Methods for reducing the risk of major adverse cardiovascular events in primary prevention subjects

Abstract

The present invention relates to methods for reducing the risk of major adverse cardiovascular events (MACE) in a subject, including administering an RNAi agent, RNAi agents and pharmaceutical compositions for use in such methods, and uses of RNAi agents for the manufacture of medicaments for such methods. Provided herein are particularly effective methods for preventing cardiovascular death, non-fatal myocardial infarction (MI), non-fatal ischemic stroke, or emergency coronary revascularization in subjects who have not experienced a major atherosclerotic cardiovascular disease (ASCVD) event, particularly those at high risk for a first MACE.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Patent Application No. 63 / 483,116, filed February 3, 2023, the entire disclosure of which is incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, the entire contents of which are incorporated herein by reference. The XML copy, created on February 1, 2024, is named PAT059477-WO-PCT_SL.xml and is 25 KB in size.

[0003] The present invention relates to methods for reducing the risk of major adverse cardiovascular events (MACE) in a subject, including administering an RNAi agent, and to RNAi agents and pharmaceutical compositions for use in such methods. Provided herein are methods that are particularly effective for preventing cardiovascular death, nonfatal myocardial infarction (MI), nonfatal ischemic stroke, or urgent coronary revascularization (4P-MACE) in subjects who have not experienced a major atherosclerotic cardiovascular disease (ASCVD) event but are at high risk for a first MACE. [Background technology]

[0004] Although significant improvements have been made in recent decades, cardiovascular disease (CVD) remains the leading cause of death worldwide, accounting for over 17 million deaths annually (WHO 2019). In 2019, 1,055,000 coronary events are expected to occur in the United States (US), including 720,000 new coronary events and 335,000 recurrent coronary events (Benjamin et al. 2019). These facts support the importance of preventing a first major ASCVD event in patients at high CV risk.

[0005] Long-term exposure to high low-density lipoprotein cholesterol (LDL-C) increases cumulative plaque burden and ASCVD risk, and lifetime exposure to high LDL-C, particularly long-term exposure to persistently elevated LDL-C, can cause acute coronary syndromes early in life and is a major risk factor for developing ASCVD. Thus, there is an unmet need to reduce the risk of major adverse cardiovascular events (MACE) in subjects (also known as primary prevention), particularly if the subject has not experienced a major ASCVD event, for example, if the subject has not experienced a major ASCVD event within about 12 months prior to administration of an RNAi agent as described herein. Summary of the Invention

[0006] Thus, the present invention addresses this need by providing a method for reducing the risk of major adverse cardiovascular events (MACE) in subjects who have not experienced a major atherosclerotic cardiovascular disease (ASCVD) event; MACE was defined as cardiovascular death, nonfatal myocardial infarction (MI), nonfatal ischemic stroke, or emergency coronary revascularization. The method includes administering to the subject an interfering ribonucleic acid (RNAi) agent, wherein the RNAi agent: an antisense strand comprising the nucleotide sequence 5'-asCfsaAfAfAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 3), and a sense strand comprising the nucleotide sequence 5'-csusagacCfuGfudTuugcuuuugu-3' (SEQ ID NO: 4), In the sequence, a, g, c, and u are 2'-O-methyl (2'-OMe) modified A, G, C, and U nucleotides, respectively; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, and U modified nucleotides, respectively; dT is a deoxy-thymine nucleotide, and s is a phosphorothioate linkage; and The sense strand is conjugated to at least one ligand.

[0007] The invention also provides RNAi agents for use in such methods, and pharmaceutical compositions comprising RNAi agents for use in such methods.

[0008] The invention also provides the use of an RNAi agent for the manufacture of a medicament for the method of the invention.

[0009] The inventors have found that administering an RNAi agent (e.g., inclisiran) to a subject as defined in the claims reduces the risk of 4P-MACE (first occurrence of CV death, non-fatal MI, non-fatal ischemic stroke, or emergency coronary revascularization) in subjects treated with the RNAi agent who are at high CV risk, with no prior major ASCVD events and LDL-C greater than or equal to 70 mg / dL (1.81 mmol / L) and less than 190 mg / dL (4.91 mmol / L). These methods are particularly useful for reducing the risk of 4P-MACE in subjects at high risk for first MACE. The inventors have also found that administering an RNAi agent (e.g., inclisiran) to a subject as defined herein reduces the risk of 3P-MACE (CV death, non-fatal MI, or non-fatal ischemic stroke) or CV death in the same subjects.

[0010] Embodiments of the present invention will now be described, by way of example only, with reference to the following drawings: [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows an overview of the screening algorithm for the screening strategy used in Example 1. [Figure 2] FIG. 2 is a diagram of the study design used in Example 1. [Figure 3] FIG. 3 illustrates the laboratory procedures for the analysis of the primary and secondary endpoints in the study according to Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0012] Treatment and prevention methods Cardiovascular disease (including ASCVD) or cardiovascular death is due to potentially modifiable risk factors. Elevated LDL-C is a major risk factor for developing ASCVD in a subject and leads to the onset of ASCVD. Atherosclerosis is a chronic, progressive disease. Atherosclerotic plaques form when fatty and fibrous materials accumulate in the innermost layer of arteries. ASCVD includes (1) coronary artery disease (ischemic heart disease); acute coronary syndromes (MI, unstable angina), and stable angina; (2) carotid and cerebral artery disease: ischemic stroke, transient ischemic attack (TIA); and (3) peripheral artery disease.

[0013] Long-term exposure to high LDL-C increases cumulative plaque burden and the risk of ASCVD, and lifetime exposure to high LDL-C, especially long-term exposure to persistently elevated LDL-C, can cause acute coronary syndrome early in life and is a major risk factor for the development of ASCVD.Therefore, there is an unmet need to reduce the risk of MACE in subjects, especially if the subject has not experienced a major ASCVD event or if the subject has not experienced a major ASCVD event within about 12 months before administration of an RNAi agent.The present invention addresses this need.

[0014] In some embodiments, the LDL-C level of the subject is reduced after administering RNAi agent.As described above, LDL-C is one of the most easily modifiable risk factors of ASCVD;However, despite the widespread use of statins, 4 out of 5 patients do not reach the LDL-C target recommended by guidelines.Inhibition of PCSK9 provides an effective approach to reduce LDL-C.

[0015] In one aspect, the present invention provides a method for reducing the risk of major adverse cardiovascular events (MACE) in subjects who have not experienced major atherosclerotic cardiovascular disease (ASCVD) events, comprising administering an interfering ribonucleic acid (RNAi) agent to the subject. The present invention is based, at least in part, on the use of small interfering RNA (siRNA) molecules (a type of RNAi agent) as a means of reducing PCSK9 protein levels and subsequently reducing LDL-C levels. Through RNA interference, siRNA molecules can bind to an RNA-induced silencing complex (RISC) in cells and cleave the messenger RNA (mRNA) encoding PCSK9. The cleaved mRNA is degraded, making it unavailable for protein translation, resulting in a decrease in PCSK9 protein levels and, consequently, a decrease in LDL-C levels. The inventors have found that administering an RNAi agent to reduce LDL-C levels in subjects who have not experienced major ASCVD events is effective in reducing the subject's risk of 4P-MACE. 4P-MACE is defined as a composite of cardiovascular death, non-fatal myocardial infarction (MI), non-fatal ischemic stroke, or urgent coronary revascularization (i.e., subjects undergoing urgent coronary revascularization, e.g., as required for subjects with coronary artery disease (CAD)). As one of skill in the art will recognize, in the context of the present invention, "composite" means the occurrence of any one of the specified component events.

[0016] Accordingly, the present invention provides a method for reducing the risk of a major adverse cardiovascular event (MACE), where MACE is cardiovascular death, non-fatal myocardial infarction (MI), non-fatal ischemic stroke, or emergency coronary revascularization, in a subject who has not experienced an ASCVD event, the method comprising administering to the subject an interfering ribonucleic acid (RNAi) agent as described herein.

[0017] Major adverse cardiovascular events (MACE) As will be apparent to one of skill in the art, a reduction in the risk of an event, e.g., a major adverse cardiovascular event (MACE), in a subject can also be described as: (a) a method for preventing an event (e.g., a MACE) in a subject; (b) Primary prevention method of events (e.g., MACE) in subjects; (c) a method for reducing the incidence of an event (e.g., a MACE) in a subject; or (d) A method of delaying the occurrence of an event (e.g., a MACE) in a subject.

[0018] All references herein to "reducing the risk" of an event may also be described as "preventing" such an event in all aspects of the invention, as will be apparent to those skilled in the art.

[0019] As will also be apparent to one of skill in the art, the reduction in risk of an event in a subject is typically assessed relative to subjects not receiving the RNAi agent, e.g., subjects receiving a placebo.

[0020] The methods of the invention, which involve administering an RNAi agent to a subject, are effective in reducing the risk of MACE in subjects who do not experience a major atherosclerotic cardiovascular disease (ASCVD) event, compared to subjects who do not receive the RNAi agent.

[0021] In some embodiments, the risk of 4P-MACE is reduced. 4P-MACE is a composite of cardiovascular death, non-fatal myocardial infarction (MI), non-fatal ischemic stroke, or emergency coronary revascularization. Reducing the risk of 4P-MACE means reducing the risk of any one of these events in a subject.

[0022] The present invention also provides a method for reducing the risk of cardiovascular death, non-fatal myocardial infarction (MI), non-fatal ischemic stroke, or emergency coronary revascularization in a subject.

[0023] The present invention also provides a method for reducing the risk of other types of MACE in subjects who have not experienced a major atherosclerotic cardiovascular disease (ASCVD) event. For example, the inventors have found that administering an RNAi agent (e.g., inclisiran) to a subject as defined herein reduces the risk of 3P-MACE (cardiovascular death, non-fatal myocardial infarction, or non-fatal ischemic stroke) or the risk of cardiovascular death.

[0024] The reduction in the risk of MACE in a subject can be quantified using the hazard rate of MACE (e.g., 4P-MACE or 3P-MACE), which is the probability that a MACE will occur in the next time interval, if the MACE has not already occurred, divided by the length of that time interval.

[0025] The reduction in the risk of MACE in a subject can also be quantified by determining the mean time to onset of MACE (e.g., 4P-MACE or 3P-MACE) in subjects administered an RNAi agent according to the methods of the invention, compared to the mean time to onset of MACE in subjects not administered the RNAi agent. For example, if the mean time to onset of MACE in subjects administered an RNAi agent according to the methods of the invention is longer than the mean time to onset of MACE in subjects not administered the RNAi agent, the risk of MACE is reduced.

[0026] In some embodiments, a reduced risk of MACE (herein, MACE is cardiovascular death, non-fatal myocardial infarction (MI), non-fatal ischemic stroke, or emergency coronary revascularization) in subjects who do not experience a major atherosclerotic cardiovascular disease (ASCVD) event is defined by a reduction in the hazard rate in subjects administered the RNAi agent compared to subjects not administered the RNAi agent. In some embodiments, the risk is defined by about a 20% reduction in the hazard rate in subjects administered the RNAi agent compared to subjects not administered the RNAi agent.

[0027] Reducing the risk of other events In a further embodiment that can be combined with any of the other embodiments described herein, the present invention also provides methods for reducing the risk of a major atherosclerotic cardiovascular disease (ASCVD) event in a subject who has not experienced such an event, as further described below.These methods include administering to a subject an RNAi agent described herein, which is effective in reducing the risk of a major atherosclerotic cardiovascular disease (ASCVD) event in a subject who has not experienced such an event, compared to a subject who has not been administered the RNAi agent.

[0028] The exemplary events according to these aspects of the present invention are described below.Those skilled in the art can determine whether the risk of an event is reduced by, for example, determining the mean time to the first occurrence of the event in the subject that is administered with RNAi agent according to the present invention, compared with the mean time to the first occurrence of the event in the comparable subject that is not administered with RNAi agent.These methods include administering the RNAi agent described herein to subject.

[0029] In some embodiments, the present invention provides methods for reducing the risk of a CV event, such as a first or recurrent non-fatal MI, a non-fatal ischemic stroke, an emergency revascularization procedure, hospitalization for unstable angina, hospitalization for heart failure, or a major adverse lower extremity event (MALE).

[0030] In some aspects, the present invention provides methods for reducing the risk of emergency coronary revascularization.

[0031] In some aspects, the present invention provides methods for reducing the risk of all-cause mortality.

[0032] In some embodiments, the present invention provides methods for reducing the risk of CV death, non-fatal MI, non-fatal ischemic stroke, and emergency coronary revascularization (first or recurrent).

[0033] In some embodiments, the present invention provides methods for reducing the risk of CV death, non-fatal MI, and non-fatal ischemic stroke (first or recurrent).

[0034] In some aspects, the invention provides, for example, methods of reducing the risk of a coronary heart disease (CHD) event, e.g., a CHD event being CHD death, a non-fatal MI, unstable angina requiring hospitalization, or emergency coronary revascularization.

[0035] In some embodiments, the present invention provides methods for reducing the risk of CV death.

[0036] In some embodiments, the present invention provides methods for reducing the risk of fatal or non-fatal MI.

[0037] In some aspects, the present invention provides methods for reducing the risk of fatal or non-fatal ischemic stroke.

[0038] In some embodiments, the present invention provides methods for reducing the risk of all-cause mortality, non-fatal MI, or non-fatal ischemic stroke.

[0039] In some embodiments, the present invention provides methods for reducing the risk of all-cause mortality, non-fatal MI, non-fatal ischemic stroke, or emergency coronary revascularization.

[0040] In some aspects, the present invention provides methods for reducing the risk of a major adverse lower extremity event (MALE), for example, a MALE being acute lower extremity ischemia, lower extremity amputation due to ischemia, or urgent lower extremity revascularization for ischemia.

[0041] In some aspects, the invention provides methods of reducing the risk of first or recurrent all-cause revascularization, for example, where the revascularization is coronary, carotid, or peripheral revascularization.

[0042] In some embodiments, the present invention provides methods for reducing the risk of first or recurrent total coronary events, eg, CHD death, MI, hospitalization for unstable angina, and / or urgent / elective revascularization.

[0043] In some aspects, the present invention provides methods for reducing the risk of first or recurrent all-cause CV hospitalization, eg, hospitalization due to myocardial infarction and hospitalization due to unstable angina.

[0044] In some aspects, the present invention provides methods for reducing the risk of new or recurrent venous thromboembolism resulting in hospitalization or death.

[0045] In a further aspect, the present invention provides a method for reducing the level of LDL-C and other lipid parameters in a subject compared to baseline. For example, the lipid parameters can include one or more of: total cholesterol, high-density lipoprotein cholesterol (HDL-C), very-low-density lipoprotein cholesterol (VLDL-C), non-high-density lipoprotein cholesterol (non-HDL-C), triglycerides, and lipoprotein(a) (Lp(a)). The reduction in the level of LDL-C or other lipid parameters in a subject can be determined by comparing the percent change or absolute change in the level of LDL-C and other lipid parameters compared to baseline at various time points, e.g., time points (e.g., V1, V2, V3, etc.), as shown in the assessment schedule (activity schedule) of Example 1.

[0046] In exemplary embodiments of all aspects of the invention, subjects administered an RNAi agent have a low risk of an adverse event (AE) or serious adverse event (SAE). For example, a subject administered an RNAi agent has a low risk of an AE or SAE if the incidence or overall incidence of an AE or SAE in subjects administered an RNAi agent in accordance with the invention is no greater than the incidence or overall incidence of AEs or SAEs in subjects not administered the RNAi agent, e.g., subjects administered a placebo.

[0047] Reduction of LDL-C and PCSK9 levels In some embodiments of the invention, administration of an RNAi agent reduces LDL-C levels in a subject. In exemplary embodiments, administration of an RNAi agent reduces the level of LDL-C in a subject by more than about 20%, or more than about 25%, or more than about 30%, or more than about 35%, or more than about 40%, or more than about 45%, or more than about 50%, or more than about 55%, or more than about 60% compared to baseline LDL-C levels.

[0048] In some embodiments, the reduction in LDL-C levels following administration of the RNAi agent can be maintained for about 30 days or more, 60 days or more, 90 days or more, 180 days or more, 1 year or more, 2 years or more, 3 years or more, 4 years or more, 5 years or more, or 6 years or more, or for any period therebetween.

[0049] In an exemplary embodiment, these reductions in LDL-C levels can be achieved, for example, by administering about 284 mg of inclisiran (equivalent to about 300 mg of inclisiran sodium) subcutaneously, which method comprises: (a) a loading phase involving the administration of inclisiran as two doses spaced approximately 90 days (3 months) apart, followed by (b) a maintenance phase comprising the administration of one or more doses of inclisiran, each dose spaced apart by approximately 180 days (6 months). As explained further below, this time interval can be varied to account for missed doses.

[0050] Administration of the RNAi agent also reduces the level of PCSK9 in the subject. In some embodiments, administration of the RNAi agent can reduce the level of PCSK9 by more than about 25%, or more than about 30%, or more than about 35%, or more than about 40%, or more than about 45%, or more than about 50%, or more than about 55%, or more than about 60%, or more than about 65%, or more than about 70%, or more than about 75%, or more than about 80%, or more than about 85%, or more than about 90%, or more than about 95%, compared to the baseline level of PCSK9, or compared to the level of PCSK9 before administration of the RNAi agent.

[0051] In some embodiments, the reduction in the level of PCSK9 following administration of the RNAi agent can be maintained for about 30 days or more, 60 days or more, 90 days or more, 180 days or more, 1 year or more, 2 years or more, 3 years or more, 4 years or more, 5 years or more, or 6 years or more, or for any period therebetween.

[0052] In an exemplary embodiment, these reductions in PCSK9 levels can be achieved, for example, by administering about 284 mg of inclisiran subcutaneously (equivalent to about 300 mg of inclisiran sodium), which method comprises: (a) a loading phase involving the administration of inclisiran as two doses spaced approximately 90 days (3 months) apart, followed by (b) a maintenance phase comprising the administration of one or more doses of inclisiran, each dose spaced apart by approximately 180 days (6 months).

[0053] Treatment target The methods of the present invention are useful for reducing the risk of MACE in subjects who have not experienced a major atherosclerotic cardiovascular disease (ASCVD) event.

[0054] Subjects at high risk of first MACE These methods are particularly useful for reducing the risk of MACE in subjects at high risk for a first MACE.

[0055] High risk of a first MACE is defined as any one of the following: a. Evidence of atherosclerotic CAD on CT or invasive coronary angiogram defined as coronary artery stenosis ≥ 20% and < 50% in the left main coronary artery or stenosis ≥ 20% and < 70% in the main epicardial coronary arteries; b. Coronary artery calcium (CAC) score of 100 Agatston units or greater obtained by CT scan; c.High 10-year ASCVD risk of 20% or more (assessed by pooled cohort equation); d. Moderate 10-year ASCVD risk of 7.5% to <20% (assessed by pooled cohort equations) with at least two of the following risk-enhancing factors: i. First-degree relatives with a history of early ASCVD (men under 55 years of age; women under 65 years of age); ii. Early puberty before age 40; iii. History of maternal pregnancy complications (defined as a history of either preeclampsia, gestational hypertension, or gestational diabetes); iv. South Asian; v. chronic inflammatory disease (defined as any of rheumatoid arthritis, systemic lupus erythematosus, psoriasis covering 10% or more of the body surface area, Crohn's disease, ulcerative colitis, or HIV / AIDS); vi. High-sensitivity C-reactive protein (hsCRP) ≥ 2 mg / L in the absence of an underlying acute or inflammatory condition (including any of the chronic inflammatory diseases listed in criterion dv above); vii. Lipoprotein(a) ≥ 50 mg / dL or ≥ 125 nmol / L; viii. Estimated glomerular filtration rate (eGFR) is 60 mL / min / 1.73 m 2 less than; ix. Ankle-brachial index (ABI) less than 0.9 and no symptoms of intermittent claudication; or x.Metabolic syndrome.

[0056] One of ordinary skill in the art can determine whether a subject is at high risk for a first MACE as defined by the above criteria.

[0057] For example, Achenbach and Raggi (2010) review exemplary methods for determining evidence of atherosclerotic CAD on CT or invasive coronary angiograms and determining a CAC score obtained by CT scan.

[0058] The pooled cohort equations for assessing ASCVD risk are described in further detail below.

[0059] The diagnostic criteria for metabolic syndrome are also described in more detail below.

[0060] The levels of high-sensitivity C-reactive protein (hsCRP), lipoprotein(a), estimated glomerular filtration rate (eGFR), and LDL-C in a subject can be determined by one of skill in the art using routine tests, such as commercially available immunoassays or commercially available tests (e.g., lipid panels), to measure the levels of these substances in the subject (e.g., in serum obtained from the subject).

[0061] One skilled in the art can also calculate a subject's Ankle Brachial Index (ABI), for example, by measuring blood pressure in the arteries of the subject's ankle and arm.

[0062] In some embodiments, the subject has not experienced a MACE at any time prior to administration of the RNAi agent.

[0063] In some embodiments, the subject has an LDL-C level greater than or equal to 70 mg / dL (1.81 mmol / L) and less than 190 mg / dL (4.91 mmol / L).

[0064] In some embodiments, the subject is a mammal, e.g., a primate. In preferred embodiments of all aspects of the invention, the subject is a human. In some embodiments, the subject is referred to as a patient.

[0065] In some embodiments, the subject is an adult (eg, an adult human) aged, for example, 40 years or older, and optionally less than 80 years.

[0066] Background lipid-lowering therapy, additional PCSK9-targeted therapy, other therapies In some embodiments, the subject is or has been treated with background lipid-lowering therapy (LLT). In other embodiments, the subject is or has not been treated with background lipid-lowering therapy (LLT). As described herein, the subject can be maintained at background LLT while administering an RNAi agent to the subject. The background LLT can be a statin and / or ezetimibe. The background statin LLT can be medium-intensity or high-intensity.

[0067] In some embodiments, the subject has not been previously treated with an additional PCSK9-targeted therapy prior to administration of the RNAi agent. In other embodiments, the subject has been previously treated with an additional PCSK9-targeted therapy prior to administration of the RNAi agent. Exemplary additional PCSK9-targeted therapies include PCSK9-blocking monoclonal antibodies (mAbs) that lower LDL-C levels, such as alirocumab or evolocumab, which result in lower LDL-C levels.

[0068] The RNAi agent (e.g., inclisiran) can be administered shortly after the last administration of the monoclonal antibody PCSK9 inhibitor. Preferably, the RNAi agent (e.g., inclisiran) is administered within two weeks of the last administration of the monoclonal antibody PCSK9 inhibitor.

[0069] In some embodiments, the subject has not been treated with lomitapide and / or mipomersen.

[0070] Pooled cohort equation Pooled cohort equations (PCEs) were introduced in the 2013 ACC / AHA guidelines on CV risk as a sex- and race-specific tool for estimating 10-year absolute rates of ASCVD events in primary prevention populations (Goff et al., 2014). These equations were derived from extensive data from several large, racially and geographically diverse, recent NHLBI-sponsored cohort studies, including the Atherosclerosis Risk in Communities (ARIC) study, the Cardiovascular Health Study, and the Coronary Artery Risk Development in Young Adults (CARDIA) study, combined with applicable data from the Framingham Original and Offspring Study cohort. Risk estimates are based on a combination of established CV risk factors prospectively examined in the specific cohorts selected for PCE derivation. Where appropriate, an ASCVD risk score is calculated using the PCE equation on the eCRF for inclusion criteria assessment.

[0071] The variables included in the PCE are: Current age ·Gender (male, female) Race (White, African American, Other) Smoking status (current, former, never) Systolic blood pressure (mmHg) Treatment status for hypertension (yes, no) History of diabetes (yes, no) Total cholesterol and HDL cholesterol levels (mg / dL or mmol / L)

[0072] Further information on the ASCVD risk tool can be found in Lloyd-Jones et al., 2018. This tool estimates 10-year ASCVD risk for adults aged 40–79 years.

[0073] Clinical diagnostic criteria for metabolic syndrome Metabolic syndrome is a risk factor for ASCVD and is diagnosed by the presence of any three of the following five risk factors:

[0074] [Table 1]

[0075] These risk factors can be measured or determined in a subject by methods known to those of skill in the art.

[0076] Primary prevention As noted above, the methods of the present invention are useful for reducing the risk of MACE in subjects who have not experienced a major atherosclerotic cardiovascular disease (ASCVD) event, also known by those skilled in the art as "primary prevention."

[0077] A major ASCVD event can be defined as any one of the following: a. Acute coronary syndrome (ACS) at any time prior to administration of the RNAi agent, e.g., ACS within about 12 months prior to administration of the RNAi agent; b. Myocardial infarction at any time point before administration of the RNAi agent; c. Ischemic stroke at any time prior to administration of the RNAi agent; or d. Symptomatic peripheral arterial disease (PAD) as evidenced by, for example, either intermittent claudication, previous revascularization, or amputation due to atherosclerosis at any time prior to administration of the RNAi agent.

[0078] In an exemplary embodiment, the subject has not experienced major ASCVD within about 12 months prior to administration of the RNAi agent.

[0079] Additional factors may indicate that a subject is not suitable for the methods of the invention. The following embodiments refer to these factors to define subjects that are particularly suitable for the methods of the invention.

[0080] In some embodiments, the subject has no history of ischemic revascularization in the coronary or extracoronary arterial bed prior to administration of the RNAi agent.

[0081] In some embodiments, the subject is free of coronary atherosclerosis by CT angiogram or invasive coronary angiogram for two years prior to administration of the RNAi agent.

[0082] In some embodiments, the subject has not had a coronary artery calcium (CAC) score of 0 obtained in the two years prior to administration of the RNAi agent.

[0083] In some embodiments, the subject does not have active liver disease or liver dysfunction, as identified, for example, as having aspartate aminotransferase (AST) or alanine aminotransferase (ALT) greater than or equal to three times the upper limit of normal (ULN).

[0084] Target subgroups The methods of the present invention may be particularly suitable or effective for particular subgroups of subjects.

[0085] In some embodiments, the subject may have ASCVD, an equivalent of ASCVD risk, a high risk of cardiovascular disease (CVD) in which low-density lipoprotein cholesterol (LDL-C) needs to be lowered, heterozygous familial hypercholesterolemia, or homozygous familial hypercholesterolemia, or otherwise have a disease that would benefit from LDL-C lowering, or a combination thereof.

[0086] In certain embodiments, a subject may be at "equivalent risk to ASCVD" if they have one or more of symptomatic atherosclerosis, type 2 diabetes, familial hypercholesterolemia, including subjects with a target LDL-C of less than 100 mg / dL and a 10-year risk of a cardiovascular event as assessed by a Framingham risk score (score > 20%) or equivalent. In some embodiments, a subject may have heterozygous familial hypercholesterolemia (HeFH).

[0087] The Framingham risk score is a method known to those skilled in the art that is used to estimate one-year cardiovascular disease risk, taking into account factors such as age, HDL-C level, total cholesterol level, systolic blood pressure, and smoking and diabetes history, and is further described in D'agostino et al. 2008.

[0088] Pooled cohort equations can also be used to estimate ASCVD risk.

[0089] In some embodiments, the subject may be under 65 years of age, between 65 and 74 years of age, or at least 75 years of age. The subject may be male or female.

[0090] In some embodiments, the subject is 2 Less than 30-35kg / m 2 Less than or at least 35 kg / m 2 have a baseline body mass index (BMI) of

[0091] In some embodiments, the subject has or has had diabetes.

[0092] In some embodiments, the subject is a current or former smoker.

[0093] In some embodiments, the subject has or has had hypertension.

[0094] In some embodiments, the subject has a baseline LDL-C level of less than 100 mg / dL, or at least 100 mg / dL.

[0095] In some embodiments, the subject has a baseline triglyceride level of less than 150 mg / dL, between 150 and 200 mg / dL, or at least 200 mg / dL.

[0096] In some embodiments, the subject has a baseline high-sensitivity C-reactive protein (hs-CRP) level of less than or at least 2 mg / dL.

[0097] In some embodiments, the subject is on high-intensity or moderate-intensity baseline statin treatment, or is statin-naive.

[0098] In some embodiments, the subject may have a risk of developing heart health problems, which may include: acute coronary syndrome; a history of myocardial infarction, stable or unstable angina, coronary artery or other arterial revascularization, stroke, transient ischemic attack, or peripheral arterial disease of atherosclerotic origin; being male; having a family history of heart disease, ASCVD, or ASCVD-like risk; smoking; being physically inactive; having high blood pressure; having high blood cholesterol; having diabetes or prediabetes; being overweight or obese; having a history of preeclampsia during pregnancy; being unable to control stress and / or anger; being postmenopausal; having an unhealthy diet, for example, a diet high in salt, saturated fat, trans fat, cholesterol, and / or refined sugar; being 55 years or older; having sleep apnea syndrome; having anemia; or a combination thereof.Therefore, this method can be applied to, for example, a young subject with a family history of heart disease, or a subject who smokes but does not have high blood pressure.

[0099] Other interventions The subject treated by the method of the present invention can be treated with background LLT throughout the period that RNAi agent is administered.In addition, additional risk-based cardiovascular disease (CVD) prevention interventions can be implemented in the subject, such as education on healthy eating, exercise, and weight management, or other lifestyle changes.The subject can also be receiving statin therapy at the maximum tolerated dose.The maximum dose of statin is known in the art.

[0100] Such healthy diets are known in the art, such as diets designed to improve lipid levels. For example, diets designed to improve lipid levels can include eating lean meat; removing fat from meat; not eating fried foods and not using high-fat sauces; not eating egg yolks; using low-fat dairy products such as skim milk and 1% milk, low-fat frozen yogurt, low-fat ice cream, and low-fat cheese; and eating foods that are sources of fiber, such as fruits and vegetables.

[0101] The subject may require further reduction in LDL-C levels.

[0102] RNAi agents The RNAi agent used in the methods of the present invention is a double-stranded RNAi agent that is capable of inhibiting the expression of proprotein convertase subtilisin kexin 9 (PCSK9) in cells.

[0103] In some aspects of the invention, the RNAi agent is a double-stranded ribonucleic acid comprising a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 1 and the sense strand comprises the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the antisense strand comprises the nucleotide sequence of SEQ ID NO: 3 and the sense strand comprises the nucleotide sequence of SEQ ID NO: 4.

[0104] In some embodiments of the invention, the RNAi agent comprises an antisense strand comprising the nucleotide sequence 5'-asCfsaAfAfAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 3), and a sense strand comprising the nucleotide sequence 5'-csusagacCfuGfudTuugcuuuugu-3' (SEQ ID NO: 4), In the sequence, a, g, c, and u are 2'-O-methyl (2'-OMe) modified A, G, C, and U nucleotides, respectively; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, and U modified nucleotides, respectively; dT is a deoxy-thymine nucleotide, and s is a phosphorothioate linkage; and the sense strand is conjugated to at least one ligand.

[0105] In some embodiments of the invention, the RNAi agent comprises an antisense strand consisting of the nucleotide sequence 5'-asCfsaAfAfAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 3), and a sense strand consisting of the nucleotide sequence 5'-csusagacCfuGfudTuugcuuuugu-3' (SEQ ID NO: 4), In the sequence, a, g, c, and u are 2'-O-methyl (2'-OMe) modified A, G, C, and U nucleotides, respectively; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, and U modified nucleotides, respectively; dT is a deoxythymine nucleotide, and s is a phosphorothioate linkage; and the sense strand is conjugated to at least one ligand.

[0106] In some embodiments, the ligand has the following structure: [ka] This structure represents a triantennary N-acetylgalactosamine (GalNAc) that promotes the uptake of RNAi agents by hepatocytes. In hepatocytes, RNAi agents utilize the RNA interference mechanism to induce catalytic degradation of PCSK9 mRNA. This increases the recycling and expression of LDL-C receptors on the hepatocyte surface, resulting in increased LDL-C uptake and reduced circulating LDL-C levels.

[0107] In a further embodiment, the ligand is conjugated to the 3' end of the sense strand.

[0108] RNAi agents have the following structural formula: [ka] (wherein X is O or S) In a preferred embodiment, X is O.

[0109] Preferably, the RNAi agent is inclisiran. Inclisiran is a preferred RNAi agent in all aspects of the present invention. Inclisiran is a double-stranded RNAi agent that is a small interfering RNA (siRNA) directed against PCSK9 (proprotein convertase subtilisin kexin type 9) mRNA. As described above, inclisiran is conjugated to triantennary N-acetylgalactosamine (GalNAc) at the 3' end of the sense strand to facilitate uptake by hepatocytes. With one exception, the 2' ribose moiety of inclisiran exists as a 2'-F or 2'-OMe ribonucleotide. In addition, the six terminal phosphodiester backbones exist as phosphorothioate linkages, as shown below.

[0110] Inclisiran is described in Recommended INN: List 76, pages 504-506 of WHO Drug Information, Vol. 30, No. 3, 2016, which is incorporated by reference.

[0111] The chemical name for inclisilane is: [(2S,4R)-1-{1-[(2-acetamido-2-deoxy-β-D-galactopyranosyl)oxy]-16,16-bis({3-[(3-{5-[(2-acetamido-2-deoxy-β-D-galactopyranosyl)oxy]pentanamido}propyl)amino]-3-oxopropoxy}methyl)-5,11,18-trioxo-14-oxa-6,10,17-triazanonacosan-29-yl}-4-hydroxypyrrolidin-2-yl]methyl hydrogen All-P-ambo-2'-O-methyl-P-thiocytidyl- (3'→5')-2'-O-methyl-P-thiouridylyl- (3'→5')-2'-O-methyladenylyl- (3'→5')-2'-O-methylguanylyl- (3'→5')-2'-O-methyladenylyl- (3'→5')-2'-O-methylcytidylyl- (3'→5')-2'-deoxy-2'-fluorocytidylyl- (3'→5')-2'-O-methyluridylyl- (3'→5')-2'-deoxy-2'-fluoroguanylyl- (3'→5')-2'-O-methyluridylyl- (3'→5')-Thymidylyl- (3'→5')-2'-O-methyluridylyl- (3'→5')-2'-O-methyluridylyl- (3'→5')-2'-O-methylguanylyl- (3'→5')-2'-O-methylcytidylyl- (3'→5')-2'-O-methyluridylyl- (3'→5')-2'-O-methyluridylyl- (3'→5')-2'-O-methyluridylyl- (3'→5')-2'-O-methyluridylyl- (3'→5')-2'-O-methylguanylyl- (3'→5')-2'-O-methyl-3'-uridylic acid and All-P-ambo-2'-O-methyl-P-thioadenylyl- (5'→3')-2'-O-methyl-P-thioadenylyl- (5'→3')-2'-O-methylguanylyl- (5'→3')-2'-O-methyladenylyl- (5'→3')-2'-O-methyluridylyl- (5'→3')-2'-deoxy-2'-fluorocytidylyl- (5'→3')-2'-O-methyluridylyl- (5'→3')-2'-deoxy-2'-fluoroguanylyl- (5'→3')-2'-O-methylguanylyl- (5'→3')-2'-deoxy-2'-fluoroadenylyl- (5'→3')-2'-O-methylcytidylyl- (5'→3')-2'-deoxy-2'-fluoroadenylyl- (5'→3')-2'-O-methyladenylyl- (5'→3')-2'-deoxy-2'-fluoroadenylyl- (5'→3')-2'-O-methyladenylyl- (5'→3')-2'-deoxy-2'-fluorocytidylyl- (5'→3')-2'-O-methylguanylyl- (5'→3')-2'-deoxy-2'-fluoroadenylyl- (5'→3')-2'-deoxy-2'-fluoroadenylyl- (5'→3')-2'-deoxy-2'-fluoroadenylyl- (5'→3')-2'-O-methyl-P-thioadenylyl- (5'→3')-2'-Deoxy-2'-fluoro-P-thiocytidylyl- It is a double strand of (5'→3')-2'-O-methyladenosine.

[0112] In the chemical names of inclisilanes (above), the notation (3'→5') or (5'→3') refers to the direction of the internucleoside bond (i.e., the direction of the interconnecting phosphate bond, e.g., phosphodiester or phosphorothioate, respectively), as in the IUPAC rules for nucleic acids, and not to the end of the chain (e.g., antisense or sense strand) having the third or fifth carbon in the sugar ring at its end.

[0113] The molecular formula of inclisilane is C 529 H 707 F 12 N 176 O 316 P 43 It's S6.

[0114] The structure of inclisilane is shown in the following structural formula: [ka]

[0115] The illustration below is another representation of the structure of inclisilane: [ka] Abbreviations: Af = adenine 2'-F ribonucleotide; Cf = cytosine 2'-F ribonucleotide; Gf = guanine 2'-F ribonucleotide; Am = adenine 2'-OMe ribonucleotide; Cm = cytosine 2'-OMe ribonucleotide; Gm = guanine 2'-OMe ribonucleotide; Um = uracil 2'-OMe ribonucleotide; s is a phosphorothioate bond.

[0116] Pharmaceutical Compositions and Administration Salt Form Each dose of an RNAi agent administered can include a salt form of the RNAi agent.

[0117] As used herein, the term "salt" or "salts" refers to acid addition salts or base addition salts of the RNAi agents described herein. "Salt" specifically includes "pharmaceutically acceptable salts." The term "pharmaceutically acceptable salts" refers to salts that retain the biological effectiveness and properties of the RNAi agents described herein and are typically biologically and otherwise acceptable. In many cases, the RNAi agents described herein can form base salts due to the presence of acidic protons, for example, as found in carboxylic acids or phosphoric acids or other groups similar thereto.

[0118] Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.

[0119] Inorganic bases from which salts can be derived can include, for example, ammonium salts and metals from columns I-XII of the periodic table. In particular embodiments, salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium, potassium, sodium, calcium, and magnesium salts.

[0120] Organic bases from which salts can be derived can include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, etc. Particular organic amines include isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.

[0121] In another aspect, the RNAi agents described herein are in the form of a salt of sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, copper, isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine, or tromethamine.

[0122] In some embodiments, the RNAi agent is present in the administered dose as a salt, for example, a sodium salt or a potassium salt. Sodium salts are the preferred salt form of the RNAi agent, but other pharmaceutically acceptable salts, for example, potassium salts or ammonium salts, can also be used. Preferably, the RNAi agent is an inclisiran salt (e.g., inclisiran sodium, inclisiran potassium, or inclisiran ammonium). More preferably, the RNAi agent is inclisiran sodium (also known as inclisiran sodium salt), but other pharmaceutically acceptable salts can also be used.

[0123] The molecular formula of inclisilane sodium is C 529 H 664 F 12 N 176 Na 43 O 316 P43 S6 and has a molecular weight of 17,284.72 g / mol. Inclisilane sodium has the following structural formula: [ka] Abbreviations: Af = adenine 2'-F ribonucleotide; Cf = cytosine 2'-F ribonucleotide; Gf = guanine 2'-F ribonucleotide; Am = adenine 2'-OMe ribonucleotide; Cm = cytosine 2'-OMe ribonucleotide; Gm = guanine 2'-OMe ribonucleotide; Um = uracil 2'-OMe ribonucleotide; s is a phosphorothioate bond.

[0124] RNAi agents and pharmaceutical compositions for use The present invention also provides an RNAi agent for use in any of the methods of the present invention. Also provided are pharmaceutical compositions comprising an RNAi agent for use in any of the methods of the present invention.

[0125] In addition to the RNAi agent or its salt, these pharmaceutical compositions may also contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other substances known to those skilled in the art. Such materials are non-toxic and do not interfere with the effectiveness of the RNAi agent. In a preferred embodiment, the pharmaceutical composition for use contains water for injection. In a further preferred embodiment, the pharmaceutical composition for use contains water for injection and sodium hydroxide and / or phosphoric acid.

[0126] Dosage and Administration Schedule dose The methods of the present invention include administering an RNAi agent in one or more doses. The RNAi agent may be provided in a therapeutically or prophylactically effective amount. In all aspects of the present invention, those skilled in the art will recognize that administering an RNAi agent can include administering a therapeutically or prophylactically effective amount of the RNAi agent to a subject in each dose.

[0127] Reference to "therapeutically effective amount" or "prophylactically effective amount" refers to administering an appropriate amount of RNAi agent according to the method of the present invention to achieve the effect of reducing the risk of major adverse cardiovascular events (MACE) in subjects who have not experienced major atherosclerotic cardiovascular disease (ASCVD) events.It will also be clear to those skilled in the art that the term "therapeutically effective amount" or "prophylactically effective amount" can refer to the effect of reducing the risk of events described in further aspects of the invention described herein, such as first or recurrent non-fatal MI, non-fatal ischemic stroke, emergency revascularization, hospitalization due to unstable angina, hospitalization due to heart failure, or major adverse lower extremity events (MALE), in subjects who have not experienced major atherosclerotic cardiovascular disease (ASCVD) events.

[0128] Each dose administered to a subject can contain about 100 mg to about 500 mg of the inclisiran salt. In some embodiments, each dose administered to a subject contains about 100 mg, 200 mg, 300 mg, 400 mg, or 500 mg of the inclisiran salt. Preferably, the inclisiran salt is inclisiran sodium.

[0129] Each dose administered to a subject may contain about 94 mg to about 473 mg of inclisiran. In certain embodiments, each dose administered to a subject contains about 95 mg of inclisiran (equivalent to about 100 mg of inclisiran sodium), 189 mg of inclisiran (equivalent to about 200 mg of inclisiran sodium), 284 mg of inclisiran (equivalent to about 300 mg of inclisiran sodium), 388 mg of inclisiran (equivalent to about 400 mg of inclisiran sodium), or about 473 mg of inclisiran (equivalent to about 500 mg of inclisiran sodium). Inclisiran may also be included in these doses in other salt forms, such as equivalent amounts of inclisiran salts, as described above.

[0130] In some embodiments, each dose administered to a subject contains about 284 mg of inclisiran (or an equivalent amount of inclisiran salt). Preferably, each dose contains about 284 mg of inclisiran (equivalent to about 300 mg of inclisiran sodium). This amount of inclisiran is considered an exemplary therapeutically effective amount or an exemplary prophylactically effective amount.

[0131] In a preferred embodiment, a dose of about 284 mg of inclisiran (equivalent to about 300 mg of inclisiran sodium) is administered initially as a single subcutaneous injection, and again at 3 months, and then every 6 months thereafter.

[0132] Loading and maintenance periods In some embodiments, administration of the RNAi agent comprises a loading phase followed by a maintenance phase.

[0133] The loading phase can include administering the RNAi agent in one or more doses, e.g., administering the RNAi agent in at least two doses spaced apart in time. Preferably, the loading phase includes administering the RNAi agent in two doses spaced apart in time.

[0134] The administration of each dose during the loading phase can be spaced apart by about 30 days (1 month) to 270 days (9 months). In some embodiments, the administration of each dose during the loading phase can be spaced apart by about 90 days (3 months) to 180 days (6 months). For example, the administration of each dose during the loading phase can be spaced apart by about 30 days (1 month), about 60 days (2 months), about 90 days (3 months), about 120 days (4 months), about 150 days (5 months), or about 180 days (6 months). Preferably, the administration of each dose during the loading phase is spaced apart by about 90 days (3 months).

[0135] In some embodiments, administration of the maintenance phase comprises administering the RNAi agent in one or more doses, each dose spaced apart in time. In exemplary embodiments, the time interval between administration of each dose is about 90 days (3 months) to about 270 days (9 months). Preferably, the time interval is about 180 days (6 months). In even more preferred embodiments, the time interval between administration of each dose in the maintenance phase is approximately equal—for example, the time interval between the first and second maintenance doses can be about 180 days (6 months), and the time interval between the second and third maintenance doses can be about 180 days (6 months).

[0136] In some embodiments, the time interval between administration of each dose is about 90 days (3 months) to about 270 days (9 months). For example, this time interval can be the interval between administration of each dose in the maintenance phase, or the interval between two separate doses from the loading and maintenance phases, respectively. In some embodiments, the first dose of the maintenance phase is administered about 180 days (6 months) after the last dose of the loading phase.

[0137] The duration of the maintenance phase can be at least 1 year. Preferably, the duration of the maintenance phase is at least 2 years, at least 3 years, at least 4 years, at least 5 years, or at least 6 years. The methods of the present invention are particularly useful for reducing the risk of MACE in subjects who have not experienced a major atherosclerotic cardiovascular disease (ASCVD) event by effectively and sustainably lowering LDL-C levels in the subject when the RNAi agent is administered during a long-term maintenance phase (e.g., at least 4 years, at least 5 years, or at least 6 years). Administering 284 mg of inclisiran in the form of 300 mg of inclisiran sodium to a subject initially, followed by another administration at 3 months, and then every 6 months thereafter, has been shown in the ORION-3 open-label extension study to effectively and sustainably lower a subject's low-density lipoprotein cholesterol (LDL-C) over 4 years of treatment. Reductions in LDL-C levels were sustained over the 4-year study period: subjects treated with inclisiran achieved a mean reduction of 47.5% in LDL-C from baseline (ORION-1 Day 1) to Day 210 (95% Cl: -50.69, -44.27), and a time-average reduction of 44.2% in LDL-C over 4 years with twice-yearly dosing.

[0138] Thus, the RNAi agent can be administered to a subject initially at 3 months, and then every 6 months thereafter. In some embodiments, the administration of the RNAi agent comprises: (a) a loading phase comprising administering two doses of the RNAi agent approximately 90 days (3 months) apart, followed by (b) a maintenance phase comprising one or more doses of the RNAi agent, each administered about 180 days (6 months) apart. As explained further below, the time interval can be varied to account for missed doses.

[0139] Forgotten dose In the methods of the invention, the time at which an RNAi agent is intended or expected to be administered according to a particular (original) administration schedule (e.g., initially, again at 3 months, and then every 6 months) is also referred to as planned administration.

[0140] If any scheduled administration is forgotten for less than 90 days (3 months), the missed scheduled administration of the RNAi agent will be administered immediately. In this situation, the next dose of the RNAi agent will usually be administered according to the original administration schedule.

[0141] If the scheduled dose is missed for more than 90 days (3 months), the dosing schedule is resumed by administering the RNAi agent in a new loading phase comprising a first dose and a second dose approximately 90 days (3 months) after the first dose, followed by a maintenance phase comprising administration of one dose approximately every 180 days (6 months) (i.e., the first dose of the maintenance phase is administered approximately 180 days (6 months) after the second dose of the loading phase).

[0142] Route of administration Considering the mechanism of action of the RNAi agent of the present invention, subcutaneous administration of the RNAi agent is particularly suitable.Preferably, the RNAi agent is administered by subcutaneous (sc), preferably by injection into the abdomen, upper arm or thigh.Administration by subcutaneous injection into the abdomen is particularly preferred.

[0143] The RNAi agent for subcutaneous administration can be contained in a pharmaceutical composition, which can be formulated based on a delivery method that can be easily achieved by those skilled in the art.

[0144] The RNAi agent can be administered in a volume of about 1.5mL of sterile aqueous solution.Preferably, the sterile aqueous solution has a pH of about 7.0.The sterile aqueous solution can contain sodium hydroxide and / or phosphoric acid for pH adjustment to a target pH of about 7.0.The sterile aqueous solution containing the RNAi agent is typically contained in a pre-filled syringe for subcutaneous administration.The RNAi agent in the pharmaceutical composition can be contained in water for injection.

[0145] The RNAi agent can also be administered intravenously (iv). In some embodiments, the RNAi agent is administered to a subject subcutaneously and intravenously, for example, the RNAi agent is administered subcutaneously in one dose and intravenously in another dose. Those skilled in the art can prepare an aqueous solution suitable for subcutaneous and / or intravenous administration (e.g., the RNAi agent can be contained in a sterile aqueous solution of about pH 7.0), and the RNAi agent can be administered subcutaneously and / or intravenously. Examples of routine techniques and protocols are described in Remington: The Science and Practice of Pharmacy, 23rd Edition, 2020.

[0146] In certain embodiments in which the RNAi agent is administered intravenously, each dose administered to a subject may contain about 100 mg to about 500 mg of inclisiran salt. In some embodiments, each dose administered to a subject may contain about 100 mg, 200 mg, 300 mg, 400 mg, or 500 mg of inclisiran salt. Preferably, the inclisiran salt is inclisiran sodium. Each dose administered to a subject may contain about 94 mg to about 473 mg of inclisiran. In certain embodiments, each dose administered to a subject contains about 95 mg of inclisiran (equivalent to about 100 mg of inclisiran sodium), 189 mg of inclisiran (equivalent to about 200 mg of inclisiran sodium), 284 mg of inclisiran (equivalent to about 300 mg of inclisiran sodium), 388 mg of inclisiran (equivalent to about 400 mg of inclisiran sodium), or about 473 mg of inclisiran (equivalent to about 500 mg of inclisiran sodium). Inclisiran may also be included in these doses in other salt forms, such as equivalent amounts of inclisiran salts as described above.

[0147] In certain embodiments in which the RNAi agent is administered intravenously, each dose administered to a subject contains about 284 mg of inclisiran (or an equivalent amount of inclisiran salt). Preferably, each dose contains about 284 mg of inclisiran (equivalent to about 300 mg of inclisiran sodium). In some embodiments, the method comprises administering the RNAi agent intravenously during the loading phase and administering the RNAi agent subcutaneously during the maintenance phase. In other embodiments, the method comprises administering the RNAi agent intravenously during the first dose of the loading phase, administering the RNAi agent subcutaneously during each subsequent dose during the loading phase, and administering the RNAi agent subcutaneously during each dose during the maintenance phase.

[0148] In further embodiments, the method comprises administering the RNAi agent intravenously at the first dose of the loading phase and subcutaneously at each subsequent dose. In embodiments where the method comprises administering the RNAi agent intravenously at the first dose of the loading phase and subcutaneously at each subsequent dose, the doses in the loading phase can be spaced apart by about 30 days (1 month) to 270 days (9 months). In some of these embodiments, the doses in the loading phase can be spaced apart by about 90 days (3 months) to 180 days (6 months). For example, the doses in the loading phase can be spaced apart by about 30 days (1 month), about 60 days (2 months), about 90 days (3 months), about 120 days (4 months), about 150 days (5 months), or about 180 days (6 months). Preferably, the doses in the loading phase are spaced apart by about 90 days (3 months).

[0149] The RNAi agent can be administered outpatient and is typically administered by a medical professional.

[0150] Illustrative Embodiments The following exemplary embodiments of the present invention are provided.

[0151] Embodiment 1. A method of reducing the risk of a major adverse cardiovascular event (MACE) in a subject who has not experienced a major atherosclerotic cardiovascular disease (ASCVD) event, comprising: MACE was defined as cardiovascular death, nonfatal myocardial infarction (MI), nonfatal ischemic stroke, or emergency coronary revascularization. The method includes subcutaneously administering inclisiran to a subject, the administration comprising: (a) a loading phase involving the administration of inclisiran as two doses spaced approximately 90 days (3 months) apart, followed by (b) a maintenance phase comprising the administration of one or more doses of inclisiran, each dose spaced apart by about 180 days (6 months); and Each dose contains about 284 mg of inclisiran (equivalent to about 300 mg of inclisiran sodium).

[0152] Embodiment 2. A method of reducing the risk of a major adverse cardiovascular event (MACE) in a subject who has not experienced a major atherosclerotic cardiovascular disease (ASCVD) event, comprising: MACE was defined as cardiovascular death, nonfatal myocardial infarction (MI), nonfatal ischemic stroke, or emergency coronary revascularization. The primary ASCVD events are: a. Acute coronary syndrome (ACS) at any time prior to administration of the RNAi agent, e.g., ACS within about 12 months prior to administration of the RNAi agent; b. Myocardial infarction at any time point before administration of the RNAi agent; c. Ischemic stroke at any time prior to administration of the RNAi agent; or d. Symptomatic peripheral arterial disease (PAD) at any time prior to administration of the RNAi agent, as evidenced by, for example, intermittent claudication, previous revascularization, or amputation due to atherosclerosis; The method includes subcutaneously administering inclisiran to a subject, the administration comprising: (a) a loading phase involving the administration of inclisiran as two doses spaced approximately 90 days (3 months) apart, followed by (b) a maintenance phase comprising administering one or more doses of inclisiran, each dose spaced apart by about 180 days (6 months); and Each dose contains about 284 mg of inclisiran (equivalent to about 300 mg of inclisiran sodium).

[0153] General Provisions The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of those skilled in the art.

[0154] The term "comprising" encompasses "including" and "consisting", for example, a composition "comprising" X may consist solely of X or may include additional items, e.g., X+Y.

[0155] The term "about" in relation to a numerical value x is arbitrary and means, for example, x±10%, unless otherwise specified.

[0156] The term "baseline" may refer to a treatment-free state, such as before treatment is first administered or initiated.

[0157] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0158] Unless specifically stated otherwise or clear from the context, the term "or" as used herein is understood to be inclusive and encompasses both "or" and "and." All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains and are commonly used in the art to which this application pertains. Publications and other references referred to herein to describe the background of the invention and to provide further details regarding its practice are incorporated herein by reference. [Example]

[0159] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0160] Example 1. Study Evaluating the Effect of Inclisiran on the Prevention of Major Adverse Cardiovascular Events in High-Risk Primary Prevention Patients A randomized, double-blind, placebo-controlled, multicenter study will be conducted to evaluate the effect of inclisiran on the prevention of major adverse cardiovascular events in high-risk primary prevention patients.

[0161] This is a phase III trial designed to test the hypothesis that treatment with inclisiran in the form of 300 milligrams (mg) inclisiran sodium administered subcutaneously (sc) initially (day 1), at day 90, and every 6 months thereafter in patients at high cardiovascular (CV) risk who have not previously experienced a major atherosclerotic cardiovascular disease (ASCVD) event will significantly reduce the risk of 4-point major adverse cardiovascular events (4P-MACE), defined as a composite of CV death, nonfatal myocardial infarction (MI), nonfatal ischemic stroke, and urgent coronary revascularization, compared with placebo.

[0162] method The objective of this study was to evaluate 300 mg of inclisiran sodium sc (equivalent to 284 mg of inclisiran) compared with placebo for reducing the risk of 4P-MACE in adult patients at high risk for a first major adverse cardiovascular event. Randomized participants received the study drug (inclisiran or placebo) subcutaneously on Day 1, Day 90, and every 6 months thereafter. The total study duration is expected to be approximately 6.3 years, and the study will continue until at least 853 participants in both treatment arms experience a Clinical Endpoint Committee (CEC)-confirmed primary 4P-MACE (a composite of CV death, nonfatal MI, nonfatal ischemic stroke, and urgent coronary revascularization) and at least 638 participants experience a CEC-confirmed 3P-MACE (a composite of CV death, nonfatal myocardial infarction, and nonfatal ischemic stroke), and all participants have a minimum of 3 years of follow-up (during the double-blind period).

[0163] Inclisiran is expected to reduce the hazard rate of 4P-MACE endpoints by at least 20% on average compared with placebo.

[0164] Additionally, inclisiran is expected to reduce the hazard rate of 3P-MACE endpoints by at least 20% on average compared with placebo.

[0165] Validity of efficacy evaluation 4P-MACE, 3P-MACE, and cardiovascular death are well-defined standard clinical assessments in clinical trials aimed at demonstrating CV risk reduction. Evaluation of events and laboratory tests related to secondary endpoints are consistent with the expected efficacy of inclisiran.

[0166] [Table 2]

[0167] [Table 3]

[0168] Primary endpoint The primary endpoint was time to first occurrence of 4P-MACE, a composite of CV death, nonfatal MI, nonfatal ischemic stroke, and urgent coronary revascularization.

[0169] Secondary endpoints Secondary efficacy endpoints considered were: Time to first occurrence of 3P-MACE (CV death, non-fatal MI, and non-fatal ischemic stroke) Time to occurrence of 4P-MACE (first and recurrent CV death, non-fatal MI, non-fatal ischemic stroke, and urgent coronary revascularization) Time to occurrence of 3P-MACE (first and recurrent CV death, nonfatal MI, and nonfatal ischemic stroke) Time to CV death Time to all-cause mortality

[0170] Exploratory Endpoints Other cardiovascular (CV) endpoints evaluated include: ·CV death ·Non-lethal MI Non-fatal ischemic stroke Emergency coronary revascularization Composite of all-cause mortality, nonfatal MI, and nonfatal ischemic stroke Composite of all-cause mortality, nonfatal MI, nonfatal ischemic stroke, and emergency coronary revascularization Composite of coronary events (CHD death, MI, unstable angina requiring hospitalization, and urgent / elective revascularization) Major adverse events (MALE) ·All-cause emergency revascularization All-cause cardiovascular hospitalizations, including hospitalizations due to MI and unstable angina Venous thromboembolism resulting in hospitalization or death All CV events (first and recurrent non-fatal myocardial infarction, non-fatal ischemic stroke, hospitalization for unstable angina, hospitalization for heart failure or MALE)

[0171] Other efficacy evaluations Other efficacy assessments will include: total cholesterol, LDL-C, HDL-C, VLDL-C, non-HDL-C, Lp(a), and triglycerides. Participants will be required to fast for 10 hours prior to the lipid / lipoprotein assessment visit. A lipid panel will be assessed at all study visits post-randomization (Day 1) according to the assessment schedule.

[0172] Study patient population The trial will enroll 14,000 patients at high CV risk who have an LDL-C level greater than or equal to 70 mg / dL (1.81 mmol / L) and less than 190 mg / dL (4.91 mmol / L) and who have not experienced a major ASCVD event.

[0173] Recruitment criteria Participants eligible for this study must meet all of the following criteria: 1. Signed informed consent must be obtained before participating in the study 2. Male or female aged 40 to under 80 3. High risk of first MACE (i.e., no prior major ASCVD event), defined as any of the following: Evidence of atherosclerotic CAD on CT or invasive coronary angiography, defined as a coronary artery stenosis of 20% or more but less than 50% in the left main coronary artery or a stenosis of 20% or more but less than 70% in the main epicardial coronary artery b. A coronary artery calcium (CAC) score obtained by CT scan of 100 Agatston units or greater, determined at any time before the screening visit c. High 10-year ASCVD risk of 20% or more (assessed by pooled cohort equations) d. Intermediate 10-year ASCVD risk of 7.5% to <20% (assessed by pooled cohort equations) with at least two of the following risk-enhancing factors: i. First-degree relatives with a history of early ASCVD (men under 55 years old, women under 65 years old) ii. Early retirement history under the age of 40 iii. History of maternal pregnancy complications (defined as a history of either preeclampsia, gestational hypertension, or gestational diabetes) iv. South Asian v. Chronic inflammatory disease (defined as any of the following: rheumatoid arthritis, systemic lupus erythematosus, psoriasis covering 10% or more of the body surface area, Crohn's disease, ulcerative colitis, or HIV / AIDS) vi. High-sensitivity C-reactive protein (hsCRP) ≥ 2 mg / L documented in the 12 months prior to the screening visit; no underlying acute or inflammatory conditions (including any of the chronic inflammatory diseases listed in criterion 3d.v. above) present vii. Lipoprotein(a) ≥ 50 mg / dL or 125 nmol / L - determined at any time prior to the screening visit viii. Estimated glomerular filtration rate (eGFR) is 60 mL / min / 1.73 m 2 Less than - recorded between screening and randomization visits ix. Ankle-brachial index (ABI) less than 0.9 and no symptoms of intermittent claudication x. Metabolic syndrome: Increased waist circumference, elevated triglycerides, elevated blood pressure, elevated blood glucose, and decreased HDL-C are factors - a total of 3 or more of these factors recorded between the screening visit and the randomization visit 4. If background LLT, the dose must be stable for at least 4 weeks prior to the screening visit and participants must be willing to continue this background therapy for the entire study period 5. LDL-C ≥ 70 mg / dL (1.81 mmol / L) and < 190 mg / dL (4.91 mmol / L) at the screening visit

[0174] Exclusion criteria Patients who meet any of the following exclusion criteria will be excluded from the study: 1. History of a major ASCVD event defined as any one of the following: Acute coronary syndrome (ACS) within 12 months prior to randomization, or b. Previous myocardial infarction at any time before randomization, or c. Previous ischemic stroke at any time before randomization, or d. Symptomatic peripheral arterial disease (PAD) as evidenced by either intermittent claudication, previous revascularization, or amputation due to atherosclerosis at any time before randomization 2. History or planned revascularization due to ischemia in the coronary or extracoronary arterial bed before randomization 3. No coronary atherosclerosis as determined by CT angiography or invasive coronary angiography within 2 years prior to randomization 4. Coronary artery calcium (CAC) score of 0 in the 2 years prior to randomization 5. Active liver disease or liver dysfunction defined as AST or ALT ≥ 3x ULN by central laboratory testing at the screening visit 6.Current kidney dialysis or planned kidney transplant 7. Previous (within 90 days prior to the screening visit), current treatment, or planned treatment with a PCSK9-targeting mAb (e.g., evolocumab, alirocumab) 8. Prior exposure to inclisiran or other non-mAb PCSK-targeted therapy, either as an investigational or commercially available drug within 2 years prior to randomization 9. Severe concomitant non-cardiovascular disease predicted to reduce life expectancy to less than 5 years 10. Use of other investigational drugs within 5 half-lives of enrollment, or within 30 days (e.g., for small molecules), or until expected pharmacodynamic effects return to baseline (e.g., for biologics), whichever is longer. 11. History of hypersensitivity to the investigational drug or its excipients, or to drugs of a similar chemical class 12. Any surgical or medical condition that, in the opinion of the investigator, may place the participant at high risk in the study or is likely to prevent the participant from complying with or completing the study requirements. 13. Unwilling or unable (e.g., physical or cognitive) to comply with study procedures (including compliance with study visits, fasting blood draws, and adherence to study treatment dosing schedules) and study drug administration (injections) and schedule. 14. Pregnant or nursing (lactating) women 15. Females of childbearing potential, defined as any woman who is physiologically capable of becoming pregnant unless she uses effective contraception while receiving study treatment 16. Anyone directly involved in the implementation of this protocol, including research study staff and their families

[0175] An overview of the screening algorithm for the screening strategy is shown in Figure 1.

[0176] Test treatment Study design A diagram of the study design is shown in Figure 2.

[0177] [Table 4]

[0178] Additional study treatment The study did not include any treatment other than the investigational drug and the control drug.

[0179] treatment group Participants will be assigned in a 1:1 ratio to one of the following two treatment groups at the Day 1 (baseline) visit: Subcutaneous administration of inclisiran sodium 300 mg (equivalent to approximately 284 mg inclisiran) Subcutaneous administration of a matched placebo

[0180] Each participant will receive one subcutaneous injection of blinded inclisiran or placebo on day 1, a second injection of blinded inclisiran or placebo at month 3, and then injections of blinded inclisiran or placebo every six months thereafter.

[0181] Treatment duration The expected duration of the study is approximately 6.3 years. However, because this is an event-driven trial, treatment duration will vary depending on when participants enter the study. The study will be completed when at least 853 participants in both treatment arms experience a primary 4P-MACE confirmed by the Clinical Endpoint Committee (CEC), at least 638 participants experience a 3P-MACE confirmed by the CEC, and all randomized participants complete 3 years of follow-up. Participants may discontinue the study drug early for safety reasons and / or at the discretion of the investigator or participant. In such cases, participants will continue to be followed in the study unless they withdraw their informed consent.

[0182] Prescribing and administering the study treatment The investigational and control drugs will not be provided to participants. The administration of the investigational drug will be performed by the investigator or a qualified medical professional.

[0183] Participants will receive a single subcutaneous injection of 300 mg of inclisiran sodium or placebo at predefined times as described in the activity schedule. The injection will be administered after all other study assessments have been completed at the clinic visit.

[0184] The preferred injection site is the abdomen. Alternative injection sites include the upper arm or thigh. Injections should not be given into areas of active skin disease or injury, such as sunburn, skin rash, inflammation, or skin infection.

[0185] Scientific basis for study design This study aims to evaluate the efficacy and safety of inclisiran sodium 300 mg in reducing the risk of 4P-MACE in participants at high CV risk, without a previous major ASCVD event (i.e., primary prevention), and with high LDL-C levels despite guideline-recommended LLT. This study design is most appropriate to answer the clinical questions of interest above. The LDL-C entry criterion of 70 mg / dL (1.81 mmol / L) or higher is based on several lipid treatment guidelines for patients at high ASCVD risk (Grundy et al., 2019, Mach et al., 2020).

[0186] Dosage justification The established dosing regimen for inclisiran sodium in adults is 300 mg subcutaneously on Day 1, Month 3 (Day 90), and every 6 months thereafter. This dose and dosing regimen has been extensively tested in a Phase 3 clinical program that exposed 1,833 patients to inclisiran and will be used in this study to provide maximum efficacy with an acceptable safety profile. Furthermore, this dose and dosing regimen is approved for LDL-C reduction in the United States, Europe, and other countries worldwide (Leqvio USPI, Leqvio SmPC).

[0187] This dose and dosing regimen were selected based on data from a Phase II dose-finding study (ORION-1), which tested different doses of inclisiran sodium, up to 500 mg, administered as single or multiple doses 90 days apart (Ray et al., 2019). This study found that a dosing regimen including two induction doses of 300 mg inclisiran sodium resulted in the greatest reduction from baseline in LDL-C (52.6%) by Day 180. This dose and dosing regimen also resulted in the greatest reduction from baseline in PCSK9 (69.1%) by Day 180. To provide further evidence for the 300 mg dose, a Phase I study (ALN-PCSSC-001) tested doses up to 900 mg, but this study did not show any clinically significant additional benefit above 300 mg (Fitzgerald et al., 2017). Furthermore, a 300 mg dose of inclisiran sodium was tested in participants with mild, moderate, and severe renal impairment, as well as mild and moderate hepatic impairment, and no clinically meaningful differences in LDL-C and PCSK9 reductions were observed compared to those observed in other participant populations, and no dose adjustment was necessary. Therefore, this dosing regimen is effective in patients, including those with renal and hepatic impairment.

[0188] Three large-scale pivotal Phase III clinical trials (ORION-9, -10, -11) in adults with ASCVD, ASCVD-risk equivalents, and HeFH (Wright et al., 2021) demonstrated that the dosing regimen used in this study (300 mg subcutaneous inclisiran sodium on day 1, month 3 (day 90), and every 6 months thereafter) resulted in placebo-adjusted reductions in LDL-C of 48% to 52% from baseline at day 510, with time-adjusted mean reductions of 44% to 54%, sustained over 18 months (Raal et al., 2020, Ray et al., 2020). This dose and dosing regimen was well tolerated and had a safety profile similar to placebo, with the exception of more injection-site reactions associated with inclisiran compared with placebo.

[0189] Treatment will continue for the entire course of the study, which is expected to be approximately 6.3 years. The study will be completed when the target number of events is reached and minimum follow-up has begun.

[0190] Rationale for choosing background therapy Participants were required to have received standard-of-care background LLT according to local clinical practice guidelines. Although clinical practice guidelines vary by country, the current protocol uses several general principles and is consistent with the most recent US-based guidelines for the management of blood cholesterol (Grundy et al., 2019).

[0191] More specifically, patients entering the study with CAD (inclusion criterion 3a), a CAC score of 100 Agatston units or more (inclusion criterion 3b), or evidence of diabetes were required to be on at least moderate-intensity statin therapy for at least 4 weeks prior to the screening visit.

[0192] According to the most recent US-based guidelines (Grundy et al. 2019), to maximize ASCVD risk reduction, high-intensity statin therapy is strongly recommended for the following categories of patients: Patients enrolled in the study with a high 10-year ASCVD risk of 20% or more (inclusion criterion 3c); Patients with an intermediate 10-year ASCVD risk (≥7.5% and <20%) plus two risk-enhancing factors that would reclassify the patient to a high 10-year ASCVD risk category according to the guidelines (inclusion criterion 3d); and Patients with diabetes and multiple ASCVD risk factors (assessed by clinical judgment), regardless of other inclusion criteria.

[0193] These recommendations for high-intensity statin therapy can be adapted to local practice guidelines and clinical judgment, where applicable.

[0194] In all cases, any LLT dose must be stable for at least 4 weeks prior to the screening visit, cannot include PCSK9-targeted therapy, and participants must be willing to remain on this background LLT for the entire duration of the study.

[0195] During the conduct of the trial, the sponsor will ensure that approximately 70% of randomized participants enter the trial with a statin as background therapy.

[0196] Rationale for comparator choice (comparator / placebo) This study is a placebo-controlled trial added to standard of care to provide solid evidence of the effects of inclisiran on clinical events, safety, and tolerability. The use of placebo as a comparator is justified because all randomized participants will be treated according to local clinical practice guidelines before entering the study and throughout the duration of the study. All participants will receive standard of care, including but not limited to dietary advice, antihypertensive medications, antihyperglycemic medications (if applicable), and antiplatelet therapy provided by the investigator, as clinically appropriate.

[0197] Combination therapy Combination lipid-lowering therapy Participants are required to keep their background LLT unchanged (i.e., no change in medication or dose) during the study. A change in background LLT is only permitted in the following circumstances: Interruption, dose reduction, or discontinuation of background LLT may be performed in the event of an AE requiring such a change.

[0198] If LDL-C increases above a predefined threshold, dose escalation or addition of another background LLT is permitted.

[0199] Prohibited drugs The treatments listed in the table below are not permitted for use after screening.

[0200] [Table 5]

[0201] [Table 6]

[0202] [Table 7]

[0203] analysis Primary endpoint / estimate analysis The primary objective of this trial is to demonstrate the superiority of inclisiran compared with placebo in reducing the risk of 4P-MACE (CV death, nonfatal MI, nonfatal ischemic stroke, and urgent coronary revascularization).

[0204] Definition of the primary endpoint The primary endpoint of this study was the time to the first occurrence of any component of 4P-MACE, a composite endpoint consisting of CV death, nonfatal MI, nonfatal ischemic stroke, and urgent coronary revascularization.

[0205] Time to event will be calculated as the number of days from randomization to the onset of the primary endpoint event.

[0206] Statistical models, hypotheses, and analytical methods The primary statistical hypotheses to be tested are: H0:λ inclisiran / λ placebo ≧1 vs. H a :λ inclisiran / λ placebo <1, In the formula, λ inclisiran and λ placebo are the hazard rates of the first 4P-MACE in the inclisiran and placebo groups, respectively. inclisiran / λ placebois the hazard ratio (HR) for inclisiran versus placebo.

[0207] The primary efficacy endpoint will be analyzed using a Cox proportional hazards model stratified by statin treatment domain and baseline status (yes / no), with treatment group as the only factor in the model. Estimated HRs and corresponding unadjusted two-sided 95% confidence intervals from the Cox regression model are provided separately. The primary hypothesis will be examined by a log-rank test stratified by statin treatment domain and baseline status (yes / no). A p-value will be provided to test the above hypothesis. The superiority of inclisiran over placebo can be claimed if the p-value is less than or equal to the corresponding alpha level of the current stage.

[0208] The overall type I error rate for the study for testing the primary and secondary endpoints will be controlled at 0.025 (one-sided). The significance level used for the final analysis will be adjusted to take into account the alpha level used for the interim efficacy analysis.

[0209] Supplementary analysis Survival functions for each treatment group were estimated by the Kaplan-Meier method, and Kaplan-Meier or cumulative incidence curves are presented separately for each treatment group for the composite primary endpoint and its components.

[0210] The frequency and proportion of participants who reached the composite primary endpoint will be provided by treatment group. Components of the composite primary endpoint will also be summarized narratively to assess their contribution to the overall treatment effect. Treatment comparisons based on time to fatal and nonfatal MI or fatal and nonfatal stroke will be analyzed using Cox regression models similar to the primary analysis.

[0211] The following supplementary analyses of the primary endpoint can also be performed: Correction of the primary analysis using the investigator-reported primary endpoint (i.e., time to first investigator-reported 4P-MACE). Modification of the primary analysis using covariate-adjusted analyses (e.g., adjustment for baseline risk estimates based on pooled cohort equations). The primary endpoint will be analyzed using a Fine-Gray subdistribution hazards model, with non-CV death modeled as a competing risk. Cumulative incidence functions considering non-CV death as a competing risk will also be presented. Correction of the primary analysis to classify deaths due to unknown causes as CV deaths.

[0212] If necessary, additional supplementary analyses may be planned and documented in the Statistical Analysis Plan (SAP).

[0213] Subgroup analysis Subgroup analyses can be performed to assess the homogeneity of treatment effects across demographic and baseline characteristics. Subgroups under consideration include: Age group (under 65, between 65 and 75, and over 75) ·sex Race ·region Baseline BMI (30 kg / m 2 Less than 30-35kg / m 2 Less than 35 kg / m 2 (End) Baseline eGFR (30 mL / min / 1.73 m 2 Less than 30-60mL / min / 1.73m 2 Less than 60 mL / min / 1.73 m 2 (End) Diabetes at baseline (Y / N) Regular smokers at baseline (Y / N) Hypertension at baseline (Y / N) Baseline LDL-C (<100 mg / dL and ≥100 mg / dL) Baseline Lp(a) (median, quartiles, and specific cutoffs determined by SAP before database lock) Baseline triglycerides (less than 150 mg / dL, 150-200 mg / dL, and 200 mg / dL or greater) Baseline hsCRP (<2mg / dL and ≥2mg / dL) Baseline statin intensity (high, medium, no statin)

[0214] Estimated HRs and two-sided 95% confidence intervals for each subgroup are derived based on Cox proportional hazards models with treatment, subgroup, and treatment by subgroup as covariates. Nominal p-values ​​for treatment by subgroup interactions are also presented. No adjustment for multiple comparisons is made. Additionally, the frequency and proportion of subjects reaching the composite primary endpoint are presented by treatment arm for each subgroup.

[0215] Secondary endpoints / estimated analysis Efficacy and / or pharmacodynamic endpoints For efficacy, five secondary endpoints are considered: Time to first occurrence of 3P-MACE (CV death, non-fatal MI, and non-fatal ischemic stroke) Time to occurrence of 4P-MACE (first and recurrent CV death, non-fatal MI, non-fatal ischemic stroke, and urgent coronary revascularization) Time to occurrence of 3P-MACE (first and recurrent CV death, nonfatal MI, and nonfatal ischemic stroke) Time to CV death Time to all-cause mortality

[0216] Unless otherwise specified, the endpoints determined above will be used in the analysis.

[0217] The corresponding hypotheses for the secondary endpoints are as follows: Time to first occurrence of 3P-MACE H1:λ inclisiran / λ placebo ≧1 vs. H a1 :λ inclisiran / λ placebo <1 Time to occurrence of 4P-MACE (initial and recurrent) H2: Inclisiran / placebo rate ratio ≥ 1 vs. Ha2 : inclisiran / placebo rate ratio <1 Time to occurrence of 3P-MACE (initial and recurrent) H3: Inclisiran / placebo rate ratio ≥ 1 vs. H a3 : inclisiran / placebo rate ratio <1 Time to CV death H4:λ inclisiran / λ placebo ≧1 vs. H a4 :λ inclisiran / λ placebo <1 Time to all-cause mortality H5:λ inclisiran / λ placebo ≧1 vs. H a5 :λ inclisiran / λ placebo <1

[0218] After successfully rejecting the primary endpoint hypothesis (H0), H1 and H2 are tested using the Benjamini-Hochberg procedure at the alpha level available at the current stage. If both H1 and H2 are rejected, H3, H4, and H5 are tested sequentially under a hierarchical testing procedure at the alpha levels available at the current stage. The testing procedure to be followed is graphically depicted in Figure 3. Note: Alpha is passed to test H3 only if both H1 and H2 are rejected.

[0219] The secondary efficacy endpoints of time to first occurrence of 3P-MACE, time to CV death, and time to all-cause mortality will be analyzed using the same Cox regression model as the primary efficacy endpoint above. Estimated HRs and corresponding unadjusted two-sided 95% confidence intervals from the Cox regression model are provided separately. Nominal p-values ​​for testing H1, H4, and H5 are also provided.

[0220] The secondary efficacy endpoints of time to occurrence of 4P-MACE and time to occurrence of 3P-MACE will be analyzed using the semiparametric proportional rate model (abbreviated as LWYY model) proposed by Lin et al. 2000. We provide the estimated rate ratios and their 95% confidence intervals, as well as the nominal p-values ​​for testing the corresponding hypotheses H2 and H3.

[0221] Safety Endpoints The study will involve targeted collection of AEs, including all SAEs, all AEs leading to discontinuation of study drug, and AEs of special interest (new-onset diabetes in participants without diabetes at baseline). In addition, liver function test results will be collected during follow-up for participants with AST or ALT >1x ULN at the screening visit (estimated to be approximately 5% of the study population) and for a randomly selected subset (approximately 15% of the study population) until the EOS visit.

[0222] The treatment period will be from the date of the first dose of study treatment until 180 days after the date of the last actual dose of study treatment.

[0223] result The trial is expected to confirm that 300 mg of inclisiran sodium, equivalent to 284 mg of inclisiran, administered sc (for example, on day 1, day 90, and every six months thereafter, is effective, compared with placebo, in reducing the risk of 4P-MACE in adult patients who have not experienced a major ASCVD event.

[0224] The study will also determine that inclisiran sodium 300 mg, equivalent to 284 mg of inclisiran, administered sc (for example, on days 1 and 90, and then every six months), is effective compared to placebo in reducing the risk of 3P-MACE (CV death, nonfatal MI, or nonfatal ischemic stroke), CV death, and all-cause mortality in adult patients who have not experienced a major ASCVD event.

[0225] The trial will also confirm that inclisiran sodium 300 mg, equivalent to 284 mg of inclisiran, administered sc (for example, on days 1, 90, and every six months thereafter, is effective, compared with placebo, in reducing the risk of CV death in adult patients who have not experienced a major ASCVD event.

[0226] The trial will also confirm that inclisiran sodium 300 mg, equivalent to 284 mg of inclisiran, administered subcutaneously, for example, on days 1 and 90 and every six months thereafter, is effective, compared with placebo, in reducing the risk of all-cause mortality in adult patients who have not experienced a major ASCVD event.

[0227] References Achenbach S, Raggi P (2010) Imaging of coronary atherosclerosis by computed tomography;European Heart Journal,Volume 31,Issue 12,June 2010,Pages 1442-1448,https: / / doi.org / 10.1093 / eurheartj / ehq150 Benjamin EJ,Muntner P,Alonso A,et al(2019)Heart Disease and Stroke Statistics - 2019 Update: A report from the American Heart Association.Circulation;139(10):e56-e528. D'agostino RB, Vasan RS, Pencina MJ, Wolf PA, Cobain M, Massaro JM, Kannel WB. General cardiovascular risk profile for use in primary care.Circulation.2008 Feb 12;117:743-53.PMID:18212285. Fitzgerald K,White S,Borodovsky A,et al(2017)A highly durable RNAi therapeutic inhibitor of PCSK9.N Engl J Med;376(1):41-51. Goff DC,Lloyd-Jones DM,Bennett G,et al(2014)2013 ACC / AHA guideline on the assessment of cardiovascular risk: a report of the American College of Cardiology / American Heart Association Task Force on Practice Guidelines.Circulation;129(25 Suppl 2):S49-73. Grundy SM,Brewer HB,Cleeman JI,et al(2004)Definition of metabolic syndrome: Report of the National Heart,Lung,and Blood Institute / American Heart Association conference on scientific issues related to definition.Circulation;109(3):433-8. Grundy SM,Stone NJ,Bailey AL,et al(2019)2018 AHA / ACC / AACVPR / AAPA / ABC / ACPM / ADA / AGS / APhA / ASPC / NLA / PCNA guideline on the management of blood cholesterol: A report of the American College of Cardiology / American Heart Association Task Force on clinical practice guidelines.J Am Coll Cardiol;73(24):3158-3209. Leqvio SmPC(2020)Summary of Product Characteristics.Leqvio(inclisiran)284 mg solution for injection.Novartis Europharm Limited,Dublin,Ireland.Updated 19 Aug 2022. Leqvio USPI(2021).Leqvio(inclisiran)284 mg injection for subcutaneous use.US Prescribing Information.Novartis Pharmaceuticals Corporation,East Hanover,NJ.Revised Dec 2021. Lin DY,Wei LJ,Yang I,et al(2000)Semiparametric regression for the mean and rate functions of recurrent events.J R Statist Soc B;62(4):711-30. Lloyd-Jones DM,Braun LT,Ndumele CE,Smith SC Jr,Sperling LS,Virani SS,Blumenthal RS.Use of risk assessment tools to guide decision-making in the primary prevention of atherosclerotic cardiovascular disease: a special report from the American Heart Association and American College of Cardiology;JACC Nov 2018,25711;DOI:10.1016 / j.jacc.2018.11.005 Mach F,Baigent C,Catapano AL,et al(2020)2019 ESC / EAS Guidelines for the management of dyslipidaemias: lipid modification to reduce cardiovascular risk.Eur Heart J;41(1):111-88. Raal FJ,Kallend D,Ray KK,et al(2020)Inclisiran for the treatment of heterozygous familial hypercholesterolemia.N Engl J Med;382(16):1520-30. Randomized Trial Assessing the Effects of Inclisiran on Clinical Outcomes Among People With Cardiovascular Disease(ORION-4).Available from: / / clinicaltrials.gov / ct2 / show / NCT03705234(Accessed 29 Jun 2022). Ray KK,Stoekenbroek RM,Kallend D,et al(2019)Effect of 1 or 2 doses of inclisiran on low-density lipoprotein cholesterol levels: One-year follow-up of the ORION-1 randomized clinical trial.JAMA Cardiol;4(11):1067-75. Ray KK,Wright RS,Kallend D,et al(2020)Two phase 3 trials of inclisiran in patients with elevated LDL cholesterol.N Engl J Med;382(16):1507-19. Recommended INN: List 76 from the WHO Drug Information, Vol.30, No.3, 2016 pages 504-506, inclisiran. Remington: The Science and Practice of Pharmacy, 23rd Edition, 2020 Study of Inclisiran to Prevent Cardiovascular (CV) Events in Participants With Established Cardiovascular Disease (VICTORION-2P). Available from: / / clinicaltrials.gov / ct2 / show / NCT05030428 (Accessed 29 Jun 2022). WHO(2019)Cardiovascular diseases (CVDs). Available from: / / who.int / health-topics / cardiovascular-diseases#tab=tab_1 (Accessed 29 Jun 2022). Wright RS, Ray KK, Raal FJ, et al(2021)Pooled patient-level analysis of inclisiran trials in patients with familial hypercholesterolemia or atherosclerosis. J Am Coll Cardiol;77(9):1182-93.

[0228] Sequence Sequence number 1 5’-ACAAAAGCAAAACAGGUCUAGAA-3’ Sequence number 2 5’-CUAGACCUGUUUUGCUUUUGU-3’ Sequence number 3 5'-asCfsaAfAfAfgCfaAfaAfcAfgGfuCfuagsasa-3' (wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, or U; Af, Gf, Cf, or Uf are 2'-fluoro A, G, C, or U; and s is a phosphorothioate bond) SEQ ID NO:4 5'-csusagacCfuGfudTuugcuuuugu-3' (wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, or U; Af, Gf, Cf, or Uf are 2'-fluoro A, G, C, or U; dT is 2'-deoxythymidine; and s is a phosphorothioate linkage)

Claims

1. 1. A method of reducing the risk of a major adverse cardiovascular event (MACE) in a subject who has not experienced a major atherosclerotic cardiovascular disease (ASCVD) event, comprising: the MACE is cardiovascular death, non-fatal myocardial infarction (MI), non-fatal ischemic stroke, or emergency coronary revascularization; The method includes administering to the subject an interfering ribonucleic acid (RNAi) agent, wherein the RNAi agent: an antisense strand comprising the nucleotide sequence 5'-asCfsaAfAfAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 3), and a sense strand comprising the nucleotide sequence 5'-csusagacCfuGfudTuugcuuuugu-3' (SEQ ID NO:4), In the sequence, a, g, c, and u are 2'-O-methyl (2'-OMe) modified A, G, C, and U nucleotides, respectively; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, and U modified nucleotides, respectively; dT is a deoxy-thymine nucleotide, and s is a phosphorothioate linkage; and The method, wherein the sense strand is conjugated to at least one ligand.

2. The ligand has the following structure: 【Chemistry 1】 The method of claim 1 , comprising:

3. The method of claim 1 or claim 2, wherein the ligand is conjugated to the 3' end of the sense strand.

4. The RNAi agent has the following structural formula: 【Chemistry 2】 (wherein X is O or S) 4. The method of claim 3, wherein the ligand is conjugated as shown in

5. 5. The method of claim 4, wherein X is O.

6. The method of any one of claims 1 to 5, wherein the subject is at high risk for a first MACE.

7. The high risk of the first MACE is: a) Evidence of atherosclerotic CAD on CT or invasive coronary angiogram defined as coronary artery stenosis of 20% or more and less than 50% in the left main coronary artery or stenosis of ≥20% and <70% in the main epicardial coronary arteries; b) a coronary artery calcium (CAC) score of 100 Agatston units or greater obtained by CT scan; c) high 10-year ASCVD risk of 20% or more (assessed by pooled cohort equation); d) Moderate 10-year ASCVD risk of 7.5% to <20% (assessed by pooled cohort equations) with at least two of the following risk-enhancing factors: i. First-degree relatives with a history of early ASCVD (men under 55 years of age; women under 65 years of age); ii. History of early closure before age 40; iii. History of maternal pregnancy complications (defined as a history of either preeclampsia, gestational hypertension, or gestational diabetes); iv. South Asian; v. Chronic inflammatory disease (defined as any of rheumatoid arthritis, systemic lupus erythematosus, psoriasis covering 10% or more of the body surface area), Crohn's disease, ulcerative colitis, or HIV / AIDS); vi. High-sensitivity C-reactive protein (hsCRP) ≥ 2 mg / L (documented in the 12 months prior to the screening visit) in the absence of an underlying acute or inflammatory condition (including any of the chronic inflammatory diseases listed in criterion 3d.v. above); vii. Lipoprotein(a) greater than or equal to 50 mg / dL or 125 nmol / L; viii. Estimated glomerular filtration rate (eGFR) of 60 mL / min / 1.73 m ２ less than; ix. Ankle-brachial index (ABI) less than 0.9 and no symptoms of intermittent claudication; or x. Metabolic syndrome 7. The method of claim 6, wherein the method is defined as any one of:

8. 8. The method of any one of claims 1 to 7, wherein the subject has an LDL-C level of 70 mg / dL (1.81 mmol / L) or greater and less than 190 mg / dL (4.91 mmol / L).

9. The method of any one of claims 1 to 8, wherein the subject is being or has been treated with background lipid-lowering therapy (LLT).

10. The method of any one of claims 1 to 9, wherein the subject is not previously or has not been treated with background lipid-lowering therapy (LLT).

11. The method of any one of claims 1 to 10, wherein the subject has not been treated with an additional PCSK9-targeted therapy prior to administration of the RNAi agent.

12. The method of any one of claims 1 to 10, wherein the subject has been treated with an additional PCSK9-targeted therapy prior to administration of the RNAi agent.

13. The method of any one of claims 1 to 12, wherein the subject has not experienced a MACE at any time prior to administration of the RNAi agent.

14. The main ASCVD events are: a. acute coronary syndrome (ACS) at any time prior to administration of the RNAi agent, e.g., ACS within about 12 months prior to administration of the RNAi agent; b. Myocardial infarction at any time prior to administration of the RNAi agent; c. ischemic stroke at any time prior to administration of the RNAi agent; or d. Symptomatic peripheral arterial disease (PAD), as evidenced by, for example, intermittent claudication, previous revascularization, or amputation due to atherosclerosis at any time prior to administration of the RNAi agent. The method according to any one of claims 1 to 13, wherein the method is defined as any one of

15. The method of any one of claims 1 to 14, wherein the subject has no history of ischemic revascularization in the coronary or extracoronary arterial bed prior to administration of the RNAi agent.

16. 16. The method of any one of claims 1-15, wherein the subject is free of coronary atherosclerosis by CT angiography or invasive coronary angiography within the two years prior to administration of the RNAi agent.

17. 17. The method of any one of claims 1-16, wherein the subject has not had a coronary artery calcium (CAC) score of 0 obtained in the two years prior to administration of the RNAi agent.

18. 18. The method of any one of claims 1 to 17, wherein the subject does not have active liver disease or liver dysfunction, as identified, for example, as having aspartate aminotransferase (AST) or alanine aminotransferase (ALT) greater than or equal to three times the upper limit of normal (ULN).

19. 19. The method of any one of claims 1 to 18, wherein the subject is receiving a diet designed to improve lipid levels, a maximum tolerated dose of statin therapy, and / or low-density lipoprotein-lowering therapy.

20. 20. The method of any one of claims 1-19, wherein administration of the RNAi agent reduces low-density lipoprotein cholesterol (LDL-C) levels in the subject.

21. The method according to any one of claims 1 to 20, comprising a loading phase and a maintenance phase.

22. 22. The method of claim 21, wherein the loading phase comprises administering the RNAi agent in one or more doses.

23. 23. The method of claim 21 or 22, wherein the loading phase comprises administering the RNAi agent in at least two doses spaced apart in time.

24. 24. The method of any one of claims 21 to 23, wherein the loading phase comprises administering the RNAi agent as two doses spaced apart in time.

25. 25. The method of claim 23 or 24, wherein the administration of each dose during the loading phase is spaced apart by a time interval of about 90 days (3 months) to 180 days (6 months).

26. 26. The method of any one of claims 23 to 25, wherein the administration of each dose during the loading phase is separated by a time interval of about 90 days (3 months).

27. 27. The method of any one of claims 21-26, wherein the maintenance phase comprises administering the RNAi agent in one or more doses, each spaced apart in time.

28. 28. The method of any one of claims 21 to 27, wherein the time interval between administration of each dose during the maintenance phase is from about 90 days (3 months) to about 270 days (9 months).

29. 29. The method of any one of claims 21 to 28, wherein the time interval between administration of each dose in the maintenance phase is about 180 days (6 months).

30. 30. The method of any one of claims 1-29, wherein the RNAi agent is administered initially, then again at 3 months, and every 6 months thereafter.

31. 31. The method of any one of claims 21 to 30, wherein the duration of the maintenance phase is at least 1 year, preferably the duration of the maintenance phase is at least 2 years, at least 3 years, at least 4 years, at least 5 years, or at least 6 years.

32. 32. The method of any one of claims 1-31, comprising administering the RNAi agent subcutaneously (e.g., intraabdominally, intrabrachially, or intrathigh) and / or intravenously.

33. 33. The method of any one of claims 21-32, comprising administering the RNAi agent intravenously during the loading phase and administering the RNAi agent subcutaneously during the maintenance phase.

34. 33. The method of any one of claims 21-32, comprising administering the RNAi agent intravenously for a first dose during the loading phase, and administering the RNAi agent subcutaneously for each subsequent dose.

35. the RNAi agent is inclisiran, and the method comprises: (a) a loading phase comprising administering inclisiran as two doses spaced approximately 90 days (3 months) apart, followed by 35. The method of any one of claims 21 to 34, comprising (b) a maintenance phase comprising administering one or more doses of inclisiran, each dose separated by a time interval of about 180 days (6 months).

36. 36. The method of claim 35, comprising administering inclisiran subcutaneously.

37. 37. The method of any one of claims 1-36, wherein the RNAi agent is administered in a sterile aqueous solution, for example, in a volume of about 1.5 mL.

38. 38. The method of claim 37, wherein the sterile aqueous solution comprises sodium hydroxide and / or phosphoric acid.

39. 39. The method of claim 37 or 38, wherein the sterile aqueous solution has a pH of about 7.

0.

40. 40. The method of any one of claims 37 to 39, wherein the sterile aqueous solution is contained in a pre-filled syringe for subcutaneous administration.

41. 41. The method of any one of claims 1 to 40, wherein the RNAi agent is inclisiran.

42. 42. The method of any one of claims 4 to 41, wherein each dose contains about 284 mg of inclisiran (equivalent to the corresponding amount of inclisiran salt).

43. 42. The method of any one of claims 4 to 41, wherein each dose contains about 284 mg of inclisiran (equivalent to about 300 mg of inclisiran sodium).

44. 44. The method of any one of claims 1 to 43, wherein a dose of 284 mg of inclisiran (equivalent to approximately 300 mg of inclisiran sodium) is administered initially as a single subcutaneous injection, and again at 3 months, and then every 6 months.

45. 45. An RNAi agent for use in the method of any one of claims 1 to 44.

46. A pharmaceutical composition comprising an RNAi agent for use in the method of any one of claims 1 to 44.

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