CN103212075B - A kind of eye drops containing VEGF antagonist - Google Patents

Abstract

The invention relates to eye drops containing a VEGF antagonist, which contain a low-concentration VEGF antagonist, in particular contain 0.05-9.0mg/ml of VEGF antagonist.

Description

A kind of eye drops containing VEGF antagonist

technical field

The invention relates to the field of pharmaceutical preparations, in particular to an eye drop containing a VEGF antagonist.

Background technique

The incidence of ocular surface diseases such as corneal neovascularization, neovascular glaucoma, pterygium, and chronic conjunctivitis has a certain relationship with the generation of neovascularization. Overexpression of VEGF can induce ocular surface neovascularization. Corneal neovascularization is not an independent corneal disease, but a pathological change. There are many reasons for corneal neovascularization, such as immune inflammation, infection, degeneration, trauma, improper use of contact lenses, and iatrogenic diseases. If the corneal neovascularization caused by the above reasons is not treated in time, it will eventually lead to corneal blindness. Corneal neovascularization is the main cause of vision loss and blindness worldwide. About 4.14% of ophthalmic patients in the United States suffer from corneal neovascularization. There are 1.4 million new corneal neovascularization patients in the United States every year, and 12% of them will cause vision loss. There are about 170,000 new corneal neovascularization patients with vision loss in the United States every year. There is no epidemiological survey on corneal neovascularization in China. If corneal neovascularization accounts for 4% of ophthalmic patients in the United States, 12% of them will cause vision loss. In 2009, there were 62.83 million ophthalmic patients nationwide, and there are about 300,000 in China every year. of new patients suffer from vision impairment due to corneal neogenesis. Progression of corneal neovascularization in patients can lead to further deterioration of vision, and eventually require corneal transplantation to restore vision. Therefore, such patients need effective anti-neovascular treatment to prevent further loss of vision and avoid corneal transplantation. Doctors have no effective treatment for corneal neovascularization.

Biopharmaceuticals, especially recombinant proteins, have many characteristics completely different from small molecular chemical molecules, and their unstable degradation reaction is a multi-step reaction. Due to the limitations of current analytical methods, it is difficult to accurately measure these multi-step degradations that occur in higher-order structures, and its stability, especially its preservation under conventional conditions, is a big challenge. The eye is one of the most important organs of the human body: nearly 80% of the information of the brain comes from the eye; at the same time, due to its special physiological structure, the eye is a relatively weak tissue, which has very high requirements for viscosity and osmotic pressure. Therefore, the requirements for ophthalmic preparations, especially ophthalmic preparations adopted when there is trauma, are extremely strict.

The biological effects of VEGF (vascular endothelial cell growth factor) are all mediated by its specific receptor VEGFR (vascular endothelial cell growth factor receptor), which can lead to ligand-mediated dimerization, Receptor dimerization promotes autophosphorylation and dephosphorylation of adjacent receptor subunits, triggering signal transduction. VEGFR are all tyrosine protein kinases, which can be divided into fins-like tyrosine kinase-1 (VEGFR-1/Flt-1), kinase insertion region receptor (VEGFR-2/Flk-1/ KDR), fins-like tyrosine kinase-4 (VEGFR-3/Flt-4) and some low molecular weight VEGFR (neuropilin-1). Flt-1 and KDR are mainly distributed on vascular endothelial cells, while Flt-4 is mainly distributed on lymphatic endothelial cells. Flt-1 and KDR are mainly combined with VEGF. The binding force between Flt-1 and VEGF is higher than that of KDR. They are all glycosylated transmembrane receptors and directly participate in the signal transmission of VEGF into cells. The combination of Flt-1 and VEGF can promote the formation of vascular endothelial cells and regulate the permeability of blood vessels; the combination of KDR and VEGF can promote the proliferation and maturation of vascular endothelial cells. VEGF antagonists, especially a class of artificially designed targeted genetically engineered proteins, such as Lucentis, Avastin, VEGF-trap, etc., can effectively block signal transmission mediated by vascular endothelial growth factor (VEGF) and inhibit pathological changes The growth of new blood vessels, and used to treat tumors, eyes and other diseases caused by new blood vessels, but the preparations of the above-mentioned drugs are in the form of injections, and the way of administration is direct injection into the vitreous or systemic administration. In other words, its compliance is poor and there are many inconveniences. Therefore, the researchers began to study a kind of pharmaceutical preparation form containing VEGF antagonist that has good patient compliance and is convenient and effective. US7303748 discloses an eye drop containing VEGF-trap, and its preparation prescription: 39.4-103.06mg/ml VEGF-trap, 5 mM phosphoric acid, 5 mM citric acid, 100 mM sodium chloride, 0.005% Tween-20. However, the concentration of the eye drops is relatively high. Due to the instability of biological agents, many patients receiving anti-vascular therapy on the ocular surface are unwilling to be hospitalized. High-concentration biological agents can easily cause inconvenience in storage and use. Ocular surface administration is a special route of administration. For pH, osmotic pressure and local irritation, bacteriostats and antibacterial agents cannot be added. The above problems make the drugs used for ocular surface anti-neovascular treatment expensive and have limited effect . Therefore, providing a kind of eye drops with low concentration and definite curative effect of inhibiting neovascularization, and using it to treat various ocular surface diseases such as corneal neovascularization, neovascular glaucoma, pterygium, and chronic conjunctivitis, is necessary for satisfying clinical conditions. Requirements have significant meaning.

Contents of the invention

One of the technical problems to be solved by the present invention is to provide an eye drop containing VEGF antagonist with small concentration but definite curative effect.

In order to solve the above technical problems, the present invention provides the following technical solutions:

One aspect of the present invention provides an eye drop containing VEGF antagonist, the eye drop contains 0.05-9.0mg/ml of VEGF antagonist; preferably contains 0.1-1.0mg/ml of VEGF antagonist; more preferably contains 0.5mg/ml VEGF antagonist; wherein the VEGF antagonist is preferably a fusion protein containing fragments of FLT-1 and KDR.

The above-mentioned VEGF antagonist preferably has a fusion protein with one of the following structures:

a. FP1, a protein composed of the 2nd immunoglobulin-like region of FLT-1 and the 3rd immunoglobulin-like region of KDR fused with the Fc fragment of human immunoglobulin: FLTd2-KDRd3-Fc;

b. FP2, a protein composed of the 1st immunoglobulin-like region of KDR, the 2nd immunoglobulin-like region of FLT-1 and the 3rd immunoglobulin-like region of KDR and the Fc fragment of human immunoglobulin:

KDRd1-FLTd2-KDRd3-Fc;

c. FP3, a protein fused by the 2nd immunoglobulin-like region of FLT-1 and the 3rd-4th immunoglobulin-like region of KDR with the Fc fragment of human immunoglobulin: FLTd2-KDRd3, 4-Fc;

d. FP4, a protein fused with the 2nd immunoglobulin-like region of FLT-1, the 3rd immunoglobulin-like region of KDR and the 4th immunoglobulin-like region of FLT-1 and the Fc fragment of human immunoglobulin :

FLTd2-KDRd3-FLTd4-Fc;

e. FP5, a protein composed of the 2nd immunoglobulin-like region of FLT-1 and the 3rd-5th immunoglobulin-like region of KDR and the Fc fragment of human immunoglobulin: FLTd2-KDRd3, 4, 5-Fc ;or

f.FP6, which is fused with the 2nd immunoglobulin-like domain of FLT-1, the 3rd immunoglobulin-like domain of KDR and the 4th-5th immunoglobulin-like domain of FLT-1 with the Fc fragment of human immunoglobulin protein:

FLTd2-KDRd3-FLTd4,5-Fc;

g. FP7, a protein fused by the 2nd immunoglobulin-like domain of FLT-1 and the 3rd-4th immunoglobulin-like domain of KDR: FLTd2-KDRd3, 4;

h. FP8, a protein fused from the second immunoglobulin-like domain of FLT-1 and the third immunoglobulin-like domain of KDR: FLTd2-KDRd3.

Amino acids FLT-1D2, FLT-1D4, KDRD1, KDRD3, and KDRD4 of the above FLT-1 and KDR immunoglobulin-like regions are shown in Sequence Table 1-5, the amino acid sequence of FP3 protein is shown in Sequence Table 6, and the amino acid sequence of FP1 protein is shown in Sequence List 7, the amino acid sequence of FP7 protein is shown in Sequence Table 8, and the amino acid sequence of FP8 protein is shown in Sequence Table 9.

In the eye drops provided by the invention, one or more of the following components can also be contained:

(a) 5-100mM buffer solution, wherein the acid is selected from one or more of Tris-HCl, citric acid, phosphoric acid, sodium hydrogen phosphate, sodium dihydrogen phosphate, acetic acid, succinic acid, hydrochloric acid;

(b) 5-500mM basic amino acid is selected from one or a combination of lysine, arginine, and histidine;

(c) 0.1-30% salt osmotic pressure regulator, wherein the sugar is selected from one or more of sucrose, trehalose, mannitol, glycerin, propylene glycol, sorbitol, and the salt is selected from sodium chloride or other One or a combination of pharmaceutically acceptable salts;

(d) 0.005-0.1% of one or more surfactants or co-solvents selected from polyethylene glycol, Tween 20, Tween 80, propylene glycol, dimethyl sulfoxide or other pharmaceutically acceptable surface one or more of the active agents;

The pH of the solution prepared by one or more of the above components is 6-8.3.

The VEGF antagonist described in the above eye drops is most preferably a fusion protein such as SEQ ID No:6.

The present invention further provides eye drops containing the following components, the specific components are as follows:

(a) 0.05-9mg/ml fusion protein such as SEQ ID No: 6;

(b) 5-250 mM citric acid;

(c) one or both of 5-500 mM arginine or histidine;

(d) 4-30% sucrose or trehalose;

(e) 0.01-0.1% surfactant or co-solvent, one or both selected from polyethylene glycol or Tween 20;

(f) Adjust the pH to 7.5-8.3.

The present invention further provides eye drops containing the following components, the specific components are as follows:

(a) 0.1-1 mg/ml fusion protein such as SEQ ID No: 6;

(b) 10-50 mM citric acid;

(c) one or both of 50-100 mM arginine or histidine;

(d) 5-20% sucrose;

(e) 0.01-0.1% surfactant or co-solvent, one or both selected from polyethylene glycol or Tween 20;

(f) Adjust the pH to 7.5-8.3.

The present invention further provides eye drops containing the following components, the specific components are as follows:

(a) 0.5 mg/ml fusion protein as SEQ ID No: 6;

(b) 50 mM citric acid;

(c) Arginine at 250 mM;

(d) 12.5% sucrose;

(e) 0.05% Tween-20;

(f) Adjust the pH to 7.5-8.3.

The present invention also provides the application of the above-mentioned eye drops in the preparation of medicines for the treatment of ocular surface diseases caused by angiogenesis or growth; Any of the pterygium or its complications.

The technical contents disclosed in Chinese patents ZL200510073595.4 and ZL200610066257.2 are referred to in this application; the technical contents in Chinese unpublished patent CN 201010267503.7 are also referred to in this application.

Compared with the prior art, the present invention has the advantages that the content of the fusion protein in the eye drops is extremely low, and the curative effect is definite, and the production cost can be greatly saved on the basis of the high stability of the low-concentration eye drops.

detailed description

The following examples are only used as a further explanation of the present invention, and cannot be regarded as limiting the protection scope of the present invention.

Example 1, Conbercept Eye Drops Study on Corneal Neovascularization Induced by Alkali Burn

Drugs: Chlortetracycline Hydrochloride Eye Ointment, specification batch number: 2.0g/bottle, 411002, valid until December 2014, Chongqing Kerui Pharmaceutical Co., Ltd.; lidocaine hydrochloride injection, specification batch number: 5ml/bottle, 0.1g/bottle, Valid until April 2012, Tianjin Pharmaceutical Jiaozuo Co., Ltd.

Reagent: sodium hydroxide (NaOH), specification batch number: 500g/bottle, 20091223, Chengdu Kelong Chemical Reagent Factory.

Samples tested:

A, Conbercept eye drops (prepared according to Example 3) 10mg/ml, colorless transparent liquid, 1ml/bottle, batch number: FR1108001, stored at 2-8°C, added dropwise to the surface of the eye when used;

B, Conbercept eye drops (prepared according to Example 3) 9mg/ml, colorless transparent liquid, 1ml/bottle, batch number: FR1108002, stored at 2-8°C, added dropwise to the surface of the eye when used;

C, dexamethasone, colorless transparent liquid, 1ml/bottle, batch number: FR1108003, stored at 2-8°C, drop onto the surface of the eye when used;

D, Conbercept eye drops (prepared according to Example 3) 0.1mg/ml, colorless transparent liquid, 1ml/bottle, batch number: FR1108004, stored at 2-8°C, added dropwise to the surface of the eye when used;

E, Conbercept preparation buffer (buffer) not included (prepared according to Example 16), colorless transparent liquid, 1ml/bottle, batch number: FR1108005, stored at 2-8°C, added dropwise to the surface of the eye when used;

F, Conbercept eye drops (prepared according to Example 3) 5mg/ml, colorless transparent liquid, 1ml/bottle, batch number: FR1108006, stored at 2-8°C, added dropwise to the surface of the eye when used;

All are provided by the Preparation Room of the Chinese Medicine Research Department of the Enterprise Technology Center of Kanghong Pharmaceutical Group.

Experimental method and results:

Get 48 healthy New Zealand rabbits without eye disease, weigh their body weight, anesthetize with 3% pentobarbital sodium (1ml/kg), and apply local anesthetic lidocaine hydrochloride solution on the eye surface, the dose is 20 μ l/eye; preparation 9mm diameter filter paper, soak in 1mol/L NaOH solution for about 10s, place the filter paper on the dry filter paper with tweezers to absorb excess NaOH solution, place the filter paper soaked in NaOH in the center of the cornea of both eyes of the rabbit for 60s Take it off, quickly take the washing bottle and rinse the cornea with about 20ml of normal saline, and give prophylactic antibiotics to prevent infection (chlortetracycline hydrochloride eye ointment), 3 times/2 days.

Immediately after modeling, it was divided into burn injury, A (Conbercept eye drops (prepared according to Example 3) 10mg/ml), B (Conbercept eye drops (prepared according to Example 3) 9mg/ml), C (dexamethasone), D (Conbercept eye drops (prepared according to Example 3) 0.1mg/ml), E (preparation buffer solution not containing Conbercept (prepared according to Example 16)) and F (conbercept eye drops (prepared according to Example 3) 5 mg/ml) seven groups, the day of modeling was 0 day, from the first day, groups A to F were given corresponding drugs, and the administration frequency was 6 times /day, the administration dose was 50 μl/eye each time, and the administration was continued for 10 days, and another 2 New Zealand rabbits were selected as the normal group. At the same time of administration every day, observe the growth state of corneal NV and whether there is an inflammatory reaction in the eye.

On the 10th day of administration, the animals were anesthetized with 3% pentobarbital sodium solution (1ml/kg), and the local anesthetic lidocaine hydrochloride solution was administered to the surface of the eyes, the dose was 20 μl/eye, and the slit lamp 10 times objective lens Observe the clockwise direction of the rabbit cornea NV, and take pictures under the 10x and 16x objective lenses. The collected images were corrected by the number of hours in Photoshop CS, and the area of corneal neogenesis was processed by Image Pro Plus; the area formula: S=C/12×3.1416×[R ² -(RL) ² ], C means that there is no NV at the edge of the cornea in the picture The number of hours to the point of no NV growth, R indicates the length from the cornea-sclera contact edge to the cornea center in the picture, L indicates the cornea-sclera contact edge NV root to the cornea NV end in the picture For NV length, the length of the longest vessel is taken in each hour. All statistical analysis of data was performed by T-test analysis of variance. data to , is the mean, and s is the standard deviation.

Through general ocular surface observation, it was found that the vascular network congestion of the corneal edge of each group was obvious on the first and second day of burn; on the third day of burn, there was NV growth on the edge of the cornea; The preparation buffer solution containing Conbercept has obvious NV growth on the corneal surface, dexamethasone, Conbercept Eye Drops 10mg/ml, Conbercept Eye Drops 9mg/ml and Conbercept Eye Drops 0.1 The growth of NV in the mg/ml group was not obvious; on the 7th day, the growth of NV on the corneal surface of each group was obvious, and the corneal NV in the burn group and the preparation buffer solution group without Conbercept grew toward the burned cornea, and part of the NV had grown to the edge of the burned cornea , while dexamethasone, Conbercept eye drops 10mg/ml, Conbercept eye drops 9mg/ml, Conbercept eye drops 5mg/ml and Conbercept eye drops 0.1mg/ml There were few NVs growing into the burnt cornea in the burn group; on the 10th day, NVs had grown into the burnt cornea in the burn group and the preparation buffer solution group without conbercept, while dexamethasone, conbercept 10mg/ml, Conbercept eye drops 9 mg/ml, Conbercept 5 mg/ml and Conbercept 0.1 mg/ml groups had less NV growth into the burned cornea.

Effect of corneal neovascularization area: The data analysis results of corneal NV area on the 10th day showed that compared with the alkali burn group and the preparation buffer solution group without Conbercept, dexamethasone, Conbercept 10mg/ml, and Compaq 9 mg/ml of Heptrop Eye Drops, 5 mg/ml of Conbercept and 0.1 mg/ml of Conbercept can significantly inhibit the growth of corneal NV and reduce the area of NV, which is statistically significant (P<0.05), among which dexamethasone Conbercept 0.1mg/ml inhibited NV growth best (P<0.01). Although the preparation buffer solution without conbercept had no inhibitory effect on corneal neovascularization, and the NV area was larger than that of the burn group, but there was no significant difference. The results are shown in Table 1.

Table 1 Effects of various drugs on corneal NV in rabbits induced by alkali burn

Note: The negative control is buffer solution without drugs, and the positive control is dexamethasone. Compared with burn group, ^* P<0.05, ^** P<0.01; compared with group B, ^# P<0.05, ^## P<0.01 ; Compared with group E, ^P<0.05, ^^P<0.01.

Example 2 Effect of low-dose Conbercept eye drops on corneal NV growth caused by alkali burn

Drugs: Chlortetracycline Hydrochloride Eye Ointment, specification batch number: 2.0g/bottle, 411002, valid until December 2014, Chongqing Kerui Pharmaceutical Co., Ltd.; lidocaine hydrochloride injection, specification batch number: 5ml/bottle, 0.1g/bottle, Valid until April 2012, Tianjin Pharmaceutical Jiaozuo Co., Ltd.

Reagent: sodium hydroxide (NaOH), specification batch number: 500g/bottle, 20091223, Chengdu Kelong Chemical Reagent Factory.

Samples tested:

A, Conbercept Eye Drops (prepared according to Example 3) 0.5mg/ml, colorless transparent liquid, 800 μl/bottle, batch number: 20111001, stored at 2-8°C, added dropwise to the surface of the eye when used;

B, Conbercept eye drops (prepared according to Example 3) 0.1 mg/ml, colorless transparent liquid, 800 μl/bottle, batch number: 20111001, stored at 2-8°C, added dropwise to the surface of the eye when used;

C, preparation buffer solution without Conbercept, colorless transparent liquid, 800μl/bottle, batch number: 20111001, stored at 2-8°C, and added dropwise to the surface of the eye when used;

D, Conbercept eye drops (prepared according to Example 3) 0.01 mg/ml, colorless transparent liquid, 800 μl/bottle, batch number: 20111001, stored at 2-8°C, added dropwise to the surface of the eye when used;

E, dexamethasone, colorless transparent liquid, 800μl/bottle, batch number: 20111001, stored at 2-8°C, drop onto the surface of the eye when used;

F, Conbercept eye drops (prepared according to Example 3) 0.05 mg/ml, colorless transparent liquid, 800 μl/bottle, batch number: 20111001, stored at 2-8°C, added dropwise to the surface of the eye when used;

G, Conbercept eye drops (prepared according to Example 3) 1mg/ml, colorless transparent liquid, 800 μl/bottle, batch number: 20111001, stored at 2-8°C, added dropwise to the surface of the eye when used;

All are provided by the Preparation Room of the Chinese Medicine Research Department of the Enterprise Technology Center of Kanghong Pharmaceutical Group.

Experimental method and results:

The test is divided into A (Conbercept eye drops (prepared according to Example 3) 0.5mg/ml), B (Conbercept eye drops (prepared according to Example 3) 0.1mg/ml), C (not Conbercept-containing formulation buffer (prepared according to Example 16) buffer), D (Conbercept eye drops (prepared according to Example 3) 0.01mg/ml), E (dexamethasone), F ( Conbercept eye drops (prepared according to Example 3) 0.05mg/ml) and G (Conbercept eye drops (prepared according to Example 3) 1mg/ml) eight groups.

Test method operation is the same as embodiment 1.

The observation of the ocular surface showed that the vascular network of the corneal edge of the burnt eyes in each group was obviously congested and some eyelids were red and swollen on the first and second day of burn; There were NV growth on the corneal surface, but some rabbit eyelids appeared red and swollen; on the 7th day, the NV growth on the corneal surface of each group was obvious, with obvious centripetal growth, while the corneal NV in the dexamethasone group was less, but the rabbit eyelids were red and swollen It was more serious; on the 10th day, corneal NV growth was obvious in each group containing Conbercept, and some corneal neovascularization had grown into the burn site, while NV growth into the corneal burn site was less in the dexamethasone group, and there was no eyelid redness and swelling in the rabbits Recovery, while each group containing Conbercept did not have redness and swelling of rabbit eyelids.

Effect of corneal neovascularization area: The data analysis results of corneal NV area on the 10th day showed that compared with the preparation buffer solution group without conbercept, dexamethasone and conbercept eye drops 0.5mg/ml could significantly inhibit Corneal NV grew and reduced NV area, which was statistically significant (P<0.05). Conbercept eye drops 1mg/ml and Conbercept eye drops 0.1mg/ml can also inhibit the growth of corneal NV. However, Conbercept eye drops 0.01mg/ml had no inhibitory effect on corneal neovascularization, and the NV area was larger than that of the burn group, but there was no significant difference. See Table 2.

Table 2 Effects of various drugs on corneal NV in rabbits caused by alkali burn

Note: Buffer control, ^* P<005, ^** P<001.

Embodiment 3, the preparation of Conbercept (FP3 fusion protein) eye drops

prescription:

FP3 fusion protein 10mg/ml, 9mg/ml, 5mg/ml, 1mg/ml, 0.5mg/ml, 0.1mg/ml, 0.05mg/ml or 0.01mg/ml

Preparation method: After thawing the concentrated FP3 fusion protein stock solution after changing the liquid, in a clean bench (sterile cabinet) of class C, add filter-sterilized solution containing 55mM citric acid, 12.5% sucrose, 250mM Arginine and 0.05% Tween 20 buffer, filter, adjust FP3 fusion protein to 10mg/ml, 9mg/ml, 5mg/ml, 1mg/ml, 0.5mg/ml, 0.1mg/ml, 0.05mg /ml or 0.01mg/ml, pH 7.5-8.3, aseptically dispensed into eye drop containers and stored at 2-8°C.

Embodiment 4, the preparation of Conbercept (FP3 fusion protein) eye drops

prescription:

Preparation method: After thawing the concentrated FP3 fusion protein stock solution after liquid exchange, in a C-level clean bench (sterile cabinet), add filter-sterilized solution containing 10mM citric acid, 8.0% sucrose, 5mM The buffer solution of arginine and 0.05% Tween 20 is filtered and adjusted to 0.5 mg/ml of FP3 fusion protein, the pH is 7.5-8.3, and aseptically dispensed into containers for eye drops and stored in 2-8 ℃.

The preparation of embodiment 5 Conbercept (FP3 fusion protein) eye drops

prescription:

Preparation method: After thawing the concentrated FP3 fusion protein stock solution after liquid exchange, in a C-level clean bench (sterile cabinet), add filter-sterilized solution containing 100mM citric acid, 20.0% sucrose, 250mM Arginine and 0.10% Tween 20 buffer solution, filter, adjust FP3 fusion protein to 1.0 mg/ml, pH 7.5-8.3, aseptically dispense into eye drop containers and store at 2-8°C .

The preparation of embodiment 6 Conbercept (FP3 fusion protein) eye drops

prescription:

Preparation method: After thawing the concentrated FP3 fusion protein stock solution after changing the solution, in a clean bench (sterile cabinet) of class C, add filter-sterilized solution containing 5mM sodium dihydrogen phosphate and 10.0% seaweed according to the aseptic method. The buffer solution of sugar, 100mM arginine and 0.01% PEG400, filter, adjust the FP3 fusion protein to 0.1mg/ml, the pH is 7.5-8.3, aseptically dispense it into a container containing eye drops, and store it in 2- 8°C.

The preparation of embodiment 7 Conbercept (FP3 fusion protein) eye drops

prescription:

Preparation method: After thawing the FP3 fusion protein stock solution after concentrated liquid replacement, in a clean bench (sterile cabinet) of class C, add filter-sterilized solution containing 5mM citric acid, 4.0% sucrose, 4.0 % sodium chloride, 100mM arginine, 100mM histidine, 0.05% Tween 20, 0.05% PEG400 buffer, filter, adjust FP3 fusion protein to 0.05mg/ml, pH 7.5-8.3, sterile Put it in a container containing eye drops and store it at 2-8°C.

The preparation of embodiment 8 Conbercept (FP3 fusion protein) eye drops

prescription:

Preparation method: After thawing the concentrated FP3 fusion protein stock solution after liquid exchange, in a C-level clean bench (sterile cabinet), add filter-sterilized solution containing 10mM citric acid, 30.0% sucrose, 500mM Arginine, 0.1% Tween 20 buffer, filter, adjust FP3 fusion protein to 5.0 mg/ml, pH 7.5-8.3, aseptically dispense into eye drops containers, store in 2-8 ℃.

The preparation of embodiment 9 Conbercept (FP3 fusion protein) eye drops

prescription:

Preparation method: After thawing the concentrated FP3 fusion protein stock solution after liquid exchange, in a C-level clean bench (sterile cabinet), add filter-sterilized solution containing 10mM citric acid, 5.0% sucrose, and 100mM Arginine, 0.05% Tween 20 buffer, filter, adjust FP3 fusion protein to 0.5mg/ml, pH 7.5-8.3, aseptically dispense into eye drop containers, store in 2-8 ℃.

The preparation of embodiment 10 Conbercept (FP3 fusion protein) eye drops

prescription:

Preparation method: After thawing the concentrated FP3 fusion protein stock solution after changing the liquid, in a clean bench (sterile cabinet) of class C, add filter-sterilized solution containing 250mM citric acid, 8.0% sucrose, 100mM Histidine, 0.10% Tween 20 buffer, filter, adjust FP3 fusion protein to 0.5mg/ml, pH 7.5-8.3, aseptically dispense into eye drop containers, store in 2-8 ℃.

The preparation of embodiment 13 Conbercept (FP3 fusion protein) eye drops

prescription:

Preparation method: After thawing the concentrated FP3 fusion protein stock solution after liquid exchange, in a C-level clean bench (sterile cabinet), add filter-sterilized solution containing 10mM citric acid, 5.0% sucrose, and 100mM Arginine, 0.05% Tween 20 buffer, filter, adjust FP3 fusion protein to 0.5mg/ml, pH 7.5-8.3, aseptically dispense into eye drop containers, store in 2-8 ℃.

The preparation of embodiment 14 series fusion protein eye drops

prescription:

Preparation method: After thawing the original solution of various fusion proteins (FP1, FP2, FP4, FP5, FP6, FP7, FP8) after concentrated liquid exchange, in a clean bench (sterile cabinet) of class C, perform aseptic operation Add filter-sterilized buffer solution containing 55mM citric acid, 12.5% sucrose, 250mM arginine and 0.05% Tween 20, filter, adjust the fusion protein to 9mg/ml, 5mg/ml, 1mg/ml, 0.5mg respectively /ml, 0.1mg/ml or 0.05mg/ml, pH 6.0-8.3, aseptically dispensed into eye drop containers and stored at 2-8°C.

The preparation of embodiment 15FP1 fusion protein eye drops

prescription:

Preparation method: After thawing the concentrated FP1 fusion protein stock solution after changing the liquid, in a clean bench (sterile cabinet) of class C, add filter-sterilized solution containing 5mM phosphoric acid, 5mM citric acid, and 100mM chlorine according to the aseptic method. NaCl and 0.005% Tween 20 buffer solution, filtered, adjusted fusion protein to 1.0 mg/ml, pH 6.0-8.0, aseptically dispensed into eye drop containers and stored at 2-8°C.

Embodiment 16 does not contain the formulation buffer preparation of Conbercept (FP3 fusion protein)

prescription:

Preparation method: add filter-sterilized buffer solution containing 55mM citric acid, 12.5% sucrose, 250mM arginine and 0.05% Tween 20 according to the aseptic method, filter, adjust the pH to 7.5-8.3 respectively, and sterile Dispense it into a container for eye drops and store it at 2-8°C.

Example 17 Stability of 0.1mg/ml Conbercept (FP3 Fusion Protein) Eye Drops at 25°C

1. Prepare 0.1mg/ml Conbercept (FP3 fusion protein) eye drops according to Example 3

2. Take the above-mentioned 0.1mg/ml Conbercept (FP3 fusion protein) eye drops, reserve a sample at 25°C, , Samples were assayed in December and stability was determined by SEC-HPLC. The results show that the formulation can effectively inhibit the formation of polymers, the purity of the product does not decrease basically, and the affinity between the fusion protein and VEGF hardly changes.

The specific results are shown in Table 3.

Table 3.0.1mg/ml FP3 fusion protein stability at 25°C

time (month) Exterior pH value polymer(%) Affinity (pM) 0 qualified 7.2 0.01 10.6 1 qualified 7.4 0.03 10.5 2 qualified 7.5 0.03 10.4 3 qualified 7.5 0.05 10.4 6 qualified 7.7 0.07 10.2 9 qualified 7.6 0.08 10.3 12 qualified 7.5 0.09 10.1

Example 18 Stability of 10mg/ml Conbercept (FP3 Fusion Protein) Eye Drops at 4°C

1. Prepare 10mg/ml Conbercept (FP3 fusion protein) eye drops according to Example 3

2. Take the above-mentioned 10mg/ml Conbercept (FP3 fusion protein) eye drops, keep the sample at 4°C, and put them at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, Samples were assayed in December and stability was determined by SEC-HPLC. The results show that the formulation can effectively inhibit the formation of polymers, the polymers in the product increase slowly, and the affinity between the fusion protein and VEGF is almost unchanged. The specific results are shown in Table 4.

Table 4. Stability of 10mg/ml FP3 fusion protein at 4°C

Claims (4)

1. An eye drop containing a VEGF antagonist, characterized in that it contains (a) fusion protein such as SEQ ID No:6 of 0.1-1mg/ml; (b) 10-50 mM citric acid; (c) one or both of 50-100 mM arginine or histidine; (d) 5-20% sucrose; (e) 0.01-0.1% surfactant or co-solvent, one or both selected from polyethylene glycol or Tween 20; (f) Adjust the pH to 7.5-8.3. 2. The eye drop according to claim 1, characterized in that it contains (a) fusion protein such as SEQ ID No:6 of 0.5mg/ml; (b) 10 mM citric acid; (c) 100 mM arginine; (d) 5% sucrose; (e) 0.05% Tween-20; (f) Adjust the pH to 7.5-8.3. 3. Use of the eye drop according to any one of claims 1-2 in the preparation of a medicament for treating ocular surface diseases caused by angiogenesis or growth. 4. The purposes of the eye drops according to claim 3 in the preparation of medicines for the treatment of ocular surface diseases caused by angiogenesis or growth, characterized in that the ocular surface diseases are neovascularization after keratoplasty, corneal neovascularization , Ocular surface neovascularization or any one of pterygium or its complications.