WO2018195912A1 - 一种眼用药物组合物及其用途 - Google Patents

一种眼用药物组合物及其用途 Download PDF

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WO2018195912A1
WO2018195912A1 PCT/CN2017/082417 CN2017082417W WO2018195912A1 WO 2018195912 A1 WO2018195912 A1 WO 2018195912A1 CN 2017082417 W CN2017082417 W CN 2017082417W WO 2018195912 A1 WO2018195912 A1 WO 2018195912A1
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pharmaceutical composition
ophthalmic pharmaceutical
ophthalmic
group
seq
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PCT/CN2017/082417
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English (en)
French (fr)
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罗师平
胡红群
陈蕞
蔡明文
周建鹰
宋晓琦
陈思宇
石东红
徐一清
周群敏
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苏州思坦维生物技术股份有限公司
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Priority to CN201780047157.1A priority Critical patent/CN109562166B/zh
Priority to PCT/CN2017/082417 priority patent/WO2018195912A1/zh
Publication of WO2018195912A1 publication Critical patent/WO2018195912A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions

Definitions

  • 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.
  • 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.
  • VEGF vascular endothelial growth factor
  • VRF vascular permeability factor
  • VEGF by specifically interacting with VEGF receptors (VEGF-R1, VEGF-R2) on vascular endothelial cells
  • VEGF receptors VEGF-R1, VEGF-R2
  • VEGF-R1 VEGF receptor 2
  • VEGF-R2 VEGF receptor 2
  • 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.
  • 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.
  • 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.
  • 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.
  • VEGF inhibitor-listed drugs approved by the US FDA for the treatment of angiogenesis-related fundus diseases mainly include the following three categories:
  • 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.
  • Bevacizumab is a full-length antibody with a molecular weight of approximately 149 kd
  • ranibizumab is a Fab fragment with a molecular weight of approximately 48 kd.
  • 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.
  • VEGF receptor-Fc fusion protein (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.
  • the VEGF inhibitor drugs currently approved worldwide for the treatment of fundus diseases 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.
  • 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.
  • 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.
  • 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.
  • the fundus injection is required for the treatment of ophthalmic indications such as AMD/DME.
  • 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.
  • 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 present invention provides an ophthalmic pharmaceutical composition
  • 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.
  • the heavy 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.
  • SEQ ID NO: 1-8 are shown in the following table:
  • 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.
  • 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
  • a stabilizer for at least one of mannitol selected from the group consisting of sucrose, sorbitol, glycerin, and trehalose
  • 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.
  • 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
  • the antibody or derivative thereof has a protein concentration in the formulation of from 0.1 to 50 mg/ml.
  • the antibody or derivative thereof has a protein concentration in the pharmaceutical composition of from 1 to 10 mg/ml.
  • the ophthalmic pharmaceutical composition has an osmotic pressure of from 285 to 310 mOsmol/kg, preferably 300 mOsmol/kg.
  • 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.
  • 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.
  • 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.
  • the ophthalmic pharmaceutical composition is used in a dose of 0.005 mg / 50 ⁇ L / eye to 2 mg / 50 ⁇ L / eye, more preferably used in a dose of 0.05 mg / 50 ul / eye to 0.5 mg / 50 ul / eye .
  • the ophthalmic pharmaceutical composition is administered by intravitreal injection.
  • 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.
  • 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.
  • AMD age-related macular degeneration
  • CNV choroidal neovascularization
  • cystic macular edema edema
  • diabetes Macular edema adenocclusion
  • corneal neovascularization corneal transplantation
  • neovascular glaucoma preferably age-related macular degeneration or diabetic retinopathy.
  • 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.
  • 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.
  • the ophthalmic pharmaceutical composition is administered at a dose of 0.05 mg / 50 ⁇ L / eye to 0.5 mg / 50 ⁇ L / eye.
  • the administration is a single or multiple administrations.
  • the ophthalmic pharmaceutical composition is administered by intravitreal injection.
  • the administration time of the ophthalmic pharmaceutical composition is administered once every 1 to 3 months.
  • monoclonal antibody 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.
  • humanized monoclonal antibody 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.
  • CDRs complementarity-determining regions
  • antibody and immunoglobulin 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.
  • VH variable region
  • 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.
  • variable 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 ⁇ -sheet configuration and are joined by three CDRs forming a linker, in some cases formable Part of the ⁇ -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).
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • 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.
  • 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.
  • 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 ⁇ m in each preparation is less than 6000, and the number of particles having a diameter > 25 ⁇ m The number of particles smaller than 600; or diameter > 10 ⁇ m is less than 25 particles/ml, and the number of particles having a diameter > 25 ⁇ m 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.
  • pharmaceutically acceptable 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.
  • 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.
  • a pharmaceutically acceptable carrier or a component thereof examples 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.
  • 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
  • the present invention provides an antibody use concentration, a drug use dose, and a frequency of use in an ophthalmic pharmaceutical composition.
  • the concentration of the antibody is determined to be 0.5mg/ml-20mg/ml, and the dosage and specification are generally between 0.005mg/50 ⁇ L/eye to 2mg/50 ⁇ L/eye, at regular intervals (such as every 1 time). Up to 3 months) intravitreal injection once.
  • 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.
  • 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:
  • Example 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.
  • the vehicle control group served as a negative control group.
  • Example 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.
  • the light gray column is the insoluble particle detection value after one hour of hPV19K monoclonal antibody injection containing different formulations
  • 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
  • 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 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.
  • a indicates p ⁇ 0.05.
  • b indicates p ⁇ 0.05.
  • 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.
  • Example 1 In vitro detection of proliferation of human umbilical vein endothelial cells (HUVEC) by samples containing hPV19K monoclonal antibody
  • 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 diluent DMEM-Medium culture solution containing 10% FBS
  • 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 ⁇ l/well, and incubated at 37 ° C in a 5% carbon dioxide incubator. 30 minutes.
  • Human Umbilical Vein Endothelial Cells were resuspended to 1.0 ⁇ 10 4 /ml with a dilution solution, and added to the above 96-well cell culture plate at 50 ⁇ l/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 ⁇ l/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.
  • HUVEC Human Umbilical Vein Endothelial Cells
  • 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.
  • 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).
  • 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.
  • 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 ⁇ m, and the number of particles having a diameter > 25 ⁇ m is 0.3 particles/ Ml.
  • the insoluble fine particles were further detected to be 312 particles/ml of particles having a diameter of >10 ⁇ m, and the number of particles having a diameter of >25 ⁇ m was 11 particles/ml.
  • hPV19K monoclonal antibody injection for fundus administration containing 8 different optimized formulas (code: YF20161201-YF20161208) was further designed.
  • 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 ⁇ g/ml, PBS, 100 ⁇ l/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 ⁇ g/ml.
  • PBST PBS-0.1% Tween20 solution
  • the total number of eyes in each group is 6; "+, ++, +++, ++++" indicate slight, mild, moderate, and severe, respectively.
  • 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.
  • the plate was coated with recombinant human VEGF165 protein (0.1 ⁇ g/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.
  • 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.
  • 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.
  • VEGF-R recombinant receptor
  • a 96-well plate (2 ⁇ g/ml, 50 ⁇ l/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 ⁇ g/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
  • 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/50 ⁇ L/eye, 1mg/50 ⁇ L/eye, single intravenous bolus for intravenous group).
  • 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.
  • 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 ratio of the ratio to the average AUC last is 1:1.59, 1:2.02, respectively.
  • 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 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
  • CNV model choroidal neovascularization
  • 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).
  • FFA fundus fluorescein angiography
  • 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 ⁇ L/eye, both eyes).
  • the test sample (hPV19K monoclonal antibody ophthalmic injection) or the commercially available control ranibizumab (Lucentis) and Compaqip.
  • ranibizumab injection control group 0.5 mg/eye
  • D15, D24 and D31 were used to perform fundus photography, fundus fluorescein angiography (FFA) and optical coherence tomography (OCT).
  • FFA fundus fluorescein angiography
  • OCT optical coherence tomography
  • 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.
  • 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.
  • 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).
  • 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.
  • 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
  • medium and high dose group ranibizumab injection group
  • 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).
  • 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 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.
  • 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.

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Abstract

一种眼用药物组合物，其包含能够拮抗抑制血管内皮生长因子与其受体结合的抗体或其衍生体，以及用于眼科治疗剂的一种或多种药学上可接受的辅料，所述抗体或其衍生体的轻链抗原互补决定区具有选自如SEQ ID NO：1，SEQ ID NO：2及SEQ ID NO：3所示的氨基酸序列；其重链抗原互补决定区具有选自如SEQ ID NO：4，SEQ ID NO：5及SEQ ID NO：6所示的氨基酸序列，以及该眼用药物组合物在制备用于治疗与血管生成相关的眼部疾病或病症的药物中的用途。

Description

一种眼用药物组合物及其用途 技术领域
本发明属于生物技术-单克隆抗体领域，具体地，本发明涉及一种眼用药物组合物及其用途。
背景技术
血管新生或增生(angiogenesis)在生物学上是指体内已存在的血管(如毛细血管和微小动、静脉)通过出芽或***的方式而产生新的血管的过程。
血管新生在维持机体许多正常生理过程如组织胚胎发育、外伤伤口的癒合与修复等是有益的和必需的；但过度的血管新生或增生也会导致机体发生病变。如体内如肿瘤的增生扩散转移与复发、老年黄斑变性(又称年龄相关性黄斑变性，age-related macular degeneration，AMD)眼底病、糖尿病性黄斑水肿(diabetic macular edema，DME)、炎症反应及自身免疫疾病等都与血管新生或增生密切相关。
体内血管能够增生与增长的关键是其内衬的血管内皮细胞具有不断***增生及定向迁移植入已有的血管管壁的能力。目前已知的最重要及最强烈的促血管内皮细胞***新生或血管增生的物质是血管内皮细胞生长因子(vascular endothelial growth factor，VEGF)。VEGF又称为血管渗透因子(vascular permeability factor，VRF)，编码VEGF/VRF蛋白的cDNA已由两研究组于1989年同时在美国Science杂志上公开报道。
VEGF通过特异性地与血管内皮细胞上的VEGF受体(VEGF-R1，VEGF-R2) 结合而发挥促血管内皮细胞生长迁移、血管新生及增加血管通透性的作用。VEGF及其受体介导的血管增生之重要性已在VEGF基因剔除小鼠的研究中被充分证实：因为只要当VEGF基因中的一份被敲除后，小鼠胚胎在仅发育至11至12天时就会因血管增生受阻及异常而死亡。
VEGF及其受体介导的促血管新生及促血管渗透在老年黄斑变性、糖尿病性黄斑水肿等致盲性眼底疾病的发生与病理进展中也起着关键的作用。老年黄斑变性大多发生于50多岁以上，患病率随年龄增长而增高，该病的临床症状表现为中心视力减退、病程进展迅速，是导致老年人失明的重要疾病。而糖尿病性黄斑水肿则是当今危害数以千万计的中年工薪阶层(working-class)视力的重大疾病。
老年黄斑变性及糖尿病性黄斑水肿的主要病理表现为黄斑及周边组织萎缩变性、视细胞破坏、玻璃膜疣形成，严重者发生渗出性黄斑变性，伴随视网膜下新生血管形成、出血以及渗出。以往对老年黄斑变性及糖尿病性黄斑水肿等致盲性眼底疾病常采用激光治疗和光动力治疗手段，虽可暂时缓解症状，但复发率高，且并不能阻止病程进展。
2005年以来，临床上采用各种VEGF抑制剂来治疗渗出性黄斑变性及糖尿病性黄斑水肿，均取得比较理想的疗效，现已被美国国家眼科研究所(National Eye Institute，NEI)推荐为常规疗法。
到目前为止，经美国FDA批准用于治疗血管新生相关眼底疾病的VEGF抑制剂上市药物主要包括以下三大类：
(一)哌加他尼钠注射液(Pegaptanib，商品名为Macugen)，由美国Eyetech制药公司(Eyetech Pharmaceuticals，Inc.)和辉瑞公司(Pfizer Inc.)合作开发， 于2004年12月获美国FDA批准上市。哌加他尼钠为PEG修饰的单链RNA类似物，其独特的三维结构使其能特异结合VEGF并抑制VEGF的活性。
(二)拮抗VEGF的抗体或抗体衍生物。目前该类药物中获美国FDA批准上市的仅有Genentech公司开发的雷珠单抗(Ranibizumab，中文商品名：诺适得，英文商品名：Lucentis)，用于治疗包括老年黄斑变性及糖尿病性黄斑水肿等致盲性眼底疾病。雷珠单抗与Genentech公司早先开发上市的适应症为治疗多种肿瘤的另一个抗体药物贝伐珠单抗(Bevacizumab，商品名Avastin)药物同源：两者都是由鼠源单抗A4.6.1衍生而来，其可变区同源性在99％以上。贝伐单抗是全长抗体，分子量约149kd，而雷珠单抗是Fab片段，分子量约48kd。与Avastin一样，雷珠单抗可与VEGF高亲和力结合，竞争性阻断VEGF信号通路，抑制新生血管形成及促进黄斑区渗液吸收。Genentech公司发起的临床研究表明患者接受雷珠单抗治疗2年后，95％的“湿性”AMD患眼的视力稳定或提高。凭此优异疗效，雷珠单抗于2006年6月获美国FDA批准上市。雷珠单抗其后在美国进一步获批用于治疗糖尿病性黄斑水肿、视网膜静脉阻塞继发黄斑水肿、病理性近视继发脉络膜新生血管等多个适应症，雷珠单抗目前已成为治疗AMD等与血管新生相关眼底疾病的主流药物。
在中国，雷珠单抗于2011年获中国药监局批准上市用于治疗AMD。
(三)VEGF受体-Fc融合蛋白。该类药物由VEGF受体胞膜外区与人免疫球蛋-Fc段融合而成，其代表性药物为美国再生元(Regeneron)公司开发的阿柏西普(Aflibercept，商品名：Eylea)。阿柏西普由VEGF-R1受体胞膜外区第2结构域、VEGF-R2胞膜外区第3结构域、人免疫球蛋白IgG1的Fc段融合而成。VEGF-Trap与VEGF的亲和力高于其受体与配体的自然亲和力，可竞争性阻抑 人体内的VEGF-R与VEGF的结合。阿柏西普自2011年以来已先后获美国FDA批准用于治疗包括老年黄斑变性及糖尿病性黄斑水肿等致盲性眼底疾病，且目前在临床适应症的获批范围及全球多个市场销售领域都在赶超雷珠单抗。
中国成都康宏药业集团股份有限公司开发的康柏西普(Conbercept)也是VEGF受体-Fc融合蛋白。康柏西普在药物结构上与阿柏西普非常相似，由VEGF-R1胞膜外第2结构域、VEGF-R2胞膜外第3和4结构域、人免疫球蛋白IgG1的Fc段融合而成。康柏西普于2013年获中国CFDA批准用于治疗老年眼底黄斑变性等疾病，目前也正在美国开展临床III期研究。
综上所述，目前世界范围内正式获批用于治疗AMD/DME等眼底疾病的VEGF抑制剂类药物均属VEGF单抗的Fab片段或VEGF受体-抗体Fc融合蛋白。单抗-Fab片段如雷珠单抗因其分子量较小，眼底注射后具有易快速穿透到病灶而发挥药效的优点；但单抗-Fab片段在眼内半衰期仅为2-3天，短于完整的抗体，从而增加了给药次数和患者的经济负担。VEGF受体-Fc融合蛋白药物如阿柏西普及康柏西普因其与配体结合亲和力高于其天然受体(VEGFR-1，VEGFR-2)；且眼底注射后，其在眼内半衰期为3-5天，稍长于雷珠单抗，故具有药效高、用药频率低等优点。但阿柏西普及康柏西普因其同时还可与VEGF-B、VEGF-C、PLGF(Placental growth factor)等其他生长因子结合，从而增加了与治疗目的(特异性阻断VEGF介导)不一致的药物不良反应发生的潜在风险。
与Fab片段或Fc融合蛋白相比，完整的、全长的抗体如Avastin兼具Fab段和Fc段双重优势，具有体内半衰期长、用药频率低等优势，临床上可望以更少的剂量或更低的注射频率用于治疗AMD/DME等血管新生性眼病。近年来临床前及临床研究结果数据均显示，全长抗体Avastin用于治疗AMD/DME的效果 其实并不亚于雷珠单抗或阿柏西普。但Avastin并未获FDA或其他国家的药监部门批准用于治疗恶性肿瘤之外的其他适应症如眼科适应症；Avastin在治疗AMD/DME的眼科使用属于“非标签使用”或“标签外使用”。用于***适应症的药物和用于治疗AMD/DME等眼科适应症药物在药物配方、剂量、制造与检测放行标准及产品包装等都有很多不同之处。用于治疗AMD/DME等眼科适应症药物需眼底注射，药物除需严格保持无菌之外，其pH及渗透压应接近于泪液，且不溶性微粒物质要求控制在非常低的数量下；而用于***的Avastin在其制造及包装上并没有考虑眼科用药的这些特点与要求。此外，临床上患者或医生为了降低眼底注射用药的成本费用，更是常将大包装规格的Avastin(每瓶规格为4ml，或20ml)分装成小份供患者分享使用，患者接受眼底注射分装的药物后常发生感染或炎症的安全风险。
抗血管增生药物治疗AMD/DME的市场惠及千万个患者，且患者需常年或反复多次给药，现仅有的抗血管增生眼科药物雷珠单抗、阿柏西普及康柏西普还未满足市场需求；而另一方面，目前上市的这三个药物也不是对所有的AMD/DME患者都有疗效。因此，临床上及市场上也都仍然需要再研发出新的或更安全有效的抗血管增生眼科药物。
发明内容
本发明提供了一种眼用药物组合物，其包含能够拮抗抑制血管内皮生长因子与其受体结合的抗体或其衍生体，以及用于眼科治疗剂的一种或多种药学上可接受的辅料，所述抗体或其衍生体的轻链抗原互补决定区具有选自如SEQ ID NO：1，SEQ ID NO：2及SEQ ID NO：3所示的氨基酸序列；其重链抗原互补 决定区具有选自如SEQ ID NO：4，SEQ ID NO：5及SEQ ID NO：6所示的氨基酸序列。在所述眼用药物组合物的一个优选实施方案中，其中所述抗体或其衍生体的轻链可变区具有SEQ ID NO：7所示的氨基酸序列，其重链可变区具有SEQ ID NO：8所示的氨基酸序列。其中，SEQ ID NO：1-8如下表所示：
表1：氨基酸序列
Figure PCTCN2017082417-appb-000001
在所述眼用药物组合物的一个优选实施方案中，基于所述眼用药物组合物的总体积，其中所述一种或多种药学上可接受的辅料含有0.01-0.15％(m/v，g/ml)的选自聚山梨醇酯(吐温)、聚乙二醇、丙二醇及它们的组合中的至少一种的有机共溶剂；和1-10％(m/v，g/ml)的选自蔗糖、山梨醇、甘油、海藻糖 和甘露醇中的至少一种的稳定剂。
优选地，基于所述眼用药物组合物的总体积，所述一种或多种药学上可接受的辅料含有0.01-0.02％(m/v，g/ml)的聚山梨醇酯和7-9％(m/v，g/ml)的蔗糖。
优选地，基于所述眼用药物组合物的总体积，所述一种或多种药学上可接受的辅料含有0.03-0.04％(m/v，g/ml)的聚山梨醇酯和0-5％(m/v，g/ml)的蔗糖
在所述眼用药物组合物的一个优选实施方案中，其中所述抗体或其衍生体在所述制剂中的蛋白浓度为0.1-50mg/ml。
优选地，其中所述抗体或其衍生体在所述药物组合物中的蛋白浓度为1-10mg/ml。
优选地，所述眼用药物组合物的渗透压为285-310mOsmol/kg，优选300mOsmol/kg。
优选地，所述眼用药物组合物的pH值为5.5-6.5，更优选pH值为6.0。
本发明还提供了上述眼用药物组合物在制备用于治疗与血管生成相关的眼部疾病或病症的药物中的用途。
在所述用途中，其中所述与血管生成相关的眼部疾病或病症选自年龄相关性黄斑变性、糖尿病性黄斑水肿、糖尿病性视网膜病变、病理性近视继发脉络膜新生血管、新生血管性青光眼、增生性玻璃体视网膜病变、视网膜血管阻塞、特发性脉络膜视网膜炎、眼组织胞浆菌病、眼部肿瘤、眼外伤、脉络膜新生血管形成、囊性黄斑水肿、角膜新生血管形成、角膜移植、和慢性结膜炎中的一种或多种。
在所述用途中，其中所述与血管生成相关的眼部疾病或病症的结果包括选 自平均脉络膜血管新生渗漏减少、平均视力改善、平均中央凹视网膜厚度减少、平均黄斑大小减少和平均损伤大小减少中的一种或多种的症状改善。
在所述用途中，其中所述眼用药物组合物的使用剂量为0.005mg/50μL/眼至2mg/50μL/眼，更优选的使用剂量为0.05mg/50ul/眼至0.5mg/50ul/眼。
优选地，其中所述眼用药物组合物的施用方式为通过玻璃体内注射。
优选地，其中所述眼用药物组合物的施用时间为每间隔1至3个月施用一次。
本发明还提供了一种治疗受试者的与血管生成相关的眼部疾病或病症的方法，其包括向受试者眼内局部施用上述眼用药物组合物。
在所述方法中，其中，所述与血管生成相关的眼部疾病或病症选自年龄相关性黄斑变性(AMD)，糖尿病性视网膜病变，脉络膜新生血管形成(CNV)，囊性黄斑水肿，糖尿病性黄斑水肿，视网膜血管闭塞，角膜新生血管形成，角膜移植，新生血管性青光眼和慢性结膜炎，优选为年龄相关性黄斑变性或糖尿病性视网膜病变。
优选地，所述治疗的结果包括选自平均脉络膜血管新生渗漏减少、平均视力改善、平均中央凹视网膜厚度减少、平均黄斑大小减少和平均损伤大小减少中的一种或多种的症状改善。
优选地，所述眼用药物组合物的施用剂量为0.05mg/50μL/眼至0.5mg/50μL/眼。
优选地，所述施用为单次或多次施用。
优选地，所述眼用药物组合物的施用方式为通过玻璃体内注射。
优选地，所述眼用药物组合物的施用时间为每间隔1至3个月施用一次。
本文所采用的术语“单克隆抗体(单抗)”指从一纯系细胞得到的免疫球蛋 白，具有相同的结构和化学特性，对单一抗原决定簇有特异性。单克隆抗体与常规多克隆抗体制剂(通常是具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性外，单克隆抗体的好处还在于它们是通过杂交瘤或重组工程细胞培养获得，不会混杂有其它免疫球蛋白。修饰语“单克隆”表示了抗体的特性，是从均一的抗体群中获得的，这不应被解释成需要用任何特殊方法来生产抗体。
本文所采用的术语“人源化单克隆抗体”系将鼠源单克隆抗体的氨基酸序列除保留互补决定区(complementarity-determining regions，CDR)外，其它序列(包括可变区中的框架区序列)全部或大部分替换成人免疫球蛋白的氨基酸序列，以达到通过基因工程手段最大限度地降低鼠源性单克隆抗体的免疫原性。
本文所用的术语“抗体”和“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)。其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
本文所用的术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中成为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(Framework regions，FR)。抗体重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形 成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位。抗体恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性(antibody-dependent cellular cytotoxicity，ADCC)或补体介导毒性(complement-dependent cytotoxicity，CDC)。
本发明提供的眼用药物组合物含有药学上有效量的如本发明中描述的人源化抗体或其衍生体以及药学上可接受的合适载体及其他组分，使该生物制剂符合眼科用制剂的特殊剂量规格及技术要求。其中技术要求的一大指标是该眼科用制剂渗透压应与泪液等渗，且不含或含极少量的不溶性颗粒物质，以用于眼睛玻璃体内注射或滴眼给药。
在本发明的具体实施例中，描述了对该眼用药物组合物的不溶性微粒、渗透压、pH值和注射体积的规定。通常，用于临床使用的制剂中的融合蛋白的浓度为0.01mg/mL至100mg/mL，具体剂量取决于制剂的形式，临床需要等。通常，玻璃体内注射包括给予约0.01mg至10mg；本发明的该眼用注射液中的微粒数量最高限度为：每支制剂中直径＞10μm的微粒个数小于6000，直径＞25μm的微粒个数小于600；或直径＞10μm的微粒个数小于25粒/ml，直径＞25μm的微粒个数小于3粒/ml。本发明的该眼用药物组合物摩尔浓度渗透压应与泪液等渗，一般为285-310mOsmol/kg；该眼用药物组合物pH应在5.5-6.5左右，以尽可能与泪液接近；本发明的该眼用药物组合物采用玻璃体内注射方式给药，要求给药体积很小，药物浓度达到一定要求。
本文所用的术语“药学上可接受的”是指当该抗体和组合物适当地给予动物或人时，它们不会产生过敏或其它不良反应。本文所用的“药学上可接受的载体”应当与本发明的抗体蛋白相容，即能与其共混而不会大幅度降低药物组 合物的效果。可作为药学上可接受的载体或其组分的一些物质的具体例子包括糖类，如乳糖、葡萄糖和蔗糖；淀粉，如玉米淀粉和土豆淀粉；植物油，如花生油、棉籽油、芝麻油、橄榄油、玉米油和可可油；多元醇，如丙二醇、甘油、山梨糖醇、甘露糖醇和聚乙二醇；海藻酸；乳化剂，如Tween；稳定剂；抗氧化剂；无热原无菌注射用水；生理盐水溶液；磷酸盐缓冲剂等。
本发明提供了眼用药物组合物中抗体使用浓度、药物使用剂量及使用频率。根据现有研究资料，确定抗体的浓度为0.5mg/ml-20mg/ml，使用剂量与规格一般为0.005mg/50μL/眼至2mg/50μL/眼之间，每隔一定时间(如每隔1到3个月)玻璃体腔注射一次。具体实施中应由医师根据患者种类、年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的给药剂量，给药时间及给药频率。
在下文的实施例及附图说明中，为表述方便，将本发明药物组合物中的所述抗体或其衍生体以代号hPV19K表示。hPV19K单抗是由两条相同的重链和两条相同的轻链组成的四聚体，其中hPV19K单抗轻链具有SEQ ID NO：9所示的氨基酸序列，hPV19K单抗重链具有SEQ ID NO：10所示的氨基酸序列。SEQ ID NO：9-10如下表2所示：
表2：hPV19K单抗轻链及重链氨基酸序列
Figure PCTCN2017082417-appb-000002
Figure PCTCN2017082417-appb-000003
附图说明
图1为本发明实施例1中体外检测分析hPV19K单抗及Avastin抑制VEGF介导的人脐静脉内皮细胞(Human Umbilical Vein Endothelial Cells，HUVEC)增殖活性的结果。其中，溶媒对照组作为阴性对照组。
图2为本发明实施例2中含不同配方的hPV19K单抗眼用注射液中不溶性微粒的检测结果。其中，浅灰柱状物为含不同配方的hPV19K单抗注射液放置一小时后的不溶性微粒检测数值，深灰柱状物为各配方注射液放置14小时后的不溶性微粒检测数值；粗线为药典规定的注射剂大输液不溶性微粒的标准线，细线为药典规定的注射剂小针剂不溶性微粒的标准线。
图3为本发明实施例2中直接ELISA法检测含不同配方的hPV19K单抗的眼用注射液的结合VEGF的相对活性。
图4为本发明实施例3中直接ELISA法检测含不同配方的hPV19K单抗的眼用注射液与市售的Avastin及康柏西普与VEGF结合的相对活性比较。
图5为本发明实施例3中竞争性ELISA法检测各VEGF抑制剂体外阻断VEGF与其受体(VEGFR-1)的结合的示意图。
图6为本发明实施例4中hPV19K单抗眼用注射液单次玻璃体腔注射给予食蟹猴后血清hPV19K单抗浓度-时间曲线(n＝6)。
图7为本发明实施例4中hPV19K单抗眼用注射液单次玻璃体腔注射给予食蟹猴后房水hPV19K单抗浓度-时间曲线(n＝12)。
图8为本发明实施例4中hPV19K单抗眼用注射液单次玻璃体腔注射给予食蟹猴后玻璃体液中hPV19K单抗浓度-时间曲线(n＝12)。
图9为本发明实施例5中hPV19K单抗眼用注射液玻璃体腔单次注射前后食蟹猴眼底照相和荧光造影中晚期图片。
图10为本发明实施例5中hPV19K单抗眼用注射液玻璃体腔单次注射对食蟹猴脉络膜新生血管4级光斑改善率的检测结果图，与同期溶媒对照组比较，a表示p＜0.05。
图11A、11B及11C为本发明实施例5中hPV19K单抗眼用注射液玻璃体腔单次注射对食蟹猴脉络膜新生血管4级光斑渗漏面积及改善率结果图。
其中，图11A为给药前后各组动物每眼荧光渗漏平均面积变化结果，与同期溶媒对照组比较，a表示p＜0.05；
图11B为D24、D31各组动物每眼荧光渗漏平均面积减少量，与同期溶媒 对照组比较，a表示p＜0.05；与同期供试品高剂量组比较，d表示p＜0.05；与同期雷珠单抗注射液组比较，e表示＜0.05；
图11C为D24、D31各组动物每眼荧光渗漏面积改善率，与同期溶媒对照组比较，a表示p＜0.05；与同期供试品低剂量组比较，b表示p≤0.05；与同期供试品中剂量组比较，c表示p＜0.05；与同期雷珠单抗注射液组比较，e表示p＜0.05。
图12A、12B及12C为本发明实施例5中hPV19K单抗眼用注射液玻璃体腔单次注射对食蟹猴视网膜下的异常高反射信号物质的光学相干断层扫描结果。
其中，图12A为给药前后各组动物每眼SHRM平均高度变化，与同期溶媒对照组比较，a表示p＜0.05；
图12B为D24、D31各组动物每眼SHRM平均高度减少量，与同期溶媒对照组比较，a表示p＜0.05；
图12C为D24、D31各组动物每眼SHRM平均高度改善率，与同期溶媒对照组比较，a表示p＜0.05；与同期供试品中剂量组比较，c表示p＜0.05。
具体实施方式
下面将结合实施实例来进一步描述本发明，这些实施例只是为了起说明作用，而不是用来限制本发明。
实施例1.体外检测含hPV19K单抗的样品抑制人脐静脉内皮细胞(HUVEC)增殖的活性
在本研究实施例中，分别用稀释液(含10％FBS的DMEM-Medium培养液)将hPV19K抗体参比品、hPV19K抗体供试样品和Avastin对照抗体样品，配制 成2000ng/ml的溶液，随后用含6ng/ml rhVEGF165的稀释液连续3倍梯度稀释，共10个浓度梯度。以稀释液作为阴性对照，含6ng/ml rhVEGF165的稀释液作为阳性对照，将阴性对照、阳性对照、系列稀释样品按50μl/孔加入96孔白色细胞培养板中，37℃5％二氧化碳培养箱孵育30分钟。
来源于人脐静脉的血管内皮细胞(Human Umbilical Vein Endothelial Cells，HUVEC)用稀释液重悬至1.0×104/ml，按50μl/孔加入上述96孔细胞培养板中，外圈用无菌水封闭，37℃5％二氧化碳培养箱孵育72小时。按顺序加入新鲜配制的CellTier-Glo增殖试剂(美国Promega公司产品)100μl/孔，室温避光显色15min，以荧光酶标仪读取RLU读数，以参比品或待测样品的浓度作为横坐标、RLU读数的平均数作为纵坐标作图，结果如图1。经计算，hPV19K抗体参比品、hPV19K供试样品和Avastin对照品的IC50分别为16.9ng/ml、16.8ng/ml和89.6ng/ml，对于体外抑制VEGF介导的人脐静脉内皮细胞(HUVEC)的增殖活性，hPV19K抗体显著强于Avastin(平均强6.7倍)。
实施例2.眼用hPV19K单抗药物制剂的开发
为降低眼科用hPV19K单抗注射剂中不溶性微粒数量，防止眼部非特异性炎症的发生，在hPV19K单抗药物制剂开发中特意加入糖类(如蔗糖)、乳化剂(如聚山梨醇酯，吐温80或吐温20)等载体以减少抗体大分子的相互聚集。在该药物制剂开发中初步设计了8个不同的配方，将hPV19K单抗分别溶于含有不同浓度的吐温和蔗糖的不同配方的液体中，各放置1小时和14小时后检测各溶液中的不溶性微粒。图2为含不同配方的hPV19K单抗药物制剂中的不溶性微粒检测结果。
之后，对含配方8的hPV19K单抗药物制剂样品中的氯化钠含量和缓冲液的比例分别进行调整获得注射液制剂初步样品。经检测，该注射液制剂样品的渗透压为300mOsmol/kg；pH为5.5-6.5；不溶性微粒为：直径＞10μm的微粒个数为5.8粒/ml，直径＞25μm的微粒个数为0.3粒/ml。其后将注射液制剂样品于25℃下放置6个月后，不溶性微粒再检测结果为直径＞10μm的微粒个数为312粒/ml，直径＞25μm的微粒个数为11粒/ml。
在上述研究结果基础上，再进一步设计出含8种不同优化配方(代号：YF20161201-YF20161208)的拟用于眼底给药的hPV19K单抗注射液。
表3拟用于眼底给药的hPV19K单抗制剂中不同优化配方组分及其含量
Figure PCTCN2017082417-appb-000004
经紫外吸收法测得各优化配方制剂中的hPV19K抗体蛋白浓度在9.81mg/ml-10.01mg/ml范围内，SEC-HPLC测定hPV19K单抗纯度为96.60％-96.70％，由此可知上述含不同优化配方的hPV19K单抗眼用注射液的蛋 白浓度和纯度基本相同。
之后利用直接ELISA法检测hPV19K单抗眼用注射液中的抗体相对结合活性。其检测步骤如下：以重组人VEGF165蛋白(0.1μg/ml，PBS，100μl/孔)包被酶标板，37℃包被2小时或4℃过夜；5％脱脂牛奶37℃放置1小时或4℃封闭过夜。经PBS-0.1％Tween20液(PBST)洗涤后分别加入hPV19K单抗参考品、含不同优化配方的hPV19K单抗眼用注射液样品，参考品及各样品的起始浓度为5μg/ml，进行连续3倍梯度稀释，共12个浓度梯度，37℃孵育1小时；经PBST洗涤后，加入辣根过氧化物酶(HRP)-标记的羊抗人IgG(购自上海西唐生物公司)，37℃孵育1小时；再经PBST充分洗涤后，加入邻苯二胺(OPD)-0.1％H2O2底物液显色10-15min，以0.1M HCl终止反应。在MK3-Multiskan酶标仪(美国Thermo Scientific公司产品)中读取492nm处OD值，结果如图3，经计算含不同配方的hPV19K单抗眼用注射液的相对结合活性保持在84.19％-100.83％之间。
其后，选择单抗相对结合活性大于90％的5个优化配方的hPV19K单抗眼用注射液(YF20161202、YF20161205、YF20161206、YF20161207、YF20161208)给予新西兰兔眼底注射，进行动物眼部毒性观察和比较实验。
该实验具体过程如下：
选择检疫合格、体重相近的15只雄性新西兰兔，按体重随机分成5组，每组3只动物，分组当天记为D-1。第二天(记为D1)各组动物分别双眼玻璃体腔单次注射含不同配方的hPV19K单抗眼用注射液(10mg/ml，50μl/眼)。在筛选时(D-1)、给药前(D1)、D3、D8、D15进行一般眼科检查。下表4为各组实验动物的观察结果。
表4不同优化配方hPV19K单抗眼用注射液给予新西兰兔一般眼科检查结果
Figure PCTCN2017082417-appb-000005
Figure PCTCN2017082417-appb-000006
注：各组眼睛总数为6；“+、++、+++、++++”分别表示轻微、轻度、中度、重度
经比较，发现配方1、配方3及配方4的兔眼部安全毒理表现相对较好。其中，配方4(10mM磷酸盐，8.8mM冰醋酸，44mM NaCl，0.03％吐温20，5％蔗糖，1-20mg/ml hPV19K单抗)作为本发明的最优选眼用制剂，用于开展下述实施例3-5的研究。
实施例3.体外检测优选的hPV19K单抗眼用药物制剂的生物活性
1)直接ELISA法检测与比较本发明优选的hPV19K单抗眼用药物制剂、市售的Avastin及康柏西普结合VEGF的生物活性
以重组人VEGF165蛋白(0.1μg/ml，pH9.6，0.1M NaHCO3液)包被酶标板，37℃包被2小时或4℃过夜；2％BSA4℃封闭过夜。经PBST洗涤后分别加入hPV19K单抗眼用注射液、市售的Avastin、市售的康柏西普，各样品的起始浓度为1000ng/ml，并进行连续两倍梯度稀释，37℃孵育2小时；经PBST洗涤后，加入HRP-标记的羊抗人IgG(购自上海西唐生物公司西唐生物)，37℃孵育1小时；再经PBST充分洗涤后，加入OPD-0.1％H2O2底物液显色10-15min，以0.1M HCl终止反应。在酶标仪中读取492nm处OD值。
ELISA检测结果如图4，hPV19K单抗眼用注射液保持与人VEGF165蛋白高度结合的活性，且其结合活力比Avastin和康柏西普高4-8倍。
2)竞争性ELISA法检测与比较本发明优选的hPV19K单抗眼用药物制剂、市售的Avastin、Lucentis及康柏西普体外阻断VEGF与其重组受体(VEGF-R)的结合
用重组可溶性人VEGFR1蛋白(美国R&D公司产品)包被96-孔板(2μg/ml，50μl/孔)，4℃过夜；经PBST漂洗及2％BSA室温封闭后，分别加入0.1μg/ml的生物素标记的VEGF165与不同浓度的本发明优选的hPV19K单抗眼用注射液样品、市售的Avastin、市售的Lucentis及市售的康柏西普，37℃孵育2h；经PBST洗脱后，加入HRP标记的Avidin(1：5000)，37℃孵育1h；经PBST洗脱后，加入OPD-3％双氧水，室温10min至显色；加入0.1M HCl终止反应，以酶标仪测定492nm波长处各孔的吸光值。
图5为该竞争性ELISA检测结果，如图所示：本发明优选的hPV19K单抗眼用注射液样品及市售的几个VEGF阻断剂体外均可特异阻断VEGF与其受体(VEGF-R)的结合，根据检测曲线可测算hPV19K单抗眼用注射液样品的IC50值＜1nM，其体外活性优于市售的Avastin、雷珠单抗(Lucentis)及康柏西普。
实施例4.优选的hPV19K单抗药物眼用制剂单次玻璃体腔注射给予食蟹猴药代动力学研究
试验选用食蟹猴18只，雌雄各半，分性别随机分成3组(玻璃体注射低、高剂量组及静脉注射组)。玻璃体注射低、高剂量组分别单次双眼玻璃体注射给予hPV19K单抗眼用注射液供试品(供试品0.5mg/50μL/眼、1mg/50μL/眼，静脉组单次静脉推注给予供试品2mg/只)。玻璃体注射给药组动物给药前，药后2、6、12、24、48、72h，5、7、9、11、14、17、21、24、28天采集全血，静脉 组动物给药前，药后2min、30min、2、6、12、24、48、72h，5、7、11、14、17、21、24、28天采集全血，分离血清，ELISA测定血清中药物浓度，采用Winnonlin 6.4非房室模型(NCA)计算药代动力学参数。玻璃体给药组动物于给药后1h，3、7、14、28天各采集约0.1mL房水和玻璃体液，ELISA测定hPV19K单抗浓度，采用Winnonlin 6.4非房室模型(NCA)计算药代动力学参数。
试验期间各动物均未见异常临床表现，各组动物血清、房水中及玻璃体药物代谢动力学参数统计结果分别见表5、表6及表7。
表5 hPV19K单抗单次玻璃体腔注射给予食蟹猴血清药物代谢动力学参数
Figure PCTCN2017082417-appb-000007
表6 hPV19K单抗单次玻璃体腔注射给予食蟹猴房水中药物代谢动力学参数
Figure PCTCN2017082417-appb-000008
表7 hPV19K单抗单次玻璃体腔注射给予食蟹猴房玻璃体液中药物代谢动力学参数
Figure PCTCN2017082417-appb-000009
图6、图7及8分别为hPV19K单抗眼用注射剂给药后，食蟹猴血清、房水及玻璃体液中hPV19K单抗浓度与时间图。
结果如上述图表所示，食蟹猴血清、房水及玻璃体中药物峰浓度及血清药物暴露量均与给药剂量正相关。血清平均Cmax之比及平均AUClast之比分别为1∶1.94、1∶1.99，房水平均Cmax之比及平均AUClast之比分别为1∶2.28、1∶3.16，玻璃体平均Cmax之比及平均AUClast之比分别为1∶1.59、1∶2.02。
单次双眼玻璃体注射给药(1mg/眼，2mg/只)的绝对生物利用度(玻璃体注射高剂量组AUC(0-408h)/静脉组AUC(0-408h)为44.90％。
单次玻璃体腔注射给药后，供试品(hPV19K单抗眼用注射剂)低、高剂量组动物玻璃体液、房水与血清中平均药物峰浓度(Cmax)比值分别为300.21∶101.42∶1、245.32∶118.73∶1；药物暴露量AUClast比值分别为151.02∶44.78∶1、153.13∶71.05∶1，药物在玻璃体中的暴露量高于房水，远高于血清。
实施例5.hPV19K单抗眼用药物制剂单次玻璃体腔注射对食蟹猴脉络膜新生血管的抑制作用
步骤1：食蟹猴脉络膜新生血管模型(choroidal neovascularization CNV)建立
从54只食蟹猴中选取50只经筛选合格的动物进行双眼眼底激光光凝，以诱导脉络膜血管新生(CNV模型)，每只眼激光灼烧数量为6-8个，所有动物造模当天记为D1；造模后2周(D15)，通过眼底荧光造影(FFA)检查评估动物的眼底荧光渗漏情况，评估时将食蟹猴CNV的损伤程度分为4级。D17挑选36个有4级渗漏光斑的动物入组，根据4级光斑平均渗漏面积和4级光斑率进行平均分组，保证在分组时各组动物眼底平均渗漏面积和4级光斑率无显著差异。
步骤2：hPV19K单抗眼用药物制剂治疗食蟹猴脉络膜新生血管
将上述形成CNV的食蟹猴随机分成6组(每组6只，雌雄各3只)，入组当天(D17)各组动物分别单次经玻璃体腔注射(50μL/眼，双眼给药)供试品(hPV19K单抗眼用注射剂)或市售的对照品雷珠单抗(Lucentis)及康柏西普。
各组动物给药分组如下：
1)hPV19K单抗眼用注射液溶媒照品组；
2)hPV19K单抗眼用注射液供试品低剂量组(0.05mg/眼)；
3)hPV19K单抗眼用注射液供试品中剂量组(0.15mg/眼)；
4)hPV19K单抗眼用注射液供试品高剂量组(0.5mg/眼)；
5)市售的雷珠单抗注射液对照品组(0.5mg/眼)；
6)市售的康柏西普眼用注射液对照品组(0.5mg/眼)。
造模前、D15、D24和D31对各组动物进行眼底照相、眼底血管荧光造影(FFA)及光相干断层成像(OCT)检测。根据眼底荧光渗漏情况对食蟹猴CNV进行分级和测量。测定OCT图像中视网膜下高反射信号物质(Subretinal hyperreflective material，SHRM)的高度。第33天(D33)试验结束。
试验结果：
1)荧光造影检查(FFA)
所有动物在造模前进行了眼底照相和荧光造影检查，结果均无明显异常。造模后D15、D24和D31进行了眼底照相和荧光造影检查，图9为hPV19K单抗眼用注射液组、雷珠单抗注射液组和康柏西普眼用注射液组给药前后食蟹猴眼底照相和荧光造影中晚期图片。各组动物眼底照相除可见激光光凝斑及光凝造成的损伤，未见其他异常。
同时，对造模后D15、D24和D31的眼底荧光造影检查图片进行阅图评级及相关测量，计算4级光斑数、4级光斑率及4级光斑渗漏面积。
a.4级光斑数及4级光斑改善率
造模后2周(D15)，各组间4级光斑数均无显著差异(p＞0.05)。给药后7天(D24)，溶媒对照品组，供试品(hPV19K单抗眼用注射液)低、中、高剂量组，雷珠单抗注射液组和康柏西普眼用注射液组4级光斑数(个)分别为4.8±2.8、1.8±2.1、2.4±2.4、1.3±2.7、1.9±2.3和0.8±1.4，4级光斑改善率(％)分别为16.7±38.9、69.9±33.4、66.2±38.4、82.4±36.69.1±35.3和87.4±25.5。给药后14天(D31)，溶媒对照品组，供试品(hPV19K单抗眼用注射液)低、中、高剂量组，雷珠单抗注射液组和康柏西普眼用注射液组4级光斑数(个)分别为4.7±2.9、1.8±2.1、1.3±1.7、1.0±2.2、1.4±1.9和0.8±1.3，4级光斑改善率(％)分别为20.8±39.6、69.9±33.4、78.8±31.4、85.9±28.8、77.1±28.1和87.4±24.0。各组动物每眼4级光斑改善率示意图见图10。
给药后7天和14天，溶媒对照品组4级光斑数未见明显变化，4级光斑改善率不明显，供试品(hPV19K单抗眼用注射液)低、中、高剂量组，雷珠单抗注射液组和康柏西普眼用注射液组4级光斑数均显著低于溶媒对照品组 (p＜0.05)；各组动物4级光斑改善率均显著高于溶媒对照品组(p＜0.05)。
b.4级光斑平均渗漏面积、平均渗漏面积减少量及改善率
给药前(造模后2周，D15)、给药后7天(D24)和14天(D31)，各组动物每眼荧光渗漏平均面积、减少量及荧光渗漏改善率(均数±标准差)如下表：
表8各组动物每眼荧光渗漏平均面积、平均渗漏面积减少量及改善率
Figure PCTCN2017082417-appb-000010
造模后2周(D15)，溶媒对照品组，供试品(hPV19K单抗眼用注射液)低、中、高剂量组，雷珠单抗注射液组和康柏西普眼用注射液组每眼荧光渗漏平均面积(mm2)均值无显著差异(p＞0.05)。
给药后7天和14天，溶媒对照品组荧光渗漏面积变化不明显，供试品(hPV19K单抗眼用注射液)低、中、高剂量组，雷珠单抗注射液组及康柏西普眼用注射液组荧光渗漏面积明显减少，各组动物每眼荧光渗漏平均面积减少量和荧光渗漏改善率(％)均显著高于溶媒对照品组(p＜0.05)。各组动物每眼荧光渗漏面积及改善情况见图11。给药后7天供试品(hPV19K单抗眼用注射液)高剂量组每眼荧光渗漏平均面积减少量及改善率高于(p＞0.05)雷珠单抗注射液组、供试品(hPV19K单抗眼用注射液)低、中剂量组。给药后14天，供试品(hPV19K单抗眼用注射液)高剂量组每眼荧光渗漏平均面积减少量及改善率高于(p＞0.05)雷珠单抗注射液组、康柏西普眼用注射液组及供试品低、中剂量组。
2)光学相干断层扫描(OCT)
所有动物在造模前进行光学相干断层扫描(OCT)检查，结果显示所有动物均无明显异常。造模后D15、D24和D31进行了OCT检查，并根据D15荧光造影检查结果测定了4级光斑对应的OCT图像中视网膜色素上皮层以上感光视网膜下的异常高反射信号物质(Subretinal hyperreflective material，SHRM)的最高高度。
给药前(造模后2周，D15)、7天(D24)和14天(D31)，各组动物每眼SHRM平均高度、减少量及改善率(均数±标准差)如表9。
表9各组动物每眼SHRM平均高度、平均高度减少量及改善率
Figure PCTCN2017082417-appb-000011
造模后2周(D15)，溶媒对照品组，供试品(hPV19K单抗眼用注射液)低、中、高剂量组，雷珠单抗注射液组和康柏西普眼用注射液组每组动物每眼 SHRM平均高度(μm)均值分别为212.7±39.4、250.5±70.6、246.3±78.9、249.5±53.5、246.1±69.5和241.9±89.2，各组间无显著差异(p＞0.05)。
给药后7天和14天，溶媒对照品SHRM高度变化不明显，其他各组均明显减少。给药后7天(D24)，供试品(hPV19K单抗眼用注射液)低、中、高剂量组，雷珠单抗注射液组和康柏西普眼用注射液组动物每眼SHRM平均高度均显著低于溶媒对照品组(p＜0.05)；各组每眼SHRM平均高度减少量及SHRM高度改善率(％)均显著高于溶媒对照品组(p＜0.05)。
给药后14天(D31)，供试品(hPV19K单抗眼用注射液)低、中、高剂量组，雷珠单抗注射液组和康柏西普眼用注射液组每眼SHRM平均高度均显著低于溶媒对照品组(p＜0.05)；各组每眼SHRM平均高度减少量及SHRM高度改善率(％)均显著高于溶媒对照品组(p＜0.05)。供试品(hPV19K单抗眼用注射液)高剂量组每眼SHRM平均高度减少量及改善率略高于(p＞0.05)雷珠单抗注射液组、康柏西普眼用注射液组、供试品低、中剂量组。
综合分析上述荧光造影检查及光学相干断层扫描结果表明，各剂量组的hPV19K单抗眼用注射液均可明显抑制食蟹猴脉络膜新生血管，减少4级光斑数、改善4级光斑渗漏面积及SHRM高度。此外，单次仅注射1/10剂量(0.05mg/眼)的hPV19K单抗眼用液，其抑制食蟹猴脉络膜血管新生及渗漏的疗效就与注射0.05mg/眼的市售对照品雷珠单抗或注射0.05mg/眼的疗效相近，显示优良的体内活性。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。

Claims (15)

  1. 一种眼用药物组合物，其包含能够拮抗抑制血管内皮生长因子与其受体结合的抗体或其衍生体，以及用于眼科治疗剂的一种或多种药学上可接受的辅料，所述抗体或其衍生体的轻链抗原互补决定区具有选自如SEQ ID NO：1，SEQ ID NO：2及SEQ ID NO：3所示的氨基酸序列；其重链抗原互补决定区具有选自如SEQ ID NO：4，SEQ ID NO：5及SEQ ID NO：6所示的氨基酸序列。
  2. 根据权利要求1所述的眼用药物组合物，其中所述抗体或其衍生体的轻链可变区具有SEQ ID NO：7所示的氨基酸序列，其重链可变区具有SEQ ID NO：8所示的氨基酸序列。
  3. 根据权利要求1或2所述的眼用药物组合物，基于所述眼用药物组合物的总体积，其中所述一种或多种药学上可接受的辅料含有以克每毫升计0.01-0.15％的选自聚山梨醇酯、聚乙二醇、丙二醇及它们的组合中的至少一种的有机共溶剂；和以克每毫升计1-10％的选自蔗糖、山梨醇、甘油、海藻糖和甘露醇中的至少一种的稳定剂。
  4. 根据权利要求3所述的眼用药物组合物，基于所述眼用药物组合物的总体积，其中所述一种或多种药学上可接受的辅料含有以克每毫升计0.01-0.02％的聚山梨醇酯和以克每毫升计7-9％的蔗糖。
  5. 根据权利要求3所述的眼用药物组合物，基于所述眼用药物组合物的总体积，其中所述一种或多种药学上可接受的辅料含有以克每毫升计0.03-0.04％的聚山梨醇酯和以克每毫升计0-5％的蔗糖。
  6. 根据权利要求1所述的眼用药物组合物，其中所述抗体或其衍生体在所述药物组合物中的蛋白浓度为0.1-50mg/ml。
  7. 根据权利要求6所述的眼用药物组合物，其中所述抗体或其衍生体在所述药物组合物中的蛋白浓度为1-20mg/ml，优选1-10mg/ml。
  8. 根据权利要求1所述的眼用药物组合物，其中所述眼用药物组合物的渗透压为285-310mOsmol/kg，优选300mOsmol/kg。
  9. 根据权利要求1所述的眼用药物组合物，其中所述眼用药物组合物的pH值为5.5-6.5，优选pH值为6.0。
  10. 权利要求1至9中任一项所述的眼用药物组合物在制备用于治疗与血管生成相关的眼部疾病或病症的药物中的用途。
  11. 根据权利要求10所述的用途，其中所述与血管生成相关的眼部疾病或病症选自年龄相关性黄斑变性、糖尿病性黄斑水肿、糖尿病性视网膜病变、病理性近视继发脉络膜新生血管、新生血管性青光眼、增生性玻璃体视网膜病变、视网膜血管阻塞、特发性脉络膜视网膜炎、眼组织胞浆菌病、眼部肿瘤、眼外伤、脉络膜新生血管形成、囊性黄斑水肿、角膜新生血管形成、角膜移植、和慢性结膜炎中的一种或多种。
  12. 根据权利要求11所述的用途，其中所述治疗与血管生成相关的眼部疾病或病症的结果包括选自平均脉络膜血管新生渗漏减少、平均视力改善、平均中央凹视网膜厚度减少、平均黄斑大小减少和平均损伤大小减少中的一种或多种的症状改善。
  13. 根据权利要求10至12中任一项所述的用途，其中所述眼用药物组合物的使用剂量为0.005mg/50μL/眼至2mg/50μL/眼，优选的使用剂量为0.05mg/50ul/眼至0.5mg/50ul/眼；
    优选地，所述眼用药物组合物的施用方式为通过玻璃体内注射；
    优选地，所述眼用药物组合物的施用时间为每间隔1至3个月施用一次。
  14. 一种治疗受试者的与血管生成相关的眼部疾病或病症的方法，其包括向受试者眼内局部施用如权利要求1-9任一项所述的眼用药物组合物。
  15. 根据权利要求14所述的方法，其中，所述与血管生成相关的眼部疾病或病症选自年龄相关性黄斑变性(AMD)，糖尿病性视网膜病变，脉络膜新生血管形成(CNV)，囊性黄斑水肿，糖尿病性黄斑水肿，视网膜血管闭塞，角膜新生血管形成，角膜移植，新生血管性青光眼和慢性结膜炎，优选为年龄相关性黄斑变性或糖尿病性视网膜病变；
    优选地，所述治疗的结果包括选自平均脉络膜血管新生渗漏减少、平均视力改善、平均中央凹视网膜厚度减少、平均黄斑大小减少和平均损伤大小减少中的一种或多种的症状改善；
    优选地，所述眼用药物组合物的施用剂量为0.05mg/50μL/眼至0.5mg/50μL/眼；
    优选地，所述施用为单次或多次施用；
    优选地，所述眼用药物组合物的施用方式为通过玻璃体内注射；
    优选地，所述眼用药物组合物的施用时间为每间隔1至3个月施用一次。
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