CN109562166B - Medicinal composition for eyes and application thereof - Google Patents

Abstract

An ophthalmic pharmaceutical composition comprising an antibody or a derivative thereof capable of antagonizing and inhibiting the binding of vascular endothelial growth factor to its receptor, and one or more pharmaceutically acceptable excipients for ophthalmic therapeutic agents, wherein the light chain antigen complementary determining region of the antibody or the derivative thereof has an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; and the heavy chain antigen complementary determining region thereof has an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. The ophthalmic pharmaceutical composition is used in the preparation of a medicament for treating angiogenesis-related ophthalmic diseases or conditions.

Description

Medicinal composition for eyes and application thereof

Technical Field

The invention belongs to the field of biotechnology-monoclonal antibodies, and particularly relates to an ophthalmic pharmaceutical composition and application thereof.

Background

Angiogenesis or hyperplasia (angiogenesis) refers to the process by which blood vessels (such as capillaries and minute arteries and veins) existing in the body generate new blood vessels by budding or splitting.

Angiogenesis is beneficial and necessary in maintaining many of the normal physiological processes of the body such as tissue embryonic development, healing and repair of traumatic wounds, and the like; however, excessive angiogenesis or hyperplasia can also lead to pathological changes in the body. For example, in vivo, proliferation, diffusion, metastasis and recurrence of tumor, age-related macular degeneration (AMD), ocular fundus disease, Diabetic Macular Edema (DME), inflammatory response, autoimmune diseases, and the like are closely related to angiogenesis or hyperplasia.

The key to the proliferation and growth of blood vessels in vivo is the ability of the lined vascular endothelial cells to divide, proliferate and migrate directionally into the wall of the existing blood vessel. The most important and most potent vascular endothelial cell division-promoting or vascular proliferation substance known to date is vascular endothelial cell growth factor (VEGF). VEGF, also known as vascular permeability factor (VRF), encodes a VEGF/VRF protein as reported by two research groups in 1989 and in the journal of American Science.

VEGF acts to promote the growth and migration of vascular endothelial cells, angiogenesis and increase vascular permeability by binding specifically to VEGF receptors (VEGF-R1, VEGF-R2) on vascular endothelial cells. The importance of VEGF and its receptor-mediated vascular proliferation has been well documented in studies with VEGF knockout mice: because mouse embryos die from the inhibition and abnormality of vascular proliferation when only one portion of the VEGF gene is knocked out, when only 11 to 12 days have elapsed.

VEGF and its receptor mediated angiogenesis and vascular infiltration promotion play a key role in the occurrence and pathological progression of blindness-induced fundus diseases such as age-related macular degeneration, diabetic macular edema, and the like. The age-related macular degeneration mostly occurs in more than 50 years old, the prevalence rate increases with the age, and the clinical symptoms of the disease are manifested as central hypopsia and rapid disease progression, and the disease is an important disease causing blindness of the elderly. Diabetic macular edema is a serious disease that endangers tens of millions of middle-aged salary vision today.

The main pathological manifestations of age-related macular degeneration and diabetic macular edema are atrophic degeneration of macula and peripheral tissues, destruction of visual cells, drusen formation, and exudative macular degeneration in severe cases, accompanied by subretinal neovascularization, bleeding and exudation. In the past, laser therapy and photodynamic therapy are commonly adopted for blindness fundus diseases caused by age-related macular degeneration, diabetic macular edema and the like, although the symptoms can be temporarily relieved, the recurrence rate is high, and the disease course cannot be prevented from progressing.

Since 2005, various VEGF inhibitors have been clinically used to treat exudative macular degeneration and diabetic macular edema with a more desirable therapeutic effect, and the conventional therapy has been recommended by the National Eye Institute (NEI).

To date, the drugs on the market for VEGF inhibitors approved by the FDA in the united states for the treatment of ocular fundus diseases associated with angiogenesis mainly include the following three major classes:

pegaptanib sodium injection (Pegaptanib, trade name Macugen), was co-developed by Eyetech Pharmaceuticals, Inc, and Pfizer Inc, in 2004 and obtained us FDA approval for marketing at 12 months. The pegaptanib sodium is a single-stranded RNA analog modified by PEG, and the unique three-dimensional structure of the pegaptanib sodium enables the pegaptanib sodium to specifically bind to VEGF and inhibit the activity of the VEGF.

(II) an antibody or antibody derivative that antagonizes VEGF. Only Ranibizumab (Chinese trade name: Norethic, English trade name: Lucentis) developed by Genentech company which is currently approved by the US FDA to be marketed in the medicines is used for treating blindness-causing fundus diseases such as age-related macular degeneration and diabetic macular edema. Ranibizumab is homologous to another antibody drug Bevacizumab (Bevacizumab, trade name Avastin) drug that has been previously developed by Genentech corporation for marketing indications: both are derived from murine mAb A4.6.1, with homology of the variable region above 99%. Bevacizumab is a full length antibody with a molecular weight of about 149kd, whereas ranibizumab is an Fab fragment with a molecular weight of about 48 kd. Like Avastin, ranibizumab can be combined with VEGF with high affinity, competitively block VEGF signal pathway, inhibit neovascularization and promote the absorption of the exudate in the macular region. Clinical studies initiated by Genentech showed that the visual acuity of 95% of "wet" AMD affected eyes stabilized or improved after 2 years of patient treatment with ranibizumab. Because of this excellent therapeutic effect, ranibizumab was approved by the FDA in the united states and marketed in 6 months 2006. Ranibizumab is further approved in the United states for treating a plurality of indications such as diabetic macular edema, macular edema secondary to retinal vein occlusion, choroidal neovascularization secondary to pathological myopia and the like, and is a mainstream medicament for treating fundus diseases related to angiogenesis such as AMD at present.

In china, ranibizumab was approved by the chinese drug administration in 2011 for the treatment of AMD.

(III) VEGF receptor-Fc fusion proteins. The medicine is formed by fusing a VEGF receptor extracellular domain and a human immunoglobulin-Fc segment, and a representative medicine is Aflibercept (trade name: Eylea) developed by American Regeneron company. The aflibercept is formed by fusing the 2 nd domain of the extracellular region of a VEGF-R1 receptor, the 3 rd domain of the extracellular region of a VEGF-R2 receptor and the Fc segment of human immunoglobulin IgG 1. The affinity of VEGF-Trap to VEGF is higher than the natural affinity of its receptor to ligand, and the binding of VEGF-R to VEGF in human body can be competitively inhibited. Aflibercept has been approved by the FDA in the united states since 2011 for the treatment of blindness-causing fundus diseases including age-related macular degeneration and diabetic macular edema, and is now overtaking ranibizumab in the approved range of clinical indications and in multiple marketing fields around the world.

Cupresscept (Conbercept) developed by Chengdu Kanghong pharmaceutical group GmbH in China is also a VEGF receptor-Fc fusion protein. The combretastatin cypress western-style product is very similar to aflibercept in medicine structure and is formed by fusing VEGF-R1 extramural 2-th domain, VEGF-R2 extramural 3-th and 4-th domains and Fc segment of human immunoglobulin IgG 1. Cupressus occidentalis is approved by China CFDA in 2013 to treat diseases such as senile fundus macular degeneration, and clinical phase III research is also being carried out in the United states at present.

In summary, VEGF inhibitor drugs that are currently approved worldwide for the treatment of fundus diseases such as AMD/DME all belong to the Fab fragment of VEGF mab or VEGF receptor-antibody Fc fusion protein. The monoclonal antibody-Fab fragment such as ranibizumab has the advantage of easy and rapid penetration to focus after fundus injection to exert drug effect due to small molecular weight; however, the half-life of the monoclonal antibody-Fab fragment in the eye is only 2-3 days, which is shorter than that of the intact antibody, thereby increasing the number of administrations and the economic burden on patients. VEGF receptor-Fc fusion protein drugs such as aflibercept and combretastatin have higher binding affinity for their ligands than their natural receptors (VEGFR-1, VEGFR-2); after fundus injection, the half-life period in eyes is 3-5 days, which is slightly longer than that of ranibizumab, so the injection has the advantages of high drug effect, low drug administration frequency and the like. However, aflibercept and combretastatin are also combined with other growth factors such as VEGF-B, VEGF-C, PLGF (central growth factor) so as to increase the potential risk of adverse drug reactions inconsistent with the treatment purpose (specifically blocking VEGF mediation).

Compared with Fab fragment or Fc fusion protein, the complete and full-length antibody such as Avastin has the advantages of both Fab fragment and Fc fragment, has the advantages of long half-life in vivo, low administration frequency and the like, and is clinically expected to be used for treating angiogenesis eye diseases such as AMD/DME and the like with lower dose or lower injection frequency. Both pre-clinical and clinical study data in recent years show that the full-length antibody, Avastin, is not really inferior to ranibizumab or aflibercept for its efficacy in the treatment of AMD/DME. Avastin, however, has not been approved by the FDA or other drug administration for the treatment of other indications than malignancies, such as ophthalmic indications; the ophthalmic use of Avastin in the treatment of AMD/DME is referred to as "off-label use" or "off-label use". There are many differences in drug formulations, dosages, manufacturing and testing release criteria, product packaging, etc. for the treatment of oncological indications and ophthalmic indications such as AMD/DME. Drugs for treating ophthalmic indications such as AMD/DME require fundus injection, the pH and osmotic pressure of the drug should be close to that of tears, and insoluble particulate matter should be controlled at very low amounts, except that sterility is strictly maintained; avastin for the treatment of tumors does not take into account these features and requirements of ophthalmic drugs in its manufacture and packaging. In addition, in order to reduce the cost of fundus injection, patients or doctors usually package large-package Avastin (4 ml or 20ml in each bottle) into small parts for sharing, and the patients often have the safety risk of infection or inflammation after receiving the packaged drugs for fundus injection.

The anti-angiogenesis drugs for treating AMD/DME are commercially available for tens of millions of patients, and the patients need to be repeatedly administrated for a plurality of times throughout the year, and the existing anti-angiogenesis ophthalmic drugs of ranibizumab, aflibercept and combaiccept do not meet the market demand; on the other hand, the three drugs currently on the market are not effective in all AMD/DME patients. Therefore, there is still a need to develop new or safer and more effective anti-angiogenesis ophthalmic drugs in clinic and on the market.

Disclosure of Invention

The invention provides an ophthalmic pharmaceutical composition, which comprises an antibody or a derivative thereof capable of antagonizing and inhibiting the binding of vascular endothelial growth factor and a receptor thereof, and one or more pharmaceutically acceptable auxiliary materials for 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 the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; the heavy chain antigen complementarity determining region has a sequence selected from 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 the derivative thereof has the amino acid sequence of SEQ ID NO: 7, and the heavy chain variable region thereof has the amino acid sequence shown in SEQ ID NO: 8. Wherein, 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, wherein the one or more pharmaceutically acceptable excipients comprise 0.01-0.15% (m/v, g/ml) of an organic co-solvent selected from at least one of polysorbate (tween), polyethylene glycol, propylene glycol and combinations thereof, based on the total volume of the ophthalmic pharmaceutical composition; and 1-10% (m/v, g/ml) of at least one stabilizer selected from sucrose, sorbitol, glycerol, trehalose, and mannitol.

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

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

In a preferred embodiment of said ophthalmic pharmaceutical composition, wherein said antibody or derivative thereof is present in said formulation in a protein concentration of 0.1-50 mg/ml.

Preferably, wherein the protein concentration of said antibody or derivative thereof in said pharmaceutical composition is 1-10 mg/ml.

Preferably, the osmotic pressure of the ophthalmic drug composition is 285-310mOsmol/kg, preferably 300 mOsmol/kg.

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

The invention also provides the application of the ophthalmic medicine composition in preparing medicines for treating eye diseases or symptoms related to angiogenesis.

In the use, wherein the ocular disease or disorder associated with angiogenesis is selected from one or more of age-related macular degeneration, diabetic macular edema, diabetic retinopathy, choroidal neovascularization secondary to pathological myopia, neovascular glaucoma, proliferative vitreoretinopathy, retinal vessel occlusion, idiopathic chorioretinitis, ocular histoplasmosis, ocular tumors, ocular trauma, choroidal neovascularization, cystic macular edema, corneal neovascularization, corneal transplantation, and chronic conjunctivitis.

In the use, wherein the outcome of the angiogenesis-related ocular disease or disorder comprises an improvement in a symptom selected from one or more of a decrease in mean choroidal neovascularization leakage, an improvement in mean vision, a decrease in mean foveal retinal thickness, a decrease in mean macular size, and a decrease in mean lesion size.

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

Preferably, wherein the mode of administration of the ophthalmic pharmaceutical composition is by intravitreal injection.

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

The present invention also provides a method of treating an angiogenesis-related ocular disease or disorder in a subject comprising topically administering the above-described ophthalmic pharmaceutical composition into the eye of the subject.

In the method, wherein the ocular disease or disorder associated with angiogenesis is selected from the group consisting of age-related macular degeneration (AMD), diabetic retinopathy, Choroidal Neovascularization (CNV), cystic macular edema, diabetic macular edema, retinal vessel 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 an improvement in a symptom selected from one or more of a decrease in mean choroidal neovascularization leakage, an improvement in mean vision, 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 0.05mg/50 μ L/eye to 0.5mg/50 μ L/eye.

Preferably, the administration is a single or multiple administration.

Preferably, the mode of administration of the ophthalmic pharmaceutical composition is by intravitreal injection.

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

The term "monoclonal antibody (mab)" as used herein refers to an immunoglobulin derived from a pure line of cells, having the same structural and chemical properties, and being 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, and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

The term "humanized monoclonal antibody" as used herein is a monoclonal antibody in which the amino acid sequence of a murine monoclonal antibody is replaced, in whole or in large part, with the amino acid sequence of an adult immunoglobulin, except for the complementary-determining regions (CDRs), including the framework regions in the variable region, to minimize the immunogenicity of the murine monoclonal antibody by genetic engineering means.

The terms "antibody" and "immunoglobulin" as used herein are heterotetrameric proteins of about 150000 daltons having the same structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at one end. Followed by a plurality of constant regions. Each light chain has a variable domain (VL) at one end and a constant domain 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 the variable region of the heavy chain. Particular amino acid residues form the 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 results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments in the light and heavy chain variable regions that become Complementarity Determining Regions (CDRs) or hypervariable regions. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of the heavy and light chains of an antibody each comprise four FR regions, which are in a substantially β -sheet configuration, connected by three CDRs which form a connecting loop, and in some cases may form part of a β -sheet structure. The CDRs in each chain are held together tightly by the FR regions and form the antigen binding site of the antibody with the CDRs of the other chain. Antibody constant regions are not directly involved in binding an antibody to an antigen, but they exhibit different effector functions, such as participation in antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) of an antibody.

The invention provides an ophthalmic pharmaceutical composition comprising a pharmaceutically effective amount of the humanized antibody or the derivative thereof as described in the invention, a pharmaceutically acceptable carrier and other components, so that the biological preparation meets the special dosage specification and technical requirements of ophthalmic preparations. One of the major indicators of the technical requirements is that the ophthalmic preparation should have an osmotic pressure isotonic with tear fluid and contain no or very little insoluble particulate matter for intravitreal injection or eye drop administration to the eye.

In specific embodiments of the present invention, the specification of insoluble microparticles, osmotic pressure, pH and injection volume of the ophthalmic pharmaceutical composition is described. Generally, the concentration of the fusion protein in a preparation for clinical use is 0.01mg/mL to 100mg/mL, and the specific dose depends on the form of the preparation, clinical needs, and the like. Typically, intravitreal injections include administration of about 0.01mg to 10 mg; the maximum limit of the number of the particles in the ophthalmic injection of the invention is as follows: the number of particles with the diameter of more than 10 mu m in each preparation is less than 6000, and the number of particles with the diameter of more than 25 mu m in each preparation is less than 600; or the number of particles with the diameter of more than 10 mu m is less than 25 particles/ml, and the number of particles with the diameter of more than 25 mu m is less than 3 particles/ml. The osmolarity osmotic pressure of the ophthalmic drug composition of the invention should be isotonic with the tears, generally 285-310 mOsmol/kg; the pH value of the ophthalmic medicine composition is about 5.5-6.5 so as to be as close to the lacrimal fluid as possible; the ophthalmic medicine composition is administrated by adopting an intravitreal injection mode, the administration volume is required to be small, and the medicine concentration meets certain requirements.

The term "pharmaceutically acceptable" as used herein means that the antibodies and compositions do not produce allergic or other untoward reactions when properly administered to an animal or human. As used herein, a "pharmaceutically acceptable carrier" should be compatible with, i.e., capable of being blended with, the antibody proteins of the present invention without substantially reducing the effectiveness of the pharmaceutical composition. Specific examples of some substances that may serve as pharmaceutically acceptable carriers or components 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; polyhydric alcohols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as Tween; a stabilizer; an antioxidant; pyrogen-free sterile water for injection; a physiological saline solution; phosphate buffers, and the like.

The invention provides the use concentration of an antibody in an ophthalmic pharmaceutical composition, the use dosage of a medicament and the use frequency. According to the existing research data, the concentration of the antibody is determined to be 0.5mg/ml-20mg/ml, the dosage and specification are generally 0.005mg/50 muL/eye to 2mg/50 muL/eye, and the injection is performed in a vitreous cavity at regular intervals (such as every 1 to 3 months). The dosage, time and frequency of administration of the drug to be administered to a patient will be determined by the physician in the practice of the invention, depending on the type, age, weight and general condition of the patient, the mode of administration and the like.

In the following examples and the description of the drawings, the antibody or its derivative in the pharmaceutical composition of the present invention is represented by the symbol hPV19K for convenience of description. hPV19K monoclonal antibody is a tetramer consisting of two identical heavy chains and two identical light chains, wherein hPV19K monoclonal antibody light chain has the amino acid sequence of SEQ ID NO: 9, hPV19K monoclonal antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 10, or a pharmaceutically acceptable salt thereof. SEQ ID NO: 9-10 are shown in Table 2 below:

table 2: hPV19K monoclonal antibody light chain and heavy chain amino acid sequence

Drawings

FIG. 1 is a graph showing the results of in vitro assay hPV19K monoclonal antibody and Avastin inhibiting VEGF-mediated proliferation activity of Human Umbilical Vein Endothelial Cells (HUVEC) in example 1 of the present invention. Wherein the vehicle control group is used as a negative control group.

FIG. 2 shows the results of the detection of insoluble particles in the ophthalmic injection solution containing hPV19K mAbs with different formulations in example 2 of the present invention. Wherein the light gray column is an insoluble particle detection value of hPV19K monoclonal antibody injection containing different formulas after being placed for one hour, and the dark gray column is an insoluble particle detection value of each formula after being placed for 14 hours; the thick line is the standard line of the insoluble particles of the large infusion solution of the injection specified by the pharmacopoeia, and the thin line is the standard line of the insoluble particles of the small infusion solution of the injection specified by the pharmacopoeia.

FIG. 3 is a graph showing the relative activities of different formulations of hPV19K in detecting VEGF-binding activity of ophthalmic injection solutions containing different formulations of the monoclonal antibody according to example 2 of the present invention by direct ELISA.

FIG. 4 is a graph showing the relative activities of different formulations of hPV19K in the direct ELISA method of example 3 of the present invention in comparison with the binding activities of commercial Avastin and combaici cypress to VEGF.

FIG. 5 is a schematic diagram of the competitive ELISA method for detecting in vitro blockade of VEGF binding to its receptor (VEGFR-1) for each VEGF inhibitor in example 3 of the present invention.

Fig. 6 is a graph showing the concentration-time curve of the hPV19K mab-containing ophthalmic injection solution of example 4 of the present invention (n ═ 6) in serum hPV19K mab after a single intravitreal injection administered to cynomolgus monkeys.

Fig. 7 is a plot of aqueous humor hPV19K concentration versus time (n-12) of an hPV19K ophthalmic injection solution administered to cynomolgus monkeys by single intravitreal injection in example 4 of the present invention.

Fig. 8 is a plot of the concentration of hPV19K mab in the vitreous humor of a cynomolgus monkey, in which hPV19K mab is present, versus time (n-12) after a single intravitreal injection of the ophthalmic injection solution of hPV K in example 4 of the present invention.

FIG. 9 is a photograph of fundus oculi of cynomolgus monkey taken before and after a single injection of hPV19K monoclonal antibody for eye injection in vitreous chamber of the present invention in example 5 and a middle-late stage photograph taken by fluorescence angiography.

Fig. 10 is a graph showing the results of detecting the 4-stage flare improvement rate of the cynomolgus monkey choroidal neovascularization by single intravitreal injection of hPV19K monoclonal antibody ophthalmic injection solution in example 5 of the present invention, wherein a represents p <0.05 compared with the contemporary vehicle control group.

Fig. 11A, 11B and 11C are graphs showing the results of single intravitreal injection of the hPV19K mab ophthalmic injection solution of example 5 of the present invention on the leakage area and improvement rate of cynomolgus monkey choroidal neovascularization at level 4 light spots.

Wherein, fig. 11A is the mean change in area of fluorescence leakage per eye of each group of animals before and after administration, a represents p <0.05 compared to the vehicle control group at the same time;

FIG. 11B is the mean decrease in fluorescence leakage per eye for animals in groups D24 and D31, a indicating p <0.05 compared to the vehicle control group at the same time; d represents p <0.05 compared to the high dose group of the contemporary test article; e represents <0.05 compared to the contemporary ranibizumab injection group;

FIG. 11C is a graph showing the improvement in fluorescence leakage area per eye for animals in groups D24 and D31, where a represents p <0.05 compared to a contemporary vehicle control group; b represents that p is less than or equal to 0.05 when compared with the low dose group of the test article in the same period; c represents p <0.05 compared to the dose group in the contemporary test article; compared with the contemporary ranibizumab injection group, e represents p < 0.05.

FIGS. 12A, 12B and 12C are Optical Coherence Tomography (OCT) results of single intravitreal injection of hPV19K mab in example 5 of this invention for abnormal high-reflectance signaling under cynomolgus monkey retina.

Wherein, figure 12A is the average height change per eye of SHRM for each group of animals before and after dosing, a represents p <0.05 compared to a contemporary vehicle control group;

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

FIG. 12C is the average height improvement per eye of SHRM in D24 and D31 groups compared to the vehicle control group at the same time, a indicates p < 0.05; c indicates p <0.05 compared to the dose group in the contemporary test article.

Detailed Description

The invention will now be further described with reference to examples, which are intended to be illustrative only and are not intended to be limiting.

Example 1 in vitro assay of the Activity of a sample containing hPV19K monoclonal antibody to inhibit proliferation of Human Umbilical Vein Endothelial Cells (HUVEC)

In the present study example, hPV19K antibody reference, hPV19K antibody test sample, and Avastin control antibody sample were prepared as 2000ng/ml solutions using dilutions (DMEM-Medium culture solution containing 10% FBS), respectively, and then serially diluted 3-fold in a dilution containing 6ng/ml rhVEGF165 for 10 concentration gradients. The dilution was used as a negative control, the dilution containing 6ng/ml rhVEGF165 was used as a positive control, and the negative control, the positive control, and the serial diluted samples were added to a 96-well white cell culture plate at 50. mu.l/well, and incubated in a 5% carbon dioxide incubator at 37 ℃ for 30 minutes.

Vascular Endothelial Cells (HUVEC) derived from Human Umbilical veins were resuspended at 1.0X 104/ml in a diluent, 50. mu.l/well in the above 96-well cell culture plate, the outer ring was sealed with sterile water, and incubated at 37 ℃ for 72 hours in a 5% carbon dioxide incubator. The results are shown in FIG. 1, where 100. mu.l/well of freshly prepared CellTier-Glo proliferation reagent (Promega, USA) was added in this order, color development was carried out at room temperature for 15min in the dark, RLU readings were taken with a fluorescence microplate reader, the concentration of the reference or sample to be tested was plotted as the abscissa, and the average of the RLU readings was plotted as the ordinate. The IC50 of the hPV19K antibody reference, hPV19K test sample, and Avastin control were calculated to be 16.9ng/ml, 16.8ng/ml, and 89.6ng/ml, respectively, and the hPV19K antibody was significantly stronger than Avastin (6.7-fold stronger on average) for inhibiting VEGF-mediated proliferation activity of Human Umbilical Vein Endothelial Cells (HUVECs) in vitro.

EXAMPLE 2 development of pharmaceutical formulations of hPV19K monoclonal antibody for eye

In order to reduce the number of insoluble particles in an ophthalmic hPV19K monoclonal antibody injection and prevent non-specific inflammation of eyes, carriers such as saccharides (such as sucrose) and emulsifiers (such as polysorbate, tween 80 or tween 20) are added intentionally in the development of a hPV19K monoclonal antibody pharmaceutical preparation to reduce mutual aggregation of antibody macromolecules. 8 different formulas are preliminarily designed in the development of the pharmaceutical preparation, hPV19K monoclonal antibody is respectively dissolved in liquids of different formulas containing different concentrations of Tween and sucrose, and insoluble particles in each solution are detected after the solutions are respectively placed for 1 hour and 14 hours. FIG. 2 shows the results of the detection of insoluble particles in pharmaceutical preparations containing hPV19K mAbs with different formulations.

And then, respectively adjusting the sodium chloride content and the ratio of the buffer solution in the hPV19K monoclonal antibody drug preparation sample containing the formula 8 to obtain an injection preparation primary sample. The osmotic pressure of the injection preparation sample is detected to be 300 mOsmol/kg; the pH value is 5.5-6.5; the insoluble microparticles were: the number of particles with a diameter of >10 μm was 5.8 particles/ml and the number of particles with a diameter of >25 μm was 0.3 particles/ml. Thereafter, after the injection preparation sample was left at 25 ℃ for 6 months, the number of insoluble microparticles measured again was 312 particles/ml for those having a diameter of >10 μm and 11 particles/ml for those having a diameter of >25 μm.

On the basis of the research results, hPV19K monoclonal antibody injection which contains 8 different optimized formulas (code number: YF20161201-YF20161208) and is intended for fundus drug administration is further designed.

TABLE 3 formulation of hPV19K mAbs intended for ocular fundus drug delivery and its optimized formulation components and contents

The protein concentration of the hPV19K antibody in each optimized formula preparation is measured to be within the range of 9.81mg/ml-10.01mg/ml by an ultraviolet absorption method, and the purity of the hPV19K monoclonal antibody is measured to be 96.60% -96.70% by SEC-HPLC, so that the protein concentration and the purity of the hPV19K monoclonal antibody ophthalmic injection containing different optimized formulas are basically the same.

And then detecting the relative binding activity of the antibody in hPV19K monoclonal antibody eye injection by using a direct ELISA method. The detection steps are as follows: recombinant human VEGF165 protein (0.1. mu.g/ml, PBS, 100. mu.l/well) is used for coating the ELISA plate, and the ELISA plate is coated for 2 hours at 37 ℃ or is coated overnight at 4 ℃; 5% skimmed milk was left at 37 ℃ for 1 hour or blocked overnight at 4 ℃. Washing with PBS-0.1% Tween20 solution (PBST), adding hPV19K monoclonal antibody reference substance and hPV19K monoclonal antibody eye injection sample containing different optimized formulations, wherein the initial concentration of the reference substance and each sample is 5 μ g/ml, continuously diluting with 3-fold gradient, and obtaining 12 concentration gradientIncubating at 37 ℃ for 1 hour; after PBST washing, horseradish peroxidase (HRP) -labeled goat anti-human IgG (purchased from Shanghai West Tang Biotech) was added and incubated at 37 ℃ for 1 hour; after PBST washing, o-phenylenediamine (OPD) -0.1% H was added₂O₂The substrate solution was developed for 10-15min and the reaction was stopped with 0.1M HCl. The OD at 492nm was read in MK3-Multiskan microplate reader (Thermo Scientific Co., USA), and the relative binding activity of hPV19K monoclonal antibody ophthalmic injection containing different formulations was calculated to be maintained between 84.19% and 100.83% as shown in FIG. 3.

Thereafter, 5 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 and administered to New Zealand rabbit for fundus injection for animal eye toxicity observation and comparison experiments.

The experiment specifically comprises the following steps:

selecting 15 male New Zealand rabbits with qualified quarantine and similar weight, randomly dividing the rabbits into 5 groups according to the weight, wherein each group comprises 3 animals, and the animals are marked as D-1 on the same day. On the next day (D1), each group of animals received a single injection of hPV19K monoclonal antibody eye injections (10mg/ml, 50. mu.l/eye) in each eye in a single intravitreal injection. General ophthalmic examinations were performed at screening (D-1), before administration (D1), D3, D8, D15. Table 4 below is an observation result of each group of experimental animals.

TABLE 4 general ophthalmic examination results of New Zealand rabbits administered with different optimized formulations hPV19K monoclonal antibody ophthalmic injection

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

Through comparison, the safe toxicological performance of the eyes of the rabbits according to the formulas 1, 3 and 4 is relatively good. Of these, formulation 4(10mM phosphate, 8.8mM glacial acetic acid, 44mM NaCl, 0.03% Tween20, 5% sucrose, 1-20mg/ml hPV19K mab) was the most preferred ophthalmic formulation of the present invention for carrying out the studies of examples 3-5 below.

Example 3 in vitro testing of the biological Activity of a preferred ophthalmic pharmaceutical formulation of hPV19K mAb

1) Direct ELISA method for detecting and comparing biological activity of hPV19K monoclonal antibody eye medicine preparation, commercial Avastin and combaici-cept combined VEGF

Recombinant human VEGF165 protein (0.1. mu.g/ml, pH 9.6, 0.1M NaHCO)₃Liquid) coated with an ELISA plate, and coated for 2 hours at 37 ℃ or overnight at 4 ℃; blocking with 2% BSA at 4 ℃ overnight. After PBST washing, hPV19K monoclonal antibody eye injection, commercial Avastin and commercial combaici cypress are respectively added, the initial concentration of each sample is 1000ng/ml, and the samples are continuously diluted by two times of gradient and incubated for 2 hours at 37 ℃; after PBST washing, HRP-labeled goat anti-human IgG (purchased from West Tang Biopsis, Shanghai) was added and incubated at 37 ℃ for 1 hour; after PBST washing, OPD-0.1% H was added₂O₂The substrate solution was developed for 10-15min and the reaction was stopped with 0.1M HCl. OD at 492nm was read in a microplate reader.

ELISA detection results are shown in FIG. 4, and hPV19K monoclonal antibody eye injection maintains high binding activity with human VEGF165 protein, and the binding activity is 4-8 times higher than that of Avastin and combretastatin.

2) Detection and comparison of hPV19K monoclonal antibody eye medicine preparation, commercial Avastin, Lucentis and Corbescept in vitro blocking of VEGF binding with its recombinant receptor (VEGF-R) by competitive ELISA method

Coating 96-well plates (2. mu.g/ml, 50. mu.l/well) with recombinant soluble human VEGFR1 protein (R & D, USA), overnight at 4 ℃; after PBST rinsing and 2% BSA room temperature blocking, respectively adding 0.1. mu.g/ml biotin-labeled VEGF165 and different concentrations of the hPV19K monoclonal antibody ophthalmic injection sample, commercially available Avastin, commercially available Lucentis and commercially available combaicipu of the invention, and incubating for 2h at 37 ℃; after elution by PBST, HRP-labeled Avidin (1: 5000) was added and incubated at 37 ℃ for 1 h; eluting with PBST, adding OPD-3% hydrogen peroxide, and developing at room temperature for 10 min; the reaction was stopped by adding 0.1M HCl and the absorbance of each well was measured at a wavelength of 492nm using a microplate reader.

FIG. 5 shows the results of the competitive ELISA assay, as shown in the figure: the preferable hPV19K monoclonal antibody eye injection sample and a plurality of commercially available VEGF blockers can specifically block the combination of VEGF and a receptor thereof (VEGF-R) in vitro, the IC50 value of the hPV19K monoclonal antibody eye injection sample can be calculated to be less than 1nM according to a detection curve, and the in vitro activity of the injection sample is superior to that of commercially available Avastin, ranibizumab (Lucentis) and combretacept.

Example 4A Single Subsomatic Administration of a preferred pharmaceutical ophthalmic formulation hPV19K in a vitreous Chamber of Single injections into cynomolgus monkey

18 cynomolgus monkeys were selected for testing, half male and half female, and were randomly divided into 3 groups (vitreous injection low, high dose and intravenous injection groups) by sex. The vitreous injection is low in dosage and high in dosage, and the test sample of the ophthalmic injection hPV19K monoclonal antibody is respectively given by single double-eye vitreous injection (the test sample is 0.5mg/50 mu L/eye, 1mg/50 mu L/eye, and the test sample is given by single intravenous injection of the intravenous group by 2 mg/eye). Before administration, 2, 6, 12, 24, 48, 72h, 5, 7, 9, 11, 14, 17, 21, 24 and 28 days after administration of the animals in the group of vitreous injection, and before administration, 2min, 30min, 2, 6, 12, 24, 48, 72h, 5, 7, 11, 14, 17, 21, 24 and 28 days after administration of the animals in the group of intravenous injection, whole blood was collected, serum was separated, drug concentration in serum was measured by ELISA, and pharmacokinetic parameters were calculated by using Winnolin 6.4 non-compartmental model (NCA). Animals in the vitreous administration group collected about 0.1mL of aqueous humor and vitreous humor 1h, 3, 7, 14, and 28 days after administration, and the pharmacokinetic parameters were calculated using Winnolin 6.4 non-compartmental (NCA) model, in which hPV19K monoclonal antibody concentration was measured by ELISA.

No abnormal clinical manifestations were observed in any animal during the test period, and the statistical results of the pharmacokinetic parameters of serum, aqueous humor and vitreous humor of each group of animals are shown in tables 5, 6 and 7, respectively.

TABLE 5 hPV19 single intravitreal injection of 19K monoclonal antibodies to serum pharmacokinetic parameters in cynomolgus monkeys

TABLE 6 hPV19 single intravitreal injection of 19K monoclonal antibodies to pharmacokinetic parameters in aqueous humor of cynomolgus monkeys

TABLE 7 hPV19 Single intravitreal injection of 19K monoclonal antibodies to pharmacokinetic parameters in the vitreous humor of cynomolgus monkey Room

FIG. 6, FIG. 7 and FIG. 8 are graphs showing the concentration and time course of hPV19K mab in cynomolgus monkey serum, aqueous humor and vitreous humor after hPV19K mab eye injection is administered.

As shown in the above chart, the peak concentrations of the drug in the serum, aqueous humor and vitreous humor of the cynomolgus monkey and the amount of the drug exposed in the serum were positively correlated with the administered dose. Mean serum C_maxRatio of the sum of the mean AUC_lastThe ratio of the water to the water is 1:1.94, 1:1.99 respectively, and the average of the water to the room temperature is C_maxRatio of the sum of the mean AUC_lastThe ratio of the glass bodies is 1:2.28, 1:3.16 respectively, and the average C of the glass bodies_maxRatio of the sum of the mean AUC_lastThe ratio of the two is 1:1.59 and 1:2.02 respectively.

The absolute bioavailability (AUC (0-408h) in the group of high dose intravitreal injections/AUC (0-408h) in the venous group) was 44.90% for a single administration (1 mg/eye, 2 mg/injection) by intravitreal injection.

After single intravitreal injection, the test sample (hPV19K monoclonal antibody eye injection) is used for low and high dose animals, and the average peak concentration (C) of the drug in vitreous humor, aqueous humor and serum is_max) The ratios are 300.21:101.42:1, 245.32:118.73:1 respectively; AUC of drug exposure_lastThe ratio is 151.02:44.78:1 and 153.13:71.05:1 respectively, and the exposure of the medicine in the vitreous body is higher than that of aqueous humor and far higher than that of serum.

Example 5.hPV19 monoclonal antibody 19K ophthalmic pharmaceutical preparation Single intravitreal injection inhibition of cynomolgus monkey choroidal neovascularization

Step 1: establishment of cynomolgus monkey Choroidal Neovascularization (CNV)

Selecting 50 screened qualified animals from 54 cynomolgus monkeys, performing binocular fundus laser photocoagulation to induce choroidal neovascularization (CNV model), wherein the number of laser burns in each eye is 6-8, and the number of laser burns in all animals is recorded as D1 on the day of modeling; the fundus fluorescence leakage of the animals was evaluated by Fundus Fluorography (FFA) examination 2 weeks after molding (D15), and the injury degree of cynomolgus monkey CNV was classified into 4 stages at the time of evaluation. D17, selecting 36 animals with 4-level leakage spots into groups, and averagely grouping according to the average leakage area of the 4-level spots and the 4-level spot rate to ensure that the average leakage area of the eyeground of each group of animals and the 4-level spot rate have no significant difference in grouping.

Step 2: hPV19K monoclonal antibody eye medicine preparation for treating crab eating monkey choroidal neovascularization

The CNV-forming cynomolgus monkeys were randomly divided into 6 groups (6 animals per group, 3 animals per female and male), and on the day of group entry (D17), animals of each group were each injected once into the vitreous cavity (50. mu.L/eye, both eyes) with the test product (hPV19K monoclonal antibody eye injection) or the commercial control products ranibizumab (Lucentis) and combaicipu.

The groups of animals were dosed as follows:

1) hPV19 (vehicle control group) of eye injection of 19K monoclonal antibody;

2) hPV19 eye injection of 19K monoclonal antibody in low dose (0.05 mg/eye);

3) hPV19K monoclonal antibody eye injection test sample dosage group (0.15 mg/eye);

4) hPV19 eye injection of 19K monoclonal antibody in high dose group (0.5 mg/eye);

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

6) a control group (0.5 mg/eye) of commercially available ophthalmic injection of Corbina cypress and cypress.

Fundus photography, fundus angiogram (FFA), and Optical Coherence Tomography (OCT) measurements were performed on each group of animals before molding, D15, D24, and D31. And (4) grading and measuring the CNV of the cynomolgus monkey according to the condition of fundus fluorescence leakage. The height of Subretinal High Reflective Material (SHRM) in the OCT image was determined. The experiment was ended on day 33 (D33).

And (3) test results:

1) fluoroscopy (FFA)

All animals were examined by fundus photography and fluoroscopy before molding, and no obvious abnormality was observed in the results. Fundus photography and fluorography examination were performed after the model was made in D15, D24 and D31, and FIG. 9 is a photograph of fundus photography and middle and late stage fluorography of cynomolgus monkey before and after the administration of the hPV19K monoclonal antibody injection solution group, the ranibizumab injection solution group and the Corpaticep injection solution group. The damage caused by visible laser photocoagulation spots and photocoagulation is removed by photographing the fundus of the eyes of each group of animals, and no other abnormality is seen.

Meanwhile, reading grading and related measurement are carried out on fundus fluorography pictures of D15, D24 and D31 after modeling, and the 4-level facula number, the 4-level facula rate and the 4-level facula leakage area are calculated.

a.4-order spot number and 4-order spot improvement rate

No significant difference in the number of 4-stage spots was observed between groups 2 weeks after molding (D15) (p > 0.05). 7 days after administration (D24), the vehicle control group, the test article (hPV19K monoclonal antibody ophthalmic injection) low, medium and high dose groups, the 4-grade spot numbers (number) of the ranibizumab ophthalmic injection group and the combispu ophthalmic injection group are respectively 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-grade spot improvement rates (%) are respectively 16.7 +/-38.9, 69.9 +/-33.4, 66.2 +/-38.4, 82.4 +/-36.69.1 +/-35.3 and 87.4 +/-25.5. 14 days after administration (D31), the vehicle control group, the test article (hPV19K monoclonal antibody ophthalmic injection) low, medium and high dose groups, the 4-grade spot numbers (number) of the ranibizumab ophthalmic injection group and the combisipu ophthalmic injection group are respectively 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, and the 4-grade spot improvement rates (%) are respectively 20.8 + -39.6, 69.9 + -33.4, 78.8 + -31.4, 85.9 + -28.8, 77.1 + -28.1 and 87.4 + -24.0. The 4-order spot improvement per eye for each group of animals is shown schematically in fig. 10.

7 days and 14 days after administration, the number of spots of 4 grades in the solvent control group is not obviously changed, the improvement rate of the 4 grades of spots is not obvious, the number of the spots of the test sample (hPV19K monoclonal antibody eye injection) in the low, medium and high dose groups is obviously lower than that of the solvent control group (p <0.05) in the 4 grades of the ranibizumab injection group and the combaicipu injection group; the improvement rate of 4-grade faculae of each group of animals is obviously higher than that of a vehicle control product group (p < 0.05).

b.4-level light spot average leakage area, average leakage area reduction and improvement rate

The mean area of fluorescence leakage per eye, the amount of decrease, and the rate of improvement in fluorescence leakage (mean standard deviation) for each group of animals before (2 weeks after molding, D15), 7 days after (D24), and 14 days (D31) were as follows:

TABLE 8 mean area of fluorescence leakage per eye, mean leakage area reduction and improvement rate for each group of animals

2 weeks after molding (D15), the vehicle control group, the test product (hPV19K monoclonal antibody eye injection) low, medium and high dose groups, and the mean value of fluorescence leakage average area (mm2) of each eye of the ranibizumab injection group and the combaicipip eye injection group has no significant difference (p is more than 0.05).

7 days and 14 days after administration, the change of the fluorescence leakage area of the solvent control group is not obvious, the fluorescence leakage area of the test article (hPV19K monoclonal antibody eye injection) is obviously reduced in the low, medium and high dose groups, the fluorescence leakage area of the ranibizumab injection group and the fluorescence leakage improvement rate (%) of the combaici cypriped eye injection group are obviously reduced, and the average reduction amount of the fluorescence leakage area of each eye and the fluorescence leakage improvement rate (%) of each group of animals are obviously higher than those of the solvent control group (p < 0.05). The fluorescence leakage area per eye and improvement for each group of animals is shown in FIG. 11. The average reduction amount and improvement rate of fluorescence leakage per eye of the test article (hPV19K monoclonal antibody eye injection) high-dose group 7 days after administration are higher than those of (p >0.05) ranibizumab injection group and the test article (hPV19K monoclonal antibody eye injection) low-dose and medium-dose groups. 14 days after administration, the average decrease amount and improvement rate of fluorescence leakage per eye of the test article (hPV19K monoclonal antibody eye injection) high dose group are higher than those of (p >0.05) ranibizumab injection group, combaicipu monoclonal eye injection group and test article low and medium dose group.

2) Optical Coherence Tomography (OCT)

All animals were examined by Optical Coherence Tomography (OCT) prior to molding and the results showed no obvious abnormalities in all animals. After the model is manufactured, OCT examinations are performed on D15, D24, and D31, and the maximum height of abnormal high reflectance signal material (SHRM) under the photosensitive retina above the retinal pigment epithelium layer in the OCT image corresponding to the 4-order spot is determined from the D15 fluorescence angiography examination result.

The average height, reduction and improvement (mean standard deviation) of the SHRM per eye of each group of animals before dosing (2 weeks after molding, D15), 7 days (D24) and 14 days (D31) are shown in Table 9.

TABLE 9 average height per eye SHRM, average height reduction and improvement rate for each group of animals

2 weeks after molding (D15), the vehicle control group, the test product (hPV19K monoclonal antibody ophthalmic injection) low, medium and high dose groups, the average height per eye (mum) of SHRM of each group of animals in the ranibizumab injection group and the Corbescept ophthalmic injection group are 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 no significant difference (p >0.05) exists among the groups.

The vehicle control SHRM was not significantly changed at 7 and 14 days after administration, and was significantly reduced in each of the other groups. 7 days after administration (D24), the test product (hPV19K monoclonal antibody eye injection) is in low, medium and high dose groups, and the average height of SHRM per eye of the ranibizumab injection group and the combaici cypress eye injection group is significantly lower than that of the vehicle control group (p < 0.05); the average height reduction of SHRM per eye and the improvement rate (%) of SHRM height of each group are significantly higher than those of the vehicle control group (p < 0.05).

At 14 days after administration (D31), the test article (hPV19K monoclonal antibody eye injection) is in low, medium and high dose groups, and the average height of SHRM per eye of the ranibizumab injection group and the combaici cypress eye injection group is significantly lower than that of the vehicle control group (p < 0.05); the average height reduction of SHRM per eye and the improvement rate (%) of SHRM height of each group are significantly higher than those of the vehicle control group (p < 0.05). The average height reduction amount and the improvement rate of the SHRM per eye of the test article (hPV19K monoclonal antibody eye injection) high dose group are slightly higher than (p >0.05) ranibizumab injection group, combaicipip eye injection group and test article low and medium dose group.

The comprehensive analysis of the fluorescence contrast examination and the optical coherence tomography results shows that the hPV19K monoclonal antibody eye injection in each dosage group can obviously inhibit the cynomolgus monkey choroidal neovascularization, reduce the number of 4-level light spots, and improve the 4-level light spot leakage area and the SHRM height. In addition, the curative effect of the hPV19K monoclonal antibody eye solution which is injected with 1/10 dose (0.05 mg/eye) in a single time for inhibiting the choroidal angiogenesis and leakage of the cynomolgus monkey is similar to the curative effect of a commercial reference product ranibizumab injected with 0.05 mg/eye or the curative effect of the commercial reference product ranibizumab injected with 0.05 mg/eye, and the ophthalmic solution shows excellent in-vivo activity.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

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Claims (8)

1. An ophthalmic pharmaceutical preparation for treating an ocular disease or condition related to angiogenesis, comprising an antibody capable of antagonizing and inhibiting the binding of vascular endothelial growth factor to its receptor, and one or more of an ophthalmic therapeutic agent A pharmaceutically acceptable adjuvant, the light chain of the antibody is the amino acid sequence shown in SEQ ID NO: 9; the heavy chain is the amino acid sequence shown in SEQ ID NO: 10; the antibody is composed of two identical A tetramer composed of the heavy chain and two identical light chains; The adjuvant is a combination of polysorbate and sucrose, and based on the total volume of the ophthalmic pharmaceutical preparation, the adjuvant contains 0.03-0.04% polysorbate in grams per milliliter and 0-5% in grams per milliliter. sucrose; The ophthalmic pharmaceutical preparation further comprises a buffer reagent; the buffer reagent is a combination of disodium hydrogen phosphate dodecahydrate and glacial acetic acid; The antibody concentration of the antibody in the pharmaceutical preparation is 1-20 mg/ml; The osmotic pressure of the ophthalmic pharmaceutical preparation is 285-310 mOsmol/kg; The pH of the ophthalmic pharmaceutical preparation is 5.5-6.5. 2. Use of the ophthalmic pharmaceutical preparation of claim 1 in the preparation of a medicament for the treatment of an ocular disease or condition associated with angiogenesis. 3. Use according to claim 2, 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, choroidal neovascularization secondary to pathological myopia , neovascular glaucoma, proliferative vitreoretinopathy, retinal vascular occlusion, idiopathic chorioretinitis, ocular histoplasmosis, ocular tumors, choroidal neovascularization, cystic macular edema, corneal neovascularization, corneal neovascularization One or more of transplantation, and chronic conjunctivitis. 4. The use according to claim 3, wherein the result of the treatment of an ocular disease or condition associated with angiogenesis comprises a reduction in mean choroidal neovascular leakage, mean improvement in visual acuity, mean reduction in foveal retinal thickness, mean Symptomatic improvement in one or more of reduction in macular size and reduction in mean lesion size. 5. The use according to any one of claims 2 to 4, wherein the ophthalmic pharmaceutical formulation is used in a dose of 0.005 mg/50 μL/eye to 2 mg/50 μL/eye. 6. The use according to any one of claims 2 to 4, wherein the ophthalmic pharmaceutical formulation is used in a dose of 0.05 mg/50 μL/eye to 0.5 mg/50 μL/eye. 7. The use of any one of claims 2 to 4, wherein the ophthalmic pharmaceutical formulation is administered by intravitreal injection. 8. The use according to any one of claims 2 to 4, wherein the ophthalmic pharmaceutical formulation is administered every 1 to 3 months.

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