Method for preventing or treating cholesterol-related diseases by using Anti-PCSK9 antibody

Abstract

To provide a method for preventing or treating a cholesterol-related disease using an anti-PCSK9 antibody and / or an antibody fragment thereof and a pharmaceutical preparation comprising the same.SOLUTION: The present invention relates to an anti-PCSK9 antibody or a liquid preparation thereof for preventing or treating the above disease.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims the benefit of Chinese Patent Application No. 201910884902.9 filed on September 19, 2019, and Chinese Patent Application No. 202010424274.9 filed on May 19, 2020, the contents of which are incorporated by reference in their entirety.

[0002] The present invention relates to the field of antibody therapy, and in particular to a method for preventing or treating cholesterol-related diseases using an antibody that specifically binds to proprotein convertase subtilisin / kexin type 9 (PCSK9) (hereinafter referred to as an anti-PCSK9 antibody) and / or an antibody fragment thereof, and a pharmaceutical preparation containing the same. The present invention also relates to an anti-PCSK9 antibody or a liquid formulation thereof for preventing or treating the disease. [Background technology]

[0003] The incidence and mortality rates of coronary atherosclerotic heart disease and other atherosclerotic vascular diseases in China are both on the rise. Hypercholesterolemia is one of the known risk factors for coronary atherosclerotic heart disease, and a growing body of data has demonstrated that aggressive treatment of hypercholesterolemia is associated with a reduced risk of coronary atherosclerotic cardiovascular endpoint events. This relationship is particularly pronounced in patients at very high risk of cardiovascular events, such as those with coronary heart disease.

[0004] Currently, statins are the foundation of cholesterol-lowering therapy, playing an important role in the primary and secondary prevention of coronary atherosclerotic heart disease. However, current lipid-lowering therapies fail to meet clinical needs. Although statins can reduce cardiovascular mortality, they have certain limitations. First, statins can only reduce low-density lipoprotein cholesterol (LDL-C) by 40% to 55% at maximum, and even doubling the dose only reduces LDL-C by approximately 6%. Furthermore, safety is a key concern for the Chinese population when using high-intensity statins. A growing body of research has shown that high-intensity statin therapy is associated with a higher risk of myopathy and elevated liver enzymes, which is more pronounced in the Chinese population. The HPS2-THRIVE study found that when using moderate-intensity statin therapy, the incidence of adverse liver reactions in Chinese patients was significantly higher than in European patients, the rate of liver enzyme elevations (>3 times the upper limit of normal) was 10 times higher than in European patients, and the risk of muscle disease was also 10 times higher than in Europeans (Zhao Jing et al., Chinese Guide to the Prevention and Treatment of Blood Lipid Disorders in Adults (2016 Revised Edition). Chinese Journal of Circulation 31(10):937-953, 2016.). Currently, there is no safety data on high-intensity statin therapy in Chinese patients.

[0005] Therefore, the development of new, effective cholesterol-lowering therapies based on statins is essential, and this will have important clinical implications for reducing cardiovascular endpoints in high-risk groups. Currently, cholesterol-lowering drugs with various mechanisms of action are being marketed or investigated. Among these, anti-PCSK-9 monoclonal antibodies (mAbs) have attracted widespread attention due to their favorable safety profile and therapeutic efficacy [Zhao Jing et al., Chinese Guide to the Prevention and Treatment of Adult Hemolipid Disorders (2016 revised edition), Chinese Journal of Circulation 31(10):937-953, 2016]. PCSK-9 mAbs can inhibit PCSK-9 and block the binding of plasma PCSK-9 to the low-density lipoprotein receptor (LDLR), thereby preventing LDLR endocytosis and degradation, increasing the expression and abundance of LDLR on the cell surface and increasing LDLR reuptake into LDL-C, ultimately reducing circulating LDL-C levels and achieving a direct effect of lowering blood lipids.

[0006] PCSK-9 monoclonal antibodies have remarkable therapeutic effects, a favorable safety profile, and are easy to administer via subcutaneous injection. However, the current administration cycle for PCSK-9 monoclonal antibodies is once every 2 or 4 weeks. Patient compliance needs to be improved, and there remains a clinical unmet need.

[0007] Currently, there remains a need for improved methods of treating or preventing sterol-related disorders that have longer dosing intervals, are easier to administer to patients, improve patient compliance, and / or have fewer side effects or greater safety and / or are easier to administer. Summary of the Invention [Problem to be solved by the invention]

[0008] 1. Preventive or therapeutic methods In one aspect, the invention relates to a method of lowering cholesterol levels in a subject, comprising administering to the subject an anti-PCSK9 antibody or antigen-binding fragment thereof at a dose of between about 15 mg and 3500 mg. In another aspect, the invention relates to a method for preventing or treating a disorder associated with elevated LDL-cholesterol levels in a subject, comprising administering to the subject an anti-PCSK9 antibody or antigen-binding fragment thereof at a dose of between about 15 mg and 3500 mg. In one aspect, the invention relates to a method for preventing or treating a cholesterol-related disease, such as hypercholesterolemia and / or hyperlipidemia, comprising administering to a subject an anti-PCSK9 antibody or antigen-binding fragment thereof at a dose of between about 15 mg and 3500 mg.

[0009] In some embodiments of the above-described methods, the anti-PCSK9 antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises a combination of an HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 10, an HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17, and an HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 19, and the VL comprises a combination of an LCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 4, an LCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 5, and an LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 6.

[0010] In some embodiments of the above methods, the anti-PCSK9 antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 30, and the light chain variable region VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24.

[0011] In some embodiments of the above methods, the anti-PCSK9 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:41, and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:35. In some embodiments of the above methods, the anti-PCSK9 antibody or antigen-binding fragment thereof is ADI-10087, disclosed in PCT patent application PCT / CN2017 / 118050. In some embodiments of the above methods, the cholesterol is LDL-cholesterol.

[0012] In some embodiments of the above-described methods, the anti-PCSK9 antibody or antigen-binding fragment thereof is administered to a subject at a dose of about 25 mg to about 3000 mg, about 50 mg to about 2500 mg, about 75 mg to about 2000 mg, about 100 mg to about 2000 mg, about 200 mg to about 2000 mg, about 250 mg to about 1500 mg, about 300 mg to about 1000 mg, about 450 mg to about 1000 mg, about 600 mg to about 1000 mg, about 350 mg to about 900 mg, about 400 mg to about 800 mg, about 450 mg to about 700 mg, about 300 mg to about 600 mg, or about 450 mg to about 600 mg.

[0013] In some embodiments, the anti-PCSK9 antibody or antigen-binding fragment thereof is administered to a subject at a dose of about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 140 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 420 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 900 mg, about 1000 mg, about 1200 mg, about 1500 mg, about 2000 mg, about 2500 mg, about 3000 mg, or about 3500 mg.

[0014] In some embodiments of the above methods, the anti-PCSK9 antibody or antigen-binding fragment thereof is administered parenterally. In some embodiments of the above methods, the anti-PCSK9 antibody or antigen-binding fragment thereof is administered subcutaneously. In some embodiments of the above methods, the anti-PCSK9 antibody or antigen-binding fragment thereof is administered intravenously.

[0015] In some embodiments of the above methods, the subject's LDL-cholesterol level is still reduced by >30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, or >90% 4 weeks after administration, 5 weeks after administration, 6 weeks after administration, 7 weeks after administration, 8 weeks after administration, 9 weeks after administration, 10 weeks after administration, 3 months after administration, 4 months after administration, or 5 months after administration compared to the subject's LDL-cholesterol level before administration.

[0016] In some embodiments of the above methods, the subject's serum free PCSK-9 level is still reduced by >30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, or >90% 4 weeks after administration, 5 weeks after administration, 6 weeks after administration, 7 weeks after administration, 8 weeks after administration, 9 weeks after administration, 10 weeks after administration, 3 months after administration, 4 months after administration, or 5 months after administration compared to the subject's serum free PCSK-9 level before administration.

[0017] In some embodiments of the above methods, the subject's total cholesterol level is still reduced by >30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, or >90% four weeks after administration, five weeks after administration, six weeks after administration, seven weeks after administration, eight weeks after administration, nine weeks after administration, ten weeks after administration, three months after administration, four months after administration, or five months after administration compared to the subject's total cholesterol level before administration.

[0018] In some embodiments of the above methods, the subject's apolipoprotein B levels are still reduced by >30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85% or >90% 4 weeks after administration, 5 weeks after administration, 6 weeks after administration, 7 weeks after administration, 8 weeks after administration, 9 weeks after administration, 10 weeks after administration, 3 months after administration, 4 months after administration or 5 months after administration compared to the subject's apolipoprotein B levels before administration.

[0019] In some embodiments of the above methods, the subject's non-high density lipoprotein cholesterol level is still reduced by >30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, or >90% four weeks after administration, five weeks after administration, six weeks after administration, seven weeks after administration, eight weeks after administration, nine weeks after administration, ten weeks after administration, three months after administration, four months after administration, or five months after administration compared to the subject's non-high density lipoprotein cholesterol level before administration.

[0020] In some embodiments of the above methods, the subject's lipoprotein a levels are still reduced by >20%, >30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, or >90% 4 weeks after administration, 5 weeks after administration, 6 weeks after administration, 7 weeks after administration, 8 weeks after administration, 9 weeks after administration, 10 weeks after administration, 3 months after administration, 4 months after administration, or 5 months after administration compared to the subject's lipoprotein a levels before administration.

[0021] In some embodiments of the above methods, the subject's apolipoprotein B / apolipoprotein A1 ratio level is still reduced by >20%, >30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85% or >90% 4 weeks after administration, 5 weeks after administration, 6 weeks after administration, 7 weeks after administration, 8 weeks after administration, 9 weeks after administration, 10 weeks after administration, 3 months after administration, 4 months after administration or 5 months after administration compared to the subject's apolipoprotein B / apolipoprotein A1 ratio level before administration.

[0022] In some embodiments of the above methods, the cholesterol or LDL-cholesterol is serum LDL-cholesterol.

[0023] In some embodiments of the above methods, the anti-PCSK9 antibody or antigen-binding fragment thereof is administered at intervals of once every four weeks (Q4W) or more, e.g., once every five weeks (Q5W), once every six weeks (Q6W), once every seven weeks (Q7W), once every eight weeks (Q8W), once every nine weeks (Q9W), once every ten weeks (Q10W), once every 11 weeks (Q11W), once every 12 weeks (Q12W), once every four months, once every five months, once every six months, once every seven months, or once yearly.

[0024] In some embodiments of the above methods, after administration, the subject does not experience a serious adverse event, particularly a serious adverse event associated with the anti-PCSK9 antibody or antigen-binding fragment thereof. In some embodiments of the above methods, after administration, the subject has an incidence of adverse events comparable to subjects receiving a placebo.

[0025] In some embodiments of the above methods, the subject is a human. In some embodiments of the above methods, the cholesterol-related disease is selected from homozygous familial hypercholesterolemia, heterozygous familial hypercholesterolemia, and non-familial hypercholesterolemia.

[0026] In some specific embodiments of the above methods, the methods comprise administering ADI-10087 subcutaneously or intravenously to the subject at a dose of about 300 mg to 1000 mg (e.g., about 300 mg, about 350 mg, about 400 mg, about 420 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 900 mg, or about 1000 mg) once every four or six weeks, which still reduces the subject's LDL-cholesterol levels by >50% four or six weeks after administration compared to the subject's LDL-cholesterol levels before administration.

[0027] In some specific embodiments of the above methods, the methods include administering ADI-10087 subcutaneously or intravenously to the subject at a dose of about 450 mg to 1000 mg (e.g., about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 900 mg, or about 1000 mg) once every four or six weeks, which still reduces the subject's LDL-cholesterol levels by >50% four or six weeks after administration compared to the subject's LDL-cholesterol levels before administration.

[0028] In some specific embodiments of the above methods, the methods include administering ADI-10087 subcutaneously or intravenously to the subject at a dose of about 600 mg to 1000 mg (e.g., about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 900 mg, or about 1000 mg) once every six or eight weeks, which still reduces the subject's LDL-cholesterol levels by >50% six or eight weeks after administration compared to the subject's LDL-cholesterol levels before administration.

[0029] In some specific embodiments of the above methods, the methods include administering ADI-10087 subcutaneously or intravenously to the subject at a dose of about 300 mg to 1000 mg (e.g., about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 900 mg, or about 1000 mg) once every four or six weeks, which still reduces the subject's LDL-cholesterol level by >50% four or six weeks after administration and still reduces the subject's serum free PCSK-9 level by >60% four or six weeks after administration compared to before administration.

[0030] In some specific embodiments of the above methods, the methods include administering ADI-10087 subcutaneously or intravenously to the subject at a dose of about 450 mg to 1000 mg (e.g., about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 900 mg, or about 1000 mg) once every four or six weeks, which still reduces the subject's LDL-cholesterol level by >50% four or six weeks after administration and still reduces the subject's serum free PCSK-9 level by >80% four or six weeks after administration compared to before administration.

[0031] In some specific embodiments of the above methods, the methods include administering ADI-10087 subcutaneously or intravenously to the subject at a dose of about 600 mg to 1000 mg (e.g., about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 900 mg, or about 1000 mg) once every 6 or 8 weeks, which still reduces the subject's LDL-cholesterol level by >50% 6 or 8 weeks after administration and still reduces the subject's serum free PCSK-9 level by >50% 8 weeks after administration compared to before administration.

[0032] In some specific embodiments of the above methods, the anti-PCSK9 antibody or antigen-binding fragment thereof is administered in a formulation described below.

[0033] In some specific embodiments of the above methods, the anti-PCSK9 antibody or antigen-binding fragment thereof is administered in a liquid antibody formulation, wherein the liquid antibody formulation comprises: (i) about 100 mg / mL to about 200 mg / mL of an anti-PCSK-9 antibody or antigen-binding fragment thereof; (ii) about 0.2 mg / mL to 10 mg / mL of histidine; (iii) about 1% to 6% sorbitol, and (iv) about 0.05 mg / mL to 1 mg / mL of polysorbate-80 or polysorbate-20 The liquid formulation has a pH of about 5.0 to 6.0.

[0034] In some specific embodiments of the above methods, the anti-PCSK9 antibody or antigen-binding fragment thereof is administered in a liquid antibody formulation, wherein the liquid antibody formulation comprises: (i) about 100 mg / mL to about 200 mg / mL of an anti-PCSK-9 antibody or antigen-binding fragment thereof; (ii) about 0.2 mg / mL to 10 mg / mL of histidine; (iii) about 1% to 6% sorbitol and about 50 mmol / L to 180 mmol / L arginine or an arginine salt, and (iv) about 0.05 mg / mL to 1 mg / mL of polysorbate-80 or polysorbate-20 The liquid formulation has a pH of about 5.0 to 6.0.

[0035] In some specific embodiments of the above methods, the anti-PCSK9 antibody or antigen-binding fragment thereof is administered in a liquid antibody formulation, wherein the liquid antibody formulation contains water as a solvent and has the following composition: Anti-PCSK9 antibody or its antigen-binding fragment 150mg / mL Histidine 1.5g / L Arginine 90mmol / L Sorbitol 3% (w / v) Polysorbate 80 0.3g / L and the pH of the liquid formulation is about 5.5.

[0036] In some specific embodiments, the liquid antibody formulation comprises water as a solvent and has the following composition: ADI-10087 150mg / mL Histidine 1.5g / L Arginine 90mmol / L Sorbitol 3% (w / v) Polysorbate 80 0.3g / L and the pH of the liquid formulation is about 5.5.

[0037] It should be noted that the antibody or antigen-binding fragment thereof used in the present invention, its sequence, preparation, biological activity, etc. have already been disclosed in the applicant's other PCT patent application PCT / CN2017 / 118050 (publication number WO2018 / 113781), the entire contents of which are incorporated herein by reference. For ease of correspondence and reference, the sequences and their numbers disclosed herein (including the sequence listing) are directly quoted from patent application PCT / CN2017 / 118050 and have not been renumbered.

[0038] II. Antibody formulations of the present invention The methods of the present invention can use the antibody formulation described in 201810450088.5, the entire contents of which are incorporated herein by reference. The anti-PCSK9 antibody formulation used in the present invention (hereinafter abbreviated as the "antibody formulation of the present invention") is described in detail below. In some specific embodiments of the above methods, the anti-PCSK9 antibody or antigen-binding fragment thereof is administered in a formulation described below.

[0039] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) an anti-PCSK-9 antibody or fragment thereof (e.g., an antigen-binding fragment) as defined herein; (ii) a buffering agent; (iii) a viscosity reducing agent, and (iv) surfactants (hereinafter referred to as "the liquid antibody formulation of the present invention") can be used.

[0040] In some embodiments, the anti-PCSK9 antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises three complementarity-determining regions (CDRs) contained in the VH set forth in SEQ ID NO: 30, and the VL comprises three complementarity-determining regions (CDRs) contained in the VL set forth in SEQ ID NO: 24.

[0041] In some embodiments of the above-described methods, the anti-PCSK9 antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises a combination of an HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 10, an HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17, and an HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 19, and the VL comprises a combination of an LCDR1, an LCDR2, and an LCDR3 comprising or consisting of the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.

[0042] In some embodiments of the above methods, the anti-PCSK9 antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region VH comprises or consists of the amino acid sequence of SEQ ID NO: 30, and the light chain variable region VL comprises or consists of the amino acid sequence of SEQ ID NO: 24.

[0043] In some embodiments of the above methods, the anti-PCSK9 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO: 41 and the light chain comprises or consists of the amino acid sequence of SEQ ID NO: 35. In some embodiments of the above method, the anti-PCSK9 antibody is ADI-10087, disclosed in Chinese patent application PCT / CN2017 / 118050.

[0044] In one embodiment, the concentration of the anti-PCSK9 antibody or antigen-binding fragment thereof in the liquid antibody formulation of the present invention is about 50 mg / mL to about 200 mg / mL. In another embodiment, the concentration of the anti-PCSK9 antibody or antigen-binding fragment thereof in the liquid antibody formulation of the present invention is about 100 mg / mL to about 200 mg / mL. In another embodiment, the concentration of the anti-PCSK9 antibody or antigen-binding fragment thereof in the liquid antibody formulation of the present invention is about 100 mg / mL. In another embodiment, the concentration of the anti-PCSK9 antibody or antigen-binding fragment thereof in the liquid antibody formulation of the present invention is about 125 mg / mL. In another embodiment, the concentration of the anti-PCSK9 antibody or antigen-binding fragment thereof in the liquid antibody formulation of the present invention is about 150 mg / mL. In another embodiment, the concentration of the anti-PCSK9 antibody or antigen-binding fragment thereof in the liquid antibody formulation of the present invention is about 175 mg / mL. In another embodiment, the concentration of the anti-PCSK9 antibody or antigen-binding fragment thereof in the liquid antibody formulation of the present invention is about 200 mg / mL.

[0045] In one embodiment, the anti-PCSK9 antibody is any antibody that binds to a PCSK9 protein (e.g., human PCSK9), such as a polyclonal antibody, a monoclonal antibody, or a combination of both. Preferably, in one embodiment, the anti-PCSK9 antibody is a monoclonal antibody. In one embodiment, the anti-PCSK9 antibody or antigen-binding fragment thereof is an anti-PCSK9 antibody or antigen-binding fragment thereof defined herein.

[0046] In one embodiment, the concentration of the buffering agent in the liquid antibody formulation of the present invention is about 0.01 mg / mL to 50 mg / mL. In one embodiment, the concentration of the buffering agent in the liquid antibody formulation of the present invention is about 0.1 mg / mL to 50 mg / mL. In one embodiment, the concentration of the buffering agent in the liquid antibody formulation of the present invention is about 0.2 mg / mL to 10 mg / mL. In one embodiment, the concentration of the buffering agent in the liquid antibody formulation of the present invention is about 0.5 mg / mL to 2.5 mg / mL. In one embodiment, the concentration of the buffering agent in the liquid antibody formulation of the present invention is about 1.0 mg / mL to 2.0 mg / mL. In one embodiment, the concentration of the buffering agent in the liquid antibody formulation of the present invention is about 1.5 mg / mL.

[0047] In one embodiment, the buffering agent is selected from histidine, glutamate, phosphate, acetate, citrate, and tris(hydroxymethyl)aminomethane, hi one embodiment, the buffering agent is histidine.

[0048] In one embodiment, the concentration of the viscosity-lowering agent in the liquid antibody formulation of the present invention is about 10 mmol / L to 1000 mmol / L. In one embodiment, the concentration of the viscosity-lowering agent in the liquid antibody formulation of the present invention is about 20 mmol / L to 500 mmol / L. In one embodiment, the concentration of the viscosity-lowering agent in the liquid antibody formulation of the present invention is about 50 mmol / L to 300 mmol / L. In one embodiment, the concentration of the viscosity-lowering agent in the liquid antibody formulation of the present invention is about 50 mmol / L to 200 mmol / L.

[0049] In one embodiment, the viscosity-lowering agent is selected from a sugar alcohol (e.g., sorbitol), arginine, arginine hydrochloride, sodium thiocyanate, ammonium thiocyanate, ammonium sulfate, ammonium chloride, calcium chloride, zinc chloride, sodium acetate, and combinations thereof. In one embodiment, the viscosity-lowering agent is sorbitol. In one embodiment, the viscosity-lowering agent is sorbitol at a concentration of about 1% to 6% (w / v). In one embodiment, the viscosity-lowering agent is sorbitol at a concentration of about 1% to 6% (w / v), and the liquid formulation does not contain other viscosity-lowering agents. In one embodiment, the viscosity-lowering agent is a combination of sorbitol and arginine or an arginine salt (preferably arginine hydrochloride). In one embodiment, the viscosity-lowering agent is a combination of sorbitol and arginine or an arginine salt (preferably arginine hydrochloride), wherein the concentration of sorbitol is about 1% to 6% (w / v), preferably about 2% to 4% (w / v), and more preferably about 3% (w / v), and the concentration of arginine or an arginine salt (preferably arginine hydrochloride) is about 50 mmol / L to 180 mmol / L, preferably about 70 mmol / L to 150 mmol / L, and more preferably about 90 mmol / L. In one embodiment, the viscosity-lowering agent is a combination of sorbitol and arginine or an arginine salt (preferably arginine hydrochloride), the concentration of sorbitol is about 1% to 6% (w / v), preferably about 2% to 4% (w / v), and more preferably about 3% (w / v), the concentration of arginine or an arginine salt is about 50 mmol / L to 180 mmol / L, preferably about 70 mmol / L to 150 mmol / L, and more preferably about 90 mmol / L, and the liquid formulation does not contain any other viscosity-lowering agents.

[0050] In one embodiment, the concentration of the surfactant is about 0.01 mg / mL to 5 mg / mL. In one embodiment, the concentration of the surfactant is about 0.05 mg / mL to 1 mg / mL. In one embodiment, the concentration of the surfactant is about 0.1 mg / mL to 0.5 mg / mL. In one embodiment, the concentration of the surfactant is about 0.3 mg / mL.

[0051] In one embodiment, the surfactant is a non-ionic surfactant, such as pluronics, polysorbate-80, polysorbate-60, polysorbate-40, or polysorbate-20.

[0052] In one embodiment, the liquid formulation of the present invention comprises a solvent, preferably selected from water for injection, organic solvents for injection (including but not limited to oil for injection, ethanol, propylene glycol, etc.), or combinations thereof.

[0053] In one embodiment, the pH of the liquid formulation is about 5.0 to 6.0. In one embodiment, the pH of the liquid formulation is about 5.2 to 5.8. In one embodiment, the pH of the liquid formulation is about 5.4 to 5.6. In one embodiment, the pH of the liquid formulation is about 5.5.

[0054] In one embodiment, the viscosity of the liquid formulation at 25°C is about 1.0 to 20 centipoise. In one embodiment, the viscosity of the liquid formulation at 25°C is about 1.0 to 10 centipoise. In one embodiment, the viscosity of the liquid formulation at 25°C is about 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0 centipoise. In one embodiment, the viscosity of the liquid formulation at 25°C is about 5.0 to 7.0 centipoise. In one embodiment, the viscosity of the liquid formulation at 25°C is about 5.2 centipoise. In one embodiment, the viscosity of the liquid formulation at 25°C is about 6.0 centipoise. In one embodiment, the liquid formulation is a pharmaceutical formulation, preferably an injection, more preferably a subcutaneous injection.

[0055] In a preferred embodiment, the liquid antibody formulation of the present invention comprises: (i) about 100 mg / mL to about 200 mg / mL of an anti-PCSK-9 antibody or antigen-binding fragment thereof; (ii) about 0.2 mg / mL to 10 mg / mL of histidine; (iii) about 1% to 6% (w / v) sorbitol, and (iv) about 0.05 mg / mL to 1 mg / mL of polysorbate-80 or polysorbate-20 The liquid formulation has a pH of about 5.0 to 6.0. In a more preferred embodiment, the liquid antibody formulation of the present invention further comprises water for injection.

[0056] In a preferred embodiment, the liquid antibody formulation of the present invention comprises: (i) about 100 mg / mL to about 200 mg / mL of an anti-PCSK-9 antibody or antigen-binding fragment thereof; (ii) about 0.2 mg / mL to 10 mg / mL of histidine; (iii) about 1% to 6% sorbitol and about 50 mmol / L to 180 mmol / L arginine or an arginine salt, and (iv) about 0.05 mg / mL to 1 mg / mL of polysorbate-80 or polysorbate-20 The liquid formulation has a pH of about 5.0 to 6.0.

[0057] In a more preferred embodiment, the liquid antibody formulation of the present invention further comprises water for injection. In some specific embodiments, the liquid antibody formulation comprises water as a solvent and has the following composition: Anti-PCSK9 antibody or its antigen-binding fragment 150mg / mL Histidine 1.5g / L Arginine 90mmol / L Sorbitol 3% (w / v) Polysorbate 80 0.3g / L and the pH of the liquid formulation is about 5.5.

[0058] In some specific embodiments, the liquid antibody formulation comprises water as a solvent and has the following composition: ADI-10087 150mg / mL Histidine 1.5g / L Arginine 90mmol / L Sorbitol 3% (w / v) Polysorbate 80 0.3g / L and the pH of the liquid formulation is about 5.5. In a more preferred embodiment, the liquid antibody formulation of the present invention further comprises water for injection.

[0059] In one embodiment, a liquid formulation comprising an anti-PCSK9 antibody of the invention is stored at about −80° C. to about 45° C., e.g., −80° C., about −30° C., about −20° C., about 0° C., about 5° C., about 25° C., about 35° C., about 37° C., about 42° C., or about 45° C., for at least 14 days, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or longer, and the purity of the anti-PCSK-9 antibody or antigen-binding fragment thereof, as detected by SEC-HPLC or non-reducing CE-SDS, has decreased by 10% or less, e.g., 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% or less.

[0060] In one embodiment, a liquid formulation comprising an anti-PCSK9 antibody of the invention is stored at about −80° C. to about 45° C., e.g., −80° C., about −30° C., about −20° C., about 0° C., about 5° C., about 25° C., about 35° C., about 37° C., about 42° C., or about 45° C., for at least 14 days, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or longer, and the change in charge heterogeneity of the anti-PCSK-9 antibody or antigen-binding fragment thereof, as detected by CEX-HPLC, is 10% or less, e.g., 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% or less.

[0061] The formulations of the present invention have high antibody concentrations while possessing low viscosities suitable for administration, particularly subcutaneous administration, high physical and chemical stability, low generation of aggregates and particles during storage, and low change in charge heterogeneity, which are highly beneficial for the production of complete, efficient, and consistent clinical drugs.

[0062] The present inventors have surprisingly found that sorbitol can be used as a viscosity-lowering agent for anti-PCSK9 antibody liquid formulations, that a low-viscosity, high-concentration liquid formulation of an anti-PCSK-9 antibody or its antigen-binding fragment can be obtained without adding any other viscosity-lowering agent, and that the liquid formulation has high stability.The present inventors have also surprisingly found that a combination of sorbitol and arginine, as a viscosity-lowering agent, can lower the viscosity of an anti-PCSK-9 antibody liquid formulation, allowing a high-concentration liquid formulation of an anti-PCSK-9 antibody or its antigen-binding fragment to have a lower viscosity that makes it easier to subcutaneously inject, and can improve the stability of the PCSK-9 antibody liquid formulation.

[0063] In another aspect, the present invention provides a solid formulation obtained by lyophilizing the liquid formulation described above, which can be reconstituted with a suitable solvent prior to use to form the liquid formulation of the present invention.

[0064] 3. Anti-PCSK9 antibodies or their preparations for treating or preventing diseases The present invention relates to the above-described anti-PCSK9 antibody or antigen-binding fragment thereof, or a liquid antibody formulation thereof, for use in the aforementioned method of the present invention. It should be understood that in the above solutions relating to liquid formulations, the dosage refers to the amount of anti-PCSK9 antibody in the formulation.

[0065] 4. Single-dose drug units, drug kits, or their use in the manufacture of pharmaceuticals In other embodiments, the present invention relates to a single pharmaceutical dosage unit comprising an anti-PCSK9 antibody or antigen-binding fragment thereof at a dose of about 15 mg to 3500 mg, preferably about 25 mg to about 3000 mg, about 50 mg to about 2500 mg, about 75 mg to about 2000 mg, about 100 mg to about 2000 mg, about 200 mg to about 2000 mg, about 250 mg to about 1500 mg, about 300 mg to about 1000 mg, about 450 mg to about 1000 mg, about 600 mg to about 1000 mg, about 350 mg to about 900 mg, about 400 mg to about 800 mg, about 450 mg to about 700 mg, about 300 mg to about 600 mg, or about 450 mg to about 600 mg.

[0066] In some embodiments, the anti-PCSK9 antibody or antigen-binding fragment thereof comprises a heavy chain variable region, VH, and a light chain variable region, VL, (a) the VH is (i) a combination of an HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 10, an HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17, and an HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 19, or (ii) comprises three complementarity-determining regions (CDRs) contained in VH set forth in SEQ ID NO: 30; (b) the VL is (i) a combination of an LCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 4, an LCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 5, and an LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 6, or (ii) It contains three complementarity-determining regions (CDRs) contained in the VL shown in SEQ ID NO: 24.

[0067] In some embodiments, the anti-PCSK9 antibody or antigen-binding fragment thereof comprises a heavy chain variable region, VH, and a light chain variable region, VL, (a) the heavy chain variable region VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 30; (b) the light chain variable region VL comprises or consists of the amino acid sequence set forth in SEQ ID NO:24.

[0068] In some embodiments, the anti-PCSK9 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain: (a) the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 41; (b) the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35; Preferably, the anti-PCSK9 antibody is ADI-10087.

[0069] In another aspect, the invention relates to a pharmaceutical kit comprising an anti-PCSK9 antibody or antigen-binding fragment thereof according to any one of the single pharmaceutical dosage unit embodiments above. In another aspect, the present invention relates to the use of a single drug dosage unit as described above or a drug kit as described above in the manufacture of a medicament for lowering cholesterol levels in a subject.

[0070] In another aspect, the present invention relates to the use of a single drug dose unit as described above or a drug kit as described above in the manufacture of a medicament for preventing or treating a disorder associated with elevated LDL-cholesterol levels in a subject.

[0071] In another aspect, the present invention relates to the use of a single drug dose unit as described above or a drug kit as described above in the manufacture of a medicament for preventing or treating a cholesterol-related disease, such as hypercholesterolemia and / or hyperlipidemia.

[0072] It should be understood that the present application also includes technical solutions obtained by combining the technical features described in the first to third parts above with the technical features of the technical solutions in the fourth part above. [Brief explanation of the drawings]

[0073] [Figure 1] The graph shows the mean free concentration-time curve of ADI-10087 after administration of ADI-10087 to subjects in each dose group. Upper panel: linear coordinates, lower panel: semi-logarithmic coordinates. Error bars are expressed as mean ± standard deviation, and each dose group corresponds to a line. [Figure 2] The mean total concentration-time curves of ADI-10087 after administration to subjects in each dose group are shown. Upper panel: linear coordinates, lower panel: semi-logarithmic coordinates. Error bars are expressed as mean ± standard deviation, and each dose group corresponds to a line. [Figure 3] The distribution of total and free ADI-10087 Cmax following subcutaneous injection of 25 mg to 600 mg of ADI-10087 into the abdomen of subjects (scatter plot). The broken lines represent the median values ​​for each dose group, the scattered dots represent individual values, the solid lines represent the corresponding parameters for free ADI-10087, and the dotted lines represent the corresponding parameters for total ADI-10087. [Figure 4]The distribution of AUC0-last for total and free ADI-10087 after subcutaneous injection of 25 mg to 600 mg of ADI-10087 into the abdomen of subjects (scatter plot). The broken lines represent the median values ​​for each dose group, the scattered dots represent individual values, the solid lines represent the corresponding parameters for free ADI-10087, and the dotted lines represent the corresponding parameters for total ADI-10087. [Figure 5] The distribution of AUC0-∞ of total and free ADI-10087 after subcutaneous injection of 25 mg to 600 mg of ADI-10087 into the abdomen of subjects (scatter plot). The broken lines in the plot represent the median values ​​for each dose group, the scattered dots represent individual values, the solid lines represent the corresponding parameters for free ADI-10087, and the dotted lines represent the corresponding parameters for total ADI-10087. [Figure 6] The percent change from baseline in serum free PCSK-9 concentration after a single dose is shown. [Figure 7] 1 shows a least squares mean number plot of change from baseline in LDL-C after a single dose. [Figure 8] Percent change in mean LDL-C concentrations relative to baseline after a single dose is shown. [Figure 9] 1 shows the mean concentration-time diagram of LDL-C after a single dose. [Figure 10] The percent change in total cholesterol concentration relative to baseline after a single dose is shown. [Figure 11] Percent change in ApoB concentration relative to baseline after a single dose is shown. [Figure 12] The percentage change in non-HDL-C concentration after a single administration relative to baseline is shown. [Figure 13] Percent change from baseline in lipoprotein a (Lp(a)) concentrations after a single dose is shown. [Figure 14] The percent change from baseline in serum free PCSK-9 concentrations after multiple doses is shown. [Figure 15] Percent change in LDL-C levels relative to baseline after multiple doses is shown. [Figure 16] Changes in LDL-C levels relative to baseline after multiple doses are shown. [Figure 17] Percent change in Lp(a) levels relative to baseline after multiple doses is shown. [Figure 18] Percent change in ApoB levels relative to baseline after multiple doses is shown. [Figure 19] The percentage change from baseline in non-HDL-C levels after multiple doses is shown. [Figure 20] Percent change in total cholesterol TC levels relative to baseline after multiple doses is shown. [Figure 21] Percent change in ApoB / ApoA1 levels relative to baseline after multiple doses is shown. DETAILED DESCRIPTION OF THE INVENTION

[0074] definition The terms used in the present invention have the definitions listed below. If not defined in this application, the definitions in patent application PCT / CN2017 / 118050 preferably apply. If not defined in either, the terms used in the present invention have the meanings commonly understood in the art.

[0075] The following definitions are used for the purpose of interpretation herein, and unless inconsistent, terms used in the singular will also include the plural and vice versa. It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the term "and / or" should be understood to refer to any one of selectable options or two or more of selectable options.

[0076] As used herein, the term "comprise" or "comprises" means including the stated element, integer, or step, but not excluding other elements, integers, or steps. As used herein, the terms "comprise" or "comprises" also include cases consisting of the stated element, integer, or step, unless otherwise specified. For example, when referring to "comprising" an antibody variable region of a specific sequence, it is intended to include an antibody variable region consisting of that sequence.

[0077] Examples of "antibodies and antigen-binding fragments thereof" applicable to the present invention include, but are not limited to, polyclonal antibodies, monoclonal antibodies, monovalent antibodies, bispecific antibodies, heteroconjugate antibodies, multispecific antibodies, recombinant antibodies, heteroantibodies, heteroconjugate antibodies, chimeric antibodies, humanized (especially CDR-grafted) antibodies, deimmunized antibodies, or antibodies of human origin, Fab fragments, Fab' fragments, F(ab')2 fragments, fragments produced by an Fab expression library, Fd, Fv, disulfide-linked Fv (dsFv), single-chain antibodies (e.g., scFv), diabodies or trabodies (Holliger P. et al., (1993) Proc. Natl. Acad. Sci. USA 90(14), 6444-6448), nanobodies (also called single-domain antibodies), anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies against an antibody of the present invention), and epitope-binding fragments of any one of the above.

[0078] An "individual" or "subject" includes a mammal. Non-limiting examples of mammals include livestock (e.g., cows, goats (caprines), cats, dogs, horses), primates (e.g., humans, non-human primates such as monkeys), rabbits, and rodents (e.g., mice, rats). In some embodiments, the individual or subject is human, including a child, adolescent, or adult.

[0079] The term "combination therapy" refers to the administration of two or more therapeutic agents to treat cholesterol-related diseases as described herein. Such administration includes the co-administration of these therapeutic agents at substantially the same time, for example, in the form of a single capsule having a fixed ratio of active ingredients. Alternatively, such administration includes the co-administration of each active ingredient in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). The powders and / or liquids can be reconstituted or diluted to the desired dose before administration. Furthermore, such administration includes the sequential use of each type of therapeutic agent at substantially the same time or at different times. In either case, the treatment plan provides the beneficial effect of the pharmaceutical combination in treating the disorders or conditions described herein.

[0080] As used herein, the term "treatment" refers to the alleviation, interruption, slowing, amelioration, cessation, reduction, or reversal of the progression or severity of an existing symptom, disorder, condition, or disease. As used herein, the term "prevention" includes the inhibition of the onset or progression of a disease, disorder, or symptom associated with a particular disease or disorder. In some embodiments, subjects with a family history are candidates for a preventative regimen. In general, the term "prevention" refers to the administration of a drug before a disorder or symptom occurs, particularly in subjects at risk.

[0081] The term "cholesterol-related disease" includes any one or more of hypercholesterolemia, hyperlipidemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular diseases, Alzheimer's disease, and general dyslipidemia (e.g., manifested as elevated total serum cholesterol, elevated LDL, elevated triglycerides, elevated VLDL, and / or decreased HDL). Some non-limiting examples of primary and secondary dyslipidemias that can be treated with anti-PCSK9 antibodies (alone or in combination with one or more other drugs) include metabolic syndrome, diabetes mellitus, familial combined hyperlipidemia, familial hypertriglyceridemia, familial hypercholesterolemias, heterozygous hypercholesterolemia, homozygous hypercholesterolemia, familial defective apoplipoprotein B-100, polygenic hypercholesterolemia, homozygous familial hypercholesterolemia, heterozygous familial hypercholesterolemia, non-familial hypercholesterolemia, remnant removal disease, hepatic lipase deficiency, and dyslipidemia. deficiency, dietary indiscretion, hypothyroidism, medications (including estrogen and progesterone therapy, beta-blockers and thiazide diuretics), nephrotic syndrome, chronic renal failureThis includes dyslipidemias secondary to any of the following: liver cirrhosis, primary renal failure, Cushing's syndrome, primary biliary cirrhosis, glycogen storage diseases, hepatoma, cholestasis, acromegaly, insulinoma, isolated growth hormone deficiency, and alcohol-induced hypertriglyceridemia.

[0082] The term "hypercholesterolemia," as used herein, refers to a disorder in which cholesterol levels are elevated above a certain level. In some embodiments, LDL-cholesterol levels are elevated above a certain level. In some embodiments, serum LDL-cholesterol levels are elevated above a certain level.

[0083] As used herein, the term "vector" refers to a nucleic acid molecule that propagates another nucleic acid to which it is linked. The term includes a vector as a structure capable of autonomous replication of a nucleic acid, as well as a vector that is integrated into the genome of a host cell into which it has been introduced. Some vectors are capable of directing the expression of a nucleic acid to which it is operably linked. Such vectors are referred to herein as "expression vectors."

[0084] The term "effective amount" refers to an amount or dosage that produces the desired effect in a treated patient after administration of one or more doses of the formulation, antibody, or fragment of the present invention to the patient. The effective amount can be easily determined by a skilled physician, taking into account several factors, such as the mammalian species, the size, age, and health condition of the subject, the disease being treated, the degree or severity of the disease, the response of the individual patient, the antibody to be administered, the mode of administration, the bioavailability characteristics of the administered formulation, the selected dosing regimen, and the use of combination therapy.

[0085] A "therapeutically effective amount" refers to an amount effective to achieve a desired therapeutic result at a given dose over a given period of time. The therapeutically effective amount of the formulation, antibody or antibody fragment, or conjugate or composition of the present invention may vary depending on several factors, such as the disease state, the individual's age, sex, and weight, and the ability of the antibody or antibody portion to elicit a desired response in an individual. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the formulation, antibody or antibody fragment, or conjugate or composition thereof do not outweigh the beneficial therapeutic effect.

[0086] A "prophylactically effective amount" refers to an amount effective to achieve a desired prophylactic result at a given dose over a given period of time. Generally, a prophylactic dose is administered prior to or at an earlier stage of disease in a subject, and therefore the prophylactically effective amount is less than the therapeutically effective amount. "Single drug dosage unit" refers to a single drug dosage form administered to a patient at a scheduled time, and includes injectable solutions, tablets, lyophilized powders, and the like.

[0087] As used herein, the term "formulation" refers to a composition suitable for administration to animals, preferably mammals (including humans), comprising at least one active ingredient and at least one inactive ingredient. A "liquid formulation" refers to a formulation in liquid form. The liquid formulation of the present invention comprises (i) an anti-PCSK-9 antibody or fragment thereof (preferably an antigen-binding fragment), (ii) a buffer, (iii) a viscosity-reducing agent, (iv) a surfactant, and (v) a solvent. The composition of the formulation of the present invention can be as shown in the above-mentioned liquid formulation embodiments. The liquid formulation of the present invention is preferably an injection, more preferably a subcutaneous injection.

[0088] As used herein, the term "buffer" refers to a pH buffer. Preferably, the buffer can maintain the pH of the liquid formulation of the present invention at about 5.0 to 6.0, preferably about 5.2 to 5.8, more preferably about 5.4 to 5.6, and most preferably about 5.5. The concentration of the buffer in the liquid formulation is, for example, about 0.01 mg / mL to 50 mg / mL, about 0.1 mg / mL to 50 mg / mL, about 0.2 mg / mL to 10 mg / mL, about 0.5 mg / mL to 2.5 mg / mL, about 1.0 mg / mL to 2.0 mg / mL, or about 1.5 mg / mL. Preferably, the buffer is selected from histidine, glutamate, phosphate, acetate, citrate, and tris(hydroxymethyl)aminomethane.

[0089] As used herein, the term "viscosity-lowering agent" refers to a substance capable of reducing the viscosity of an anti-PCSK-9 antibody liquid formulation. The concentration of the viscosity-lowering agent in the liquid formulation is, for example, about 10 mmol / L to 1000 mmol / L, about 20 mmol / L to 500 mmol / L, about 50 mmol / L to 300 mmol / L, or about 50 mmol / L to 200 mmol / L. Preferably, the viscosity-lowering agent is selected from sugar alcohols, arginine, arginine hydrochloride, sodium thiocyanate, ammonium thiocyanate, ammonium sulfate, ammonium chloride, calcium chloride, zinc chloride, sodium acetate, and combinations thereof. More preferably, the viscosity-lowering agent is sorbitol, or a combination of sorbitol and arginine or an arginine salt (preferably arginine hydrochloride). Preferably, the concentration of sorbitol in the liquid formulation is about 1% to 6% (w / v), and the concentration of arginine or an arginine salt (preferably arginine hydrochloride) in the liquid formulation is about 50 mmol / L to 180 mmol / L, preferably about 70 mmol / L to 150 mmol / L, and more preferably about 90 mmol / L.

[0090] As used herein, a "surfactant" refers to a substance whose addition in small amounts can significantly change the interfacial state of a solution system. The concentration of the surfactant in a liquid formulation is, for example, about 0.01 mg / mL to 5 mg / mL, about 0.05 mg / mL to 1 mg / mL, or about 0.1 mg / mL to 0.5 mg / mL, more preferably about 0.3 mg / mL. Preferably, the surfactant is a nonionic surfactant, such as pluronics, polysorbate-80, polysorbate-60, polysorbate-40, or polysorbate-20.

[0091] The term "solvent" as used herein refers to the liquid used to dissolve or suspend active ingredients and inactive ingredients to form a liquid formulation.Solvents useful in the present invention include, but are not limited to, water for injection, organic solvent for injection (including but not limited to, oil for injection, ethanol, propylene glycol, etc.), or combinations thereof.

[0092] The term "sugar alcohol" as used herein refers to the corresponding polyol obtained by reducing a monosaccharide, such as by catalytic hydrogenation. Sugar alcohols include, but are not limited to, sorbitol, mannitol, erythritol, maltitol, lactitol, xylitol, etc.

[0093] The term "about," when used in conjunction with a numerical value, is meant to include numbers in a range of 10% less than the stated numerical value as a lower limit, and 10% more than the stated numerical value as an upper limit. As used herein, "w / v" refers to "weight / volume", for example, "1% w / v" is 1 g / 100 mL = 0.01 g / mL = 10 mg / mL.

[0094] In this specification, unless expressly stated otherwise, "administration" and "dosing" can be used interchangeably. Herein, a drug kit may also be referred to as a drug set box.

[0095] The present invention is further illustrated by the following examples, which should not be construed as limiting the invention. Abbreviations: SEC-HPLC: Size-exclusion high-performance liquid chromatography CE-SDS: Sodium lauryl sulfate capillary gel electrophoresis CZE: Capillary Zone Electrophoresis SC: subcutaneous injection IV: Intravenous injection PK: Pharmacokinetics LDL-C: low-density lipoprotein cholesterol [Example]

[0096] Example 1: Preparation of anti-PCSK9 antibody liquid formulation (Formulation A, i.e., test drug) An anti-PCSK9 liquid formulation was formulated according to Formulation A at a concentration of 150 mg / mL. Prescription A Anti-PCSK9 antibody 150mg / mL (ADI-10087) Histidine 1.5g / L Arginine 90mmol / L Sorbitol 3% (w / v) Polysorbate 80 0.3g / L pH 5.5

[0097] Anti-PCSK9 antibody (ADI-10087) obtained according to patent application PCT / CN2017 / 118050 was added to an ultrafiltration centrifuge tube (molecular weight cutoff: 30 kD). After centrifugation and concentration, an aqueous solution of Formulation A (1.5 g / L histidine, 90 mmol / L arginine, 3% sorbitol, pH 5.5) without polysorbate 80 and anti-PCSK9 antibody was added, followed by dilution and concentration. This process was repeated until the protein was completely substituted. After adjusting the substituted protein concentration to 150 mg / mL, 1 / 100th the volume of an aqueous solution of polysorbate 80 (30 g / L) was added, resulting in a final polysorbate 80 concentration of 0.3 g / L. This semi-finished product was sterile filtered and dispensed into vials. The finished product was then sealed with rubber stoppers and aluminum plastic caps.

[0098] Example 2: Preparation of a comparative anti-PCSK9 antibody liquid formulation (Formulation B) Comparative Example: A liquid formulation of anti-PCSK9 is prepared according to Formulation B at a concentration of 150 mg / mL. Prescription B Anti-PCSK9 antibody 150mg / mL (ADI-10087) Histidine 1.5g / L Arginine 180mmol / L Polysorbate 80 0.3g / L pH 5.5

[0099] Anti-PCSK9 antibody (ADI-10087) obtained according to patent application PCT / CN2017 / 118050 was added to an ultrafiltration centrifuge tube (molecular weight cutoff: 30 kD). After centrifugation and concentration, polysorbate 80 and an aqueous solution of Formulation B (1.5 g / L histidine, 180 mmol / L arginine, pH 5.5) without anti-PCSK9 antibody were added, diluted, and concentrated. This process was repeated until the substitution was complete. After adjusting the protein concentration to 150 mg / mL, 1 / 100th the volume of an aqueous solution of polysorbate 80 (30 g / L) was added, resulting in a final polysorbate 80 concentration of 0.3 g / L. This semi-finished product was sterile filtered and then dispensed into vials. The finished product was obtained by sealing with rubber stoppers and aluminum plastic caps.

[0100] Example 3: Viscosity measurement of anti-PCSK9 antibody liquid formulations Viscosity measurements were performed at room temperature (20–25°C) using a cone-and-plate viscometer (Brookfield, CAP1000+ / 2000+; see http: / / www.sinoinstrument.com / product_details-4-83-401-60.html for details). The rotor and speed were selected (rotor: cp-40, rotation speed: 25 rpm). The rotor was attached to the coupling nut, adjusted to its position, and the sample was added. The Run button was pressed to perform the sample viscosity measurement. Based on the above experiment, the viscosity of formulation A was determined to be 5.2 centipoise, and the viscosity of formulation B was determined to be 6.0 centipoise. Surprisingly, the viscosity of formulation A was significantly lower than that of formulation B, indicating that the combination of arginine and sorbitol is more advantageous for reducing the viscosity of high-concentration antibody formulations.

[0101] Example 4: Variation in protein purity of anti-PCSK9 antibody liquid formulations Accelerated stability experiments were conducted to investigate the stability of Formulations A and B. The finished Formulations A and B were dispensed into vials and capped with plugs, and accelerated stability studies were conducted under conditions of 40°C ± 2°C / 60% RH ± 5% RH for a period of two months. During this period, changes in protein purity were measured using the following SEC-HPLC method and non-reducing CE-SDS method.

[0102] SEC-HPLC method: The sample concentration was diluted to 2 mg / mL and analyzed by the area normalization method using a hydrophilic silica gel volume exclusion column TSKG 3000 SWxl, a protein injection amount of 100 μg, a mobile phase of 20 mmol / L Na2HPO4·12H2O, 150 mmol / L NaCl, 200 mmol / L arginine, pH 6.8, a flow rate of 0.5 mL / min, a detection wavelength of 280 nm, and a column temperature of 25°C.

[0103] CE-SDS method: Approximately 100 μg of sample was mixed with a pH 7.0 sample buffer solution to a total volume of 95 μl, and 5 μl of β-mercaptoethanol was added to the reduced sample, and 5 μl of 250 mmol / L NEM was added to the non-reduced sample. After mixing, the mixture was heated at 70°C for 10 minutes and then measured.

[0104] In the SEC-HPLC method, the change in protein purity was measured based on the percentage decrease in the SEC main peak. The protein purity of Formulation A decreased by 2.6% at 1 month and 3.7% at 2 months. The protein purity of Formulation B decreased by 4.1% at 1 month and 7.0% at 2 months. The change in protein purity was measured based on the percentage decrease in non-reducing CE-SDS. The protein purity of Formulation A decreased by 0.4% at 2 weeks and 2.0% at 1 month. The protein purity of Formulation B decreased by 2.5% at 2 weeks and 7.3% at 1 month. It can be seen that the rate of purity change for Formulation A is significantly slower than that for Formulation B. Compared to Formulation B, Formulation A has higher stability.

[0105] Example 5: Measuring charge heterogeneity of anti-PCSK9 antibody liquid formulations Samples were taken at different time points from Formulations A and B, which underwent accelerated stability testing in Example 4, and charge heterogeneity measurements were performed. A Beckman Uncoated Capillary capillary and a Beckman PA800 plus capillary electrophoresis system were used, with a sampling voltage of 0.5 psi, a sampling time of 10.0 s, a separation voltage of 30 kV, and an analysis time of 30 min. The peak areas of the acidic components, basic components, and main peak were calculated as percentages of the total peak area using the area normalization method. Formulation A showed a 0% decrease in the main CZE peak after 2 weeks and a 2.9% decrease after 1 month. Formulation B, on the other hand, showed a 2.0% decrease in the main CZE peak after 2 weeks and a 7.6% decrease after 1 month. It can be seen that the rate of change in charge heterogeneity of formulation A is significantly slower than that of formulation B, and the combination of arginine and sorbitol results in less change in charge heterogeneity of the formulation.

[0106] Example 6: Preparation of placebo (control drug) Prepare an antibody-free placebo according to Formulation C using water as the solvent. Prescription C Histidine 1.5g / L Arginine 90mmol / L Sorbitol 3% (w / v) Polysorbate 80 0.3g / L pH 5.5

[0107] Example 7: Analysis method for detecting blood samples for pharmacokinetics of ADI-10087 (1) ADI-10087 Total PK Method: ADI-10087 total PK is a quantitative electrochemiluminescence method for detecting total ADI-10087 in human serum. An ADI-10087 neutralizing antibody is coated onto an MSD plate, followed by the addition of a serum sample containing neutralized ADI-10087 after oxidation. The serum sample is then detected with a ruthenium-labeled anti-ADI-10087 polyclonal antibody. The ruthenium-labeled anti-ADI-10087 polyclonal antibody forms an immune complex with the drug and the ADI-10087 neutralizing antibody coated on the plate. After appropriate incubation and washing, Read Buffer is added, and the plate is placed in the MSD plate reader, triggering an electrocatalytic chemiluminescence reaction to generate a luminescent signal. The intensity of the chemiluminescent signal is directly proportional to the ADI-10087 content in the sample.

[0108] (2) ADI-10087 free PK method: Based on the principle of ELISA, ADI-10087 neutralizing antibody is coated onto an ELISA plate and allowed to bind. Serum samples are then added, allowing ADI-10087 in the serum sample to specifically bind to the coated antigen. Biotin-labeled ADI-10087 neutralizing antibody is then added for capture, and SA-HRP is added for detection. The corresponding TMB substrate for HRP is then added, and the color depth is directly proportional to the amount of free ADI-10087. The concentration of ADI-10087 monoclonal antibody in clinical serum can be detected by measuring the OD450nm / 620nm readings on a microplate reader.

[0109] Example 8: ADI-10087 PCSK-9 detection method Based on the ELISA principle, ADI-10087 is coated onto an ELISA plate and allowed to bind, followed by the addition of serum samples. PCSK-9 in the serum samples specifically binds to the coated ADI-10087, followed by the addition of goat anti-human PCSK-9 polyclonal antibodies, followed by the addition of goat IgG HRP-conjugate antibodies for detection. The corresponding TMB substrate for the HRP enzyme is then added, and the color depth is directly proportional to the PCSK-9 content. The PCSK-9 concentration in clinical serum can be detected by measuring the OD450nm / 620nm readings on a microplate reader.

[0110] Example 9: Method for detecting ADI-10087 anti-drug antibodies A semi-quantitative electrochemiluminescence assay was used to detect ADI-10087 anti-drug antibodies (ADA) in human serum. After acid digestion of a human serum sample, free ADA was captured by ADI-10087 coated on an ELISA plate, which was then acid-digested again. The eluted ADA was mixed with biotin- and ruthenium-labeled ADI-10087 and incubated to form an immune complex, which then bound to a streptavidin-coated MSD plate. After appropriate incubation and washing, Read Buffer was added, and the plate was placed on an MSD plate reader, triggering an electrocatalytic chemiluminescence reaction to generate a luminescent signal. The intensity of the chemiluminescent signal was directly proportional to the ADA content of the sample.

[0111] Example 10: Method for detecting ADI-10087 neutralizing antibodies Electrochemiluminescence was used to determine whether samples determined to be ADA-positive contained ADI-10087 neutralizing antibodies (NAb). After acid digestion of human serum samples, free NAb was captured by ADI-10087 coated on an ELISA plate, then acid digested again. The eluted NAb was mixed with ruthenium-labeled ADI-10087 and incubated to form an immune complex. The eluted NAb was then added to an MSD plate pre-coated with biotin-labeled streptavidin for PCSK-9 and incubated together. Free ruthenium-labeled drug in the analyte binds to the biotin-labeled PCSK-9 pre-coated on the plate. After appropriate incubation and washing, Read Buffer was added, and the plate was placed on the MSD plate reader, triggering the electrocatalytic chemiluminescence reaction to generate a luminescent signal. The intensity of the chemiluminescent signal was negatively correlated with the NAb content of the sample.

[0112] Example 11: Evaluation of safety and tolerability after a single dose of ADI-10087 in healthy adult Chinese subjects Study design: A total of 58 subjects will be assigned to eight escalating dose cohorts (25 mg SC, 75 mg SC, 75 mg IV, 150 mg SC, 300 mg SC, 450 mg SC, 450 mg IV, and 600 mg SC). The 25 mg SC cohort will include two subjects who will receive a single dose of ADI-10087 and will be added to subsequent dose cohorts after completing a 14-day safety evaluation. For subsequent 75 mg SC to 600 mg SC cohorts, each cohort will include 8 subjects, of which the first 2 subjects in the 75 mg SC cohort will receive open-label ADI-10087 75 mg SC, the last 6 subjects will be randomized 2:1 to receive ADI-10087 (N=4) or placebo (N=2), and the other 6 cohort subjects will be randomized 3:1 to receive a single dose of ADI-10087 (N=6) or placebo (N=2).

[0113] Two subjects in the 25 mg SC cohort may be added to the 75 mg SC cohort after completing 14 days of post-dose safety evaluations. All subjects in the 75 mg SC cohort may be added to the 150 mg SC cohort after completing 14 days of post-dose safety evaluations. After adding to the 150 mg SC cohort, both subjects may be added to the 75 mg IV cohort. All subjects in the 150 mg SC cohort may be added to the 300 mg SC cohort after completing 14 days of post-dose safety evaluations. All subjects in the 300 mg SC cohort may be added to the 450 mg SC cohort after completing 14 days of post-dose safety evaluations. All subjects in the 450 mg SC cohort may be added to the 600 mg SC cohort after completing 14 days of post-dose safety evaluations. After adding to the 600 mg SC cohort, both subjects may be added to the 450 mg IV cohort. Each subject will be followed through study day 85 (12 weeks) to assess safety, tolerability, PK / PD, and immunogenicity of the medication.

[0114] Subject demographic characteristics In the safety analysis set (SS), of the 58 subjects, 44 (75.9%) were male, with a mean age of 30.1 ± 5.74 years, of which the minimum age was 19 years and the maximum age was 44 years. The mean weight was 67.20 ± 9.419 kg, of which the minimum weight was 50.9 kg and the maximum weight was 92.4 kg. The mean height was 169.12 ± 7.468 cm, of which the minimum height was 151.0 cm and the maximum height was 186.0 cm. The mean BMI was 23.44 ± 2.309 kg / m 2 Among them, the minimum BMI is 19.3 kg / m 2 and the maximum BMI was 27.4 kg / m 2Fifty-seven subjects were of Han ethnicity (98.3%). Of the 44 subjects in the ADI-10087 group, 43 (97.7%) were of Han ethnicity, with a mean age of 30.3 ± 5.71 years, a mean height of 168.36 ± 6.992 cm, a mean weight of 66.17 ± 8.289 kg, and a mean BMI of 23.31 ± 2.175 kg / m 2 Of the 14 subjects in the placebo group, 14 (100%) were Han Chinese, with a mean age of 29.8 ± 6.03 years, a mean height of 171.50 ± 8.716 cm, a mean weight of 70.43 ± 12.122 kg, and a mean BMI of 23.82 ± 2.741 kg / m 2 The demographic characteristics of both subjects met the inclusion criteria of this study, and the demographic characteristics of the two sets of subjects were similar.

[0115] Drugs used in the study The test drug used in the study was the formulation prepared as described in Example 1, and the control drug used in the study was the placebo prepared as described in Example 6.

[0116] Administration method Subjects in cohorts 1-8 will receive a single dose of ADI-10087 at 25 mg SC, 75 mg SC, 75 mg IV, 150 mg SC, 300 mg SC, 450 mg SC, 450 mg IV, or 600 mg SC or placebo, respectively.

[0117] Subcutaneous (SC) administration method: The study drug or placebo was injected subcutaneously into the subject's abdomen. The investigator administered a single abdominal injection based on the actual injection volume of the study drug, with each subcutaneous injection volume being 1 mL. For the 25 mg and 75 mg groups, the drug solution was drawn into a 1 mL syringe (2 mL needle), the air was expelled, and then the syringe was replaced with a 1 mL syringe. For the 150 mg and higher groups, the drug solution was drawn into a 2 mL syringe (2 mL needle), the air was expelled, and then the syringe was replaced with a 1 mL syringe.

[0118] Intravenous (IV) administration: (1) 75 mg IV group: 1 mL of 150 mg of the study drug injection was taken and added to a standardized 100 mL intravenous infusion bag of 0.9% sodium chloride sterile saline. The intravenous administration volume was 50 mL, and the infusion time was 30 minutes. (2) 450 mg IV group: 1 mL of 900 mg of the study drug was taken and added to a standardized 200 mL IV bag of sterile 0.9% sodium chloride saline. The intravenous infusion volume was 100 mL, and the infusion time was 1 hour.

[0119] Adverse events Adverse events occurring during the study period will be recorded and coded using MedDRA (version 21.0) and reported using system organ classes (SOCs) and preferred terms (PTs). Drug-related adverse events include three categories: "definitely related," "probably related," and "undetermined." Events with missing relevant data will also be defined as drug-related adverse events.

[0120] AEs are grouped by treatment group by SOC and PT within the SOC. Results are displayed in descending order of incidence both between and within SOCs. For AE summaries, the incidence of an AE is the number of subjects who reported the AE, not the number of AEs reported. When calculating the number of subjects and percentages, if the same AE occurs multiple times in the same subject, it is calculated only once for this AE.

[0121] Screening AEs are those that occurred up to the first study dose. Treatment-emergent adverse events (TEAEs) are defined as AEs that worsen or occur within 12 weeks (including day 85) after the first study dose and after the last study dose. If the stage of an AE cannot be determined due to missing time variables, any AE will be defined as a TEAE. Drug-related treatment-emergent adverse events—SOC and PT classification and summary are shown in Table 1 .

[0122] [Table 1]

[0123] The incidence of treatment-emergency adverse events in the ADI-10087 group was 52.3%, and the incidence of drug-related treatment-emergency adverse events was 38.6%. The incidence of treatment-emergency adverse events in the placebo group was 57.1%, and the incidence of drug-related treatment-emergency adverse events was 50.0%. The incidence of at least one adverse event of special interest (hepatic events, muscle events, allergic reactions, and injection site reactions) in the ADI-10087 and placebo groups was 13.6% and 28.6%, respectively. There were no serious adverse events, study drug-related serious adverse events, adverse events leading to withdrawal from the study, or adverse events leading to death. The most common drug-related treatment-emergency adverse events in the ADI-10087 group were upper respiratory tract infection (20.5%) and elevated blood creatine phosphokinase (6.8%). The most common drug-related treatment-emergency adverse event in the placebo group was upper respiratory tract infection (14.3%). All drug-related adverse events in the ADI-10087 group were mild to moderate and did not require special treatment or improved after corrective treatment. No adverse events occurred in the SC group, particularly at doses up to 450 mg.

[0124] Immunogenicity assessment Immunogenicity assessment will be based on the Immunogenicity Analysis Set (IS), with the evaluation indices being anti-ADI-10087 antibodies (ADA) and neutralizing antibodies (NAb). The proportion of subjects who developed anti-ADI-10087 antibodies and neutralizing antibodies (NAb) for each dose group will be compiled, and the subjects who tested positive for ADI-10087 antibodies (ADA) / neutralizing antibodies (NAb) after administration will be listed.

[0125] Immunogenicity was measured by measuring the production of anti-drug antibodies (ADA) and neutralizing antibodies (NAb) in serum. (1) Sampling times for the detection of anti-drug antibodies and neutralizing antibodies: 1 hour before administration, 336 hours (day 15), 672 hours (day 29), 1344 hours (day 57), and 2016 hours (day 85) after administration. Blood sampling times for immunogenicity: pre-dose (within 1 hour), 336 hours (±12 hours) after administration (day 15), 672 hours (±24 hours) after administration (day 29), 1344 hours (±48 hours) after administration (day 57), and 2016 hours (±48 hours) after administration (day 85).

[0126] At baseline, one subject (1.7%) in the placebo group tested positive for anti-ADI-10087 antibodies (ADA) before administration, but no subjects tested positive for ADA after administration. Neutralizing antibodies (Nab) were detected in this subject, and the results were negative. Therefore, neither ADA nor Nab were positive in either the ADI-10087 group or the placebo group after administration, indicating a favorable safety profile.

[0127] Example 12: Evaluation of the pharmacokinetics of ADI-10087 after a single dose in healthy adult Chinese subjects In the study described in Example 11 above, PK blood samples were collected 1 hour pre-dose, 4 hours post-dose, and at 24 hours (Day 2), 48 hours (Day 3), 72 hours (Day 4), 96 hours (Day 5), 144 hours (Day 7), 168 hours (Day 8), 240 hours (Day 11), 336 hours (Day 15), 504 hours (Day 22), 672 hours (Day 29), 840 hours (Day 36), 1008 hours (Day 43), 1344 hours (Day 57), 1680 hours (Day 71), and 2016 hours (Day 85) post-dose.

[0128] To fully reflect the distribution and elimination characteristics of drugs in the human body, WinNonlin Professional software was used to perform non-compartmental analysis (NCA) to calculate T according to the actual blood sampling time. max , C max ,AUC,V,t 1 / 2 Pharmacokinetic parameters of total and free ADI-10087, including CL, were calculated, and key parameters are shown in Tables 2 and 3 below. [Table 2] [Table 3]

[0129] The mean total concentration-time curves of ADI-10087 after administration of ADI-10087 to subjects in each dose group are shown in Figure 2, and the mean free concentration-time curves of ADI-10087 in each dose group are shown in Figure 1.

[0130] Pharmacokinetic evaluation was performed based on the pharmacokinetic concentration set (PKCS), and data from 44 subjects were included in the PKCS set and analyzed. The mean apparent volume of distribution (V) of total ADI-10087 was measured after a single subcutaneous injection of doses ranging from 25 mg to 600 mg in the abdomen. z / F ) was approximately 3.2 L to 7.4 L, the mean apparent clearance (CL / F) was approximately 13.0 mL / h to 40.2 mL / h, and the mean t 1 / 2 The mean V of free ADI-10087 was approximately 5 to 7 days. z / F The average CL / F was approximately 4.0 L to 8.6 L, the average CL / F was 17.4 mL / h to 74.6 mL / h, and the average t 1 / 2 The Vz / F and CL / F decreased with increasing dose, and the t 1 / 2 was gradually extended.

[0131] After a single subcutaneous injection of 25 mg to 600 mg of ADI-10087 in the abdomen, the T of total ADI-10087 in serum was measured. max The T of free ADI-10087 is approximately 3-6 days. max The absorption time of ADI-10087 in the abdomen was approximately 2.5 to 10 days. It has been shown that the absorption time of ADI-10087 in the abdomen is slow, and that as the dose increases, a longer time is required to reach the peak absorption. Within the dose range of 25 mg to 600 mg of ADI-10087 administered as a single dose, the C of total and free ADI-10087 was max , AUC 0-last and AUC 0-∞ The rate of increase in each of the doses was greater than that of the dose, and the drug exhibited nonlinear kinetics.

[0132] Mean V of total ADI-10087 after single intravenous infusion of 75 mg and 450 mg doses z are approximately 2.6L and 2.1L, respectively, and the average V ss were approximately 3.0 L and 4.3 L, respectively, and the average CL was approximately 17.3 mL / h and 9.81 mL / h, respectively, and the average t 1 / 2 The mean V of free ADI-10087 was approximately 4 and 6 days, respectively. z are approximately 2.3L and 2.1L, respectively, and the average V ss were approximately 2.5 L and 3.0 L, respectively, and the average CL was 24.1 mL / h and 9.41 mL / h, respectively, and the average t 1 / 2 were approximately 3 and 7 days, respectively. Similarly, with increasing dose, CL decreased and t 1 / 2 Within the dose range of 75 mg to 450 mg, the absolute bioavailability of ADI-10087 administered subcutaneously in the abdomen was approximately 58% (based on total ADI-10087).

[0133] AUC corresponding to total and free ADI-10087 after subcutaneous injection or intravenous infusion of 75 mg and 450 mg ADI-10087 into the abdomen of healthy subjects, respectively 0-∞ See Table 4 for the geometric mean values ​​and corresponding estimates of absolute bioavailability for subcutaneous administration. [Table 4]

[0134] As can be seen, when assessed based on total ADI-10087, the absolute bioavailability of the 75 mg and 450 mg doses administered subcutaneously to healthy subjects was similar, 57.8% and 57.5%, respectively, demonstrating that the absolute bioavailability of the subcutaneous doses is nearly consistent within the dose range of 75 mg to 450 mg. When assessed based on free ADI-10087, the absolute bioavailability of 75 mg and 450 mg doses administered subcutaneously to healthy subjects was 33.8% and 49.2%, respectively, which was slightly lower than the results for total ADI-10087. PK parameters (C max , AUC 0-last and AUC 0-∞ ) and dose correlation is analyzed.

[0135] C of total and free ADI-10087 after subcutaneous injection of doses of 25 mg to 600 mg into the abdomen of healthy subjects max , AUC 0-last and AUC 0-inf The scatter plots of the changes in C as a function of dose are shown in Figures 3 to 5, respectively. As can be seen from the results, the C of total ADI-10087 and free ADI-10087 increased significantly within the dose range of 25 mg to 600 mg. max The C and AUC increased with increasing dose, but the rate of increase was greater than the rate of dose increase, indicating that ADI-10087 exhibited nonlinear kinetic properties. For example, the mean C of total ADI-10087 increased with increasing dose (1:3:6:12:18:24) from 25 mg to 600 mg. max The increase ratios were approximately 1:3.4:8.0:13:22:29, and the mean AUC 0-last The ratios of increase in C for free ADI-10087 were approximately 1:4.2:11:26:46:82. Due to the low concentrations, PK parameters could not be reported for the 25 mg group relative to free ADI-10087. Within the dose range of 75 mg to 600 mg (with an increase factor of 1:2:4:6:8), the mean C for free ADI-10087 was max The increase ratios were approximately 1:2.9:5.4:10:12, and the mean AUC 0-last The increase ratio was approximately 1:3.9:12:21:34.

[0136] Only two doses, 75 mg and 450 mg, were evaluated by intravenous infusion. The mean C of total ADI-10087 was significantly increased with a 6-fold increase in intravenous dose. maxand AUC 0-last increased by 4.6 and 11-fold, respectively, and the mean C max and AUC 0-last increased by 6.1 and 17 times, respectively.

[0137] Example 13: Evaluation of the pharmacodynamics of a single dose of ADI-10087 in healthy adult Chinese subjects In the study described in Example 11 above, blood samples were collected 1 hour pre-dose, 4 hours post-dose, and 24 hours (day 2), 48 hours (day 3), 72 hours (day 4), 96 hours (day 5), 144 hours (day 7), 168 hours (day 8), 240 hours (day 11), 336 hours (day 15), 504 hours (day 22), 672 hours (day 29), 840 hours (day 36), 1008 hours (day 43), 1344 hours (day 57), 1680 hours (day 71), and 2016 hours (day 85) post-dose. Using the methods disclosed herein or known in the art, levels of PCSK-9, LDL-C, total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), lipoprotein a (Lp(a)), very-low-density lipoprotein cholesterol (VLDL-C), non-high-density lipoprotein cholesterol (non-HDL-C), apolipoprotein A1 (ApoA1), and apolipoprotein B (ApoB) were measured and graphs were plotted.

[0138] (1) Change from baseline in serum free PCSK-9 concentration The central laboratory collected blood samples at designated time points to detect serum free PCSK-9 concentrations. Pharmacodynamic evaluation was performed based on the Pharmacodynamic Analysis Set (PDS), which compiled and listed the subjects' serum free PCSK-9 concentrations observed at each time point during each visit and the percent change from baseline. Baseline was defined as the observed PCSK-9 concentration before administration. Percent change from baseline (%) = 100 x (observed post-administration concentration - baseline value) / baseline value. Drug concentration-time curves (including linear and logarithmic concentration graphs) were plotted for each dose group to determine the percent change from baseline in the mean PCSK-9 concentration of each dose group.

[0139] Figure 6 shows the percent change from baseline in serum free PCSK-9 concentrations. As can be seen from the results, the mean duration of PCSK-9 reduction increased with increasing dose. At 24 hours post-dose, the maximum mean reduction in PCSK-9 levels for each group was 90%-100%. At 2 weeks post-dose, the ADI-10087 75 mg SC group and the 150 mg and higher dose groups experienced an average reduction in PCSK-9 levels of >70%. At 4 weeks post-dose, the ADI-10087 300 mg and higher SC and IV dose groups experienced an average reduction in PCSK-9 levels of >60%. At 6 weeks post-dose, the ADI-10087 450 mg SC, 450 mg IV, and 600 mg SC groups experienced an average reduction in PCSK-9 levels of >80%. At 8 weeks post-dose, the ADI-10087 600 mg SC group experienced an average reduction in PCSK-9 levels of >50%.

[0140] (2) Least squares mean number of changes from baseline in LDL-C At each evaluation time point, the change from baseline in serum LDL-C for each study drug dose group compared with the placebo group was analyzed using a mixed-effects model for repeated measures (MMRM). The analysis results included least-squares means of change, mean differences between groups, 95% confidence intervals, and P values. The model analysis results showed that within one month of dosing, most study drug dose groups had P values ​​of <0.05 compared with the placebo group. Over time, the lower dose groups had P values ​​of >0.05 compared with the placebo group. By day 29, the 150 mg and higher dose groups had P values ​​of <0.05 compared with the placebo group. By day 57, only the 450 mg and higher dose groups had P values ​​of <0.05 compared with the placebo group. By day 71, only the 600 mg dose group had P values ​​of <0.05 compared with the placebo group. At day 85, all study drug dose groups had P values ​​>0.05 compared with the placebo group. Figure 7 plots the least squares mean changes in LDL-C from baseline at each treatment effect assessment time point for each dose group.

[0141] (3) Percent change in mean LDL-C concentration from baseline The percent change in mean LDL-C concentrations relative to baseline is shown in Figure 8. Results indicate that the duration of serum LDL-C reduction is dose-dependent. The maximum LDL-C reduction was first observed on Day 5. By Day 15, each dose group approached maximum reduction. The maximum mean LDL-C reduction in each subcutaneous ADI-10087 group ranged from 51.7% to 72.1%. On Day 7 after administration, LDL-C in the 75 mg and 150 mg ADI-10087 groups decreased by an average of >50%, and the >50% reduction in LDL-C in the 75 mg group was maintained through Day 15, and in the 150 mg group, it was maintained through Day 22. At day 11 after administration, LDL-C in the 300 mg and higher ADI-10087 dose groups was reduced by an average of >50% and was sustained through at least day 43 (week 6). At day 57 (week 8) after administration, LDL-C in the ADI-10087 600 mg SC group was reduced by an average of >50%. At day 71 (week 10) after administration, LDL-C in the ADI-10087 600 mg SC group was reduced by an average of >30%. When compared with evolocumab as a control, it was found that at the same dose, ADI-10087 had a greater maximum LDL-C reduction rate and duration than evolocumab.

[0142] (4) Area under the curve for LDL-C levels The mean LDL-C concentration-time diagram is shown in FIG. The area under the curve for LDL-C levels was calculated using the trapezoidal method. For subjects who withdrew early, the trapezoidal method was also used to accumulate the area under the curve using the concentration data before withdrawal. The geometric mean for the LDL-C area under the curve in the test drug group was 3522.433, with a geometric coefficient of variation of 44.3. The geometric mean for the placebo group was 5609.877, with a geometric coefficient of variation of 20.1. The 75 mg IV dose of the test drug group had the largest area under the curve, with a geometric mean of 4709.056 and a geometric coefficient of variation of 18.0. The 450 mg IV dose had the smallest area under the curve, with a geometric mean of 1978.572 and a geometric coefficient of variation of 99.0.

[0143] (5) Percent change in total cholesterol (TC) concentration from baseline The percent change in total cholesterol (TC) concentration relative to baseline is shown in Figure 10. As can be seen from the results, the mean duration of total cholesterol reduction increased with increasing dose, with the ADI-10087 75 mg and higher dose groups achieving a maximum mean reduction in total cholesterol of between 37.9% and 48.6%, approaching the maximum reduction on day 11 after administration.

[0144] (6) Percent change from baseline in apolipoprotein B (ApoB) concentration The percent change in apolipoprotein B (ApoB) concentration relative to baseline is shown in Figure 11. As can be seen from the results, the duration of ApoB reduction increased with increasing dose, with the maximum mean percent reduction in total cholesterol in the 75 mg and higher ADI-10087 dose groups ranging from 52.1% to 62.8%, with the maximum reduction approaching on day 11 after administration.

[0145] (7) Percent change from baseline in non-high-density apolipoprotein cholesterol (Non-HDL-C) levels The percent change in non-high-density apolipoprotein cholesterol (Non-HDL-C) levels relative to baseline is shown in Figure 12. As can be seen from the results, the duration of Non-HDL-C reduction increased with increasing dose, with the ADI-10087 75 mg and higher dose groups achieving a maximum mean reduction in total cholesterol of between 56.8% and 69.7%, with the maximum reduction approaching on day 11 after administration.

[0146] (8) Percent change from baseline in mean lipoprotein a (LP(a)) concentration The percent change in mean lipoprotein a (LP(a)) concentrations from baseline is shown in Figure 13. On day 7 after administration, Lp(a) decreased in each ADI-10087 dose group, with the maximum mean decrease in each group ranging from 24.9% to 62.3%.

[0147] Example 14: Evaluation of the pharmacokinetics of ADI-10087 following multiple repeated doses in Chinese hypercholesterolemic subjects 1. Selection of research subject Selected subjects must simultaneously meet all of the following criteria: (1) Provide signed and dated informed consent. (2) Male or female, aged 18 years or older or 70 years or older at the time of screening. (3) 18 kg / m² <BMI<30kg / m2であること。 (4) Diagnosed with hyperlipidemia and treated with a stable dose of moderate or higher strength statins for at least 4 weeks (see the Chinese Guide for the Prevention and Treatment of Blood Lipid Dyslipidemia in Adults 2016 for details). (5) Fasting LDL-C ≤ 100 mg / dL (2.6 mmol / L) ≤ 220 mg / dL (5.7 mmol / L) at screening. (6) Fasting triglycerides ≤ 400 mg (4.5 mmol / L) at screening; and (7) The subject demonstrated willingness to cooperate with all steps of the study and completion of the study intervention cycle.

[0148] 2. Study Design A total of 60 patients who met the inclusion criteria were randomly assigned to the following dose groups: 75 mg or 140 mg of ADI-10087 administered subcutaneously every two weeks or placebo; 300 mg or 420 mg of ADI-10087 administered subcutaneously every four weeks or placebo; or 450 mg or 600 mg of ADI-10087 administered subcutaneously every six weeks or placebo. Each dose of ADI-10087 was randomized 4:1 to placebo. Treatment lasted for 12 weeks, after which the results were analyzed.

[0149] 3. Drugs used in the study The test drug used in the study was the formulation prepared as described in Example 1, and the control drug used in the study was the placebo prepared as described in Example 6.

[0150] 4. Administration method The test drug or placebo is injected subcutaneously into the abdomen.

[0151] 5. Specimen collection and processing PK / PD (PCSK9) blood sampling time: 75mg Q2W, 140mg Q2W dosing schedule: First dose: before administration (within 1 hour), 4 hours (±10 minutes) after administration, 24 hours (±1 hour) (day 1), 48 hours (±2 hours) (day 2), 72 hours (±3 hours) (day 3), 96 hours (±4 hours) (day 4), 144 hours (±6 hours) (day 6), 168 hours (±6 hours) (day 7), 240 hours (±12 hours) (day 10), 288 hours (±12 hours) (day 12). 2nd / 3rd / 4th / 5th dose: Before administration (within 1 hour). Sixth dose: before administration (within 1 hour), 4 hours (±10 minutes) after administration, 24 hours (±1 hour) (Day 1), 48 hours (±2 hours) (Day 2), 72 hours (±3 hours) (Day 3), 96 hours (±4 hours) (Day 4), 144 hours (±6 hours) (Day 6), 168 hours (±6 hours) (Day 7), 240 hours (±12 hours) (Day 10), 288 hours (±12 hours) (Day 12), 336 hours (±12 hours) (Day 14). If a subject is unable to complete the study and is terminated early and withdrawn from the study, a PK / PD (PCSK9) blood sample will be collected during the early termination visit. PK / PD (PCSK9) will be performed once during safety follow-up. 300mg Q4W, 420mg Q4W dosing schedule: First dose: before administration (within 1 hour), 4 hours (±10 minutes) after administration, 24 hours (±1 hour) (day 1), 48 hours (±2 hours) (day 2), 72 hours (±3 hours) (day 3), 96 hours (±4 hours) (day 4), 144 hours (±6 hours) (day 6), 168 hours (±6 hours) (day 7), 240 hours (±12 hours) (day 10), 336 hours (±12 hours) (day 14), 504 hours (±24 hours) (day 21). Second dose: within 1 hour before administration. Third dose: before administration (within 1 hour), 4 hours (±10 minutes) after administration, 24 hours (±1 hour) (Day 1), 48 hours (±2 hours) (Day 2), 72 hours (±3 hours) (Day 3), 96 hours (±4 hours) (Day 4), 144 hours (±6 hours) (Day 6), 168 hours (±6 hours) (Day 7), 240 hours (±12 hours) (Day 10), 336 hours (±12 hours) (Day 14), 504 hours (±24 hours) (Day 21), 672 hours (±24 hours) (Day 28). If a subject is unable to complete the study and is terminated early and withdrawn from the study, PK / PD blood samples will be collected during the early termination visit. One PK / PD blood sample will be collected during safety follow-up.

[0152] 450mg Q6W, 600mg Q6W dosing regimen: First dose: before administration (within 1 hour), 4 hours (±10 minutes) after administration, 24 hours (±1 hour) (Day 1), 48 hours (±2 hours) (Day 2), 72 hours (±3 hours) (Day 3), 96 hours (±4 hours) (Day 4), 144 hours (±6 hours) (Day 6), 168 hours (±6 hours) (Day 7), 240 hours (±12 hours) (Day 10), 336 hours (±12 hours) (Day 14), 504 hours (±24 hours) (Day 21), 672 hours (±24 hours) (Day 28), 840 hours (±24 hours) (Day 35). Second dose: before administration (within 1 hour), 4 hours (±10 minutes) after administration, 24 hours (±1 hour) (Day 1), 48 hours (±2 hours) (Day 2), 72 hours (±3 hours) (Day 3), 96 hours (±4 hours) (Day 4), 144 hours (±6 hours) (Day 6), 168 hours (±6 hours) (Day 7), 240 hours (±12 hours) (Day 10), 336 hours (±12 hours) (Day 14), 504 hours (±24 hours) (Day 21), 672 hours (±24 hours) (Day 28), 840 hours (±24 hours) (Day 35), 1008 hours (±48 hours) (Day 42). For the 600 mg dose group, one PK / PD blood sample will be collected at 1344 hours (±48 hours) after the second dose (Day 56). If a subject is unable to complete the study and is terminated early and withdrawn from the study, PK / PD blood samples will be collected during the early termination visit. One PK / PD blood sample will be collected during safety follow-up. PK / PD (PCSK9) blood samples must be tested at a central laboratory. Blood lipids during the screening period will be tested at a local laboratory, and blood lipids from the baseline period will be tested at a central laboratory. 5 mL of whole blood will be collected using a clotting-promoting vacuum collection tube, and serum will be separated, aliquoted, frozen, and stored for PK / PD (PCSK9) analysis. Blood lipid detection and blood sampling are performed as described above.

[0153] 6. Pharmacokinetic results Pkanalix 2019R2 (Lixoft, Antony, France) software was employed to calculate the pharmacokinetic parameters of the free concentration of ADI-10087 according to the actual blood sampling time by non-compartmental model analysis (NCA). After multiple subcutaneous injections of different doses of ADI-10087 in hypercholesterolemic patients, the mean pharmacokinetic parameters for each dose group at the first dose and steady-state dosing of ADI-10087 are summarized in Tables 5 and 6. [Table 5-1] [Table 5-2] [Table 6-1] [Table 6-2]

[0154] Within the first dose range of ADI-10087 from 75 mg to 600 mg, the C max , AUC 0-inf The rate of increase in each of the doses was greater than that of the dose, and the drug exhibited nonlinear kinetics. In a PK study of the first and steady-state cycles, the median time to peak of ADI-10087 (T) was measured after subcutaneous injection of 75 mg to 600 mg of ADI-10087 in the abdomen at different dosing intervals. max ) ranged from 70.7 h to 239 h (2.9 to 10 days), and the median time to peak of ADI-10087 after subcutaneous injection of 75 mg to 600 mg of ADI-10087 in the abdomen at different dosing intervals after steady-state cyclic administration (T max ) ranged from 95.1 h to 155 h (3.96 to 6.46 days). As can be seen from the results, absorption of ADI-10087 in the abdomen takes time, and as the dose increases, a longer time is required to reach peak absorption.

[0155] After subcutaneous injection of 75 mg to 600 mg of ADI-10087 into the abdomen in the first cycle, the mean Vz / F of ADI-10087 was approximately 5.14 L to 33.4 L, the mean CL / F was approximately 0.0163 L / h to 0.242 L / h, and the mean t 1 / 2 The mean Vz / F of ADI-10087 ranged from approximately 5.13 L to 12.2 L, the mean CL / F ranged from approximately 0.0124 L / h to 0.091 L / h, and the mean t 1 / 2 The time to administration was approximately 98.6 hours to 430 hours (4.10 to 17.9 days). With increasing dose, Vz / F and CL / F decreased, and t 1 / 2 was gradually extended.

[0156] When administered once every two weeks, the 336 h concentrations (C 336h ) were 3.44 and 3.83, respectively, and the AUC 0-336h The average accumulation ratios of were 3.20 and 3.64, respectively. When administered once every 4 weeks, the 672 h concentrations (C 672h ) were 2.03 and 1.47, respectively, and the AUC 0-672h The average accumulation ratios of α, β, and β were 1.34 and 1.28, respectively.

[0157] When administered once every 6 weeks, the 1008h concentrations (C 1008h ) were 1.18 and 0.983, respectively, and the AUC 0-1008h The mean accumulation rates of ADI-10087 were 1.02 and 0.986, respectively. With increasing dose and prolonging the interval between dosing cycles, the accumulation rate of ADI-10087 decreased, and the results are shown in Table 7. [Table 7]

[0158] Example 15: Evaluation of the Pharmacodynamics of Multiple Repeat Doses of ADI-10087 in Chinese Hypercholesterolemic Subjects The selection of researchers, study design, drugs frequently used in the study, administration methods, and sample collection and processing were the same as in Example 14. Pharmacodynamic results Pharmacodynamic evaluation was performed based on the Pharmacodynamic Analysis Set (PDS), which monitored changes in serum free PCSK9 levels relative to the baseline before each dose and summarized the observed values ​​and percent change (%) from baseline for each subject's serum free PCSK9 level at each time point during the visit. The PDS set included 60 hypercholesterolemic subjects (48 in the ADI-10087 group and 12 in the placebo group) and analyzed serum PCSK9 data after multiple repeated doses of ADI-10087 at different dosing intervals. The PDS set included patients who had received at least one dose of the investigational drug and had at least one effective test result after dosing.

[0159] 1. Percent change in serum free PCSK9 concentration after administration compared to baseline Figure 14 shows the percent change in serum free PCSK9 concentration relative to baseline after administration. As can be seen from the results, there was no significant change in PCSK9 levels in the placebo group. For the ADI-10087 group, after the first administration of ADI-10087, PCSK9 levels in the 75mg to 600mg dose groups generally reached a maximum reduction rate at approximately 24 hours, and the time to reach the maximum reduction rate was consistent across dose groups. After the first administration of ADI-10087, PCSK9 levels in the 75mg dose group reached a maximum reduction rate of approximately 78%, while those in the 140mg to 600mg dose groups reached a maximum reduction rate of >90%. The higher the dose, the greater the PCSK9 reduction rate. PCSK9 was completely suppressed until the dose reached 300mg or higher. After the time to reach the maximum reduction rate, PCSK9 began to rebound, with the magnitude of the rebound decreasing with the dose. After multiple consecutive doses of ADI-10087, PCSK9 levels were able to maintain a consistent reduction rate. For the 2-week dosing cohort, PCSK9 levels in the 75 mg dose group were maintained at levels equivalent to baseline, while PCSK9 levels in the 140 mg dose group were able to maintain a reduction rate of approximately 80%. For the 4-week and 6-week dosing cohorts, PCSK9 levels in both the 300 mg and 600 mg dose groups were able to maintain a reduction rate of more than 80%.

[0160] 2.Comparison of percentage change from baseline in LDL-C at the same dosing interval In the 2-week dosing interval group, LDL-C levels in the 75mg Q2W group were significantly reduced compared to baseline after the first dose, with a maximum reduction of approximately 51.09% before the second dose (Day 11). There was no significant rebound between 4 and 336 hours after the final dose, and LDL-C levels remained significantly reduced compared to baseline (-61.323%) 336 hours after the final dose. LDL-C levels in the 140mg Q2W group were also significantly reduced after the first dose, with a maximum reduction of approximately 45.99% before the second dose (Day 13). There was a slight rebound between 4 and 336 hours after the final dose, but the magnitude of the rebound was small, and LDL-C levels remained reduced by 54.296% to 60.991% compared to baseline.

[0161] Four-week dosing interval group: LDL-C levels in both the 300mg Q4W and 420mg Q4W groups after the first dose were significantly reduced compared to baseline, reaching a maximum reduction rate of 59.8% and 71.363% before the second dose on D29, respectively. The LDL-C reduction rate in the 420mg Q4W group before each dose was significantly greater than that of the 300mg Q4W group. No significant rebound was observed after the final dose in either group. 672 hours after the final dose, the LDL-C reduction rate in the 450mg Q4W group was still 72.256% (compared to 60.136% in the 300mg Q4W group).

[0162] Six-week dosing interval group: After the first administration, the LDL-C levels of both the 450mg Q6W group and the 600mg Q6W group were significantly reduced compared to baseline, and the reduction rate of 600mg Q6W was significant (maximum reduction rate of approximately 71.875%). After the second administration (i.e., after the final administration), the LDL-C reduction rate of 600mg Q6W was still superior to that of 450mg Q6W, with a slight rebound at 672 hours after the second administration, but the extent was small (approximately 2.9%). At 1008 hours after the second administration, the LDL-C levels of the 450mg Q6W group and the 600mg Q6W group were still significantly reduced compared to baseline, by 62.883% and 56.516%, respectively. Furthermore, the results of the study in Example 13 revealed that the higher the dose, the longer the duration of LDL-C reduction and PCSK9 inhibition. At the same time, after 8 weeks of administration at 600 mg (1344 hours after the second administration), LDL-C levels were still observed to be reduced by 43.455% compared to baseline. This indicates that ADI-10087 can be administered over a long period of time, from 4 to 8 weeks. The percentage change in LDL-C concentration from baseline before and after each administration is shown in Figure 15.

[0163] 3. Percent change from baseline in LDL-C levels at 6, 12, and 14 weeks After 6 weeks of continuous administration, compared with the placebo group, LDL-C in each dose group of ADI-10087 significantly decreased compared to baseline: 75 mg Q2W group: -52.385%, 140 mg Q2W group: -52.523%, 450 mg Q6W group: -60.353%; 600 mg Q6W group: -62.175%, respectively. The percentage decrease in LDL-C compared to baseline increased in a dose-dependent manner, i.e., the higher the dose, the greater the mean decrease in LDL-C. After 12 weeks of continuous administration, there was no significant rebound in LDL-C in each dose group of ADI-10087 compared to the baseline reduction rate 6 weeks after administration. Compared with the placebo group, the percentage change in LDL-C from baseline in each dose group was: 75mg Q2W group: -58.913%, 140mg Q2W group: -51.886%, 300mg Q4W group: -57.726%, 420mg Q4W group: -69.846%, 450mg Q6W group: -60.473%, and 600mg Q6W group: -54.106%, respectively. Of these, LDL-C in the 420mg Q4W group achieved a reduction rate of -72.256% (placebo -2.410%). After 14 weeks of treatment with 600 mg Q6W, i.e., 8 weeks after the second dose, LDL-C reduction from baseline was still -43.455% (compared to 16.980% in the placebo group). The change from baseline in LDL-C levels before and after each dose is shown in Figure 16.

[0164] 4. Analysis of percent change from baseline in Lp(a) levels at 6, 12, and 14 weeks After 6 weeks of continuous administration, the mean reduction in Lp(a) from baseline in each ADI-10087 dose group ranged from -8.720% to -22.436% compared with the placebo group, of which the reductions in the 75 mg Q2W and 450 mg Q6W groups were most significant, at -22.436% and -21.914%, respectively. After 12 weeks of continuous administration, the mean reduction in Lp(a) from baseline in each ADI-10087 dose group compared with the placebo group was -2.088% to -28.638%, of which the reductions in Lp(a) in the 75mg Q2W and 450mg Q6W groups were most significant, -28.638% and -27.362%, respectively, while the reductions in Lp(a) in the 600mg Q6W and 420mg Q4W groups were smaller, ranging from 2% to 7%. After 14 weeks of continuous administration (8 weeks after the second dose) in the 600 mg Q6W group, the reduction in Lp(a) levels from baseline reached -24.520% compared to the placebo group. The percentage change in Lp(a) levels from baseline before and after each dose is shown in Figure 17.

[0165] 5. Analysis of percent change from baseline in ApoB levels at 6, 12, and 14 weeks After 6 weeks of continuous administration, ApoB significantly decreased compared to baseline compared with the placebo group. The mean ApoB reduction rate for each dose group of ADI-100876 ranged from -57.118% to -61.414%. At the same dosing interval, the higher the dose, the greater the ApoB reduction rate. After 12 weeks of continuous administration, compared with the placebo group, the mean reduction in ApoB from baseline in each dose group of ADI-10087 was between -53.761% and -69.573%, which was basically consistent with the reduction in ApoB and LDL-C. Of these, ApoB in the 420mg Q4W group was reduced by approximately 70% from baseline. After 14 weeks of continuous dosing in the 600 mg Q6W group (8 weeks after the second dose), the percent reduction in ApoB levels from baseline was -61.729% compared to the placebo group. The percent change in ApoB levels from baseline before and after each dose is shown in Figure 18.

[0166] 6. Analysis of percent change in non-HDL-C levels from baseline at 12 and 14 weeks After 12 weeks of continuous administration, the mean reduction in non-HDL-C from baseline in each ADI-10087 dose group was 55% or more compared to the placebo group, with the maximum reduction reaching 75.394% (420 mg Q4W group). After 14 weeks of continuous administration (8 weeks after the second dose) in the 600 mg Q6W group, the reduction in non-HDL-C levels from baseline was -61.958% compared to the placebo group. The percent change in non-HDL-C levels from baseline before and after each dose is shown in Figure 19.

[0167] 7. Analysis of percent change in TC levels from baseline at 12 and 14 weeks After 12 weeks of continuous administration, the mean reduction in TC from baseline for each ADI-10087 dose group was between 40% and 57%, with the greatest reduction of approximately 56% in the 420 mg Q4W group. TC in the placebo group did not change significantly from baseline (increased approximately 0.5%). After 14 weeks of continuous dosing in the 600 mg Q6W group (8 weeks after the second dose), the percent decrease in TC from baseline was 36.184%, while the placebo group's TC increased by 10.635% from baseline. The percent change in TC levels from baseline before and after each dose is shown in Figure 20.

[0168] 8. Analysis of percent change in ApoB / ApoA1 levels from baseline at 12 and 14 weeks After 12 weeks of continuous administration, the mean reduction in ApoB / ApoA1 from baseline in each dose group of ADI-10087 was greater than 50% compared to the placebo group, with the maximum reduction reaching -68.476% (420 mg Q4W group). After 14 weeks of continuous dosing in the 600 mg Q6W group (8 weeks after the second dose), the percent reduction in ApoB / ApoA1 levels from baseline was -53.656% compared to the placebo group. The percent change in ApoB / ApoA1 levels from baseline before and after each dose is shown in Figure 21.

[0169] 9. Conclusion of treatment effect analysis In the safety analysis set, 48 subjects in the ADI-10087 group and 12 subjects in the placebo group were included in the treatment efficacy analysis. At week 12, the mean reduction in LDL-C levels from baseline in each dose group ranged from 54.3% to 72.26%. In particular, in the 600 mg Q6W cohort, a 56.52% (-72.50%, -40.54%) reduction in LDL-C was observed, and this effect persisted until week 14 (8 weeks after the last dose), at which point the reduction from baseline was still 43.46% (-60.96%, -25.96%). At week 12, the mean reduction in Lp(a) from baseline ranged from 24.04% to 50.59%. Other lipids also decreased to some extent in each dose group compared with the placebo group. Therefore, ADI-10087 has favorable PK / PD (LDL-C) properties and can be administered over a long interval of 4 to 8 weeks.

[0170] Example 16: Safety evaluation of multiple repeated doses of ADI-10087 in Chinese subjects with hypercholesterolemia The results of this study demonstrated that ADI-10087 was highly safe when administered subcutaneously multiple times in Chinese subjects with hypercholesterolemia. The incidence of treatment-related adverse events (TEAEs) during treatment in the ADI-10087 group was 70.8%, with a 31.3% incidence of study drug-related TEAEs. The incidence of TEAEs in the placebo group was 75.0%, with a 41.7% incidence of study drug-related TEAEs. The incidence rates of at least one adverse event of particular interest in the ADI-10087 and placebo groups (allergic reactions, injection site reactions, elevated aminotransferases, liver damage, and muscle events) were 10.4% and 25%, respectively. There were no serious adverse events, study drug-related serious adverse events, adverse events leading to withdrawal from the study, or adverse events leading to death. All drug-related adverse events in the ADI-10087 group were mild to moderate in severity and did not require special treatment or improved after corrective treatment, demonstrating a favorable safety profile. A summary of adverse events during the study is shown in Table 8 below. [Table 8-1] [Table 8-2]

[0171] While exemplary embodiments of the present invention have been described above, those skilled in the art should understand that these disclosures are exemplary only and that various other substitutions, adaptations and modifications can be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments enumerated herein.

Claims

1. A method of lowering cholesterol levels in a subject, comprising administering to the subject an anti-PCSK9 antibody or antigen-binding fragment thereof at a dose of about 15 mg to 3500 mg.

2. A method of preventing or treating a disorder associated with elevated LDL-cholesterol levels in a subject, comprising administering to the subject an anti-PCSK9 antibody or antigen-binding fragment thereof at a dose of about 15 mg to 3500 mg.

3. A method for preventing or treating a cholesterol-related disease, such as hypercholesterolemia and / or hyperlipidemia, comprising administering to a subject an anti-PCSK9 antibody or antigen-binding fragment thereof at a dose of about 15 mg to 3500 mg.

4. The anti-PCSK9 antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL; (a) the VH is (i) a combination of an HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 10, an HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17, and an HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 19; or (ii) comprising three complementarity-determining regions (CDRs) contained in VH as set forth in SEQ ID NO: 30; (b) the VL is (i) a combination of an LCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 4, an LCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 5, and an LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 6; or (ii) The method according to any one of claims 1 to 3, comprising three complementarity-determining regions (CDRs) contained in the VL shown in SEQ ID NO:

24.

5. The anti-PCSK9 antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL; (a) the heavy chain variable region VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 30; (b) the light chain variable region VL comprises or consists of the amino acid sequence shown in SEQ ID NO:

24.

6. the anti-PCSK9 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain; (a) the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 41; (b) the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35; The method of any one of claims 1 to 5, wherein the anti-PCSK9 antibody is ADI-10087.

7. The method according to any one of claims 1 to 6, wherein the cholesterol is LDL-cholesterol.

8. 8. The method of claim 1, wherein the anti-PCSK9 antibody or antigen-binding fragment thereof is administered to a subject at a dose of about 25 mg to about 3000 mg, about 50 mg to about 2500 mg, about 75 mg to about 2000 mg, about 100 mg to about 2000 mg, about 200 mg to about 2000 mg, about 250 mg to about 1500 mg, about 300 mg to about 1000 mg, about 450 mg to about 1000 mg, about 600 mg to about 1000 mg, about 350 mg to about 900 mg, about 400 mg to about 800 mg, about 450 mg to about 700 mg, about 300 mg to about 600 mg, or about 450 mg to about 600 mg, wherein the dose is preferably 450 mg or 600 mg.

9. The method of any one of claims 1 to 8, wherein the anti-PCSK9 antibody or antigen-binding fragment thereof is administered parenterally, for example, subcutaneously or intravenously.

10. 10. The method of any one of claims 1 to 9, wherein the method still reduces the subject's LDL-cholesterol levels by >30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, or >90% four weeks after administration, five weeks after administration, six weeks after administration, seven weeks after administration, eight weeks after administration, nine weeks after administration, ten weeks after administration, three months after administration, four months after administration, or five months after administration, compared to the subject's LDL-cholesterol levels before administration.

11. The method of any one of claims 1 to 10, wherein the subject's serum free PCSK-9 level is still reduced by >30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, or >90% after 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 3 months, 4 months, or 5 months compared to the subject's serum free PCSK-9 level before administration.

12. 12. The method of any one of claims 1 to 11, wherein the method still reduces the subject's total cholesterol level by >30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, or >90% 4 weeks after administration, 5 weeks after administration, 6 weeks after administration, 7 weeks after administration, 8 weeks after administration, 9 weeks after administration, 10 weeks after administration, 3 months after administration, 4 months after administration, or 5 months after administration, compared to the subject's total cholesterol level before administration.

13. 13. The method of any one of claims 1 to 12, wherein the subject's apolipoprotein B levels are still reduced by >30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, or >90% 4 weeks after administration, 5 weeks after administration, 6 weeks after administration, 7 weeks after administration, 8 weeks after administration, 9 weeks after administration, 10 weeks after administration, 3 months after administration, 4 months after administration, or 5 months after administration, compared to the subject's apolipoprotein B levels before administration.

14. 14. The method of any one of claims 1 to 13, wherein the method still reduces the subject's non-high density lipoprotein cholesterol level by >30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, or >90% 4 weeks after administration, 5 weeks after administration, 6 weeks after administration, 7 weeks after administration, 8 weeks after administration, 9 weeks after administration, 10 weeks after administration, 3 months after administration, 4 months after administration, or 5 months after administration, compared to the subject's non-high density lipoprotein cholesterol level before administration.

15. 15. The method of any one of claims 1 to 14, wherein the method still reduces the subject's lipoprotein a levels by >20%, >30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, or >90% 4 weeks after administration, 5 weeks after administration, 6 weeks after administration, 7 weeks after administration, 8 weeks after administration, 9 weeks after administration, 10 weeks after administration, 3 months after administration, 4 months after administration, or 5 months after administration, compared to the subject's lipoprotein a levels before administration.

16. 16. The method of any one of claims 1 to 15, wherein the subject's ApoB / ApoA1 levels are still reduced by >20%, >30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, or >90% 4 weeks after administration, 5 weeks after administration, 6 weeks after administration, 7 weeks after administration, 8 weeks after administration, 9 weeks after administration, 10 weeks after administration, 3 months after administration, 4 months after administration, or 5 months after administration, compared to the subject's ApoB / ApoA1 levels before administration.

17. The method of any one of claims 1 to 16, wherein the cholesterol or LDL-cholesterol is serum LDL-cholesterol.

18. The method of any one of claims 1 to 17, wherein the anti-PCSK9 antibody or antigen-binding fragment thereof is administered at intervals of once every four weeks (Q4W) or more, for example, once every four weeks (Q4W), once every five weeks (Q5W), once every six weeks (Q6W), once every seven weeks (Q7W), once every eight weeks (Q8W), once every nine weeks (Q9W), once every ten weeks (Q10W), once every 11 weeks (Q11W), once every 12 weeks (Q12W), once every four months, once every five months, once every six months, once every seven months, or once a year, preferably once every four weeks (Q4W), once every six weeks (Q6W), or once every eight weeks (Q8W).

19. 19. The method of any one of claims 1 to 18, wherein after administration, the subject does not experience any serious adverse events, particularly serious adverse events associated with the anti-PCSK9 antibody or antigen-binding fragment thereof.

20. 20. The method of any one of claims 1 to 19, wherein after administration, the incidence of adverse events in the subject is comparable to subjects receiving a placebo.

21. 21. The method of any one of claims 1 to 20, wherein the cholesterol-related disease is selected from homozygous familial hypercholesterolemia, heterozygous familial hypercholesterolemia, and non-familial hypercholesterolemia.

22. The anti-PCSK9 antibody or antigen-binding fragment thereof is administered in a liquid antibody formulation, the liquid antibody formulation comprising: (i) about 100 mg / mL to about 200 mg / mL of an anti-PCSK-9 antibody or antigen-binding fragment thereof; (ii) about 0.2 mg / mL to 10 mg / mL of histidine; (iii) about 1% to 6% sorbitol and / or about 50 mmol / L to 180 mmol / L arginine or an arginine salt, and (iv) about 0.05 mg / mL to 1 mg / mL of polysorbate-80 or polysorbate-20 22. The method of any one of claims 1 to 21, wherein the pH of the liquid formulation is about 5.0 to 6.

0.

23. The anti-PCSK9 antibody or antigen-binding fragment thereof is administered in a liquid antibody formulation, the liquid antibody formulation containing water as a solvent and having the following composition: Anti-PCSK9 antibody or antigen-binding fragment thereof 150 mg / mL Histidine 1.5g / L Arginine 90mmol / L Sorbitol 3% (w / v) Polysorbate 80 0.3g / L wherein the pH of the liquid formulation is about 5.

5.

23. The method according to any one of claims 1 to 22.

24. The anti-PCSK9 antibody or antigen-binding fragment thereof is contained in a dose of about 15 mg to 3500 mg, preferably about 25 mg to about 3000 mg, about 50 mg to about 2500 mg, about 75 mg to about 2000 mg, about 100 mg to about 2000 mg, about 200 mg to about 2000 mg, about 250 mg to about 1500 mg, about 300 mg to about 1000 mg, about 450 mg to about 1000 mg, about 600 mg to about 1500 mg, about 1000 mg to about 1500 mg, about 1500 mg to about 2000 mg, about 1500 mg to about 2500 mg, about 25 ... and a single drug dosage unit comprising an anti-PCSK9 antibody or antigen-binding fragment thereof at a dose of about 1000 mg to about 1000 mg, about 350 mg to about 900 mg, about 400 mg to about 800 mg, about 450 mg to about 700 mg, about 300 mg to about 600 mg, or about 450 mg to about 600 mg, more preferably an anti-PCSK9 antibody or antigen-binding fragment thereof at a dose of 450 mg or 600 mg.

25. The anti-PCSK9 antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL; (a) the VH is (i) a combination of an HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 10, an HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17, and an HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 19; or (ii) comprising three complementarity-determining regions (CDRs) contained in VH as set forth in SEQ ID NO: 30; (b) the VL is (i) a combination of an LCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 4, an LCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 5, and an LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 6; or (ii) The single drug dose unit of claim 24, comprising three complementarity determining regions (CDRs) contained in the VL set forth in SEQ ID NO:

24.

26. The anti-PCSK9 antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL; (a) the heavy chain variable region VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 30; 26. The single pharmaceutical dose unit of claim 25, wherein (b) the light chain variable region VL comprises or consists of the amino acid sequence set forth in SEQ ID NO:

24.

27. the anti-PCSK9 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain; (a) the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 41; (b) the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35; 27. The single drug dosage unit of claim 26, wherein the anti-PCSK9 antibody is ADI-10087.

28. A pharmaceutical kit comprising the anti-PCSK9 antibody or antigen-binding fragment thereof according to any one of claims 24 to 27.

29. Use of a single drug dosage unit according to any one of claims 24 to 27 or a drug kit according to claim 28 in the manufacture of a medicament for lowering cholesterol levels in a subject.

30. Use of a single pharmaceutical dosage unit according to any one of claims 24 to 27 or a pharmaceutical kit according to claim 28 in the manufacture of a medicament for preventing or treating a disorder associated with elevated LDL-cholesterol levels in a subject.

31. Use of a single drug dosage unit according to any one of claims 24 to 27 or a drug kit according to claim 28 in the manufacture of a medicament for preventing or treating a cholesterol-related disease, such as hypercholesterolemia and / or hyperlipidemia.

Images