w / v;

[0094] Osmotic pressure regulator (sodium chloride), 0.2% w / v, adjust the osmotic pressure molarity to 276mOsm / kg;

[0095] pH adjuster, sodium hydroxide, 8% w / v, adjusted the pH to 6.47.

[0096] This embodiment provides a method for preparing polypeptide eye drops for preventing or treating posterior ocular neovascular diseases, which specifically comprises the following steps:

[0097] (1) Preparation of excipient solution: Take 100 mL of ultrapure water and add the prescribed amount of sodium dihydrogen phosphate, benzalkonium chloride, polysorbate 80, poloxamer 407, and tyloxapol in sequence. Stir continuously until completely dissolved. Filter with a 0.22 μm filter membrane and prepare solution A.

[0098] (2) Preparation of 2-hydroxypropyl β-cyclodextrin solution: Take 100 mL of solution A and place it in a water bath stirring pot and heat it to 60°C. Turn on the stirring and add 2-hydroxypropyl β-cyclodextrin in several times. After it is completely dispersed and dissolved, take it out and cool it down. It is used as solution B for later use.

[0099] (3) Adjusting pH and osmotic pressure: Detect the pH and osmotic pressure of Solution B, adjust the pH to 7.5 and the osmotic pressure to 250 using a pH regulator and an osmotic pressure regulator, filter with a 0.22 μm filter membrane, and prepare Solution C for later use;

[0100] (4) Preparation of polypeptide eye drops: Weigh 2000 mg of the acetate salt form of the fusion polypeptide AJ007 and add it to 100 mL of solution C. Shake continuously to allow the fusion polypeptide AJ007 or its pharmaceutically acceptable salt form to be dispersed and dissolved completely. This is the AJ007 polypeptide eye drop with a concentration of 20 mg / mL. The osmotic pressure molar concentration was measured to be 276 mOsm / kg; the pH value was 6.47.

[0101] Example 3

[0102] This example provides a comparative experiment on the ocular tissue distribution of the polypeptide eye drops (AJ007 eye drops) and the multifunctional fusion polypeptide VI (HM-1) in Example 2, as follows:

[0103] Drug solution: polypeptide eye drops (AJ007 eye drops), in which the fusion polypeptide AJ007 is 20 mg / mL; multifunctional fusion polypeptide VI (HM-1) solution, in which the multifunctional fusion polypeptide VI (HM-1) is 20 mg / mL.

[0104] Animal grouping: 24 female C57BL / 6J mice were randomly divided into 6 time point groups: 0h, 1h, 3h, 7h, 12h, and 24h, with 2 mice in each group.

[0105] Experimental process: All mice in the eye drop group were given bilateral eye drops once, with 5 μL of the corresponding solution in each eye. At 0h, 1h, 3h, 7h, 12h, and 24h after the administration, the eyeballs of the mice in the corresponding groups were removed for blood collection. After the whole blood was taken out, it was immediately centrifuged at 6000rpm for 5min, and then the supernatant was collected and stored as the sample to be tested. In addition, the vitreous body, retina, and RPE / choroid complex were separated from the eyeball under a stereomicroscope at the same time. After being diluted with PBS at a ratio of 1:10, they were ground and centrifuged at 12000rpm for 5min, and the supernatant was collected as the sample to be tested. The concentration of the fusion polypeptide AJ007 or the multifunctional fusion polypeptide VI (HM-1) in each tissue was subsequently detected by ELISA. The ELISA method is as follows:

[0106] (1) Coating: HM-1 was diluted to a certain concentration of 1 μg / mL using coating solution (CBS), added to the ELISA plate, 100 μL per well, and incubated at 4°C overnight;

[0107] (2) Washing: Discard the coating solution and add 265 μL of PBST to each well. Wash three times for 3 minutes each time and pat dry.

[0108] (3) Blocking: Add 200 μL of 1% BSA blocking solution to each well, place in a 37°C water bath for 1.5 h, repeat the plate washing process, and pat dry;

[0109] (4) Add the sample to be tested and the antibody: first add 100 μL of the sample to be tested to the ELISA plate, then add 100 μL of HM-1 monoclonal antibody dilution at a certain concentration, set up 2 to 3 replicate wells, shake and mix on a shaker at 37°C for 2 minutes, then move to a 37°C water bath for 2 hours. After sufficient reaction, remove the plate and repeat the washing process;

[0110] (5) Reaction with enzyme-labeled secondary antibody: Add a certain concentration of HRP enzyme-labeled goat anti-mouse IgG, 100 μL per well, and incubate at 37°C for 1.5 h. Repeat the plate washing process and pat dry.

[0111] (6) Add substrate: Add 100 μL of TMB substrate colorimetric solution to each well and develop the color at room temperature in the dark for a certain period of time;

[0112] (7) Termination: Add 50 μL of stop solution to each well to terminate the reaction.

[0113] (8) Detection: Read the OD450nm value of each well using an enzyme-labeled instrument.

[0114] Experimental results and conclusions:

[0115] Comparing the drug-dose curves of HM-1 and AJ007 eye drops (Figures 1 and 2) reveals that only a minimal amount of active molecule from HM-1 reaches the target site of wAMD lesions—the RPE / choroid complex—and the duration of this activity is short. However, at the same concentration, at least 100 times more active molecule from AJ007 eye drops reaches the RPE / choroid complex, and a significant amount remains at this site even after 24 hours, significantly improving bioavailability. Multifunctional fusion polypeptide VI (HM-1) eye drops cannot pass through the blood-ocular barrier to reach the RPE / choroid complex, and therefore cannot have a therapeutic effect on improving CNV leakage. However, by adding TAT peptide, more active molecules can be promoted to reach the RPE / choroid complex. Compared with the general transmembrane peptides connecting small molecules or large molecules, the effect of reaching the posterior segment of the eye is only within several times (CN107129521A, CN114073774A, CN114668717A), which has a great improvement.

[0116] Example 4

[0117] This example provides a comparative experiment on the efficacy of the polypeptide eye drops (AJ007 eye drops) and the multifunctional fusion polypeptide VI (HM-1) in Example 2, as follows:

[0118] Animal groups: 56 female C57BL / 6J mice were randomly divided into a complete blank group, a model + solvent (a control solution containing equal amounts of all excipients but not the fusion peptide AJ007) control group, a model + positive drug aflibercept vitreous injection (10 mg / mL) group, a model + low-dose HM-1 (20 mg / mL) group, a model + high-dose HM-1 (80 mg / mL) group, a model + low-dose AJ007 eye drops (20 mg / mL) group, a model + medium-dose AJ007 eye drops (40 mg / mL) group, and a model + high-dose AJ007 eye drops (80 mg / mL) group, with 7 mice in each group.

[0119] Experimental Procedure: Mice were first mydriaticized with compound tropicamide eye drops and anesthetized with an intraperitoneal injection of 4% chloral hydrate solution at 0.2 mL / 20 g of mouse body weight. After anesthesia, sodium hyaluronate gel was applied to the eyes for eye protection, and a coverslip was placed. The left eye was laser-induced with a 532 nm argon-ion laser at a power of 120 mW, a burst time of 0.01 s, and a spot size of 100 μm. Drug administration began on the day of modeling, and each group received drug administration according to a different route. Only the left eye was administered. Mice in the eye drop group received eye drops five times daily, 1 hour apart, with 5 μL of the corresponding drug solution each time, for 7 days. Mice in the intravitreal injection group were anesthetized on the day of modeling and then administered intravitreally. Each mouse received 1 μL of the corresponding drug solution using a microinjection needle. After modeling, an FFA examination was performed using a fundus fluorescence angiography system on the third day after modeling to evaluate the CNV leakage level; the mice were killed on the seventh day after modeling, and FITC-dextran was injected into the ventricle and then the RPE-choroid / sclera complex was fluorescently stained to detect changes in CNV area.

[0120] Experimental results and conclusions: The results of FFA examination showed that neither low-dose nor high-dose HM-1 could significantly improve laser-induced CNV leakage, while low-dose AJ007 eye drops could significantly improve CNV leakage, which was significantly better than low-dose and high-dose HM-1, and better than medium-dose and high-dose AJ007 eye drops, and could achieve the same efficacy as the positive drug aflibercept injected into the vitreous (Figures 3 and 4); FITC-dextran staining results after injection showed that low-dose HM-1 could not reduce the area of ​​laser-induced CNV, while high-dose HM-1 could partially reduce the area of ​​CNV. However, low-dose AJ007 eye drops could significantly reduce the area of ​​CNV, which was significantly better than high-dose HM-1, and better than medium-dose and high-dose AJ007 eye drops, and even slightly better than the positive drug aflibercept injected into the vitreous (Figures 5 and 6). Combined FFA examination and FITC-dextran staining results after injection indicate that 20 mg / mL AJ007 eye drops significantly improves neovascularization in the wAMD mouse model, significantly outperforming low-dose and high-dose HM-1. Its efficacy in this classic wAMD model is comparable to or even slightly better than the currently most effective clinical treatment for wAMD, intravitreal aflibercept.

[0121] Example 5

[0122] This example provides a comparative experiment on the ocular tissue distribution of the polypeptide eye drops (AJ007 eye drops) in Example 2, multifunctional fusion polypeptide VI (HM-1) at equimolar concentrations, multifunctional fusion polypeptide VI preparations (HM-1 preparations, polypeptide is HM-1, and the other excipients are the same as those of AJ007 eye drops), and fusion polypeptide AJ007 preparations (TAT-HM-1, polypeptide is AJ007, pure water is the solvent) after eye drops, and multifunctional fusion polypeptide VI (HM-1) after vitreous injection, as follows:

[0123] Animal grouping: 70 female C57BL / 6J mice were randomly divided into 7 time point groups: 0 h, 10 min, 1 h, 3 h, 7 h, 12 h, and 24 h, with 2 mice in each group.

[0124] Experimental Procedure: All mice in the eye drop administration group received a single bilateral eye drop, with 5 μL of the corresponding drug solution in each eye. The HM-1 intravitreal injection group received 5 μL of the corresponding drug solution in each eye. At seven time points after administration (0 h, 10 min, 1 h, 3 h, 7 h, 12 h, and 24 h), the eyeballs of the mice in the corresponding groups were enucleated and blood was collected. The blood was immediately centrifuged at 6000 rpm for 5 min, and the supernatant was collected and stored as the sample for testing. Furthermore, under a stereomicroscope, the vitreous, retina, and RPE / choroid complex were isolated from the eyeballs. The samples were diluted 1:10 with PBS, ground, and centrifuged at 12000 rpm for 5 min. The supernatant was collected and used as the sample for testing. The concentration of HM-1 or the fusion peptide AJ007 in each tissue was subsequently determined by ELISA.

[0125] Experimental results and conclusions:

[0126] By comparing the drug-time curves of AJ007 eye drops with equimolar concentrations of HM-1, HM-1 preparations, TAT-HM-1 after eye drops and HM-1 vitreous injection (Figures 7, 8, 9, 10, and 11), it can be found that after eye drops of either HM-1 or HM-1 preparations, only less than 10 ng / mL of active molecules can reach the target site of wAMD lesions - the RPE / choroid complex, and the maintenance time is also very short; however, after HM-1 is linked to TAT, 843.28 ng / mL of active molecules can reach the RPE / choroid complex, which is much higher than the concentration of HM-1 and HM-1 preparation groups, but the maintenance time is also shorter; after HM-1 vitreous injection, Approximately 5729.7 ng / mL of active molecules can reach the RPE / choroid complex, which is a higher concentration than that of the TAT-HM-1 group, but the maintenance time is shorter. In contrast, after instillation of AJ007 eye drops, approximately 6291.38 ng / mL of active molecules can reach the RPE / choroid complex, which is much higher than that of the HM-1, HM-1 preparation, and TAT-HM-1 eye drops groups, and slightly higher than the HM-1 vitreous injection group. In addition, the maintenance time is also longer than that of the HM-1 vitreous injection group. This shows that the AJ007 eye drops made after optimization and modification can enable a large amount of active molecules to reach the RPE / choroid complex, providing pharmacokinetic data support for the efficacy of effectively improving the abnormal neovascularization of the choroid.

[0127] Example 6

[0128] This example provides a comparative experiment on the efficacy of the polypeptide eye drops (AJ007 eye drops) in Example 2, the multifunctional fusion polypeptide VI (HM-1) at an equimolar concentration, the multifunctional fusion polypeptide VI preparation (HM-1 preparation), the fusion polypeptide AJ007 (TAT-HM-1) after eye drops, and the multifunctional fusion polypeptide VI (HM-1) after vitreous injection, as follows:

[0129] Animal groups: 42 female C57BL / 6J mice were randomly divided into model + vehicle control group, model + positive drug aflibercept vitreous 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 vitreous injection (8.76 mg / mL) group, with 6 mice in each group.

[0130] Experimental Procedure: Mice were first mydriaticized with compound tropicamide eye drops and anesthetized with an intraperitoneal injection of 4% chloral hydrate solution at a rate of 0.2 mL / 20 g mouse body weight. After anesthesia, sodium hyaluronate gel was applied to the eye for protection, and a coverslip was placed. Laser modeling was performed in the left eye using a 532 nm argon ion laser with a power of 120 mW, a burst time of 0.01 s, and a spot size of 100 μm. Drug administration began on the day of modeling. Each group received drug administration according to a different route, with only the left eye inoculated. Mice in the eye drop group received 5 μL of the corresponding drug solution five times daily, 1 hour apart, for 7 days. Mice in the intravitreal injection group were anesthetized on the day of modeling and then administered intravitreally. Each mouse received the corresponding drug solution (1 μL per eye in the aflibercept group and 5 μL per eye in the HM-1 group) using a microinjection needle. After modeling, an FFA examination was performed using a fundus fluorescence angiography system on the third day after modeling to evaluate the CNV leakage level; the mice were killed on the seventh day after modeling, and FITC-dextran was injected into the ventricle and then the RPE-choroid / sclera complex was fluorescently stained to detect changes in CNV area.

[0131] Experimental results and conclusion: The results of FFA examination showed that HM-1, HM-1 preparation, and TAT-HM-1 eye drops could not significantly improve laser-induced CNV leakage, while HM-1 intravitreal injection and AJ007 eye drops could significantly improve CNV leakage, and the effect was comparable to that of the positive drug aflibercept injected into the vitreous (Figures 12 and 13). Among them, the efficacy of AJ007 eye drops was slightly better than that of the positive drug aflibercept injected into the vitreous; the results of FITC-dextran staining after injection also showed that HM-1, HM-1 preparation, and TAT-HM-1 eye drops could not reduce the area of ​​laser-induced CNV, while HM-1 intravitreal injection and AJ007 eye drops could significantly reduce the area of ​​CNV, and the efficacy was even slightly better than that of the positive drug aflibercept injected into the vitreous (Figures 14 and 15). Combined FFA examination and FITC-dextran staining after injection revealed that neither HM-1, nor HM-1 formulation, nor HM-1 conjugated to TAT, was effective in ameliorating neovascularization in the wAMD mouse model. However, after HM-1 conjugated to TAT and then formulated into a formulation, AJ007 eye drops, administered in this classic wAMD model, demonstrated efficacy comparable to, or even slightly superior to, intravitreal injection of an equimolar concentration of HM-1. The efficacy was comparable to, or even slightly superior to, intravitreal injection of aflibercept, the current most effective clinical treatment for wAMD. Given that patient compliance with eye drops is significantly better than with intravitreal injection, the polypeptide eye drops of the present invention possess significant clinical potential, providing a superior option for the clinical treatment of wAMD patients and potentially generating significant economic benefits.

[0132] According to the results of the above examples, the present application successfully optimized and upgraded the molecule by connecting the cell-penetrating peptide TAT to the front end of the multifunctional fusion polypeptide VI (HM-1) sequence with strong neovascularization inhibition ability and preparing it into eye drops with selected excipients. The ocular pharmacokinetics and efficacy results of the mouse laser-induced AMD model showed that AJ007 eye drops significantly improved the tissue concentration and efficacy results in the retina and choroid compared to the original HM-1 molecule. Its efficacy is even slightly better than that of aflibercept, the best drug currently used clinically for wAMD. Overall, it achieves a 1+1>2 effect. The effect of this eye drop is significantly better than that of the eye drops connected to the cell-penetrating peptide in other comparative documents. In addition, considering that the clinical administration method of aflibercept is the invasive method of intravitreal injection, it is obvious that the non-invasive eye drop method of the polypeptide eye drops is safer and more convenient for patients, avoiding the various serious side effects caused by intravitreal injection, and has better clinical treatment potential.

Claims

1. A 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 neovascularization diseases of the eye.

3. The use according to claim 2, characterized in that: The posterior segment neovascular disease includes one or more of 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 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 ocular segment neovascularization disease contains at least the fusion polypeptide AJ007 and / or a pharmaceutically acceptable carrier thereof.

6. A polypeptide eye drop for preventing or treating posterior ocular neovascularization disease, characterized in that: The polypeptide eye drops include the fusion polypeptide AJ007 or a pharmaceutically acceptable salt form thereof according to claim 1, and auxiliary materials.

7. The polypeptide eye drops for preventing or treating posterior ocular neovascularization diseases according to claim 6, characterized in that: The polypeptide eye drops, each 100 mL, include the following components: Fusion polypeptide AJ007 or a pharmaceutically acceptable salt thereof, 0-50% w / v; 2-Hydroxypropyl-β-cyclodextrin, 5-10% w / v; Polysorbate 80, 0.1-5% w / v; Tyloxapol, 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 regulator, 0-1% w / v, adjusting the osmotic pressure molar concentration to 250-350mOsm / kg; pH adjuster, 0-1% w / v, adjusts pH to 6.0-8.

0.

8. The polypeptide eye drops for preventing or treating posterior ocular neovascularization diseases according to claim 7, characterized in that: The pharmaceutically acceptable salt form of the fusion polypeptide AJ007 includes: one or more of acetate, trifluoroacetate, hydrochloride, ammonium salt, sodium salt, pamoate, citrate, and salicylate; and / or the preservative includes one or more of benzalkonium chloride, benzalkonium bromide cationic surfactant, benzyl alcohol, chlorobutanol, and sorbic acid; and / or the osmotic pressure regulator includes one or more of sodium chloride, boric acid, and glucose; And / or the pH adjuster includes one or more of phosphate buffer, borate buffer, hydrochloric acid, and sodium hydroxide.

9. The polypeptide eye drops for preventing or treating posterior ocular neovascularization diseases according to claim 8, characterized in that: The pharmaceutically acceptable salt form of the fusion polypeptide AJ007 includes 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 a polypeptide eye drop for preventing or treating posterior ocular neovascularization disease according to any one of claims 6 to 9, characterized in that: The steps include: (1) Preparation of excipient solution: Take ultrapure water, add sodium dihydrogen phosphate, benzalkonium chloride, polysorbate 80, poloxamer 407, and tyloxapol in the prescribed amount in sequence, and continue stirring until completely dissolved; filter with a 0.22 μm filter membrane and use as solution A for later use; (2) Preparation of 2-hydroxypropyl β-cyclodextrin solution: Take solution A and place it in a water bath stirring pot and heat it to 60°C. Turn on the stirring and add 2-hydroxypropyl β-cyclodextrin in several times. After it is completely dispersed and dissolved, take it out and cool it. Use it as solution B for later use; (3) Adjusting pH and osmotic pressure: Detect the pH value and osmotic pressure of Solution B, adjust the pH and osmotic pressure using a pH adjuster and an osmotic pressure adjuster, filter with a 0.22 μm filter membrane, and prepare Solution C for later use; (4) Preparation of polypeptide eye drops: Weigh the fusion polypeptide AJ007 or its pharmaceutically acceptable salt form and add it to liquid C. Shake continuously to allow the fusion polypeptide AJ007 or its pharmaceutically acceptable salt form to be dispersed and dissolved completely. This is polypeptide eye drops.

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