Fusion polypeptide AJ007 and application thereof

Abstract

The invention discloses a fusion polypeptide AJ007 and application thereof, and belongs to the technical field of ophthalmic drugs. The fusion polypeptide AJ007 is formed by connecting a TAT cell-penetrating peptide to the front end of a polypeptide HM-1 molecule for inhibiting neovascularization. The fusion polypeptide AJ007 is used for preparing the polypeptide eye drops, the fusion polypeptide AJ007 is prepared into the eye drops by selecting the prescription provided by the invention, and the eye drops can be used for preventing or treating wet age-related macular degeneration diseases. The administration mode of the eye drops can effectively avoid the problems of various serious side effects and poor patient tolerance caused by clinical intraeyeball vitreous injection, has the advantage of noninvasive treatment, and shows an excellent drug effect of improving choroidal neovascularization abnormal hyperplasia in a laser-induced mouse wAMD model; good clinical treatment potential is realized.

Description

Technical Field

[0001] The present application belongs to the technical field of ophthalmic drugs, 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 segment neovascular diseases of the eye. Background Art

[0002] With the increase of age, the aggravation of myopia or the development of related metabolic diseases, when fundus diseases such as pathological myopia, age-related macular degeneration, diabetic retinopathy occur, some unnecessary blood vessels will be generated in the fundus. These new blood vessels will invade the choroidal blood vessels, retinal photoreceptor cells and other normal fundus tissues, causing visual impairment and even 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). Among them, wet age-related macular degeneration (wAMD) is an important cause of low vision and blindness in the population, and its incidence is increasing year by year. Choroidal neovascularization (CNV) is the main pathological basis and primary cause of its damage to visual function. Therefore, it is very necessary to develop suitable drugs to alleviate patients' vision loss by improving CNV.

[0004] Vascular endothelial growth factor (VEGF) has been shown to play an important role in the formation of CNV. Therefore, the current clinical drugs for wet age-related macular degeneration are all antibodies against VEGF, such as ranibizumab, aflibercept, and conbercept. These drugs have a certain effect on improving the vision of patients with wet age-related macular degeneration, but these antibody drugs have a large molecular weight and cannot pass through the blood-ocular barrier to reach the retina and choroid. Therefore, they are clinically treated by intravitreal injection. However, intravitreal injection can cause multiple serious side effects such as intraocular hemorrhage, intraocular infection, retinal detachment, intraocular inflammation, cataracts, and increased intraocular pressure. Patients have poor compliance and tolerance. Therefore, more appropriate treatment options are needed clinically to avoid the occurrence of these serious side effects. In addition, this type of antibody drugs directly neutralizes 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 visual impairment. Therefore, it is more appropriate to target VEGF receptors or other signal pathways that inhibit angiogenesis in the choroid for treatment.

[0005] In the applicant's prior Chinese invention patent with the authorization announcement number CN104045718B, a multifunctional fusion polypeptide VI (HM-1) is disclosed, and its amino acid sequence is: Arg-Gly-Ala-Asp-Arg-Ala-Gly-Gly-Gly-Gly-Arg-Gly-Asp (SEQ ID NO: 1). The multifunctional fusion polypeptide VI has a strong ability to inhibit new blood vessels, which mainly inhibits the formation of blood vessels by inhibiting the VEGFR2 and αvβ3 signaling pathways, and further verifies that the multifunctional fusion polypeptide VI can treat rat choroidal neovascularization. However, the multifunctional fusion polypeptide VI still needs to be administered by intravitreal injection, which is not a good way for patient compliance in clinical practice and will also cause 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. Summary of the invention

[0007] 1. Problem to be solved

[0008] 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 by intravitreal injection, which is prone to cause serious side effects, etc., and provides a fusion polypeptide AJ007 and its application. 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 inhibition ability, which enhances 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 for compatibility, and prepared into 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.

[0009] 2. Technical solution

[0010] In order to solve the above problems, the technical solutions adopted in this application are as follows:

[0011] 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), and is connected to the cell-penetrating peptide TAT at the front end of the multifunctional fusion polypeptide VI (HM-1) sequence previously disclosed by the applicant with strong angiogenesis inhibitory ability, thereby enhancing 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.

[0012] 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 segment neovascularization diseases of the eye, wherein the drug can inhibit the formation of new blood vessels.

[0013] Furthermore, the above-mentioned drug for preventing or treating posterior segment neovascularization disease at least contains the fusion polypeptide AJ007 and / or its pharmaceutically acceptable carrier.

[0014] Furthermore, the above-mentioned drug for preventing or treating posterior segment neovascularization disease of the eye includes: eye drops containing the fusion polypeptide AJ007 and a pharmaceutically acceptable carrier thereof.

[0015] 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.

[0016] Furthermore, the above-mentioned posterior segment neovascular disease includes: wet age-related macular degeneration.

[0017] The present application also provides a polypeptide eye drop for preventing or treating posterior segment neovascularization diseases of the eye, wherein the polypeptide eye drop comprises the above-mentioned fusion polypeptide AJ007 or a pharmaceutically acceptable salt form thereof, and an auxiliary material.

[0018] Furthermore, the above-mentioned polypeptide eye drops for preventing or treating posterior ocular neovascularization diseases include the following components per 100 mL:

[0019] Fusion polypeptide AJ007 or a pharmaceutically acceptable salt thereof, 0-50% w / v;

[0020] 2-Hydroxypropyl-β-cyclodextrin, 5-10% w / v;

[0021] Polysorbate 80, 0.1-5% w / v;

[0022] Tyloxapol, 0.05-5% w / v;

[0023] Poloxamer 407, 0.05-10% w / v;

[0024] Sodium dihydrogen phosphate, 0-1% w / v;

[0025] Preservatives, 0-0.005% w / v;

[0026] Osmotic pressure regulator, 0-1% w / v, adjusting the osmotic pressure molar concentration to 250-350mOsm / kg;

[0027] pH adjuster, 0-1% w / v, adjusting pH to 6.0-8.0;

[0028] 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 is used 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 have a better effect of penetrating the blood-ocular barrier or the sclera to reach the posterior segment of the eye. By optimizing the front end of the multifunctional fusion polypeptide VI (HM-1) sequence with strong angiogenesis inhibitory ability, connecting the cell-penetrating peptide TAT and the excipient combination selected in the present application, the fusion polypeptide AJ007 with anti-angiogenesis function is delivered to the posterior segment of the eye from the retina to the choroid, and the effect of improving CNV by eye drop administration can be achieved.

[0029] Furthermore, the above-mentioned polypeptide eye drops for preventing or treating posterior ocular neovascularization diseases include the following components per 100 mL:

[0030] Fusion polypeptide AJ007 or a pharmaceutically acceptable salt thereof, 2% w / v;

[0031] 2-Hydroxypropyl-β-cyclodextrin, 8.41% w / v;

[0032] Polysorbate 80, 1% w / v;

[0033] Tyloxapol, 0.1% w / v;

[0034] Poloxamer 407, 2% w / v;

[0035] Sodium dihydrogen phosphate, 0.142% w / v;

[0036] Preservative, 0.005% w / v;

[0037] Osmotic pressure regulator, adjust the osmotic pressure molar concentration to 276mOsm / kg;

[0038] pH adjuster, adjust the pH value to 6.47.

[0039] Further, 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. Further, the pharmaceutically acceptable salt form of the fusion polypeptide AJ007 includes acetate.

[0040] 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, accounting for 0.005% w / v.

[0041] Furthermore, the osmotic pressure regulator includes: one or more of sodium chloride, boric acid, and glucose. Furthermore, the osmotic pressure regulator includes: sodium chloride, accounting for 0.2% w / v.

[0042] Furthermore, the pH adjuster includes: one or more of phosphate buffer, borate buffer, hydrochloric acid, and sodium hydroxide. Furthermore, the pH adjuster includes: sodium hydroxide, accounting for 0.8% w / v.

[0043] 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.

[0044] Furthermore, the above-mentioned posterior segment neovascular disease includes: wet age-related macular degeneration.

[0045] The present application also provides a method for preparing the above-mentioned polypeptide eye drops for preventing or treating posterior ocular neovascularization diseases, which specifically comprises the following steps:

[0046] (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;

[0047] (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;

[0048] (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;

[0049] (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.

[0050] 3. Beneficial effects

[0051] Compared with the prior art, the present application has the following beneficial effects:

[0052] (1) The present application provides a fusion polypeptide AJ007, which is connected to the cell-penetrating peptide TAT at the front end of the multifunctional fusion polypeptide VI (HM-1) sequence previously disclosed by the applicant with strong angiogenesis inhibitory ability, thereby enhancing its ability to penetrate the blood-ocular barrier or the sclera to reach the posterior segment of the eye. It can penetrate the blood-ocular barrier or the sclera to reach the choroid. Moreover, it can be seen from Example 3 that after the original multifunctional fusion polypeptide VI (HM-1) is administered by eye drops, only less than 10 ng / mL of active molecules can reach the target lesion site of wAMD - the RPE / choroid complex, while the fusion polypeptide AJ007 has 843.28 ng / mL of active molecules that can reach the RPE / choroid complex, which is only several times the effect of general transmembrane peptides connecting small molecules or large molecules to reach the posterior segment of the eye (such as Chinese invention patents with publication numbers CN107129521A, CN114073774A, CN114668717A, etc.), which has a great improvement. Therefore, this superiority is beyond normal expectations, indicating the particularity and creativity of the peptide.

[0053] (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 a polypeptide API and selecting suitable excipients. By optimizing the two aspects of connecting the cell-penetrating peptide TAT and the excipient compatibility selected in the present application to the front end of the multifunctional fusion polypeptide VI (HM-1) sequence with strong neovascularization inhibition ability, 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. By comparing the ocular tissue drug distribution and mouse laser-induced CNV efficacy experiments with the original multifunctional fusion polypeptide VI (HM-1), the experimental results of ocular tissue drug distribution show that the polypeptide eye drops of the present application have at least 100 times more active molecules than the multifunctional fusion polypeptide VI (HM-1) to reach the target location of wAMD lesions—RPE / choroidal complex. The efficacy experiment results show that the polypeptide eye drops of the present application have a very effective CNV leakage improvement effect compared to the original multifunctional fusion polypeptide VI (HM-1) molecule, and even have non-inferiority compared to the current best clinical wAMD treatment drug aflibercept, which is difficult to achieve for all other peptide eye drops. It is more advanced in technology and overcomes the shortcomings of the previous eye drops with limited efficacy in treating fundus diseases. Therefore, it has a strong clinical potential for treating wAMD, and is expected to change the current situation that wAMD patients must undergo intraocular vitreous injection as a treatment method, which can significantly increase patient compliance and provide wAMD patients with a treatment option with significantly improved compliance. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 This is the drug-time curve result of the original multifunctional fusion polypeptide VI (HM-1) eye drops.

[0055] Figure 2 This is the drug-time curve result of the polypeptide eye drops of the present application.

[0056] Figure 3 It is a schematic diagram of FFA angiography after administration of the polypeptide eye drops of the present application to a wAMD model mouse.

[0057] Figure 4 It is a statistical graph of CNV leakage area of ​​FFA angiography after administration of the polypeptide eye drops of the present application to a wAMD model mouse.

[0058] Figure 5 It is a schematic diagram of FITC-dextran staining after administering the polypeptide eye drops of the present application to a wAMD model mouse.

[0059] Figure 6 It is a statistical graph of CNV area of ​​FITC-dextran spread staining after administration of the polypeptide eye drops of the present application to a wAMD model mouse.

[0060] Figure 7 This is the drug-time curve result of eye drops of original multifunctional fusion polypeptide VI (HM-1) with equimolar concentration.

[0061] Figure 8 This is the drug-time curve result of eye drops of the original multifunctional fusion polypeptide VI (HM-1) preparation with equimolar concentration.

[0062] Fig. 9 This is the drug-time curve result of equimolar concentration of fusion polypeptide AJ007 eye drops.

[0063] Fig.10 This is the drug-time curve result of the polypeptide eye drops of the present application with equimolar concentration.

[0064] Fig.11 This is the drug-time curve result of intravitreal injection of original multifunctional fusion polypeptide VI (HM-1) with equimolar concentration.

[0065] Fig.12 This is a schematic diagram of FFA angiography of wAMD model mice given equimolar concentrations of various drug solutions.

[0066] Fig.13 This is a statistical graph of CNV leakage area obtained by FFA angiography when equimolar concentrations of various drug solutions were administered to a wAMD mouse model.

[0067] Fig.14This is a schematic diagram of FITC-dextran staining of equimolar concentrations of various drug solutions administered to the wAMD model of mice.

[0068] Fig.15 The CNV area statistics of FITC-dextran staining in wAMD mice were given equimolar concentrations of various drug solutions. DETAILED DESCRIPTION

[0069] The present application is further described below in conjunction with specific embodiments.

[0070] It should be noted that the terms such as "upper", "lower", "left", "right", "middle", etc. 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.

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled 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.

[0072] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0073] As used herein, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement or value. The degree of flexibility for a particular variable can be easily determined by one skilled in the art.

[0074] 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" expressly includes only A, only B, only C, and combinations of each thereof.

[0075] Concentration, amount and other numerical data can be presented in range format herein.It should be understood that such range format is only used for convenience and simplicity, and should be flexibly interpreted as not only including the numerical value clearly described as range limit, but also including all single numerical values ​​or sub-ranges contained in the range, just as each numerical value and sub-range 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 single numerals (such as 2,3,4) and sub-ranges (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 the above-mentioned values ​​and ranges.In addition, no matter how the breadth of the described range or feature is, this explanation should be applicable.

[0076] Example 1

[0077] This embodiment provides a fusion polypeptide AJ007.

[0078] 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 announcement 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 choroidal neovascularization and leakage caused by laser induction in mice.

[0079] Example 2

[0080] This example provides the use of the fusion polypeptide AJ007 described in Example 1 in the preparation of a drug for preventing or treating posterior ocular neovascularization, which is a polypeptide eye drop for preventing or treating posterior ocular neovascularization. This example also provides polypeptide eye drops for preventing or treating posterior ocular neovascularization and a preparation method thereof.

[0081] The polypeptide eye drops provided in this embodiment for preventing or treating posterior ocular neovascularization diseases include the following components per 100 mL:

[0082] acetate form of fusion peptide AJ007, 2% w / v;

[0083] 2-Hydroxypropyl-β-cyclodextrin, 8.41% w / v;

[0084] Polysorbate 80, 1% w / v;

[0085] Tyloxapol, 0.1% w / v;

[0086] Poloxamer 407, 2% w / v;

[0087] Sodium dihydrogen phosphate, 0.142% w / v;

[0088] Preservative (benzalkonium chloride), 0.005% w / v;

[0089] Osmotic pressure regulator (sodium chloride), 0.2% w / v, adjust the osmotic pressure molar concentration to 276mOsm / kg;

[0090] pH adjuster, sodium hydroxide, 8% w / v, adjusted pH to 6.47.

[0091] The present embodiment provides a method for preparing a polypeptide eye drop for preventing or treating posterior ocular neovascularization diseases, which specifically comprises the following steps:

[0092] (1) Preparation of excipient solution: Take 100 mL of ultrapure water, add the prescribed amount of sodium dihydrogen phosphate, benzalkonium chloride, polysorbate 80, poloxamer 407, and tyloxapol in sequence, and continue stirring until completely dissolved; filter with a 0.22 μm filter membrane and use as solution A for later use;

[0093] (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. It is used as solution B for standby use;

[0094] (3) Adjusting pH and osmotic pressure: Detect the pH value and osmotic pressure of Solution B, adjust the pH to 7.5 and the osmotic pressure to 250 using a pH adjuster and an osmotic pressure adjuster, filter with a 0.22 μm filter membrane, and prepare Solution C for later use;

[0095] (4) Preparation of polypeptide eye drops: Weigh 2000 mg of the acetate form of the fusion polypeptide AJ007 and add it to 100 mL of liquid C. Shake continuously to allow the fusion polypeptide AJ007 or its pharmaceutically acceptable salt form to be dispersed and dissolved completely, to obtain 20 mg / mL AJ007 polypeptide eye drops. The osmotic pressure molar concentration was measured to be 276 mOsm / kg; the pH value was 6.47.

[0096] Example 3

[0097] This example provides a comparative experiment on the ocular tissue distribution of the polypeptide eye drops (AJ007 eye drops) in Example 2 and the multifunctional fusion polypeptide VI (HM-1), as follows:

[0098] 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.

[0099] 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.

[0100] 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 to collect blood. After the whole blood was taken out, it was immediately centrifuged at 6000rpm for 5min, and then the supernatant was collected and saved 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, diluted with PBS at a ratio of 1:10, 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 multifunctional fusion polypeptide VI (HM-1) in each tissue was subsequently detected by ELISA. The ELISA method is as follows:

[0101] (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;

[0102] (2) Washing: discard the coating solution, add 265 μL PBST solution to each well, wash three times, 3 min each time, and pat dry;

[0103] (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;

[0104] (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 a certain concentration of HM-1 monoclonal antibody dilution, set up 2 to 3 replicate wells, shake and mix on a shaker at 37°C for 2 min, then move to a 37°C water bath for 2 h, take out after sufficient reaction, and repeat the plate washing process;

[0105] (5) Reaction with enzyme-labeled secondary antibody: Add a certain concentration of HRP enzyme-labeled goat anti-mouse IgG, 100 μL per well, incubate at 37°C for 1.5 h. Repeat the plate washing process and pat dry;

[0106] (6) Adding substrate: Add 100 μL of TMB substrate colorimetric solution to each well and color develop at room temperature in the dark for a certain period of time;

[0107] (7) Termination: Add 50 μL of stop solution to each well to terminate the reaction.

[0108] (8) Detection: Read the OD450 nm value of each well using an ELISA reader.

[0109] Experimental results and conclusions:

[0110] By comparing the drug-time curves of HM-1 eye drops and AJ007 eye drops ( Figure 1 and Figure 2 ), it can be found that only a very small amount of active molecules can reach the target location of wAMD lesions - RPE / choroid complex after HM-1 eye drops, and the maintenance time is also short. However, after the same concentration of AJ007 eye drops, at least 100 times more active molecules can reach the RPE / choroid complex, and at 24h, there are still many active molecules present in this part, which greatly improves the bioavailability. Multifunctional fusion polypeptide VI (HM-1) eye drops cannot reach the RPE / choroid complex through the blood-ocular barrier, so it cannot play a therapeutic effect of improving CNV leakage, and by adding TAT peptide, more active molecules can be promoted to reach the RPE / choroid complex, which is only within several times of the effect of reaching the posterior segment of the eye by connecting small molecules or macromolecules with general transmembrane peptides (CN107129521A, CN114073774A, CN114668717A). Compared with it, there is a great improvement.

[0111] Example 4

[0112] This example provides a comparative experiment of the efficacy of the polypeptide eye drops (AJ007 eye drops) and the multifunctional fusion polypeptide VI (HM-1) in Example 2, as follows:

[0113] Animal grouping: 56 female C57BL / 6J mice were randomly divided into a complete blank group, a model + solvent (a control solution containing all equal amounts of excipients but not containing the fusion polypeptide 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.

[0114] Experimental process: The mice were first dilated with compound tropicamide eye drops, and anesthetized with 4% chloral hydrate solution at 0.2mL / 20g of mouse body weight. After anesthesia, sodium hyaluronate gel was applied to protect the eyes, and a cover glass was attached. The left eye was laser modeled with a 532nm argon ion laser, with a power of 120mW, a burst time of 0.01s, and a spot size of 100μm. The drug was administered on the day after modeling, and each group was administered according to different administration methods. Only the left eye was given. The mice in the eye drop group were given eye drops 5 times a day, with an interval of 1h each time, and 5μL of the corresponding drug solution was dropped each time for 7 days; the mice in the vitreous injection group were anesthetized on the day after modeling and then given vitreous injections. Each mouse was injected with 1μL 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 stained with fluorescent staining to detect changes in CNV area.

[0115] 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 of the eyeball ( Figure 3 and Figure 4 ); The results of FITC-dextran staining 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. The effect was also better than medium-dose and high-dose AJ007 eye drops, and the efficacy was even slightly better than the positive drug aflibercept injected into the vitreous of the eyeball ( Figure 5 and Figure 6). The results of FFA examination and FITC-dextran staining after injection show that 20 mg / mL AJ007 eye drops has a very significant effect on improving the abnormal neovascularization of the wAMD model in mice, which is significantly better than the efficacy of low-dose and high-dose HM-1. The efficacy in this classic wAMD model is comparable to or even slightly better than the best clinical treatment for wAMD, intravitreal injection of aflibercept.

[0116] Example 5

[0117] This example provides a comparative experiment on the distribution of eye tissues after eye drops of the polypeptide eye drops (AJ007 eye drops) in Example 2 and equimolar concentrations of multifunctional fusion polypeptide VI (HM-1), multifunctional fusion polypeptide VI preparation (HM-1 preparation, polypeptide is HM-1, and the other auxiliary materials are the same as AJ007 eye drops), fusion polypeptide AJ007 preparation (TAT-HM-1, polypeptide is AJ007, pure water is solvent) and after vitreous injection of multifunctional fusion polypeptide VI (HM-1), as follows:

[0118] Animal grouping: 70 female C57BL / 6J mice were randomly divided into 7 time point groups, namely 0h, 10min, 1h, 3h, 7h, 12h, and 24h, with 2 mice in each group.

[0119] Experimental process: All mice in the eye drop administration group were given one bilateral eye drop, with 5 μL of the corresponding solution in each eye, while the HM-1 vitreous injection group was injected with 5 μL of the corresponding solution in each eye. At 0h, 10min, 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 quickly centrifuged at 6000rpm for 5min, and then the supernatant was collected and saved as the sample to be tested; in addition, the vitreous, retina, and RPE / choroid complex were separated from the eyeball under a stereo microscope, diluted with PBS at a ratio of 1:10, ground, and centrifuged at 12000rpm for 5min, and the supernatant was collected as the sample to be tested. The concentration of HM-1 or fusion polypeptide AJ007 in each tissue was subsequently detected by ELISA.

[0120] Experimental results and conclusions:

[0121] By comparing the drug-time curves of AJ007 eye drops and HM-1 of equal molar concentration, HM-1 preparation, TAT-HM-1 eye drops and HM-1 vitreous injection ( Figure 7 , Figure 8 , Fig. 9 , Fig.10 , Fig.11), it can be found that no matter HM-1 or HM-1 preparation, only less than 10ng / mL of active molecules can reach the target site of wAMD lesions - RPE / choroid complex after eye drops, and the maintenance time is also very short; however, after HM-1 is connected to TAT, 843.28ng / mL of active molecules can reach the RPE / choroid complex, which is much higher than the concentration of HM-1 and HM-1 preparation group, but the maintenance time is also shorter; after HM-1 vitreous injection, about 5729.7ng / mL of active molecules can reach the RPE / choroid complex, which is higher than TA T-HM-1 group, but the maintenance time was shorter; in contrast, after AJ007 eye drops, about 6291.38 ng / mL of active molecules could reach the RPE / choroid complex, which was much higher than that of HM-1, HM-1 preparation, and TAT-HM-1 eye drops groups, and slightly higher than that of HM-1 vitreous injection group. In addition, the maintenance time was also longer than that of HM-1 vitreous injection group. This shows that AJ007 eye drops made after optimization and modification can enable a large number of active molecules to reach the RPE / choroid complex, providing data support for the pharmacokinetic part of the efficacy for effectively improving the abnormal neovascularization at the choroid.

[0122] Example 6

[0123] This example provides a comparative experiment of the efficacy of the polypeptide eye drops (AJ007 eye drops) in Example 2 with the multifunctional fusion polypeptide VI (HM-1) of equal molar 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:

[0124] Animal grouping: 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.

[0125] Experimental process: The mice were first dilated with compound tropicamide eye drops, and anesthetized by intraperitoneal injection of 4% chloral hydrate solution at 0.2mL / 20g of mouse body weight. After anesthesia, sodium hyaluronate gel was applied to protect the eyes, and a cover glass was attached. The left eye was laser modeled with a 532nm argon ion laser, with a power of 120mW, a burst time of 0.01s, and a spot size of 100μm. The drug was administered on the day after modeling, and each group was administered according to different administration methods. Only the left eye was given. The mice in the eye drop group were given eye drops 5 times a day, with an interval of 1h each time, and 5μL of the corresponding drug solution was dropped each time for 7 days; the mice in the vitreous injection group were anesthetized on the day after modeling and then given vitreous injection. Each mouse was injected with a microinjection needle with the corresponding drug solution (1μL was injected into each eye in the aflibercept group, and 5μL was injected into each eye in the HM-1 group). 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 stained with fluorescent staining to detect changes in CNV area.

[0126] Experimental results and conclusions: 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, which was comparable to the efficacy of intravitreal injection of the positive drug aflibercept ( Fig.12 and 13 ), among which AJ007 eye drops had a slightly better efficacy than aflibercept after intravitreal injection; 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 aflibercept after intravitreal injection of the positive drug ( Fig.14 and 15). The results of combined FFA examination and FITC-dextran staining after injection show that neither HM-1 nor HM-1 preparation nor HM-1 connected to TAT has the effect of improving the abnormal neovascularization of the mouse wAMD model. However, after HM-1 is connected to TAT and then made into a preparation, that is, AJ007 eye drops are administered by eye drops, the efficacy of which on the classic wAMD model is comparable to that of HM-1 injected into the vitreous of the eyeball at an equal molar concentration, or even slightly better. Even compared with the current best treatment for wAMD in clinical practice - intravitreal injection of aflibercept, the efficacy is comparable to or even slightly better. Considering that the patient compliance of the eye drop administration method is significantly better than that of the vitreous injection administration method, the polypeptide eye drops of the present invention have good clinical potential, provide a better choice for the clinical treatment of wAMD patients, and can generate good economic benefits.

[0127] According to the results of the above embodiments, the present application successfully optimized and upgraded the molecule by connecting the cell-penetrating peptide TAT and the selected excipients to the front end of the multifunctional fusion polypeptide VI (HM-1) sequence with strong angiogenesis inhibition ability. The results of ocular pharmacokinetics and the efficacy 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 with the original HM-1 molecule, and its efficacy was even slightly better than that of Aflibercept, the best clinical drug for wAMD. Overall, the effect of 1+1>2 was achieved, and the effect of the eye drops was 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 of Aflibercept is an invasive method of intravitreal injection, it is obvious that the non-invasive eye drops of the polypeptide eye drops are safer and more convenient for patients, avoiding various serious side effects caused by intravitreal injection, and have 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.

Images