Double stranded RNAi agents and compositions for reducing expression of angiopoietin-like 3 (ANGPTL3) and methods of use thereof

Abstract

Disclosed are certain double-stranded RNA (dsRNA) or siRNA agents for inhibiting angiopoietin-like 3 gene expression, compositions comprising the angiopoietin-like 3 agents, and methods of using the same. The double-stranded RNA (dsRNA) or siRNA agents are useful, inter alia, for treating or preventing cardiovascular diseases and metabolic conditions, and for treating heart failure and chronic kidney disease.

Description

Technical Field

[0001] This technical field relates to certain double-stranded RNA (dsRNA) or siRNA agents for inhibiting angiopoietin-like 3 gene expression, compositions containing angiopoietin-like 3 agents, and methods of using them.

[0002] sequence list

[0003] This application is submitted together with an electronic sequence listing. The sequence listing is provided with the filename “201286-PCT01-NP_SL.xml” (Homo sapiens angiopoietin-like 3 (ANGPTL3) transcript GenBank NM_014495.4 (SEQ ID NO:1)), created on January 31, 2025, and is 193,866 bytes in size. The information in the electronic sequence listing is incorporated herein by reference in its entirety. Although each sequence in the appended sequence listing is identified as “RNA” or “DNA” upon request, in practice, these sequences may be modified by any combination of chemical modifications. Those skilled in the art will readily understand that the use of names such as “RNA” or “DNA” to describe modified oligonucleotides is arbitrary in certain circumstances. For example, an oligonucleotide containing a nucleoside with a 2'-OH sugar moiety and a thymine base can be described as DNA with a modified sugar (replacing one 2'-H in the DNA with 2'-OH) or RNA with a modified base (replacing uracil in the RNA with thymine (methylated uracil)). Therefore, the nucleic acid sequences provided herein (including, but not limited to, those in the sequence listing) are intended to cover nucleic acids containing any combination of native or modified RNA and / or DNA, unless otherwise stated, including, but not limited to, such nucleic acids with modified nucleobases. Background Technology

[0004] Angiopoietin-like 3 (also known as ANGPTL3, ANGPL3, ANG3, or angiopoietin-like protein 3) is an angiopoietin protein encoded by the human angiopoietin-like 3 gene, reported to be involved in regulating lipid metabolism. ANGPTL3 is a 460-amino acid polypeptide composed of a signal peptide, an N-terminal coiled-coil domain, and a C-terminal fibrinogen (FBN)-like domain. ANGPTL3 is known to be primarily produced in human hepatocytes and secreted into circulation after synthesis. ANGPTL3 acts as an inhibitor of lipoprotein lipase (which catalyzes the hydrolysis of triglycerides) and endothelial lipase (which hydrolyzes lipoprotein phospholipids). Inhibition of these enzymes leads to elevated levels of triglycerides, high-density lipoprotein (HDL), and phospholipids in plasma. Furthermore, loss-of-function mutations in ANGPTL3 result in familial hypoβ-lipoproteinemia, characterized by low levels of triglycerides and low-density lipoprotein (LDL-C) in plasma. In humans, loss of ANGPTL3 function is also associated with a reduced risk of atherosclerotic cardiovascular disease.

[0005] It has been reported that effective therapeutic agents targeting ANGPTL3 have the potential to have beneficial effects on the treatment (including preventative treatment) of cardiovascular metabolic diseases such as hypertriglyceridemia, obesity, hyperlipidemia, lipid and / or cholesterol metabolism disorders, atherosclerosis, type 1 diabetes, cardiovascular disease, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, homozygous and heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia, and other metabolic-related conditions and diseases. Certain double-stranded RNA (dsRNA) or siRNA agents have been identified as capable of inhibiting the expression of the ANGPTL3 gene (see, for example, WO 2012 / 17784, WO 2016 / 168286, WO 2016 / 154127, WO 2019 / 055633 and WO 2021 / 188795), but the specific ANGPTL3 siRNA agents disclosed herein have not been previously disclosed or are known, and are capable of achieving highly effective ANGPTL3-specific inhibition of ANGPTL3 gene expression.

[0006] The potential of effective treatments targeting ANGPTL3 to have a beneficial effect on the treatment of heart failure (including HFpEF and HFrEF) or chronic kidney disease (CKD) has never been described before. Summary of the Invention

[0007] There is a need for novel ANGPTL3-specific RNA interference (RNAi) agents (e.g., double-stranded RNA (dsRNA) or siRNA agents) capable of inhibiting ANGPTL3 gene expression in vitro and / or in vivo using the ANGPTL3 siRNA agents and compositions containing ANGPTL3 siRNA agents described herein. The ANGPTL3 siRNA agents described herein selectively and effectively reduce or inhibit ANGPTL3 gene expression, thereby reducing triglyceride (TG) levels and / or cholesterol in subjects (e.g., human or animal subjects).

[0008] The described ANGPTL3 siRNA agents can be used in methods of therapeutic treatment (including prophylactic and preventative treatment) of symptoms and diseases associated with elevated TG levels and / or elevated cholesterol levels, including but not limited to hypertriglyceridemia, obesity, hyperlipidemia, lipid and / or cholesterol metabolism disorders, atherosclerosis, type 1 diabetes, cardiovascular disease, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, homozygous and heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia, and other metabolic-related conditions and diseases. The ANGPTL3 siRNA agents disclosed herein selectively reduce ANGPTL3 gene expression, which can lead to a reduction in TG and / or cholesterol levels in subjects, among other things. The methods disclosed herein involve administering one or more ANGPTL3 siRNA agents to subjects (e.g., human or animal subjects) using any suitable method known in the art, such as subcutaneous injection or intravenous administration.

[0009] The aim is to provide a method for treating symptoms and diseases associated with elevated triglyceride (TG) levels and / or elevated cholesterol levels, including but not limited to hypertriglyceridemia, obesity, hyperlipidemia, lipid and / or cholesterol metabolism disorders, atherosclerosis, type 1 diabetes, cardiovascular disease, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, homozygous and heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia, and other metabolic-related conditions and diseases. The method comprises administering a therapeutically effective amount of double-stranded siRNA to a subject, the double-stranded siRNA comprising an antisense strand and a sense strand, the antisense strand comprising a [missing information - likely a SEQ ID NO: ] The ANGPTL3 nucleic acid, with its 1-base sequence, is complementary to an equal-length portion of the nucleotide sequence, thereby treating hypertriglyceridemia, obesity, hyperlipidemia, lipid and / or cholesterol metabolism disorders, atherosclerosis, type I diabetes, cardiovascular disease, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, homozygous and heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia, and other metabolic-related conditions and diseases.

[0010] The aim is to provide a method for treating heart failure, the method comprising administering to a subject a therapeutically effective amount of double-stranded siRNA comprising an antisense strand and a sense strand, the antisense strand comprising a nucleobase sequence complementary to an equal-length portion of an ANGPTL3 nucleic acid having the nucleobase sequence of SEQ ID NO: 1, thereby treating heart failure.

[0011] Another objective is to provide a method for treating chronic kidney disease, the method comprising administering to a subject a therapeutically effective amount of double-stranded siRNA comprising an antisense strand and a sense strand, the antisense strand comprising a nucleotide sequence complementary to an equal-length portion of an ANGPTL3 nucleic acid having the nucleotide sequence of SEQ ID NO: 1, thereby treating chronic kidney disease.

[0012] In one embodiment, the ANGPTL3 siRNA agent is conjugate 1, which is a double-stranded RNA (dsRNA) or siRNA capable of inhibiting human ANGPTL3 gene expression, comprising (i) a first (sense) strand and (ii) a second (antisense) strand, wherein the first (sense) strand has the following sequence:

[0013]

[0014] The second (antisense) chain has the following sequence:

[0015]

[0016] NAG is a trianticular ligand portion attached to the 5' end of the first chain and having the following structure:

[0017] .

[0018] In one embodiment, the ANGPTL3 siRNA agent is conjugate 2, which is a double-stranded RNA (dsRNA) or siRNA capable of inhibiting human ANGPTL3 gene expression, comprising (i) a first (sense) strand and (ii) a second (antisense) strand, wherein the first (sense) strand has the following sequence:

[0019]

[0020] The second (antisense) chain has the following sequence:

[0021]

[0022] Wherein [M] is the trianticular ligand portion connected to the 5' end of the first chain and having the following structure:

[0023] .

[0024] Therefore, the terminal thiophosphate group of the ligand moiety [M] is directly (through the “free” bond indicated by “*”) bonded to the 5' position of the 5' terminal nucleotide of the first chain (i.e., bonded to the 5'-carbon of the 2'-O-methylribose moiety of the mC residue at the 5' end of the first chain).

[0025] The sequence is displayed in the standard 5' to 3' orientation.

[0026] In the context of the above expression, the following abbreviations are used:

[0027]

[0028] In another embodiment, a chiral enriched population of double-stranded siRNAs is also provided, wherein the population is enriched with double-stranded siRNAs comprising at least one thiophosphate nucleoside bond having a specific stereochemical configuration, or a polynucleotide conjugate comprising said double-stranded siRNA.

[0029] A chiral enrichment population can enrich double-stranded siRNAs containing at least one (Sp) or (Rp) conformation of a specific phosphate thioester nucleoside bond, or polynucleotide conjugates containing said double-stranded siRNAs. A chiral enrichment population can enrich double-stranded siRNAs having a specific, independently selected stereochemical conformation at each phosphate thioester nucleoside bond, or polynucleotide conjugates containing said double-stranded siRNAs. A chiral enrichment population can enrich double-stranded siRNAs having an (Rp) conformation at one specific phosphate thioester nucleoside bond and an (Sp) conformation at each of the remaining phosphate thioester nucleoside bonds, or polynucleotide conjugates containing said double-stranded siRNAs. A chiral enrichment population can enrich double-stranded siRNAs having at least two or at least three consecutive phosphate thioester nucleoside bonds in Sp, Sp, and Rp conformations in the 5' to 3' direction, or polynucleotide conjugates containing said double-stranded siRNAs.

[0030] In a population of double-stranded siRNAs containing double-stranded or polynucleotide conjugates of the present invention, all phosphate thioester nucleoside bonds of the double-stranded siRNA may be stereo-random.

[0031] In another embodiment, a pharmaceutical composition is also provided comprising double-stranded siRNA, a polynucleotide conjugate, and a pharmaceutically acceptable diluent or carrier.

[0032] Pharmaceutically acceptable diluents may be water or phosphate-buffered saline. The pharmaceutical composition may consist essentially of the double-stranded siRNA, the polynucleotide conjugate, and water or phosphate-buffered saline.

[0033] In another embodiment, a method is also provided, comprising administering the double-stranded siRNA, the polynucleotide conjugate, or the pharmaceutical composition to a subject.

[0034] In one embodiment, a method of treating ANGPTL3-related disease is provided, comprising administering a therapeutically effective amount of conjugate 2 or the pharmaceutical composition to a subject suffering from ANGPTL3-related disease, thereby treating ANGPTL3-related disease.

[0035] Diseases associated with ANGPTL3 may include hypertriglyceridemia, obesity, hyperlipidemia, lipid and / or cholesterol metabolism disorders, atherosclerosis, type 2 diabetes, cardiovascular disease, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, homozygous and heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia, and other metabolic-related conditions and diseases.

[0036] In another embodiment, a method for treating heart failure is provided, comprising administering to a subject a therapeutically effective amount of double-stranded siRNA comprising an antisense strand and a sense strand, wherein the antisense strand comprises a nucleobase sequence that is at least 80% complementary to an equal-length portion of an ANGPTL3 nucleic acid having the nucleobase sequence of SEQ ID NO: 1, thereby treating heart failure.

[0037] In yet another embodiment, a method for treating heart failure is provided, comprising administering to a subject a therapeutically effective amount of double-stranded siRNA comprising an antisense strand and a sense strand, the antisense strand comprising a nucleobase sequence that is at least 85% complementary to an equal-length portion of an ANGPTL3 nucleic acid having the nucleobase sequence of SEQ ID NO: 1, thereby treating heart failure.

[0038] In yet another embodiment, a method for treating heart failure is provided, comprising administering to a subject a therapeutically effective amount of double-stranded siRNA comprising an antisense strand and a sense strand, the antisense strand comprising a nucleobase sequence that is at least 90% complementary to an equal-length portion of an ANGPTL3 nucleic acid having the nucleobase sequence of SEQ ID NO: 1, thereby treating heart failure.

[0039] In yet another embodiment, a method for treating heart failure is provided, comprising administering to a subject a therapeutically effective amount of double-stranded siRNA comprising an antisense strand and a sense strand, the antisense strand comprising a nucleobase sequence that is at least 95% complementary to an equal-length portion of an ANGPTL3 nucleic acid having the nucleobase sequence of SEQ ID NO: 1, thereby treating heart failure.

[0040] In yet another embodiment, a method for treating heart failure is provided, comprising administering to a subject a therapeutically effective amount of double-stranded siRNA comprising an antisense strand and a sense strand, the antisense strand comprising a nucleobase sequence 100% complementary to an equal-length portion of an ANGPTL3 nucleic acid having the nucleobase sequence of SEQ ID NO: 1, thereby treating heart failure.

[0041] In yet another embodiment, a method for treating heart failure is provided, comprising administering to a subject a therapeutically effective amount of conjugate 2 or the pharmaceutical composition.

[0042] In yet another embodiment, a method for treating heart failure is provided, comprising administering to a subject a therapeutically effective amount of conjugate 1 or the pharmaceutical composition.

[0043] In another embodiment, a method for treating chronic kidney disease (CKD) is provided, comprising administering to a subject a therapeutically effective amount of double-stranded siRNA comprising an antisense strand and a sense strand, wherein the antisense strand comprises a nucleotide sequence that is at least 80% complementary to an equal-length portion of an ANGPTL3 nucleic acid having the nucleotide sequence of SEQ ID NO: 1, thereby treating CKD.

[0044] In yet another embodiment, a method for treating chronic kidney disease (CKD) is provided, comprising administering to a subject a therapeutically effective amount of double-stranded siRNA comprising an antisense strand and a sense strand, wherein the antisense strand comprises a nucleotide sequence that is at least 85% complementary to an equal-length portion of an ANGPTL3 nucleic acid having the nucleotide sequence of SEQ ID NO: 1, thereby treating CKD.

[0045] In yet another embodiment, a method for treating chronic kidney disease (CKD) is provided, comprising administering to a subject a therapeutically effective amount of double-stranded siRNA comprising an antisense strand and a sense strand, wherein the antisense strand comprises a nucleobase sequence that is at least 90% complementary to an equal-length portion of an ANGPTL3 nucleic acid having the nucleobase sequence of SEQ ID NO: 1, thereby treating CKD.

[0046] In yet another embodiment, a method for treating chronic kidney disease (CKD) is provided, comprising administering to a subject a therapeutically effective amount of double-stranded siRNA comprising an antisense strand and a sense strand, wherein the antisense strand comprises a nucleobase sequence that is at least 95% complementary to an equal-length portion of an ANGPTL3 nucleic acid having the nucleobase sequence of SEQ ID NO: 1, thereby treating CKD.

[0047] In yet another embodiment, a method for treating chronic kidney disease (CKD) is provided, comprising administering to a subject a therapeutically effective amount of double-stranded siRNA comprising an antisense strand and a sense strand, wherein the antisense strand comprises a nucleobase sequence that is 100% complementary to an equal-length portion of an ANGPTL3 nucleic acid having the nucleobase sequence of SEQ ID NO: 1, thereby treating CKD.

[0048] In yet another embodiment, a method for treating chronic kidney disease (CKD) is provided, comprising administering to a subject a therapeutically effective amount of conjugate 2 or the pharmaceutical composition.

[0049] In yet another embodiment, a method for treating chronic kidney disease (CKD) is provided, comprising administering to a subject a therapeutically effective amount of conjugate 1 or the pharmaceutical composition.

[0050] Brief description of the attached figures

[0051] Figure 1 Five-fold concentration assay of siRNA conjugates 2 and 1 targeting ANGPTL3 in primary human hepatocytes.

[0052] Figure 2 Five-fold concentration assay of siRNA conjugates 2 and 1 targeting ANGPTL3 in primary cynomolgus monkey hepatocytes.

[0053] Figure 3 Baseline-corrected circulating human ANGPTL3 protein in humanized mice treated with a single dose of conjugate 2.

[0054] Figure 4 Human ANGPTL3 mRNA expression in the liver of humanized mice treated with a single dose of conjugate 2 on day 49.

[0055] Figure 5 Human ANGPTL3 mRNA expression in the liver of humanized mice treated with single doses of conjugate 3 and conjugate 4, respectively, was observed on day 49.

[0056] Figure 6 Summary of an illustrative study of Example 5 (the in vivo therapeutic effect of conjugate 1 in an obese HFpEF ZSF1 rat model).

[0057] Figure 7 Body weight (in grams) at the end of the 12-week treatment period in Example 5.

[0058] Figure 8 ANGPTL3 protein and TG plasma levels in ZSF-1 rats after administration of conjugate 1 in Example 5.

[0059] Figure 9a Heart content in ZSF-1 rats after administration of conjugate 1 (lipomics analysis).

[0060] Figure 9b Liver content of ZSF-1 rats after administration of conjugate 1 (lipomics analysis).

[0061] Figure 10 Diastolic function (IVRT and NT-proBNP) in ZSF-1 rats after administration of conjugate 1.

[0062] Figure 11 Tissue proteomics of ZSF-1 rats after administration of conjugate 1.

[0063] Figure 12 Kidney health in ZSF-1 rats after administration of conjugate 1, as represented by UACR, podocyte health, and glomerular sclerosis.

[0064] Figure 13 Proton density fat fraction (PDFF) results at baseline and endpoint in HfpEF NHP after ANGPTL3 inhibition treatment.

[0065] Figure 14 Extracellular volume (ECV) results at baseline and endpoint in HfpEF NHP after ANGPTL3 inhibition treatment.

[0066] Figure 15 Left atrial volume index (LAVi) in HfpEF NHP after ANGPTL3 suppression treatment.

[0067] Figure 16 Plasma ANGPTL3 levels in HfpEF NHP after ANGPTL3 inhibition treatment.

[0068] Figure 17 Plasma TG levels in HFpEF NHP after ANGPTL3 inhibition treatment. Detailed Implementation

[0069] While this detailed description and its specific examples illustrate embodiments, they are intended for illustrative purposes only. Therefore, the illustrative embodiments described herein do not constitute any limitation. Furthermore, it should be understood that various features described in the context of individual embodiments for clarity may also be combined to form a single embodiment. Conversely, various features described in the context of individual embodiments for brevity may also be combined to form sub-combinations.

[0070] The following lists the definitions of various terms used in this specification and claims.

[0071] It should be understood that the general description above and the specific description below are exemplary and explanatory only, and not restrictive. In this document, unless otherwise specified, the use of the singular includes the plural. As used herein, unless otherwise specified, the use of “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including” and “containing” is non-restrictive. Similarly, unless otherwise specified, terms such as “element” or “component” cover elements and components that comprise one unit as well as elements and components that comprise more than one subunit.

[0072] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions thereof (including, but not limited to, patents, patent applications, articles, books, and monographs) referenced in this application are expressly incorporated herein by reference into the portions thereof discussed herein and in their entirety.

[0073] Unless specifically defined, the nomenclature, procedures, and techniques used in connection with analytical chemistry, synthetic organic chemistry, and pharmaceutical and pharmaceutical chemistry as described herein are those well-known and commonly used in the field.

[0074] The description of embodiments of this disclosure is not intended to be exhaustive or to limit this disclosure to the exact forms disclosed. Although specific embodiments and examples of this disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of this disclosure, as will be recognized by those skilled in the art. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially simultaneously. The teachings of this disclosure provided herein can be suitably applied to other procedures or methods. The various embodiments described herein may be combined to provide further embodiments. If necessary, aspects of this disclosure may be modified to employ the compositions, functions, and concepts described herein to provide yet another embodiment of this disclosure. Furthermore, due to considerations of biological functional equivalence, some changes may be made to the protein structure without affecting the type or amount of biological or chemical action. These and other changes may be made to this disclosure according to specific embodiments. All such modifications are intended to be included within the scope of the appended claims.

[0075] Numerical ranges include the numbers that define the range. Where a range of values ​​is provided, it should be understood that, unless the context explicitly specifies otherwise, each intermediate value (accurate to one-tenth of the lower limit unit) between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value within the range and any other stated value or intermediate value within the range is covered within this disclosure. The upper and lower limits of these smaller ranges may be independently included or excluded from the range, and each range that includes any limit, excludes neither limit, or includes both limits is covered within this disclosure, but is subject to any express exclusions within the range. Where the range includes one or two limits, ranges that exclude either or both of these included limits are also included in this disclosure.

[0076] The term "about" generally refers to the acceptable degree of error in a measured quantity, taking into account the nature or precision of the measurement. An exemplary degree of error is within 20% of a given value or range of values, typically within 10%, and more usually within 5%. Preferably, the term "about" should be understood herein as plus or minus (±) 5% of the number used with it, more preferably ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.1%.

[0077] The term "composed of" refers to the compositions, methods and their respective components described herein, excluding any elements not listed in the description of this invention.

[0078] Implementations described herein as “comprising” one or more features may also be considered as disclosing corresponding implementations that are “composed of these features” and / or “substantially composed of these features”.

[0079] Concentrations, amounts, volumes, percentages, and other values ​​may be presented in range form throughout this document. It should also be understood that this range format is for convenience and brevity only and should be flexibly interpreted to include not only the values ​​explicitly listed as range limits, but also all individual values ​​or subranges covered within that range, as if each value and subrange were explicitly listed.

[0080] The polynucleotides of the present invention can be prepared by any means known in the art. For example, large quantities of polynucleotides can be produced by replication in suitable host cells. The polynucleotides of the present invention are preferably prepared by chemical synthesis, for example by the phosphoramidite method or the triester method, and can be performed on a commercially available automated oligonucleotide synthesizer. Double-stranded oligonucleotides can be obtained from chemically synthesized single-stranded products by the following steps: synthesizing complementary strands and annealing the strands together under appropriate conditions, or adding complementary strands using DNA polymerase and appropriate primer sequences.

[0081] Methods for determining the percentage sequence identity of nucleic acids are known in the art. For example, when assessing nucleic acid sequence identity, a sequence having a defined number of consecutive nucleotides can be compared with the corresponding portion of the nucleic acid sequence of the present invention (having the same number of consecutive nucleotides). Tools known in the art for determining the percentage sequence identity of nucleic acids include Nucleotide BLAST (described below).

[0082] The terms “reduction,” “decrease,” “reduction,” or “inhibition” are used herein to refer to a reduction in a statistically significant amount. The terms “reduction,” “decrease,” “reduction,” or “inhibition” generally mean a reduction of at least 10% compared to a reference level (e.g., without a given treatment), and may include, for example, a reduction of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, “reduction” or “inhibition” includes complete inhibition or reduction compared to a reference level. “Complete inhibition” is 100% inhibition (i.e., elimination) compared to a reference level.

[0083] The terms “increase,” “enhance,” “enhance,” or “activate” are used in this document to refer to an increase that is statistically significant. The terms “increase,” “enhance,” “enhance,” or “activate” may mean an increase of at least 25%, at least 50%, for example, an increase of at least about 50%, or at least about 75%, or at least about 80%, or at least about 90%, at least about 95%, or at least about 98%, or at least about 99%, or at least about 100%, or at least about 250% or more, or an increase of at least about 1.5 times, or at least about 2 times, or at least about 2.5 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times, or any increase between 1.5 times and 10 times or greater, compared to the reference level.

[0084] As used herein, a "chiral enrichment population" refers to a group of molecules of the same molecular formula, wherein the number or percentage of molecules within the population containing a specific stereochemical configuration at a particular chiral center is greater than the expected number or percentage of molecules within the population containing the same specific stereochemical configuration at the same particular chiral center if that particular chiral center were stereorandom. A chiral enrichment population of molecules having multiple chiral centers per molecule may contain one or more stereorandom chiral centers. In some embodiments, the molecule is a modified oligonucleotide. In some embodiments, the molecule is an oligomeric compound containing a modified oligonucleotide.

[0085] As used herein, "complementary" in oligonucleotides means that when the nucleotide sequence of an oligonucleotide is aligned in the opposite direction to the nucleotide sequence of another nucleic acid, at least 70% of the nucleotide bases of the oligonucleotide are hydrogen-bonded to the nucleotides of the other nucleic acid or one or more regions thereof. "Complementary region" in oligonucleotide terms means that when the nucleotide sequence of an oligonucleotide is aligned in the opposite direction to the nucleotide sequence of another nucleic acid, i.e., when the nucleotide sequences are reverse complementary, at least 70% of the nucleotide bases of that region are hydrogen-bonded to the nucleotides of the other nucleic acid or one or more regions thereof. For the avoidance of doubt, all references herein to the complementarity of an oligonucleotide (e.g., the antisense strand of the double-stranded oligonucleotide of the present invention) with a target sequence mean that the nucleotide sequence of the oligonucleotide (e.g., the antisense strand of the double-stranded oligonucleotide of the present invention) is reverse complementary to the target sequence.

[0086] Complementary nucleobases refer to nucleobases that can form hydrogen bonds with each other. Complementary nucleobase pairs include adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), and 5-methylcytosine (mC) and guanine (G). Certain modified nucleobases that pair with native nucleobases or other modified nucleobases are known in the art and are not considered complementary nucleobases as defined herein unless otherwise stated. For example, inosine may pair with adenosine, cytosine, or uracil, but is not considered complementary. Complementary oligonucleotides and / or nucleic acids do not need to be complementary at every nucleoside. Instead, some mismatches are tolerable. As used herein, “perfectly complementary” or “100% complementary” with respect to oligonucleotides means that the oligonucleotide is complementary to another oligonucleotide or nucleic acid at every nucleoside.

[0087] As used herein, a "conjugation group" refers to a group of atoms directly attached to an oligonucleotide. A conjugation group includes a conjugation moiety and a conjugation linker that connects the conjugation moiety to the oligonucleotide.

[0088] As used in this article, "conjugation linker" refers to a single bond that connects a conjugation portion to an oligonucleotide or a group of atoms containing at least one bond.

[0089] As used herein, "conjugated moiety" refers to a group of atoms that, compared to an identical molecule without the conjugated moiety, alter one or more properties of the molecule, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge, and clearance.

[0090] As used herein, "nucleoside bond" refers to a covalent bond between adjacent nucleosides in an oligonucleotide. As used herein, "modified nucleoside bond" refers to any nucleoside bond other than a phosphodiester nucleoside bond.

[0091] As used in this article, "motif" refers to the pattern of unmodified and / or modified sugar moieties, nucleobases, and / or nucleoside bonds in an oligonucleotide.

[0092] As used herein, “modified nucleoside” means a nucleoside that contains modified nucleobases and / or modified sugar moieties.

[0093] As used herein, “nucleobase” means an unmodified nucleobase or a modified nucleobase. A nucleobase is a heterocyclic moiety. As used herein, “unmodified nucleobase” is adenine (A), thymine (T), cytosine (C), uracil (U), or guanine (G). As used herein, “modified nucleobase” is a group other than the unmodified A, T, C, U, or G that can pair with at least one other nucleobase. “5-methylcytosine” is a modified nucleobase. A universal base is a modified nucleobase that can pair with any of the five unmodified nucleobases.

[0094] As used herein, “nucleobase sequence” means the sequence of consecutive nucleobases in a nucleic acid or oligonucleotide, regardless of any sugar or nucleoside internucleotide modifications. For example, but not limited to, oligomeric compounds having the nucleobase sequence “ATCGATCG” encompass any compound having that nucleobase sequence, whether modified or unmodified, including but not limited to such compounds containing RNA bases, such as those having the sequence “AUCGAUCG”, and compounds having some DNA bases and some RNA bases, such as “AUCGATCG”, and compounds having other modified nucleobases, such as “AT…”. m "CGAUCG", among which m C indicates a cytosine base containing a methyl group at position 5. Finally, for clarity, unless otherwise stated, the phrase “nucleobase sequence of SEQ ID NO: X” refers only to the nucleobase sequence in SEQ ID NO: X, without regard to any sugar or nucleoside inter-bond modifications also described in that SEQ ID.

[0095] As used herein, "nucleoside" refers to a compound or fragment of a compound containing a nucleobase and a sugar moiety. The nucleobase and sugar moiety are either unmodified or modified independently.

[0096] As used herein, “oligomer” means double-stranded oligonucleotide and optionally one or more additional features, such as a second oligonucleotide.

[0097] As used herein, “polynucleotide conjugate” means a double-stranded oligonucleotide and one or more optional additional features, such as conjugation groups or terminal groups.

[0098] A double-stranded oligonucleotide comprises a first oligonucleotide chain that pairs with a second oligonucleotide chain, the second oligonucleotide chain being complementary to the first oligonucleotide chain. A double-stranded oligonucleotide comprises a sense oligonucleotide or chain (also referred to in the art as a guest oligonucleotide or guest chain) and an antisense oligonucleotide or chain (also referred to in the art as a guide oligonucleotide or guide chain).

[0099] As used herein, “antisense activity” means any detectable and / or measurable change attributable to hybridization of the antisense strand with its target nucleic acid. In some embodiments, antisense activity is a reduction in the amount or expression of the target nucleic acid or the protein encoded by the target nucleic acid compared to the target nucleic acid or target protein levels in the absence of the antisense strand. In some embodiments, antisense activity is the regulation of target precursor mRNA (pre-mRNA) splicing.

[0100] As used herein, "antisense agent" means antisense strand and optionally one or more additional features, such as sense strand. Therefore, the double-stranded oligonucleotide of the present invention can be described as an antisense agent.

[0101] As used herein, "antense compound" means an antisense chain and optionally one or more additional features, such as conjugated groups. An antisense agent may comprise an antisense compound and a sense chain.

[0102] As used herein, “justice compound” means justice chain and one or more optional additional features, such as conjugation groups.

[0103] As used herein, "antisense oligonucleotide" means an oligonucleotide capable of hybridizing with a target nucleic acid and possessing at least one antisense activity, including the oligonucleotide moiety of an antisense compound. Antisense oligonucleotides include, but are not limited to, antisense RNAi oligonucleotides and antisense RNase H oligonucleotides. The double-stranded oligonucleotides of the present invention are typically siRNAs and therefore include antisense RNAi oligonucleotides.

[0104] As used in this article, "positive oligonucleotide" or "positive chain" refers to an oligonucleotide that can hybridize with an antisense oligonucleotide, including the oligonucleotide moiety of a positive compound.

[0105] As used herein, "oligonucleotide" means a chain of linked nucleosides connected by nucleotide bonds, wherein each nucleoside and nucleotide bond may be modified or unmodified. Unless otherwise stated, an oligonucleotide consists of 8-50 linked nucleosides. As used herein, "modified oligonucleotide" means an oligonucleotide in which at least one nucleoside or nucleotide bond is modified. As used herein, "unmodified oligonucleotide" means an oligonucleotide that does not contain any nucleoside or nucleotide modifications.

[0106] As used in this article, "stable phosphate group" means a 5'-phosphate analog that is metabolically more stable than the 5'-phosphate naturally present on DNA or RNA.

[0107] As used herein, “standard cell assay” means the assay described in the examples and its reasonable variations.

[0108] As used herein, in the context of a group of molecules with the same molecular formula, "stereorandom chiral center" means a chiral center with a random stereochemical configuration. For example, in a group of molecules containing a stereorandom chiral center, the number of molecules with the (S) configuration of that stereorandom chiral center may, but is not necessarily, the same as the number of molecules with the (R) configuration of that stereorandom chiral center. The configuration is considered random when the stereochemical configuration of the chiral center is due to a synthetic method not intended to control the stereochemical configuration. In some embodiments, the stereorandom chiral center is a stereorandom phosphate thioester nucleoside bond.

[0109] As used herein, “target nucleic acid” and “target RNA” refer to nucleic acids to which oligomeric compounds are designed to affect. Unless otherwise stated, target RNA refers to RNA transcripts and includes precursor mRNA and mRNA.

[0110] As used in this article, "target region" refers to the portion of the oligomeric compound that is designed to hybridize with the target nucleic acid.

[0111] As used in this article, "terminal group" refers to a chemical group or atomic group covalently attached to the end of an oligonucleotide.

[0112] As used herein, “hybridization” means the annealing of oligonucleotides and / or nucleic acids. While not limited to a specific mechanism, the most common hybridization mechanisms involve hydrogen bonding between complementary nucleobases, which can be Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonding. In some embodiments, complementary nucleic acid molecules include, but are not limited to, antisense compounds and nucleic acid targets.

[0113] As used herein, "RNAi agent" means an antisense agent that acts at least partially through RISC or Ago2 to regulate a target nucleic acid and / or the protein encoded by the target nucleic acid. RNAi agents include, but are not limited to, double-stranded siRNA and single-stranded RNAi (ssRNA). i RNAi agents include microRNAs, including microRNA mimics. RNAi agents may contain conjugation groups and / or terminal groups. In some embodiments, RNAi agents regulate the amount and / or activity of target nucleic acids. The term RNAi agent excludes antisense agents that act via RNase H.

[0114] As used herein, "RNase H agent" means an antisense agent that acts via RNase H to regulate a target nucleic acid and / or the protein encoded by the target nucleic acid. In some embodiments, the RNase H agent is single-stranded. In some embodiments, the RNase H agent is double-stranded. RNase H compounds may contain conjugation groups and / or terminal groups. In some embodiments, the RNase H agent regulates the amount and / or activity of the target nucleic acid. The term RNase H agent excludes antisense agents that act primarily via RISC / Ago2.

[0115] As used herein, “reduced” or “inhibited” ANGPTL3 means that the expression and / or protein levels of ANGPTL3 RNA are lower in the presence of the oligomers or oligomers described herein compared to the expression and / or protein levels of ANGPTL3 RNA in the absence of the oligomers or oligomers described herein.

[0116] As used herein, "pharmaceutically acceptable carrier or diluent" means any substance suitable for administration to animals. Certain such carriers enable pharmaceutical compositions to be formulated, for example, into tablets, pills, sugar-coated pills, capsules, liquids, gels, syrups, slurries, suspensions, and lozenges for oral ingestion by a subject. In some embodiments, a pharmaceutically acceptable carrier or diluent is sterile water, sterile saline, sterile buffer solutions, or sterile artificial cerebrospinal fluid.

[0117] As used herein, "pharmaceutically acceptable salt" means a compound that is physiologically and pharmaceutically acceptable. A pharmaceutically acceptable salt retains the desired biological activity of the parent compound without conferring undesirable toxicological effects. For example, the term "pharmaceutically acceptable" can refer to salts, excipients, carriers, diluents, etc., approved by federal or state regulatory agencies or listed in the United States Pharmacopeia, the European Pharmacopoeia, or other recognized pharmacopoeias.

[0118] As used herein, "pharmaceutical composition" means a mixture of substances suitable for administration to a subject. For example, a pharmaceutical composition may comprise an oligomeric compound and a sterile aqueous solution. In some embodiments, the pharmaceutical composition exhibits activity in free uptake assays in certain cell lines.

[0119] As used herein, "prodrug" means a therapeutic agent in its first form in vitro, which is converted into a second form within an animal or its cells. Typically, the conversion of a prodrug in an animal is facilitated by enzymes (e.g., endogenous enzymes or viral enzymes) or chemicals present in cells or tissues and / or by physiological conditions. In some embodiments, the first form of the prodrug is less active than the second form.

[0120] As used herein, “treatment” means improving a subject’s disease or condition by administering the oligomers or oligomers described herein. In some embodiments, treatment improves the subject’s symptoms relative to the same symptoms without treatment. In some embodiments, treatment reduces the severity or frequency of symptoms, or delays the onset of symptoms, slows the progression of symptoms, or slows the severity or frequency of symptoms.

[0121] As used herein, "therapeutic effective amount" means the amount of a drug or composition that has been observed to provide a therapeutic benefit to animals. For example, a therapeutic effective amount may be observed to improve the symptoms of a disease.

[0122] The terms “individual,” “subject,” and “patient” are used interchangeably herein and refer to a mammalian subject to whom diagnosis, prognosis, disease monitoring, management, treatment, and / or treatment optimization is desired. Mammals can be (but are not limited to) humans, non-human primates, mice, rats, dogs, cats, horses, or cattle. In a preferred embodiment, the individual, subject, or patient is a human. An “individual” can be an adult, adolescent, or infant. An “individual” can be male or female.

[0123] A “subject” who “needs” treatment for a specific condition can be an individual who has the condition, has been diagnosed with the condition, or is at risk of developing the condition.

[0124] Subjects may be subjects who have been previously diagnosed or identified as having or having a condition requiring treatment, or one or more complications or symptoms related to that condition, and optionally have received treatment for the condition or one or more complications or symptoms related to said condition as defined herein. Alternatively, subjects may also be subjects who have not been previously diagnosed with a condition as defined herein, or one or more symptoms or complications related to said condition. For example, a subject may be a subject exhibiting a condition or one or more risk factors for one or more symptoms or complications related to said condition, or a subject not exhibiting risk factors.

[0125] As used herein, the term "healthy individual" refers to an individual or group of individuals in a healthy state, such as an individual who does not exhibit any symptoms of disease, has not been diagnosed with a disease, and / or is unlikely to develop a disease (e.g., a thrombotic event). Preferably, the healthy individual is not taking any medications that affect hemostasis and has not been diagnosed with any other disease. One or more healthy individuals may have similar sex, age, and / or body mass index (BMI) compared to the test individual. The application of standard statistical methods used in medicine allows for the determination of normal expression levels in healthy individuals and significant deviations from such normal levels.

[0126] In this article, the terms "control group" and "reference group" are used interchangeably.

[0127] Conjugate 2 is provided, comprising (i) a first (positive) chain and (ii) a second (antisense) chain, wherein the first (positive) chain has the following sequence:

[0128]

[0129] The second (antisense) chain has the following sequence:

[0130]

[0131] Where [M] is the trianticular ligand portion connected to the 5' end of the second chain and having the following structure:

[0132] .

[0133] Therefore, the terminal thiophosphate group of the ligand moiety [M] is directly (through the “free” bond indicated by “*”) bonded to the 5' position of the 5' terminal nucleotide of the first chain (i.e., bonded to the 5'-carbon of the 2'-O-methylribose moiety of the mC residue at the 5' end of the first chain).

[0134] The sequence is displayed in the standard 5' to 3' orientation.

[0135] In the context of the above expression, the following abbreviations are used:

[0136]

[0137] In one embodiment, a method for preparing a conjugate (I) or a pharmaceutically acceptable salt of a conjugate (I) is provided, as well as an intermediate for preparing the conjugate.

[0138] Another implementation is the product that can be obtained through any of the methods or examples disclosed herein.

[0139] Pharmaceutical and pharmaceutical uses

[0140] It is believed that conjugate 2 and its pharmaceutically acceptable salts can be used to prevent or treat cardiovascular metabolic diseases in mammals (especially humans), such as hypertriglyceridemia, obesity, hyperlipidemia, lipid and / or cholesterol metabolism disorders, atherosclerosis, type I diabetes, cardiovascular disease, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, homozygous and heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia, and other metabolic-related conditions and diseases.

[0141] It is believed that conjugate 2 and its pharmaceutically acceptable salts can be used to prevent or treat heart failure in mammals (especially humans).

[0142] It is believed that conjugate 1 and its pharmaceutically acceptable salts can be used to prevent or treat heart failure in mammals (especially humans).

[0143] It is believed that conjugate 2 and its pharmaceutically acceptable salts can be used to prevent or treat chronic kidney disease in mammals, especially humans.

[0144] It is believed that conjugate 1 and its pharmaceutically acceptable salts can be used to prevent or treat chronic kidney disease in mammals (especially humans).

[0145] To avoid ambiguity, as used herein, the term “treatment” includes both therapeutic and / or preventative treatment.

[0146] When the compounds or salts described herein are administered as a therapy to treat a disease, the “therapeutic effective amount” is an amount sufficient to alleviate or completely relieve symptoms or other adverse effects of the disease, cure the disease, reverse disease progression, completely stop or slow disease progression, or reduce the risk of disease exacerbation.

[0147] Therefore, the compounds described herein are suitable for therapeutic and / or preventative treatment of these conditions.

[0148] The compounds described herein have the following advantages: compared with compounds known in the prior art, they are more effective, less toxic, more selective, more potent, produce fewer side effects, are more easily absorbed, and / or have better pharmacokinetic characteristics (e.g., higher oral bioavailability and / or lower clearance).

[0149] For the above-mentioned therapeutic indications, the dosage will vary depending on the compound used, the route of administration, and the desired treatment. However, generally speaking, satisfactory results are obtained when the compound is administered in solid form at doses ranging from 1 mg to 2000 mg daily.

[0150] Conjugate 2 and its pharmaceutically acceptable salt can be used alone or in the form of a suitable pharmaceutical composition, wherein the compound or derivative is mixed with a pharmaceutically acceptable adjuvant, diluent, or carrier. Therefore, another aspect relates to a pharmaceutical composition comprising conjugate 2 or its pharmaceutically acceptable salt, said conjugate 2 or its pharmaceutically acceptable salt being mixed with a pharmaceutically acceptable adjuvant, diluent, or carrier.

[0151] Conjugate 1 and its pharmaceutically acceptable salts can be used alone or in the form of suitable pharmaceutical compositions, wherein the compound or derivative is mixed with a pharmaceutically acceptable adjuvant, diluent, or carrier. Therefore, another aspect relates to a pharmaceutical composition comprising conjugate 1 or its pharmaceutically acceptable salts, said conjugate 1 or its pharmaceutically acceptable salts being mixed with a pharmaceutically acceptable adjuvant, diluent, or carrier.

[0152] In one embodiment, conjugate 2 or a pharmaceutically acceptable salt thereof is provided for the treatment, particularly for the prevention or treatment of cardiovascular metabolic diseases in mammals (especially humans), such as hypertriglyceridemia, obesity, hyperlipidemia, lipid and / or cholesterol metabolism disorders, atherosclerosis, type I diabetes, cardiovascular disease, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, homozygous and heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia, and other metabolic-related conditions and diseases.

[0153] In one embodiment, conjugate 2 or a pharmaceutically acceptable salt thereof is provided for the treatment, particularly for the prevention or treatment of heart failure in mammals (especially humans).

[0154] In one embodiment, conjugate 1 or a pharmaceutically acceptable salt thereof is provided for the treatment, particularly for the prevention or treatment of heart failure in mammals (especially humans).

[0155] In one embodiment, conjugate 2 or a pharmaceutically acceptable salt thereof is provided for the treatment, particularly for the prevention or treatment of chronic kidney disease in mammals (especially humans).

[0156] In one embodiment, conjugate 1 or a pharmaceutically acceptable salt thereof is provided for the treatment, particularly for the prevention or treatment of chronic kidney disease in mammals (especially humans).

[0157] These and other embodiments are described in more detail below, and further aspects will be apparent to those skilled in the art upon reading this specification.

[0158] Combination therapy

[0159] Conjugate 2 or its pharmaceutically acceptable salt may also be used in combination with other compounds used to treat the above conditions.

[0160] In another embodiment, a combination therapy is provided, wherein conjugate 2 or a pharmaceutically acceptable salt thereof is administered simultaneously, sequentially, or in combination with a second active ingredient to treat one or more of the conditions listed above. This combination may be used in conjunction with one or more other active ingredients.

[0161] When used in combination therapy, it is anticipated that conjugate 2 or a pharmaceutically acceptable salt thereof may be administered with other active ingredients in a single composition, in completely separate compositions, or in combination thereof. It is also anticipated that the active ingredients may be administered in parallel, simultaneously, sequentially, or separately. The specific composition and frequency of administration for combination therapy will depend on a variety of factors, including, for example, the route of administration, the condition being treated, the species of the patient, any potential interactions between the active ingredients when combined as a single composition, any interactions between the active ingredients when administered to animal patients, and a variety of other factors known to physicians (in the case of human patients), veterinarians (in the case of non-human patients), and others skilled in the art.

[0162] Pharmaceutical Composition

[0163] A method for treating ANGPTL3-related diseases is provided, comprising administering a therapeutically effective amount of an ANGPTL3 siRNA agent to a subject suffering from an ANGPTL3-related disease, thereby treating the ANGPTL3-related disease.

[0164] Typically, the pharmaceutical composition further comprises a pharmaceutically acceptable diluent or carrier. A pharmaceutically acceptable diluent may be water or saline. The pharmaceutical composition may comprise, or consist of, a sterile saline solution and an ANGPTL3 siRNA agent, wherein the ANGPTL3 siRNA agent comprises an antisense strand having a nucleobase sequence complementary to the ANGPTL3 target nucleic acid. The sterile saline solution is preferably pharmaceutical grade saline.

[0165] The pharmaceutical composition may comprise, or consist of, an ANGPTL3 siRNA agent and sterile water, or the ANGPTL3 siRNA agent and sterile water, wherein the ANGPTL3 siRNA agent comprises an antisense strand having a nucleobase sequence complementary to the ANGPTL3 target nucleic acid. Preferably, the sterile water is pharmaceutical grade water, such as water for injection. The saline may be phosphate-buffered saline (PBS), preferably sterile PBS.

[0166] The pharmaceutical composition may comprise an ANGPTL3 siRNA agent and one or more excipients, said ANGPTL3 siRNA agent comprising an antisense strand having a nucleobase sequence complementary to the ANGPTL3 target nucleic acid. Excipients may be selected from water, salt solutions, alcohols, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silica, viscous paraffin, hydroxymethyl cellulose, and polyvinylpyrrolidone.

[0167] In some embodiments, the ANGPTL3 siRNA agent (which comprises an antisense strand having a nucleobase sequence complementary to the ANGPTL3 target nucleic acid) may be mixed with pharmaceutically acceptable active and / or inert substances for use in the preparation of pharmaceutical compositions or formulations. The compositions and methods used to formulate pharmaceutical compositions depend on a variety of criteria, including but not limited to route of administration, disease severity, or dosage.

[0168] Pharmaceutical compositions comprising an ANGPTL3 siRNA agent (which comprises an antisense strand having a nucleobase sequence complementary to the ANGPTL3 target nucleic acid) encompass any pharmaceutically acceptable salt of the ANGPTL3 siRNA agent. Pharmaceutical compositions comprising an ANGPTL3 siRNA agent (which comprises an antisense strand having a nucleobase sequence complementary to the ANGPTL3 target nucleic acid) generally provide (directly or indirectly) its biologically active metabolites or residues after administration to animals (including humans). Therefore, this disclosure also relates, for example, to pharmaceutically acceptable salts of ANGPTL3 siRNA agents, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

[0169] Pharmaceutical compositions may comprise a cosolvent system. Some such cosolvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In some embodiments, such cosolvent systems are used for hydrophobic compounds. A non-limiting example of such a cosolvent system is the VPD cosolvent system, which comprises 3% w / v benzyl alcohol and 8% w / v nonpolar surfactant polysorbate 80. TM And a 65% w / v polyethylene glycol 300 anhydrous ethanol solution. The proportions of these co-solvent systems can be varied significantly without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components can be changed: for example, other surfactants can be used instead of polysorbate 80. TM The fraction size of polyethylene glycol can vary; other biocompatible polymers can replace polyethylene glycol, such as polyvinylpyrrolidone; and other sugars or polysaccharides can replace dextrose.

[0170] Pharmaceutical compositions can be prepared for injection (e.g., intravenous, subcutaneous, etc.). In some such embodiments, the pharmaceutical composition comprises a carrier and is formulated in an aqueous solution, such as water or a physiologically compatible buffer, such as Hanks' solution, Ringer's solution, or physiological saline buffer. In some embodiments, other components are included (e.g., components that aid solubility or act as preservatives). In some embodiments, an injectable suspension is prepared using a suitable liquid carrier, suspending agent, etc. Some pharmaceutical compositions for injection are presented in unit dosage forms, such as in ampoules or multi-dose containers. Some pharmaceutical compositions for injection are suspensions, solutions, or emulsions in oily or aqueous media and may contain formulations such as suspending agents, stabilizers, and / or dispersants. Some solvents suitable for pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes.

[0171] Under certain conditions, some of the compounds disclosed herein act as acids. Although such compounds can be drawn or described in protonated (free acid) or ionized and associated with a cation (salt) form, aqueous solutions of such compounds exist in equilibrium between these forms. For example, the phosphate bonds of oligonucleotides in aqueous solutions exist in equilibrium between free acid, anionic, and salt forms. Unless otherwise stated, the compounds described herein are intended to include all such forms. Furthermore, some oligonucleotides have multiple such bonds, each in equilibrium. Thus, oligonucleotides in solution exist as a collection of forms in equilibrium at multiple locations. The term "oligonucleotide" is intended to include all such forms. The drawn structures necessarily depict a single form. However, unless otherwise stated, such drawings are also intended to include the corresponding forms. In this document, structures depicting compounds as free acids, followed by the term "or salts thereof," explicitly include all such forms that can be fully or partially protonated / deprotonated / associated with a cation. In some cases, one or more specific cations are identified.

[0172] ANGPTL3 siRNA (containing an antisense strand with a nucleobase sequence complementary to the ANGPTL3 target nucleic acid) can be dissolved in aqueous solution with sodium. ANGPTL3 siRNA (containing an antisense strand with a nucleobase sequence complementary to the ANGPTL3 target nucleic acid) can be dissolved in aqueous solution with potassium. ANGPTL3 siRNA (containing an antisense strand with a nucleobase sequence complementary to the ANGPTL3 target nucleic acid) can be dissolved in PBS. ANGPTL3 siRNA (containing an antisense strand with a nucleobase sequence complementary to the ANGPTL3 target nucleic acid) can be dissolved in water. The pH of the solution can be adjusted to the desired pH using NaOH and / or HCl.

[0173] Preparation of compounds

[0174] Example 1

[0175] Conjugate 1 (WO2019 / 055633: AD05488 (Guide: AM07235-AS, Passerby: AM07234-SS)):

[0176]

[0177]

[0178] Single-chain synthesis, Example 1

[0179] Unless otherwise stated, all synthetic reactions were carried out under an inert atmosphere. In the following examples, when the source of the starting material is not specified, it should be understood that the starting material is a known compound (e.g., a commercially available compound from a supplier such as Sigma-Aldrich) and / or can be prepared according to known methods, such as those described in the literature and patent publications described herein.

[0180] Nucleotide phosphoramides were purchased from Sigma-Aldrich or WuXi. Linker phosphoramides were purchased from Glen Research or WuXi. The 5'-amino modifier C6 was obtained from Glen Research. UV purity was determined using ion-pair LCMS and is given at 260 nm. Yields are given based on initial resin loading and the oligonucleotide content of the final product (calculated from UV absorption).

[0181] General synthesis procedure, Example 1

[0182] Use this program unless otherwise specified.

[0183] Oligonucleotides SEQ ID NO: 2, 3 were synthesized on the K&A system at a scale of 10 μmol using CUTAG CPG support (Sigma-Aldrich, 25-35 μmol / g).

[0184] Phosphorous amides were dissolved in DNA-grade acetonitrile (ACN) to a final concentration of 0.1 M (3 equivalents) before use, except for GalNAc phosphorous amides, which were dissolved in anhydrous DCM (dry DCM) at 0.2 M. Detrimethylation was performed using 3 vol-% dichloroacetic acid in DCM (contact time 5 x 35 s). Bis-detrimethylation was used to deprotect dianthind and trianthind linkers. BTT (0.3 M in ACN) was used as the coupling activator.

[0185] The phosphorus amide recycling time for all 2'-modified building blocks was 10 min (single coupling), and for GalNAc phosphorus amide (SEQ ID no 1) it was 4 x 10 min (four couplings). DDTT was dissolved in pyridine (0.2 M) and used as the thiolation agent with a contact time of 5 min. The oxidant solution, purchased from Sigma-Aldrich, was used directly with a contact time of 9 s. Equal volumes of Cap A (9.1 vol-% acetic anhydride in tetrahydrofuran (THF)) and Cap B (THF / N-methylimidazolium / pyridine 80:10:10 vol-%) were mixed in situ for capping (contact time 50 s). After synthesis, the cyanoethyl skeleton was removed with 20 vol-% diethylamine in ACN (contact time 7 x 1 min). Oligonucleotides were cleaved on a solid support and further deprotected by treatment with 10 mL of ammonia (40% aq.) solution at 55°C for 18 hours, and then dried on a Speedvac.

[0186] Purification procedure, Example 1

[0187] Single-stranded oligonucleotides were purified using ion-pair HPLC or reversed-phase HPLC.

[0188] The identity and purity of the oligonucleotides were confirmed by liquid chromatography-mass spectrometry (LC-MS) using a Waters Corporation Acquity I-class LC system (BioAccord) equipped with a PDA and coupled to RDa via heated electrospray ionization. Analytical runs were performed at 80°C on an ACQUITY PREMIER BEH C18 1.7 μm 2.1 x 100 mm (130 Å) column using a gradient of 35–50% B (100% ACN) in A (10 mM TBAA in 10 mM 10 mM TBAA in 90% H2O) for 10 min at a flow rate of 0.5 mL / min.

[0189] Oligonucleotide SEQ ID NO: 2 was purified by RP HPLC (XBridge C18, 5μm 19x150mm, A: 50 mM NH4HCO3 in water (pH 8), B: ACN) with gradients of 5-12% B for 16 min and 12-30% B for 3 min. Further purification was performed using the same column and 60 mM DBuAA in A: (H2O / ACN 95 / 5) at pH 7.0 and 60 mM DBuAA in B: ACN with gradients of 10-40% for 12 min and 40-80% for 5 min. The UV purity was 95.0%; calculated m / z (ESI-; TOF) value was 2158.91 (4-); measured value: 2158.91.

[0190] Oligonucleotide SEQ ID NO: 3 was purified by RP HPLC (XBridge C18, 5μm 19x150mm, A: 60 mM DBuAA in (H2O / ACN 95 / 5), pH 7.0, B: 60 mM DbuAA in ACN), with gradients of 20-50% for 12 min and 50-80% for 0.5 min. The UV purity was 90.2%; the calculated m / z (ESI-; TOF) value was 1728.22 (4-); the measured value was 1728.23.

[0191] Double-stranded siRNA synthesis-annealing, Example 1

[0192] The amount of single chains was quantified at 260 nm using UV spectrophotometry, and the solution was normalized to 2 mM using the calculated extinction coefficient.

[0193] For siRNA compounds, single strands were mixed in equimolar amounts, heated to 95°C and held for 5 min, and then cooled to room temperature over one hour.

[0194] Double-stranded siRNAs were analyzed using UPLC-MS. Analysis of double-stranded siRNAs was performed on an ACQUITY PREMIER BEHAmide 1.7 μm 2.1 x 100 mm, 300 Å (30°C, flow rate 0.5 mL / min) using a gradient of 15–60% B for 5 min, where A: 70 / 30 ACN / 25 mM ammonium acetate in water, and B: 30 / 70 ACN / 25 mM ammonium acetate in water.

[0195] The final siRNA was freeze-dried and redissolved in 1X PBS to a concentration of 10 mg / mL.

[0196] Conjugate 1: Theoretical quality: 15564.6, measured deconvolution quality: 15564.7.

[0197] Example 2

[0198] Conjugate 2:

[0199]

[0200]

[0201] Building block synthesis, Example 2

[0202] The synthesis of [M] phosphoramide derivatives (ST41-phos) and ST23 (ST23-phos) and their precursor compounds can be carried out as described in WO2017 / 174657.

[0203] ST41-phos:

[0204]

[0205] ST23-phos:

[0206]

[0207] Synthesis of oligonucleotides, Example 2

[0208] Oligonucleotides were synthesized using the standard phosphoramidite chemistry method on an AKTA oligopilot 100 synthesizer. Commercially available base-loaded solid-phase support (Nitto phase HL250, Kinovate, CA), 2'OMe nucleotide phosphoramidite, and 2'F nucleotide phosphoramidite (all standard protected, ChemGenes) were used, following the manufacturer's recommended procedure.

[0209] Auxiliary reagents were purchased from EMP Biotech. The synthesis was performed using a 0.1 M solution of phosphorous amide in anhydrous acetonitrile, with benzylthiotetrazole (BTT) as the activator (0.3 M in acetonitrile). The coupling time was 10 min. Cap / OX / Cap or Cap / Thio / Cap cycles were applied (Cap: Ac₂O / NMI / dimethylpyridine / acetonitrile; oxidant: 0.1 M MI₂ in pyridine / H₂O). The thiophosphate was introduced using 0.2 M XH (0.2 M xanthane hydride in pyridine). DMT cleavage was achieved by treatment with 3% dichloroacetic acid in toluene. After the programmed synthesis cycle, the cells were washed with diethylamine (DEA). All other reagents and solvents were commercially available and used at standard reagent quality.

[0210] All oligonucleotides were synthesized in DMT-off mode. Single strands were cleaved from the support by treatment with 40% methylamine aqueous solution (90 min, RT). The resulting crude oligonucleotides were concentrated to a concentration of approximately 10 mg / mL under reduced pressure and purified by ion-exchange chromatography with a sodium chloride gradient on an AKTAPure HPLC system (SourceQ. 7.5 mL. GE Healthcare). Fractions containing the product were combined and desalted by UFDF (Sartorius Sartoflow Smart, Sartocon Slice200 Hydrosart 2k, with a 10-fold volume replacement with sterile water).

[0211] To demonstrate purity, all final single-stranded products were analyzed by AEX-HPLC (DNA Pac PA200 4.0x250mm & DNAPac PA200 Guard 4x50mm, 80 °C, using a gradient of 25-70% B (10% MeCN, 20 mM TRIS in water, 0.4 M LiClO4, pH = 7.4) in A (10% MeCN, 20 mM TRIS in water), for 10 min at a flow rate of 1 mL / min). Purity is given as %FLP (% full-length product), which is the percentage of the UV area under the specified product signal in the UV trajectory of the final product AEX-HPLC analysis. The identities of each single-chain product were confirmed by UPLC-MS analysis (Acquity UPLCBEH C18 2.1x50 mm, 1.7 µm Acquity BEH C18 1.7 µM Vanguard Pre-Col, 60°C, using a gradient of 10-22% B (100 mM HFIP, 15 mM TEA in MeOH) in A (100 mM HFIP, 15 mM TEA in water), for 5 min at a flow rate of 0.3 mL / min).

[0212] Single-chain oligonucleotides:

[0213]

[0214] Double chain formation, Example 2

[0215] Each single strand was dissolved in H₂O at a concentration of 60 OD / mL. The solutions of both individual oligonucleotides were added together to the reaction vessel. Titration was performed for easier reaction monitoring. The first strand was added in 25% excess relative to the second strand, determined by UV absorption at 260 nm. The reaction mixture was heated to 80°C and held for 5 min, then slowly cooled to room temperature. Double strand formation was monitored by ion-pair reversed-phase HPLC (X Bridge BEH C18 2.1x50 mm 2.5µm, XPVanGuard Cartridges, 15 °C, using a gradient of 5-45% B (100 mM HFIP, 15 mM TEA in MeOH) in A (100 mM HFIP, 15 mM TEA in water), over 15 min at a flow rate of 0.3 mL / min). The required amount of the second strand was calculated based on the UV area of ​​the remaining single strand and added to the reaction mixture. The reaction was reheated to 80°C and slowly cooled to room temperature. Repeat the process until less than 10% of residual single strands are detected.

[0216] Nucleic acid conjugates:

[0217]

[0218] Abbreviations used in the examples:

[0219]

[0220] [M] is:

[0221]

[0222] [M] is bonded via the "free" key indicated by "*".

[0223] NAG is:

[0224]

[0225] NAG is bonded via "free" bonds indicated by "*".

[0226] Example 3

[0227] In vitro test of GalNAc-conjugated siRNA inhibiting ANGPTL3 mRNA expression in primary hepatocytes

[0228] List of abbreviations for Example 3:

[0229]

[0230]

[0231] Test Project

[0232] The GalNAc-conjugated siRNA tested was:

[0233] Conjugate 1 (Example 1) and Conjugate 2 (Example 2).

[0234] Cell culture

[0235] Primary human hepatocytes (lot number Hu8264) and cynomolgus monkey hepatocytes (Macaca fascicularis, lot number CY411) were obtained from Life Technologies. As described by the manufacturer, the primary hepatocytes were thawed and plated in Williams' E medium (Life Technologies) supplemented with 5% fetal bovine serum, 1 µM dexamethasone in DMSO (DMSO final concentration = 0.01%), and 3.6% v / v thaw / plate Cocktail-A (Thermo Fisher Scientific, CM3000), and cultured at 37°C in a 5% CO2 atmosphere.

[0236] Primary hepatocytes were treated with GalNAc-conjugated siRNA.

[0237] Primary hepatocytes were seeded in type I collagen-coated 96-well plates (Life Technologies) at densities of 35,000 human cells and 45,000 cynomolgus monkey cells per well. GalNAc-conjugated siRNA conjugates 1 and 2 were serially diluted in triplicate in previously defined media to achieve final concentrations of 100, 20, 4, 0.8, and 0.16 nM for human cells and 100, 10, 1, 0.1, and 0.01 nM for cynomolgus monkey cells. The plates were then incubated at 37°C in a 5% CO2 atmosphere for 24 hours. Cells were subsequently lysed, and RNA was isolated from the cells as described in Section 4.4.

[0238] Quantitative RT-PCR

[0239] Twenty-four hours after treatment with GalNAc-conjugated siRNA conjugates 1 and 2, total RNA was extracted using an InviTrap HTS 96-well kit (Invitek Molecular GmbH, Berlin, Germany) according to the manufacturer's instructions, and eluted with 2 × 30 µl of elution buffer. The eluted RNA was stored at -80°C until analysis by qPCR.

[0240] For qPCR, mix 10 µl of RNA with the Takyon 5x Master mix (Eurogentec, Seraing, Belgium) and the primer / probe set for ANGPTL3 or PPIB as listed in Table 1 (BioTez GmbH, Berlin, Germany or Eurogentec, Seraing, Belgium):

[0241] Table 1: Primer and probe sequences used for RT-qPCR.

[0242]

[0243] RT-qPCR reactions were performed using a QuantStudio 6 Flex cycler (Applied Biosystems, ThermoFisher Scientific, Massachusetts, USA) with the standard RT-qPCR protocol (48°C for 30 min, 95°C for 3 min, 40 cycles: 95°C for 15 seconds, followed by 60°C for 1 min) at a rate of 20 ml per sample.

[0244] Data uses comparison C T Law (also known as 2) -DDCt (Livak and Schmittgen, 2001; and Schmittgen and Livak, 2008) Calculations were performed. In short, the amount of ANGPTL3 mRNA was first normalized relative to the endogenous housekeeping gene PPIB, and then normalized relative to untreated control cells, according to the following formula:

[0245] .

[0246] Data are expressed as the mean ± SD of three replicates.

[0247] result

[0248] Unless otherwise stated, all figures presented in this article refer to the mean ± standard deviation (SD). Some tables present calculations using standard spreadsheet software (Graph Pad Prism 9, Microsoft). ® EXCEL ® The calculations for some parameters used more digits than the tabulated data. Therefore, recalculating parameters based on the tabulated data can lead to minor changes in some cases. Since the original data remained unchanged and is therefore numerically correct, this has no significant impact on the research results.

[0249] To assess the effects of conjugate 1 and conjugate 2 on ANGPTL3 mRNA levels in vitro, primary human hepatocytes and cynomolgus monkey hepatocytes were treated with five different concentrations for 24 hours. RNA was then isolated, and the residual ANGPTL3 transcript levels were quantified by qPCR. Normalization was performed using untreated cells. Figure 1 The experimental results of human primary hepatocytes were shown, and Figure 2 The experimental results of cynomolgus monkey liver cells are shown.

[0250] like Figure 1 As shown, conjugate 2 induced dose-dependent ANGPTL3 knockdown in human hepatocytes, with a knockdown of over 50% at 100 nM, while conjugate 1 showed knockdowns of over 50% at both 100 nM and 20 nM. The maximum knockdowns of conjugate 2 and conjugate 1 measured in this experiment were 59 ± 3% and 60.3 ± 2%, respectively.

[0251] In cynomolgus monkey liver cells ( Figure 2 The tested compounds generally performed more effectively, consistent with previous observations (data not shown), therefore a 10-fold dilution series was used in this experiment. Conjugate 2 showed a knockdown level exceeding 50% at 1 nM, and conjugate 1 showed a knockdown level exceeding 50% at 10 nM. Maximum knockdown levels were observed for both at 100 nM (85.4 ± 1% for conjugate 2 vs. 80.4 ± 1% for conjugate 1).

[0252] Data are presented as mean ± SD of three replicates. The corresponding gene expression levels are listed in Table 2.

[0253] Table 2: ANGPTL3 mRNA expression levels in primary hepatocytes relative to PPIB mRNA normalization

[0254]

[0255] Reference compounds 3 and 4

[0256] Conjugate 3 (WO2012 / 177784: AD-52988.1 (Guide: A-108397.1, Passerby: A-108396.1)):

[0257]

[0258]

[0259] Conjugate 4 (WO2012 / 177784: AD-45887.1 (Guide chain: A-96144.1, Passenger chain: A-96143.1)):

[0260]

[0261]

[0262] In the context of the above expression, the following abbreviations are used:

[0263]

[0264] L96 is a trianticular ligand portion with the following structure:

[0265]

[0266] Therefore, the terminal thiophosphate group of the ligand portion L96 is directly (via the "free" bond indicated by "*") bonded to the 3' position of the 3' terminal nucleotide of the first chain.

[0267] Single-chain synthesis

[0268] Unless otherwise stated, all synthetic reactions were carried out under an inert atmosphere. In the following examples, when the source of the starting material is not specified, it should be understood that the starting material is a known compound (e.g., a commercially available compound from a supplier such as Sigma-Aldrich) and / or can be prepared according to known methods, such as those described in the literature and patent publications described herein.

[0269] Nucleotide phosphoramides were purchased from Sigma-Aldrich or WuXi. Linker phosphoramides were purchased from Glen Research or WuXi. The 5'-amino modifier C6 was obtained from Glen Research. UV purity was determined using ion-pair LCMS and is given at 260 nm. Yields are given based on initial resin loading and the oligonucleotide content of the final product (calculated from UV absorption).

[0270] General synthesis program

[0271] Use this program unless otherwise specified.

[0272] Oligonucleotides SEQ ID NO: 7, 9 were synthesized at a scale of 32 µmole on an ÄKTA OligoPilot Plus 10 synthesizer (GE Healthcare) using a standard synthetic cycle: detriphenylmethylation (3% dichloroacetic acid in toluene), coupling (coupling agent: 0.25 M 5-[3,5-bis(trifluoromethyl)phenyl]-1H-tetrazole solution in acetonitrile), capping (Cap A: 20% N-methylimidazolium and 80% acetonitrile; Cap B: 20% pyridine, 20% acetic anhydride and 60% acetonitrile), oxidation (0.05 M iodine in pyridine and water) or thiolation (0.2 M hydroxanthogen in pyridine), and solid support (UNY Primer Support 5G ~353 μmole / g, GE Healthcare). All fully protected β-cyanoethylphosphoramide monomers were dissolved in anhydrous acetonitrile (0.1 M) under argon atmosphere immediately before use. The phosphorus amide recycling / coupling time for DNA monomers was 5 min, and for RNA monomers, it was extended to 10 min. Stepwise coupling efficiency and overall yield were determined by automated triphenylmethyl cation absorption monitoring, with all synthesized oligonucleotides exceeding 98%. At the end of solid-phase synthesis, the oligonucleotides bound to the solid support were treated with 10 mL of ammonia (aq. 40%) solution at 55°C for 18 h and subsequently dried on a Speedvac.

[0273] Oligonucleotides SEQ ID NO: 6 and 8 were synthesized at a scale of 13 µmole on an ÄKTA OligoPilot Plus 10 synthesizer (GE Healthcare) using the standard synthesis cycle described above and a solid support NittoPhase preloaded with L96 GalNac (loading amount 0.153 mmol / g).

[0274] Purification procedure

[0275] Single-stranded oligonucleotides were purified using ion-pair HPLC or reversed-phase HPLC.

[0276] The identity and purity of the oligonucleotides were confirmed by liquid chromatography-mass spectrometry (LC-MS) using a Waters Corporation Acquity I-class LC system (BioAccord), equipped with a PDA and coupled to RDa via heated electrospray ionization. Analytical runs were performed at 80°C on an ACQUITY PREMIER BEH C18 1.7 μm 2.1 x 100 mm (130 Å) column using a gradient of 35–50% B (100% ACN) in A (10 mM TBAA in 90% H₂O) for 10 min at a flow rate of 0.5 mL / min.

[0277] Oligonucleotide SEQ ID NO: 6 was purified by RP HPLC (XBridge C18, 5μm 19x150mm, A: 50 mM NH4HCO3 in water (pH 8), B: ACN), with a gradient of 5% B for 1 min, 5-20% B for 11 min, and 20-90% B for 3 min. The UV purity was 94%; the calculated m / z (ESI-; TOF) value was 1717.19 (5-); the measured value was 1718.0.

[0278] Oligonucleotide SEQ ID NO: 7 was purified by IP HPLC (XBridge C18, 5μm 19x150mm, A: 60mM DBuAA (H2O / ACN 95 / 5) pH7, B: 60 mM DBuAA in ACN), with a gradient of 5% B for 1 min, 20-40% B for 12 min, and 50-80% B for 0.5 min. The UV purity was 95.6%; the calculated m / z (ESI-; TOF) value was 1879.0 (4-); the measured value was 1878.98.

[0279] Oligonucleotide SEQ ID NO: 8 was purified by RP HPLC (XBridge C18, 5μm 19x150mm, A: 50 mM NH4HCO3 in water (pH 8), B: ACN) with a gradient of 5% B for 1 min, 5-18% B for 11 min, and 18-90% B for 3 min. The UV purity was 96.5%; the calculated m / z (ESI-; TOF) value was 1709.59 (5-); the measured value was 1710.3.

[0280] Oligonucleotide SEQ ID NO: 9 was purified by IP HPLC (XBridge C18, 5μm 19x150mm, A: 60mM DBuAA (H2O / ACN 95 / 5) pH7, B: 60 mM DBuAA in ACN), with a gradient of 5% B for 1 min, 20-40% B for 12 min, and 40-80% B for 0.5 min. The UV purity was 96.3%; the calculated m / z (ESI-; TOF) value was 2243.27(3-); the measured value was 2243.26.

[0281] Double-stranded siRNA synthesis-annealing

[0282] The amount of single chains was quantified at 260 nm using UV spectrophotometry, and the solution was normalized to 2 mM using the calculated extinction coefficient.

[0283] For siRNA compounds, single strands were mixed in equimolar amounts, heated to 95°C and held for 5 min, and then cooled to room temperature over 1 h.

[0284] Double-stranded siRNAs were analyzed using UPLC-MS. Analysis of double-stranded siRNAs was performed on an ACQUITY PREMIER BEHAmide 1.7 μm 2.1 x 100 mm, 300 Å (30°C, flow rate 0.5 mL / min) using a gradient: 15–60% B for 5 min, where A: 70 / 30 ACN / 25 mM ammonium acetate in water, and B: 30 / 70 ACN / 25 mM ammonium acetate in water.

[0285] The final siRNA was freeze-dried and redissolved in 1X PBS to a concentration of 10 mg / mL.

[0286] Conjugate 3: Theoretical quality: 16118.8, measured deconvolution quality: 16118.6.

[0287] Conjugate 4: Theoretical quality: 15293.2, measured deconvolution quality: 15293.1.

[0288] Example 4

[0289] In vivo ANGPTL3 knockdown was performed using an AAV-based humanized ANGPTL3 model.

[0290] This study aimed to characterize the role of conjugate 2 in knocking down (KD) human ANGPTL3 mRNA in the liver in vivo and reducing circulating ANGPTL3 protein, and to compare it with conjugates 3 and 4.

[0291] List of abbreviations for Example 4:

[0292]

[0293] Test formulation

[0294]

[0295]

[0296] In vivo testing system

[0297]

[0298] Research Design

[0299]

[0300]

[0301] In-body design

[0302] Mice arrived at the testing facility at 8 weeks of age. All mice were fed a standard diet from arrival until the end of the study. After acclimatization for 6 weeks or 6 days, mice were injected with AAV via the tail vein. Two weeks after AAV injection, each group was treated with a single subcutaneous injection of the designated treatment. Blood samples were collected from the tail vein into EDTA glass capillaries (REF#164113 Micro Haematocrit Tubes, Vitrex Medical) on days 0, 7, 22, 30, 42, and 49. The study was terminated after the last blood sample collection.

[0303] At termination, all animals were anesthetized with isoflurane (Abbot, Stockholm, Sweden) using an vaporizer (Univentor 400, Agnthos, Sweden), and blood was collected from the orbital plexus into tubes containing EDTA as an anticoagulant (REF#15.1671.101, Multivette 600K3E, Sarstedt). Blood samples were centrifuged at 10,000 xg for 4 min at 4°C. The resulting plasma was aliquoted and stored at -20°C until further analysis. Subsequently, anesthetized mice were euthanized by dissection of the heart. The left lateral lobe of the liver was collected, flash-frozen in liquid nitrogen, and stored at -80°C until subsequent analysis.

[0304] Generation of AAV-ANGPTL3

[0305] A liver-targeting AAV vector genome construct carrying the liver-specific hAAT-hApoE promoter (a chimeric promoter with human apolipoprotein E and α1-antitrypsin gene enhancer / promoter) has been previously reported (Liu, 2023#499). The human ANGPTL3 open reading frame (ORF) sequence was synthesized using GenScripts and primer pairs. 3' (SEQ ID NO: 21) and (SEQ ID NO: 22) The ORF was amplified by PCR. The PCR product was then purified using the QIAquick PCR purification kit, digested with EcoRI and NheI, and purified by gel extraction using the QIAquick gel extraction kit. The digested ANGPTL3 ORF was cloned into the EcoRI and SpeI sites of the AAV-hAAT-hApoE-FGF21 mutant vector plasmid, resulting in the introduction of the human ANGPTL3 ORF under the control of the liver-specific hAAT-hApoE promoter. The resulting plasmid was named pAAV-hAAT-hApoE-ANGPTL3_noUTRs, and the inserted fragment was confirmed by sequencing using the following primers.

[0306]

[0307] The full sequence of pAAV-hAAT-hApoE-ANGPTL3_noUTRs is as follows:

[0308]

[0309]

[0310]

[0311]

[0312] Generation of recombinant AAV vector: Ad293 cells (Agilent, Santa Clara, CA, USA) were plated in Gibco DMEM in a five-layer culture chamber, which was supplemented with 10% Gibco FBS and 1% Gibco penicillin-streptomycin (Thermo Fisher Scientific, Waltham, MA, USA). When these cells reached 60-85% confluence under a microscope, they were transfected with a tri-plasmid using polyethyleneimine (Polyscience, Torrance, CA, USA). The tri-plasmid consisted of pHelper containing the adenovirus E2A and E4 genes, pRep2Cap8 encoding the AAV2 Rep protein and the AAV8 serotype capsid, and pAAV-hAAT-hApoE-ANGPTL3_noUTRs in a ratio of 2:1.4:1 ((Liu, 2023 #499)). Seventy-two hours post-transfection, cells were collected and lysed using three freeze-thaw cycles, followed by benzonase treatment at 37°C for 1 hour. The supernatant was further purified by ultracentrifugation based on an iodixanol gradient. The titer of the AAV vector preparation used for in vivo studies was quantified by quantitative PCR. Because the recombinant AAV8 vector was generated using a standard, unhelper-free tri-plasmid transfection system, it does not express AAV8 or adenovirus helper proteins and cannot replicate in transduced hepatocytes.

[0313] Human ANGPTL3 mRNA analysis

[0314] RNA was isolated from approximately 20 mg of frozen liver using the RNeasy Mini kit (Qiagen, CAT#74104) following the manufacturer's instructions. High-Capacity RNA-to-cDNA technology from Applied Biosystems was used. TM The kit (ThermoFisher cat# 4387406) generated cDNA and was used for real-time PCR using QuantStudio 7 Flex (Applied Biosystems). ANGPTL3 gene expression was measured using the Applied Biosystems TaqMan gene expression assay (ThermoFisher, ANGPTL3: FAM-MGB Hs00205581_m1 cat# 4331182), and the mouse RPLPO primer and probe set (Sigma Aldrich) was used as a housekeeping gene.

[0315] Forward primer: ,

[0316] Reverse primer: and

[0317] FAM-TAM labeled probe: (SEQ ID NO:34).

[0318] Real-time PCR was performed on a Quantstudio 7 flex, and the PCR cycle number (Ct) was determined using Quantstudio Real-time PCR software (Applied Biosystems). ANGPTL3 gene expression levels were normalized against the housekeeping gene RPLP0.

[0319] Human ANGPTL3 mRNA analysis

[0320] Human ANGPTL3 in mouse plasma was measured using the Human Angiopoietin-like 3 Quantikine ELISA Kit (DANL30, R&D Systems, Minneapolis, MN). Prior to use, this ELISA assay was demonstrated to be non-cross-reactive with endogenous mouse ANGPTL3 present in plasma samples (BS003151-48). Human ANGPTL3 in mouse plasma samples was analyzed according to the manufacturer's instructions with the following modifications: the calibration curve was extended to four points to cover 0.02–10 ng / mL, and plasma samples were diluted 25, 50, or 100-fold prior to analysis. Calibration standards were analyzed in duplicate, and the study sample was analyzed as a single sample. The ELISA plate was read from an absorbance reader capable of measuring absorbance at 450 nm, with the calibration wavelength set to 540 nm (e.g., iX3, Molecular Devices). The standard curve response was plotted against logarithmic concentration in linearly scaled absorbance units and fitted using a 4-parameter function (SoftMax Pro). To control for inter-plate variations, duplicate runner quality control samples were prepared at the beginning and end of each analysis plate. ANGPTL3 levels were reported as ng / mL plasma.

[0321] Data Analysis

[0322] Plasma ANGPTL3 protein data were baseline-corrected individually for each animal. The mean and standard error were calculated for each group daily. The normality of the data was examined using the one-sample Kolmogorov-Smirnov test. Log-transformed data are recommended and used in subsequent tests. For human ANGPTL3 mRNA expression data, the mean and standard error were calculated for each group. The normality of the data was examined using the one-sample Kolmogorov-Smirnov test. Log-transformed data are recommended and used in subsequent tests. For all analyses, multiple comparisons were performed using the Tukey post-hoc test. Calculations were performed in MATLAB (R2022b; MathWorks). Specifically, the normality was tested using the kstest function, and multiple comparisons were performed using the anova1 and multiply-compare functions.

[0323] result

[0324] For conjugate 2, the maximum reduction in baseline-corrected ANGPTL3 protein concentration was 37%, 58%, and 86% at single doses of 0.25, 1, and 3 mg / kg, respectively (Table 3). Figure 3 For the treatment groups, the maximum reduction was observed between 22 and 30 days after administration. Baseline-corrected ANGPTL3 protein concentrations in the placebo group decreased over time, with a maximum reduction of 37% on day 42 and a reduction of 22% at the end of the study (day 49). No statistically significant reduction in baseline-corrected ANGPTL3 protein concentrations was observed in either the 0.25 mg / kg or 1 mg / kg groups compared to placebo (P < 0.05). The high-dose 3 mg / kg group showed a statistically significant reduction in baseline-corrected ANGPTL3 protein concentrations at all measurement time points compared to placebo (P < 0.05). In the 1 mg / kg group, baseline-corrected ANGPTL3 protein concentrations began to recover to baseline and reached the same level as the placebo group on day 49, but did not fully recover to baseline. In the 3 mg / kg group, baseline-corrected ANGPTL3 protein concentrations did not fully recover to baseline during the 7-week study period. At the end of the study on day 49, compared with placebo, the knockdown of human ANGPTL3 mRNA in the liver was 56% in the 3 mg / kg high-dose group (P < 0.005, compared with all other groups). No knockdown of human ANGPTL3 mRNA in the liver was observed in the 0.25 mg / kg and 1 mg / kg dose groups on day 49 (Table 4;). Figure 4For conjugate 3, the maximum baseline-corrected reduction in ANGPTL3 protein concentration was 31%, 28%, and 43% at single doses of 0.25, 1, and 3 mg / kg, respectively (Table 3). For conjugate 4, the maximum baseline-corrected reduction in ANGPTL3 protein concentration was 0%, 20%, and 30% at single doses of 0.25, 1, and 3 mg / kg, respectively (Table 3). No knockdown of human ANGPTL3 mRNA in the liver was observed in any dose group of conjugate 3 or conjugate 4 on day 49 (Table 4). Figure 5 ).

[0325] Table 3. Percentage of circulating human ANGPTL3 protein (baseline) in humanized mice treated with a single dose of conjugate 2, conjugate 3, or conjugate 4.

[0326]

[0327]

[0328]

[0329] ANGPTL3, angiopoietin-like protein 3; mRNA, messenger ribonucleic acid; N, quantity.

[0330] Table 4. Expression of human ANGPTL3 mRNA in the liver of humanized mice treated with a single dose of conjugate 2, conjugate 3, or conjugate 4 on day 49.

[0331]

[0332]

[0333]

[0334] ANGPTL3, angiopoietin-like protein 3; mRNA, messenger ribonucleic acid; N, quantity.

[0335] Example 5

[0336] In vivo therapeutic effect of conjugate 1 in an obese HFpEF ZSF1 rat model.

[0337]

[0338] The effect of conjugate 1 on reducing ANGPTL3 protein was investigated using a ZSF-1 obese rat model of heart failure with preserved ejection fraction (HFpEF). Once secreted into circulation, ANGPTL3 acts as an inhibitor of lipoprotein lipase (LPL) and thus plays an important role in regulating total circulating plasma triglyceride levels (Prog Lipid Res. 2022;85101140). Known for its similarity to the favorable metabolic profile observed in individuals with ANGPTL3 protein deficiency (The Journal of clinical endocrinology and metabolism. 2012;97(7):E1266-75), pharmacological reduction of circulating ANGPTL3 protein has been shown to result in clinically significant and well-tolerated reductions in cardiovascular risk factors (Evinacumab. (EVKEEZA®) Prescribing Information. Regeneron Pharmaceuticals, Inc. 2023, Circulation 2019. p. E987-8). Data from observational studies (Arterioscler ThrombVasc Biol. 2018; 38(2): 464-72) also suggest that this reduction may have potential clinical benefits in patients with metabolically driven heart failure.

[0339] ZSF-1 rats are hybrids of two different leptin receptor mutants, and their offspring can be either obese or lean. Obese ZSF-1 rats spontaneously develop obesity, hypertension, type 2 diabetes, hyperlipidemia, left ventricular (LV) dysfunction, nephropathy, and insulin resistance. Lean ZSF-1 rats have hypertension but do not show any other pathological conditions. Obese ZSF-1 rats with unilateral nephrectomy were treated with either conjugate 1 or the mediator for 12 weeks and given a high-salt diet (to induce the HFpEF phenotype). This study used non-invasive imaging—echocardiography—as the "gold standard" for assessing cardiac function. The conclusion is that ANGPTL3 protein reduction decreases circulating triglyceride (TG) levels, reduces cardiac TG content, and reduces the development of diastolic dysfunction in obese ZSF-1 rats with HFpEF (as shown by NT-proBNP and IVRT results), without significantly increasing hepatic TG.

[0340] Obese unilaterally nephrectomized (Unx) ZSF-1 rats (n=8) were treated with either conjugate 1 or the mediator for 12 weeks on a high-salt (HS) diet. Lean ZSF-1 rats (n=8) served as the control group. Blood samples were collected before the start of treatment and after 12 weeks of treatment for analysis of NT-proBNP, ANGPTL3, and circulating lipids and lipoproteins. At the end of treatment, tissue samples from the heart, kidneys, and liver were collected for analysis. Non-invasive imaging—echocardiography—was used as the gold standard for assessing cardiac function. Isovolumetric relaxation time (IVRT) was the primary endpoint.

[0341] The study design for each rat is summarized in... Figure 6 Rats were grouped into groups of four, with four rats per cage, and had free access to a high-salt diet or Purina pelleted food and regular tap water throughout the study period. Rats were weighed weekly. Rats were randomly assigned to groups based on IVRT, blood glucose, UACR, and body weight. After an acclimatization period, baseline echocardiography and baseline blood sample collection began.

[0342] Research Design

[0343]

[0344] Table 5 Grouping, Dosage, and Number of Animals

[0345]

[0346] The study design for each rat is summarized in... Figure 6 Rats were grouped in groups of four, with four rats per cage, and had free access to a high-salt diet or Purina pelleted feed and regular tap water throughout the study. Rats were weighed weekly. Rats were randomly assigned to groups based on IVRT, blood glucose, UACR, and body weight. After an acclimatization period, baseline echocardiography and baseline blood sampling were initiated. Obese ZSF1 rats with unilateral nephrectomy on a high-salt diet were treated with either conjugate 1 or the mediator for 12 weeks (n=8). Lean ZSF1 rats (n=8) served as the control group. Treatment (PBS or conjugate 1) was administered subcutaneously at weeks 1, 4, 7, and 10 of the study. Blood samples were collected before treatment, every three weeks, and after week 12 for analysis of NT-proBNP, ANGPTL3, and circulating lipids. At the end of the study, blood and tissue samples from the heart, kidneys, and liver were collected for analysis. This study used non-invasive imaging—echocardiography—as the "gold standard" for assessing cardiac function. Isovolumetric relaxation time (IVRT from echocardiography) was the primary endpoint. The 25th (obese Unx+HS+AD), 30th, and 32nd (obese Unx+HS) animals died before the finish line.

[0347] Experimental Procedure

[0348] Tail vein blood collection

[0349] At baseline, every three weeks, and at the endpoint (e.g.) Figure 1 (as described), via a butterfly catheter (21G, BD Valu-Set) TM Blood samples were collected from the tail vein of conscious rats in Plymouth, UK, and placed in EDTA-coated vials (MiniCollect, K3EDTA, Kremsmünster, Austria).

[0350] plasma analysis

[0351] Blood was centrifuged at 5000 xg for 10 min. EDTA-plasma was used to analyze NT-proBNP and ANGPTL3. Determinations were performed according to the manufacturer's instructions.

[0352] Metabolic cage

[0353] Animals were placed in metabolic cages for urine sampling and a fasting period for subsequent blood collection. From an animal welfare perspective, a shorter duration (e.g., 4 hours) is recommended as it is less challenging for rats (less stress and less impact on body weight, food and water intake). During weeks -1, 7, 10, and 12 of the study, animals were placed in metabolic cages for 4 hours with free access to water but no food. For the fasting period during blood collection in week 4, animals were placed in plastic containers for 4 hours with free access to water.

[0354] Echocardiography

[0355] Animal preparation for echocardiography

[0356] Non-invasive imaging—echocardiography—was performed three times: at baseline, at week 9, and at week 12 of the study. Animals were initially anesthetized in a gas chamber with isoflurane gas (Isoba® vet, Schering-Plough Ltd., England) (5%). Gas concentrations were established by applying O2 (200 mL / min) and air (500 mL / min) streams to the isoflurane solution. During the experiment, animals were maintained anesthetized by inhaling isoflurane gas (approximately 2.2%) through a face mask. After shaving, Veet was used… ® Use hair removal cream to remove hair from the chest and left back. Rinse the skin thoroughly with lukewarm tap water.

[0357] Echocardiography protocol

[0358] Echocardiography was performed using a high-resolution ultrasound scanner (Vevo 3100, Visualsonics Inc.) equipped with an MX201 solid-state transducer. Long-axis images of the heart were displayed using the parasternal long-axis (LAX) view. Short-axis (SAX) images were then acquired at the mid-ventricular level, with the right ventricle (RV) facing left of the field of view (FOV) and the papillary muscles on the right. B-mode and M-mode dynamic images (loop) were acquired.

[0359] To assess diastolic function (including isovolumetric relaxation time (IVRT)), pulsed-wave Doppler flow spectrum analysis was performed on the mitral valve. The image was acquired via a modified apical four-chamber view. Tissue Doppler imaging (TDI) of the septal mitral annulus was performed from the same section to assess myocardial longitudinal velocity.

[0360] Termination and organ sampling

[0361] At the end point, the heart, lungs, spleen, liver, and one kidney are removed and weighed, and the tibia is dissected and its length is measured.

[0362] Rat ANGPTL3 ELISA

[0363] This method was fully automated using the Beckman Coulter Motoman Robotic System (FLIPR), which included a Biomek FX Span-8 / 96-head Biotek plate washer, a Cytomat incubator (shaking), a Carousel, and an MDParadigm reader. A mouse / rat angiopoietin-like protein 3 ELISA assay kit (ANGPTL3, Cat NoRAG011R, Czech Republic) was used.

[0364] Robotic assay: Samples were pre-diluted from 10 μL to a total of 800-fold (method alone) in two steps. Standards were serially diluted (2-fold) and transferred to pre-diluted plates. 100 μL of pre-diluted sample / standard was pipetted into an ELISA plate. Incubated at 37°C for 1 hour. The ELISA plate was washed (ELISA 3, 5 washes). 100 μL of detection antibody was added. The plate was incubated at 37°C for 1 hour. The plate was washed (ELISA 3, 5 washes). 100 μL of HRP conjugate was added. The plate was incubated at 37°C for 1 hour. The plate was washed (ELISA 3, 5 washes). 100 μL of TMB substrate was added. The plate was incubated at room temperature in the dark for 15 minutes. 100 μL of stop solution was added. The absorbance was read at 450 nm, with a reference wavelength of 540 nm (within 20 minutes).

[0365] Rat NT-proBNP MSD ELISA assay

[0366] This method was fully automated in the Beckman Coulter SCARA robotic system (Lambda), including a Biomek NX Span-8, Biomek FX 96 / 96-heads, a Biotek plate washer, a Cytomat incubator, plate racks (hotels), an MD Paradigm reader, and an MSD S600. A rat NT-proBNP kit (Cat No K153JKD-1) was used.

[0367] Robotic assay: Samples were pre-diluted (3-fold) in Diluent 34 containing a protease inhibitor. MSD plates were washed (3 times with wash buffer). 50 μL of standards, controls, and diluted samples were dispensed. The plates were incubated for 1 hour with vigorous shaking (500 rpm) at room temperature. The plates were washed (3 times with wash buffer). 25 μL of detection antibody was dispensed. The plates were incubated for 2 hours with vigorous shaking (500 rpm) at room temperature. The plates were washed (3 times with wash buffer). 150 μL of read buffer was dispensed. The plates were read within 2 minutes using an MSD Sector Imager S600.

[0368] Normal-phase HPLC of plasma TG

[0369] Plasma triglycerides were extracted using liquid / liquid extraction (Lofgren L, 2012). The liquid extract was evaporated under a nitrogen stream and redissolved in heptane:isopropanol [9:1]. Analyzed using normal-phase HPLC as previously described (JChromatogr B Biomed Sci Appl. 1998; 708(1-2): 21-6). Quantification was performed against an external calibration curve.

[0370] TG content in the liver

[0371] Liver fragments (approximately 50 mg) were homogenized with isopropanol (2-isopropanol) to release triglycerides from the tissue. The triglycerides in the sample were then enzymatically hydrolyzed into glycerol and fatty acids. Glycerol produced hydrogen peroxide through two enzymatic steps, which promoted the formation of a red quinone imine derivative. The color intensity was directly proportional to the amount of triglycerides (glycerol) in the sample and was determined colorimetrically at a wavelength of 500 nm.

[0372] Lipomics of TG content in the heart

[0373] Pre-weighed frozen ZSF1 heart samples were homogenized by bead milling in a 10X (v / w) 3:1:6 IPA:water:EtOAc mixture containing a 1:100 EquiSplash (Avanti) internal standard mixture. A portion of the clarified supernatant was dried by speedvac and then resuspended in an equal volume of 9:1 MeOH:toluene (maintaining a 10X ratio). 1 μL of the sample was analyzed using a C18 LCMS-based method.

[0374] Mass spectrometry: Non-targeted (full scan) mass spectrometry analysis was performed using Thermo Tribrid ID-X, with metabolomics scans ranging from 50 to 950 m / z and lipidomics scans ranging from 50 to 1700 m / z. Individual samples were acquired only in MS1 ​​mode using a polarity switching method.

[0375] Data Analysis: Thermo raw files underwent feature detection, alignment, identification, and normalization analysis in MS-DIAL (≥v. 4.8). Peak areas were normalized relative to EquiSplash standards (lipomics) and total ion currents (metabolomics). Features were filtered in R based on QC / blank signal >3-fold and QC RSD <30%. Statistical analysis was performed using the MetaboAnalyst R package (v. 3.2.0) and R language (v. 4.1.0). In short, data were normalized to the median, logarithmically transformed, and scaled using the Pareto method. Fold changes were calculated using the data before column-wise normalization (i.e., the original scale).

[0376] Proteomics

[0377] Proteomics data acquisition: Mouse heart tissue was lysed and bead-milled in metabolite extraction buffer (IPA:H2O:EtOAc=6:1:3). The protein precipitate, along with the residual buffer, was used for proteomic characterization. High-pH reverse-phase peptide fractionation (Thermo 84868) was performed for peptide fractionation. LC-MS / MS analysis was performed on a timsTOF Pro mass spectrometer (Bruker) coupled with an Evosep LC system and a nanoliter electrospray ionization source (Captive Spray Source, Bruker). Data-dependent acquisition (DDA) and data-independent acquisition (DIA)-PASEF modes were used to obtain peptide data. The merged DDA and DIA raw acquisition files were analyzed using Spectronaut (Biognosys AG) software (Version 18) and the Pulsar search engine (SN14.10.201222) to build libraries using the UniProt rat proteome database (UP000002494_20231024).

[0378] Proteomics data analysis: Protein strength was analyzed using R (v. 4.2.3) and the DEP package (v. 1.20.0). In short, the data were normalized using the SVM method, and missing values ​​were imputed using the missForest method (v. 1.5). Finally, differential analyses were performed using linear models and empirical Bayesian statistics. PCA was calculated using the imputed values. Enrichment analysis was performed using the clusterProfiler package (v. 4.6.2).

[0379] Electron microscopy of podocyte health

[0380] Kidney slices were fixed in a mixture of 2.5% glutaraldehyde and 1% paraformaldehyde (both from Sigma-Aldrich) in 0.1 M phosphate buffer (pH 7.4) and stored at 4°C. Electron microscopy analysis was performed in the electron microscopy laboratory at Huddinge University Hospital (Stockholm, Sweden).

[0381] Data Analysis

[0382] Statistical evaluation was performed using the Holm-Sidak multiple comparison test or the unpaired t-test. Data are expressed as mean ± SD. Other cases are indicated in the footnotes of the figures.

[0383] Histological readings: glomerular score, podocyte stress and TEM (glomerular basement membrane GBM thickness and cleft / µm).

[0384] Determinations for analyzing urine samples: U-albumin and U-creatinine were determined in a Pentra 400. U-albumin was determined by colorimetry: Albumin CP ABX Pentra, ref A11A01664. U-creatinine was determined by the Jaffe reaction (picric acid): Creatinine 120CP, Horiba ABX A11A01933. The required volume for U-albumin and creatinine analysis is approximately 50 µL.

[0385] result

[0386] weight

[0387] like Figure 7As shown, weight changes were observed between lean and obese rats. No weight changes were observed in obese animals treated with conjugate 1 (C1) compared to the obese control group. Solid black circles represent individual animals. An asterisk indicates a significant change between comparison groups. Insignificant (ns) indicates no significant change between comparison groups. Holm-Sidak multiple comparison test was used. Lean (N=8), Obese Unx+HS (N=6), Obese Unx+HS+C1 (N=7). Lean – Control animals; Obese Unx+HS – Unilateral nephrectomy + high-salt diet induced HFpEF; Obese Unx+HS+AD – Unilateral nephrectomy + high-salt diet induced HFpEF + Conjugate 1 treatment.

[0388] ANGPTL3 protein and TG plasma levels

[0389] like Figure 8 As shown, compared with the control group, conjugate 1 reduced circulating ANGPTL3 protein levels by more than 95% in the obese Unx+HS+AD group. This change was also observed in the reduction of circulating plasma triglyceride (TG) levels, which were reduced to almost baseline levels after treatment. Black solid circles represent individual animals. Asterisks indicate significant changes between comparison groups. Holm-Sidak multiple comparison test was used. Lean (N=8), obese Unx+HS (N=6), obese Unx+HS+AD (N=7). Lean - control animals, obese Unx+HS - unilateral nephrectomy + high-salt diet induced HFpEF; obese Unx+HS+AD, unilateral nephrectomy + high-salt diet induced HFpEF + conjugate 1 treatment.

[0390] TG content in the heart and liver of ZSF-1 rats (lipomic analysis)

[0391] Figure 9a The results showed that, compared with the control group, conjugate 1 significantly reduced the TG content in cardiac tissue (lipomics analysis) in the obese Unx+HS+AD group (tissue triglycerides 51:4, 51 carbons, 4 double bonds). Figure 9bAs shown, compared with the control group, ANGPTL3 gene silencing did not affect liver TG content in the obese Unx+HS+AD group. Solid black circles represent individual animals. Asterisks indicate significant changes between comparison groups. Insignificant (ns) indicates no significant change between comparison groups. Holm-Sidak multiple comparison test was used. Lean (N=8), obese Unx+HS (N=6), obese Unx+HS+C1 (N=7). Lean – control animals; obese Unx+HS – unilateral nephrectomy + high-salt diet induced HFpEF; obese Unx+HS+AD – unilateral nephrectomy + high-salt diet induced HFpEF + conjugate 1 treatment.

[0392] NT-proBNP and IVRT

[0393] At the functional level, prevention of diastolic dysfunction is demonstrated by a reduction in isovolumetric relaxation time and a decrease in circulating N-terminal pro-B-type natriuretic peptide levels. Figure 10 NT-proBNP is a biomarker of myocardial stretch, and isovolumetric relaxation time is an echocardiographic parameter of diastolic function. Solid black circles represent individual animals. An asterisk indicates a significant change between comparison groups. Insignificant (ns) indicates no significant change between comparison groups. Holm-Sidak multiple comparison test was used. Lean (N=8), obese Unx+HS (N=6), obese Unx+HS+C1 (N=7). Lean – control animals; obese Unx+HS – unilateral nephrectomy + high-salt diet induced HFpEF; obese Unx+HS+AD – unilateral nephrectomy + high-salt diet induced HFpEF + conjugate 1 treatment.

[0394] Proteomics data (heart tissue)

[0395] Proteomic data from cardiac tissue showed changes in proteins involved in fatty acid metabolism. This was confirmed using a multi-pathway platform. Specifically, ANGPTL3 inhibition altered the concentrations of various apolipoproteins. These results indicate that treatment enhances lipid degradation in cardiac tissue.

[0396] A) Volcano plot shows proteins with significant changes (Adj p value < .1 & |log2(FC)|>0.5).

[0397] B) Significantly enriched KEGG pathway. C) Apolipoprotein levels in different treatment groups ("***"=0.001, "**"=0.01, "*"=0.05). Lean-control animals, obese Unx+HS-unilateral nephrectomy + high-salt diet induced HFpEF; obese Unx+HS+ARO-unilateral nephrectomy + high-salt diet induced HFpEF + conjugate 1 treatment.

[0398] Kidney health

[0399] like Figure 8 As shown, ANGPTL3 inhibition prevented kidney damage in ZSF-1 rats with CKD, as indicated by UACR, podocyte injury, and glomerular sclerosis. Rats used for CKD indication were not given a high-salt diet. They received a Purina diet during the study period. Black solid circles, triangles, and squares represent individual animals. Asterisks indicate significant changes between comparison groups. Holm-Sidak multiple comparison test was used. The number of N varies depending on the assay performed. Lean – control animals, obese Unx – unilateral nephrectomy-induced CKD; obese Unx + ARO, unilateral nephrectomy-induced CKD + conjugate 1 treatment.

[0400] Treatment with conjugate 1 showed that reducing ANGPTL3 protein improved cardiovascular and metabolic outcomes in ZSF-1 HFpEF rats by decreasing circulating TG levels, reducing cardiac TG content, and reducing the development of diastolic dysfunction. It is hypothesized that targeting ANGPTL3 mRNA with conjugate 2 or any other siRNA agent that inhibits ANGPTL3 and thus silences hepatic ANGPTL3 gene expression would enhance LPL enzyme activity, promote TG-rich lipoprotein hydrolysis, reduce circulating TG, and improve cardiac and renal outcomes.

[0401] Example 6

[0402] The therapeutic effect of conjugate 1 in cynomolgus monkeys (Macaca fascicularis) with HFpEF.

[0403] The aim of this study was to examine the long-term effects of targeted inhibition of conjugate 1 on cardiac-renal function indicators and biomarkers in the most relevant animal model reflecting the expected human population. Because cynomolgus monkeys are closely related to humans both phylogenetically and physiologically, they were chosen over other lower mammal species as the experimental species to obtain more meaningful and translatable results. The long-term efficacy of conjugate 1 in treating heart failure was explored in a cynomolgus monkey model of heart failure with preserved ejection fraction (HFpEF), a model very close to the adult HFpEF phenotype.

[0404] Research Design

[0405] Group 1: Age-matched healthy controls (4 subjects)

[0406] Group 2: HFpEF PBS control (10 subjects)

[0407] Group 3: HFpEF conjugate 1 subcutaneous dose 3 mg / kg (10 subjects)

[0408] Group 4: HFpEF empagliflozin oral dose 3 mg / kg (10 subjects)

[0409] Dosing was administered on day 1 of week 1 and day 84 of week 12. This was followed by a 12-week post-dosing observation / washout period.

[0410] Food and water

[0411] The monkeys were provided with three meals a day, consisting of the following: 100.0 g of standard monkey formula (extruded pellets) at 9:00-10:00 AM; one regular apple (150 g) at 2:00-3:00 PM; and 100 g of high-fat diet (HFD) at 4:00-5:00 PM. Water was provided freely. All monkeys were fasted overnight. After each feeding, all leftover food was removed, and the amount of food consumed was determined by weighing the remaining food.

[0412] Two-dimensional (2D) echocardiography

[0413] Two-dimensional echocardiography (2D echo) was performed using a Mindray M9CV echocardiography system (Mindray, Shenzhen, China) with an SP5-1s (1.1-4.4MHz) phased array transducer, under sedation with intramuscular (IM) administration of 10 mg / kg ketamine. Animals were fasted for 4-6 hours to minimize the risk of vomiting and pulmonary aspiration during anesthesia induction and recovery. Nevertheless, unrestricted drinking water was provided during fasting. Ketamine was administered intramuscularly at 10 mg / kg, and the fur on the left chest of the animal was thoroughly shaved using a power shaver and razor blade. The monkey was properly positioned on an examination table or operating table that had been cleaned with 70% alcohol and covered with sterile disposable surgical drapes. The animal was kept warm on a heating pad / blanket throughout the procedure. Typically, the animal was placed in the left lateral decubitus position for echocardiography.

[0414] MRI scan preparation

[0415] Before each scan, the monkeys were fasted overnight or fasted for at least 6 hours before imaging.

[0416] Sedation was induced by intramuscular injection of ketamine (5–10 mg / kg, IM), and atropine hydrochloride (0.02–0.04 mg / kg, IM) was administered to suppress respiratory spasm and salivation. Endotracheal intubation was then performed, with a peripheral intravenous (IV) access established and ECG leads connected to the animal's chest wall. The animal was then transferred to an MRI bed for ECG and vital sign monitoring. The animal was connected to a mechanical ventilator, and anesthesia was induced and maintained using isoflurane (1–3%) in medical air. The animal was positioned supine with its head forward on the MRI bed, and cardiac imaging was performed using an abdominal coil. Each scan lasted no more than four (4) hours. The animal was kept warm during the scan. Heart rate, oxygen saturation, noninvasive blood pressure, respiratory rate, end-tidal CO2 level, and body temperature were recorded / monitored throughout the scan to ensure adequate anesthesia. ECG monitoring was continued.

[0417] MRI procedure

[0418] All animals underwent CMR using a 3.0 Tesla magnetic resonance imaging system (MagnetomPrisma; Siemens Healthineers, Erlangen, Germany) equipped with a 32-channel phased array body coil. The following MRI sequences were performed twice throughout the study (at baseline and week 25): proton density fractional fat (PDFF) imaging; regardsone loading perfusion imaging; and extracellular volume (ECV) scanning. Pulse sequence parameters were recorded and described in the MRI report. Animals were mechanically ventilated at a rate of 20–30 breaths per minute during scans. Breath-holding was performed during inspiration to mitigate the impact of respiration on vascular image quality. This was achieved only by shutting off the ventilator for no more than 10 seconds during pulse sequences. Oxygen flow was maintained during breath-holding (when the ventilator was shut off).

[0419] Proton density fat fraction (PDFF) imaging

[0420] Myocardial proton density fat fraction (mPDFF) was measured using a 2D, ECG-triggered, multi-echo segmented gradient echo sequence. An ECG trigger delay was used to ensure data acquisition occurred during the quiescent phase of the cardiac cycle to minimize cardiac motion. Respiratory motion was controlled by a single breath-hold. mPDFF was measured using VivoQuant (V2.5) software by placing regions of interest (ROIs) at different myocardial locations.

[0421] Regadoxone loading perfusion imaging and extracellular volume (ECV) scan

[0422] For stress imaging, resting first-pass perfusion imaging was initiated with a bolus injection of MRI contrast agent (0.075 mmol / kg gadoteryl meglumine injection; 15 ml: 5.654 g (a sterile solution containing 377 mg / ml gadoteryl meglumine; Jiangsu Hengrui Medicine Co., Ltd.)). The perfusion sequence was a 2D saturated recovery single-shot gradient echo sequence, repeated 70 times. A total of three slices with the same pre-contrast T1 mapping were scanned, with a temporal resolution of one RR interval (approximately 500 ms). Cine images were acquired along the 2nd, 3rd, and 4th cavities after the perfusion images. Approximately 15 min later, a bolus injection of retardon (5 μg / kg; Nanjing Hairong Pharmaceutical Co., Ltd.) was administered to induce vasodilation, and another first-pass perfusion imaging (0.075 mmol / kg gadoteryl meglumine injection) was performed 1 min after retardon injection for stress perfusion imaging. Ten minutes after the second contrast agent injection, post-contrast MOLLI T1 maps were acquired on the same three short-axis planes used for pre-contrast T1 scans using a 4(1)3(1) protocol. Delayed gadolinium-enhanced images were obtained 15 minutes after contrast agent injection using ECG-gated breath-hold inversion recovery Turbo FLASH. All CMR images were prospectively analyzed. Blood samples were obtained from all monkeys immediately prior to each CMR study for hematocrit (Hct) determination. Cine images were analyzed using Medis software (Medis Medical Imaging Systems BV, Leiden, Netherlands).

[0423] Pre- and post-contrast T1 images were automatically generated using the inline processing function of the MRI system prototype, and global or regional ECV values ​​were calculated using Hct values. If motion existed between the pre- and post-contrast T1 series, pre- and post-contrast MOLLI were manually registered. First-pass perfusion imaging was analyzed using a custom software, Perfusion Imaging Toolkit (PIT). Quantitative perfusion maps at three slices were generated under both resting and loading conditions. Subsequently, myocardial perfusion reserve was calculated as the ratio of loading perfusion to resting perfusion.

[0424] Blood samples for pharmacokinetic (PK) characterization, target biomarkers, and other exploratory pharmacodynamic (PD) biomarkers.

[0425] Approximately 6 mL of whole blood was collected directly from the peripheral veins of unsedated / alert monkeys into K2EDTA tubes for use in the following target biomarkers. EDTA plasma was separated by centrifugation at 2,500 xg for 10 minutes at 4°C. The collected plasma was aliquoted into cryovials.

[0426] Samples were frozen at -80°C. Frozen tubes were labeled with the study number, animal number, group number, experiment date, and time after drug administration. One aliquot of the sample used for pharmacokinetic analysis was analyzed by KBI, and another aliquot was frozen at -80°C as a backup. Samples used for target-binding biomarkers, ANGPTL3, and TG were preserved and analyzed.

[0427] Proton density fat fraction (PDFF) imaging

[0428] like Figure 13 As shown, animals treated with conjugate 1 (ANGPTL3 siRNA inhibitor) exhibited a significant reduction in cardiac fat fraction. Similar results were observed as expected with the positive control SGLT2 inhibitor (empagliflozin).

[0429] Extracellular volume (ECV) scan

[0430] like Figure 14 As shown, ANGPTL3 inhibition significantly reduced extracellular volume. This parameter is associated with survival in patients with heart failure.

[0431] Left atrial volume index

[0432] Left atrial volume index (LAVi) is a key parameter for diagnosing HfpEF and predicting poor prognosis in these patients (J Cardiovasc Med (Hagerstown). 2018 June;19(6):304-309). Figure 15 As shown, inhibition of ANGPTL3 (rather than SGLT2 inhibition) resulted in a significant reduction in LAVi.

[0433] Plasma ANGPTL3 levels

[0434] like Figure 16 As shown, the ANGPTL3 circulation level was significantly reduced after treatment.

[0435] Plasma triglyceride (TG) levels

[0436] As a result of ANGPTL3 inhibition, TG cycling levels were significantly reduced, such as Figure 17 As shown, treatment with the SGLT2 inhibitor empagliflozin had no effect on reducing circulating TG levels.

Claims

1. An ANGPTL3 siRNA agent or a pharmaceutically acceptable salt thereof, said ANGPTL3 siRNA agent comprising (i) a first (sense) strand and (ii) a second (antisense) strand, the first (sense) strand having the following sequence: The second (antisense) chain has the following sequence: Wherein [M] is a trianticular ligand portion connected to the 5' end of the first chain and having the following structure: 。 2. The ANGPTL3 siRNA agent according to claim 1, or a pharmaceutically acceptable salt thereof, is used as a medicine.

3. A pharmaceutical composition comprising the ANGPTL3 siRNA agent according to claim 1 or a pharmaceutically acceptable salt thereof, optionally mixed with a pharmaceutically acceptable adjuvant, diluent or carrier.

4. A method for treating cardiovascular disease or metabolic condition or reducing its risk, the method comprising administering to a person suffering from or at risk of said disease or condition a therapeutically effective amount of the ANGPTL3 siRNA agent according to claim 1 or a pharmaceutically acceptable salt thereof.

5. The method according to claim 1, wherein the disease is hypertriglyceridemia, obesity, hyperlipidemia, lipid and / or cholesterol metabolism disorders, atherosclerosis, type I diabetes, cardiovascular disease, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, homozygous and heterozygous familial hypercholesterolemia, or statin-resistant hypercholesterolemia.

6. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, used as a medicine.

7. The compound of claim 1 or a pharmaceutically acceptable salt thereof, for the treatment of hypertriglyceridemia, obesity, hyperlipidemia, lipid and / or cholesterol metabolism disorders, atherosclerosis, type I diabetes, cardiovascular disease, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, homozygous and heterozygous familial hypercholesterolemia, or statin-resistant hypercholesterolemia.

8. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment or prevention of cardiovascular diseases or metabolic conditions.

9. A method for treating heart failure or reducing its risk, the method comprising administering to a person suffering from or at risk of heart failure a therapeutically effective amount of a double-stranded siRNA or a pharmaceutically acceptable salt thereof, said double-stranded siRNA comprising an antisense strand and a sense strand, said antisense strand comprising a nucleobase sequence that is at least 80% complementary to an equal-length portion of an ANGPTL3 nucleic acid having the nucleobase sequence of SEQ ID NO:

1.

10. The method of claim 9, wherein the double-stranded siRNA comprises an antisense strand and a sense strand, the antisense strand comprising a nucleobase sequence that is at least 90% complementary to an equal-length portion of an ANGPTL3 nucleic acid having the nucleobase sequence of SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof.

11. The method of claim 9, wherein the double-stranded siRNA comprises an antisense strand and a sense strand, the antisense strand comprising a nucleobase sequence that is 100% complementary to an equal-length portion of an ANGPTL3 nucleic acid having the nucleobase sequence of SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof.

12. The method of claim 9, wherein the double-stranded siRNA is an ANGPTL3 siRNA agent or a pharmaceutically acceptable salt thereof, the ANGPTL3 siRNA agent comprising (i) a first (sense) strand and (ii) a second (antisense) strand, the first (sense) strand having the following sequence: The second (antisense) chain has the following sequence: Wherein [M] is a trianticular ligand portion connected to the 5' end of the first chain and having the following structure: 。 13. The method of claim 9, wherein the double-stranded siRNA is an ANGPTL3 siRNA agent or a pharmaceutically acceptable salt thereof, the ANGPTL3 siRNA agent comprising (i) a first (sense) strand and (ii) a second (antisense) strand, the first (sense) strand having the following sequence: The second (antisense) chain has the following sequence: NAG is a trianticular ligand portion connected to the 5' end of the first chain and having the following structure: 。 14. A method of treating chronic kidney disease or reducing its risk, the method comprising administering to a person suffering from or at risk of chronic kidney disease a therapeutically effective amount of double-stranded siRNA or a pharmaceutically acceptable salt thereof, said double-stranded siRNA comprising an antisense strand and a sense strand, said antisense strand comprising a nucleobase sequence that is at least 80% complementary to an equal-length portion of an ANGPTL3 nucleic acid having the nucleobase sequence of SEQ ID NO:

1.

15. The method of claim 14, wherein the double-stranded siRNA comprises an antisense strand and a sense strand, the antisense strand comprising a nucleobase sequence that is at least 90% complementary to an equal-length portion of an ANGPTL3 nucleic acid having the nucleobase sequence of SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof.

16. The method of claim 14, wherein the double-stranded siRNA comprises an antisense strand and a sense strand, the antisense strand comprising a nucleobase sequence that is 100% complementary to an equal-length portion of an ANGPTL3 nucleic acid having the nucleobase sequence of SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof.

17. The method of claim 14, wherein the double-stranded siRNA is an ANGPTL3 siRNA agent or a pharmaceutically acceptable salt thereof, the ANGPTL3 siRNA agent comprising (i) a first (sense) strand and (ii) a second (antisense) strand, the first (sense) strand having the following sequence: The second (antisense) chain has the following sequence: NAG is a trianticular ligand portion connected to the 5' end of the first chain and having the following structure: 。 18. The method of claim 14, wherein the double-stranded siRNA is an ANGPTL3 siRNA agent or a pharmaceutically acceptable salt thereof, the ANGPTL3 siRNA agent comprising (i) a first (sense) strand and (ii) a second (antisense) strand, the first (sense) strand having the following sequence: The second (antisense) chain has the following sequence: NAG is a trianticular ligand portion connected to the 5' end of the first chain and having the following structure: 。

Images