WO2018195912A1 - Ophthalmic pharmaceutical composition and use thereof - Google Patents

Abstract

Disclosed are an ophthalmic pharmaceutical composition comprising an antibody or a derivative thereof capable of antagonistically inhibiting the binding of a vascular endothelial growth factor to the receptor thereof, and one or more pharmaceutically acceptable excipients for use in an ophthalmic therapeutic agent, wherein the light chain antigen complementarity determining region of the antibody or the derivative thereof has an amino acid sequence selected from one as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and the heavy chain antigen complementarity determining region of the antibody or the derivative thereof has an amino acid sequence selected from one as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; and the use of the ophthalmic pharmaceutical composition in the preparation of a drug for treating an ocular disease or condition associated with angiogenesis.

Description

Ophthalmic pharmaceutical composition and use thereof Technical field

The present invention is in the field of biotechnology-monoclonal antibodies, and in particular, the invention relates to an ophthalmic pharmaceutical composition and use thereof.

Background technique

Angiogenesis or angiogenesis is the process by which blood vessels (such as capillaries and tiny movements, veins) that are already present in the body produce new blood vessels by budding or dividing.

Angiogenesis is beneficial and necessary to maintain many normal physiological processes in the body such as tissue embryo development, healing and repair of traumatic wounds; however, excessive angiogenesis or hyperplasia can also cause disease in the body. Such as the proliferation and recurrence of tumors such as tumors, age-related macular degeneration (AMD), fundus diseases, diabetic macular edema (DME), inflammatory response and self Immune diseases and the like are closely related to angiogenesis or hyperplasia.

The key to the proliferation and growth of blood vessels in the body is that the lining of the vascular endothelial cells has the ability to continuously divide and proliferate and orientate and implant into the existing vessel wall. The most important and strong substance known to promote vascular endothelial cell division or angiogenesis is vascular endothelial growth factor (VEGF). VEGF, also known as vascular permeability factor (VRF), has been published by the two research groups in 1989 and in the journal Science in the United States.

VEGF by specifically interacting with VEGF receptors (VEGF-R1, VEGF-R2) on vascular endothelial cells In combination, it plays a role in promoting vascular endothelial cell growth and migration, angiogenesis, and increasing vascular permeability. The importance of VEGF and its receptor-mediated angiogenesis has been well documented in studies of VEGF knockout mice: because as soon as one of the VEGF genes is knocked out, mouse embryos only develop to 11 At 12 days, it will die due to obstruction and abnormality of vascular proliferation.

VEGF and its receptor-mediated angiogenesis and vascular invasion also play a key role in the occurrence and pathological progression of blinding fundus diseases such as age-related macular degeneration and diabetic macular edema. Most of the age-related macular degeneration occurs in more than 50 years old, and the prevalence increases with age. The clinical symptoms of the disease are central vision loss and rapid progression of the disease, which is an important disease leading to blindness in the elderly. Diabetic macular edema is a major disease that currently jeopardizes tens of millions of middle-aged working-class vision.

The main pathological manifestations of age-related macular degeneration and diabetic macular edema are atrophy and degeneration of macular and peripheral tissues, destruction of visual cells, formation of drusen, and exudative macular degeneration in severe cases, accompanied by subretinal neovascularization, hemorrhage, and exudation. In the past, laser treatment and photodynamic therapy were often used for blinding fundus diseases such as age-related macular degeneration and diabetic macular edema. Although the symptoms can be temporarily relieved, the recurrence rate is high and does not prevent the progression of the disease.

Since 2005, various VEGF inhibitors have been used clinically to treat exudative macular degeneration and diabetic macular edema. They have achieved satisfactory results and have been recommended as routine by the National Eye Institute (NEI). therapy.

So far, VEGF inhibitor-listed drugs approved by the US FDA for the treatment of angiogenesis-related fundus diseases mainly include the following three categories:

(1) Pegaptanib injection (Pegaptanib, trade name Macugen), developed by Eyetech Pharmaceuticals, Inc. and Pfizer Inc. It was approved by the US FDA in December 2004. Pegaptanib sodium is a PEG-modified single-stranded RNA analog with a unique three-dimensional structure that allows it to specifically bind to VEGF and inhibit VEGF activity.

(2) An antibody or antibody derivative that antagonizes VEGF. Currently, the only drugs approved by the US FDA for this class of drugs are Ranibizumab (Chinese name: Novo, English trade name: Lucentis), which is used to treat age-related macular degeneration and diabetic macula. Blind fundus diseases such as edema. The indications for the initial development of ranibizumab and Genentech are another antibody for bevacizumab (Bevacizumab, trade name Avastin) for the treatment of multiple tumors: both are derived from murine monoclonal antibody A4. Derived from 6.1, its variable region homology is above 99%. Bevacizumab is a full-length antibody with a molecular weight of approximately 149 kd, while ranibizumab is a Fab fragment with a molecular weight of approximately 48 kd. Like Avastin, ranibizumab binds with high affinity to VEGF, competitively blocks VEGF signaling, inhibits neovascularization and promotes exudate absorption in the macular area. Clinical studies initiated by Genentech have shown that after 2 years of treatment with ranibizumab, 95% of the "wet" AMD eyes have stable or improved vision. Based on this excellent efficacy, Raychemizumab was approved by the US FDA in June 2006. Ramizumab was subsequently approved in the United States for the treatment of diabetic macular edema, retinal vein occlusion secondary to macular edema, pathological myopia secondary to choroidal neovascularization, etc., and ranibizumab has now become a treatment for AMD. Mainstream drugs that are associated with angiogenesis and fundus diseases.

In China, Raychem monoclonal antibody was approved by the China Food and Drug Administration for the treatment of AMD in 2011.

(iii) VEGF receptor-Fc fusion protein. The drug is composed of a VEGF receptor extracellular domain and a human immunoglobulin-Fc segment. A representative drug is Aflibercept (trade name: Eylea) developed by Regeneron. Aboxicept is fused from the second domain of the extracellular domain of the VEGF-R1 receptor, the third domain of the extracellular domain of VEGF-R2, and the Fc segment of human immunoglobulin IgG1. The affinity of VEGF-Trap to VEGF is higher than the natural affinity of its receptor and ligand, and it can be competitively suppressed. Binding of VEGF-R to VEGF in humans. Absap has been approved by the US FDA for the treatment of blinding fundus diseases including age-related macular degeneration and diabetic macular edema since 2011, and is currently in the approved scope of clinical indications and sales in multiple markets worldwide. Are catching up with Raymondumab.

Conbercept developed by Chengdu Kanghong Pharmaceutical Group Co., Ltd. is also a VEGF receptor-Fc fusion protein. Composcipal is very similar in structure to aboxicept, consisting of the VEGF-R1 extracellular domain 2 domain, the VEGF-R2 extracellular domain 3 and 4 domains, and the human immunoglobulin IgG1 Fc fragment fusion. Made. Compaqip was approved by the Chinese CFDA in 2013 for the treatment of diseases such as age-related macular degeneration in the elderly, and is currently undergoing clinical phase III studies in the United States.

In summary, the VEGF inhibitor drugs currently approved worldwide for the treatment of fundus diseases such as AMD/DME are Fab fragments of VEGF monoclonal antibodies or VEGF receptor-antibody Fc fusion proteins. The monoclonal antibody-Fab fragment, such as ranibizumab, has the advantage of being relatively easy to penetrate quickly into the lesion after the injection of the fundus because of its small molecular weight; however, the half-life of the monoclonal antibody-Fab fragment in the eye is only 2-3 days. Shorter than intact antibodies, thereby increasing the number of doses and the economic burden on the patient. VEGF receptor-Fc fusion protein drugs such as Abbasi popular Compaqip because its binding affinity to ligands is higher than its natural receptor (VEGFR-1, VEGFR-2); and its intraocular half-life after fundus injection It is 3-5 days, slightly longer than Raymond monoclonal antibody, so it has the advantages of high efficacy and low frequency of drug use. However, Abbey's popularity of Composcipation is also associated with other growth factors such as VEGF-B, VEGF-C, and PLGF (Placental growth factor), thereby increasing the therapeutic purpose (specifically blocking VEGF-mediated). The potential risk of inconsistent adverse drug reactions.

Compared with Fab fragments or Fc fusion proteins, intact, full-length antibodies such as Avastin have the dual advantages of Fab segment and Fc segment, and have the advantages of long half-life in vivo and low frequency of administration. It is expected to be clinically less dosed or Lower injection frequency is used to treat angiogenic eye diseases such as AMD/DME. In recent years, data from preclinical and clinical studies have shown that the full-length antibody Avastin is used to treat AMD/DME. In fact, it is not inferior to rapizumab or abashi. However, Avastin has not been approved by the FDA or other national drug regulatory authorities for the treatment of other indications other than malignant tumors such as ophthalmic indications; Avastin's ophthalmic use in the treatment of AMD/DME is “non-label use” or “out-of-label use” ". Drugs used to treat tumor indications and ophthalmic indications for the treatment of AMD/DME have many differences in drug formulation, dosage, manufacturing and testing release standards, and product packaging. For the treatment of ophthalmic indications such as AMD/DME, the fundus injection is required. In addition to strict maintenance of the sterility, the pH and osmotic pressure should be close to the tears, and the insoluble particulate matter should be controlled at a very low level. Avastin for the treatment of tumors does not consider these characteristics and requirements for ophthalmic use in its manufacture and packaging. In addition, in order to reduce the cost of fundus injection, clinical patients or doctors often divide the large-package Avastin (4ml, or 20ml per bottle) into small portions for patients to share, and patients receive fundus injection. The risk of infection or inflammation often occurs after the drug is loaded.

The market for anti-angiogenic drugs for AMD/DME benefits millions of patients, and patients need to be administered all year round or repeatedly. The only anti-angiogenic ophthalmic drug, ranibizumab, and Abbasi popular Compaqip The market demand is not met; on the other hand, the three drugs currently on the market are not effective for all AMD/DME patients. Therefore, there is still a need to develop new or safer and effective anti-angiogenic ophthalmic drugs both clinically and in the market.

Summary of the invention

The present invention provides an ophthalmic pharmaceutical composition comprising an antibody or a derivative thereof capable of antagonizing the inhibition of binding of a vascular endothelial growth factor to its receptor, and one or more pharmaceutically acceptable excipients for an ophthalmic therapeutic agent The light chain antigen complementarity determining region of the antibody or its derivative has an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; The decision region has an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. In a preferred embodiment of the ophthalmic pharmaceutical composition, wherein the light chain variable region of the antibody or derivative thereof has the amino acid sequence set forth in SEQ ID NO: 7, the heavy chain variable region thereof has the SEQ ID NO: The amino acid sequence shown in 8. Among them, SEQ ID NO: 1-8 are shown in the following table:

Table 1: Amino acid sequence

In a preferred embodiment of the ophthalmic pharmaceutical composition, based on the total volume of the ophthalmic pharmaceutical composition, wherein the one or more pharmaceutically acceptable excipients comprise from 0.01 to 0.15% (m/v) , g/ml) of an organic cosolvent selected from the group consisting of polysorbate (Tween), polyethylene glycol, propylene glycol, and combinations thereof; and 1-10% (m/v, g/ml) ) selected from the group consisting of sucrose, sorbitol, glycerin, and trehalose And a stabilizer for at least one of mannitol.

Preferably, the one or more pharmaceutically acceptable excipients comprise from 0.01 to 0.02% (m/v, g/ml) of polysorbate and 7-based, based on the total volume of the ophthalmic pharmaceutical composition. 9% (m/v, g/ml) of sucrose.

Preferably, the one or more pharmaceutically acceptable excipients comprise from 0.03 to 0.04% (m/v, g/ml) of polysorbate and 0-based on the total volume of the ophthalmic pharmaceutical composition. 5% (m/v, g/ml) of sucrose

In a preferred embodiment of the ophthalmic pharmaceutical composition, wherein the antibody or derivative thereof has a protein concentration in the formulation of from 0.1 to 50 mg/ml.

Preferably, wherein the antibody or derivative thereof has a protein concentration in the pharmaceutical composition of from 1 to 10 mg/ml.

Preferably, the ophthalmic pharmaceutical composition has an osmotic pressure of from 285 to 310 mOsmol/kg, preferably 300 mOsmol/kg.

Preferably, the ophthalmic pharmaceutical composition has a pH of from 5.5 to 6.5, more preferably a pH of 6.0.

The invention also provides the use of the above ophthalmic pharmaceutical composition for the manufacture of a medicament for the treatment of an ocular disease or condition associated with angiogenesis.

In the use, the ocular disease or condition associated with angiogenesis is selected from the group consisting of age-related macular degeneration, diabetic macular edema, diabetic retinopathy, pathological myopia secondary to choroidal neovascularization, neovascular glaucoma Proliferative vitreoretinopathy, retinal vascular occlusion, idiopathic chorioretinitis, ocular histoplasmosis, ocular tumor, ocular trauma, choroidal neovascularization, cystic macular edema, corneal neovascularization, corneal transplantation, And one or more of chronic conjunctivitis.

In the use, the result of the ocular disease or condition associated with angiogenesis comprises selection Symptoms of one or more of the reduction in mean choroidal neovascular leakage, mean visual acuity improvement, mean foveal retinal thickness reduction, mean macular size reduction, and mean lesion size reduction.

In the use, wherein the ophthalmic pharmaceutical composition is used in a dose of 0.005 mg / 50 μL / eye to 2 mg / 50 μL / eye, more preferably used in a dose of 0.05 mg / 50 ul / eye to 0.5 mg / 50 ul / eye .

Preferably, wherein the ophthalmic pharmaceutical composition is administered by intravitreal injection.

Preferably, wherein the administration time of the ophthalmic pharmaceutical composition is administered once every 1 to 3 months.

The invention also provides a method of treating an ocular disease or condition associated with angiogenesis in a subject comprising topically administering to the eye of the subject an ophthalmic pharmaceutical composition as described above.

In the method, wherein the ocular disease or condition associated with angiogenesis is selected from the group consisting of age-related macular degeneration (AMD), diabetic retinopathy, choroidal neovascularization (CNV), cystic macular edema, diabetes Macular edema, retinal vascular occlusion, corneal neovascularization, corneal transplantation, neovascular glaucoma and chronic conjunctivitis, preferably age-related macular degeneration or diabetic retinopathy.

Preferably, the result of the treatment comprises a symptom improvement selected from one or more of a reduction in mean choroidal neovascular leakage, an improvement in mean visual acuity, a decrease in mean foveal retinal thickness, a decrease in mean macular size, and a decrease in mean lesion size.

Preferably, the ophthalmic pharmaceutical composition is administered at a dose of 0.05 mg / 50 μL / eye to 0.5 mg / 50 μL / eye.

Preferably, the administration is a single or multiple administrations.

Preferably, the ophthalmic pharmaceutical composition is administered by intravitreal injection.

Preferably, the administration time of the ophthalmic pharmaceutical composition is administered once every 1 to 3 months.

The term "monoclonal antibody (mAb)" as used herein refers to an immunoglobulin obtained from a pure lineage cell. White, with the same structure and chemical properties, specific for a single antigenic determinant. Monoclonal antibodies differ from conventional polyclonal antibody preparations (typically having different antibodies directed against different determinants), each monoclonal antibody being directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are also advantageous in that they are obtained by hybridoma or recombinant engineered cell culture without intermixing with other immunoglobulins. The modifier "monoclonal" indicates the identity of the antibody and is obtained from a homogeneous population of antibodies, which should not be construed as requiring any particular method for producing the antibody.

The term "humanized monoclonal antibody" as used herein refers to the sequence of the framework region in the variable region, except for the complementarity-determining regions (CDRs) of the amino acid sequence of the murine monoclonal antibody. The amino acid sequence of all or most of the adult immunoglobulin is replaced to minimize the immunogenicity of the murine monoclonal antibody by genetic engineering.

The terms "antibody" and "immunoglobulin" as used herein are isomeric polysaccharide proteins of the same structural feature of about 150,000 daltons consisting of two identical light chains (L) and two identical heavy chains ( H) Composition. Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. One end of each heavy chain has a variable region (VH). This is followed by a plurality of constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain . Particular amino acid residues form an interface between the variable regions of the light and heavy chains.

The term "variable" as used herein means that certain portions of the variable regions of an antibody differ in sequence, which form the binding and specificity of various specific antibodies to their particular antigen. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in the light and heavy chain variable regions into three segments in the complementarity determining region (CDR) or hypervariable region. The more conservative part of the variable region is called the framework regions (FR). The variable regions of the antibody heavy and light chains each comprise four FR regions which are substantially in a β-sheet configuration and are joined by three CDRs forming a linker, in some cases formable Part of the β-fold structure. The CDRs in each chain are closely joined together by the FR region and together with the CDRs of the other chain form the antigen binding site of the antibody. Antibody constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity. , CDC).

The ophthalmic pharmaceutical composition of the present invention comprises a pharmaceutically effective amount of a humanized antibody or a derivative thereof as described in the present invention, and a pharmaceutically acceptable carrier and other components, such that the biological preparation conforms to an ophthalmic preparation Special dosage specifications and technical requirements. A major indicator of the technical requirements is that the osmotic pressure of the ophthalmic preparation should be isotonic with tears, and does not contain or contain a very small amount of insoluble particulate matter for intravitreal injection or eye drop administration.

In a particular embodiment of the invention, provisions are made for insoluble particulates, osmotic pressure, pH and injection volume of the ophthalmic pharmaceutical composition. Generally, the concentration of the fusion protein in the preparation for clinical use is from 0.01 mg/mL to 100 mg/mL, depending on the form of the preparation, clinical needs, and the like. In general, intravitreal injection includes administration of about 0.01 mg to 10 mg; the maximum number of microparticles in the ophthalmic injection of the present invention is: the number of particles having a diameter > 10 μm in each preparation is less than 6000, and the number of particles having a diameter > 25 μm The number of particles smaller than 600; or diameter > 10 μm is less than 25 particles/ml, and the number of particles having a diameter > 25 μm is less than 3 particles/ml. The osmolality of the ophthalmic pharmaceutical composition of the present invention should be isotonic with tear fluid, generally 285-310 mOsmol/kg; the pH of the ophthalmic pharmaceutical composition should be about 5.5-6.5, as close as possible to the tear; the present invention The ophthalmic pharmaceutical composition is administered by intravitreal injection, and the administration volume is required to be small, and the drug concentration reaches a certain requirement.

The term "pharmaceutically acceptable" as used herein means that when the antibody and composition are suitably administered to an animal or a human, they do not produce an allergic or other untoward reaction. As used herein, a "pharmaceutically acceptable carrier" should be compatible with the antibody proteins of the present invention, i.e., can be blended therewith without substantially reducing the drug group. The effect of the compound. Specific examples of some substances which can be used as a pharmaceutically acceptable carrier or a component thereof include sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil , corn oil and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as Tween; stabilizers; antioxidants; pyrogen-free sterile water for injection; Physiological saline solution; phosphate buffer and the like.

The present invention provides an antibody use concentration, a drug use dose, and a frequency of use in an ophthalmic pharmaceutical composition. According to the existing research data, the concentration of the antibody is determined to be 0.5mg/ml-20mg/ml, and the dosage and specification are generally between 0.005mg/50μL/eye to 2mg/50μL/eye, at regular intervals (such as every 1 time). Up to 3 months) intravitreal injection once. In the specific implementation, the physician should determine the dosage, the administration time and the frequency of administration which are beneficial to the patient according to the patient's type, age, body weight, and general disease state, mode of administration and the like.

In the following examples and description of the accompanying drawings, the antibody or derivative thereof in the pharmaceutical composition of the present invention is represented by the code hPV19K for convenience of presentation. The hPV19K mAb is a tetramer composed of two identical heavy chains and two identical light chains, wherein the hPV19K mAb light chain has the amino acid sequence set forth in SEQ ID NO: 9, and the hPV19K mAb heavy chain has the SEQ ID NO: The amino acid sequence shown in 10. SEQ ID NOs: 9-10 are shown in Table 2 below:

Table 2: hPV19K mAb light and heavy chain amino acid sequences

DRAWINGS

1 is a result of in vitro detection and analysis of hPV19K monoclonal antibody and Avastin inhibiting VEGF-mediated proliferation of human Umbilical Vein Endothelial Cells (HUVEC) in Example 1 of the present invention. Among them, the vehicle control group served as a negative control group.

2 is a test result of insoluble microparticles in an ophthalmic injection of hPV19K monoclonal antibody containing different formulations in Example 2 of the present invention. Among them, the light gray column is the insoluble particle detection value after one hour of hPV19K monoclonal antibody injection containing different formulations, and the dark gray column is the insoluble particle detection value after 14 hours of each formula injection; the thick line is Pharmacopoeia The injection line is a standard line for infusion of insoluble particles, and the thin line is the standard line for injectable small injection insoluble particles specified by the Pharmacopoeia.

Figure 3 is a diagram showing the relative activity of VEGF binding of ophthalmic injections containing different formulations of hPV19K monoclonal antibody by direct ELISA in Example 2 of the present invention.

Figure 4 is a comparison of the relative activities of ophthalmic injections containing different formulations of hPV19K monoclonal antibody with commercially available Avastin and Compaqep with VEGF by direct ELISA in Example 3.

Figure 5 is a schematic diagram showing the binding of each VEGF inhibitor to block the binding of VEGF to its receptor (VEGFR-1) in vitro by competitive ELISA in Example 3.

Figure 6 is a concentration-time curve of serum hPV19K monoclonal antibody (n=6) after administration of a single intravitreal injection of hPV19K monoclonal antibody to cynomolgus monkey in the fourth embodiment of the present invention.

Fig. 7 is a concentration-time curve (n=12) of hPV19K monoclonal antibody in aqueous humor of a cynomolgus monkey administered by single intravitreal injection of hPV19K monoclonal antibody ophthalmic injection in Example 4 of the present invention.

Figure 8 is a concentration-time curve (n=12) of hPV19K mAb in vitreous humor after administration of single intravitreal injection of hPV19K monoclonal antibody ophthalmic injection in Example 4 of the present invention.

Figure 9 is a mid-stage photograph of the fundus photography and fluorescein imaging of the cynomolgus monkey before and after a single injection of the hPV19K monoclonal antibody ophthalmic injection in Example 5 of the present invention.

Figure 10 is a graph showing the detection rate of choroidal neovascularization level 4 spot of cynomolgus monkey by single injection of hPV19K monoclonal antibody ophthalmic injection in Example 5 of the present invention, and a is p<0.05 compared with the vehicle control group in the same period. .

11A, 11B and 11C are graphs showing the results of the fluoroscopy area and the improvement rate of the choroidal neovascularization of the cynomolgus monkey in a single injection of the hPV19K monoclonal antibody ophthalmic solution in the fifth embodiment of the present invention.

11A is a graph showing changes in the average area of fluorescence leakage per eye of each group of animals before and after administration, and a is p<0.05 compared with the vehicle control group at the same time;

Figure 11B shows the average area of fluorescence leakage per eye of each group of D24 and D31, and the same period of time. Compared with the control group, a indicates p<0.05; compared with the high-dose group for the same period, d indicates p<0.05; compared with the same period of the ranibizumab injection group, e indicates <0.05;

Figure 11C shows the improvement rate of fluorescence leakage area per eye of each group of D24 and D31. Compared with the vehicle control group, a indicates p<0.05. Compared with the low-dose group for the same period, b indicates p≤0.05. In the test medium dose group, c indicates p<0.05; compared with the ranibizumab injection group, e indicates p<0.05.

12A, 12B and 12C are optical coherence tomography results of abnormally high-reflecting signal substances in the retina of cynomolgus monkeys in a single intravitreal injection of hPV19K monoclonal antibody ophthalmic injection in Example 5 of the present invention.

12A is the average height change of SHRM per eye of each group of animals before and after administration, and a is p<0.05 compared with the vehicle control group at the same time;

Fig. 12B is the average height reduction of SHRM per eye of each group of D24 and D31, and a is p<0.05 compared with the vehicle control group at the same time;

Fig. 12C shows the average height improvement rate of SHRM per eye of each group of D24 and D31. Compared with the vehicle control group, a indicates p<0.05. Compared with the dose group in the same period, c indicates p<0.05.

detailed description

The invention is further described in the following examples, which are intended to be illustrative only and not to limit the invention.

Example 1. In vitro detection of proliferation of human umbilical vein endothelial cells (HUVEC) by samples containing hPV19K monoclonal antibody

In the examples of the present study, the hPV19K antibody reference product, the hPV19K antibody test sample and the Avastin control antibody sample were prepared by using a diluent (DMEM-Medium culture solution containing 10% FBS), respectively. A 2000 ng/ml solution was then serially diluted 3 folds with a dilution containing 6 ng/ml rhVEGF165 for a total of 10 concentration gradients. The dilution was used as a negative control, and the dilution containing 6 ng/ml rhVEGF165 was used as a positive control. The negative control, positive control, and serial dilution samples were added to a 96-well white cell culture plate at 50 μl/well, and incubated at 37 ° C in a 5% carbon dioxide incubator. 30 minutes.

Human Umbilical Vein Endothelial Cells (HUVEC) were resuspended to 1.0×10 ⁴ /ml with a dilution solution, and added to the above 96-well cell culture plate at 50 μl/well, and sterile water was used for the outer ring. Closed and incubated for 72 hours at 37 ° C in a 5% carbon dioxide incubator. 100 μl/well of freshly prepared CellTier-Glo proliferation reagent (Promega, USA) was added in order, and the color was read at room temperature for 15 min. The RLU reading was read by a fluorescent microplate reader, and the concentration of the reference sample or the sample to be tested was used as a horizontal The average of the coordinates and RLU readings is plotted as the ordinate, and the results are shown in Figure 1. Calculated, hPV19K antibodies reference product, hPV19K Avastin test sample and the reference were IC ₅₀ 16.9ng / ml, 16.8ng / ml and 89.6ng / ml, for in vitro inhibition of VEGF-mediated human umbilical vein endothelial cells ( The proliferative activity of HUVEC) was significantly stronger than that of Avastin (average 6.7-fold).

Example 2. Development of ophthalmic hPV19K monoclonal antibody preparation

In order to reduce the amount of insoluble microparticles in ophthalmic hPV19K monoclonal antibody injection, to prevent the occurrence of non-specific inflammation in the eye, deliberately add sugar (such as sucrose) and emulsifier (such as polysorbate, Tween) in the development of hPV19K monoclonal antibody preparation. Carriers such as 80 or Tween 20) reduce the mutual aggregation of antibody macromolecules. In the development of the pharmaceutical preparation, eight different formulations were preliminarily designed. The hPV19K monoclonal antibody was dissolved in different formulations containing different concentrations of Tween and sucrose, and the insolubility in each solution was measured after 1 hour and 14 hours. particle. Figure 2 shows the results of insoluble particulate detection in hPV19K monoclonal antibody formulations containing different formulations.

Thereafter, the sodium chloride content and the ratio of the buffer in the sample of the hPV19K monoclonal antibody containing Formulation 8 were separately adjusted to obtain a preliminary sample of the injection preparation. After testing, the osmotic pressure of the sample of the injection preparation is 300 mOsmol/kg; the pH is 5.5-6.5; the insoluble particles are: 5.8 particles/ml of particles having a diameter > 10 μm, and the number of particles having a diameter > 25 μm is 0.3 particles/ Ml. Thereafter, after the injection preparation sample was allowed to stand at 25 ° C for 6 months, the insoluble fine particles were further detected to be 312 particles/ml of particles having a diameter of >10 μm, and the number of particles having a diameter of >25 μm was 11 particles/ml.

Based on the above research results, hPV19K monoclonal antibody injection for fundus administration containing 8 different optimized formulas (code: YF20161201-YF20161208) was further designed.

Table 3 Different optimized formulation components and their contents in hPV19K monoclonal antibody preparations for fundus administration

The concentration of hPV19K antibody in each optimized formulation was determined by UV absorption method to be in the range of 9.81 mg/ml-10.01 mg/ml, and the purity of hPV19K monoclonal antibody was 96.60%-96.70% by SEC-HPLC. Optimized formula of hPV19K monoclonal antibody for ophthalmic injection of eggs The white concentration and purity are basically the same.

The relative binding activity of the antibody in the hPV19K monoclonal antibody ophthalmic injection was then determined by direct ELISA. The detection procedure was as follows: coating the plate with recombinant human VEGF165 protein (0.1 μg/ml, PBS, 100 μl/well), coating at 37 ° C for 2 hours or 4 ° C overnight; 5% skim milk at 37 ° C for 1 hour or 4 °C closed overnight. After washing with PBS-0.1% Tween20 solution (PBST), the hPV19K monoclonal antibody reference product and the hPV19K monoclonal antibody ophthalmic injection sample containing different optimized formulas were added, and the reference product and the initial concentration of each sample were 5 μg/ml. 3 times gradient dilution, a total of 12 concentration gradients, incubation at 37 ° C for 1 hour; after washing with PBST, horseradish peroxidase (HRP)-labeled goat anti-human IgG (purchased from Shanghai Xitang Biological Co., Ltd.), 37 Incubate for 1 hour at ° C; after thorough washing with PBST, add o-phenylenediamine (OPD)-0.1% H ₂ O ₂ substrate solution for 10-15 min, and terminate the reaction with 0.1 M HCl. The OD value at 492 nm was read in a MK3-Multiskan microplate reader (product of Thermo Scientific, USA). The results are shown in Fig. 3. The relative binding activity of the hPV19K monoclonal antibody ophthalmic injection containing different formulations was calculated to be 84.19%-100.83. %between.

Thereafter, the five optimized formulations of hPV19K monoclonal antibody ophthalmic injection (YF20161202, YF20161205, YF20161206, YF20161207, YF20161208) with a monoclonal antibody relative binding activity greater than 90% were selected for injection into the fundus of New Zealand rabbits for observation and comparison of ocular toxicity in animals. experiment.

The specific process of the experiment is as follows:

Fifteen male New Zealand rabbits with quarantine and similar body weight were randomly divided into 5 groups according to their body weight, 3 animals in each group, and D-1 was recorded on the day of grouping. On the second day (denoted as D1), each group of animals was injected with a single injection of hPV19K monoclonal antibody ophthalmic solution (10 mg/ml, 50 μl/eye) containing different formulations. General ophthalmic examinations were performed at screening (D-1), before administration (D1), D3, D8, and D15. Table 4 below shows the observation results of each group of experimental animals.

Table 4 Different optimized formula hPV19K monoclonal antibody ophthalmic injection for general eye examination results in New Zealand rabbits

Note: The total number of eyes in each group is 6; "+, ++, +++, ++++" indicate slight, mild, moderate, and severe, respectively.

By comparison, it was found that the safety and toxicological performance of rabbit eyes in Formulation 1, Formulation 3 and Formulation 4 was relatively good. Among them, Formulation 4 (10 mM phosphate, 8.8 mM glacial acetic acid, 44 mM NaCl, 0.03% Tween 20, 5% sucrose, 1-20 mg/ml hPV19K mAb) is the most preferred ophthalmic preparation of the present invention, and is used for carrying out The study of Examples 3-5 is described.

Example 3. In vitro detection of bioactivity of preferred hPV19K monoclonal antibody ophthalmic pharmaceutical preparations

1) Direct ELISA assay and comparison of the preferred hPV19K monoclonal antibody ophthalmic pharmaceutical preparations of the present invention, commercially available Avastin and Composine combined with VEGF bioactivity

The plate was coated with recombinant human VEGF165 protein (0.1 μg/ml, pH 9.6, 0.1 M NaHCO ₃ solution), coated at 37 ° C for 2 hours or 4 ° C overnight; 2% BSA was blocked at 4 ° C overnight. After washing with PBST, hPV19K monoclonal antibody ophthalmic injection, commercially available Avastin, and commercially available Composip, each with a starting concentration of 1000 ng/ml, were serially diluted twice and incubated at 37 °C. After washing with PBST, HRP-labeled goat anti-human IgG (purchased from Xitang Biotechnology Co., Ltd., Shanghai Xitang Biotechnology Co., Ltd.) was added, and incubated at 37 ° C for 1 hour; after thorough washing with PBST, OPD-0.1% H ₂ O was added. _{2 The} substrate solution was developed for 10-15 min and quenched with 0.1 M HCl. The OD value at 492 nm was read in a microplate reader.

The results of the ELISA assay are shown in Figure 4. The hPV19K monoclonal antibody ophthalmic injection maintains high binding activity to human VEGF165 protein, and its binding activity is 4-8 times higher than that of Avastin and Composcipal.

2) Competitive ELISA assay and comparison The preferred hPV19K monoclonal antibody ophthalmic preparations of the present invention, commercially available Avastin, Lucentis and Compaqip in vitro block the binding of VEGF to its recombinant receptor (VEGF-R).

A 96-well plate (2 μg/ml, 50 μl/well) was coated with recombinant soluble human VEGFR1 protein (product of R&D, USA) at 4 ° C overnight; after PBST rinse and 2% BSA at room temperature, 0.1 μg/ml was added. Biotinylated VEGF165 was incubated with different concentrations of the preferred hPV19K monoclonal antibody ophthalmic injection samples of the present invention, commercially available Avastin, commercially available Lucentis, and commercially available Compaqip, for 2 h at 37 ° C; after elution with PBST Add HRP-labeled Avidin (1:5000), incubate at 37 °C for 1 h; after elution with PBST, add OPD-3% hydrogen peroxide, room temperature for 10 min to color; add 0.1 M HCl to stop the reaction, determine the wavelength of 492 nm by microplate reader The absorbance of each hole.

Figure 5 is the result of the competitive ELISA assay, as shown in the figure: the preferred hPV19K monoclonal antibody ophthalmic injection sample of the present invention and several commercially available VEGF blocking agents can specifically block VEGF and its receptor (VEGF- in vitro). The combination of R), based on the detection curve, can be used to measure the IC ₅₀ value of hPV19K monoclonal antibody ophthalmic injection sample <1 nM, and its in vitro activity is better than the commercially available Avastin, ranibizumab (Lucentis) and Compaqip.

Example 4. Pharmacokinetic study of cynomolgus monkeys administered by single intravitreal injection of a preferred hPV19K monoclonal antibody ophthalmic preparation

Eighteen cynomolgus monkeys, half male and half female, were randomly divided into three groups (intravitreal low-dose, high-dose group and intravenous group). The low- and high-dose groups of vitreous injection were given a single intraocular vitreous injection of hPV19K monoclonal antibody for ophthalmic injection (for the test sample 0.5mg/50μL/eye, 1mg/50μL/eye, single intravenous bolus for intravenous group). Sample 2mg / only). Whole blood, veins were collected at 2, 6, 12, 24, 48, 72, 5, 7, 9, 11, 14, 17, 21, 24, 28 days after administration of the vitreous injection group. Before the administration of the animals, whole blood was collected at 2 min, 30 min, 2, 6, 12, 24, 48, 72 h, 5, 7, 11, 14, 17, 21, 24, 28 days after the drug, serum was separated, and serum was determined by ELISA. The drug concentration was calculated using the Winnonlin 6.4 non-compartmental model (NCA). In the vitreous group, about 0.1 mL of aqueous humor and vitreous humor were collected at 1 hour, 3, 7, 14, and 28 days after administration. The concentration of hPV19K monoclonal antibody was determined by ELISA, and the drug was calculated by Winnonlin 6.4 non-compartmental model (NCA). Kinetic parameters.

No abnormal clinical manifestations were observed in each animal during the experiment. The statistical results of serum, aqueous humor and vitreous pharmacokinetic parameters of each group were shown in Table 5, Table 6 and Table 7, respectively.

Table 5 Serum pharmacokinetic parameters of cynomolgus monkeys administered by single intravitreal injection of hPV19K monoclonal antibody

Table 6 Pharmacokinetic parameters of a single injection of hPV19K monoclonal antibody in aqueous humor of cynomolgus monkeys

Table 7 Pharmacokinetic parameters of vitreous humor in cynomolgus monkeys treated with single intravitreal injection of hPV19K monoclonal antibody

Figure 6, Figure 7 and Figure 8 show the concentration and timing of hPV19K monoclonal antibody in serum, aqueous humor and vitreous humor of cynomolgus monkeys after administration of hPV19K monoclonal antibody for ophthalmic injection.

Results As shown in the above graph, the drug peak concentration and serum drug exposure in cynomolgus monkey serum, aqueous humor and vitreous were positively correlated with the dose administered. The average ratio of serum C _max and AUC _last of average 1:1.94,1:1.99 respectively, the average C _max ratio of the aqueous and the average AUC _last was 1:2.28,1:3.16 respectively, mean C _max of the glass body The ratio of the ratio to the average AUC _last is 1:1.59, 1:2.02, respectively.

The absolute bioavailability of a single binocular vitreous injection (1 mg/eye, 2 mg/only) (vitreous injection high dose group AUC (0-408 h) / intravenous group AUC (0-408 h) was 44.90%.

After a single intravitreal injection, the average drug peak concentration (C _max ) ratio of vitreous humor, aqueous humor and serum in the low- and high-dose groups of the test article (hPV19K monoclonal antibody for ophthalmic injection) was 300.21:101.42:1, respectively. 245.32 _: 118.73 _: 1; the drug exposure AUC _last ratio was 151.02 _: 44.78 _: 1, 153.13 _: 71.05 _: 1, the exposure of the drug in the vitreous was higher than that of aqueous humor, much higher than serum.

Example 5. Inhibition of choroidal neovascularization in cynomolgus monkey by single intravitreal injection of hPV19K monoclonal antibody ophthalmic drug preparation

Step 1: Establishment of choroidal neovascularization (CNV) in cynomolgus monkeys

From the 54 cynomolgus monkeys, 50 screened animals were selected for bilateral fundus laser photocoagulation to induce choroidal neovascularization (CNV model). The number of laser burns per eye was 6-8, and all animals were modeled on the same day. It was recorded as D1; 2 weeks after model establishment (D15), the fundus fluorescence leakage was evaluated by fundus fluorescein angiography (FFA). The degree of damage of CNV in cynomolgus monkeys was divided into 4 grades. D17 selected 36 animals with 4 leaking spots, and averaged according to the average leakage area of the 4th spot and the 4 spot rate to ensure the average leakage area and the 4 spot rate of each group of animals at the time of grouping. Significant differences.

Step 2: hPV19K monoclonal antibody ophthalmic preparation for treatment of choroidal neonatal choroidal neovascularization

The above-mentioned CNV-forming cynomolgus monkeys were randomly divided into 6 groups (6 in each group, 3 in each group), and each group of animals on the day of enrollment (D17) was injected intravitreally (50 μL/eye, both eyes). The test sample (hPV19K monoclonal antibody ophthalmic injection) or the commercially available control ranibizumab (Lucentis) and Compaqip.

Groups of animals were grouped as follows:

1) hPV19K monoclonal antibody ophthalmic injection solvent vehicle group;

2) hPV19K monoclonal antibody ophthalmic injection for the low dose group (0.05mg/eye);

3) hPV19K monoclonal antibody ophthalmic injection for the test medium (0.15mg / eye);

4) hPV19K monoclonal antibody ophthalmic injection for the high dose group (0.5mg / eye);

5) Commercially available ranibizumab injection control group (0.5 mg/eye);

6) Commercially available Compaqip ophthalmic injection control group (0.5 mg/eye).

Before the model was established, D15, D24 and D31 were used to perform fundus photography, fundus fluorescein angiography (FFA) and optical coherence tomography (OCT). The cynomolgus monkey CNV was graded and measured according to the fundus fluorescence leakage. The height of the subretinal hyperreflective material (SHRM) in the OCT image was measured. The 33rd day (D33) test was completed.

test results:

1) Fluorescence contrast examination (FFA)

All animals underwent fundus photography and fluoroscopy before the modeling, and the results were not abnormal. Fundus photography and fluoroscopy were performed on D15, D24 and D31 after modeling. Figure 9 shows the hPV19K monoclonal antibody ophthalmic injection group, the ranibizumab injection group and the Compaqic eye injection group. The late picture of the fundus photography and fluorescein imaging of the crab monkey. In the fundus photography of each group of animals, except for the damage caused by laser light condensation and photocoagulation, no other abnormalities were observed.

At the same time, the fundus fluorescein image of D15, D24 and D31 after modeling was evaluated and the related measurements were taken. The number of spot 4, the spot of 4 and the spot of 4 spot were calculated.

A.4 spot number and 4 spot improvement rate

Two weeks after model establishment (D15), there was no significant difference in the number of 4-level spots between groups (p>0.05). 7 days after administration (D24), vehicle control group, test article (hPV19K monoclonal antibody ophthalmic injection) low, medium and high dose group, ranibizumab injection group and Compaqip eye injection The number of 4 spot spots in the group was 4.8±2.8, 1.8±2.1, 2.4±2.4, 1.3±2.7, 1.9±2.3 and 0.8±1.4, and the 4-level spot improvement rate (%) was 16.7±38.9 and 69.9±, respectively. 33.4, 66.2 ± 38.4, 82.4 ± 36.69.1 ± 35.3 and 87.4 ± 25.5. 14 days after administration (D31), vehicle control group, test article (hPV19K monoclonal antibody ophthalmic injection) low, medium and high dose group, ranibizumab injection group and Compaqip eye injection The number of spot 4 spots was 4.7±2.9, 1.8±2.1, 1.3±1.7, 1.0±2.2, 1.4±1.9 and 0.8±1.3, respectively. The improvement rate of the 4th spot (%) was 20.8±39.6 and 69.9±, respectively. 33.4, 78.8 ± 31.4, 85.9 ± 28.8, 77.1 ± 28.1 and 87.4 ± 24.0. A schematic diagram of the improvement rate of the 4-level spot per eye of each group of animals is shown in Fig. 10.

At 7 and 14 days after administration, there was no significant change in the number of spots in the vehicle control group, and the improvement rate of the level 4 spot was not obvious. The test article (hPV19K monoclonal antibody ophthalmic injection) was low, medium and high dose groups. The number of spot 4 in the Raychemumab injection group and the Compaqer eye group was significantly lower than that in the vehicle control group. (p<0.05); the level 4 spot improvement rate of each group of animals was significantly higher than that of the vehicle control group (p<0.05).

b. Level 4 spot average leakage area, average leakage area reduction and improvement rate

Before administration (2 weeks after model establishment, D15), 7 days after administration (D24) and 14 days (D31), the average area of fluorescence leakage per eye, reduction amount and fluorescence leakage improvement rate of each group (mean ± standard deviation) as shown in the following table:

Table 8 Average area of fluorescence leakage per eye, reduction of average leakage area and improvement rate of each group of animals

2 weeks after model establishment (D15), vehicle control group, test sample (hPV19K monoclonal antibody ophthalmic injection) low, medium and high dose group, ranibizumab injection group and Compaqima eye injection There was no significant difference in the mean area of fluorescence leakage per eye (mm ² ) between the groups (p>0.05).

At 7 and 14 days after administration, the fluorescence leakage area of the vehicle control group did not change significantly. The test article (hPV19K monoclonal antibody ophthalmic injection) low, medium and high dose group, ranibizumab injection group and Kang The fluorescence leakage area of the Percyepone injection group was significantly reduced. The average area of fluorescence leakage per eye and the improvement rate of fluorescence leakage (%) were significantly higher in the groups than in the vehicle control group (p<0.05). The fluorescence leakage area and improvement of each eye of each group of animals are shown in Fig. 11. The average area of fluorescence leakage per eye and the improvement rate of the high-dose group in the high-dose group 7 days after administration were higher than (p>0.05), the ranibizumab injection group and the test sample. (hPV19K monoclonal antibody ophthalmic injection) low, medium dose group. 14 days after administration, the average area of fluorescence leakage per eye and the improvement rate of the high-dose group (hPV19K monoclonal antibody for ophthalmic injection) were higher than (p>0.05), ranibizumab injection group, Compaq Xipu Ophthalmic Injection Group and low and medium dose groups for the test.

2) Optical coherence tomography (OCT)

All animals underwent optical coherence tomography (OCT) before modeling, and all animals showed no significant abnormalities. After modeling, D15, D24 and D31 were examined by OCT, and the subretinal hyperreflective material (SHRM) under the retinal pigment epithelial layer was measured according to the D15 fluoroscopy results. The highest height.

Before administration (2 weeks after model establishment, D15), 7 days (D24), and 14 days (D31), the average height, reduction, and improvement rate (mean±standard deviation) of SHRM per eye of each group of animals were as shown in Table 9.

Table 9 SHRM average height, average height reduction and improvement rate of each group of animals

2 weeks after model establishment (D15), vehicle control group, test sample (hPV19K monoclonal antibody ophthalmic injection) low, medium and high dose group, ranibizumab injection group and Compaqima eye injection Group of animals per eye The mean values of SHRM mean height (μm) were 212.7±39.4, 250.5±70.6, 246.3±78.9, 249.5±53.5, 246.1±69.5 and 241.9±89.2, respectively, and there was no significant difference between the groups (p>0.05).

At 7 and 14 days after administration, the height of SHRM in the vehicle control substance was not significantly changed, and the other groups were significantly reduced. 7 days after administration (D24), the test article (hPV19K monoclonal antibody ophthalmic injection) low, medium and high dose group, ranibizumab injection group and Compaqip eye injection group animals SHRM per eye The average height was significantly lower than the vehicle control group (p<0.05); the average SHRM height reduction and SHRM height improvement rate (%) in each group were significantly higher than the vehicle control group (p<0.05).

14 days after administration (D31), the test article (hPV19K monoclonal antibody ophthalmic injection) low, medium and high dose group, ranibizumab injection group and Compaqip eye injection group average SHRM per eye The heights were significantly lower than the vehicle control group (p<0.05); the average SHRM height reduction and SHRM height improvement rate (%) in each group were significantly higher than the vehicle control group (p<0.05). The average height reduction and improvement rate of SHRM per eye in the high-dose group of the test sample (hPV19K monoclonal antibody ophthalmic injection) was slightly higher than (p>0.05) the ranibizumab injection group and the Compaqip eye injection group. , for the test sample low, medium dose group.

Comprehensive analysis of the above-mentioned fluorescein spectroscopy and optical coherence tomography showed that hPV19K monoclonal antibody ophthalmic injection in each dose group can significantly inhibit choroidal neovascularization in cynomolgus monkeys, reduce the number of 4 spot spots, and improve the level 4 spot leakage. SHRM height. In addition, a single dose of only 1/10 dose (0.05mg / eye) of hPV19K monoclonal antibody ophthalmic solution, its inhibition of choroidal choroidal angiogenesis and leakage is the same as the injection of 0.05mg / eye of the commercially available control The efficacy of benizumab or injection of 0.05 mg/eye was similar, showing excellent in vivo activity.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solutions of the present invention within the scope of the technical idea of the present invention. These simple variants All fall within the scope of protection of the present invention.

Claims (15)

1. An ophthalmic pharmaceutical composition comprising an antibody or a derivative thereof capable of antagonizing the inhibition of binding of a vascular endothelial growth factor to its receptor, and one or more pharmaceutically acceptable excipients for use in an ophthalmic therapeutic agent, said antibody Or the light chain antigen complementarity determining region of the derivative thereof has an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; the heavy chain antigen complementarity determining region thereof has a selected from the group consisting of SEQ ID NO : 4, the amino acid sequences shown by SEQ ID NO: 5 and SEQ ID NO: 6.
2. The ophthalmic pharmaceutical composition according to claim 1, wherein the light chain variable region of the antibody or a derivative thereof has the amino acid sequence of SEQ ID NO: 7, and the heavy chain variable region thereof has SEQ ID NO: The amino acid sequence shown in 8.
3. The ophthalmic pharmaceutical composition according to claim 1 or 2, based on the total volume of the ophthalmic pharmaceutical composition, wherein the one or more pharmaceutically acceptable excipients contain 0.01 to 0.15 in grams per milliliter % of an organic cosolvent selected from the group consisting of polysorbate, polyethylene glycol, propylene glycol, and combinations thereof; and 1-10% by weight per gram selected from the group consisting of sucrose, sorbitol, glycerin, seaweed A stabilizer for at least one of a sugar and mannitol.
4. The ophthalmic pharmaceutical composition according to claim 3, based on the total volume of the ophthalmic pharmaceutical composition, wherein the one or more pharmaceutically acceptable excipients contain 0.01 to 0.02% per gram of gram. Polysorbate and 7-9% sucrose in grams per milliliter.
5. The ophthalmic pharmaceutical composition according to claim 3, based on the total volume of the ophthalmic pharmaceutical composition, wherein the one or more pharmaceutically acceptable excipients comprise 0.03-0.04% in grams per milliliter Polysorbate and 0-5% sucrose in grams per milliliter.
6. The ophthalmic pharmaceutical composition according to claim 1, wherein the antibody or a derivative thereof has a protein concentration in the pharmaceutical composition of 0.1 to 50 mg/ml.
7. The ophthalmic pharmaceutical composition according to claim 6, wherein the antibody or a derivative thereof has a protein concentration in the pharmaceutical composition of from 1 to 20 mg/ml, preferably from 1 to 10 mg/ml.
8. The ophthalmic pharmaceutical composition according to claim 1, wherein the ophthalmic pharmaceutical composition has an osmotic pressure of from 285 to 310 mOsmol/kg, preferably 300 mOsmol/kg.
9. The ophthalmic pharmaceutical composition according to claim 1, wherein the ophthalmic pharmaceutical composition has a pH of from 5.5 to 6.5, preferably a pH of 6.0.
10. Use of an ophthalmic pharmaceutical composition according to any one of claims 1 to 9 for the manufacture of a medicament for the treatment of an ocular disease or condition associated with angiogenesis.
11. The use according to claim 10, wherein the ocular disease or condition associated with angiogenesis is selected from the group consisting of age-related macular degeneration, diabetic macular edema, diabetic retinopathy, pathological myopia secondary to choroidal neovascularization, neonatalization Vascular glaucoma, proliferative vitreoretinopathy, retinal vascular occlusion, idiopathic chorioretinitis, ocular histoplasmosis, ocular tumor, ocular trauma, choroidal neovascularization, cystic macular edema, corneal neovascularization, One or more of corneal transplantation, and chronic conjunctivitis.
12. The use according to claim 11, wherein the result of treating an ocular disease or condition associated with angiogenesis comprises a reduction in mean choroidal neovascular leakage, an improvement in mean visual acuity, a reduction in mean foveal thickness, and an average macular size. Symptoms of one or more of reduced and reduced lesion size reduction.
13. The use according to any one of claims 10 to 12, wherein the ophthalmic pharmaceutical composition is used in a dose of 0.005 mg / 50 μL / eye to 2 mg / 50 μL / eye, preferably used at a dose of 0.05 mg / 50 ul / Eye to 0.5mg/50ul/eye;

    Preferably, the ophthalmic pharmaceutical composition is administered by intravitreal injection;

    Preferably, the administration time of the ophthalmic pharmaceutical composition is administered once every 1 to 3 months.
14. A method of treating an ocular disease or condition associated with angiogenesis in a subject, comprising topically administering to the eye of the subject an ophthalmic pharmaceutical composition according to any one of claims 1-9.
15. The method according to claim 14, wherein the ocular disease or condition associated with angiogenesis is selected from the group consisting of age-related macular degeneration (AMD), diabetic retinopathy, choroidal neovascularization (CNV), cystic macula Edema, diabetic macular edema, retinal vascular occlusion, corneal neovascularization, corneal transplantation, neovascular glaucoma and chronic conjunctivitis, preferably age-related macular degeneration or diabetic retinopathy;

    Preferably, the result of the treatment comprises a symptom improvement selected from one or more of a reduction in mean choroidal neovascular leakage, improvement in mean visual acuity, reduction in mean foveal thickness, reduction in mean macular size, and reduction in mean lesion size;

    Preferably, the ophthalmic pharmaceutical composition is administered at a dose of 0.05 mg / 50 μL / eye to 0.5 mg / 50 μL / eye;

    Preferably, the administration is single or multiple administrations;

    Preferably, the ophthalmic pharmaceutical composition is administered by intravitreal injection;

    Preferably, the administration time of the ophthalmic pharmaceutical composition is administered once every 1 to 3 months.

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