RU2846536C1 - Use of anti-pcsk9 antibodies for preventing or treating diseases associated with high cholesterol - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to methods for preventing or treating diseases associated with high cholesterol. Disclosed are a method for lowering cholesterol in a subject and a method for preventing or treating a disease associated with high cholesterol, selected from hypercholesterolemia and hyperlipidemia, comprising administering to subject from 75 mg to 600 mg of an anti-PCSK9 antibody containing a heavy chain and a light chain with SEQ ID NO: 41 and SEQ ID NO: 35, respectively. Anti-PCSK9 antibody is administered in an interval equal to or greater than once every two weeks.

EFFECT: invention provides improved method of treating or preventing diseases associated with high cholesterol, characterized by a long interval between administrations, causing fewer side effects.

25 cl, 21 dwg, 8 tbl, 16 ex

Description

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

AREA OF TECHNOLOGY

The present invention relates to the field of therapy using antibodies, in particular to the use of an antibody specifically binding to proprotein convertase subtilisin/kexin type 9 (PCSK9) (hereinafter referred to as an anti-PCSK9 antibody) and/or a fragment of such an antibody, as well as a pharmaceutical preparation containing it, for the prevention or treatment of diseases associated with high cholesterol. In addition, the present invention relates to the use of an anti-PCSK9 antibody or a liquid preparation thereof for the prevention or treatment of the above-mentioned diseases.

LEVEL OF TECHNOLOGY

The incidence and mortality of coronary atherosclerotic heart disease and other atherosclerotic vascular diseases have been increasing in China in recent years. Hypercholesterolemia is a known risk factor for coronary atherosclerotic heart disease, and increasing evidence suggests that aggressive treatment of hypercholesterolemia is associated with a reduced risk of final cardiovascular events related to coronary atherosclerosis. This relationship is particularly evident in patients with high risk of cardiovascular events, such as those with coronary artery disease.

Currently, the mainstay of cholesterol-lowering drugs are statins, which play an important role in the primary and secondary prevention of coronary atherosclerotic heart disease. However, current lipid-lowering therapy does not meet the clinical needs. Although statins can reduce cardiovascular mortality, statin therapy has certain limitations. First, statins reduce low-density lipoprotein cholesterol (LDL-C, LDL-C) by no more than 40% to 55%, and doubling the dose can reduce LDL-C by only about 6%. In addition, high-intensity statin therapy has raised safety concerns in the Chinese population. An increasing number of studies have shown that high-intensity statin therapy is associated with an increased incidence of myopathy and an increased risk of elevated liver function tests, and this association is stronger in the Chinese population than in other populations. The HPS2-THRIVE study showed that in the case of moderate-intensity statin therapy, the frequency of adverse liver reactions in Chinese patients is significantly higher than in European patients. Thus, the frequency of increased liver enzyme activity (>3 times the upper limit of normal) in Chinese patients is 10 times higher than in European patients. The risk of myopathy in the Chinese population is also 10 times higher than in European patients (Zhao Shuiping, et al, Guidelines for the Prevention and Treatment of Dyslipidemia in Chinese Adults (2016 Revised Edition), China Circulation Journal 31 (10): 937-953, 2016). There are currently no data on the safety of high-intensity statin therapy in the Chinese population.

In view of the above, it seems necessary to continue the development of new effective cholesterol-lowering drugs based on statins, which is of great clinical significance for reducing the incidence of final cardiovascular events in high-risk populations. Currently, cholesterol-lowering drugs with different mechanisms of action have been launched on the market or are under development. Among them are monoclonal antibodies against PCSK-9, which have attracted much attention due to their favorable safety and efficacy profile (Zhao Shuiping, et al., Guidelines for the Prevention and Treatment of Dyslipidemia in Chinese Adults (2016, revised edition). Chinese Circulation Journal 31 (10): 937-953, 2016). The anti-PCSK-9 monoclonal antibody is able to inhibit PCSK-9, block the binding of plasma PCSK-9 to low-density lipoprotein receptors (LDL receptors; LDLRs), further prevent the endocytosis and degradation of LDLRs, increase the expression level and number of LDL receptors on the cell surface, increase the reuptake of LDL-C by LDLRs, and have a direct effect on lowering blood lipid levels.

The anti-PCSK-9 monoclonal antibody has significant therapeutic effect and is also safe and convenient to administer (it is administered by subcutaneous injection). However, the anti-PCSK-9 monoclonal antibody must be administered at intervals of 2 or 4 weeks in a single treatment cycle. There is an unmet clinical need to improve patient compliance.

There also remains a need for an improved method for the treatment or prevention of diseases associated with high cholesterol that would be characterized by a longer interval between administrations, which would be more convenient for patients and would increase their compliance with therapy; and/or a method that causes fewer side effects or is safer; and/or a method that would have a more convenient route of administration.

ESSENCE OF THE INVENTION

I. Method of prevention or treatment

In one aspect, the present invention relates to a method for lowering cholesterol levels in a subject, comprising administering to the subject an anti-PCSK9 antibody or antigen-binding fragment thereof at a dose of from about 15 mg to 3500 mg.

In another aspect, the present invention relates to a method for preventing or treating a disease associated with elevated LDL cholesterol in a subject, comprising administering to the subject an anti-PCSK9 antibody or antigen-binding fragment thereof at a dose of from about 15 mg to 3500 mg.

In one aspect, the present invention relates to a method for preventing or treating a disease associated with elevated cholesterol levels, such as hypercholesterolemia and/or hyperlipidemia, comprising administering to a subject an anti-PCSK9 antibody or antigen-binding fragment thereof at a dose of from about 15 mg to 3500 mg.

In some embodiments of the methods mentioned above, the anti-PCSK9 antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL, wherein VH comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence presented in SEQ ID NO: 10, HCDR2 comprises or consists of the amino acid sequence presented in SEQ ID NO: 17, and HCDR3 comprises or consists of the amino acid sequence presented in SEQ ID NO: 19; VL comprises a combination of LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence presented in SEQ ID NO: 4, LCDR2 comprises or consists of the amino acid sequence presented in SEQ ID NO: 5, and LCDR3 comprises or consists of the amino acid sequence presented in SEQ ID NO: 6.

In some embodiments of the methods mentioned above, 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 presented in SEQ ID NO: 30; the light chain variable region VL comprises or consists of the amino acid sequence presented in SEQ ID NO: 24.

In some embodiments of the methods mentioned above, 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; the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35.

In some embodiments of the methods mentioned above, the anti-PCSK9 antibody is the antibody ADI-10087 disclosed in PCT patent application PCT/CN2017/118050.

In some embodiments of the methods mentioned above, the cholesterol is LDL cholesterol.

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

In some embodiments, an 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.

In some embodiments of the methods mentioned above, the anti-PCSK9 antibody or antigen-binding fragment thereof is administered parenterally.

In some embodiments of the methods mentioned above, the anti-PCSK9 antibody or antigen-binding fragment thereof is administered subcutaneously.

In some embodiments of the methods mentioned above, the anti-PCSK9 antibody or antigen-binding fragment thereof is administered intravenously.

In some embodiments of the methods mentioned above, the LDL cholesterol level of the subject remains reduced by >30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, or >90% at 4 weeks post-administration, 5 weeks post-administration, 6 weeks post-administration, 7 weeks post-administration, 8 weeks post-administration, 9 weeks post-administration, 10 weeks post-administration, 3 months post-administration, 4 months post-administration, or 5 months post-administration compared to the LDL cholesterol level of the subject prior to administration.

In some embodiments of the methods mentioned above, the level of free PCSK-9 in the serum of the subject remains reduced by >30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, or >90% at 4 weeks post-administration, 5 weeks post-administration, 6 weeks post-administration, 7 weeks post-administration, 8 weeks post-administration, 9 weeks post-administration, 10 weeks post-administration, 3 months post-administration, 4 months post-administration, or 5 months post-administration compared to the level of free PCSK-9 in the serum of the subject prior to administration.

In some embodiments of the methods mentioned above, the total cholesterol level in the subject remains reduced by >30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, or >90% at 4 weeks post-administration, 5 weeks post-administration, 6 weeks post-administration, 7 weeks post-administration, 8 weeks post-administration, 9 weeks post-administration, 10 weeks post-administration, 3 months post-administration, 4 months post-administration, or 5 months post-administration compared to the total cholesterol level in the subject prior to administration.

In some embodiments of the methods mentioned above, the level of apolipoprotein B in the subject remains reduced by >30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, or >90% at 4 weeks post-administration, 5 weeks post-administration, 6 weeks post-administration, 7 weeks post-administration, 8 weeks post-administration, 9 weeks post-administration, 10 weeks post-administration, 3 months post-administration, 4 months post-administration, or 5 months post-administration compared to the level of apolipoprotein B in the subject prior to administration.

In some embodiments of the methods mentioned above, the level of non-HDL cholesterol in the subject remains reduced by >30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, or >90% at 4 weeks post-administration, 5 weeks post-administration, 6 weeks post-administration, 7 weeks post-administration, 8 weeks post-administration, 9 weeks post-administration, 10 weeks post-administration, 3 months post-administration, 4 months post-administration, or 5 months post-administration compared to the level of non-HDL cholesterol in the subject before administration.

In some embodiments of the methods mentioned above, the lipoprotein (a) level in the subject remains reduced by >20%, >30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, or >90% at 4 weeks post-administration, 5 weeks post-administration, 6 weeks post-administration, 7 weeks post-administration, 8 weeks post-administration, 9 weeks post-administration, 10 weeks post-administration, 3 months post-administration, 4 months post-administration, or 5 months post-administration compared to the lipoprotein (a) level in the subject prior to administration.

In some embodiments of the methods mentioned above, the apolipoprotein B/apolipoprotein A1 ratio in the subject remains reduced by >20%, >30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, or >90% at 4 weeks post-administration, 5 weeks post-administration, 6 weeks post-administration, 7 weeks post-administration, 8 weeks post-administration, 9 weeks post-administration, 10 weeks post-administration, 3 months post-administration, 4 months post-administration, or 5 months post-administration compared to the apolipoprotein B/apolipoprotein A1 ratio in the subject prior to administration.

In some embodiments of the methods mentioned above, the cholesterol or LDL cholesterol is serum LDL cholesterol.

In some embodiments of the methods mentioned above, the anti-PCSK9 antibody or antigen-binding fragment thereof is administered once over a period equal to or greater than four weeks (p/4 weeks), such as once every five weeks (p/5 weeks), once every six weeks (p/6 weeks), once every seven weeks (p/7 weeks), once every eight weeks (p/8 weeks), once every nine weeks (p/9 weeks), once every ten weeks (p/10 weeks), once every eleven weeks (p/11 weeks), once every twelve weeks (p/12 weeks), once every 4 months, once every 5 months, once every 6 months, once every 7 months, or once a year.

In some embodiments of the methods mentioned above, the subject does not develop severe adverse events following administration, in particular severe adverse events associated with the anti-PCSK9 antibody or antigen-binding fragment thereof.

In some embodiments of the methods mentioned above, the incidence of adverse events in a subject is comparable to the incidence of such events in a subject who received a placebo.

In some embodiments of the methods mentioned above, the subject is a human.

In some embodiments of the methods mentioned above, the disease associated with elevated cholesterol levels is selected from homozygous familial hypercholesterolemia, heterozygous familial hypercholesterolemia, and non-familial hypercholesterolemia.

In some embodiments of the methods mentioned above, the method comprises subcutaneously or intravenously administering to a subject the antibody ADI-10087 once every four weeks or every six weeks 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), and wherein the subject's LDL-cholesterol level four or six weeks after administration remains reduced by >50% compared to the subject's LDL-cholesterol level before administration.

In some embodiments of the methods mentioned above, the method comprises subcutaneously or intravenously administering to a subject the antibody ADI-10087 once every four weeks or every six weeks 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), and wherein the subject's LDL cholesterol level four or six weeks after administration remains reduced by >50% compared to the subject's LDL cholesterol level before administration.

In some embodiments of the methods mentioned above, the method comprises subcutaneously or intravenously administering to a subject the antibody ADI-10087 once every six weeks or every eight weeks 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), and wherein the subject's LDL cholesterol level six or eight weeks after administration remains reduced by >50% compared to the subject's LDL cholesterol level before administration.

In some embodiments of the methods mentioned above, the method comprises subcutaneously or intravenously administering to a subject the antibody ADI-10087 once every four weeks or every six weeks 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), and wherein the subject's LDL-cholesterol level four or six weeks after administration remains reduced by >50%, and the subject's serum free PCSK-9 level four or six weeks after administration remains reduced by >60%, compared to the corresponding levels in the subject before administration.

In some embodiments of the methods mentioned above, the method comprises subcutaneously or intravenously administering to a subject the antibody ADI-10087 once every four weeks or every six weeks 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), and wherein the subject's LDL-cholesterol level four or six weeks after administration remains reduced by >50%, and the subject's serum free PCSK-9 level four or six weeks after administration remains reduced by >80%, compared to the corresponding levels in the subject before administration.

In some embodiments of the methods mentioned above, the method comprises subcutaneously or intravenously administering to a subject the antibody ADI-10087 once every six weeks or every eight weeks 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), and wherein the subject's LDL cholesterol level six or eight weeks after administration remains reduced by >50%, and the subject's serum free PCSK-9 level eight weeks after administration remains reduced by >50%, compared to the corresponding levels in the subject before administration.

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

In some specific embodiments of the methods mentioned above, the anti-PCSK9 antibody or antigen-binding fragment thereof is administered in a liquid antibody preparation, wherein the liquid antibody preparation comprises:

(i) from about 100 mg/mL to about 200 mg/mL of an anti-PC8K-9 antibody or antigen-binding fragment thereof;

(ii) from about 0.2 mg/ml to 10 mg/ml histidine;

(iii) about 1% to 6% sorbitol; And

(iv) about 0.05 mg/ml to 1 mg/ml polysorbate-80 or polysorbate-20;

wherein the pH of the liquid preparation is from about 5.0 to 6.0.

In some specific embodiments of the methods mentioned above, the anti-PCSK9 antibody or antigen-binding fragment thereof is administered in a liquid antibody preparation, wherein the liquid antibody preparation comprises:

(i) from about 100 mg/mL to about 200 mg/mL of an anti-PC8K-9 antibody or antigen-binding fragment thereof;

(ii) from about 0.2 mg/ml to 10 mg/ml histidine;

(iii) about 1% to 6% sorbitol and about 50 mmol/L to 180 mmol/L arginine or arginine salt; And

(iv) about 0.05 mg/ml to 1 mg/ml polysorbate-80 or polysorbate-20;

wherein the pH of the liquid preparation is from about 5.0 to 6.0.

In some specific embodiments of the methods mentioned above, the anti-PCSK9 antibody or antigen-binding fragment thereof is administered in a liquid antibody preparation, and wherein the liquid antibody preparation comprises water as a solvent and the following components:

Anti-PCSK9 antibody or its antigen-binding fragment 150 mg/ml Histidine 1.5 g/l Arginine 90 mmol/l Sorbitol 3% (w/v) Polysorbate-80 0.3 g/l

and the pH of the liquid preparation is about 5.5.

In some specific embodiments of the invention, the liquid antibody preparation comprises water as a solvent and the following components:

ADI-10087 150 mg/ml Histidine 1.5 g/l Arginine 90 mmol/l Sorbitol 3% (w/v) Polysorbate-80 0.3 g/l

and the pH of the liquid preparation is about 5.5.

It should be noted that the antibody or antigen-binding fragments thereof, sequence, formulation, biological activity, etc. described in the present invention have been disclosed in another PCT patent application PCT/CN2017/118050 (Publication No. WO2018/113781) filed by the same applicant, and the contents of this application are incorporated herein by reference in their entirety. For ease of reference and reference, the sequences disclosed in this document (including the sequence listing) and their numbering are directly cited from the patent application PCT/CN2017/118050 without changing the numbering.

II. The antibody preparation disclosed herein

The methods of the present invention may utilize the antibody preparations described in application 201810450088.5, which is incorporated herein by reference in its entirety. The anti-PCSK9 antibody preparation used in the present invention (hereinafter referred to as the "antibody preparation disclosed in this document") is also described in detail below.

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

In one aspect, the present invention may employ the following liquid antibody preparation (hereinafter referred to as the "liquid antibody preparation disclosed herein"), which comprises:

(i) an anti-PCSK9 antibody or fragment thereof (e.g., an antigen-binding fragment) as defined herein;

(ii) buffer;

(iii) a viscosity inhibitor; and

(iv) surfactant.

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

In some embodiments of the methods mentioned above, the anti-PCSK9 antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL, wherein VH comprises a combination of HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence presented in SEQ ID NO: 10, HCDR2 comprises or consists of the amino acid sequence presented in SEQ ID NO: 17, and HCDR3 comprises or consists of the amino acid sequence presented in SEQ ID NO: 19; VL comprises a combination of LCDR1, LCDR2 and LCDR3, which comprise or consist of the amino acid sequences presented in SEQ ID NOs: 4, 5 and 6, respectively.

In some embodiments of the methods mentioned above, 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; the light chain variable region VL comprises or consists of the amino acid sequence of SEQ ID NO: 24.

In some embodiments of the methods mentioned above, 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; the light chain comprises or consists of the amino acid sequence of SEQ ID NO: 35.

In some embodiments of the methods mentioned above, the anti-PCSK9 antibody is the ADI-10087 antibody disclosed in Chinese Patent Application PCT/CN2017/118050.

In one embodiment, the concentration of the anti-PCSK9 antibody or antigen-binding fragment thereof in the liquid antibody formulation disclosed herein is from 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 disclosed herein is from 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 disclosed herein 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 disclosed herein 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 disclosed herein 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 disclosed herein 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 disclosed herein is about 200 mg/mL.

In one embodiment, the anti-PCSK9 antibody is any antibody that binds to a PCSK9 protein (such as human PCSK9), such as a polyclonal antibody, a monoclonal antibody, or a combination of both. In one preferred 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 as defined herein.

In one embodiment, the buffer concentration in the liquid antibody formulation disclosed herein is from about 0.01 mg/mL to 50 mg/mL. In one embodiment, the buffer concentration in the liquid antibody formulation disclosed herein is from about 0.1 mg/mL to 50 mg/mL. In one embodiment, the buffer concentration in the liquid antibody formulation disclosed herein is from about 0.2 mg/mL to 10 mg/mL. In one embodiment, the buffer concentration in the liquid antibody formulation disclosed herein is from about 0.5 mg/mL to 2.5 mg/mL. In one embodiment, the buffer concentration in the liquid antibody formulation disclosed herein is from about 1.0 mg/mL to 2.0 mg/mL. In one embodiment of the invention, the concentration of the buffer in the liquid antibody formulation disclosed herein is about 1.5 mg/mL.

In one embodiment of the invention, the buffer is selected from histidine, glutamate, phosphate, acetate, citrate, and tris(hydroxymethyl)aminomethane. In one embodiment of the invention, the buffer is histidine.

In one embodiment, the concentration of the viscosity inhibitor in the liquid antibody formulation disclosed herein is from about 10 mmol/L to 1000 mmol/L. In one embodiment, the concentration of the viscosity inhibitor in the liquid antibody formulation disclosed herein is from about 20 mmol/L to 500 mmol/L. In one embodiment, the concentration of the viscosity inhibitor in the liquid antibody formulation disclosed herein is from about 50 mmol/L to 300 mmol/L. In one embodiment, the concentration of the viscosity inhibitor in the liquid antibody formulation disclosed herein is from about 50 mmol/L to 200 mmol/L.

In one embodiment, the viscosity inhibitor is selected from sugar alcohols (such as 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 inhibitor is sorbitol. In one embodiment, the viscosity inhibitor is sorbitol at a concentration of about 1% to 6% (w/v). In one embodiment, the viscosity inhibitor is sorbitol at a concentration of about 1% to 6% (w/v), and the liquid preparation does not contain other viscosity inhibitors. In one embodiment, the viscosity inhibitor is a combination of sorbitol and arginine or an arginine salt (preferably arginine hydrochloride). In one embodiment of the invention, the viscosity inhibitor is a combination of sorbitol and arginine or an arginine salt (preferably arginine hydrochloride), wherein the concentration of sorbitol is from about 1% to 6% (w/v), preferably from 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 from about 50 mmol/L to 180 mmol/L, preferably from about 70 mmol/L to 150 mmol/L and, more preferably, about 90 mmol/L. In one embodiment of the invention, the viscosity inhibitor is a combination of sorbitol and arginine or an arginine salt (preferably arginine hydrochloride), wherein the concentration of sorbitol is from about 1% to 6% (w/v), preferably from about 2% to 4% (w/v) and, more preferably, about 3% (w/v), and the concentration of arginine or an arginine salt is from about 50 mmol/l to 180 mmol/l, preferably from about 70 mmol/l to 150 mmol/l and, more preferably, about 90 mmol/l, and wherein the liquid preparation does not contain other viscosity inhibitors.

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

In one embodiment of the invention, the surfactant is a nonionic surfactant. In one embodiment of the invention, the surfactant is, for example, Pluronic, Polysorbate-80, Polysorbate-60, Polysorbate-40, or Polysorbate-20.

In one embodiment of the invention, the liquid preparation disclosed in the present invention comprises a solvent, wherein the solvent is preferably selected from water for injection, organic solvents for injection (including, but not limited to, oil for injection, ethanol, propylene glycol, and the like) and combinations thereof.

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

In one embodiment, the viscosity of the liquid preparation at 25°C is from about 1.0 to 20 centipoise. In one embodiment, the viscosity of the liquid preparation at 25°C is from about 1.0 to 10 centipoise. In one embodiment, the viscosity of the liquid preparation at 25°C is about 1.0 centipoise, 2.0 centipoise, 3.0 centipoise, 4.0 centipoise, 5.0 centipoise, 6.0 centipoise, 7.0 centipoise, 8.0 centipoise, 9.0 centipoise, or 10.0 centipoise. In one embodiment, the viscosity of the liquid preparation at 25°C is from about 5.0 to 7.0 centipoise. In one embodiment, the viscosity of the liquid preparation at 25°C is about 5.2 centipoise. In one embodiment of the invention, the viscosity of the liquid preparation at 25°C is about 6.0 centipoise.

In one embodiment of the invention, the liquid preparation is a pharmaceutical preparation, preferably an injectable form, and more preferably a subcutaneous injection form.

In one preferred embodiment of the invention, the liquid antibody preparation disclosed herein comprises:

(i) from about 100 mg/mL to about 200 mg/mL of an anti-PC8K-9 antibody or antigen-binding fragment thereof;

(ii) from about 0.2 mg/ml to 10 mg/ml histidine;

(iii) from about 1% to 6% (w/v) sorbitol; and

(iv) about 0.05 mg/ml to 1 mg/ml polysorbate-80 or polysorbate-20;

wherein the pH of the liquid preparation is from about 5.0 to 6.0.

In one more preferred embodiment of the invention, the liquid antibody preparation disclosed herein also comprises water for injection.

In one preferred embodiment of the invention, the liquid antibody preparation disclosed herein comprises:

(i) from about 100 mg/mL to about 200 mg/mL of an anti-PC8K-9 antibody or antigen-binding fragment thereof;

(ii) from about 0.2 mg/ml to 10 mg/ml histidine;

(iii) about 1% to 6% sorbitol and about 50 mmol/L to 180 mmol/L arginine or arginine salt; And

(iv) about 0.05 mg/ml to 1 mg/ml polysorbate-80 or polysorbate-20;

wherein the pH of the liquid preparation is from about 5.0 to 6.0.

In one more preferred embodiment of the invention, the liquid antibody preparation disclosed herein also comprises water for injection.

In some specific embodiments of the invention, the liquid antibody preparation comprises water as a solvent and the following components:

Anti-PCSK9 antibody or its antigen-binding fragment 150 mg/ml Histidine 1.5 g/l Arginine 90 mmol/l Sorbitol 3% (w/v) Polysorbate-80 0.3 g/l

and the pH of the liquid preparation is about 5.5.

In some specific embodiments of the invention, the liquid antibody preparation comprises water as a solvent and the following components:

ADI-10087 150 mg/ml Histidine 1.5 g/l Arginine 90 mmol/l Sorbitol 3% (w/v) Polysorbate-80 0.3 g/l

and the pH of the liquid preparation is about 5.5.

In one more preferred embodiment of the invention, the liquid antibody preparation disclosed herein also comprises water for injection.

In one embodiment, the purity of the anti-PCSK9 antibody or antigen-binding fragment thereof in a liquid formulation comprising the anti-PCSK9 antibody disclosed herein decreases by no more than 10%, such as no more than 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%, as determined by SEC-HPLC and CE-SDS under non-reducing conditions, after storage at a temperature of from about -80°C to about 45°C, such as at -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.

In one embodiment, the charge heterogeneity of the anti-PCSK9 antibody or antigen-binding fragment thereof in a liquid formulation comprising the anti-PCSK9 antibody disclosed herein changes by no more than 10%, such as no more than 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%, as determined by KOC-HPLC, after storage at a temperature of from about -80°C to about 45°C, such as at -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.

The preparation disclosed in this document contains a high concentration of antibody, has a low viscosity suitable for administration (particularly for subcutaneous administration), is characterized by high physical and chemical stability, as well as a low tendency to aggregation and sedimentation, and exhibits little change in charge heterogeneity during storage. These characteristics are very useful for the production of complete, effective and highly stable drugs for clinical use.

The inventors have unexpectedly found that sorbitol can be used as a viscosity inhibitor of a liquid preparation containing an anti-PCSK9 antibody, which allows obtaining a liquid preparation containing an anti-PCSK9 antibody or an antigen-binding fragment thereof with a low viscosity and a high concentration without adding other viscosity inhibitors; wherein the liquid preparation is characterized by high stability. In addition, the inventors have unexpectedly found that a combination of sorbitol and arginine as a viscosity inhibitor is able to further reduce the viscosity of a liquid preparation containing an anti-PCSK9 antibody and provide a liquid preparation with a high concentration containing an anti-PCSK9 antibody or an antigen-binding fragment thereof, which allows providing a viscosity suitable for subcutaneous injection; and wherein the liquid preparation containing an anti-PCSK9 antibody has higher stability.

In another aspect, the subject of the present invention is a solid preparation obtained by lyophilizing the liquid preparation mentioned above. Before use, the solid preparation can be transferred to a suitable solvent to obtain the liquid preparation disclosed in this document.

III. Anti-PCSK9 antibody or its preparation for the treatment or prevention of diseases

The present invention relates to an anti-PCSK9 antibody or antigen-binding fragment thereof, or a liquid preparation of the antibody described above, for use in the above-described methods of the present invention.

It should be understood that in the above embodiments of the invention relating to liquid preparations, the administered dose refers to the amount of anti-PCSK9 antibody in the preparation.

IV. Pharmaceutical dosage unit for single use, pharmaceutical kit or their pharmaceutical use

In another aspect, the present invention relates to a single-use pharmaceutical dosage unit that comprises an anti-PCSK9 antibody or antigen-binding fragment thereof in a dose of from about 15 mg to 3500 mg; preferably, the anti-PCSK9 antibody or antigen-binding fragment thereof in a dose of from about 25 mg to about 3000 mg, from about 50 mg to about 2500 mg, from about 75 mg to about 2000 mg, from about 100 mg to about 2000 mg, from about 200 mg to about 2000 mg, from about 250 mg to about 1500 mg, from about 300 mg to about 1000 mg, about 450 mg to about 1000 mg, from about 600 mg to about 1000 mg, from about 350 mg to about 900 mg, from about 400 mg to about 800 mg, from about 450 mg to about 700 mg, from about 300 mg to about 600 mg, or from about 450 mg to about 600 mg.

In some embodiments of the invention, an anti-PCSK9 antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL, wherein

(a) VH contains:

(i) a combination of HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 10; HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17; HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19; or

(II) three complementarity determining regions (CDRs) contained in the VH shown in SEQ ID NO: 30, and

(b) VL contains:

(i) a combination of LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4; LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5; LCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 6; or

(ii) three complementarity determining regions (CDRs) contained within the VL shown in SEQ ID NO: 24.

In some embodiments of the invention, an anti-PCSK9 antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL, wherein

(a) the heavy chain variable region VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 30; and

(b) the light chain variable region VL comprises or consists of the amino acid sequence presented in SEQ ID NO: 24.

In some embodiments, an anti-PCSK9 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein

(a) the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 41; and

(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 the ADI-10087 antibody.

In another aspect, the present invention relates to a pharmaceutical kit comprising an anti-PCSK9 antibody or antigen-binding fragment thereof, according to any of the above-described embodiments of a single-use pharmaceutical dosage unit.

In another aspect, the present invention relates to the use of a single-use pharmaceutical dosage unit as described above, or a kit of components as described above, for the preparation of a medicament for lowering cholesterol levels in a subject.

In another aspect, the present invention relates to the use of a single-use pharmaceutical dosage unit as described above, or a kit of components as described above, for the prevention or treatment of a disease associated with elevated LDL cholesterol in a subject.

In another aspect, the present invention relates to the use of a single-use pharmaceutical dosage unit as described above, or a kit of components as described above, for the prevention or treatment of a disease associated with elevated cholesterol levels, such as hypercholesterolemia and/or hyperlipidemia.

It should be understood that technical solutions based on a combination of the technical features described above in Section I and Section III, and the technical solutions described above in Section IV, are also included in this application.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

FIG. 1 shows the mean free concentration-time curves for the ADI-10087 antibody following administration of ADI-10087 to subjects in each dose group; the upper graph is presented in linear coordinates; the lower graph is presented in semi-logarithmic coordinates; error bars represent the mean ± standard deviation; one curve per dose group.

FIG. 2 shows the mean total concentration-time curves for the ADI-10087 antibody following administration of ADI-10087 to subjects in each dose group; the upper graph is presented in linear coordinates, the lower graph is presented in semi-log coordinates; error bars represent the mean ± standard deviation; one curve per dose group.

FIG. 3 shows the dependence of C _max for the total concentration of ADI-10087 and the free concentration of ADI-10087 after subcutaneous injection of ADI-10087 into the abdomen at a dose of 25-600 mg; (scatter plot) the inflection line in the graph corresponds to the median value in each dose group; each point in the scatter plot corresponds to an individual value; the solid line corresponds to the corresponding parameter for free ADI-10087, and the dotted line - for total ADI-10087.

FIG. 4 shows the AUC _0-last dependence for total ADI-10087 and free ADI-10087 following subcutaneous injection of ADI-10087 into the abdomen at a dose of 25-600 mg; (scatterplot) the inflection line in the graph corresponds to the median value in each dose group; each point in the scatterplot corresponds to an individual value; the solid line corresponds to the corresponding parameter for free ADI-10087, and the dotted line corresponds to the corresponding parameter for total ADI-10087.

FIG. 5 shows the AUC _0-∞ relationship for total ADI-10087 and free ADI-10087 following subcutaneous injection into the abdomen of ADI-10087 at doses of 25-600 mg; (scatterplot) the inflection line in the graph corresponds to the median value in each dose group; each point in the scatterplot corresponds to an individual value; the solid line corresponds to the corresponding parameter for free ADI-10087, and the dotted line corresponds to the corresponding parameter for total ADI-10087.

FIG. 6 shows the rate of change (%) of free PCSK-9 concentration in serum from baseline after a single administration.

FIG. 7 shows linear least squares plots of changes in LDL-C from baseline after a single dose.

FIG. 8 shows the mean rate of change in LDL-C concentration from baseline after a single administration.

FIG. 9 shows the mean concentration-time curves for LDL-C after a single administration.

FIG. 10 shows the rate of change in total cholesterol concentration relative to baseline after a single administration.

FIG. 11 shows the rate of change in ApoB concentration relative to baseline after a single administration.

FIG. 12 shows the rate of change in non-HDL-C concentration from baseline after a single administration.

FIG. 13 shows the rate of change in lipoprotein (a) (Lp(a)) concentration from baseline after a single administration.

FIG. 14 shows the rate of change (%) of free PCSK-9 concentration in serum from baseline after multiple administrations.

FIG. 15 shows the rate of change in LDL-C levels from baseline after multiple administrations.

FIG. 16 shows the changes in LDL-C levels from baseline after multiple administrations.

FIG. 17 shows the rate of change in lipoprotein(a) (Lp(a)) levels from baseline after multiple administrations.

FIG. 18 shows the rate of change of ApoB levels relative to baseline after multiple administrations.

FIG. 19 shows the rate of change in non-HDL-C levels from baseline after multiple administrations.

FIG. 20 shows the rate of change in total cholesterol (TC) levels relative to baseline after multiple administrations.

FIG. 21 shows the rate of change of ApoB/ApoA1 levels from baseline after multiple administrations.

DETAILED DESCRIPTION

Definitions

The terms used in the description of the present invention have the following definitions. If there is no definition in this document, it is preferable to rely on the definitions provided in the patent application PCT/CN 2017/118050. If there are no definitions in both patent applications, the terms used in the description of the present invention have the generally accepted meaning understood by those skilled in the art.

For the purpose of explaining this description, the following definitions will be used, and, depending on the context, the singular terms used may include the plural and vice versa. It should be understood that the terms used herein are intended to describe only specific embodiments of the invention and are not intended to limit its scope.

As used herein, the term "and/or" refers to any one of the options or any two or more of the options.

As used herein, the terms "comprise" or "include" are to be understood as including the described elements, integers, or steps, but not to the exclusion of any other elements, integers, or steps. As used herein, unless otherwise indicated, the terms "comprise" or "include" also include the situation where the whole consists of the described elements, integers, or steps. For example, if the variable region of an antibody is said to "comprise" a certain sequence, the variable region of the antibody consisting of the certain sequence is also intended to include.

"Antibodies or antigen-binding fragments thereof" as used herein include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, isoconjugate, multispecific, recombinant, heterogeneous, heterogeneous hybrid, chimeric, humanized (particularly CDR-grafted), deimmunized or human antibodies, Fab fragments, Fab' fragments, F(ab') ₂ fragments, Fab, Fd, Fv expression library fragments, disulfide-linked Fv (dsFv), single-chain antibodies (e.g., scFv), diabodies and tetrabodies (Holliger P. et al. (1993) Proc. Natl. Acad. Sci. USA 90 (14), 6444-6448), nanobodies (also referred to as single-domain antibodies), anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies against an antibody disclosed herein), and an epitope-binding fragment of any of the foregoing.

The term "individual" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, goats, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human, including a child, adolescent, or adult.

The term "combination therapy" means that two or more therapeutic agents are administered to treat diseases associated with elevated cholesterol levels as described herein. Such administration includes co-administration of these therapeutic agents substantially simultaneously, such as in a single capsule with a fixed ratio of the active ingredients. Alternatively, such administration includes co-administration of each active ingredient in different or separate containers (e.g., tablets, capsules, powder and liquid). The powder and/or liquid may be reconstituted or diluted to the desired dose prior to administration. In addition, such administration also includes the use of therapeutic agents of each type sequentially at approximately the same time or at different time points. In either case, the therapeutic regimen will provide the beneficial effect of the combination of drugs in treating the disorders or symptoms described herein.

As used herein, the term "treatment" (or "treat" or "providing treatment") refers to slowing, interrupting, halting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

As used herein, the term "prophylaxis" (or "prevent" or "provide prophylaxis") refers to slowing the onset or progression of symptoms of a disease or disorder, or a specific disease or disorder. In some embodiments, candidates for prophylactic therapy are subjects with a relevant family history. In general, the term "prophylaxis" (or "prevent" or "provide prophylaxis") refers to the administration of a drug before the onset of a disease or symptoms, particularly in subjects at risk.

The term "high cholesterol disease" includes one or more of the following diseases: hypercholesterolemia, hyperlipidemia, heart disease, metabolic syndrome, diabetes mellitus, coronary artery disease, stroke, cardiovascular disease, Alzheimer's disease, and global dyslipidemia (e.g., elevated serum total cholesterol, elevated LDL, elevated triglycerides, elevated VLDL, and/or decreased HDL). Non-limiting examples of primary or secondary dyslipidemias that may be treated with an anti-PCSK9 antibody (alone or in combination with one or more other medicinal products) include metabolic syndrome, diabetes mellitus, hereditary combined hyperlipidemia, hereditary hypertriglyceridemia, hereditary hypercholesterolemias including heterozygous hypercholesterolemia, homozygous hypercholesterolemia, and hereditary apolipoprotein B100 deficiency; polygenic hypercholesterolemia; homozygous familial hypercholesterolemia, heterozygous familial hypercholesterolemia, and non-familial hypercholesterolemia; hereditary dysbetalipoproteinemia; hepatic lipase deficiency; Dyslipidemia due to any of the following: poor diet, hypothyroidism, drugs (including estrogen and progesterone therapy, beta-blockers and thiazide diuretics); nephrotic syndrome, chronic renal failure, Cushing's syndrome, primary biliary cirrhosis, glycogen storage diseases, hepatoma, cholestasis, acromegaly, insulinoma, isolated growth hormone deficiency and alcohol-induced hypertriglyceridemia.

The term "hypercholesterolemia" as used herein refers to a disorder in which the concentration of cholesterol increases above a certain level. In some embodiments, the LDL cholesterol level increases above a certain level. In some embodiments, the serum LDL cholesterol level increases above a certain level.

As used herein, the term "vector" refers to a nucleic acid molecule capable of causing the proliferation of another nucleic acid to which it is linked. This term includes vectors that serve as self-replicating nucleic acid structures, as well as vectors that bind to the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors."

The term "effective amount" refers to the amount or dose of the drug, antibody or fragment described herein that produces the desired effect in a patient receiving the therapy after one or more doses have been administered. An effective amount can be readily determined by the attending physician, as one skilled in the art, by considering a variety of factors including: the species, such as mammal; the size, age and general health; the specific disease present; the extent or severity of the disease; the response of the individual patient; the specific antibody administered; the route of administration; the bioavailability parameters of the drug administered; the selected mode of administration; and the use of concomitant therapy.

The term "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic result at the desired dosage for the desired period of time. A therapeutically effective amount of a drug, antibody, or antibody fragment described herein, or a conjugate or composition thereof, may vary depending on a variety of factors, such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to produce the desired response in the individual. A therapeutically effective amount also means an amount in which any toxic or undesirable effect of the drug, antibody, or antibody fragment, or a conjugate or composition thereof, is outweighed by the therapeutically beneficial effect.

The term "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result at the required dosage for the required period of time. In general, since the prophylactic dose is administered to a subject prior to the onset of a disease or at an early stage thereof, the prophylactically effective amount will be less than the therapeutically effective amount.

The term "single-use pharmaceutical dosage unit" refers to a single-use pharmaceutical dosage form, including injectable solutions, tablets, and lyophilized powders, that is administered to a patient during a dosing regimen.

The term "preparation" as used herein refers to a composition that is suitable for administration to animals, preferably mammals (including humans), and comprises at least one active ingredient and at least one inactive ingredient. The term "liquid preparation" refers to a preparation in the form of a liquid. The liquid preparation disclosed herein comprises (i) an anti-PCSK-9 antibody or fragment thereof (preferably an antigen-binding fragment); (ii) a buffer; (iii) a viscosity inhibitor; (iv) a surfactant; and (v) a solvent. The composition of the preparation disclosed herein may correspond to the embodiments of the liquid preparations mentioned above. The liquid preparation disclosed herein is preferably an injectable form, and more preferably a subcutaneous injection form.

The term "buffer" as used in this document refers to a buffer for maintaining pH. Preferably, the buffer is capable of maintaining the pH of the liquid preparation disclosed in this document in the range of 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 preparation 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.

The term "viscosity inhibitor" as used herein refers to a substance capable of reducing the viscosity of a liquid preparation comprising an anti-PCSK9 antibody. The concentration of the viscosity inhibitor in the liquid preparation is, for example, from about 10 mmol/L to 1000 mmol/L, from about 20 mmol/L to 500 mmol/L, from about 50 mmol/L to 300 mmol/L, or from about 50 mmol/L to 200 mmol/L. Preferably, the viscosity inhibitor is selected from a sugar alcohol, arginine, arginine hydrochloride, sodium thiocyanate, ammonium thiocyanate, ammonium sulfate, ammonium chloride, calcium chloride, zinc chloride, sodium acetate, and combinations thereof. More preferably, the viscosity inhibitor is sorbitol or a combination of sorbitol and arginine or an arginine salt (preferably arginine hydrochloride). Preferably, the concentration of sorbitol in the liquid preparation is from about 1% to 6% (w/v), and the concentration of arginine or an arginine salt (preferably arginine hydrochloride) in the liquid preparation is from about 50 mmol/L to 180 mmol/L, preferably from about 70 mmol/L to 150 mmol/L, and more preferably about 90 mmol/L.

The term "surfactant" as used in this document refers to a substance that is capable of significantly changing the state of the interface in a solution system when added in a small amount. The concentration of the surfactant in the liquid preparation is, for example, from about 0.01 mg/mL to 5 mg/mL, from about 0.05 mg/mL to 1 mg/mL, from about 0.1 mg/mL to 0.5 mg/mL, and 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.

The term "solvent" as used herein refers to a liquid used to dissolve or suspend active ingredients and inactive ingredients to produce a liquid preparation. Solvents that can be used in the present invention include, but are not limited to, water for injection, organic solvents for injection (including, but not limited to, oil for injection, ethanol, and propylene glycol), and combinations thereof.

The term "sugar alcohol" as used in this document refers to the corresponding polyol which is obtained by a reduction reaction, such as catalytic hydrogenation, of a monosaccharide. Sugar alcohols include, but are not limited to, sorbitol, mannitol, erythritol, maltitol, lactitol, xylitol, etc.

The term "about" when used in combination with a numerical value is intended to cover numerical values ranging from a lower limit of 10% less than the stated numerical value to an upper limit of 10% more than the stated numerical value.

The term “w/v” used in this document means “mass/volume”, for example “1% w/v” means 1 g/100 ml = 0.01 g/ml = 10 mg/ml.

The terms "introduction provision" and "introduction" are used interchangeably in this document unless otherwise specified.

A pharmaceutical kit may also be referred to as a kit of components in this document.

The present invention will now be illustrated by way of examples, and the following examples should not be construed as limiting the present invention.

Abbreviations:

SEC-HPLC: size exclusion chromatography - high performance liquid chromatography

CE-SDS: capillary electrophoresis with sodium dodecyl sulfate

CZE: capillary zone electrophoresis

s/c: subcutaneous injection

IV: intravenous injection

PK: pharmacokinetics

LDL-C: low-density lipoprotein cholesterol

Example 1. Preparation of a liquid preparation containing anti-PCSK9 antibody (preparation A - study preparation)

A liquid preparation containing anti-PCSK9 antibody at a concentration of 150 mg/ml was prepared according to composition A.

Composition A

Anti-PCSK9 antibody (ADI-10087) 150 mg/ml Histidine 1.5 g/l Arginine 90 mmol/l Sorbitol 3% (w/v) Polysorbate-80 0.3 g/l pH 5.5

Anti-PCSK9 antibody (ADI-10087) described in patent application PCT/CN2017/118050 was added to an ultrafiltration centrifuge tube (molecular weight cutoff: 30 kDa); after concentration by centrifugation, the anti-PCSK9 antibody was diluted with an aqueous solution of Formulation A without polysorbate 80 and anti-PCSK9 antibody (histidine 1.5 g/L, arginine 90 mmol/L, sorbitol 3%, pH 5.5) and concentrated; this procedure was repeated until the solvent was completely replaced. The protein concentration in the solution after solvent replacement was adjusted to 150 mg/mL. Then, an aqueous solution of polysorbate-80 (30 g/L; 1/100 by volume) was added to adjust the final concentration of polysorbate-80 to 0.3 g/L. After aseptic filtration, the semi-finished product was poured into vials, which were then sealed with rubber stoppers and aluminum-plastic caps to obtain the finished product.

Example 2. Preparation of a comparison drug (drug B), which is a liquid drug containing an anti-PCSK9 antibody

Comparator drug (example): a drug containing anti-PCSK9 antibody in liquid at a concentration of 150 mg/ml, prepared according to composition B.

Composition B

Anti-PCSK9 antibody (ADI-10087) 150 mg/ml Histidine 1.5 g/l Arginine 180 mmol/l Polysorbate-80 0.3 g/l pH 5.5

The anti-PCSK9 antibody (ADI-10087) described in patent application PCT/CN2017/118050 was added to an ultrafiltration centrifuge tube (molecular weight cutoff: 30 kDa); after concentration by centrifugation, the anti-PCSK9 antibody was diluted with an aqueous solution of Formulation B without polysorbate 80 and anti-PCSK9 antibody (histidine 1.5 g/L, arginine 180 mmol/L, pH 5.5) and concentrated; this procedure was repeated until the solvent was completely replaced. The protein concentration in the solution after solvent replacement was adjusted to 150 mg/mL. Then, an aqueous solution of polysorbate 80 (30 g/L; 1/100 by volume) was added to adjust the final concentration of polysorbate 80 to 0.3 g/L. After filtration under aseptic conditions, the semi-finished product was poured into bottles, which were then closed with rubber stoppers and aluminum-plastic caps, and the finished product was obtained.

Example 3. Measurement of viscosity of a liquid preparation containing anti-PCSK9 antibody

The viscosity was measured using a cone and plate viscometer (Brookfield, CAP1000+/2000+. For details, please visit http://www.sinoinstrument.com/product_details-4-83-401-60.html) at room temperature from 20°C to 25°C. The rotor and rotation speed (rotor: SR-40, rotation speed: 25 rpm) were selected as follows. The samples were added after fixing the rotor with the coupling nut and adjusting the position. The Run button was pressed to start measuring the viscosity of the samples. According to the above experimental conditions, the viscosity of preparation A was 5.2 centipoise, and that of preparation B was 6.0 centipoise. Unexpectedly, the viscosity of preparation A was significantly lower than that of preparation B, which suggests that the combination of arginine and sorbitol is more preferable for reducing the viscosity of the preparation with a high antibody concentration.

Example 4. Change in protein purity in a liquid preparation containing anti-PCSK9 antibody

In order to study the stability of drug A and drug B, an accelerated stability study was conducted. The stability of drug A and drug B, filled into capped and sealed vials, was studied in an accelerated stability study at 40°C ± 2°C/60% RH ± 5% RH for 2 months. Simultaneously, the change in protein purity was determined by SEC-HPLC and CE-SDS under non-reducing conditions, as shown below.

EC-HPLC:

The sample was diluted to a concentration of 2 mg/mL; a TSKG 3000 SWxl hydrophilic silica gel size exclusion chromatography column was used; the amount of protein injected was 100 μg; the mobile phases were 20 mmol/L _Na2HPO4 • _12H2O ; 150 mmol/L NaCl, and 200 mmol/L arginine; the pH was 6.8; the flow rate was 0.5 _mL /min, the detection wavelength was 280 nm; the column temperature was 25°C; and the area normalization method was used for the calculation.

KE-DSN:

100 μg of sample was collected and added to sample buffer (pH 7.0) to obtain a total volume of 95 μl. To obtain a reduced sample, 5 μl of β-mercaptoethanol was added, and then the unreduced sample was added with 5 μl of NEM at a concentration of 250 mmol/l. After mixing, the mixture was heated at 70°C for 10 min and detection was performed.

Based on the percentage decrease in the area of the main peak of SEC obtained by SEC-HPLC, it was found that the changes in the protein purity in preparation A after 1 month were manifested as a decrease of 2.6%, and after 2 months - by 3.7%; in preparation B after 1 month they were manifested as a decrease of 4.1%, and after 2 months - by 7.0%. Based on the percentage decrease in the intensity of the bands in CE-SDS under non-reducing conditions, it was found that the changes in the protein purity in preparation A after 2 weeks were manifested as a decrease of 0.4%, and after 1 month - by 2.0%; in preparation B after 2 weeks they were manifested as a decrease of 2.5%, and after 1 month - by 7.3%. It can be seen that the rate of purity change in preparation A is noticeably lower than in preparation B. That is, the stability of preparation A is higher than the stability of preparation B.

Example 5. Analysis of charge heterogeneity in a liquid preparation containing anti-PCSK9 antibody

To analyze charge heterogeneity, samples of drug A and drug B from the accelerated stability study (Example 4) were collected at different time points. Uncoated Beckman capillaries and a Beckman PA800 plus capillary electrophoresis apparatus were used; the injection pressure was 0.5 psi, the sampling time was 10.0 s, the separation voltage was 30 kV, and the analysis time was 30 min. The area normalization method was used to calculate the peak area of the acidic components, the peak area of the basic components, and the area of the main peak as a percentage of the total peak area. According to the CZE results for drug A, the main peak decreased by 0% after 2 weeks and by 2.9% after 1 month. According to the CZE results for drug B, the main peak decreased by 2.0% after 2 weeks and by 7.6% after 1 month.

It can be seen that the rate of change of charge heterogeneity in preparation A is significantly lower than in preparation B, that is, the combination of arginine and sorbitol allows to reduce the rate of change of charge heterogeneity in the preparation.

Example 6. Placebo drug (control drug)

A placebo without antibody was prepared according to formulation C using water as the solvent.

Composition C

Histidine 1.5 g/l Arginine 90 mmol/l Sorbitol 3% (w/v) Polysorbate-80 0.3 g/l pH 5.5

Example 7. Determination of pharmacokinetic parameters in blood samples and assay method ADI-10087

(1) Method for Determining PK Parameters of Total ADI-10087: The PK parameters of total ADI-10087 were detected by a quantitative electrochemiluminescence assay for total ADI-10087 in human serum. The wells of an MSD plate were coated with neutralizing antibody to ADI-10087, then acidified serum sample containing neutralized ADI-10087 was added and detected using ruthenium-labeled polyclonal antibody to ADI-10087. The ruthenium-labeled polyclonal antibody to ADI-10087 formed an immune complex with the drug and neutralizing antibody to ADI-10087 coated on the plate. After incubation for the required time, the plate was washed, reading buffer was added, and the plate was placed in an MSD reader; Luminescence signals were generated by an electrocatalytic chemiluminescence reaction. The intensity of the chemiluminescence signals was proportional to the ADI-10087 content in the sample.

(2) Free ADI-10087 PK parameter determination method: According to the principle of ELISA method, the wells of the microplate were coated with neutralizing antibody to ADI-10087 and fixed, then serum samples were added thereto; ADI-10087 in the serum samples specifically bound to the coated antigen, then biotin-labeled neutralizing antibody to ADI-10087 was added for capture, and finally SA-HRP (streptavidin-labeled horseradish peroxidase) was added into the wells for detection. After that, TMB (tetramethylbenzidine) substrate of peroxidase (HRP) enzyme was added, the color intensity of TMB substrate was proportional to the content of free ADI-10087; and the concentration of monoclonal antibody ADI-10087 in clinical serum samples was determined by optical density (OD) values at 450 nm or 620 nm using a microplate reader.

Example 8. Method for detecting the ADI-10087 antibody against PCSK-9

According to the principle of ELISA method, the wells of the microplate were coated with ADI-10087 antibody and fixed, then serum samples were added thereto; PCSK-9 in the serum samples specifically bound to the coated ADI-10087, then goat anti-human PCSK-9 polyclonal antibody was added, and finally goat IgG HRP-conjugate was added for detection. After that, TMB substrate of HRP enzyme was added, the color intensity of TMB substrate was proportional to the content of PCSK-9; and the concentration of PCSK-9 in clinical serum samples was determined by the optical density (OD) values at a wavelength of 450 nm or 620 nm using a microplate reader.

Example 9. Method for detection of antibodies to the drug ADI-10087

Anti-drug antibodies (ADAs), i.e., ADI-10087, were detected in human serum by a semiquantitative electrochemiluminescence assay. A human serum sample was acidolyzed, free AADs were captured with ADI-10087 coated on the wells of a microplate, acidolyzed again, the eluted AADs were mixed with biotin- and ruthenium-labeled ADI-10087 for incubation to form an immune complex, which was fixed on streptavidin-coated wells of an MSD plate. After incubation for the required time, the plate was washed, reading buffer was added, and the plate was placed in an MSD reader; luminescence signals were generated by an electrocatalytic chemiluminescence reaction. The intensity of the chemiluminescence signals was positively correlated with the AAD content of the sample.

Example 10. Method for detecting neutralizing antibodies to ADI-10087

Samples positive for ALP were tested for the presence of neutralizing antibodies (nAb) to ADI-10087 using electrochemiluminescence. A human serum sample was acidolyzed, free nAb was captured with ADI-10087 coated on the wells of a microplate, acidolyzed again, the eluted nAb was mixed with ruthenium-labeled ADI-10087 and incubated to form an immune complex, which was then added to the wells of an MSD plate precoated with biotin-labeled PCSK-9 for incubation with streptavidin. Free ruthenium-labeled drug in the test solution bound to the biotin-labeled PCSK-9 precoated on the plate. After incubation for the required time, the plate was washed, reading buffer was added, and the plate was placed in an MSD reader; Luminescence signals were generated during an electrocatalytic chemiluminescence reaction. The intensity of the chemiluminescence signals was negatively correlated with the nAb content in the sample.

Example 11. Evaluation of safety and tolerability in healthy adult subjects of Chinese descent who received a single dose of ADI-10087

Study design:

The 58 subjects will be divided into 8 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 includes 2 subjects who will receive a single dose of ADI-10087. These 2 subjects, after 14 days of safety assessment, will be enrolled in clinical trials with subsequent dose cohorts. Each subsequent cohort (75 mg SC to 600 mg SC) will include 8 subjects, with the first 2 subjects in the 75 mg SC cohort entering the open-label study to receive ADI-10087 at a dose of 75 mg SC and the last 6 subjects randomly assigned in a 2:1 ratio to receive either ADI-10087 (N=4) or placebo (N=2). In the remaining 6 cohorts, subjects will be randomly assigned in a 3:1 ratio to receive a single dose of either ADI-10087 (N=6) or placebo (N=2).

The 2 subjects in the 25 mg SC cohort may be included in the 75 mg SC cohort after the 14-day post-injection safety evaluation period. All subjects in the 75 mg SC cohort may be included in the 150 mg SC cohort after the 14-day post-injection safety evaluation period. The 150 mg SC cohort may include subjects in the 75 mg IV cohort after inclusion. All subjects in the 150 mg SC cohort may be included in the 300 mg SC cohort after the 14-day post-injection safety evaluation period. All subjects in the 300 mg SC cohort may be included in the 450 mg SC cohort after the 14-day post-injection safety evaluation period. All subjects in the 450 mg SC cohort may be included in the 600 mg SC cohort after a 14-day safety period has elapsed after injection. Subjects in the 450 mg IV cohort may be included in the 600 mg SC cohort after inclusion.

Each subject will be followed until day 85 (12 weeks) to assess the safety, tolerability, PK/PD, and immunogenicity of the drug.

Demographic characteristics of subjects

In the safety analysis set (SS), which includes 5 8 subjects, 44 (75.9%) are male; the mean age is 30.1±5.74 years old with a minimum age of 19 years and a maximum age of 44 years; the mean body weight is 67.20±9.419 kg with a minimum body weight of 50.9 kg and a maximum body weight of 92.4 kg; the mean height is 169.12±7.468 cm with a minimum height of 151.0 cm and a maximum height of 186.0 cm; the mean BMI is 23.44±2.309 kg/ ^m2 with a minimum BMI of 19.3 kg/ ^m2 and a maximum BMI of 27.4 kg/ ^m2 ; 57 patients (98.3%) are of Han ethnicity. Of the 44 subjects in the ADI-10087 group, 43 (97.7%) subjects were of Han ethnicity, their mean age was 30.3±5.71 years old, their mean height was 168.36±6.992 cm, their mean body weight was 66.17±8.289 kg, and their mean BMI was 23.31±2.175 kg/ ^m2 . Of the 14 subjects in the placebo group, 14 (100%) subjects were of Han ethnicity, their mean age was 29.8±6.03 years old, their mean height was 171.50±8.716 cm, their mean body weight was 70.43±12.122 kg, and their mean BMI was 23.82±2.741 kg/ ^m2 . The demographic characteristics of the subjects were in accordance with the inclusion criteria for the study; and the demographic characteristics were similar in both groups.

The drug used in the study

The test drug used in the study was the drug prepared as described in Example 1; the control drug used in the study was a placebo prepared as described in Example 6.

Administration schemes

All subjects in each cohort of 18 were administered ADI-10087 or placebo as a single dose of 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.

Subcutaneous (SC) injection technique: Subjects received subcutaneous injections of study drug or placebo into the abdomen. Investigators administered abdominal injections as a single 1 mL subcutaneous dose via a needle, which corresponds to the actual volume of study drug injected. For injections in subjects in the 25 mg and 75 mg groups, liquid drug was withdrawn using a 1 mL syringe (2 mL needle) and then 1 mL was injected through the needle after air was removed. For injections in subjects in the 150 mg group and in the >150 mg dose groups, liquid drug was withdrawn using a 2 mL syringe (2 mL needle) and then 1 mL was injected through the needle after air was removed.

Technique of intravenous (IV) administration:

(1) 75 mg IV group: 150 mg of the study drug for injection (1 mL) was withdrawn and added to an IV infusion bag containing the marked 100 mL of sterile 0.9% sodium chloride. The volume of the IV solution was 50 mL, and the administration time was 30 minutes.

(2) 450 mg IV group: 900 mg of the study drug for injection (1 mL) was withdrawn and added to an IV infusion bag containing the marked 200 mL of sterile 0.9% sodium chloride. The volume of the IV solution was 100 mL, and the administration time was 1 hour.

Adverse events

All adverse events (AEs) occurring during the study described above were recorded and coded using MedDRA version 21.0 system organ classes (SOCs) and preferred terms (PTs). Adverse events associated with the drug were assigned to one of three categories: “certain relationship,” “possible relationship,” and “not assessable.” If relevant data are not available, the adverse event will be considered related to the drug.

AEs were presented and classified by study groups according to the SOC and PT. The results will be presented in descending order of occurrence frequency both in different SOCs and within a SOC. In the AE summary table, the incidence rate of AEs is calculated based on the number of subjects who experienced AEs, not the number of AEs reported. When calculating the number and percentage of subjects, AEs are only counted once if the same AE occurred multiple times in the same subject.

An AE occurring during the screening period is an AE that occurs before the first administration of the study drug. Adverse events associated with therapy (AEFTs) are defined as AEs that occur or worsen within 12 weeks (including day 85) from the first administration of the study drug to the last administration of the study drug. Stages of AEs that cannot be determined due to insufficient time for their occurrence are categorized as AEFTs.

Table 1 shows the classification and summary of drug-related adverse events (DRAEs) and treatment-emergent adverse events (TEAEs).

In the ADI-10087 group, the incidence of treatment-emergent adverse events was 52.3% and the incidence of treatment-emergent adverse events was 38.6%; in the placebo group, the incidence of treatment-emergent adverse events was 57.1% and the incidence of treatment-emergent adverse events was 50.0%. At least one adverse event of interest (hepatic event, muscle event, allergic reaction, and injection site reaction) was reported in 13.6% and 28.6% of subjects in the ADI-10087 and placebo groups, respectively; there were no serious adverse events, serious adverse events related to the drug, adverse events leading to withdrawal from the study, or adverse events resulting in death. The most common drug-related adverse events in the ADI-10087 group were upper respiratory tract infection (20.5%) and increased creatine phosphokinase (6.8%); the most common drug-related adverse event in the placebo group was upper respiratory tract infection (14.3%).

Adverse drug events in the ADI-10087 group were mild to moderate in severity, did not require specific treatment, and improved with corrective treatment. In particular, no adverse events were noted in the group receiving the drug at a dose of up to 450 mg s.c.

Evaluation of immunogenicity

Immunogenicity was assessed using the immunogenicity assay (IS) set, evaluating parameters such as anti-ADI-10087 antibodies (ALP) and neutralizing antibodies (nAb). The proportion of subjects developing anti-ADI-10087 antibodies and neutralizing antibodies (nAb) was determined for each group, and a list of subjects who were positive for anti-ADI-10087 antibodies (ALP)/neutralizing antibodies (nAb) was presented.

The production of anti-drug antibodies (ADAs) and neutralizing antibodies (NAbs) in serum was determined using an immunogenicity assay. (1) Sampling time points for detection of anti-drug antibodies and neutralizing antibodies:

1 hour before administration; 336 hours (day 15), 672 hours (day 29), 1344 hours (day 57), 2016 hours (day 85) after administration.

Time points for blood sampling for immunogenicity analysis: within 1 hour before administration; 336 hours (±12 h) (day 15), 672 hours (±24 h) (day 29), 1344 hours (±48 h) (day 57), 2016 hours (±48 h) (day 85) after administration.

No subjects were found to have antibodies to ADI-10087 (ALP) after drug administration, except for 1 subject (1.7%) in the placebo group who was positive for ALP at baseline before drug administration. No neutralizing antibodies (nAb) were found in this subject. Thus, no subjects in the ADI-10087 or placebo groups were found to have positive ALP or nAb after drug administration, indicating adequate safety of the drug.

Example 12. Pharmacokinetic evaluation in healthy adult subjects of Chinese origin who received a single dose of ADI-10087

In the study described above in Example 11, blood samples for PK analysis were collected at the following time points:

1 hour before administration; 4 hours after administration; 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), 2016 hours (day 85) after injection.

The pharmacokinetic parameters for total ADI-10087 and free ADI-10087 were calculated for actual blood sampling time points using non-compartmental analysis (NCA) method and using WinNonlin Professional software, including T _max , C _max , AUC, V, t _1/2 and CL, which can fully describe the distribution and elimination characteristics of the drug in the human body, and the main parameters are shown in Tables 2 and 3 below.

The mean total concentration-time curves for ADI-10087 following administration of ADI-10087 to subjects in each dose group are shown in FIG. 2, and the mean free concentration-time curves for ADI-10087 for each dose group are shown in FIG. 1.

Pharmacokinetic parameter estimates were based on the pharmacokinetic concentration-time series (PKCS) of 44 subjects. Following a single subcutaneous injection of 25-600 mg ADI-10087 into the abdomen, the mean apparent volume of distribution (V _{z /F} ) of total ADI-10087 was approximately 3.2-7.4 L, the mean apparent clearance (CL/F) was approximately 13.0-40.2 mL/h, and the mean half-life (t _1/2 ) was approximately 5-7 days; the mean V _{z /F} for free ADI-10087 was approximately 4.0-8.6 L, the mean CL/F was 17.4-74.6 mL/h, and the mean t _1/2 was approximately 3-7 days. As the dose increased, Vz/F and CL/F decreased continuously, while at _1/2 gradually increased.

Following a single subcutaneous injection of 25-600 mg of ADI-10087 into the abdomen, the T _max for total ADI-10087 in serum was approximately 3-6 days, and the T _max for free ADI-10087 in serum was approximately 2.5-10 days. It should be noted that when injected into the abdomen, ADI-10087 is not immediately absorbed into the bloodstream; however, as the dose increases, the time to reach the maximum accumulation concentration increases.

Over the single dose range of 25-600 mg C _max for ADI-10087, the increase in AUC _0-last and AUC _0-∞ values for total ADI-10087 and free ADI-10087 was greater than proportional with dose and demonstrated a non-linear kinetic relationship.

Following single intravenous administration of 75 mg and 450 mg, the mean _Vz for total ADI-10087 was approximately 2.6 L and 2.1 L, respectively; the mean _Vss was approximately 3.0 L and 4.3 L, respectively; the mean CL was approximately 17.3 mL/h and 9.81 mL/h, respectively; and the mean t _1/2 was approximately 4 days and 6 days, respectively; the mean _Vz for free ADI-10087 was approximately 2.3 L and 2.1 L, respectively; the mean _Vss was approximately 2.5 L and 3.0 L, respectively; the mean CL was approximately 24.1 mL/h and 9.41 mL/h, respectively; and the mean t _1/2 was approximately 3 days and 7 days, respectively. As the dose increased, the CL also decreased, while t _1/2 increased. The absolute bioavailability of ADI-10087 following subcutaneous injection into the abdomen was approximately 58% (based on total ADI-10087) over the dose range of 75-450 mg.

Table 4 provides the corresponding geometric mean AUC _0-∞ values for total ADI-10087 and free ADI-10087 following administration of 75 mg and 450 mg by subcutaneous injection into the abdomen or by intravenous drip, as well as the corresponding absolute bioavailability estimates for subcutaneous administration.

It can be seen that when assessed based on the total ADI-10087, its absolute bioavailability in the case of subcutaneous administration to healthy subjects of the drug at doses of 75 mg and 450 mg was approximately the same and amounted to 57.8% and 57.5%, respectively, which indicates that the absolute bioavailability in the case of subcutaneous administration in the dose range of 75-450 mg does not change significantly.

When assessed on a free ADI-10087 basis, absolute bioavailability following subcutaneous administration of 75 mg and 450 mg to healthy subjects was 33.8% and 49.2%, respectively, which was slightly lower than that assessed for total ADI-10087.

A graphical method was used to analyze the correlation between PK parameters (C _max , AUC _0-last and AUC _0-∞ ) and dose in the case of subcutaneous injections of the drug into the abdomen at doses of 25-600 mg.

FIGS. 3-5, respectively, show scatterplots of C _max , AUC _0-last , and AUC _0-inf values for total ADI-10087 and free ADI-10087, which varied with dose, following subcutaneous injection into the abdomen at doses of 25-600 mg in healthy subjects. The results show that over the dose range of 25-600 mg, C _max and AUC for total ADI-10087 and free ADI-10087 increased with increasing dose, and the increase was greater than proportional to the dose increase, and ADI-10087 exhibits a non-linear kinetic relationship. For example, the mean C _max for total ADI-10087 increased in a ratio of approximately 1:3.4:8.0:13:22:29 and the mean AUC _0-last increased in a ratio of approximately 1:4.2:11:26:46:82 over the 25-600 mg dose fold increase (1:3:6:12:18:24). In the 25 mg group, PK parameters for free ADI-10087 could not be determined due to low concentrations; over the 75,600 mg dose range (1:2:4:6:8 dosing fold increase), the mean C _max for free ADI-10087 increased in a ratio of approximately 1:2.9:5.4:10:12 and the mean AUC _0-last increased in a ratio of approximately 1:3.9:12:21:34.

Intravenous drip administration of the drug was performed only for two doses - 75 mg and 450 mg. In the case of a 6-fold increase in the dose for intravenous drip administration, the average C _max and AUC _0-last values for total ADI-10087 increased by 4.6 and 11 times, respectively; while the average C _max and AUC _0-last values for free ADI-10087 increased by 6.1 and 17 times, respectively.

Example 13. Evaluation of pharmacodynamics in healthy adult subjects of Chinese origin who received a single dose of ADI-10087

In the study described in Example 11 above, blood samples were collected at the following time points: 1 hour before administration; 4 hours after administration; 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), 2016 hours (day 85) after injection. 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), apolipoprotein B (ApoB) and similar parameters were determined according to the methods described in this document or well known in the art; and the results were plotted.

(1) Change in serum free PCSK-9 concentration from baseline

Blood samples will be collected at pre-specified time points in a central laboratory for analysis of serum free PCSK-9 concentrations. Pharmacodynamic assessment will be performed on a pharmacodynamics set (PDS) basis; the values at each time point and the rate of change (%) in serum free PCSK-9 concentration from baseline will be presented in summary tables and lists. Baseline was the pre-dose observed PCSK-9 concentration. Rate of change (%) from baseline = 100×(post-dose observed concentration minus baseline concentration)/baseline concentration. Individual serum free PCSK-9 concentrations, mean PCSK-9 concentrations for each dose group, and the rate of change from baseline will be presented as concentration-time curves (including a linear concentration plot and a log concentration plot) on a dose-group basis.

The rate of change in serum free PCSK-9 concentrations from baseline is shown in FIG. 6. The results show that: the mean duration of decline in PCSK-9 concentrations increased with increasing dose; at 24 days post-dose, the mean maximal decline in PCSK-9 in each group was 90%–100%; at 2 weeks post-dose, PCSK-9 concentrations had declined by an average of >70% in the 75 mg SC ADI-10087 group, as well as in the 150 mg and higher dose groups; at 4 weeks post-dose, PCSK-9 concentrations had declined by an average of >60% in the 300 mg ADI-10087 group, in the higher dose SC and IV groups; At 6 weeks post-administration, PCSK-9 decreased by an average of >80% in the 450 mg s.c. ADI-10087 group, the 450 mg i.v. group, and the 600 mg s.c. group; at 8 weeks post-administration, PCSK-9 decreased by an average of >50% in the 600 mg s.c. ADI-10087 group.

(2) Least squares mean changes in LDL-C from baseline

For each assessment time point, changes in serum LDL-C from baseline in each study drug dose group and in the placebo group were analyzed using a multiple-measure mixed-effects model (MMRM), and results included least-squares mean changes, mean differences between groups, 95% confidence intervals, and P-values. Results of the analysis based on this model showed that at one month after dosing, P-values for most study drug dose groups compared with placebo were <0.05. As time after dosing increased, P-values for the low-dose group compared with placebo became >0.05. By Day 29, P-values for the 150 mg and higher dose groups compared with placebo were <0.05; By Day 57, the 450 mg only and higher dose groups had P-values <0.05 compared with placebo; by Day 71, the 600 mg group had P-values <0.05 compared with placebo; by Day 85, any dose group of study drug had P-values >0.05 compared with placebo. Least squares changes in LDL-C from baseline for each assessment time point for each dose group are shown in FIG. 7.

(3) Average rate of change in LDL-C concentration relative to baseline

The mean rate of change in serum LDL-C from baseline is shown in FIG. 8. The results demonstrate that the duration of reduction in serum LDL-C was dependent on the dose of ADI-10087. Near-maximal reductions in LDL-C were first achieved by day 5. At day 15, near-maximal reductions in LDL-C were achieved in each dose group. The mean maximum reductions in LDL-C in each subcutaneous ADI-10087 group ranged from 51.7% to 72.1%. At 7 days post-dose, the mean reductions in LDL-C in the 75 mg and 150 mg ADI-10087 groups were >50%; the mean reduction in LDL-C in the 75 mg group was >50% and was maintained for 15 days; and the mean LDL-C reduction in the 150 mg group was maintained for 22 days. At day 11 after dosing, the mean LDL-C reduction in the 300 mg ADI-10087 group and in the higher dose groups was >50% and was sustained until at least day 43 (week 6). At day 57 (8 weeks) after dosing, the mean LDL-C reduction in the 600 mg SC ADI-10087 group was >50%. At day 71 (10 weeks), the mean LDL-C reduction in the 600 mg SC ADI-10087 group was >30%.

Using evolocumab as a control, the maximum LDL-C reduction and duration of such reduction were shown to be > with ADI-10087 compared to evolocumab at the same dose.

(4) Area under the curve "LDL-C level - time"

The mean LDL-C versus time plot is shown in FIG. 9.

The area under the LDL-C versus time curve was calculated using the trapezoidal method. The area under the LDL-C versus time curve for subjects who withdrew early was also calculated using the trapezoidal method based on the concentration data obtained before withdrawal. For the area under the LDL-C versus time curve, the geometric mean for all study drug dose groups was 3522.433, and the geometric coefficient of variation was 44.3; the geometric mean for all placebo groups was 5609.877, and the geometric coefficient of variation was 20.1. The area under the curve for the 75 mg IV group was higher than for the other study drug dose groups, with a geometric mean of 4709.056 and a geometric coefficient of variation of 18.0; The area under the curve for the 450 mg IV group was the lowest, the geometric mean was 1978.572, and the geometric coefficient of variation was 99.0.

(5) The rate of change in total cholesterol (TC) concentration relative to baseline

The rate of change in total cholesterol (TC) concentration from baseline is shown in FIG. 10. The results demonstrate that: the mean duration of total cholesterol reduction increased with dose. In the 75 mg ADI-10087 group and in the higher dose groups, the mean maximum total cholesterol reduction ranged from 37.9% to 48.6%; close to the maximum value achieved 11 days after administration.

(6) Rate of change in apolipoprotein B (ApoB) concentration relative to baseline

The rate of change from baseline in apolipoprotein B (ApoB) concentrations is shown in FIG. 11. The results demonstrate that: the mean duration of ApoB reduction increased with dose. In the 75 mg ADI-10087 group and in the higher dose groups, the mean maximum reduction in total cholesterol ranged from 52.1% to 62.8%; close to the maximum value achieved 11 days after administration.

(7) Rate of change in non-HDL-C concentration from baseline

The rate of change from baseline in non-high-density lipoprotein cholesterol (non-HDL-C) is shown in FIG. 12. The results demonstrate that: the mean duration of non-HDL-C reduction increased with dose. In the 75 mg ADI-10087 group and in the higher dose groups, the mean maximum reduction in total cholesterol ranged from 56.8% to 69.7%; close to the maximum achieved 11 days after administration.

(8) Rate of change in mean lipoprotein (a) concentration (Lp(a)) relative to baseline

The rate of change from baseline in mean lipoprotein (a) (Lp(a)) concentrations is shown in FIG. 13. At 7 days post-dose, Lp(a) decreased in each of the ADI-10087 dose groups; and the mean maximum decrease in each group ranged from 24.9% to 62.3%.

Example 14. Evaluation of the pharmacokinetics of ADI-10087 after repeated dosing in a population of Chinese patients with hypercholesterolemia

1. Selection of the study population

Subjects included in the study must simultaneously meet all of the following criteria:

(1) provision of signed and dated informed consent;

(2) a man or woman aged >18 and <70 years at the time of screening;

(3) BMI>18 kg/ ^m2 and <30 kg/ ^m2 ;

(4) the subject has been diagnosed with hyperlipidemia and has been treated with a statin at a moderate or higher dose for at least 4 weeks (see Guidelines for the Prevention and Treatment of Dyslipidemia in Chinese Adults 2016);

(5) fasting LDL-C >100 mg/dL (2.6 mmol/L) and <220 mg/dL (5.7 mmol/L) at screening;

(6) fasting triglycerides <400 mg/dL (4.5 mmol/L) at screening; and

(7) willingness to cooperate in order to complete all stages of the study and receive the study therapy in full.

2. Research design

A total of 60 patients who met inclusion criteria were randomly assigned equally to the following dose groups at different time points: one subcutaneous injection of ADI-10087 at a dose of 75 mg or 140 mg or placebo every 2 weeks; one subcutaneous injection of ADI-10087 at a dose of 300 mg or 420 mg or placebo every 4 weeks; or one subcutaneous injection of ADI-10087 at a dose of 450 mg or 600 mg or placebo every 6 weeks. For each dose, patients were randomized to the ADI-10087 and placebo cohorts in a 4:1 ratio. Therapy was continued for 12 weeks, after which time the analysis of results occurred.

3. Medicinal products used in the study

The test drug used in the study was the drug prepared as described in Example 1; the control drug used in the study was a placebo prepared as described in Example 6.

4. Administration schemes

Subcutaneous injection of study drug or placebo into the abdomen

5. Collection and processing of samples

Sample collection time points for PK/PD analysis (PCSK9)

• Administration regimens for groups receiving 75 mg r/2 weeks and 140 mg r/2 weeks:

^1st administration: before administration (within 1 hour); 4 hours (±10 min), 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) and 288 hours (±12 hours) (day 12) after administration;

^2nd / ^3rd / ^4th / ^5th administration: before administration (within 1 hour);

^6th administration: before administration (within 1 hour); 4 hours (±10 min), 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) and 336 hours (±12 hours) (day 14) after administration;

Blood samples for PK/PD (PCSK9) analysis were collected at the early termination visit if the subject was unable to complete the study in full and withdrew early;

1 blood sample for PK/PD (PCSK9) analysis was collected at the subsequent safety visit.

• Administration regimens for groups receiving 300 mg r/4 weeks and 420 mg r/4 weeks:

^1st administration: before administration (within 1 hour); 4 hours (±10 min), 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) and 504 hours (±24 hours) (day 21) after administration;

^2nd administration: before administration (within 1 hour);

^3rd administration: before administration (within 1 hour); 4 hours (±10 min), 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) and 672 hours (±24 hours) (day 28) after administration;

Blood samples for PK/PD analysis were collected at the early termination visit if the subject was unable to complete the study in full and dropped out early;

1 blood sample for PK/PD analysis was collected at the subsequent safety visit.

Administration regimens for groups receiving 450 mg r/6 weeks and 600 mg r/6 weeks:

^1st administration: before administration (within 1 hour); 4 hours (±10 min), 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) and 840 hours (±24 hours) (day 35) after administration;

^2nd administration: before administration (within 1 hour); 4 hours (±10 min), 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) and 1008 hours (±48 hours) (day 42) after administration;

For the 600 mg dose group, one blood sample for PK/PD analysis was collected at 1344 h (+48 h) (day 56) after the 2nd administration.

Blood samples for PK/PD analysis were collected at the early termination visit if the subject was unable to complete the study in full and dropped out early;

1 blood sample for PK/PD analysis was collected at the subsequent safety visit.

Blood samples for PK/PD (PCSK9) analysis were analyzed in a central laboratory. Blood lipid levels during the screening period were analyzed in a local laboratory and from baseline after drug initiation in a central laboratory.

A total of 5 ml of whole blood was collected into a vacuum blood collection tube with procoagulant, serum was separated, aliquoted and frozen for subsequent PK/PD (PCSK9) analysis.

Blood lipid levels were determined as described above.

6. Results of the pharmacokinetics analysis

Pharmacokinetic parameters for free ADI-10087 antibody concentrations were calculated at actual blood collection time points using non-compartmental analysis (NCA) and Pkanalix 2019R2 software (Lixoft, Antony, France).

Tables 5 and 6 show the summarized results for the mean pharmacokinetic parameters for each dose group for the first administration and for administration after reaching steady-state concentrations of ADI-10087 following multiple subcutaneous injections of ADI-10087 at different doses in patients with hypercholesterolemia.

Across the dose range, the first dose of 75-600 mg ADI-10087 resulted in greater than dose-proportional increases in ADI-10087 C _max and AUC _0-inf values and demonstrated a non-linear kinetic relationship.

In the Cycle 1 and Steady-State PK studies, the median time to peak plasma concentration (T _max ) of ADI-10087 following subcutaneous abdominal injection of 75-600 mg ADI-10087 was 70.7-239 h (2.9-10 days); following steady-state administration following subcutaneous abdominal injection of 75-600 mg ADI-10087, the median time to peak plasma concentration (T _max ) of ADI-10087 was 95.1-155 h (3.96-6.46 days). These results indicate that ADI-10087 is not immediately absorbed into the bloodstream following abdominal injection, and the time to peak plasma concentration increases as the dose increases.

Following subcutaneous injection of ADI-10087 into the abdomen at a dose of 75-600 mg in Cycle 1, the mean Vz/F of ADI-10087 was approximately 5.14-33.4 L, the mean CL/F was approximately 0.0163-0.242 L/h, and the mean t _1/2 was approximately 88.6-291 h (3.69-12.1 days); After subcutaneous injection of ADI-10087 into the abdomen at a dose of 75,600 mg in the steady state cycle, the mean Vz/F of ADI-10087 was about 5.13-12.2 L, the mean CL/F was about 0.0124-0.091 L/h, and the mean t _1/2 was about 98.6-430 h (4.10-17.9 days). As the dose increased, Vz/F and CL/F continuously decreased, t _1/2 gradually increased.

When the drug was administered every 2 weeks, the mean concentration accumulation ratios at 336 hours ( _C336h ) after administration of ADI-10087 in the 75 mg and 140 mg multiple subcutaneous injection groups were 3.44 and 3.83, respectively, and the mean AUC _0-336h accumulation ratios were 3.20 and 3.64, respectively;

when the drug was administered once every 4 weeks, the mean concentration accumulation ratios at 672 hours ( _C672h ) after administration of ADI-10087 in the groups receiving doses of 300 mg and 420 mg by multiple subcutaneous injections were 2.03 and 1.47, respectively, and the mean AUC _0-672h accumulation ratios were 1.34 and 1.28, respectively;

When the drug was administered every 6 weeks, the mean concentration accumulation ratios at 1008 hours ( _C1008h ) after ADI-10087 administration in the 450 mg and 600 mg multiple subcutaneous injection groups were 1.18 and 0.983, respectively, and the mean AUC _0-1008h accumulation ratios were 1.02 and 0.986. As the dose increased, the interval in the dosing cycle increased and the ADI-10087 accumulation ratio decreased, as shown in Table 7.

Example 15. Evaluation of the pharmacodynamics of ADI-10087 after repeated dosing in a population of Chinese patients with hypercholesterolemia

The selection of investigators, study design, drugs used in the study, administration schedules, sample collection and processing procedures were the same as in Example 14.

Results of pharmacodynamic evaluation

Pharmacodynamics were assessed using the pharmacodynamics set (PDS). Changes from baseline in serum free PCSK-9 were determined before each administration, and the observed values for each time point and the rate of change (%) in serum free PCSK-9 from baseline were presented in summary tables. The PDS set included 60 hypercholesterolemic subjects (48 in the ADI-10087 group and 12 in the placebo group). Serum PCSK9 concentration data after repeated multiple administrations of ADI-10087 at different time intervals were analyzed, and the PDS set included all patient populations who received at least one dose of study drug and for whom at least one valid post-dose assay result was available.

1. Rate of change in serum free PCSK9 concentration relative to baseline after administration

The rate of change in serum free PCSK9 concentration from baseline after administration is shown in FIG. 14. The results demonstrate that PCSK9 levels did not change statistically significantly in the placebo group. After the first administration of ADI-10087, in the ADI-10087 group: in the 75-600 mg dose groups, the maximum reduction in PCSK9 was generally achieved at about 24 hours, and the time required for maximum reduction was well correlated within each dose group. After the first administration of ADI-10087, in the 75 mg dose group, the maximum reduction in PCSK9 was about 78%, and in the 140-600 mg dose groups, the maximum reduction in PCSK9 was >90%, and the higher the dose, the more pronounced the reduction in PCSK9 was; and at a dose of 300 mg or more, the suppression of PCSK9 was almost complete; After the point of maximal decline, PCSK9 levels began to recover, and the higher the dose, the less the recovery. In the case of multiple doses of ADI-10087, PCSK9 concentrations remained at a certain reduced level; in the cohort with a 2-week dosing interval, PCSK9 concentrations in the 75 mg dose group remained at a level close to baseline, and PCSK9 concentrations in the 140 mg dose group remained at a level reduced by 80%; in the cohorts with a 4-week, 6-week dosing interval, PCSK9 concentrations in the 300-600 mg dose groups remained at a level reduced by 80% or more.

2. Comparison of percentage changes in LDL-C from baseline for the same dosing intervals

2-week dosing interval group: In the 75 mg p/2 weeks group, the LDL-C level after the first administration of the drug was significantly reduced from the baseline level; the maximum reduction before the ^2nd administration was about 51.09% (day 11); there was no obvious recovery of the LDL-C level between 4 h and 336 h after the last administration; however, 336 h after the last administration, this level remained significantly reduced (61.323%) from the baseline level. The LDL-C level in the 140 mg p/2 weeks group also showed a significant reduction after the first administration; the maximum reduction before the ^2nd administration was about 45.99% (day 13). Although there was a slight recovery at 4,336 h after the last administration and LDL-C levels increased slightly, LDL-C levels were still 54.296-60.991% lower than baseline.

Groups with 4-week administration interval: in the group receiving 300 mg r/4 weeks and in the group receiving 420 mg r/4 weeks, the LDL-C level after the first administration was significantly reduced compared with the baseline; and the maximum reduction before the second administration was observed on day 29 and was 59.8% and 71.363%, respectively; the reduction in LDL-C in the group receiving 420 mg r/4 weeks before each administration was significantly more pronounced than in the group receiving 300 mg r/4 weeks; after the last administration, no obvious recovery was observed in either group; and in the group receiving 450 mg r/4 weeks, the concentration of LDL-C remained reduced at a level of 72.256% (60.136% in the group receiving 300 mg r/4 weeks) 672 hours after the last administration.

Groups with a 6-week administration interval: in the group receiving 450 mg r / 6 weeks and in the group receiving 600 mg r / 6 weeks, the LDL-C level after the first administration was noticeably reduced compared with the baseline level, and in the group receiving 600 mg r / 6 weeks, the reduction was pronounced (the maximum reduction was about 71.875%); the reduction in the LDL-C level in the group receiving 600 mg r / 6 weeks after the second administration (i.e., after the last administration) was more pronounced than in the group receiving 450 mg r / 6 weeks; 672 hours after the second administration, some recovery of the level was observed, and the reduction was less pronounced (the difference was about 2.9%); At 1008 hours after the second administration, the LDL-C level in the groups receiving 450 mg r/6 weeks and 600 mg r/6 weeks remained reduced compared to the baseline level (62.883% and 56.516%, respectively). As can be seen from the study results presented in Example 13, the duration of the LDL-C reduction and the duration of PCSK9 suppression were expressed at higher doses of the drug: in the 600 mg group, the LDL-C level remained reduced by 43.455% compared to the baseline level at 8 weeks after administration (1344 hours after the second administration). It was shown that ADI-10087 can be administered at a longer interval of 4-8 weeks. The rate of change in LDL-C concentration relative to the baseline level before and after each administration is shown in FIG. 15.

3. Rate of change in LDL-C levels after 6 weeks, 12 weeks and 14 weeks relative to baseline

After 6 weeks of continuous administration, LDL-C levels were significantly lower from baseline in each ADI-10087 dose group compared with placebo, specifically: -52.385% in the 75 mg q2w group, -52.523% in the 140 mg q2w group, -60.353% in the 450 mg q6w group, and -62.175% in the 600 mg q6w group; the percentage reduction in LDL-C from baseline increased with increasing dose, with higher doses producing greater reductions in LDL-C.

After 12 weeks of continuous administration, the reduction in LDL-C from baseline in any ADI-10087 dose group did not demonstrate a significant recovery from the reduction at 6 weeks post-administration, and the percentage changes in LDL-C from baseline in each dose group compared to the reduction in the placebo group were: -58.913% in the 75 mg q 2 weeks group, -51.886% in the 140 mg q 2 weeks group, -57.726% in the 300 mg q 4 weeks group, -69.846% in the 420 mg q 4 weeks group, -60.473% in the 450 mg q 6 weeks group, and -54.106% in the 600 mg q 6 weeks group; Moreover, the reduction in LDL-C levels in the group receiving 420 mg r/4 weeks was 72.256% (-2.410% in the placebo group).

At 14 weeks after the start of administration, i.e., 8 weeks after the second administration, the LDL-C level in the group receiving 600 mg r/6 weeks decreased relative to the baseline level by 43.455% (-16.980% in the placebo group). The rate of change in the LDL-C level relative to the baseline level before and after each administration is shown in FIG. 16.

4. Analysis of the rate of change (%) of Lp(a) relative to baseline after 6 weeks, 12 weeks and 14 weeks

After 6 weeks of continuous administration, the mean reduction in Lp(a) in each ADI-10087 dose group ranged from 8.720% to 22.436% from baseline compared with the reduction in the placebo group; the 75 mg q2w and 450 mg q6w groups had the greatest reductions, at -22.436% and -21.914%, respectively.

After 12 weeks of continuous administration, the mean reductions in Lp(a) in each ADI-10087 dose group ranged from 2.088% to 28.638% from baseline compared with the reduction in the placebo group; the 75 mg q2w and 450 mg q6w groups had the largest reductions, ranging from 28.638% to 27.362%, respectively; and the 600 mg q6w and 420 mg q4w groups had smaller reductions, ranging from 2% to 7%.

After 14 weeks of continuous administration (8 weeks after the second administration), the Lp(a) level in the 600 mg q6w group decreased by 24.520% from baseline when compared with the decrease in the placebo group. The rate of change in Lp(a) level before and after each administration is shown in FIG. 17.

5. Analysis of the rate of change (%) of ApoB relative to baseline after 6 weeks, 12 weeks and 14 weeks

After 6 weeks of continuous administration, ApoB concentrations showed a significant reduction from baseline when compared to placebo. The mean reduction in ApoB in each ADI-100876 dose group ranged from 57.118% to 61.414%; the higher the dose, the greater the reduction in ApoB concentrations.

After 12 weeks of continuous dosing, the mean reduction in ApoB in each ADI-10087 dose group ranged from 53.761% to 69.573% from baseline when compared with the reduction in the placebo group; and the reduction in ApoB concentrations correlated with the reduction in LDL-C, with the 420 mg q4w dose group achieving a reduction in ApoB approaching 70% from baseline.

After 14 weeks of continuous administration (8 weeks after the second administration), the ApoB level in the 600 mg q6w group decreased by 61.729% from baseline compared with the decrease in the placebo group. The rate of change in ApoB level from baseline before and after each administration is shown in FIG. 18.

6. Analysis of the rate of change (%) of non-HDL-C from baseline after 12 weeks 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 greater than 55% compared with the reduction in the placebo group, and the maximum reduction was 75.394% (in the group receiving 420 mg q4w);

After 14 weeks of continuous administration (8 weeks after the second administration), the non-HDL-C level in the 600 mg r/6 week group decreased by 61.958% from baseline compared with the decrease in the placebo group. The rate of change in non-HDL-C level from baseline before and after each administration is shown in FIG. 19.

7. Analysis of the rate of change (%) of TC relative to the baseline level after 12 weeks and 14 weeks

After 12 weeks of continuous dosing, the mean reduction in TC from baseline in each ADI-10087 dose group ranged from 40% to 57%; the maximum reduction was approximately 56% in the group receiving 420 mg q4w. In the placebo group, TC concentrations did not change statistically significantly from baseline (an increase of approximately 0.5%).

After 14 weeks of continuous administration (8 weeks after the second administration), the TC concentration in the group receiving 600 mg r/6 weeks was reduced by 36.184% relative to the baseline level, while in the placebo group, the TC concentration increased by 10.635% relative to the baseline level. The rate of change in TC level relative to the baseline level before and after each administration is shown in FIG. 20.

8. Analysis of the rate of change (%) of ApoB/ApoA1 relative to baseline after 12 weeks and 14 weeks

After 12 weeks of continuous administration, the mean reduction in ApoB/ApoA1 from baseline in each ADI-10087 dose group was greater than 50% compared with the reduction in the placebo group, and the maximum reduction was 68.476% (in the group receiving 420 mg q4w).

After 14 weeks of continuous administration (8 weeks after the second administration), the ApoB/ApoA1 level in the 600 mg q6w group was reduced by 53.656% from baseline compared with the reduction in the placebo group. The rate of change in ApoB/ApoA1 level from baseline before and after each administration is shown in FIG. 21.

9. Conclusion from the analysis of effectiveness

The efficacy analysis included 48 patients in the ADI-10087 group and 12 subjects in the placebo group, who were part of the safety analysis set. At week 12, the reduction in the mean LDL-C concentration in each dose group ranged from 54.3% to 72.26% compared to baseline. In particular, it was shown that in the group receiving 600 mg r/6 weeks, the LDL-C concentration decreased by 56.52% (from 40.54% to 72.50%), and the reduction was maintained until week 14 (8 weeks after the second administration), and the reduction was 43.46% (from 25.96% to 60.96%) compared to baseline. At week 12, the mean reduction in Lp(a) compared to baseline ranged from 24.04% to 50.59%. Concentrations of other lipids in each dose group also decreased compared to the decrease in the placebo group. Thus, the PK/PD parameters (LDL-C) of ADI-10087 indicate good characteristics of the drug and demonstrate compliance with the requirements for its administration at a longer interval of 4-8 weeks.

Example 16. Safety assessment of ADI-10087 after repeated dosing in a Chinese hypercholesterolemic population

The results demonstrate that in this study, the administration of multiple repeat doses of subcutaneous injection of ADI-10087 was safe in a population of Chinese patients with hypercholesterolemia. In the ADI-10087 group, the incidence of treatment-emergent adverse events (TEAEs) was 70.8%, and the incidence of drug-related TEAEs was 31.3%; in the placebo group, the incidence of TEAEs was 75.0%, and the incidence of drug-related TEAEs was 41.7%. The incidence of at least one TEAE of interest (allergic reaction, injection site reaction, aminotransferase increase, liver injury, or muscle event) was reported in 10.4% and 25.0% of subjects in the ADI-10087 and placebo groups, respectively; There were no serious adverse events, serious adverse events related to the drug, adverse events requiring withdrawal from the study, or adverse events resulting in death. Adverse events related to the drug in the ADI-10087 group were mild to moderate in severity, did not require specific treatment, and improved after corrective treatment, indicating the safety of the drug. Data on adverse events that occurred during the study are presented in Table 8.

Illustrative embodiments of the present invention have been described above. Those skilled in the art will understand that these embodiments are provided for illustrative purposes only and that various other substitutions, adaptations and modifications thereof may be made without departing from the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments of the invention listed herein.

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SEQUENCE LIST

<110> Innovent Biologics (Suzhou) Co., Ltd.

<120> USE OF ANTI-PCSK9 ANTIBODIES FOR PREVENTION OR TREATMENT

DISEASES ASSOCIATED WITH HIGH CHOLESTEROL LEVELS

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Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

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Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

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Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

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Phe Gly Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

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Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

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Trp Ile Gly Ser Ile Asn Tyr Arg Gly Ser Thr Tyr Tyr Asn Pro Ser

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Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

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Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

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Cys Ala Arg Glu Asn Ser Gly Val Val Pro Ala Ala Gly Pro Asn Trp

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Phe Gly Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

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Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr

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Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

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Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

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His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

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Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr

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Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val

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<---

Claims (40)

1. A method for reducing cholesterol levels in a subject, comprising administering to the subject an anti-PCSK9 antibody at a dose of 75 to 600 mg, wherein the anti-PCSK9 antibody comprises a heavy chain and a light chain, wherein (a) said heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 41; and (b) said light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35; and wherein the anti-PCSK9 antibody is administered at an interval equal to or greater than once every two weeks (p/2 wk). 2. A method for the prevention or treatment of a disease associated with elevated cholesterol levels selected from hypercholesterolemia and hyperlipidemia, comprising administering to a subject an anti-PCSK9 antibody at a dose of 75 to 600 mg, wherein the anti-PCSK9 antibody comprises a heavy chain and a light chain, wherein (a) said heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 41; and (b) said light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35; wherein the anti-PCSK9 antibody is administered at an interval equal to or greater than once every two weeks (p/2 wk). 3. The method according to claim 1 or 2, wherein said antibody is administered once every two weeks (r/2 weeks), once every four weeks (r/4 weeks), once every five weeks (r/5 weeks), once every six weeks (r/6 weeks), once every seven weeks (r/7 weeks), once every eight weeks (r/8 weeks), once every nine weeks (r/9 weeks), once every ten weeks (r/10 weeks), once every eleven weeks (r/11 weeks), once every twelve weeks (r/12 weeks), once every 4 months, once every 5 months, once every 6 months, once every 7 months, or once a year. 4. The method according to claim 1 or 2, wherein said antibody is administered at an interval equal to or greater than once every four weeks (p/4 wk). 5. The method according to any one of claims 1-3, wherein said antibody is administered once every two weeks (r/2 weeks), once every four weeks (r/4 weeks), once every five weeks (r/5 weeks), or once every six weeks (r/6 weeks). 6. The method according to any one of claims 1-5, wherein the cholesterol is LDL cholesterol. 7. The method according to any one of claims 1-6, wherein the anti-PCSK9 antibody is administered to the subject at a dose of 300 to 600 mg or 450 to 600 mg. 8. The method according to claim 7, wherein the anti-PCSK9 antibody is administered to the subject at a dose of 450 or 600 mg. 9. The method according to any one of claims 1-8, wherein the anti-PCSK9 antibody is administered parenterally. 10. The method according to any one of claims 1-8, wherein the anti-PCSK9 antibody is administered subcutaneously or intravenously. 11. The method of any one of claims 1-10, wherein the subject's LDL cholesterol level remains reduced by >30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, or >90% at 4 weeks post-administration, 5 weeks post-administration, 6 weeks post-administration, 7 weeks post-administration, 8 weeks post-administration, 9 weeks post-administration, 10 weeks post-administration, 3 months post-administration, 4 months post-administration, or 5 months post-administration compared to the subject's LDL cholesterol level prior to administration. 12. The method of any one of claims 1-11, wherein the level of free PCSK-9 in the subject's serum remains reduced by >30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, or >90% at 4 weeks post-administration, 5 weeks post-administration, 6 weeks post-administration, 7 weeks post-administration, 8 weeks post-administration, 9 weeks post-administration, 10 weeks post-administration, 3 months post-administration, 4 months post-administration, or 5 months post-administration compared to the level of free PCSK-9 in the subject's serum prior to administration. 13. The method of any one of claims 1-12, wherein the subject's total cholesterol level remains reduced by >30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, or >90% at 4 weeks post-administration, 5 weeks post-administration, 6 weeks post-administration, 7 weeks post-administration, 8 weeks post-administration, 9 weeks post-administration, 10 weeks post-administration, 3 months post-administration, 4 months post-administration, or 5 months post-administration compared to the subject's total cholesterol level prior to administration. 14. The method of any one of claims 1-13, wherein the level of apolipoprotein B in the subject remains reduced by > 30%, > 35%, > 40%, > 45%, > 50%, > 55%, > 60%, > 65%, > 70%, > 75%, > 80%, > 85%, or > 90% at 4 weeks post-administration, 5 weeks post-administration, 6 weeks post-administration, 7 weeks post-administration, 8 weeks post-administration, 9 weeks post-administration, 10 weeks post-administration, 3 months post-administration, 4 months post-administration, or 5 months post-administration compared to the level of apolipoprotein B in the subject prior to administration. 15. The method of any one of claims 1-14, wherein the subject's non-HDL-C level remains reduced by >30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, or >90% at 4 weeks post-administration, 5 weeks post-administration, 6 weeks post-administration, 7 weeks post-administration, 8 weeks post-administration, 9 weeks post-administration, 10 weeks post-administration, 3 months post-administration, 4 months post-administration, or 5 months post-administration compared to the subject's non-HDL-C level before administration. 16. The method of any one of claims 1-15, wherein the lipoprotein (a) level in the subject remains reduced by >20%, >30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, or >90% at 4 weeks post-administration, 5 weeks post-administration, 6 weeks post-administration, 7 weeks post-administration, 8 weeks post-administration, 9 weeks post-administration, 10 weeks post-administration, 3 months post-administration, 4 months post-administration, or 5 months post-administration compared to the lipoprotein (a) level in the subject prior to administration. 17. The method of any one of claims 1-16, wherein the ApoB/ApoA1 level in the subject remains reduced by >20%, >30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, or >90% at 4 weeks post-administration, 5 weeks post-administration, 6 weeks post-administration, 7 weeks post-administration, 8 weeks post-administration, 9 weeks post-administration, 10 weeks post-administration, 3 months post-administration, 4 months post-administration, or 5 months post-administration compared to the ApoB/ApoA1 level in the subject prior to administration. 18. The method according to any one of claims 1-17, wherein the cholesterol or LDL cholesterol is serum LDL cholesterol. 19. The method according to any one of claims 1-18, wherein the anti-PCSK9 antibody is administered once every four weeks (r/4 wk), once every six weeks (r/6 wk), or once every eight weeks (r/8 wk). 20. The method according to any one of claims 1-19, wherein the subject does not develop severe adverse events after administration. 21. The method of any one of claims 1-19, wherein the subject does not develop severe adverse events associated with the anti-PCSK9 antibody after administration. 22. The method according to any one of claims 1-21, wherein the incidence of adverse events in the subject is comparable to the incidence of such events in the subject after administration who received a placebo. 23. The method according to any one of claims 2-22, wherein the disease associated with elevated cholesterol levels is selected from homozygous familial hypercholesterolemia, heterozygous familial hypercholesterolemia, and non-familial hypercholesterolemia. 24. The method according to any one of claims 1-23, wherein the anti-PCSK9 antibody is administered in a liquid antibody preparation comprising: (i) 100 to 200 mg/ml anti-PCSK-9 antibody; (ii) 0.2 to 10 mg/ml histidine; (iii) 1 to 6% sorbitol and/or 50 to 180 mmol/L arginine or arginine salt; And (iv) 0.05 to 1 mg/ml polysorbate-80 or polysorbate-20; and the pH of the liquid preparation is from about 5.0 to 6.0. 25. The method according to any one of claims 1-24, wherein the anti-PCSK9 antibody is administered in a liquid antibody preparation, and wherein said liquid antibody preparation contains water as a solvent and consists of the following components: anti-PCSK9 antibody 150 mg/ml histidine 1.5 g/l arginine 90 mmol/l sorbitol 3% (w/v) polysorbate-80 0.3 g/l; and the pH of the liquid preparation is 5.5.