Method for treating or preventing cholesterol-related diseases using PCSK9 inhibitor

Abstract

The present invention relates to the field of medicines, and particularly relates to a method for treating or preventing cholesterol-related diseases using a PCSK9 inhibitor. The method comprises: administering 35-420 mg of a PCSK9 antibody to a subject, thus not only reducing the frequency of drug administration, but also prolonging the duration of drug action.

Description

Methods for using PCSK9 inhibitors in treating or preventing cholesterol-related diseases Technical Field

[0001] The present invention relates to the field of medicine, and more specifically, to a method for using a PCSK9 inhibitor to treat or prevent cholesterol-related diseases. Background Art

[0002] Hyperlipidemia is a heterogeneous group of disorders characterized by excess blood lipids (i.e., cholesterol, phospholipids, and triglycerides), while hypercholesterolemia refers to elevated blood cholesterol levels. Primary hypercholesterolemia is typically caused by genetic factors (monogenic or polygenic) and environmental factors (e.g., diet and lifestyle). Treatment aims to lower low-density lipoprotein cholesterol (LDL-C) levels. Because elevated LDL-C is a significant risk factor for atherosclerotic cardiovascular disease (ASCVD), lowering LDL-C levels can significantly reduce the risk of ASCVD morbidity and mortality. Despite the availability of a variety of oral lipid-lowering drugs, less than 30% of patients at high risk for ASCVD achieve the recommended target LDL-C level (<2.6 mmol / L), and fewer than 10% of patients at very high risk achieve the target LDL-C level (<1.8 mmol / L). With the increasing incidence and prevalence of primary hypercholesterolemia, there is a huge unmet medical need to improve the efficacy, safety, and convenience of lipid-lowering therapies.

[0003] Recycling of the low-density lipoprotein receptor (LDLR) on the hepatocyte surface plays a key role in maintaining cellular and systemic cholesterol homeostasis by regulating plasma LDL-C levels. Studies have shown that proprotein convertase subtilisin / kexin type 9 (PCSK9) plays an important role in LDLR recycling and regulation. PCSK9, a member of the subtilisin family of serine proteases, is primarily expressed in the liver, kidney, and intestine. Following secretion, it causes a decrease in hepatocyte surface LDLR by binding to and targeting it for lysosomal destruction. Decreased hepatic LDLR levels lead to elevated circulating LDL-C levels. Therefore, PCSK9 may represent a novel therapeutic inhibitory target in patients with primary hypercholesterolemia (heterozygous familial and nonfamilial), mixed dyslipidemia, and homozygous familial hypercholesterolemia.

[0004] Currently, patent WO2021243002 discloses a PCSK9 inhibitor and a method for treating cholesterol-related diseases. However, the main target population in the patent is minors under the age of 18. 350-500 mg / L of evolocumab is injected every two or four weeks. The frequency of medication is high, the dosage is large, and the duration of efficacy is short.

[0005] Summary of the Invention

[0006] The present invention aims to address the deficiencies of the prior art and provides a method for using a PCSK9 inhibitor to treat or prevent cholesterol-related diseases, which can not only reduce the frequency of medication and the dosage, but also prolong the duration of drug efficacy.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] In a first aspect, the present invention provides a method for treating cholesterol-related diseases, comprising:

[0009] 35-420 mg of a PCSK9 antibody is administered to the subject, wherein the heavy chain variable region of the PCSK9 antibody comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 7, LCDR2 as shown in SEQ ID NO: 8, and LCDR3 as shown in SEQ ID NO: 9.

[0010] The heavy chain variable region of the PCSK9 antibody is shown in SEQ ID NO: 13, and the light chain variable region is shown in SEQ ID NO: 14.

[0011] As a preferred technical solution of the present invention, the PCSK9 antibody is administered in an amount of any value of 35-420 mg, for example, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg.

[0012] As a preferred technical solution of the present invention, 70 mg of PCSK9 antibody is administered to the subject.

[0013] As a preferred technical solution of the present invention, 140 mg of PCSK9 antibody is administered to the subject.

[0014] As a preferred technical solution of the present invention, 280 mg of PCSK9 antibody is administered to the subject.

[0015] As a preferred technical solution of the present invention, 420 mg of PCSK9 antibody is administered to the subject.

[0016] As a preferred technical solution of the present invention, the PCSK9 antibody is administered to the subject once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every twelve weeks, or twice every two weeks, twice every three weeks, twice every four weeks, twice every five weeks, twice every six weeks, twice every seven weeks, twice every eight weeks, twice every nine weeks, twice every ten weeks, or twice every twelve weeks.

[0017] As a preferred technical solution of the present invention, 70 mg of PCSK9 antibody is administered to the subject once a week or every two weeks.

[0018] As a preferred technical solution of the present invention, 140 mg of PCSK9 antibody is administered to the subject once every two weeks, every four weeks, every six weeks or every eight weeks, preferably 140 mg of PCSK9 antibody is administered once every four weeks.

[0019] As a preferred technical solution of the present invention, 280 mg of PCSK9 antibody is administered to the subject once every two weeks, every four weeks, every six weeks or every eight weeks.

[0020] As a preferred technical solution of the present invention, 420 mg of PCSK9 antibody is administered to the subject once every four weeks, every six weeks, every eight weeks or every twelve weeks, preferably 420 mg of PCSK9 antibody is administered once every six weeks or every eight weeks.

[0021] As a preferred technical solution of the present invention, the administration method is subcutaneous injection or intramuscular injection.

[0022] As a preferred technical solution of the present invention, after 70mg-420mg of PCSK9 antibody is administered to the subject for 1-16 weeks, the plasma curve AUC of the subject is 0-t At least greater than 5 million ng·h / mL. For example, after 140 mg of PCSK9 antibody was administered to the subject for 4 weeks, the plasma curve AUCo - t is greater than 5 million ng·h / mL; for example, after 8 weeks of administration of 420 mg of PCSK9 antibody to the subject, the plasma curve AUC 0-t It is greater than 30 million ng·h / mL.

[0023] As a preferred technical solution of the present invention, after administering 140 mg to 420 mg of the PCSK9 antibody to the subject, the time for maintaining a reduction of LDL-c by at least -50% is 25 to 60 days. The reduction time is any value from 25 to 60 days, for example, after administering 140 mg of the antibody to the subject, the time for maintaining a reduction of LDL-c by at least -50% is 27 days, 29 days, 31 days, 33 days, 35 days, 37 days, 39 days, 41 days, 43 days, 45 days, 47 days, 49 days, 51 days, 53 days, 55 days, 57 days, and 59 days. For another example, after administering 420 mg of the antibody to the subject, the time for maintaining at least a -50% reduction in LDL-c is 27 days, 29 days, 31 days, 33 days, 35 days, 37 days, 39 days, 41 days, 43 days, 45 days, 47 days, 49 days, 51 days, 53 days, 55 days, 57 days, and 59 days.

[0024] In a second aspect, the present invention provides a method for treating cholesterol-related diseases, comprising:

[0025] Subjects were administered clinical doses of statins and 35-420 mg of a PCSK9 antibody;

[0026] The heavy chain variable region of the PCSK9 antibody includes HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3; the light chain variable region includes LCDR1 as shown in SEQ ID NO: 7, LCDR2 as shown in SEQ ID NO: 8, and LCDR3 as shown in SEQ ID NO: 9.

[0027] As a preferred technical solution of the present invention, the PCSK9 antibody is administered in an amount of any value of 35-420 mg, for example, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, and 420 mg.

[0028] As a preferred technical solution of the present invention, 70 mg of PCSK9 antibody is administered to the subject.

[0029] As a preferred technical solution of the present invention, 140 mg of PCSK9 antibody is administered to the subject.

[0030] As a preferred technical solution of the present invention, 280 mg of PCSK9 antibody is administered to the subject.

[0031] As a preferred technical solution of the present invention, 420 mg of PCSK9 antibody is administered to the subject.

[0032] As a preferred technical solution of the present invention, the PCSK9 antibody is administered to the subject once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every twelve weeks, or twice every two weeks, twice every three weeks, twice every four weeks, twice every five weeks, twice every six weeks, twice every seven weeks, twice every eight weeks, twice every nine weeks, twice every ten weeks, or twice every twelve weeks.

[0033] As a preferred technical solution of the present invention, 70 mg of PCSK9 antibody is administered to the subject once a week or every two weeks.

[0034] As a preferred technical solution of the present invention, 140 mg of PCSK9 antibody is administered to the subject once every two weeks, every four weeks, every six weeks or every eight weeks.

[0035] As a preferred technical solution of the present invention, 280 mg of PCSK9 antibody is administered to the subject once every two weeks, every four weeks, every six weeks or every eight weeks.

[0036] As a preferred technical solution of the present invention, 420 mg of PCSK9 antibody is administered to the subject once every four weeks, every six weeks, every eight weeks or every twelve weeks.

[0037] As a preferred technical solution of the present invention, the PCSK9 antibody is administered by subcutaneous injection or intramuscular injection, and the statin is administered orally.

[0038] As a preferred technical solution of the present invention, the statin is selected from atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin. The statin can be appropriately adjusted according to the clinical dosage.

[0039] As a preferred technical solution of the present invention, the clinical dosage of atorvastatin is 10-80 mg, preferably 20 mg or 30 mg.

[0040] Specifically, it also includes 40 mg atorvastatin, 80 mg atorvastatin, 20 mg rosuvastatin, 40 mg rosuvastatin, 40 mg simvastatin, and 80 mg simvastatin.

[0041] As a preferred technical solution of the present invention, after 70mg-420mg of PCSK9 antibody is administered to the subject for 1-16 weeks, the plasma curve AUC of the subject is 0-inf At least greater than 4 million ng·h / mL. For example, after 140 mg of PCSK9 antibody is administered to the subject for 4 weeks, the plasma curve AUC 0-inf At least greater than 4 million ng·h / mL. For another example, after 8 weeks of administration of 420 mg of PCSK9 antibody to the subject, the plasma curve AUC 0-inf At least greater than 10 million ng·h / mL.

[0042] The antibody of the present invention is further preferably the 18.156.8 antibody in patent application number CN201710816808.0, and "18.156.8 (hIgG4)" is the 18.156.8 antibody having a human IgG4 isotype constant region.

[0043] Compared with the prior art, the present invention has the following advantages and beneficial effects: administering the PCSK9 antibody inhibitor of the present invention to a subject can not only reduce the subject's medication frequency, but also significantly reduce LDL-C, and the duration of the reduction effect is prolonged with increasing dosage.

[0044] definition

[0045] As used herein, the term "comprising" is used to mean, and is used interchangeably with, the phrase "including, but not limited to," unless the context clearly dictates otherwise.

[0046] The term "Proprotein convertase subtilisin / kexin type 9 (PCSK9)", "PCSK9" or "NARC-1" refers to a naturally occurring human proprotein convertase that belongs to the proteinase K subfamily of the secretory subtilisin protease family. PCSK9 is synthesized as a lysozyme, undergoes autocatalytic intramolecular processing in the endoplasmic reticulum, and is believed to function as a proprotein convertase. When used in this application, the term refers to any naturally occurring PCSK9, preferably any naturally occurring PCSK9 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats).

[0047] The term "PCSK9" encompasses "full-length" unprocessed PCSK9 as well as any form of PCSK9 produced by intracellular processing or any fragment thereof. The term also includes naturally occurring variants of PCSK9, e.g., splice variants, derivative variants, substitution variants, deletion variants and / or insertion variants or allelic variants, such as mutants D374Y, S127R, and F216L.

[0048] The term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, or bispecific (bivalent) antibody that can bind to a specific antigen. A natural, intact antibody contains two heavy chains and two light chains. Each heavy chain consists of a variable region (VH) and three constant regions (CH1, CH2, and CH3), with a hinge region between CH1 and CH2; each light chain consists of a variable region (VL) and a constant region (CL). Mammalian heavy chains are classified as α, δ, ε, γ, and μ, while mammalian light chains are classified as λ or K. Antibodies are "Y"-shaped, with the neck of the "Y" structure consisting of the CH2 and CH3 of two heavy chains, which are linked by disulfide bonds in the hinge region; each arm of the "Y" structure includes the variable region and CH1 of one heavy chain, which are connected to the variable region and CL of one light chain by a disulfide bond. The variable regions of the light and heavy chains determine how the antibody binds to the antigen. The variable region of each chain contains three hypervariable regions (HVRs), called complementarity-determining regions (CDRs). The light chain (L) CDR region comprises LCDR1, LCDR2, and LCDR3, and the heavy chain (H) CDR region comprises HCDR1, HCDR2, and HCDR3. The CDR boundaries of the antibodies or antigen-binding fragments thereof disclosed herein can be defined or identified using the Kabat, Chothia, IMGT, AbM, or Contact numbering systems / methods.

[0049] wherein each heavy chain complementarity determining region and / or each light chain complementarity determining region is defined according to the Kabat definition, the Chothia definition, a combination of the Kabat definition and the Chothia definition, the IMGT definition, the AbM definition, or the CDR contact definition; preferably, each CDR is defined according to the Kabat CDR definition or the Chothia CDR definition or a combination of the two.

[0050] "PCSK9 inhibitor" refers to a molecule or therapy that inhibits PCSK9 activity, thereby lowering LDL-C (and / or other lipids, such as non-HDL-C, ApoB, Lp(a) etc.) levels. For example, this can include neutralizing antibodies to PCSK9 and antisense molecules to PCSK9. PCSK9 inhibitor therapy refers to a method using a PCSK9 inhibitor.

[0051] The term "LDL-C" refers to low-density lipoprotein cholesterol. "HDL-C" refers to high-density lipoprotein cholesterol. LDL and HDL belong to five major lipoprotein groups: chylomicrons, very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) (in order from largest particles to densest / smallest particles). LDL (also known as the "bad" cholesterol containing particles) can transport lipid / sterol molecules such as cholesterol (i.e., LDL-C) to the artery wall, attracting macrophages and thus inducing atherosclerosis. In contrast, HDL (also known as the "good" cholesterol containing particles) can remove lipid molecules such as cholesterol (i.e., HDL-C) from macrophages on the artery wall. Therefore, high levels of LDL-C are a major risk factor for cardiovascular diseases (CVD), such as peripheral artery disease, coronary artery disease (CAD, such as angina pectoris, myocardial infarction (commonly known as heart disease), hyperlipidemia, hypercholesterolemia, hypertriglyceridemia), atherosclerosis, stroke, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, arrhythmias, congenital heart disease, valvular heart disease, myocarditis, aortic aneurysm, peripheral artery disease, obesity, hepatobiliary disease, nephrotic syndrome, hypothyroidism, and venous thrombosis.

[0052] The term "treating" encompasses alleviating at least one symptom or other embodiment of a disorder, or reducing the severity of the disease, etc. PCSK9 inhibitors and / or one or more other therapies that lower LDL cholesterol do not need to achieve a complete cure or eradicate every symptom or manifestation of the disease to be viable therapeutics. As recognized in the relevant art, drugs used as therapeutic agents can reduce the severity of a given disease state, but do not have to eliminate every manifestation of the disease to be considered a useful therapeutic. In some embodiments, it is sufficient to simply reduce the impact of the disease (e.g., by reducing the number of its symptoms or reducing the severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect) or reduce the likelihood of the disease occurring or worsening in the subject. Certain embodiments of the present disclosure relate to methods comprising administering to a subject a PCSK9 inhibitor (e.g., an anti-PCSK9 antibody or interfering RNA) in an amount and for a time sufficient to cause a sustained improvement compared to a baseline of an indicator reflecting the severity of the particular disorder.

[0053] The term "prevent" encompasses preventing at least one symptom or other embodiment of a disorder. According to the present disclosure, a prophylactic treatment administered in combination with a PCSK9 inhibitor and / or one or more other LDL cholesterol-lowering therapies need not be completely effective in preventing the onset of the disorder to be a viable preventative. In some embodiments, simply reducing the likelihood of the disease occurring or worsening in a subject is sufficient. In some embodiments, the development of disease symptoms is delayed by the treatment methods disclosed herein.

[0054] The term "cholesterol-related disease" includes, but is not limited to, hyperlipoproteinemia, hyperlipidemia; dyslipidemia (e.g., elevated total cholesterol, elevated LDL, elevated triglycerides, elevated VLDL and / or decreased HDL); hypercholesterolemia, mixed hyperlipidemia, heart disease, stroke, coronary heart disease, atherosclerosis, peripheral vascular disease, claudication, type II diabetes, hypertension, cardiovascular disease or symptoms, inflammation or autoimmune disease. Methods for identifying / diagnosing the above-mentioned diseases or symptoms are known in the art.

[0055] The term "subject" includes any human or non-human animal. "Non-human animals" include all vertebrates, such as mammals (including but not limited to domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., monkeys), rabbits, and rodents (e.g., mice and rats)) and non-mammals (e.g., poultry, amphibians, and reptiles). In some embodiments, the subject is a human.

[0056] The term "therapeutically effective amount" or "effective dose" refers to a dose or concentration that is effective to prevent or ameliorate symptoms associated with a disease or condition and / or reduce the severity of the disease or condition, at a desired dosage and for a desired period of time. The therapeutically effective amount of an agent, antibody, or antigen-binding fragment thereof, or composition of the invention can vary depending on a variety of factors, such as the disease state, the age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also considered to be such that any toxic or deleterious effects of the agent, antibody, or antigen-binding fragment thereof, or composition are outweighed by the therapeutically beneficial effects. DETAILED DESCRIPTION

[0057] The present invention will be further described in detail below with reference to examples, but the embodiments of the present invention are not limited thereto.

[0058] Example 1 Preparation of PCSK9 Antibody

[0059] The preparation method of the PCSK9 antibody (antibody A) in the present invention refers to the preparation method of antibody 18.156.8 in patent application number CN201710816808.0, and the preparation method of this patent is fully introduced into the present invention by reference.

[0060] Table 1 shows the amino acid and nucleic acid sequences of antibody A

[0061] Comparative Example 1 Evolocumab was purchased from Amgen Inc. at a concentration of 140 mg / ml.

[0062] Example 2

[0063] This study was a single-center, randomized (35 mg was not randomized), double-blind, placebo / A controlled, single-dose, dose-escalating Phase I clinical study was conducted. Five dose groups were set up: 35 mg, 70 mg, 140 mg, 280 mg, and 420 mg. Starting with the lowest dose of 35 mg, each group was administered. If the patient could tolerate the drug within 7 days (35 mg and 70 mg groups) or 14 days (140 mg, 280 mg, and 420 mg groups) after administration, they could enter the next dose group at the discretion of the investigator. Each volunteer received only one corresponding dose.

[0064] Up to 34 healthy volunteers, both male and female, are planned to be enrolled. The design of each dose group is as follows:

[0065] 35 mg: Two volunteers are planned to be enrolled, both receiving a single subcutaneous injection of Antibody A. They will be hospitalized for observation until Day 6 after dosing and followed up until Day 29 after discharge. This dose group will only be evaluated for tolerability, safety, PD, and immunogenicity; its PK characteristics will not be assessed.

[0066] 70 mg: Four volunteers are planned to be enrolled and randomized in a 3:1 ratio to receive a single subcutaneous injection of Antibody A or placebo. Patients will be hospitalized for observation until Day 6 after dosing and followed up until Day 29 after discharge. This group of volunteers will undergo tolerability, safety, PK, PD, and immunogenicity assessments.

[0067] 140mg: 12 volunteers are planned to be enrolled and randomly receive antibody A or A single subcutaneous injection is required. Patients must be hospitalized for observation until day 3 after administration and followed up until day 113 after discharge. Volunteers in this dose group need to undergo tolerability, safety, PK, PD, and immunogenicity evaluations.

[0068] 280 mg: Four volunteers are planned to be enrolled and randomized in a 3:1 ratio to receive a single subcutaneous injection of Antibody A or placebo. Patients will be hospitalized for observation until Day 3 after dosing and followed up until Day 29 after discharge. This dose group will only be evaluated for tolerability, safety, PD, and immunogenicity; its PK characteristics will not be assessed.

[0069] 420mg: 12 volunteers are planned to be enrolled and randomly receive antibody A or A single subcutaneous injection is required. Patients must be hospitalized for observation until day 3 after administration and followed up until day 113 after discharge. Volunteers in this dose group need to undergo tolerability, safety, PK, PD, and immunogenicity evaluations.

[0070] Number of volunteers (planning and analysis):

[0071] A maximum of 34 healthy volunteers were planned to be enrolled. A total of 34 healthy volunteers participated in the trial, including 20 in the experimental drug group, 2 in the placebo group, and 12 in the positive control group. The distribution of volunteers is as follows:

[0072] Table 2 Distribution of volunteers

[0073] Note: The case numbers are all based on antibody A or express

[0074] Inclusion Criteria: Volunteers must meet all of the following criteria to be selected:

[0075] (1) Age 18 to 45 years old (including the critical value), both men and women;

[0076] (2) Male volunteers weighed ≥50.0 kg, female volunteers weighed ≥45.0 kg, and body mass index (BMI) was between 19.0 and 26.0 kg / m2, including the critical value;

[0077] (3) Volunteers voluntarily signed a written informed consent form.

[0078] Exclusion criteria: Volunteers who meet one or more of the following criteria will be excluded:

[0079] (1) Patients who have suffered from or are currently suffering from any serious clinical disease such as circulatory system, endocrine system, nervous system, digestive system, respiratory system, genitourinary system, hematology, immunology, psychiatry and metabolic disorders, or any other disease that may interfere with the test results;

[0080] (2) Those with a history of headache or migraine;

[0081] (3) Those with a history of recurrent nausea or vomiting;

[0082] (4) history of allergy to drugs, food, or other substances;

[0083] (5) Patients who have undergone surgery within 4 weeks before the trial, or plan to undergo surgery during the study, or have undergone surgery that may affect drug absorption, distribution, metabolism, or excretion;

[0084] (6) Those who have taken any medication or health products (including Chinese herbal medicine) within 14 days before the trial;

[0085] (7) Those who have used any drugs that inhibit or induce liver metabolism of drugs within 30 days before the trial (such as inducers - barbiturates, carbamazepine, phenytoin, glucocorticoids, omeprazole; inhibitors - SSRI antidepressants, cimetidine, diltiazem, macrolides, nitroimidazoles, sedatives and hypnotics, verapamil, fluoroquinolones, antihistamines, clarithromycin, nefazodone, ritonavir and atazanavir);

[0086] (8) Participated in any clinical trial and took any clinical trial drug within 3 months before the trial, or received any treatment targeting PCSK9 target;

[0087] (9) Those who donated blood or lost a large amount of blood (≥200 mL), received blood transfusion, or used blood products within 3 months before enrollment;

[0088] (10) Pregnant or breastfeeding women;

[0089] (11) Volunteers have unprotected sexual intercourse within 2 weeks before screening, and have fertility plans and are unwilling to take one or more non-drug contraceptive measures during the trial and within 6 months after the trial;

[0090] (12) Those who have special dietary requirements and cannot follow the unified diet;

[0091] (13) Those who drank excessive amounts of tea, coffee, or caffeinated beverages (more than 8 cups, 1 cup = 250 mL) every day within 3 months before the test;

[0092] (14) Smokers or those who smoked more than 5 cigarettes per day in the three months before the trial or those who could not stop using any tobacco products during the trial;

[0093] (15) Heavy drinkers (men who drink 5 or more units of alcohol within 2 hours, women who drink 4 or more units of alcohol within 2 hours) or regular drinkers within 6 months before the trial, that is, those who drink more than 14 units of alcohol per week (1 unit = 360 mL of beer or 45 mL of 40% alcohol liquor or 150 mL of wine) or those who cannot stop using any alcoholic products during the trial;

[0094] (16) Drug abusers or those who have used soft drugs (such as marijuana) within 3 months before the test or those who have taken hard drugs (such as cocaine, phencyclidine, etc.) within 1 year before the test;

[0095] (17) patients with abnormal vital signs (systolic blood pressure <90 mmHg or >140 mmHg, diastolic blood pressure <50 mmHg or >90 mmHg; heart rate <50 bpm or >100 bpm) or abnormalities in physical examination, electrocardiogram, or laboratory tests that are clinically significant (subject to the judgment of the clinical research physician);

[0096] (18) Those who cannot tolerate venipuncture blood collection;

[0097] (19) LDL-C level <1.8mmol / L or >4.14mmol / L at screening; (20) Volunteers who may not be able to complete this study due to other reasons or who the researchers believe should not be included.

[0098] Primary objective: To evaluate the safety and tolerability of a single dose of Antibody A in the dose range of 35mg to 420mg in healthy adult Chinese volunteers.

[0099] Secondary objective: To evaluate the pharmacokinetic and pharmacodynamic characteristics of antibody A in healthy adult Chinese volunteers after a single dose of 35 mg to 420 mg, and to preliminarily evaluate the immunogenicity of a single dose.

[0100] Test article, dosage and administration method:

[0101] Name: Recombinant fully human anti-PCSK9 monoclonal antibody injection (Antibody A)

[0102] Specifications: 140mg / ml (1ml)

[0103] Dosage: 35mg, 70mg, 140mg, 280mg, 420mg

[0104] Dosage method: After the volunteers fasted for at least 10 hours overnight, a single subcutaneous injection was given in the abdomen after eating a unified breakfast on the first day of the experiment.

[0105] Duration of treatment: The study duration for all volunteers will include a screening period of 1 to 21 days, a hospitalization observation period of 3 days (140 mg, 280 mg and 420 mg groups) / 6 days (35 mg and 70 mg groups) and a follow-up observation period of 23 days (35 mg, 70 mg and 140 mg groups) / 110 days (140 mg and 420 mg groups).

[0106] Positive control:

[0107] Name: Evolumab Injection

[0108] Specifications: 140mg / ml (1ml)

[0109] Dosage: 140mg, 420mg

[0110] Dosage method: After the volunteers fasted for at least 10 hours overnight, a single subcutaneous injection was given in the abdomen after eating a unified breakfast on the first day of the experiment.

[0111] Placebo:

[0112] Name: Recombinant fully human anti-PCSK9 monoclonal antibody injection (antibody A) mimetic

[0113] Dosage method: After the volunteers fasted for at least 10 hours overnight, a single subcutaneous injection was given in the abdomen after eating a unified breakfast on the first day of the experiment.

[0114] Evaluation criteria:

[0115] Pharmacokinetics: Test drug (antibody A and ) concentration.

[0116] Pharmacodynamic variables:

[0117] serum free PCSK9 concentration;

[0118] concentrations of blood lipid markers (total cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, small dense low-density lipoprotein cholesterol, non-high-density lipoprotein cholesterol, apolipoprotein A1, apolipoprotein B, and lipoprotein a);

[0119] Safety variables: adverse events, vital signs, physical examination, 12-lead electrocardiogram, clinical laboratory tests (blood routine, urine routine, blood biochemistry, blood lipid index, coagulation function), antidrug antibodies / neutralizing antibodies.

[0120] Summary Overview:

[0121] Safety conclusion: The MTD of Antibody A was not observed in this trial, and no serious adverse events occurred. A total of 81 TEAEs occurred in 85% of the volunteers, and the incidence of TEAEs of Antibody A was The most common TEAE was increased blood triglycerides, which may not be related to the test drug. All ADRs in Antibody A were ADR has been reported, and no unexpected adverse reactions occurred. There was no significant change in ADA before and after administration, and Nab was negative. In summary, Antibody A showed good safety and tolerability after a single dose.

[0122] Pharmacokinetic results

[0123] No descriptive statistical analysis was performed for the 70 mg dose group because there were only 3 volunteers. The main pharmacokinetic parameter results of 140mg and 420mg are shown in the table below:

[0124] Table 3 Main pharmacokinetic parameters (PKPS) of each drug group

[0125] As shown in Table 3, the efficacy results of this study show that the inhibition rate and duration of PCSK9 increase with the increase of the dose of Antibody A. Whether it is 140mg or 420mg, the exposure of Antibody A is significantly higher than

[0126] Efficacy results

[0127] Table 4 Changes in blood lipid indexes compared with baseline in different dose groups (PDPS)

[0128] As shown in Table 4, Antibody A has a significant effect on lowering LDL-C, and the duration of the effect is prolonged with increasing doses. In addition, the duration of the effect of Antibody A 140mg and 420mg on lowering LDL-C, total cholesterol and ApoB is also significantly longer than that of 140mg and 420mg.

[0129] Furthermore, by comparing the duration of the LDL-C lowering effect, the duration of the LDL-C lowering effect of Antibody A 140mg was significantly longer than that of At 140 mg, the median time for the two drugs to reach the minimum percentage change in LDL-C from baseline was 22 days and 11 days, respectively, suggesting that at a dose of 140 mg, Antibody A can support dosing once every 4 weeks. In the 420 mg dose group, the duration of Antibody A's LDL-C lowering effect was significantly longer than that of Antibody A. The median time to minimum percentage change in LDL-C from baseline for the two drugs was 43 days and 29 days, respectively, suggesting that at a dose of 420 mg, antibody A can support dosing once every 6 weeks or every 8 weeks.

[0130] Example 3

[0131] This study is a multicenter, randomized, double-blind, placebo / positive-controlled, multiple-dose study conducted in patients with hypercholesterolemia and mixed hyperlipidemia to evaluate the safety and tolerability of different dosing regimens of Antibody A combined with atorvastatin, describe the pharmacokinetic characteristics in patients, and preliminarily explore the efficacy.

[0132] This study plans to enroll a total of 36 subjects, both men and women, with hypercholesterolemia and combined hyperlipidemia. Two enrollment routes are designed for this study: In Route 1, subjects who have not been taking atorvastatin regularly and meet the screening criteria will be treated with a stable dose of atorvastatin monotherapy for 4 weeks during the run-in phase. Subjects who still meet the eligibility criteria after 4 weeks will enter the double-blind treatment phase. In Route 2, subjects who have been taking atorvastatin regularly for 4 weeks or more and meet the eligibility criteria will directly enter the double-blind treatment phase. Following the double-blind treatment phase, subjects in both routes will be randomly assigned to receive Antibody A 140 mg, Antibody A 420 mg, Evolocumab 420 mg, or placebo in a 5:5:5:3 ratio.

[0133] Primary objective: To evaluate the safety, tolerability and pharmacokinetic characteristics of multiple subcutaneous injections of antibody A combined with atorvastatin at different dosing regimens.

[0134] Secondary objective: To preliminarily explore the effectiveness of different dosing regimens of Antibody A combined with Atorvastatin.

[0135] Main inclusion criteria

[0136] Inclusion criteria:

[0137] 1. Applicants must be between 18 and 65 years old (inclusive), with no gender restrictions.

[0138] 2. 18.5kg / m 2 ≤BMI≤35kg / m 2 (BMI=weight / height2).

[0139] 3. Patients diagnosed with hypercholesterolemia and / or combined hyperlipidemia according to the diagnostic criteria of the Guidelines for the Prevention and Treatment of Dyslipidemia in Adults of China (revised in 2016).

[0140] 4. When screening, any one of the following conditions will suffice:

[0141] A: Execution route 1: At screening, subjects who are receiving any type of statin therapy and whose serum LDL-C is ≥2.6mmol / L and are willing to receive a stable dose of atorvastatin (the dose is determined by the investigator): LDL-C ≥2.6mmoIL before randomization;

[0142] B: Execution route 2: Subjects who have received a stable dose of atorvastatin for 28 days or more at screening have serum LDL-C ≥ 2.6 mmol / L; and LDL-C ≥ 2.6 mmol / L is still required before randomization.

[0143] 5. Fasting triglyceride ≤ 4.5mmol / L.

[0144] 6. The subjects fully understand the content of the trial and possible adverse reactions, voluntarily participate in the trial and voluntarily sign the written informed consent, and are able to comply with the research procedures specified in the research protocol.

[0145] Exclusion criteria

[0146] 1. Patients with homozygous familial hypercholesterolemia (HOFH); other diseases that may cause secondary increases in LDL-C include Cushing's syndrome, nephrotic syndrome, myeloma, glycogen storage disease, systemic lupus erythematosus, and acute intermittent porphyria.

[0147] 2. For Route 1, atorvastatin medication compliance during the run-in period was <80% or >120%; for Route 2, atorvastatin medication compliance within 28 days before screening was <80% or >120%.

[0148] 3. Patients with heart failure (New York Heart Association NYHA heart function class II and IV), acute coronary syndrome, percutaneous coronary intervention or other serious heart diseases [such as cardiogenic shock, I-III degree atrioventricular block, bradycardia (heart rate <50 beats / min), other serious arrhythmias, etc.] in the past 6 months.

[0149] 4. Patients with severe cerebrovascular diseases (hypertensive encephalopathy, cerebrovascular injury, stroke, transient ischemic attack, etc.), or severe aortic or peripheral vascular lesions, or indications for surgical intervention.

[0150] 5. Patients with poorly controlled hypertension (SBP>160mmHg or DBP>100mmHg).

[0151] 6. Diabetic patients with the following known conditions: 1) Type 1 diabetes patients 2) Type 2 diabetes patients with poor blood sugar control (HA1c 8.0%).

[0152] 7. Patients with active viral hepatitis (including hepatitis B and hepatitis C), other severe liver diseases, or hepatic insufficiency (ALT or AST>2.5 times the upper limit of normal, TBIL2 times the upper limit of normal).

[0153] 8. Screening period eGFR <45ml / min / 1.73m 2 .

[0154] 9. People with abnormal thyroid function (thyroid-stimulating hormone TSH exceeds the normal range of the center).

[0155] 10. Creatine kinase CK>3 times the upper limit of normal during the screening period.

[0156] 11. HIV and syphilis tests were positive.

[0157] 12. Patients with active malignant tumors or a history of malignant tumors within 5 years prior to screening (except basal cell carcinoma of the skin or carcinoma in situ of the cervix).

[0158] 13. Patients who are known to be allergic to the investigational drug or any of its ingredients, or who have had severe allergic reactions to other antibody drugs.

[0159] 14. Patients with a known history of organ transplantation.

[0160] 15. Those who have received inactivated virus vaccine within 1 month before screening, received adenovirus recombinant / mRNA vaccine 3 months before screening, or plan to receive vaccination during the trial.

[0161] 16. Those who donated blood or lost a large amount of blood (>400mL) within 3 months before screening, or those who were clinically diagnosed with hypovolemia.

[0162] 17. Use of any Chinese herbal medicine or tonic known to affect lipid metabolism (such as fish oil >1000 mg / day, drugs or health products containing red yeast rice) within 30 days before receiving the study drug.

[0163] 18. Participated in other drug or device clinical trials within 3 months before screening, or received any treatment targeting proprotein convertase subtilisin / kexin type 9 (PCSK9).

[0164] 19. History of drug abuse or alcoholism within 6 months prior to screening (drinking 14 units of alcohol per week: 1 unit = 285 mL of beer, or 25 mL of liquor, or 100 mL of wine).

[0165] 20. Female subjects are breastfeeding or have a positive serum pregnancy result during the screening period or the trial.

[0166] 21. The subject has plans to donate sperm or eggs within 6 months after signing the informed consent form.

[0167] 22. The subject and his / her spouse or partner have pregnancy plans within 6 months from the date of signing the informed consent form or do not agree to adopt acceptable and effective contraceptive methods.

[0168] 23. Patients who are deemed by researchers to be unsuitable for clinical trials.

[0169] Study duration (trial process)

[0170] The longest study duration for each subject in this study was approximately 22 weeks, including a screening period (2 weeks before dosing), a run-in period (4 weeks), and a double-blind treatment period (16 weeks).

[0171] 1. Screening period (Day -43 to Day -30 or Day -15 to Day -2)

[0172] After the target subject signs the informed consent form, at least one visit is required to complete the screening assessment: medical history, medication history, demographic data, vital signs, physical examination, laboratory tests at the research center (blood routine, urine routine, blood biochemistry, blood pregnancy test (only for women of childbearing age, four blood transfusion tests, thyroid function, coagulation function), abdominal B-ultrasound, chest X-ray, 12-lead electrocardiogram). Any test results that deviate from the normal range require the research physician to make a professional judgment on whether the result is clinically significant. The researcher must determine whether the subject has been taking atorvastatin regularly for 4 weeks or more before the screening period. If not, the subject will enter the run-in period according to route 1; if so, the subject will directly enter the double-blind treatment period according to route 2.

[0173] 2. Introduction period (Day -29 to Day -30): Only for subjects in route 1

[0174] In Route 1, researchers need to determine whether the subjects meet the conditions for entering the atorvastatin lead-in period to ensure that the subjects meet the requirements of the protocol and are suitable to start taking the lead-in medication.

[0175] In Route 1, subjects who were taking stable doses of other statins (except atorvastatin) before screening and who were judged by the investigator to be suitable for drug switching will enter the lead-in period and receive an appropriate dose of atorvastatin for 4 weeks; subjects who were already taking atorvastatin before screening but did not meet the regular requirements for 4 weeks can maintain the current dose for another 4 weeks.

[0176] After 28 days of the lead-in period, the subjects will be followed up to determine whether they can enter the randomized treatment period.

[0177] 3. Double-blind treatment period

[0178] Subjects in routes 1 and 2 who met the criteria for entering the double-blind treatment period were randomly administered Antibody A1 40 mg, Antibody A4 20 mg, Evolocumab 420 mg, and placebo in a ratio of 5:5:5:3 and continued treatment for 16 weeks. The details are as follows:

[0179] Antibody A 140 mg, Evolovumab 420 mg group: designed to be administered once every other week during atorvastatin basic treatment (4 doses on Day 1, Day 29, Day 57, and Day 85);

[0180] Antibody A 420 mg group: designed to be administered once every 8 weeks during atorvastatin-based treatment (Antibody A 420 mg administered on Day 1 and Day 57; placebo administered on Day 29 and Day 85);

[0181] Placebo group: The placebo group was designed to be administered once every 4 weeks during the basic treatment with atorvastatin (placebo was administered four times on Day 1, Day 29, Day 57, and Day 85).

[0182] Blood samples were collected from subjects at the prescribed time points for pharmacokinetic testing, at the time of the first dose on Day 1 and at Day 57. Changes in blood lipid profiles compared to baseline were compared after 16 weeks.

[0183] Results Overview:

[0184] Medication compliance: The trial drug in this study was an injection. The drug was administered by the research nurse at each visit. The medication compliance of each group was controlled. The Antibody A 140 mg group and the Evolocumab 420 mg group were both administered once every 4 weeks, and the Antibody A 420 mg group was administered once every 8 weeks. All subjects completed the medication according to the protocol plan.

[0185] All subjects in this study took atorvastatin as the basic medication. The compliance of 4 subjects in the Antibody A 140 mg group, 2 subjects in the Antibody A 420 mg group, and 2 subjects in the Evolocumab 420 mg group was less than 80%, while the compliance of other subjects in the above-mentioned dosage groups was ≥80%; the compliance of all subjects in the placebo group was ≥80%.

[0186] In terms of safety, combined with the analysis results of safety indicators (adverse events, SAEs, laboratory tests, vital signs, physical examinations, 12-lead electrocardiograms, and immunogenicity), there was no significant difference in the safety of the Antibody A 140 mg group and the Antibody A 420 mg group from the Evolocumab 420 mg group or the placebo group, indicating that the two dose groups of Antibody A were safe.

[0187] Pharmacokinetic parameter analysis

[0188] The concentration of free drug in serum was determined using a validated method, and the results are shown in Table 5.

[0189] Table 5 shows the pharmacokinetic parameter set (PKPS, N=20) for single and multiple doses of Antibody A.

[0190] As shown in Table 5, at the same dose, the peak concentration and time to peak of Antibody A were similar after single and multiple dosing, and there was no accumulation of Antibody A after multiple dosing. The pharmacokinetic profile of Antibody A was similar after single and multiple dosing: the in vivo exposure of Antibody A increased with increasing dose, and the half-life showed a certain dose-dependency. Exposure was greater than that of evolocumab at both 140 mg and 420 mg.

[0191] Lipid index efficacy endpoints

[0192] After 16 weeks of double-blind treatment, the changes in various blood lipid indicators relative to baseline were evaluated. The results are shown in Table 6.

[0193] Table 6 is a summary analysis of the changes in various indicators of the subjects after treatment compared with the baseline

[0194] As shown in Table 6, the rates of change of various indicators compared with the baseline in the Antibody A 140 mg (administered once every 4 weeks) and 420 mg groups (administered once every 8 weeks) were comparable to those in the Evolocumab 420 mg (administered once every 4 weeks) group, and were significantly higher than those in the placebo group, indicating that Antibody A can support less frequent administration or a lower dosage at the same administration frequency.

[0195] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for treating or preventing cholesterol-related diseases, characterized in that: include: 35-420 mg of a PCSK9 antibody is administered to the subject, wherein the heavy chain variable region of the PCSK9 antibody includes HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3; and the light chain variable region includes LCDR1 as shown in SEQ ID NO: 7, LCDR2 as shown in SEQ ID NO: 8, and LCDR3 as shown in SEQ ID NO:

9.

2. The method according to claim 1, characterized in that: The subject was administered 70 mg, 140 mg, 210 mg, 280 mg, 350 mg, 420 mg of PCSK9 antibody.

3. The method according to claim 1, characterized in that The subject is administered 70 mg of the PCSK9 antibody once per week or every two weeks, or 140 mg of the PCSK9 antibody is administered to the subject once every two weeks, every four weeks, every six weeks or every eight weeks, or 280 mg of the PCSK9 antibody is administered to the subject once every two weeks, every four weeks, every six weeks or every eight weeks, or 420 mg of the PCSK9 antibody is administered to the subject once every four weeks, every six weeks, every eight weeks or every twelve weeks.

4. The method according to any one of claims 1 to 3, characterized in that: The administration method is subcutaneous injection or intramuscular injection.

5. The method according to any one of claims 1 to 3, characterized in that: After 1-16 weeks of administration of 70 mg-420 mg of PCSK9 antibody to the subject, the plasma curve AUC 0-t At least greater than 5 million ng·h / mL.

6. The method according to any one of claims 1 to 3, characterized in that: After administering 140 mg to 420 mg of the PCSK9 antibody to the subject, the period of maintaining at least a -50% reduction in LDL-c is 25 to 60 days.

7. A method for treating or preventing cholesterol-related diseases, characterized in that: include: Subjects were administered clinical doses of statins and 35-420 mg of PCSK9 antibodies; Wherein, the heavy chain variable region of the PCSK9 antibody includes HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2 and HCDR3 as shown in SEQ ID NO: 3; the light chain variable region includes LCDR1 as shown in SEQ ID NO: 7, LCDR2 as shown in SEQ ID NO: 8 and LCDR3 as shown in SEQ ID NO:

9.

8. The method according to claim 7, characterized in that The subject was administered 70 mg, 140 mg, 210 mg, 280 mg, 350 mg, 420 mg of PCSK9 antibody.

9. The method according to claim 7, characterized in that: The subject is administered 70 mg of the PCSK9 antibody once a week or every two weeks, or 140 mg of the PCSK9 antibody is administered to the subject once every two weeks, every four weeks, every six weeks, or every eight weeks, or 280 mg of the PCSK9 antibody is administered to the subject once every two weeks, every four weeks, every six weeks, or every eight weeks, or 420 mg of the PCSK9 antibody is administered to the subject once every four weeks, every six weeks, every eight weeks, or every twelve weeks.

10. The method according to any one of claims 7 to 9, characterized in that: The PCSK9 antibody is administered by subcutaneous injection or intramuscular injection, and the statin is administered orally.

11. The method according to any one of claims 7 to 9, characterized in that: The statin is selected from atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin. When it is atorvastatin, its clinical dose is 10-80 mg, preferably 20 mg or 30 mg.

12. The method according to any one of claims 7 to 9, characterized in that: After 1-16 weeks of administration of 70 mg-420 mg of PCSK9 antibody to the subject, the plasma curve AUC 0-t At least greater than four million ng·h / mL.

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