Therapeutic formulations of galNAc-oligonucleotide conjugates and saponins, and their use

Abstract

The present invention relates to a pharmaceutical formulation comprising a conjugate of an effector molecule and a ligand of ASGPR, wherein the ligand of ASGPR comprises at least one GalNAc moiety, and a saponin of the monodesmosidic or bidesmosidic triterpene glycoside type. The present invention also relates to a pharmaceutical composition comprising the conjugate and the saponin. Additionally, the present invention relates to a pharmaceutical formulation or composition herein for use as a medicament or for use in the treatment or prevention of a disease or health problem whose expression product is associated with any one or more of the following genes: apoB, TTR, PCSK9, ALAS1, ATS, GO, CCS, HBV X gene, HBV S gene, AAT, and LDH, and / or for use in the treatment or prevention of cancer, infectious diseases, viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, AAT-associated liver disease, acute hepatic porphyria, TTR-mediated amyloidosis, hereditary TTR amyloidosis (hATTR), complement-mediated diseases, hepatitis B infection, or autoimmune diseases. Furthermore, the present invention relates to an in vitro or ex vivo method for translocating an effector molecule of the present invention from the outside of a cell into said cell, preferably into the cytosol of said cell. The present invention also relates to an in vitro or ex vivo method for translocating a conjugate of the present invention from the outside of a cell into said cell.

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical formulation comprising a conjugate of an effector molecule and a ligand of ASGPR, wherein the ligand of ASGPR contains at least one GalNAc moiety, and a monodesmoside or videsmoside triterpene glycoside saponin. The present invention also relates to a pharmaceutical composition comprising the conjugate and the saponin. In addition, the present invention relates to pharmaceutical formulations or compositions of the present invention for use as a drug, or for use in the treatment or prevention of diseases or health problems related to one or more of the following genes: ApoB, TTR, PCSK9, ALAS1, AT3, GO, CC5, HBV X gene, HBV S gene, AAT, and LDH, and / or for use in the treatment or prevention of cancer, infectious diseases, viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, AAT-associated liver disease, acute hepatic porphyria, TTR-mediated amyloidosis, hereditary TTR amyloidosis (hATTR), complement-mediated diseases, hepatitis B infection, or autoimmune diseases. Furthermore, the present invention relates to an in vitro or ex vivo method for transferring the effector molecule of the present invention from extracellular to intracellular, preferably into the cytosol of the cell. The present invention also relates to an in vitro or ex vivo method for transferring the conjugate of the present invention from extracellular to intracellular. [Background technology]

[0002] The ability to inhibit the function of proteins, whether human or pathogenic, is essential for discovering new drugs. Conventional small molecule drugs and antibodies are strongly limited to a subset of available targets. Small molecules primarily bind to cofactor or signaling molecule sites, effectively binding to sites designed to accommodate them. Antibodies can bind to a wider variety of proteins, but are generally limited to extracellular targets.

[0003] Oligonucleotide therapy is a relatively new and rapidly developing field that aims to overcome many of the problems faced by small molecule drugs or antibody drugs by directly manipulating gene transcription and translation pathways. The potency and versatility of oligonucleotides make them attractive drug candidates, particularly because they are expected to suppress genes encoding "undruggable" proteins using classical small molecule drugs. The first oligonucleotide drugs were based on antisense techniques in which single-stranded nucleic acid molecules bind to sequences specific to complementary mRNA targets, thereby inducing double-strand degradation by the RNase H system.

[0004] In 1998, Andrew Fire and Craig Mello published a highly influential paper in Caenorhabditis elegans identifying double-stranded RNA (dsRNA) as the causative agent of post-transcriptional gene silencing (PTGS), a phenomenon they termed RNA interference (RNAi). The discovery of RNAi explained the puzzling findings of gene silencing in plants and fungi, revolutionizing biology and ultimately demonstrating that non-coding RNA is a central regulator of gene expression in multicellular organisms. Shortly thereafter, it was discovered that small dsRNA molecules (typically 15-30 bp) can catalytically induce RNAi silencing in mammalian cells without inducing a nonspecific interferon response. Targeting the RNAi pathway with small dsRNA molecules such as small interfering RNA (siRNA) and small hairpin RNA (shRNA) offers several theoretical advantages over antisense, including significantly greater efficiency (catalysis) and longer-lasting inhibition of gene expression. This can be rephrased as meaning that the frequency of administration will decrease, the dosage will decrease, and the cost will decrease. It may also mean that the lower the exposure, the lower the toxicity problems associated with siRNA.

[0005] However, before siRNA can reach its target in vivo, it faces numerous significant obstacles, including the blockage of pathways to the RNA-induced silencing complex (RISC) mechanism. Once in the bloodstream, its small size and high anionic properties make it easily degraded by endogenous nucleases and readily excreted by kidney. In addition, before reaching its target cells, siRNA must traverse the tight endothelial junctions of blood vessels and diffuse through the extracellular matrix. Due to its numerous negative charges, siRNA does not readily bind to or pass through the cell membrane, and once inside the cell, it must escape from endosomes and interact with its intracellular protein targets.

[0006] Therefore, the success of oligonucleotide (siRNA, etc.) therapy heavily depends on the effective delivery of the drug from the extracellular space to the cytosol. Consequently, considerable attention has been paid to developing delivery systems that improve oligonucleotide (siRNA, etc.) delivery. Besides viral delivery, the main methods used to improve siRNA (or other oligonucleotide) delivery to cells include the use of liposomes, cell-permeable peptides (CPPs) and their mimics, or nanoparticles (Gooding, Matt, et al. Chemical biology & drug design 80.6(2012);787-809). Safety concerns, such as the potential for insertional mutagenesis and immunotherapy, are considered to limit the future potential of viral methods.

[0007] Liposomes are the most commonly used delivery vectors for oligonucleotides such as siRNA, in which case the oligonucleotide is encapsulated within a lipid bilayer. Liposome-mediated oligonucleotide delivery faces several challenges, including oxygen radical-mediated toxicity (typical of cationic liposomes) and cytotoxicity, which affect gene regulation and inflammatory responses. Furthermore, lipid-based in vivo delivery is thought to primarily target the liver and spleen.

[0008] Cell-permeable peptides (CCPs), also known as protein transduction domains, are short peptides (usually <30 amino acid residues) with the unusual property of being able to pass through the cell membrane. CPPs can enhance the intracellular uptake of oligonucleotides by covalently bonding to them or by forming non-covalent complexes with them. The field of CPP-mediated oligonucleotide delivery is extremely complex because problems caused by both oligonucleotide technology and peptide technology, two fields that are far from mature, are combined in a single drug. Apart from the problems related to oligonucleotides, a typical problem faced with CPPs is related to the in vivo stability (or lack thereof) of the peptide chain, which often necessitates the use of non-natural peptide derivatives that may be complex to synthesize and / or may exhibit reduced cell-permeable activity.

[0009] Despite improvements in cell delivery, overcoming endosomal capture remains one of the major challenges in designing efficient CPPs and other transport systems (Gooding et al).

[0010] Nanoparticles and nanocarriers are nanoscale oligonucleotide delivery systems typically composed of a polymer, a biological stabilizer, and a cell-targeted ligand complexed with an oligonucleotide. An exemplary siRNA nanocarrier system is known as the siRNA Dynamic Poly-Conjugate. This system is based on an amphiphilic polymer linked to polyethylene glycol (PEG) as a biological stabilizer and a hepatocyte-targeted ligand, with the siRNA covalently bonded to the polymer by disulfide bonds. The target and PEG portions are bound to the polymer via maleamate bonds, forming negatively charged nanoparticles that do not bind to serum proteins. After internal migration via endocytosis, the maleamate bonds rapidly hydrolyze upon endosome acidification, exposing the cationic amine groups of the polymer and inducing endosomal escape via the proton sponge effect. Nanocarriers are also known in the form of cationic polymers such as polyethyleneimine (PEI) (sometimes combined with cyclodextrin), which can form electrostatic complexes with oligonucleotides such as siRNA, providing protection from degradation and enhanced internal migration. However, toxicity at higher concentrations resulting from membrane disruption and apoptosis induction may limit the applications of cationic polymers as therapeutic delivery agents. The complex preparation of nanoparticles and nanosupports, as well as their long-term stability, pose significant barriers to commercially viable applications.

[0011] Despite the improved delivery of oligonucleotides (such as siRNA) to cells that can be achieved by some of these delivery systems, endosomal escape remains a major barrier to the application of oligonucleotide-based therapies, such as RNAi-based therapies. Only a small portion of endocytosized oligonucleotides escape into the cytoplasm, where they exert their intended function, while the majority remain trapped in the endocytosis compartment and become inactive. Recent literature suggests that the escape rate of passive siRNA is <0.01% (Setten, Ryan L., et al., Nature Reviews Drug Discovery 18.6(2019); 421-446). Furthermore, it has been shown that a cell's ability to functionally internalize oligonucleotides to produce the desired effect (e.g., knockdown) appears to be independent of the extent to which the cell internalizes large amounts of oligonucleotides. These findings have led to the hypothesis that there are other "productive" and "unproductive" free uptake pathways, but the molecular mechanisms that distinguish these pathways remain unknown (Setten, Ryan L., John J. Rossi, and Si-ping Han. Nature Reviews Drug Discovery 18.6(2019);421-446).

[0012] Generally speaking, regardless of the underlying mechanism (improved endosomal escape, increased uptake via "productive" pathways, or another mechanism), there remains a great need for improved oligonucleotide delivery systems that result in increased potency (e.g., target knockdown), reduced toxicity, and / or reduced off-target effects.

[0013] The new strategy inevitably involves conjugating antisense oligonucleotides (ASOs) to receptor ligands to increase the potency of oligonucleotides and distributing them to selected tissues. For example, conjugating tribranched N-acetylgalactosamine (GalNAc) to oligonucleotide therapeutics results in a 10-30 fold increase in potency not only in isolated hepatocytes but also in the liver in vivo. GalNAc is found in damaged glycoproteins, where the terminal sialic acid residue is missing from the pendant oligosaccharide of the glycoprotein. The liver has very high levels of GalNAc on the surface of hepatocytes (10 per cell). 5 ~10 6 The liver functions to remove these proteins from the systemic circulation by expressing a trimer asialoglycoprotein receptor (ASGPR), preferably ASGPR1 (of the order of 1 / 2). ASGPR specifically binds to GalNAc at neutral pH to endocytose circulating macromolecules from the blood and releases GalNAc at acidic pH (approximately 5-6) to unload cargo into early endosomes. The released ASGPR is then recycled back onto the cell surface for reuse. Approximately one-third of RNAi drugs currently in clinical trials are single-molecule chemically modified RNAi triggers conjugated to a multivalent GalNAc ligand that targets ASGPR. The favorable physiological functions of the liver, the unique properties of ASGPR, the non-toxicity of GalNAc ligands, and the simplicity of GalNAc-siRNA conjugation make this an attractive method for systemic RNAi delivery to hepatocytes.

[0014] However, there remains a need for improved delivery systems that further increase the potency of ASGPRs targeting oligonucleotide systems (e.g., GalNAc-oligonucleotide conjugates), reduce toxicity, and / or minimize off-target effects. [Overview of the project] [Means for solving the problem]

[0015] A first aspect of the present invention is: - A conjugate of an effector molecule and a ligand for the asialocrycoprotein receptor (ASGPR), wherein the ligand for ASGPR comprises at least one N-acetylgalactosamine (GalNAc) moiety, preferably three or four GalNAc moieties, and more preferably (GalNAc)3-tris, or a conjugate comprising the same; - Contains a saponin which is a monodesmoside triterpene glycoside or a videsmoside triterpene glycoside. This relates to pharmaceutical formulations comprising pharmaceutically acceptable excipients and / or pharmaceutically acceptable diluents by optional selection.

[0016] One embodiment is, - A first pharmaceutical composition comprising a conjugate and optionally a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable diluent; - A pharmaceutical formulation of the present invention in the form of at least two pharmaceutical compositions, comprising a second pharmaceutical composition containing a saponin and optionally a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable diluent.

[0017] One embodiment is a pharmaceutical formulation of the present invention in the form of a single pharmaceutical composition comprising a conjugate, a saponin, and optionally a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable diluent.

[0018] A second aspect of the present invention relates to a pharmaceutical formulation of the present invention for use as a pharmaceutical agent.

[0019] A third aspect of the present invention relates to a pharmaceutical formulation of the present invention for use in the treatment or prevention of a disease or health problem in which the expression product is involved in one or more of the following genes: ApoB, TTR, PCSK9, ALAS1, AT3, GO, CC5, the X gene of HBV, the S gene of HBV, AAT, and LDH. One embodiment is a pharmaceutical formulation for use according to the present invention, in which such use is in the treatment or prevention of cancer, infectious diseases, viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, AAT-associated liver disease, acute hepatic porphyria, TTR-mediated amyloidosis, hereditary TTR amyloidosis (hATTR), complement-mediated diseases, hepatitis B infection, or autoimmune diseases.

[0020] A fourth aspect of the present invention is an in vitro or ex vivo method for transferring an effector molecule of the present invention from outside a cell into the cell, preferably into the cytosol of the cell, a) A step of providing cells, preferably selected from liver cells, virus-infected cells, and tumor cells, that express ASGPR, preferably ASGPR1, on their surface; b) A step of providing the conjugate of the present invention containing the effector molecule to be transferred; c) A step of providing the saponin of the present invention; d) The step of contacting the cells from step a) with the conjugate from step b) and the saponin from step c) in vitro or ex vivo, The present invention relates to a method for causing the transfer of the conjugate, which includes the effector molecule, from outside the cell into the cell, and thereby causing the transfer of the conjugate, which in turn causes the transfer of the effector molecule from outside the cell into the cell, preferably into the cytosol of the cell.

[0021] A fifth aspect of the present invention is an in vitro or ex vivo method for transferring the conjugate of the present invention from outside a cell into the cell, preferably into the cytosol of the cell, a) A step of providing cells, preferably selected from liver cells, virus-infected cells, and tumor cells, that express ASGPR, preferably ASGPR1, on their surface; b) A step of providing the conjugate of the present invention; c) A step of providing the saponin of the present invention; d) The step of contacting the cells from step a) with the conjugate from step b) and the saponin from step c) in vitro or ex vivo, The present invention relates to a method for causing a conjugate to move from outside the cell to inside the cell.

[0022] A sixth aspect of the present invention relates to a kit of parts comprising the pharmaceutical formulation of the present invention or the second pharmaceutical composition of the present invention, and instructions for using the pharmaceutical formulation or the second pharmaceutical composition according to the present invention, or for using an in vitro or ex vivo method according to the present invention.

[0023] definition The term "GalNAc" has its usual scientific meaning and, as used herein, refers to N-acetylgalactosamine and its IUPAC name: 2-(acetylamino)-2-deoxy-D-galactose.

[0024] The term "(GalNAc)3-tris" has its usual chemical meaning, for example, in the field of siRNA-based therapies, and herein refers to a moiety comprising three GalNAc units, each separately, preferably via at least one linker, covalently bonded to the hydroxyl group of tris(hydroxymethyl)aminomethane (tris) (IUPAC name: 2-amino-2-(hydroxymethyl)propane-1,3-diol). (GalNAc)3-tris can exist as a molecule containing a free amine, or may be further functionalized via the remaining amine-binding site to form a (GalNAc)3-tris moiety comprising, for example, a conjugate as described herein.

[0025] The term "oligonucleotide" has its usual scientific meaning and, as used herein, refers to a series of two or more nucleotides; that is, an oligonucleotide is a short oligomer composed of ribonucleotides or deoxyribonucleotides. Examples include nucleic acids such as RNA and DNA, as well as any modified RNA or DNA, such as locked nucleic acid (LNA) or 2'-O,4'-C-aminoethylene or 2'-O,4'-C-aminomethylene-bridged nucleic acid (BNA). NC There is a series of nucleic acids, including nucleotide analogs such as cross-linked nucleic acids (BNA), which are also known as nucleotides, where nucleotide is ribonucleotide or deoxyribonucleotide.

[0026] The term "RNAi-mediated gene targeting" has its usual scientific meaning and, as used herein, refers to in vivo, ex vivo, or in vitro methods that affect the function of genes within cells by transferring oligonucleotides, such as small double-stranded RNA molecules, that target mRNA involved in gene transcription into the cells. For example, small double-stranded RNA molecules can efficiently induce RNAi silencing of specific genes.

[0027] "Cross-linked nucleic acids," or simply "BNA," or "locked nucleic acids," or simply "LNA," or 2'-O,4'-C-aminoethylene or 2'-O,4'-C-aminomethylene cross-linked nucleic acids (BNA) NC The term BNA has its usual scientific meaning and, in this specification, refers to modified RNA nucleotides. BNA is also referred to as a “restricted RNA molecule” or “inaccessible RNA molecule.” BNA monomers may contain 5-membered, 6-membered, or even 7-membered crosslinked structures via “fixed” C3'-end type sugar puckering. Synthetic incorporation of crosslinks at the 2',4' positions of ribose yields 2',4'-BNA monomers. BNA monomers can be incorporated into oligonucleotide polymer structures using standard phosphoramidite chemical properties known in the art. BNA is a structurally robust oligonucleotide with increased binding affinity and stability.

[0028] The term antisense oligonucleotide has its usual scientific meaning and may be expressed herein simply as "AON" or "ASO".

[0029] The terms “BNA-based antisense oligonucleotides,” or simply “BNA-AON,” have their usual scientific meanings and, as used herein, refer to a series of antisense nucleotides in which at least one of the nucleotides is BNA.

[0030] The term "proteinic" has its usual scientific meaning and, as used herein, refers to a molecule that is protein-like (meaning the molecule possesses the physicochemical properties of a protein to some extent), of a protein, relating to a protein, containing a protein, belonging to a protein, consisting of a protein, similar to a protein, or is a protein. When the term "proteinic" is used, for example, in "proteinic molecule," it means that at least a portion of the molecule is similar to a protein or is a protein, and "protein" should be understood to include a chain of amino acid residues of at least two residues in length, and therefore includes peptides, polypeptides and proteins, as well as aggregates of protein or protein domains. In a proteinic molecule, at least two amino acid residues are linked via an amide bond, such as a peptide bond. In a proteinic molecule, the amino acid residues are natural amino acid residues and / or artificial amino acid residues, such as modified natural amino acid residues. In a preferred embodiment, a proteinic molecule is a molecule containing at least two amino acid residues, preferably 2 to about 2,000 amino acid residues. In one embodiment, a proteinic molecule is a molecule containing 2 to 20 (typical of peptides) amino acids. In one embodiment, the protein molecule is a molecule containing 21 to 1,000 amino acids (typically found in polypeptides, proteins, protein domains such as antibodies, single-domain antibodies, and ligands for receptors such as Fab, scFv, and EGF). The amino acid residues are preferably (typically) linked via peptide bonds. According to the present invention, the amino acid residues are (modified) (non)natural amino acid residues, or include them.

[0031] The terms “effector molecule” or “effector portion” have their usual scientific meaning, for example, when referring to an effector molecule as part of a covalent conjugate, and herein, for example, target molecule: refers to a molecule that selectively binds to one or more of the following: proteins, peptides, carbohydrates, sugars such as glycans, (phospho)lipids, nucleic acids such as DNA and RNA, or enzymes, and modulates the biological activity of one or more such target molecules. Effector molecules are molecules selected from one or more of the following: small molecules such as drug molecules, toxins such as protein toxins, oligonucleotides such as BNA, heterologous nucleic acids or siRNA, enzymes, peptides, proteins, or any combination thereof. Therefore, for example, an effector molecule or effector moiety is a molecule or moiety selected from one or more of the following: small molecules such as drug molecules, toxins such as protein toxins, oligonucleotides such as BNA, heterologous nucleic acids or siRNA, enzymes, peptides, proteins, or any combination thereof, and a target molecule is a molecule or moiety that selectively binds to one or more of the following: proteins, peptides, carbohydrates, sugars such as glycans, (phospho)lipids, nucleic acids such as DNA and RNA, or enzymes, and when bound to a target molecule, can regulate the biological activity of such one or more target molecules. Typically, an effector molecule can exert a biological effect inside a mammalian cell, such as a human cell, for example, in the cytosol of said cell. Therefore, typical effector molecules are drug molecules, plasmid DNA, toxins such as toxins composed of antibody-drug conjugates (ADCs), oligonucleotides such as siRNA, BNA, and nucleic acids composed of antibody-oligonucleotide conjugates (AOCs). For example, an effector molecule is a molecule that can function as a ligand that can increase or decrease (intracellular) enzyme activity, gene expression, or cell signaling.

[0032] The term “health problem” has its usual scientific meaning and, as used herein, refers to any condition of the body or part thereof, organs, muscles, veins, arteries, skin, limbs, blood, cells, etc., of a human patient or other subject, which is below optimal compared to the condition of a healthy human body or part thereof, thereby impairing the proper function and / or health of the subject, for example, impairing the normal function of the human body.

[0033] The term "GalNAc-modified oligonucleotide drug" has its usual scientific meaning and, as used herein, refers to an oligonucleotide that interferes with gene transcription, wherein one or more GalNAc units are bound to the oligonucleotide, for example, via at least one linker.

[0034] The term "locked nucleic acid," or simply "LNA," has its usual scientific meaning and, as known in the art, refers to oligonucleotides containing one or more nucleotide building blocks, in either the C3'-end conformation "β-D-LNA" or the C2'-end conformation (α-L-LNA), with the ribose moiety fixed by additional ethylene crosslinking.

[0035] "Cross-linked nucleic acid NC," or simply "BNA" NC The term "nucleotide" has its usual scientific meaning and, as known in the art, refers to an oligonucleotide containing one or more nucleotide building blocks, comprising additional 2'-O,4'-C-aminoethylene cross-linked nucleic acids with different substitutions at the N atom (methyl groups have been most commonly used to date).

[0036] The term "chemically modified, metabolically stable siRNA" has its usual scientific meaning and, as used herein, refers to siRNA molecules that have undergone chemical modifications compared to naturally occurring RNA oligonucleotides, such as modified siRNA molecules, to make them more resistant, i.e., more stable, to metabolic degradation, digestion, enzymatic lysis, etc.

[0037] The term "saponin" has its usual scientific meaning and, as used herein, refers to a group of amphiphilic glycosides comprising one or more hydrophilic glycosides combined with a lipophilic aglycone core that is a sapogenin. Saponins may be naturally occurring or synthetic (i.e., not naturally occurring). The term "saponin" includes not only naturally occurring saponins and derivatives of naturally occurring saponins, but also saponins newly synthesized by chemical and / or biotechnological synthetic routes.

[0038] The term “saponin derivative” (also known as “modified saponin”) has its usual scientific meaning and, as used herein, refers to compounds equivalent to naturally occurring saponins that have been derivatized by one or more chemical modifications, such as oxidation of a functional group, reduction of a functional group, and / or formation of a covalent bond with another molecule (also referred to as “conjugation” or “covalent conjugation”). Preferred modifications include derivatization of the aldehyde group of the aglycone core, derivatization of the carboxyl group of the sugar chain, or derivatization of the acetoxy group of the sugar chain. Typically, saponin derivatives have no natural counterparts; that is, saponin derivatives do not occur naturally from, for example, plants or trees. The term “saponin derivative” includes not only derivatives obtained by derivatizing naturally occurring saponins, but also derivatives newly synthesized by chemical and / or biotechnological synthetic routes that produce compounds equivalent to naturally occurring saponins that have been derivatized by one or more chemical modifications.

[0039] The term "aglycone core structure" has its usual scientific meaning and, as used herein, refers to the aglycone core or aglycone of a saponin to which one or two carbohydrate antennas or sugar chains (glycans, glycones) are not bound. For example, chiric acid has the aglycone core structures of SO1861, QS-7, and QS-21.

[0040] The term "glycan" has its usual scientific meaning and, as used herein, refers to any of the following: glycan, carbohydrate antenna, glycone, single sugar moiety (monosaccharide), or chain containing multiple sugar moieties (oligosaccharide, polysaccharide). A glycan may consist only of sugar moieties, or it may include further moieties such as one of the following, for example, 4E-methoxycinnamic acid, 4Z-methoxycinnamic acid, and 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid, as present in QS-21.

[0041] In the context of glycan nomenclature, the terms "Api / Xyl-" or "Api- or Xyl-" have their usual scientific meanings and, as used herein, refer to glycans that either contain an apiose (Api) moiety or a xylose (Xyl) moiety.

[0042] The terms “antibody-drug conjugate” or “ADC” have their usual scientific meanings and, as used herein, refer to any conjugate of an antibody, such as IgG, Fab, scFv, single-domain antibody, immunoglobulin, immunoglobulin fragment, or one or more Vh domains, with any molecule (e.g., active drug components, toxins, oligonucleotides, enzymes, small molecule drug compounds, etc.) that can exert a therapeutic effect when brought into contact with target cells, such as those of a human patient.

[0043] The term “antibody oligonucleotide conjugate” or “AOC” has its usual scientific meaning and, as used herein, refers to any conjugate of an antibody, such as IgG, Fab, single-domain antibody, scFv, immunoglobulin, immunoglobulin fragment, or one or more Vh domains, with any oligonucleotide molecule (for example, an oligonucleotide selected from a set of natural or synthetic nucleic acids, including DNA, modified DNA, RNA, modified RNA, synthetic nucleic acids existing as single-stranded or double-stranded molecules, such as BNA, allele-specific oligonucleotides, small interfering RNA (siRNA: silencing RNA), antisense DNA, antisense RNA, etc.) that can exert a therapeutic effect when brought into contact with target cells, such as those of a human patient.

[0044] The term “conjugate” has its usual scientific meaning and, as used herein, refers to at least one first molecule covalently bonded to at least one second molecule by chemical bond, thereby forming a covalent aggregate comprising or consisting of the first and second molecules. Typical conjugates include (GalNAc)3-tris-siRNA, ADC, AOC, and SO1861-EMCH (in which EMCH is linked to the aldehyde group of the aglycone core structure of a saponin).

[0045] The term "S" used in GalNAc-effector molecular conjugates or constructs containing a linker represents a "stabilizing linker" that is maintained intact within endosomes and cytosols.

[0046] The term "L" used in antibody saponin conjugates or constructs containing linkers represents an "unstable linker" that is cleaved under weakly acidic conditions within endosomes.

[0047] The term “part” has its usual scientific meaning and, as used herein, refers to a molecule that is bonded, linked, or conjugated to further molecules, linkers, molecular aggregates, etc., thereby forming a larger molecule, conjugate, or molecular aggregate. Typically, a part is a first molecule covalently bonded to a second molecule, and contains one or more chemical groups that are initially present on the first molecule and are present on the second molecule. For example, saporins are typical molecules and are an example of effector molecules. As part of an antibody-drug conjugate, saporins are typical effector parts in an ADC. As part of an antibody-oligonucleotide conjugate, BNA or siRNA are typical effector parts in an AOC.

[0048] The term "DAR" typically refers to the drug-antibody ratio, meaning the average ratio of drug to antibody in a given antibody-drug conjugate (ADC) formulation. In this specification, it refers to the ratio of the amount of SO1861 portion, SPT001, or bound apoBBNA to the conjugate molecule.

[0049] The terms first, second, third, etc., used herein and in the claims are used, for example, to distinguish similar elements, compositions, components in a composition, or separate process steps, and do not necessarily describe a continuous or chronological order. The terms are interchangeable under appropriate circumstances, and embodiments of the present invention may be operated in any order other than those described or illustrated herein, unless otherwise specified.

[0050] The embodiments of the inventions described herein can be operated in combination and in cooperation with each other, unless otherwise specified.

[0051] Furthermore, although various embodiments are referred to as "preferably," "e.g.," "for example," or "especially," these should be interpreted as examples of how they can be implemented, rather than limiting the scope of the present invention.

[0052] The term “comprising” as used in the claims should not be interpreted as being limited to, for example, the elements, process steps, or components of a composition listed herein, nor should it be interpreted as excluding other elements, process steps, or components of a particular composition. This should be interpreted as specifying the existence of the described features, integers, (process) steps, or components as mentioned, but not as excluding the existence or addition of one or more other features, integers, steps, or components, or groups thereof. Accordingly, the scope of the expression “a method comprising steps A and B” should not be limited to a method consisting solely of steps A and B; rather, with respect to the present invention, only the listed steps of the method are A and B, and furthermore, the claims should be interpreted as including equivalents of these process steps. Accordingly, the scope of the expression “a composition comprising components A and B” should not be limited to a composition consisting solely of components A and B; rather, with respect to the present invention, only the listed components of the composition are A and B, and furthermore, the claims should be interpreted as including equivalents of these components.

[0053] Furthermore, referring to an element or component with the indefinite article "a" or "an" does not rule out the possibility of two or more elements or components existing, unless the context clearly indicates that only one or one element or component is required. Therefore, the indefinite article "a" or "an" usually means "at least one." [Brief explanation of the drawing]

[0054] [Figure 1] Synthesis of trivalent GalNAc. [Figure 2] Synthesis of SO1861-DBCO. [Figure 3] Synthesis of monovalent GalNAc-SO1861. [Figure 4] Synthesis of trivalent GalNAc-SO1861. [Figure 5] Synthesis of monovalent GalNAc-BNA. [Figure 6] Synthesis of trivalent GalNAc-BNA. [Figure 7] Synthesis of trivalent GalNAc-BNA. [Figure 8A] Synthesis of trivalent GalNAc. [Figure 8B] Synthesis of trivalent GalNAc. [Figure 9A] Cell viability (MTS) of GalNAc-SO1861 and trivalent GalNAc-SO1861 in HepG2(A) and Huh7(B) cell lines. The legend shown next to Figure 9B also applies to Figure 9A. [Figure 9B] Cell viability (MTS) of GalNAc-SO1861 and trivalent GalNAc-SO1861 in HepG2(A) and Huh7(B) cell lines. The legend shown next to Figure 9B also applies to Figure 9A. [Figure 10A] Cell viability assay (MTS) in HepG2(A) and Huh7(B) cell lines. The legend shown next to Figure 10B also applies to Figure 10A. [Figure 10B] Cell viability assay (MTS) in HepG2(A) and Huh7(B) cell lines. The legend shown next to Figure 10B also applies to Figure 10A. [Figure 11A] Gene expression analysis in HepG2(A) and Huh7(B) cell lines. [Figure 11B] Gene expression analysis in HepG2(A) and Huh7(B) cell lines. [Figure 12A] Gene expression analysis in HepG2(A) and Huh7(B) cell lines. The legend shown next to Figure 12B also applies to Figure 12A. [Figure 12B] Gene expression analysis in HepG2(A) and Huh7(B) cell lines. The legend shown next to Figure 12B also applies to Figure 12A. [Figure 13A] Cell viability assays in HepG2(A) and Huh7(B) cell lines. The legend shown next to Figure 13B also applies to Figure 13A. [Figure 13B]Cell viability assays in HepG2(A) and Huh7(B) cell lines. The legend shown next to Figure 13B also applies to Figure 13A. [Figure 14A] Gene expression analysis in HepG2(A) and Huh7(B) cell lines. The legend shown next to Figure 14B also applies to Figure 14A. [Figure 14B] Gene expression analysis in HepG2(A) and Huh7(B) cell lines. The legend shown next to Figure 14B also applies to Figure 14A. [Figure 15A] Cell viability assays in HepG2(A) and Huh7(B) cell lines. The legend shown next to Figure 15B also applies to Figure 15A. [Figure 15B] Cell viability assays in HepG2(A) and Huh7(B) cell lines. The legend shown next to Figure 15B also applies to Figure 15A. [Figure 16A] Gene expression analysis in HepG2(A) and Huh7(B) cell lines. The legend shown next to Figure 16B also applies to Figure 16A. [Figure 16B] Gene expression analysis in HepG2(A) and Huh7(B) cell lines. The legend shown next to Figure 16B also applies to Figure 16A. [Figure 17A] Gene expression analysis in HepG2(A) and Huh7(B) cell lines. The legend shown next to Figure 17B also applies to Figure 17A. [Figure 17B] Gene expression analysis in HepG2(A) and Huh7(B) cell lines. The legend shown next to Figure 17B also applies to Figure 17A. [Figure 18A] RNA expression analysis and cell viability assay of ApoB in primary human hepatocytes. [Figure 18B] RNA expression analysis and cell viability assay of ApoB in primary human hepatocytes. [Figure 19A] RNA expression analysis and cell viability assay of ApoB in primary human hepatocytes. [Figure 19B]RNA expression analysis and cell viability assay of ApoB in primary human hepatocytes. [Figure 20] RNA expression analysis of ApoB in primary mouse hepatocytes. [Figure 21] Synthesis of (GalNAc)3-Ls-SO1861, intermediates 22 and 23. [Figure 22] Synthesis of (GalNAc)3-Ls-BNA, intermediates 25 and 26. [Figure 23] Synthesis of (GalNAc)3-Ls-BNA. [Modes for carrying out the invention]

[0055] A first objective of the present invention is to provide an improved therapeutic formulation or improved pharmaceutical composition for delivering an effector molecule conjugated to a ligand containing ASGPR, particularly ASGPR1, such as GalNAc, when considering delivery from outside the target cell to the cell, or more specifically, delivery of the effector molecule within the cytosol of the target cell. A second objective of the present invention is to provide an improved method of treating a (human) patient suffering from a disease to be treated by a conjugate containing an effector molecule and a ligand for ASGPR.

[0056] The object of the present invention is to provide a therapeutic composition, or, for example, a therapeutic formulation of two therapeutic compositions, that contains a conjugate of an ASGPR ligand covalently bound to an effector molecule, which can exert a biological effect upon delivery into a target cell containing a molecular target for the effector molecule, and that, when administered to a (human) patient requiring the conjugate, exhibits an improved or sufficient therapeutic effect at a lower dose than currently required for the conjugate. This method effectively broadens the therapeutic range of the conjugate.

[0057] At least one of the above objectives is achieved by providing a therapeutic formulation of at least two pharmaceutical compositions, or the pharmaceutical composition of the present invention.

[0058] While the present invention describes specific embodiments, it is not limited thereto and is limited only by the claims.

[0059] A first aspect of the present invention is: - A conjugate of an effector molecule and a ligand for the asialocrycoprotein receptor (ASGPR), wherein the ligand for ASGPR comprises at least one N-acetylgalactosamine (GalNAc) moiety, preferably three or four GalNAc moieties, and more preferably (GalNAc)3-tris, or a conjugate comprising the same; - Contains a saponin which is a monodesmoside triterpene glycoside or a videsmoside triterpene glycoside. This relates to pharmaceutical formulations comprising pharmaceutically acceptable excipients and / or pharmaceutically acceptable diluents by optional selection.

[0060] One embodiment is, - A first pharmaceutical composition comprising a conjugate and optionally a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable diluent; - A pharmaceutical formulation of the present invention in the form of at least two pharmaceutical compositions, comprising a second pharmaceutical composition containing a saponin and optionally a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable diluent.

[0061] One embodiment is a pharmaceutical formulation of the present invention in the form of a single pharmaceutical composition comprising a conjugate, a saponin, and optionally a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable diluent.

[0062] The inventors have surprisingly found that cells expressing ASGPR, particularly ASGPR1, on their surface exhibit improved uptake of the conjugate containing the ligand and effector molecules of ASGPR when the cells are brought into contact with both the conjugate and triterpene glycoside saponins, typically videsmoside terpenoid saponins. For example, a GalNAc-based ligand for ASGPR1, such as the (GalNAc)3 Tris conjugate, covalently binds to oligonucleotides such as gene-silencing BNA for silencing HSP27, and thus exhibits improved uptake in the presence of saponins. Herein, the inventors provide a solution to a long-standing urgent need to improve the effectiveness of delivery of oligonucleotides from extracellular to intracellular target cells, for example, to improve the intracellular delivery of oligonucleotides such as BNA and / or siRNA into the cytosol of target cells expressing ASGPR1 on their surface. Saponins applied in accordance with the present invention are known to have the ability to promote the endosomal extrusion of protein toxins at the point of contact between such saponins and toxins (which are, for example, part of an antibody-drug conjugate (ADC)) and target cells. Here, the inventors have also demonstrated that certain derivatives of such saponins maintain their ability to promote the extrusion of effector molecules from endosomes and lysosomes into the cytosol when saponins and effector molecules coexist intracellularly, i.e., within endosomes. Surprisingly, such saponin derivatives can promote the delivery of effector molecules, which consist of a conjugate to ASGPR1 that holds target cells, while reducing the cytotoxicity and hemolytic effects of such saponin derivatives compared to those observed with (naturally occurring) unmodified saponins. Such saponin derivatives are described in more detail herein.

[0063] Conjugate - Introduction The pharmaceutical formulations and specific pharmaceutical compositions of the present invention comprise a conjugate of an effector molecule and an asialoglycoprotein receptor (ASGPR), such as ASGPR1, preferably a ligand of ASGPR1. When the effector molecule is bound to the remainder of the conjugate, it is referred to herein as the “effector moiety.” The ASGPR ligand comprises at least one N-acetylgalactosamine (GalNAc) moiety. According to embodiments of the present invention, each GalNAc moiety comprises formula (I):

[0064] [ka]

[0065] As shown, the remainder of the ASGPR ligand is bound via a covalent bond to the oxygen at position "1", or, if the ASGPR ligand consists of a single GalNAc moiety, it is bound to the effector moiety.

[0066] As shown in formula (II), the ASGPR ligand is preferably the effector partial linker L E It consists of a single GalNAc section coupled to the effector section E via [a specific method / device].

[0067] [ka]

[0068] The ASGPR ligand may contain two or more GalNAc moieties, for example, two, three, four, five, or six GalNAc moieties, preferably three or four GalNAc moieties, more preferably three GalNAc moieties. In such cases, it is preferable that each GalNAc moiety is separately covalently bonded to the central bridging moiety B via the oxygen at position "1", thereby preferably the effector moiety linker L E A bridge is effectively formed between the GalNAc portion and the effector portion via this. The GalNAc portion is given by equation (III):

[0069] [Chemical formula]

[0070] (wherein n is an integer of 3 or more, preferably n is 3, L E is an effector moiety linker, and E is an effector moiety), may be directly bonded to the crosslinking moiety B. More preferably, the GalNAc moiety has the formula (IV):

[0071] [Chemical formula]

[0072] (wherein n is an integer of 3 or more, preferably n is 3, L E is an effector moiety linker, and E is an effector moiety), as shown, the GalNAc linker L GAL is bonded to the crosslinking moiety B via. L GAL may be independently selected each time it appears, but those skilled in the art will understand that when each appearance of L GAL represents the same moiety, the synthesis of the conjugate is simplified.

[0073] Conjugate-linker L E Effector moiety linker L E represents any chemical moiety suitable for covalently bonding an effector moiety to GalNAc as in formula (II), or to the crosslinking moiety B as in formulas (III) and (IV). There are no particular restrictions on the identity and size of the linker, and covalently bonding an effector molecule to GalNAc as in formula (II) or to the crosslinking moiety B as in formulas (III) and (IV) may be carried out by means of "ordinary" chemical functional groups (e.g., ester bonds), and also typically via a "click chemistry"-type linker having a long chain length, for example, resulting in a linker E L containing more than 10 or more than 20 carbon atoms. Suitable effector moiety linker L EThe related coupling reactions are described in the handbook Hermanson, Greg T., Bioconjugate techniques, Academic Press, 2013.

[0074] As will be understood by those skilled in the art, the effector part linker L E Typically, this is GalNAc or at least one first precursor L covalently bonded to the crosslinking portion B. E1 And the second precursor L is covalently bonded to the effector part. E2 This is the result of a coupling reaction (e.g., a "click chemistry" type) between the two. This principle is illustrated by the following reaction scheme of the compound of formula (II):

[0075] [ka]

[0076] In the formula, L E This is the linker in the effects section, L E1 The effector section linker L is covalently coupled to GalNAc. E It is a precursor of L E2 The effector section linker L is covalently coupled to the effector section. E It is a precursor to [the original component], and E is the effect pedal part.

[0077] The reaction scheme corresponding to the compound of formula (III) is as follows:

[0078] [ka]

[0079] In the formula, L E This is the linker in the effects section, L E1 The effector section linker L is covalently coupled to GalNAc. E It is a precursor of L E2 The effector part linker L is covalently coupled to the effector part.E It is a precursor, where n is an integer of 3 or 4 or more, preferably n is 3, B is the bridging portion, and E is the effector portion.

[0080] The reaction scheme corresponding to the compound of formula (IV) is as follows:

[0081] [ka]

[0082] In the formula, L E This is the linker in the effects section, L E1 The effector section linker L is covalently coupled to GalNAc. E It is a precursor of L E2 The effector part linker L is covalently coupled to the effector part. E It is a precursor, where n is an integer of 3 or 4 or more, preferably n is 3, E is the effector part, L GAL is a GalNAc linker, and B is the bridging portion.

[0083] Effector section Linker L E This is GalNAc or at least one first precursor L covalently bonded to the crosslinking portion. E1 And the second precursor L is covalently bonded to the effector part. E2The coupling reaction between the two is, for example, azido-alkyne cycloaddition, thiol-maleimide coupling, Staudinger reaction, nucleophilic ring-opening of strained heterocyclic electrophiles (aziridines, epoxides, cyclic sulfate esters, aziridinium ions, episulfonium ions, etc.), non-aldol type carbonyl reactions (urea, thiourea, hydrazones, oxime ethers, amides, or aromatic heterocycle formation, etc.), or addition of carbon-carbon double bonds (epoxidation, aziridination, dihydroxylation, sulfentyl halide addition, nitrosyl halide addition, or Michael addition, etc.), preferably azido-alkyne cycloaddition, thiol-maleimide coupling, Staudinger reaction, or nucleophilic ring-opening of strained heterocyclic electrophiles, and more preferably azido-alkyne cycloaddition or thiol-maleimide coupling.

[0084] According to some embodiments of the present invention, the effector partial linker L E It contains a succinimidothioether moiety. Such succinimidothioethers are, for example, the result of thiol-maleimide coupling between an N-substituted maleimide and a thiol or sulfhydryl-containing compound. This thiol-maleimide coupling is commonly known as a "click" chemistry tool in the field of bioconjugation and is described, for example, in Hermanson, Greg T. Bioconjugate techniques. Academic press, 2013, page 289. Therefore, the effector part linker L E This is a first precursor L containing GalNAc or an N-substituted maleimide, which is covalently bonded to the crosslinking portion. E1 Then, a second precursor L, which contains a thiol or its precursor, is covalently bonded to the effector portion. E2 It is preferable that the precursor is the result of a coupling reaction between the two. A suitable thiol precursor is a disulfide that can be cleaved (e.g., in situ) by reduction to the corresponding thiol.

[0085] Precursor L present in compounds of formula (V), (VII), or (VIII) E1 The preferred structure is the following terminally N-substituted maleimide:

[0086] [ka]

[0087] (wherein X represents any linker suitable for covalently bonding a terminally N-substituted maleimide to GalNAc or a bridging moiety B). As will be understood by those skilled in the art, X may result from a coupling reaction between a first moiety covalently bonded to GalNAc or the bridging moiety and a second moiety covalently bonded to the maleimide. X may include hydrazones and / or 1,2,3-triazoles. When 1,2,3-triazole is referred to in the context of linkers in this application, it is always preferable to mean 1H-1,2,3-triazole.

[0088] Precursor L present in compounds of formula (V), (VII), or (VIII) E1 The structural embodiment of the following terminally N-substituted maleimides:

[0089] [ka]

[0090] (In the formula, a and b each independently represent an integer of 0 or more, preferably a and b each independently represent an integer selected from 0, 1, 2, and 3, more preferably a and b represent 2, L E1a represents hydrazone and / or 1,2,3-triazole, preferably 1,2,3-triazole, L E1b (wherein it represents hydrazone and / or 1,2,3-triazole, preferably hydrazone);

[0091] [ka]

[0092] (In the formula, b and c each independently represent an integer greater than or equal to 0, preferably b represents an integer selected from 0, 1, 2, and 3, c represents an integer in the range of 5 to 15, more preferably b represents 2 and c represents 9, L E1a represents hydrazone and / or 1,2,3-triazole, preferably 1,2,3-triazole, L E1b (wherein it represents hydrazone and / or 1,2,3-triazole, preferably hydrazone); and

[0093] [ka]

[0094] (In the formula, c represents an integer greater than or equal to 0, preferably an integer in the range of 5 to 15, more preferably 9, L E1c (wherein represents hydrazone and / or 1,2,3-triazole, preferably 1,2,3-triazole).

[0095] L E1a , L E1b and L E1c Preferably each contains fewer than 20 carbon atoms, more preferably each independently represents a portion according to formula (XIII), (XIV), or (XV), and preferably L E1a is the 1,2,3-triazole of formula (XIII), and L E1b is the hydrazone of formula (XIV), and L E1c is the 1,2,3-triazole of formula (XV):

[0096] [ka]

[0097] Accordingly, in some embodiments, the conjugate is a compound of formula (II), (III), or (IV) as described herein, and the effector partial linker L EThis is the result of a coupling reaction between a compound of formula (V), (VII), or (VIII) and a compound of formula (VI), and the first precursor L E1 is a terminally N-substituted maleimide of formula (X), (XI), or (XII), and L E1a For example, is 1,2,3-triazole of formula (XIII), and L E1b For example, is the hydrazone of formula (XIV), and L E1c For example, this is the 1,2,3-triazole of formula (XV).

[0098] Conjugate - Bridge portion B The crosslinking portion B present in the compound of formula (III) or (IV) consists of two or more GalNAc portions, preferably three GalNAc portions, and the effector portion linker L E Represents any part suitable for covalent bonding.

[0099] According to a preferred embodiment, the crosslinked portion B is a compound of formula (XV):

[0100] [ka]

[0101] The oxygen atom of the compound of formula (XV) is either GalNAc (corresponding to the compound of formula (III)) or GalNAc linker L. GAL (corresponding to the compound of formula (IV)), the nitrogen atom of the compound of formula (XV) is bonded to the effector partial linker L E It is connected to

[0102] Those skilled in the art will understand that these compounds of formula (III) or (IV), where the crosslinked portion B is a compound of formula (XV), correspond to a conjugate in which the ASGPR ligand consists of (GalNAc)3-tris.

[0103] Conjugate-Linker L GAL GalNAc Linker L GALrepresents any chemical moiety suitable for covalently bonding GalNAc to the bridging portion, as in formula (IV). There are no particular restrictions on the identity and size of the linker.

[0104] According to the embodiment, GalNAc linker L GAL Each contains 2 to 25 carbon atoms, preferably 7 to 15 carbon atoms, more preferably 11 carbon atoms. Preferably, GalNAc linker L GAL It comprises at least one, preferably two, amide moieties. Particularly preferred is the GalNAc linker L GAL This is a compound according to formula (XVI):

[0105] [ka]

[0106] That is the case.

[0107] Conjugate - Effect section One embodiment of the present invention is a pharmaceutical formulation in which the effector molecule comprises or consists of at least one of the following: a small molecule such as a drug molecule, a toxin such as a protein toxin, an oligonucleotide such as BNA, heterologous nucleic acid or siRNA, an enzyme, a peptide, a protein, or any combination thereof, preferably a toxin, an enzyme, or an oligonucleotide. According to the present invention, the effector molecule expresses ASGPR on its surface and is a molecule that can exert a biological effect within a cell, for example, within the cytosol of the cell, including a binding partner of the effector molecule, thereby enabling the effector molecule to exert its biological effect within the cell when it is delivered to the intracellular location where the target of the effector molecule is present, preferably within the cytosol.

[0108] One embodiment of the present invention is a pharmaceutical formulation in which an ASGPR ligand, more particularly an ASGPR1 ligand, and an effector molecule, preferably a toxin or oligonucleotide, are conjugated via a covalent bond, preferably via at least one linker. Such linkers, suitable for conjugating an ASGPR ligand such as (GalNAc)3 Tris to an effector molecule such as BNA, siRNA, or a protein toxin, are described in detail above.

[0109] One embodiment is a pharmaceutical formulation of the present invention in which the effector molecule is small interfering RNA (siRNA), small hairpin RNA (shRNA), anti-hairpin microRNA (miRNA), single-stranded RNA, aptamer RNA, double-stranded RNA (dsRNA), antimicroRNA, (anti-miRNA, anti-miR), antisense oligonucleotide (ASO), mRNA, DNA, antisense DNA, locked nucleic acid (LNA), cross-linked nucleic acid (BNA), 2'-O,4'-aminoethylene cross-linked nucleic acid (BNA) NC The oligonucleotide is selected from one or more of the following: ), BNA-based siRNA, and BNA-based antisense oligonucleotide (BNA-AON). As described above, any effector molecule that has a target binding partner in a cell expressing the ligand receptor, i.e., ASGPR, which is composed of the conjugate of the present invention, is appropriately selected for incorporation into the conjugate of the present invention. Therefore, for example, ASO, siRNA, or BNA that can target genes or mRNA present in cells exposing ASGPR to its surface are suitable candidates for incorporation into the conjugate of the present invention.

[0110] One embodiment of the present invention is a pharmaceutical formulation in which the effector molecule is an oligonucleotide selected from anti-miRNA, BNA-AON, or siRNA, for example, preferably chemically modified siRNA, metabolically stable siRNA, and BNA-based siRNA selected from chemically modified and metabolically stable siRNA. Such chemically modified and / or metabolically stable siRNA moieties are known in the art and are suitable for binding to ASGPR ligands.

[0111] One embodiment of the present invention is a pharmaceutical formulation in which the effector molecule is an oligonucleotide capable of silencing any one of the following genes: HSP27, apolipoprotein B (ApoB), transthyretin (TTR), the proprotein convertase subtilisin / kexin type 9 (PCSK9), δ-aminolevulinic acid synthase 1 (ALAS1), antithrombin 3 (AT3), glycolate oxidase (GO), complement component C5 (CC5), the X gene of hepatitis B virus (HBV), the S gene of HBV, α1-antitrypsin (AAT), and lactate dehydrogenase (LDH), and / or targeting abnormal miRNAs. One embodiment of the present invention is a pharmaceutical formulation in which, when the effector molecule is present in a cell, the effector molecule is an oligonucleotide capable of silencing any one of the following genes: apolipoprotein B (ApoB), transthyretin (TTR), the proprotein convertase subtilisin / kexin type 9 (PCSK9), δ-aminolevulinic acid synthase 1 (ALAS1), antithrombin 3 (AT3), glycolate oxidase (GO), complement component C5 (CC5), the X gene of hepatitis B virus (HBV), the S gene of HBV, α1-antitrypsin (AAT), and lactate dehydrogenase (LDH), and / or, when the effector molecule is present in a cell, it can target abnormal miRNAs. According to the present invention, for example, when administered simultaneously with a saponin according to the present invention to a target requiring modification of gene or mRNA expression (level), gene silencing, etc., the effector molecule composed of the conjugate of the present invention (e.g., BNA, siRNA, ASO) can effectively target any gene or mRNA present in cells expressing ASGPR on their cell surface. Under the influence of the saponin, the therapeutic range of the conjugate containing the effector molecule and the ASGPR ligand is broadened, thereby resulting in a more potent biological effect of the effector molecule within the target cell, and / or the biological effect of the effector molecule being achieved at a lower effective dose than would be achieved when the target cell is exposed to the conjugate in the absence of the saponin or saponin derivative.According to the present invention, by contacting target cells expressing ASGPR with a conjugate containing the ligand and effector molecule of ASGPR together with a saponin or saponin derivative, it may be possible to overcome the threshold for the intracellular activity of the effector molecule. For example, in the absence of saponin, the dose of conjugate required to reach a sufficiently high concentration of the effector molecule in the cytosol of target cells may be too high to be safely administered to patients who require such treatment with the effector molecule. In the presence of saponin, the effector molecule can exert its biological effect in the cytosol of ASGPR-expressing target cells at a lower dose than that at which it would not exert an intracellular effect when the target cells are exposed to a lower dose in the absence of saponin. For example, BNA, which silences HSP27 in (liver) cells, did not silence HSP27 in ASGPR-retaining cells when a conjugate of ASGPR's GalNAc ligand and BNA was brought into contact with ASGPR-retaining cells. On the other hand, when the same cells were brought into contact with the same or a lower dose of the conjugate in the presence of a saponin or saponin derivative, for example, a saponin having a linker EMCH conjugated to the aldehyde group in the aglycone of the saponin, HSP27 was effectively silenced in the target cells (as shown in Appendix Example 2 and Figure 11). Therefore, we hereby provide molecules, pharmaceutical formulations, pharmaceutical compositions, and methods for treating (human) subjects with such molecules, formulations, and compositions, which allow for the administration of effector molecules with an improved therapeutic range to patients in need, or to administer effector molecules having an improved therapeutic range, thereby producing an improved therapeutic effect in the target cells of the conjugate according to the present invention when the effector molecule reaches its intracellular target.

[0112] One embodiment of the present invention is a pharmaceutical formulation in which the effector molecule, when present, for example inside a mammalian cell, can target mRNA involved in the expression of any one of the following proteins: HSP27, ApoB, TTR, PCSK9, ALAS1, AT3, GO, CC5, the expression product of the X gene of HBV, the expression product of the S gene of HBV, AAT, and LDH, or, when present, for example inside a mammalian cell, can weaken or restore miRNA function, such as inhibiting oncogenic miRNA (onco-miR) or suppressing the expression of onco-miR. Such proteins are typically disease-related, and for example, silencing the genes involved in the expression of such proteins can alleviate the disease or symptoms, and patients treated with a conjugate formulation comprising a ligand for saponin and ASGPR and an effector molecule capable of targeting mRNA involved in the expression of any one of these proteins benefit. Accordingly, a part of the present invention is preferably used in formulations and compositions of the present invention containing saponin to be included in RNAi-based therapeutic regimens. RNAi allows for the silencing of target genes by cleaving mRNA or inhibiting mRNA translation. Saponins, selected for RNAi therapy as described herein, help enhance the gene-silencing effector molecules by improving cytosolic delivery of the effector molecules when ASGPR-expressing target cells are exposed to both the saponin and the conjugate.

[0113] One embodiment is a pharmaceutical formulation of the present invention, wherein the effector molecule is preferably one or more selected from peptides, proteins, enzymes such as urease and Cre-recombinase, proteotoxins, ribosome-inactivating proteins, proteotoxins selected from Table A5, and / or bacterial toxins, more preferably viral toxins such as apoptin; Shiga toxin, Shiga-like toxin, Pseudomonas aeruginosaBacterial toxins such as *Aeruginosa* exotoxin (PE) or exotoxin A of PE, full-length or truncated diphtheria toxin (DT), cholera toxin; fungal toxins such as α-sarcin; plant toxins including ribosome-inactivating proteins and the A chain of type 2 ribosome-inactivating proteins, e.g., diansin (e.g., diansin-30 or diansin-32), saporin (e.g., saporin-S3 or saporin-S6), bougain or debuganing, which is a deimmunized derivative of bougain. A toxin comprising or consisting of at least one protein molecule selected from one or more of the following: Shiga-like toxin A, pokeweed antiviral protein, lysine, lysine A chain, modesine, modesine A chain, abrin, abrin A chain, volkensin, volkensin A chain, biscumin, biscumin A chain; or animal or human toxins such as frog RNase, or human-derived granzyme B or angiogenin, or any fragment or derivative thereof; preferably, the protein toxin is dianthic The compounds are and / or saporins, and / or at least one of toxin-targeting ribosomes, toxin-targeting extension factors, toxin-targeting tubulin, toxin-targeting DNA and toxin-targeting RNA, more preferably emtansine, pasdotox, mytansinoid derivative DM1, mytansinoid derivative DM4, monomethyl auristatin E (MMAE, vedotin), monomethyl auristatin F (MMAF, mahodotin), calicheamicin, N-acetyl-γ-calicheamicin, pyrrolobenzodiazepine (PBD) Merges, benzodiazepines, CC-1065 analogs, duocalmycin, doxorubicin, paclitaxel, docetaxel, cisplatin, cyclophosphamide, etoposide, docetaxel, 5-fluorouracil (5-FU), mitoxantrone, tubulicin, indolinobenzodiazepine, AZ13599185, cryptophycin, rhizoxin, methotrexate, anthracycline, camptothecin analogs, SN-38, DX-8951f, exatecan mesylate, Pseudomonas aeruginosaThe present inventors have demonstrated that when the formulations and compositions of the present invention consist of a conjugate of an ASGPR ligand and an effector molecule, they are equally suitable for improving the delivery of nucleic acid-based effector molecules when such effector molecules are brought into contact with target cells expressing ASGPR in the presence of a saponin or saponin derivative, with respect to the delivery of effector molecules such as peptides and proteins into the cytosol. For example, when target cells are brought into contact with a conjugate containing an effector molecule (i.e., a protein toxin) and an ASGPR ligand, as well as a saponin, according to the present invention, the therapeutic range of the protein toxin is broadened. When target cells are exposed to a conjugate containing saponins and toxins, the toxins exert their intracellular effects to a greater degree or at lower doses compared to the intracellular effects achieved at the same or lower doses of toxins in the absence of saponins or saponin derivatives.

[0114] saponin One embodiment is a pharmaceutical formulation of the present invention, wherein saponin is, 2α-hydroxyoleanolic acid; 16α-hydroxyoleanolic acid; Hederagenin (23-hydroxyoleanolic acid); 16α,23-hydroxyoleanolic acid; Gypsogenin; Chiral acid; Protoesigenin-21(2-methylbuta-2-enoate)-22-acetate; 23-Oxo-Baringtogenol C-21,22-Bis(2-methylbuta-2-enoate); 23-Oxo-Baringtogenol C-21(2-methylbuta-2-enoate)-16,22-diacetate; Digitogenin; 3,16,28-trihydroxyoleanane-12-ene; Gypsogenic acid; and These derivatives It includes an aglycone core structure selected from the group consisting of (referred to as "Group C" in this specification), Preferably, the saponin comprises an aglycone core structure selected from chiraic acid and gypsogenin or derivatives thereof, and more preferably, the saponin aglycone core structure is chiraic acid or a derivative thereof. Such preferred aglycones contain an aldehyde group on the C4 atom. Although not bound by any logic, such aldehyde groups, or their derivatives as described elsewhere herein, contribute to the activity of promoting endosomal escape of aglycones selected from group C, particularly triterpene glycoside-type saponins including quillaic acid and gypsogenin.

[0115] One embodiment of the present invention is a saponin conjugate in which at least one saponin belongs to the type of 12,13-dehydrooleanane containing an aldehyde group at the C4 position, and optionally contains a glucuronic acid group as a carbohydrate substituent of the C-3β-OH group of the saponin, and is a monodesmoside or bidesmoside triterpene saponin, preferably a bidesmoside triterpene saponin belonging to the type of 12,13-dehydrooleanane containing an aldehyde group at the C4 position, and containing a glucuronic acid group as a carbohydrate substituent of the C-3β-OH group of the saponin.

[0116] One embodiment is a pharmaceutical formulation of the present invention, • Saponins, Group A: GlcA-, Glc-, Gal- Rha-(1→2)-Ara-, Gal-(1→2)-[Xyl-(1→3)]-GlcA-, Glc-(1→2)-[Glc-(1→4)]-GlcA-, Glc-(1→2)-Ara-(1→3)-[Gal-(1→2)]-GlcA-、 Xyl-(1→2)-Ara-(1→3)-[Gal-(1→2)]-GlcA-、 Glc-(1→3)-Gal-(1→2)-[Xyl-(1→3)]-Glc-(1→4)-Gal-、 Rha-(1→2)-Gal-(1→3)-[Glc-(1→2)]-GlcA-、 Ara-(1→4)-Rha-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-、 Ara-(1→4)-Fuc-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-、 Ara-(1→4)-Rha-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-、 Ara-(1→4)-Fuc-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-、 Ara-(1→4)-Rha-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-、 Ara-(1→4)-Fuc-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-、 Ara-(1→4)-Rha-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-、 Ara-(1→4)-Fuc-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-、 Xyl-(1→4)-Rha-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-、 Xyl-(1→4)-Fuc-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-、 Xyl-(1→4)-Rha-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-、 Xyl-(1→4)-Fuc-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-、 Xyl-(1→4)-Rha-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-、 Xyl-(1→4)-Fuc-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-, Xyl-(1→4)-Rha-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-, Xyl-(1→4)-Fuc-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-, and These derivatives include a sugar chain bonded to an aglycone core structure selected from these derivatives, or • Saponins, Group B: Glc-, Gal- Rha-(1→2)-[Xyl-(1→4)]-Rha-, Rha-(1→2)-[Ara-(1→3)-Xyl-(1→4)]-Rha-, Ara-, Xyl-, Xyl-(1→4)-Rha-(1→2)-[R1-(→4)]-Fuc-(wherein R1 is 4E-methoxycinnamate), Xyl-(1→4)-Rha-(1→2)-[R2-(→4)]-Fuc-(wherein R2 is 4Z-methoxycinnamate), Xyl-(1→4)-[Gal-(1→3)]-Rha-(1→2)-4-OAc-Fuc-, Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-3,4-di-OAc-Fuc-, Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R3-(→4)]-3-OAc-Fuc-(wherein R3 is 4E-methoxycinnamate), Glc-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-4-OAc-Fuc-, Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-4-OAc-Fuc-, (Ara- or Xyl-)(1→3)-(Ara- or Xyl-)(1→4)-(Rha- or Fuc-)(1→2)-[4-OAc-(Rha- or Fuc-)(1→4)]-(Rha- or Fuc-), Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Qui-(1→4)]-Fuc-, Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-Fuc-, Xyl-(1→4)-[Gal-(1→3)]-Rha-(1→2)-Fuc-, Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-Fuc-, Ara / Xyl-(1→4)-Rha / Fuc-(1→4)-[Glc / Gal-(1→2)]-Fuc-, Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R4-(→4)]-Fuc-(wherein R4 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R5-(→4)]-Fuc-(wherein R5 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Rha-(1→3)]-4-OAc-Fuc-, Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4-OAc-Fuc-, 6-OAc-Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3-OAc-Rha-(1→3)]-Fuc-, Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3-OAc--Rha-(1→3)]-Fuc-, Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Qui-(1→4)]-Fuc-, Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-[Qui-(1→4)]-Fuc-, Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc-, Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3,4-di-OAc-Qui-(1→4)]-Fuc-, Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-Fuc-, 6-OAc-Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-Fuc-, Glc-(1→3)-[Xyl-(1→3)-Xyl-(1→4)]-Rha-(1→2)-Fuc-, Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc-, Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4OAc-Fuc-, Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4OAc-Fuc-, Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R6-(→4)]-Fuc- (where R6 is 5-O-[5-O-Rha-(1→2)-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R7-(→4)]-Fuc- (where R7 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R8-(→4)]-Fuc- (where R8 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R9-(→4)]-Fuc- (where R9 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R10-(→4)]-Fuc- (where R10 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R11-(→3)]-Fuc- (where R11 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R12-(→3)]-Fuc- (where R12 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Glc-(1→3)-[Glc-(1→6)]-Gal-, and these derivatives contains a sugar chain linked to an aglycone core structure selected from or · The saponin is a bidesmoside triterpenoid glycoside that contains a first sugar chain selected from group A linked to the aglycone core structure and a second sugar chain selected from group B linked to the aglycone core structure.

[0117] Such saponins exhibit a typical endosomal escape-promoting effect when the saponin and effector molecule (part of the conjugate according to the present invention containing the effector molecule and the ligand for ASGPR1, e.g., (GalNAc)3-tris) are brought into contact with target cells. Under the influence of such selected saponins containing one or two sugars selected from group C, the effector molecule in the conjugate reaches an intracellular threshold level at which the biological activity of the effector molecule in the target cell is confirmed or increased, or at which the activity of the effector molecule can be confirmed at a lower dose than the dose required for the same activity level in the absence of the saponin. Table 1 summarizes saponins having such endosomal escape-promoting activity. These saponins contain the aglycone core structure of the listed group C, contain monosaccharides or polysaccharides of group A and / or group B, and have been shown to enhance the effector molecule when the target cell is exposed to both the effector molecule and the saponin, or to have a high structural similarity to saponins in which such endosomal escape-promoting activity has been confirmed.

[0118] Accordingly, one embodiment of the present invention is a pharmaceutical formulation in which the saponin is Quillaja bark saponin, dipsacoside B, saikosaponin A, saikosaponin D, maculantoidin A, esculentoside A, phytolaccagenin, esinate, AS6.2, NP-005236, AMA-1, AMR, α-Hederin, NP-012672, NP-017777, NP-017778, NP-017774, NP-018110, NP-017772, NP-018109, NP-017888, NP-017889, NP-018108, SA1641, AE X55, NP-017674, NP-017810, AG1, NP-003881, NP-017676, NP-017677, NP-017706, NP-017705, NP-017773, NP-017 775, SA1657, AG2, SO1861, GE1741, SO1542, SO1584, SO1658, SO1674, SO1832, SO1862, SO1904, QS-7, QS1861, QS-7 api, QS1862, QS-17, QS-18, QS-21 A-apio, QS-21 A-xylo, QS-21 B-apio, QS-21 The derivatives are selected from the group consisting of B-xylo, β-escin, escin Ia, tea seed saponin I, tea seed saponin J, Assam saponin F, digitonin, primulic acid 1 and AS64R, their stereoisomers, their derivatives, and combinations thereof, preferably selected from the group consisting of QS-21, QS-21 derivatives, SO1861, SO1861 derivatives, SA1641, SA1641 derivatives, GE1741, GE1741 derivatives, and combinations thereof, more preferably selected from the group consisting of QS-21 derivatives, SO1861 derivatives, and combinations thereof, most preferably SO1861 derivatives. The inventors have found that these derivatives have an improved enhancing effect when used in combination with a conjugate composed of effector molecules as described herein. That is, these derivatives provide an endosome escape promoting effect comparable to that of underivativeized saponins, while exhibiting lower cytotoxicity and lower hemolytic activity. More structural details of saponin derivatives suitable for the context of the present invention are shown below.

[0119] One embodiment is a pharmaceutical formulation of the present invention, wherein the saponin is a saponin derivative. i. The saponin derivative comprises an aglycone core structure composed of a derivatized aldehyde group; or ii. The saponin derivative comprises a glycan containing a derivatized carboxyl group, preferably a glycan selected from group A as defined above; or iii. The saponin derivative comprises a glycan containing a derivatized acetoxy (Me(CO)O-) group, preferably a glycan selected from group B as defined above; or iv. Any combination of derivatizations i., ii., and iii. Any combination of two derivatizations from derivatizations i., ii., and iii. exists by arbitrary selection.

[0120] The inventors have demonstrated, in detail, that saponins derivatized to a single chemical group present in naturally occurring saponins, or to two such chemical groups, maintain sufficient effector molecule enhancing activity while strongly reducing both the cytotoxicity and hemolytic activity of such mono-derivatized and bi-derivatized saponins compared to the cytotoxicity and hemolytic activity of their naturally occurring unmodified counterparts. Furthermore, preferred according to the present invention are naturally occurring saponins in which an effector molecule enhancing effect is observed when both the saponin and the conjugate containing the effector molecule are brought into contact with target cells exposed to ASGPR1 on their surface.

[0121] One embodiment of the present invention is a pharmaceutical formulation comprising saponins such as SO1861, SA1657, GE1741, SA1641, QS-21, QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21 B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862, Quillaja saponin, and Saponinum album. A selection is made from the group consisting of (album), QS-18, Quil-A, Gyp1, dypsoside A, AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, SO1904, their stereoisomers, their derivatives, and combinations thereof, preferably selected from the group consisting of QS-21, QS-21 derivatives, SO1861, SO1861 derivatives, SA1641, SA1641 derivatives, GE1741, GE1741 derivatives, and combinations thereof, more preferably selected from the group consisting of QS-21 derivatives, SO1861 derivatives, and combinations thereof, most preferably SO1861 derivatives.

[0122] One embodiment is a pharmaceutical formulation of the present invention, wherein the saponin is a saponin derivative of a chiric acid or gypsogenin saponin of group C as listed above, and molecular weight 1:

[0123] [ka]

[0124] [In the formula, A1 represents hydrogen, a monosaccharide, or a linear or branched oligosaccharide, preferably a glycan selected from group A as defined above, more preferably a glycan selected from group A as defined above, and includes or consists of a glucuronic acid moiety; A2 represents hydrogen, a monosaccharide, or a linear or branched oligosaccharide, preferably a glycan selected from group B as defined above, more preferably a glycan selected from group B as defined above, and comprises at least one acetoxy (Me(CO)O-) group, for example, 1, 2, 3, or 4 acetoxy groups, and at least one of A1 and A2 is not hydrogen, preferably both A1 and A2 are oligosaccharides; Furthermore, R is represented as either a hydrogen atom in gypsogenin or a hydroxyl atom in chiral acid: Saponin derivatives correspond to the saponin represented by molecule 1, and the following derivatizations are possible: i. C of chiral acid or gypsogenin 23 The aldehyde group at the position is derivatized; ii. A1 represents a sugar chain selected from group A as defined above, and if A1 contains or consists of a glucuronic acid moiety, the carboxyl group of the glucuronic acid moiety of A1 is derivatized; and iii. If A2 represents a sugar chain selected from group B as defined above and contains at least one acetoxy group, then at least one of the single sugar moiety or two or more sugar moieties of A2, preferably all of the acetoxy groups, is derivatized.

[0125] One embodiment is a pharmaceutical formulation of the present invention in which A1 represents a glycan selected from group A as defined above and includes or consists of a glucuronic acid moiety, the carboxyl group of the glucuronic acid moiety of A1 is derivatized, and / or A2 represents a glycan selected from group B as defined above and A2 includes at least one acetoxy group, the at least one acetoxy group of A2 is derivatized.

[0126] One embodiment is a pharmaceutical formulation of the present invention, in which the saponin represented by molecule 1 is a picesmoside triterpene saponin, that is, a picesmoside triterpene glycoside type saponin.

[0127] One embodiment is a pharmaceutical formulation of the present invention in which the saponin derivative corresponds to the saponin represented by molecule 1, and the following derivatization: i. C of chiral acid or gypsogenin 23 The aldehyde group at position - Reduction to alcohol; or - Conversion to a hydrazone bond by reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), thereby yielding a saponin-Ald-EMCH such as SO1861-Ald-EMCH or QS-21-Ald-EMCH, and the maleimide group of the EMCH is optionally derivatized by forming a thioether bond with mercaptoethanol; or - Conversion to a hydrazone bond by reaction with N-[β-maleimidopropionic acid]hydrazide (BMPH), wherein the maleimide group of BMPH is derivatized by optionally forming a thioether bond with mercaptoethanol; or - The derivatization is by a conversion to a hydrazone bond via reaction with N-[κ-maleimidoundecanoic acid]hydrazide (KMUH), wherein the maleimide group of KMUH is derivatized by optionally forming a thioether bond with mercaptoethanol; ii. If A1 represents a sugar chain selected from group A as defined above, and A1 contains or consists of a glucuronic acid moiety, the carboxyl group of the glucuronic acid moiety of A1 is derivatized by conversion to an amide bond through reaction with 2-amino-2-methyl-1,3-propanediol (AMPD) or N-(2-aminoethyl)maleimide (AEM), thereby yielding saponin-Glu-AMPD such as QS-21-Glu-AMPD or SO1861-Glu-AMPD, or saponin-Glu-AEM such as QS-21-Glu-AEM or SO1861-Glu-AEM; iii. A2 represents a sugar chain selected from group B as defined above, and when A2 contains at least one acetoxy group, at least one of one sugar moiety or two or more sugar moieties of A2, preferably all acetoxy groups, are derivatized by conversion to a hydroxyl group (HO-) by deacetylation.

[0128] One embodiment is a pharmaceutical formulation of the present invention, wherein A1 is Gal-(1→2)-[Xyl-(1→3)]-GlcA, and / or A2 is Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc, preferably the saponin represented by molecule 1 is 3-O-β-D-galactopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]-β-D-glucuronopyranosylgyracic acid 28-O-β-D-glucopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)-4OAc-β-D-quinovopyranosyl-(1→4)]-β-D-fucopyranoside, more preferably SO1861, GE1741, SA1641 and / or QS-21 or derivatives thereof, most preferably SO1861 or derivatives thereof.

[0129] One embodiment is a pharmaceutical formulation of the present invention, wherein the saponin is a saponin derivative. i. The saponin derivative is - reduction to an alcohol; or - conversion to a hydrazone bond by reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), thereby resulting in a saponin-Ald-EMCH such as SO1861-Ald-EMCH or QS-21-Ald-EMCH, wherein the maleimide group of the EMCH is derivatized by optionally forming a thioether bond with mercaptoethanol; conversion to a hydrazone bond; or - Conversion to a hydrazone bond by reaction with N-[β-maleimidopropionic acid]hydrazide (BMPH), wherein the maleimide group of BMPH is derivatized by optionally forming a thioether bond with mercaptoethanol; or - Conversion to a hydrazone bond by reaction with N-[κ-maleimidoundecanoic acid]hydrazide (KMUH), wherein the maleimide group of KMUH is derivatized by optionally forming a thioether bond with mercaptoethanol. It comprises an aglycone core structure composed of aldehyde groups derivatized by; or ii. The saponin derivative comprises a sugar chain, preferably a sugar chain selected from group A as defined above, wherein the sugar chain comprises a carboxyl group of a glucuronic acid moiety that has been derivatized by conversion to an amide bond through reaction with 2-amino-2-methyl-1,3-propanediol (AMPD) or N-(2-aminoethyl)maleimide (AEM), thereby yielding a saponin-Glu-AMPD such as QS-21-Glu-AMPD or SO1861-Glu-AMPD, or a saponin-Glu-AEM such as QS-21-Glu-AEM or SO1861-Glu-AEM; or iii. The saponin derivative preferably comprises a sugar chain containing an acetoxy (Me(CO)O-) group, which is selected from group B as defined above and derivatized by conversion to a hydroxyl group (HO-) by deacetylation; or iv. The saponin derivative comprises any combination of derivatizations i., ii., and iii., or any combination of any two derivatizations from derivatizations i., ii., and iii. by any choice; Preferably, the saponin derivative contains an aglycone core structure composed of an aldehyde group that has been derivatized by conversion to a hydrazone bond through reaction with EMCH, and the maleimide group of EMCH is optionally derivatized by forming a thioether bond with mercaptoethanol.

[0130] One embodiment is a pharmaceutical formulation of the present invention, wherein the saponin is a saponin derivative. i. The saponin derivative contains an aglycone core structure composed of an aldehyde group that has been derivatized by conversion to a hydrazone bond through reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), thereby yielding a saponin-Ald-EMCH such as SO1861-Ald-EMCH or QS-21-Ald-EMCH; or ii. The saponin derivative comprises a sugar chain, preferably a sugar chain selected from group A as defined above, wherein the sugar chain comprises a carboxyl group, preferably a carboxyl group of a glucuronic acid moiety that has been derivatized by conversion to an amide bond through reaction with N-(2-aminoethyl)maleimide (AEM), thereby yielding a saponin-Glu-AEM such as QS-21-Glu-AEM or SO1861-Glu-AEM; or iii. The saponin derivative includes a combination of derivatizations i. and ii.

[0131] One embodiment is a pharmaceutical formulation of the present invention, wherein the saponin derivative comprises an aglycone core structure composed of an aldehyde group, the saponin derivative comprises a sugar chain, preferably a sugar chain selected from group A as defined above, and the sugar chain comprises a carboxyl group, preferably a carboxyl group of a glucuronic acid moiety that has been derivatized by conversion to an amide bond through reaction with N-(2-aminoethyl)maleimide (AEM).

[0132] One embodiment is a pharmaceutical formulation of the present invention, wherein the saponin comprises a molecule 2:

[0133] [ka]

[0134] Is it a saponin derivative represented by, Alternatively, a saponin derivative may be molecular 3:

[0135] [ka]

[0136] It is a saponin derivative represented by [formula].

[0137] [Table 1]

[0138] [Table 2]

[0139] [Table 3]

[0140] [Table 4]

[0141] [Table 5]

[0142] A second aspect of the present invention relates to a pharmaceutical formulation of the present invention for use as a drug. To the best of our knowledge, we are the first to provide a pharmaceutical formulation of the present invention for, for example, improving the efficacy of siRNA or BNA-based therapies. By using the pharmaceutical formulation of the present invention, the intracellular effect of an effector molecule composed of a conjugate containing an ASGPR1 ligand is enhanced. This drug is particularly suitable for treating diseases or abnormalities involving abnormal liver cells expressing ASGPR1. Abnormal liver cells are, for example, cells of patients suffering from any disease or health problem in which the expression product is involved in one or more of the following genes: HSP27, ApoB, TTR, PCSK9, ALAS1, AT3, GO, CC5, the X gene of HBV, the S gene of HBV, AAT, and LDH. Such diseases include, for example, cancer, infectious diseases, viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, AAT-associated liver disease, acute hepatic porphyria, TTR-mediated amyloidosis, hereditary TTR amyloidosis (hATTR), complement-mediated diseases, hepatitis B infection, diseases or disorders related to HSP27 expression, or autoimmune diseases.

[0143] A third aspect of the present invention relates to a pharmaceutical formulation of the present invention for use in the treatment or prevention of diseases or health problems in which the expression product is related to one or more of the following genes: HSP27, ApoB, TTR, PCSK9, ALAS1, AT3, GO, CC5, the X gene of HBV, the S gene of HBV, AAT, and LDH.

[0144] One embodiment is a pharmaceutical formulation of the present invention, or a pharmaceutical formulation for use in the present invention for the treatment or prevention of cancer, infectious diseases, viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, AAT-associated liver disease, acute hepatic porphyria, TTR-mediated amyloidosis, hereditary TTR amyloidosis (hATTR), complement-mediated diseases, hepatitis B infection, diseases or disorders related to HSP27 expression, or autoimmune diseases.

[0145] One embodiment is a pharmaceutical formulation for use according to the present invention, wherein the pharmaceutical formulation is • Contains saponin derivatives, where the saponin derivative corresponds to the saponin represented by molecule 1, and the following derivatizations: i. C of chiral acid or gypsogenin 23 The aldehyde group at position - Reduction to alcohol; or - Conversion to a hydrazone bond by reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), thereby yielding saponin-Ald-EMCH such as SO1861-Ald-EMCH or QS-21-Ald-EMCH, and the maleimide group of EMCH is optionally derivatized by forming a thioether bond with mercaptoethanol; or - Conversion to a hydrazone bond by reaction with N-[β-maleimidopropionic acid]hydrazide (BMPH), wherein the maleimide group of BMPH is derivatized by optionally forming a thioether bond with mercaptoethanol; or - The derivatization is by conversion to a hydrazone bond via reaction with N-[κ-maleimidoundecanoic acid]hydrazide (KMUH), where the maleimide group of KMUH is derivatized by optionally forming a thioether bond with mercaptoethanol; ii. If A1 represents a sugar chain selected from group A, and A1 contains or consists of a glucuronic acid moiety, the carboxyl group of the glucuronic acid moiety of A1 is derivatized by conversion to an amide bond through reaction with 2-amino-2-methyl-1,3-propanediol (AMPD) or N-(2-aminoethyl)maleimide (AEM), thereby yielding saponin-Glu-AMPD such as QS-21-Glu-AMPD or SO1861-Glu-AMPD, or saponin-Glu-AEM such as QS-21-Glu-AEM or SO1861-Glu-AEM; iii. If A2 represents a sugar chain selected from group B, and A2 contains at least one acetoxy group, then at least one of the following exists: one or more sugar moieties of A2, or all of them, are derivatized by conversion to hydroxyl groups (HO-) by deacetylation; or • A saponin represented by molecule 1, wherein A1 is Gal-(1→2)-[Xyl-(1→3)]-GlcA and / or A2 is Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc, and preferably the saponin represented by molecule 1 is 3-O-β-D-galactopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]-β-D-glucuronopyranosyl The chiric acid is 28-O-β-D-glucopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)-4OAc-β-D-quinopopyranosyl-(1→4)]-β-D-fucopyranoside, more preferably SO1861, GE1741, SA1641 and / or QS-21 or derivatives thereof, most preferably SO1861 or derivatives thereof; or • Saponin derivatives, i. Saponin derivatives - Reduction to alcohol; or - Conversion to a hydrazone bond by reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), thereby yielding saponin-Ald-EMCH such as SO1861-Ald-EMCH or QS-21-Ald-EMCH, and the maleimide group of EMCH is optionally derivatized by forming a thioether bond with mercaptoethanol; or - Conversion to a hydrazone bond by reaction with N-[β-maleimidopropionic acid]hydrazide (BMPH), wherein the maleimide group of BMPH is derivatized by optionally forming a thioether bond with mercaptoethanol; or - Conversion to a hydrazone bond by reaction with N-[κ-maleimidoundecanoic acid]hydrazide (KMUH), wherein the maleimide group of KMUH is derivatized by optionally forming a thioether bond with mercaptoethanol. It contains an aglycone core structure composed of aldehyde groups that have been derivatized by; ii. The saponin derivative comprises a sugar chain, preferably a sugar chain selected from group A, wherein the sugar chain comprises a carboxyl group of a glucuronic acid moiety that has been derivatized by conversion to an amide bond through reaction with 2-amino-2-methyl-1,3-propanediol (AMPD) or N-(2-aminoethyl)maleimide (AEM), thereby yielding saponin-Glu-AMPD such as QS-21-Glu-AMPD or SO1861-Glu-AMPD, or saponin-Glu-AEM such as QS-21-Glu-AEM or SO1861-Glu-AEM; iii. The saponin derivative contains a sugar chain preferably selected from group B, which includes an acetoxy (Me(CO)O-) group that has been derivatized by conversion to a hydroxyl group (HO-) by deacetylation; or iv. The saponin derivative comprises any combination of derivatizations i., ii., and iii., or any combination of any two derivatizations from derivatizations i., ii., and iii. by any choice; Preferably, the saponin derivative comprises an aglycone core structure composed of an aldehyde group that has been derivatized by conversion to a hydrazone bond through reaction with EMCH, and the maleimide group of EMCH is optionally derivatized by forming a thioether bond with mercaptoethanol; or • Saponin derivatives, i. The saponin derivative contains an aglycone core structure composed of an aldehyde group that has been derivatized by conversion to a hydrazone bond through reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), thereby yielding saponin-Ald-EMCH such as SO1861-Ald-EMCH or QS-21-Ald-EMCH; ii. The saponin comprises a sugar chain, preferably a sugar chain selected from group A, wherein the sugar chain comprises a carboxyl group of a glucuronic acid moiety that has been derivatized by conversion to an amide bond through reaction with N-(2-aminoethyl)maleimide (AEM), thereby yielding a saponin-Glu-AEM such as QS-21-Glu-AEM or SO1861-Glu-AEM; or iii. The saponin derivative includes a combination of derivatization i. and ii.; or • The saponin derivative comprises an aglycone core structure composed of an aldehyde group, and the saponin derivative comprises a sugar chain, preferably a sugar chain selected from group A, wherein this sugar chain comprises a carboxyl group, preferably a carboxyl group of a glucuronic acid moiety that has been derivatized by conversion to an amide bond through reaction with N-(2-aminoethyl)maleimide (AEM); or • Saponin derivatives, molecule 2:

[0146] [ka]

[0147] It is expressed as, Alternatively, a saponin derivative may be molecular 3:

[0148] [ka]

[0149] It is represented as follows.

[0150] A fourth aspect of the present invention is an in vitro or ex vivo method for transferring an effector molecule, comprising a conjugate containing a ligand for ASGPR according to the present invention, from outside a cell into a cell, preferably into the cytosol of the cell, a) A step of providing cells, preferably selected from liver cells, virus-infected cells, and tumor cells, that express ASGPR, preferably ASGPR1, on their surface; b) A step of providing the conjugate of the present invention containing the effector molecule to be transferred; c) A step of providing a saponin according to the present invention; d) A step of contacting the cells of step a) with the conjugate of step b) and the saponin of step c) in vitro or ex vivo, thereby causing the transfer of the conjugate containing the effector molecule from outside the cell into the cell, and by causing the transfer of the conjugate, causing the transfer of the effector molecule from outside the cell into the cell, preferably into the cytosol of the cell. Regarding methods including

[0151] A fifth aspect of the present invention is an in vitro or ex vivo method for transferring the conjugate of the present invention from outside a cell into said cell, a) A step of providing cells, preferably selected from liver cells, virus-infected cells, and tumor cells, that express ASGPR, preferably ASGPR1, on their surface; b) A step of providing the conjugate of the present invention; c) A step of providing the saponin of the present invention; d) A step of contacting the cells of step a) with the conjugate of step b) and the saponin of step c) in vitro or ex vivo, thereby causing the conjugate to move from outside the cell to inside the cell. Regarding methods including

[0152] One embodiment is one of two methods of the present invention, wherein the ligand for ASGPR comprises at least one N-acetylgalactosamine (GalNAc) moiety, preferably three or four GalNAc moieties, more preferably (GalNAc)3-tris, and / or the effector molecule is an oligonucleotide selected from anti-miRNA, BNA-AON, or siRNA, for example, preferably chemically modified siRNA, metabolically stable siRNA, and BNA-based siRNA selected from chemically modified and metabolically stable siRNA.

[0153] One embodiment is one of the two methods of the present invention, wherein the effector molecule comprises or consists of at least one of the following: a small molecule such as a drug molecule, a toxin such as a protein toxin, an oligonucleotide such as BNA, heterologous nucleic acid or siRNA, an enzyme, a peptide, a protein, or any combination thereof, wherein the effector molecule is preferably a toxin, an enzyme, or an oligonucleotide, and preferably the toxin is a saporin or diansin.

[0154] One embodiment is one of two methods of the present invention, wherein the saponin is derivatized SO1861 and / or derivatized QS-21, preferably SO1861-Glu-AEM or SO1861-Ald-EMCH or QS-21-Glu-AEM or QS-21-Ald-EMCH according to the above embodiments of the present invention.

[0155] One embodiment is one of two methods of the present invention, wherein the saponin is a saponin derivative corresponding to the saponin represented by molecule 1, and the derivatization is as follows: i. C of chiral acid or gypsogenin 23 The aldehyde group at position - Reduction to alcohol; or - Conversion to a hydrazone bond by reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), thereby yielding saponin-Ald-EMCH such as SO1861-Ald-EMCH or QS-21-Ald-EMCH, and the maleimide group of EMCH is optionally derivatized by forming a thioether bond with mercaptoethanol; or - Conversion to a hydrazone bond by reaction with N-[β-maleimidopropionic acid]hydrazide (BMPH), wherein the maleimide group of BMPH is derivatized by optionally forming a thioether bond with mercaptoethanol; or - The derivatization is by conversion to a hydrazone bond via reaction with N-[κ-maleimidoundecanoic acid]hydrazide (KMUH), where the maleimide group of KMUH is derivatized by optionally forming a thioether bond with mercaptoethanol; ii. If A1 represents a sugar chain selected from group A, and A1 contains or consists of a glucuronic acid moiety, the carboxyl group of the glucuronic acid moiety of A1 is derivatized by conversion to an amide bond through reaction with 2-amino-2-methyl-1,3-propanediol (AMPD) or N-(2-aminoethyl)maleimide (AEM), thereby yielding saponin-Glu-AMPD such as QS-21-Glu-AMPD or SO1861-Glu-AMPD, or saponin-Glu-AEM such as QS-21-Glu-AEM or SO1861-Glu-AEM; iii. If A2 represents a sugar chain selected from group B, and A2 contains at least one acetoxy group, then at least one of the following exists: one or more sugar moieties of A2, preferably all of them, are derivatized by conversion to a hydroxyl group (HO-) by deacetylation.

[0156] One embodiment is one of two methods of the present invention, in which, if the effector molecule is present, for example inside a mammalian cell, it can silence any one of the following genes: apolipoprotein B (ApoB), transthyretin (TTR), the proprotein convertase subtilisin / kexin type 9 (PCSK9), δ-aminolevulinic acid synthase 1 (ALAS1), antithrombin 3 (AT3), glycolate oxidase (GO), complement component C5 (CC5), the X gene of hepatitis B virus (HBV), the S gene of HBV, α1-antitrypsin (AAT), and lactate dehydrogenase (LDH), and This oligonucleotide can / or, for example, when present inside mammalian cells, target abnormal miRNAs, and / or, for example, when present inside mammalian cells, target mRNAs involved in the expression of any one of the following proteins: ApoB, TTR, PCSK9, ALAS1, AT3, GO, CC5, the expression product of the HBV X gene, the expression product of the HBV S gene, AAT, and LDH, or, for example, when present inside mammalian cells, weaken or restore miRNA function, such as inhibiting miRNA (onco-miR) or suppressing onco-miR expression.

[0157] A sixth aspect of the present invention relates to a kit of parts comprising the pharmaceutical formulation of the present invention or the second pharmaceutical composition of the present invention, and instructions for using the pharmaceutical formulation or the second pharmaceutical composition according to the present invention, or for using the method according to the present invention.

[0158] While the present invention has been described in terms of several embodiments, it is intended that substitutions, modifications, rearrangements, and equivalents thereto will become apparent to those skilled in the art upon reading the specification and examining the drawings. The present invention is not limited in any way to the exemplary embodiments. Modifications may be made without departing from the scope defined by the appended claims.

[0159] The present invention has been described above with reference to numerous exemplary embodiments. Modifications are possible and encompassed within the scope of protection as set forth in the appended claims. The present invention will be further described in the following embodiments, but should not be construed as limiting the invention in any way. [Examples]

[0160] Examples and exemplary embodiments Example 1. CD71-saporin + 4000 nM trivalent GalNAc-L-SO1861 Trivalent GalNAc is a target ligand that recognizes and binds to the ASGPR1 receptor on hepatocytes. Trivalent GalNAc was generated (Figures 1 and 8), and SO1861-EMCH was conjugated with trivalent GalNAc at a DAR=1 with respect to the bound SO1861 (unstable, underivativeized SO1861 was conjugated in the same manner as described in Figures 2 and 4, where SPT001 is synonymous with SO1861) (also referred to as trivalent GalNAc-SO1861 or (GalNAc)3-SO1861). SO1861-EMCH refers to SO1861, and is derivatized by the reaction of the aldehyde group with N-ε-maleimidocaproic acid hydrazide (EMCH) to convert it into a hydrazone bond, thereby yielding SO1861-Ald-EMCH. As shown in Figures 1 and 8, "trivalent GalNAc" is a typical (GalNAc)3 tris conjugate suitable for binding to effector molecules or saponins. For control, SO1861-EMCH was also conjugated with monovalent GalNAc (Figure 3) at DAR=1 relative to the bound SO1861 (unstable) (GalNAc-SO1861). HepG2(ASGPR1) + ;CD71 + Table A3) Cells and Huh7 (ASGPR1 + / - ;CD71 +(Table A3) Cells were treated with trivalent GalNAc-L-SO1861 and GalNAc-L-SO1861 in the presence and absence of 10 pM CD71-saporin (a monoclonal antibody OKT-9 that targets CD71 conjugated to a protein toxin saporin) within the concentration range of trivalent GalNAc-L-SO1861 and GalNAc-L-SO1861. When cells were treated in the absence of CD71-saporin, trivalent GalNAc-L-SO1861 (or trivalent GalNAc, also referred to as (GalNAc)3) as a single compound was shown to be nontoxic at a maximum of 15,000 nM (Figure 9).

[0161] Furthermore, HepG2 and Huh7 cells were treated with CD71-saporin (also known as CD71-SPRN) concentrations in combination with fixed concentrations of 1000 nM or 4000 nM trivalent GalNAc-SO1861 (DAR=1 relative to bound SO1861) (also referred to as (GalNAc)3-SPT). Target protein toxin-mediated cell death in ASGPR1 / CD71-expressing cells (HepG2 and Huh7) was measured. This revealed that low concentrations of CD71-saporin induced potent cell death in both cell lines (IC50 = 1 pM), while equivalent concentrations of CD71-saporin, CD71-saporin + 1000 nM trivalent GalNAc, or CD71-saporin + 4000 nM trivalent GalNAc could only induce cell death in both cell lines at high concentrations of CD71-saporin (IC50 = 100 pM) (Figure 10).

[0162] All of this indicates that trivalent GalNAc-SO1861 combined with low concentrations of CD71-saporin effectively induces cell death in ASGPR1 / CD71-expressing cells. Therefore, trivalent GalNAc-SO1861 effectively induces endosomal extrusion of protein toxins from ASGPR1-expressing cells.

[0163] Example 2: Trivalent GalNAc-L / S-BNA+SO1861-EMCH HSP27BNA was conjugated to trivalent GalNAc (Figures 6-7), and HepG2(ASGPR1 + ) cells or Huh7(ASPGR1+ / - The cells were treated with a fixed concentration of 4000 nM SO1861-EMCH (also known as SPT-EMCH) within the concentration range of trivalent GalNAc-L-HSP27BNA (unstable conjugation, Figure 6) or trivalent GalNAc-S-HSP27BNA (stable conjugation, Figure 7). Gene silencing of HSP27 in HepG2 and Huh7 cells was measured. Only in combination with 4000 nM SO1861-EMCH were low concentrations of trivalent GalNAc-L-HSP27BNA (HepG2:IC50=0,1nM; Huh7:IC50=3nM) or trivalent GalNAc-S-HSP27BNA (HepG2:IC50=0,1nM; Huh7:IC50=3nM) observed effective gene silencing in both HEPG2 and Huh7 cells. In contrast, equivalent concentrations of trivalent GalNAc-L-HSP27BNA (HepG2 / Huh7:IC50>2000nM) or trivalent GalNAc-S-HSP27BNA (HepG2 / Huh7:IC50>2000nM) did not show gene silencing activity in HepG2 and Huh7 cell lines (Figure 11).

[0164] Next, apoBBNA was conjugated to trivalent GalNAc using a similar method (Figures 6-7), and HepG2(ASGPR1 + ) cells or Huh7(ASPGR1 + / - ) is combined with a fixed concentration of 4000 nM SO1861-EMCH to produce trivalent GalNAc-L-apoBBNA (unstable conjugation, Figures 6, 12, 13) or trivalent GalNAc-S-apoBBNA (stable conjugation, Figures 7, 14, 15), or trivalent GalNAc-L-apoB scrambled BNA or trivalent GalNAc-S-ApoB scrambled Treatment was performed within the concentration range of BNA (in this case, scrambled apoBBNA is an antisense BNA with scrambled oligo sequences that does not bind to apoBm RNA, i.e., a non-specific control BNA). HepG2 (ASGPR1 + ) cells or Huh7(ASPGR1 + / -Gene silencing of apoB was measured in ). Effective gene silencing was observed in HepG2 cells with low concentrations of trivalent GalNAc-L-apoBBNA (IC50=10nM) or trivalent GalNAc-S-apoBBNA (IC50=10nM) only in combination with 4000nM SO1861-EMCH, whereas equivalent concentrations of trivalent GalNAc-L-apoBBNA (IC50>2000nM) or trivalent GalNAc-S-apoBBNA (IC50>2000nM) did not show gene silencing activity in HepG2 cells (Figures 12 and 14). The treatment did not affect cell line viability (Figures 13 and 15). "L" refers to an "unstable" linker, indicating that the linker is cleaved at a pH similar to that found inside cells, i.e., in endosomes and lysosomes. In contrast, "S" indicates a "stable linker," meaning the linker is not cleaved at the pH present in cells, i.e., endosomes and lysosomes. Therefore, conjugates that form a conjugate, or conjugates containing an unstable bond between any two molecules composed of a conjugate, are cleaved at the location within the unstable linker, thereby separating the two linked or bonded molecules. One example is the cleavage of hydrazone bonds under acidic conditions, which is prominent in endosomes and lysosomes of mammalian cells such as human cells.

[0165] All of this demonstrates that SO1861-EMCH can promote endosomal extrusion of trivalent GalNAc-L-BNA or trivalent GalNAc-S-BNA and target gene silencing for two independent gene targets. Gene silencing was more efficient in HepG2 cells compared to Huh7 cells, suggesting that increased uptake of GalNAc-BNA conjugates is promoted by the higher expression of ASGPR1 on the cell plasma membrane.

[0166] Example 3: Trivalent GalNAc-L / S-BNA + Trivalent GalNAc-L-SO1861 SO1861-EMCH was conjugated with trivalent GalNAc at DAR = 1 for the conjugated SO1861 (conjugated in the same manner as described in Figures 2 and 4 for the labile, un-derivatized SO1861) (trivalent GalNAc-SO1861). HepG2 (ASGPR1 + ) cells or Huh7 (ASPGR1 + / - ) cells were treated in combination with a fixed concentration of 4000 nM trivalent GalNAc-L-SO1861 (DAR = 1 for the conjugated SO1861) over a concentration range of trivalent GalNAc-L-apoBBNA (labile conjugation, Figure 16), trivalent GalNAc-S-apoBBNA (stable conjugation (Figure 17), or scrambled non-specific control conjugates (trivalent GalNAc-L-apoB scrambled BNA or trivalent GalNAc-S-apoB scrambled BNA). Gene silencing of apoB in HepG2 (ASGPR1 + ) cells and Huh7 (ASPGR1 + / - ) was measured. Effective gene silencing was observed in HepG2 cells (ASGPR1 + ) only in combination with 4000 nM trivalent GalNAc-L-SO1861 at low concentrations of trivalent GalNAc-L-apoBBNA (IC50 = 50 nM) or trivalent GalNAc-S-apoBBNA (IC50 = 50 nM). In Huh7 cells (ASGPR1 + / - ), gene silencing was poorly effective for any combination treatment (trivalent GalNAc-L-apoBBNA: IC50 = 700 nM; trivalent GalNAc-S-apoBBNA: IC50 = 700 nM). Equivalent concentrations of trivalent GalNAc-L-apoBBNA (HepG2 / Huh7: IC50 > 1000 nM) or trivalent GalNAc-S-apoBBNA (HepG2 / Huh7: IC50 > 1000 nM) showed no gene silencing activity in HepG2 and Huh7 cell lines, nor did 4000 nM trivalent GalNAc-L-SO1861, nor did scrambled trivalent GalNAc-L-apoB scrambled BNA (HepG2 / Huh7: IC50 > 1000 nM) or scrambled trivalent GalNAc-S-apoBscrambled It did not induce / promote gene silencing of BNA (HepG2 / Huh7: IC50>1000 nM) (Figure 16). The treatment did not affect the viability of both cell lines (Figure 17).

[0167] All of this indicates that very low concentrations of trivalent GalNAc-L-HSP27 BNA or trivalent GalNAc-S-HSP27 BNA in combination with trivalent GalNAc-L-SO1861 effectively induced gene silencing in ASGPR1-expressing cells, and again, a stronger effect was seen in cells with higher ASGPR1 expression.

[0168] Example 4 (GalNAc)3-BNA+SO1861-EMCH in primary human hepatocytes ApoB BNA (ApoB BNA #01, the mRNA of human apoB as the target) was conjugated to trivalent GalNAc ((GalNAc)3) (Figure 6), and primary hepatocytes (ASGPR1 + ) cells were treated in the concentration range of (GalNAc)3-ApoB BNA #01 with and without the addition of 2000 nM SO1861. Gene silencing of apoB in primary hepatocytes (ASGPR1 + ) cells was measured. Effective gene silencing was observed at a low concentration of (GalNAc)3-ApoB BNA #01 (IC50 = 8 nM) in primary hepatocytes only in the combination with 2000 nM SO1861-EMCH, while it was revealed that (GalNAc)3-ApoB BNA #01 alone had an IC50>1000 nM (Figure 18A). Next, the effect of these treatments on cell viability was measured, which revealed that (GalNAc)3-ApoB BNA #01 had an IC50>10000 nM and (GalNAc)3-ApoB BNA+SO1861-EMCH had an IC50 of approximately 500 nM (Figure 18B). All of this indicates that SO1861-EMCH can strongly promote the (GalNAc)3-targeted delivery of oligonucleotide payloads in primary human hepatocytes.

[0169] Example 5: (GalNAc)3-BNA+SO1861-EMCH in human primary hepatocytes ApoB sequence BNA (targeting both mouse and human apoB RNA), more specifically, apoB#02 BNA NC When apoB#02, apoBBNAB#02, or thiol-13-apoBBNA was conjugated to the bound apoB#02 with a linker ("Ls") at DAR=1, (GalNAc)3-apoB#02 appeared (Figures 21-23).

[0170] Human primary hepatocytes (ASGPR1 + ) were treated with (GalNAc)3-apoB#02 BNA concentration range with and without 2000 nM SO1861-EMCH. Primary hepatocytes (ASGPR1 + Gene silencing of ApoB was measured in the following conditions. Effective gene silencing was observed only with a very low concentration of (GalNAc)3-ApoBBNA#02 (IC50<0.01nM) in the 2000nM SO1861-EMCH combination, while the efficacy was inferior with equivalent concentrations of ApoBBNA#02 (IC50=200nM), (GalNAc)3-ApoBBNA#02 (IC50=5nM), or ApoBBNA#02 RNAiMAX transfection (IC50=5nM) (Figure 19A). The dotted line for ApoBBNA#02 RNAiMAX indicates the concentration at which toxicity and cell death occur. Next, we measured the effects of these treatments on cell viability, revealing that apoBBNA#02 had an IC50 > 10000, (GalNAc)3-apoBBNA#02 had an IC50 > 10000 nM, (GalNAc)3-apoBBNA#02 + SO1861-EMCH had an IC50 = 5000 nM, and apoBBNA#02 + RNAiMAX had an IC50 = 100 nM (Figure 19B). All of this indicates that SO1861-EMCH can potently promote the targeted delivery of oligonucleotide payloads to (GalNAc)3 in human primary hepatocytes.

[0171] Example 6: (GalNAc)3-BNA+SO1861-EMCH in primary mouse hepatocytes Mouse apoB sequence-targeted BNA, more specifically apoB#02 BNA NC When apoB#02, apoBBNA#02, or thiol-13-apoB BNA was conjugated to a linker ("Ls") with DAR=1 to the bound apoB#02, (GalNAc)3-apoB#02 appeared (Figures 21-23).

[0172] Primary mouse hepatocytes were treated with one of the following concentration ranges: apoB#02 BNA, (GalNAc)3-apoB#02, or (GalNAc)3-apoB#02 + 2000 nM SO1861-EMCH. ApoB RNA expression was incubated for 72 hours and then measured by qPCR. Gene expression inhibition was observed with low concentrations of (GalNAc)3-apoB#02 BNA combined with 2000 nM SO1861-EMCH (IC50 of approximately 0.03 nM) (Figure 20). When apoB#02 was treated alone, its efficacy in the repressive regulation of apoB RNA was approximately 100 to 667 times lower (apoB#02: IC50 = 20 nM), while the efficacy of (GalNAc)3-apoB#02, which contains apoB#02 with an IC50 of approximately 2 nM, was approximately 10 to 67 times lower (Figure 20).

[0173] This indicates that, only in combination with (GalNAc)3-SO1861 or SO1861-EMCH, very low concentrations of (GalNAc)3-apoB#02 BNA effectively induce repressive regulation of apoB RNA, i.e., gene silencing, in primary mouse hepatocytes.

[0174] Materials and methods Abbreviation AEM N-(2-aminoethyl)maleimidotrifluoroacetate AMPD 2-amino-2-methyl-1,3-propanediol BOP (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate DIPEA N,N-diisopropylethylamine DMF (N,N-dimethylformamide) EDCI.HCl 3-((ethylimino)methyleneamino)-N,N-dimethylpropane-1-aminium chloride EMCH.TFA N-(ε-maleimidocaproic acid)hydrazide, trifluoroacetate HATU 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate min NMM 4-methylmorpholine rt retention time TCEP Tris(2-carboxyethyl)phosphine hydrochloride Temp Temperature TFA (Trifluoroacetic Acid)

[0175] Analysis method LC-MS method 1 Apparatus: Waters IClass; Bin. Pump: UPIBSM, SM: UPISMFTN containing SO; UPCMA, PDA: UPPDATC, 210~320nm, SQD: ACQ-SQD2 ESI, Mass range depending on the molecular weight of the product: Neg or neg / pos within the range of 1500-2400 or 2000-3000; ELSD: Gas pressure 40 psi, drift tube temperature: 50°C; Column: Acquity C18, 50 × 2.1 mm, 1.7 μm, temperature: 60°C, flow rate: 0.6 mL / min, lin. Gradient according to the polarity of the product: A t0 = 2%A, t 5.0min =50%A, t 6.0min =98%A B t0 = 2%A, t 5.0min =98%A, t 6.0min =98%A Post-treatment time: 1.0 minute, Elutate A: Acetonitrile, Elutate B: 10 mM ammonium bicarbonate in water (pH=9.5).

[0176] LC-MS method 2 Apparatus: Waters IClass; Bin. Pump: UPIBSM, SM: UPISMFTN including SO; UPCMA, PDA: UPPDATC, 210~320nm, SQD: ACQ-SQD2 ESI, Mass range depending on product molecular weight: pos / neg 100~800 or neg 2000~3000; ELSD: Gas pressure 40psi, Drift tube temperature: 50℃; Column: Waters XSelect(trademark) CSH C18, 50×2.1mm, 2.5μm, Temperature: 25℃; Flow rate: 0.5mL / min, Gradient: t 0min =5%A, t 2.0min =98%A, t 2.7min =98%A, post-treatment time: 0.3 min, eluate A: acetonitrile, eluate B: 10 mM ammonium bicarbonate in water (pH=9.5).

[0177] LC-MS method 3 Apparatus: Waters IClass; Bin. Pump: UPIBSM, SM: UPISMFTN including SO; UPCMA, PDA: UPPDATC, 210~320nm, SQD: ACQ-SQD2 ESI, mass range pos / neg 10⁵~800, 500~1200 or 1500~2500 depending on the molecular weight of the product; ELSD: gas pressure 40 psi, drift tube temperature: 50℃; column: Waters XSelect(trademark) CSH C18, 50×2.1 mm, 2.5 μm, temperature: 40℃; flow rate: 0.5 mL / min, gradient: t 0min =5%A, t 2.0min =98%A, t 2.7min =98%A, post-treatment time: 0.3 minutes, eluate A: 0.1% formic acid in acetonitrile, eluate B: 0.1% formic acid in water.

[0178] LC-MS method 4 Apparatus: Waters IClass; Bin. Pump: UPIBSM, SM: UPISMFTN with SO; UPCMA, PDA: UPPDATC, 210~320nm, SQD: ACQ-SQD2 ESI, Mass range depending on product molecular weight: pos / neg 100~800 or neg 2000~3000; ELSD: Gas pressure 40psi, Drift tube temperature: 50℃ Column: Waters Acquity Shield RP18, 50×2.1mm, 1.7μm, Temperature: 25℃, Flow rate: 0.5mL / min, Gradient: t 0min =5%A, t 2.0min =98%A, t 2.7min =98%A, post-treatment time: 0.3 min, eluate A: acetonitrile, eluate B: 10 mM ammonium bicarbonate in water (pH=9.5).

[0179] Preparative separation method Preparative MP-LC method 1 Instrument model: Revelleris™ preparative MPLC; Column: Waters XSelect™ CSH C18 (145 × 25 mm, 10 μm); Flow rate: 40 mL / min; Column temperature: Room temperature; Elutate A: 10 mM ammonium bicarbonate in water pH=9.0; Elutate B: 99% acetonitrile + 1% 10 mM ammonium bicarbonate in water; Gradient: A t 0min =5%B, t 1min =5%B, t 2min =10%B, t 17min = 50%B, t 18min =100%B, t 23min =100%B A t 0min =5%B, t 1min =5%B, t 2min =20%B, t 17min =60%B, t 18min =100%B, t 23min =100%B Detection UV: 210, 235, 254 nm and ELSD.

[0180] Preparative MP-LC method 2 Machine type: Reveleris (trademark) fraction collector MPLC; Column: Phenomenex LUNA C18(3) (150×25mm, 10μm); Flow rate: 40 mL / min; Column temperature: Room temperature; Eluent A: 0.1% (v / v) formic acid in water, Eluent B: 0.1% (v / v) formic acid in acetonitrile; Gradient: A t 0min = 5%B, t 1min = 5%B, t 2min = 20%B, t 17min = 60%B, t 18min = 100%B, t 23min = 100%B B t 0min = 2%B, t 1min = 2%B, t 2min = 2%B, t 17min = 30%B, t 18min = 100%B, t 23min = 100%B C t 0min = 5%B, t 1min = 5%B, t 2min = 10%B, t 17min = 50%B, t 18min = 100%B, t 23min = 100%B D t 0min = 5%B, t 1min = 5%B, t 2min = 5%B, t 17min = 40%B, t 18min = 100%B, t 23min = 100%B ; Detection UV: 210, 235, 254 nm and ELSD.

[0181] Preparative LC-MS method 3 MS instrument model: Agilent Technologies G6130B Quadrupole; HPLC instrument model: Agilent Technologies 1290 Preparative LC; Column: Waters XSelect (trademark) CSH (C18, 150×19mm, 10μm); Flow rate: 25ml / min; Column temperature: Room temperature; Eluten A: 100% acetonitrile; Eluten B: 10mM ammonium bicarbonate in water, pH=9.0; Gradient: A t0 = 20% A, t 2.5min =20%A, t 11min =60%A, t 13min =100%A, t 17min =100%A B t0 = 5%A, t 2.5min =5%A, t 11min =40%A, t 13min =100%A, t 17min =100%A Detection: DAD (210nm); Detection: MSD (ESI pos / neg) Mass range: 100~800; Fractionation collection based on DAD.

[0182] Preparative LC-MS method 4 MS instrument model: Agilent Technologies G6130B Quadrupole; HPLC instrument model: Agilent Technologies 1290 Preparative LC; Column: Waters XBridge Protein (C4, 150×19mm, 10μm); Flow rate: 25ml / min; Column temperature: Room temperature; Elutate A: 100% acetonitrile; Elutate B: 10mM ammonium bicarbonate in water, pH=9.0; Gradient: A t0 = 2%A, t 2.5min =2%A, t 11min =30%A, t 13min =100%A, t 17min =100%A B t0 = 10% A, t 2.5min =10%A, t 11min =50%A, t 13min =100%A, t 17min =100%A C t0 = 5%A, t 2.5min =5%A, t 11min =40%A, t 13min =100%A, t 17min =100%A Detection: DAD (210nm); Detection: MSD (ESI pos / neg) Mass range: 100~800; Fractionation based on DAD

[0183] Flash chromatography Grace Reveleris X2(registered trademark) C-815 Flash; Solvent delivery system: Self-priming 3-piston pump, 4 independent channels containing up to 4 solvents in a single run, automatic switching line when solvent runs out; Maximum pump flow rate 250 mL / min; Maximum pressure 50 bar (725 psi); Detection: UV 200-400 nm, combination of up to 4 UV signals and scanning of the full UV range, ELSD; Column size: 4-330 g on the instrument, Luer type, 750 g to a maximum of 3000 g including optional holder.

[0184] Synthesis of SO1861-EMCH (SO1861-EMCH is also known as SO1861-Ald-EMCH) SO1861 (121 mg, 0.065 mmol) and EMCH.TFA (110 mg, 0.325 mmol) were mixed with methanol (extra dry, 3.00 mL) and TFA (0.020 mL, 0.260 mmol). The reaction mixture was stirred at room temperature. After 1.5 hours, the reaction mixture was subjected to preparative MP-LC. The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (120 mg, 90%) as a white, cottony solid. Purity was 96% based on LC-MS. LRMS(m / z):2069[M-1] 1- LC-MSr.t.(min):1.08 4

[0185] SO1861-EMCH-mercaptoethanol (SO1861-EMCH (blocked)) SO1861-EMCH (0.1 mg, 48 nmol) was mixed with 200 μL of mercaptoethanol (18 mg, 230 μmol), and the solution was shaken at 800 rpm for 1 hour at room temperature on a ThermoMixer C (Eppendorf). After shaking for 1 hour, the solution was diluted with methanol and dialyzed broadly against methanol for 4 hours using a regenerated cellulose membrane tube (Spectra / Por 7) containing 1 kDa MWCO. After dialyzing, aliquots were taken and analyzed by MALDI-TOF-MS. MALDI-TOF-MS (RP mode): m / z 2193Da ([M+K] + (SO1861-EMCH-mercaptoethanol), m / z 2185Da ([M+K] + (SO1861-EMCH-mercaptoethanol), m / z 2170Da ([M+Na] + (SO1861-EMCH-mercaptoethanol).

[0186] Synthesis of trivalent galNAc azide Intermediate 1: tert-butyl1-azide-17,17-bis((3-(tert-butoxy)-3-oxopropoxy)methyl)-15-oxo-3,6,9,12,19-pentaoxa-16-azadocosan-22-oate Di-tert-butyl 3,3'-((2-amino-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionate (1.27 g, 2.51 mmol) was mixed with a solution of 3-azido(PEG4)propionic acid N-hydroxysuccinimide (977 mg, 2.51 mmol) in DMF (10 mL). Next, DIPEA (657 μL, 3.77 mmol) was added, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was evaporated under vacuum, and the residue was dissolved in ethyl acetate (100 mL). The resulting solution was washed with 0.5 N potassium bisulfate solution (2 × 100 mL) and brine (100 mL), dried over Na₂SO₄, filtered, and evaporated under vacuum. The residue was purified by flash chromatography (DCM-DCM with a 10% methanol (v / v) gradient increasing from 100:0 to 0:100), and the title compound (1.27 g, 65%) was obtained as a colorless oil. Purity was 100% (ELSD) based on LC-MS. LRMS (m / z): 780 [M+1] 1+ LC-MS rt(min):2.10 2

[0187] Intermediate 2: 1-Azido-17,17-bis((2-carboxyethoxy)methyl)-15-oxo-3,6,9,12,19-pentaoxa-16-azadocosan-22-euic acid To a solution of tert-butyl 1-azide-17,17-bis((3-(tert-butoxy)-3-oxopropoxy)methyl)-15-oxo-3,6,9,12,19-pentaoxa-16-azadocosan-22-oate (1.27 g, 1.63 mmol) in DCM (5.0 mL), TFA (5.0 mL, 65 mmol) was added. The reaction mixture was stirred at room temperature. After 1.5 hours, the reaction mixture was evaporated under vacuum and then simultaneously evaporated with toluene (3 × 10 mL) and DCM (3 × 10 mL) to obtain the crude title product as a colorless oil. LRMS (m / z): 611 [M+1] 1+

[0188] Intermediate 3: Di-tert-butyl(10-(1-azido-3,6,9,12-tetraoxapentadecane-15-amide)-10-(13,13-dimethyl-5,11-dioxo-2,12-dioxa-6,10-diazatetradecyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecane-1,19-diyl)dicarbamate 1-Azido-17,17-bis((2-carboxyethoxy)methyl)-15-oxo-3,6,9,12,19-pentaoxa-16-azadocosan-22-euic acid (997 mg, 1.63 mmol), Oxyma Pure (1.04 g, 7.35 mmol), and EDCI.HCl (1.17 g, 6.12 mmol) were dissolved in DMF (10.0 mL). Next, DIPEA (1.99 mL, 11.4 mmol) was added, and then the mixture was added directly to a solution of N-BOC-1,3-propanediamine (1.07 g, 6.12 mmol) in DMF (10.0 mL). The reaction mixture was stirred overnight at room temperature. The reaction mixture was evaporated under vacuum, and the residue was dissolved in ethyl acetate (100 mL). The obtained solution was washed with 0.5N potassium bisulfate solution (100 mL), saturated sodium bicarbonate solution (2 × 100 mL), and brine (100 mL), dried over Na₂SO₄, filtered, and evaporated under vacuum. The residue was purified by flash chromatography (DCM-DCM with a 10% methanol (v / v) gradient increasing from 0:100 to 100:0, and maintained at 100:0 until the product eluted), and the title compound (1.16 g, 66%) was obtained as a yellowish viscous oil. LC-MS 99% (ELSD). LRMS (m / z): 1080 [M+1] 1+ LC-MS rt(min):1.51 3

[0189] Intermediate 4: Synthesis of 3,3'-((2-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-2-(1-azido-3,6,9,12-tetraoxapentadecane-15-amide)propane-1,3-diyl)bis(oxy))bis(N-(3-aminopropyl)propanamide)tris(2,2,2-trifluoroacetate) Di-tert-butyl(10-(1-azido-3,6,9,12-tetraoxapentadecane-15-amide)-10-(13,13-dimethyl-5,11-dioxo-2,12-dioxa-6,10-diazatetradecyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecane-1,19-diyl) dicarbamate (1.16 g, 1.08 mmol) was dissolved in DCM (10 mL) and TFA (10 mL, 131 mmol) was added. The reaction mixture was stirred at room temperature. After 2 hours, the reaction mixture was evaporated under vacuum and simultaneously evaporated with toluene (3 × 10 mL) and DCM (3 × 10 mL) to obtain the crude title product as a yellowish viscous oil. LRMS (m / z): 260 [M+3] 3+ , 390[M+2] 2+ , 780[M+1] 1+ ,

[0190] Intermediate 5: (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2,5-dioxopyrrolidine-1-yl)oxy)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyldiacetate 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid (3.00 g, 6.70 mmol, obtained according to J.Am.Chem Soc., 2014, 136, 16958-16961) and N-hydroxysuccinimide (926 mg, 8.05 mmol) were dissolved in DCM (50 mL). Next, EDCI.HCl (1.54 g, 8.05 mmol) and 4-(dimethylamino)pyridine (82 mg, 0.67 mmol) were added, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with DCM, and the resulting solution was washed with 0.5N potassium bisulfate solution (150 mL), saturated sodium bicarbonate solution (150 mL), and brine (150 mL). The mixture was dried over Na2SO4, filtered, and evaporated under vacuum to obtain the title compound (3.60 g, 99%) as a white foam. Purity was 99% (ELSD) based on LC-MS. LRMS (m / z): 545 [M+1] 1+ LC-MS rt(min):1.07 3

[0191] Intermediate 6: [(3R,6R)-3,4-bis(acetyloxy)-6-{4-[(3-{3-[2-(1-azido-3,6,9,12-tetraoxapentadecane-15-amide)-3-(2-{[3-(5-{[(2R,5R)-4,5-bis(acetyloxy)-6-[(acetyloxy)methyl]-3-acetamidooxan-2-yl]oxy}pentanamide)propyl]carbamoyl}ethoxy)-2-[(2-{[3-(5-{[(2R,5R)-4,5-bis(acetyloxy)-6-[(acetyloxy)methyl]-3-acetamidooxan-2-yl]oxy}pentanamide)propyl]carbamoyl}ethoxy)methyl]propoxy]propanamide}propyl)carbamoyl]butoxy}-5-acetamidooxan-2-yl]methylacetate 3,3'-((2-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-2-(1-azido-3,6,9,12-tetraoxapentadecane-15-amide)propane-1,3-diyl)bis(oxy))bis(N-(3-aminopropyl)propanamide)tris(2,2,2-trifluoroacetate) (1.21 g, 1.08 mmol) was dissolved in a mixture of DMF (10 mL) and DIPEA (1.69 mL, 9.70 mmol). Next, (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2,5-dioxopyrrolidine-1-yl)oxy)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyldiacetate (2.20 g, 4.04 mmol) was added, and the reaction mixture was stirred at room temperature over the weekend. The reaction mixture was then evaporated under vacuum, and the residue was purified by flash chromatography (DCM-DCM with a 30% methanol (v / v) gradient increasing from 0:100 to 100:0) to obtain the title compound (1.84 g, 83%) as a yellowish foam. LC-MS 95% (ELSD). LRMS(m / z):2068[M+1] 1+ LC-MS rt(min):1.18 3

[0192] Intermediate 7: Trivalent GalNAc-Azide [(3R,6R)-3,4-bis(acetyloxy)-6-{4-[(3-{3-[2-(1-azido-3,6,9,12-tetraoxapentadecane-15-amide)-3-(2-{[3-(5-{[(2R,5R)-4,5-bis(acetyloxy)-6-[(acetyloxy)methyl]-3-acetamidooxan-2-yl]oxy}pentanamide)propyl]carbamoyl}ethoxy)-2-[(2-{[3-(5-{[(2R,5R)-4,5-bis(acetyloxy)- 6-[(acetyloxy)methyl]-3-acetamidooxan-2-yl]oxy}pentanamide)propyl]carbamoyl}ethoxy)methyl]propoxy]propanamide}propyl)carbamoyl]butoxy}-5-acetamidooxan-2-yl]methyl acetate (300 mg, 0.145 mmol) was dissolved in a mixture of triethylamine (2.00 mL, 14.4 mmol), methanol (2.00 mL), and water (2.00 mL), and the reaction mixture was stirred at room temperature. After 2 hours, the reaction mixture was evaporated under vacuum. The residue was purified by preparative MP-LC. 2B The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (164 mg, 67%) as a white solid. Purity was 97% based on LC-MS. LRMS (m / z): 1688 [M-1] 1- LC-MS rt(min):1.99 1A

[0193] Synthesis of trivalent GalNAc-SO1861 and GalNAc-SO1861 (Figures 2 and 4) Intermediate 8: SO1861-NH2 SO1861-azide (6.89 mg, 3.20 μmol) was dissolved in a mixture of 32 mM potassium carbonate (150 μL, 4.80 μmol) and acetonitrile (150 μL). Immediately thereafter, a 1.0 M trimethylphosphine solution in THF (32 μL, 32 μmol) was added, and the resulting mixture was shaken for 1 minute and allowed to stand at room temperature. After 30 minutes, the reaction mixture was subjected to preparative MP-LC. The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (5.30 mg, 78%) as a white, cottony solid. Purity based on LC-MS was 94%. LRMS (m / z): 1062 [M-2] 2- LC-MS rt(min):2.51 1B

[0194] Intermediate 9: SO1861-DBCO SO1861-amine (48.6 mg, 22.9 μmol) and DBCO-NHS (13.0 mg, 32.4 μmol) were mixed with a solution of DIPEA (5.98 μL, 34.3 μmol) and DMF (2.0 mL). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 4 hours, the reaction mixture was diluted with a 50 mM sodium bicarbonate solution (1.00 mL, 50 μmol). The resulting mixture was shaken for 1 minute and allowed to stand at room temperature. After 30 minutes, the reaction mixture was subjected to preparative MP-LC. 1B The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (16.9 mg, 31%) as a white, cottony solid. Purity was 94% based on LC-MS. LRMS (m / z): 2412 [M-1] 1- LC-MS rt(min):2.45 1B

[0195] Synthesis of SO1861-L-trivalent GalNAc SO1861-DBCO (7.50 mg, 3.11 μmol) and trivalent GalNAc-azide (5.31 mg, 3.14 μmol) were dissolved in a mixture of water / acetonitrile (2:1, v / v, 0.90 mL). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative LC-MS. 3B The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (9.60 mg, 75%) as a white, cottony solid. Purity was 99% based on LC-MS. LRMS (m / z): 2050 [M-2] 2- LC-MS rt(min):2.04 1B

[0196] Synthesis of SO1861-L-GalNAc SO1861-DBCO (1.75 mg, 0.73 μmol) and GalNAc-PEG3-azide (0.82 mg, 2.18 μmol) were dissolved in a mixture of water / acetonitrile (1:1, v / v, 1.00 mL). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative LC-MS. 3B The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (1.63 mg, 81%) as a white, cottony solid. Purity was 100% based on LC-MS. LRMS (m / z): 2790 [M-1] 1- LC-MS rt(min):2.15 1B

[0197] Trivalent GalNAc-BNA oligo synthesis (Figures 6 and 7) Intermediate 10: 4-{2-Azatricyclo[10.4.0.0 4,9]Hexadeca-1(12),4(9),5,7,13,15-Hexaen-10-in-2-yl}-N-[2-(2-{2-[2-({4-[(E)-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide]imino}methyl]phenyl}formamide)ethoxy]ethoxy}ethoxy)ethyl]-4-oxobutanamide 4-{2-Azatricyclo[10.4.0.0 4,9 ]Hexadeca-1(12),4(9),5,7,13,15-Hexaen-10-in-2-yl}-N-{2-[2-(2-{2-[(4-formylphenyl)formamide]ethoxy}ethoxy)ethoxy]ethyl}-4-oxobutanamide (25.0 mg, 40.9 μmol) and EMCH.TFA (20.8 mg, 61.3 μmol) were dissolved in methanol (extra dry, 2.00 mL). Next, TFA (9.39 μL, 123 μmol) was added. The reaction mixture was shaken for 1 minute and allowed to stand overnight at room temperature. The reaction mixture was subjected to preparative MP-LC. 1B The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (16.2 mg, 48%) as a white solid. Purity was 91% based on LC-MS. LRMS (m / z): 410.2 [M+2] 2+ LC-MS rt(min):1.41 2

[0198] Intermediate 11: Trivalent GalNAc-L-maleimide Trivalent GalNAc-azide (20.3 mg, 12.0 μmol) and 4-{2-azatricyclo[10.4.0.0 4,9]Hexadeca-1(12),4(9),5,7,13,15-Hexaen-10-in-2-yl}-N-[2-(2-{2-[2-({4-[(E)-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide]imino}methyl]phenyl}formamide)ethoxy]ethoxy}ethoxy)ethyl]-4-oxobutanamide (11.8 mg, 14.4 μmol) was dissolved in a mixture of water / acetonitrile (2:1, v / v, 0.90 mL). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative MP-LC. 2C The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (25.6 mg, 85%) as a white solid. Purity was 88% based on LC-MS. LRMS(m / z):2101[M-405] 1- ,2304[M-202] 1- ,2507[M-1] 1- LC-MS rt(min):1.90 1B (Dual peaks due to isomers)

[0199] Intermediate 12: Trivalent GalNAc-S-maleimide Trivalent GalNAc-azide (20.3 mg, 12.0 μmol) and DBCO-maleimide (10.3 mg, 24.0 μmol) were dissolved in a mixture of water / acetonitrile (2:1, v / v, 0.90 mL). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative MP-LC. 2D The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (22.2 mg, 87%) as a white solid. Purity was 85% based on LC-MS. It contains 10% hydrolyzed maleimide. LRMS (m / z): 2115 [M-1] 1- LC-MS rt(min):1.60 1B (Dual peaks due to isomers)

[0200] Trivalent GalNAc-S-ApoB (or HSP27)BNA Oligo Synthesis A 20 mM ammonium bicarbonate solution containing 2.5 mM TCEP (1.00 mL, 2.5 μmol) was added to ApoB BNA oligodisulfide (5.00 mg, 0.686 μmol). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 1 hour, the reaction mixture was filtered using a centrifugal filter with a molecular weight cutoff of 3000 Da (5000 × g, 2 × 0.50 mL in 30 minutes). Next, the residue solution was washed twice with a 20 mM ammonium bicarbonate solution containing 2.5 mM TCEP (0.50 mL), and filtered each time under the same conditions as described above. Next, the residue solution was diluted with 20 mM ammonium bicarbonate / acetonitrile (3:1, v / v, 1.00 mL), and the resulting solution was added directly to trivalent GalNAc-S-maleimide (3.02 mg, 1.43 μmol). The reaction mixture was shaken for 1 minute and then allowed to stand at room temperature. After 1.5 hours, the reaction mixture was subjected to preparative LC-MS. 4A The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (6.75 mg, quantified) as a white, cottony solid. Purity was 94% based on LC-MS (very broad peak). LRMS (m / z): 2318 [M-4] 4- LC-MS rt(min):1.65 1A

[0201] Trivalent GalNAc-S-ApoB (or HSP27) Scrambled BNA Oligo Synthesis A 20 mM ammonium bicarbonate solution containing 2.5 mM TCEP (1.00 mL, 2.5 μmol) was added to ApoB scrambled BNA oligodisulfide (5.00 mg, 0.688 μmol). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 1 hour, the reaction mixture was filtered using a centrifugal filter with a molecular weight cutoff of 3000 Da (5000 × g, 2 × 0.50 mL in 30 minutes). Next, the residue solution was washed twice with a 20 mM ammonium bicarbonate solution containing 2.5 mM TCEP (0.50 mL), and filtered each time under the same conditions as described above. Next, the residue solution was diluted with 20 mM ammonium bicarbonate / acetonitrile (3:1, v / v, 1.00 mL), and the resulting solution was added directly to trivalent GalNAc-S-maleimide (3.09 mg, 1.46 μmol). The reaction mixture was shaken for 1 minute and then allowed to stand at room temperature. After 1.5 hours, the reaction mixture was subjected to preparative LC-MS. 4A The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (5.91 mg, 93%) as a white, cottony solid. Purity was 88% based on LC-MS (very broad peak). LRMS(m / z):2311[M-4] 4- LC-MS rt(min):1.60 1A

[0202] Trivalent GalNAc-L-apoB (or HSP27)BNA oligo synthesis A 20 mM ammonium bicarbonate solution containing 2.5 mM TCEP (1.00 mL, 2.5 μmol) was added to ApoB BNA oligodisulfide (5.00 mg, 0.686 μmol). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 1 hour, the reaction mixture was filtered using a centrifugal filter with a molecular weight cutoff of 3000 Da (5000 × g, 2 × 0.50 mL in 30 minutes). Next, the residue solution was washed twice with a 20 mM ammonium bicarbonate solution containing 2.5 mM TCEP (0.50 mL), and filtered each time under the same conditions as described above. Next, the residue solution was diluted with 20 mM ammonium bicarbonate / acetonitrile (3:1, v / v, 1.00 mL), and the resulting solution was added directly to trivalent GalNAc-L-maleimide (3.63 mg, 1.45 μmol). The reaction mixture was shaken for 1 minute and then allowed to stand at room temperature. After 1.5 hours, the reaction mixture was subjected to preparative LC-MS. 4A The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (6.68 mg, quantified) as a white, cottony solid. Purity was 99% based on LC-MS (very broad peak). LRMS (m / z): 2416 [M-4] 4- LC-MS rt(min):1.97 1A

[0203] Trivalent GalNAc-L-apoB (or HSP27) scrambled BNA oligo synthesis A 20 mM ammonium bicarbonate solution containing 2.5 mM TCEP (1.00 mL, 2.5 μmol) was added to ApoB scrambled BNA oligodisulfide (5.00 mg, 0.688 μmol). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 1 hour, the reaction mixture was filtered using a centrifugal filter with a molecular weight cutoff of 3000 Da (5000 × g, 2 × 0.50 mL in 30 minutes). Next, the residue solution was washed twice with a 20 mM ammonium bicarbonate solution containing 2.5 mM TCEP (0.50 mL), and filtered each time under the same conditions as described above. Next, the residue solution was diluted with 20 mM ammonium bicarbonate / acetonitrile (3:1, v / v, 1.00 mL), and the resulting solution was added directly to trivalent GalNAc-L-maleimide (3.68 mg, 1.47 μmol). The reaction mixture was shaken for 1 minute and then allowed to stand at room temperature. After 1.5 hours, the reaction mixture was subjected to preparative LC-MS. 4A The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (4.71 mg, 71%) as a white, cottony solid. Purity by LC-MS was 96% (very broad peak). LRMS (m / z): 2409 [M-4] 4- LC-MS rt(min):1.93 1A

[0204] Synthesis of dendron (-L-SO1861)4-trivalent GalNAc and dendron (-L-SO1861)8-trivalent GalNAc Intermediate 13: Trivalent GalNAc-amineformate Trivalent GalNAc azide (36.5 mg, 21.6 μmol) was dissolved in potassium carbonate (5.97 mg, 43.2 μmol) in water (1.00 mL) and acetonitrile (1.00 mL). Next, a 1.0 M trimethylphosphine solution in THF (216 μL, 216 μmol) was added, and the resulting mixture was shaken for 1 minute and allowed to stand at room temperature. After 45 minutes, the reaction mixture was evaporated under vacuum, and the residue was dissolved in water / acetonitrile (9:1, v / v, 1 mL). The resulting solution was then subjected to preparative MP-LC.2B The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (36.1 mg, 98%) as a white solid. Purity was 100% based on LC-MS. LRMS (m / z): 1662 [M-1] 1- LC-MS rt(min):1.62 1A

[0205] Intermediate 14: Trivalent GalNAc-DBCO Trivalent GalNAc-amine formate (17.4 mg, 10.2 μmol) and DBCO-NHS (6.14 mg, 15.3 μmol) were dissolved in a solution of NMM (2.24 μL, 20.3 μmol) in DMF (0.50 mL). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 2 hours, the reaction mixture was evaporated under vacuum, and the residue was dissolved in water / acetonitrile (8:2, v / v, 1 mL). The resulting solution was then subjected to preparative MP-LC. 2C The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (14.2 mg, 72%) as a white solid. Purity was 96% based on LC-MS. LRMS (m / z): 1950 [M-1] 1 LC-MS rt(min):1.86 1B

[0206] Intermediate 15: Dendron (-L-SO1861)8-azid Dendron (-L-SO1861)8-amine (19.6 mg, 1.08 μmol) and 2,5-dioxopyrrolidine-1-yl-1-azido-3,6,9,12-tetraoxapentadecane-15-oate (4.17 mg, 10.8 μmol) were dissolved in DMF (1.50 mL). Next, DIPEA (1.87 μL, 10.8 μmol) was added, the mixture was shaken for 1 minute, and left to stand overnight at room temperature. The reaction mixture was subjected to preparative LC-MS. 4BThe fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (11.7 mg, 59%) as a white, cottony solid. Purity was 92% based on LC-MS (very broad peak). LRMS (m / z): 2316 [M-8] 8- ):2647[M-7] 7- LC-MS rt(min):4.29 1A

[0207] Synthesis of Dendron (-L-SO1861) 4-trivalent GalNAc Dendron (-L-SO1861) 4-azide (2.50 mg, 0.266 μmol) and trivalent GalNAc-DBCO (1.56 mg, 0.799 μmol) were dissolved in a mixture of water / acetonitrile (3:1, v / v, 1.00 mL). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative LC-MS. 4B The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (2.74 mg, 91%) as a white, cottony solid. Purity by LC-MS was 86% (very broad peak). LRMS (m / z): 2832 [M-4] 4- LC-MS rt(min):4.07 1A

[0208] Synthesis of dendron (-L-SO1861)8-trivalent GalNAc Dendron (-L-SO1861)8-azide (2.50 mg, 0.135 μmol) and trivalent GalNAc-DBCO (0.79 mg, 0.405 μmol) were dissolved in a mixture of water / acetonitrile (3:1, v / v, 1.00 mL). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative LC-MS. 4B The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (2.03 mg, 74%) as a white, cottony solid. Purity was 100% based on LC-MS (very broad peak). LRMS (m / z): 2559 [M-8] 8- ,2925[M-7] 7- LC-MS rt(min):4.18 1A

[0209] Intermediate 16: Trivalent GalNAc-thioacetate Trivalent GalNAc-amine formate (18.7 mg, 11.0 μmol) and 4-nitrophenyl 3-(acetylthio)propanoate (5.90 mg, 21.9 μmol) were dissolved in a solution of NMM (2.41 μL, 21.9 μmol) in DMF (0.50 mL). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 2 hours, the reaction mixture was evaporated under vacuum, and the residue was dissolved in a mixture of 0.1% formic acid in water and 0.1% formic acid in acetonitrile (9:1, v / v, 1 mL). The resulting solution was then subjected to preparative MP-LC. 2B The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (13.0 mg, 66%) as a white solid. Purity was 100% based on LC-MS. LRMS(m / z):1749[M-43] 1- , 1792[M-1] 1 LC-MS rt(min):1.24 1B

[0210] Intermediate 17: DBCO-TCO-Trivalent GalNAc Trivalent GalNAc-thioacetate (13.0 mg, 7.25 μmol) was dissolved in methanol (0.50 mL). Next, a 1 M sodium hydroxide solution (7.98 μL, 7.98 μmol) was added. The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 30 minutes, a newly prepared trifunctional linker (Ls-t) (i.e., saponin partial linker L) was prepared. S The reaction mixture was added to a 20 mM ammonium bicarbonate / acetonitrile (3:1, v / v, 2.00 mL) solution (7.39 mg, 6.13 μmol) of the present invention (one example).

[0211] The IUPAC name for the trifunctional linker is 5,8,11,18,21,24,27-heptaoxa-2,14,30-triazatriacontanoic acid, 14-[16-(11,12-didehydrodibenz[b,f]azosin-5(6H)-yl)-13,16-dioxo-3,6,9-trioxa-12-azahexades-1-yl]-33-(2,5-dihydro-2,5-dioxo-1H-pyrrole-1-yl)-15,31-dioxo-,(1R,4E)-4-cycloocten-1-yl ester. The trifunctional linker is represented by the following formula (XVII):

[0212] [ka]

[0213] It holds.

[0214] The resulting mixture was shaken for 1 minute and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative MP-LC. 2A The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (3.83 mg, 21%) as a white solid. Purity was 89% based on LC-MS. LRMS(m / z):2409[M-546] 1- ,2955[M-1] 1- LC-MS rt(min):2.66 1B

[0215] Intermediate 18: Methyltetrazine-L-apoB BNA oligo A 20 mM ammonium bicarbonate solution containing 2.5 mM TCEP (1.00 mL, 2.5 μmol) was added to ApoB BNA oligodisulfide (5.00 mg, 0.686 μmol). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 1 hour, the reaction mixture was filtered using a centrifugal filter with a molecular weight cutoff of 3000 Da (5000 × g, 2 × 0.50 mL in 30 minutes). Next, the residue solution was washed twice with a 20 mM ammonium bicarbonate solution containing 2.5 mM TCEP (0.50 mL), and filtered each time under the same conditions as described above. Next, the residue solution was diluted with 20 mM ammonium bicarbonate (1.50 mL), and the resulting solution was added directly to a solution of (E)-1-(4-((2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl)hydrazineylidene)methyl)benzamide)-N-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)-3,6,9,12-tetraoxapentadecane-15-amide (1.36 mg, 1.73 μmol) in acetonitrile (0.5 mL). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 30 minutes, the reaction mixture was frozen and freeze-dried overnight to obtain the crude title product as a pink, cottony solid. A 20 mM ammonium bicarbonate (1.50 mL) solution was added to the crude product, and the resulting suspension was filtered through a 0.45 μm syringe filter. The filtrate was freeze-dried overnight to obtain the title product (5.44 mg, quantified) as a pink, cottony solid. Purity was 90% based on LC-MS (very broad peak). LRMS (m / z): 2648 [M-3] 3- LC-MS rt(min):0.62 4

[0216] Intermediate 19: Methyltetrazine-L-apoB scrambled BNA oligo A 20 mM ammonium bicarbonate solution containing 2.5 mM TCEP (1.00 mL, 2.5 μmol) was added to ApoB scrambled BNA oligodisulfide (5.00 mg, 0.688 μmol). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 1 hour, the reaction mixture was filtered using a centrifugal filter with a molecular weight cutoff of 3000 Da (5000 × g, 2 × 0.50 mL in 30 minutes). Next, the residue solution was washed twice with a 20 mM ammonium bicarbonate solution containing 2.5 mM TCEP (0.50 mL), and filtered each time under the same conditions as described above. Next, the residue solution was diluted with 20 mM ammonium bicarbonate (1.50 mL), and the resulting solution was added directly to a solution of (E)-1-(4-((2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl)hydrazineylidene)methyl)benzamide)-N-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)-3,6,9,12-tetraoxapentadecane-15-amide (1.34 mg, 1.70 μmol) in acetonitrile (0.5 mL). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 30 minutes, the reaction mixture was frozen and freeze-dried overnight to obtain the crude title product as a pink, cottony solid. A 20 mM ammonium bicarbonate (1.50 mL) solution was added to the crude product, and the resulting suspension was filtered through a 0.45 μm syringe filter. The filtrate was freeze-dried overnight to obtain the title product (5.48 mg, quantified) as a pink, cottony solid. Purity was 84% ​​(very broad peak) based on LC-MS. LRMS (m / z): 2639 [M-3] 3- LC-MS rt(min):0.58 4

[0217] Intermediate 20: DBCO-L-ApoB BNA Oligo-Trivalent GalNAc Methyltetrazine-L-apoB BNA oligo (5.44 mg, 0.684 μmol) and DBCO-TCO-trivalent GalNAc (2.03 mg, 0.687 μmol) were added to 20 mM ammonium bicarbonate / acetonitrile (3:1, v / v, 1.00 mL). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative LC-MS. 4C The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (2.87 mg, 39%) as a white, cottony solid. LC-MS showed multiple broad peaks with accurate m / z values. LRMS (m / z): 2175 [M-5] 5- , 2718[M-6] 4- LC-MS rt(min): 2.60~3.00 1A

[0218] Intermediate 21: DBCO-L-ApoB Scrambled BNA Oligo-Trivalent GalNAc Methyltetrazine-L-apoB scrambled BNA oligo (5.48 mg, 0.692 μmol) and DBCO-TCO-trivalent GalNAc (1.80 mg, 0.609 μmol) were mixed with 20 mM ammonium bicarbonate / acetonitrile (3:1, v / v, 1.00 mL). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative LC-MS. 4C The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (2.15 mg, 33%) as a white, cottony solid. LC-MS showed multiple broad peaks with accurate m / z values. LRMS (m / z): 2169 [M-5] 5- , 2711[M-6] 4- LC-MS rt(min):2.58~3.20 1A

[0219] Intermediate 22 (Figure 21) Trivalent GalNAc-thiol Trivalent GalNAc thioacetate (16.2 mg, 9.02 μmol) was dissolved in methanol (500 μL). Next, a 1.00 M sodium hydroxide solution (11.0 μL, 11.0 μmol) was added. The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 30 minutes, the reaction mixture was subjected to preparative MP-LC. 1A The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (13.1 mg, 83%) as a white solid. Purity was 98% based on LC-MS. LRMS (m / z): 1748 [MH] 1- LC-MS rt(min):1.78 1A

[0220] Intermediate 23: (Figure 21) DBCO-TCO-Trivalent GalNAc Trifunctional linker (Ls) (15.0 mg, 12.4 μmol) was dissolved in acetonitrile (0.50 mL). Next, a 20 mM ammonium bicarbonate solution (1.50 mL) was added, and the resulting solution was transferred directly to trivalent GalNAc-thiol (22.7 mg, 13.0 μmol). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 1 hour, the reaction mixture was frozen and freeze-dried overnight to obtain the crude title product as a white solid. Purity was 84% ​​based on LC-MS. LRMS(m / z):2409[M-546] 1- ,2955[M-1] 1- LC-MS rt(min):2.63 1B

[0221] Intermediate 25 (Figure 22): Methyltetrazine-BNA oligo Thiol-13-apoB BNA oligodisulfide (20 mg, 4.17 μmol) was added to a 20 mM ammonium bicarbonate solution containing 5.0 mM TCEP (2.00 mL, 10.0 μmol). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 1 hour, the reaction mixture was filtered using a centrifugal filter with a molecular weight cutoff of 3000 Da (5000 × g, 4 × 0.50 mL in 30 minutes). Next, the residue solution was diluted with 20 mM ammonium bicarbonate (3.00 mL), and the resulting solution was added directly to a solution of (E)-1-(4-((2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl)hydrazineylidene)methyl)benzamide)-N-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)-6,9,12-tetraoxapentadecane-15-amide (7.22 mg, 9.16 μmol) in acetonitrile (1.0 mL). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 1 hour, the reaction mixture was frozen and freeze-dried overnight to obtain the crude title product as a pink, cottony solid. 20 mM ammonium bicarbonate / acetonitrile (2:1, v / v, 2.00 mL) was added to the crude product, and the resulting solution was subjected to preparative LC-MS. 1A The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (20.3 mg, 89%) as a pink, cottony solid. Purity was 93% (broad peak) based on LC-MS. LRMS (m / z): 1817 [M-3] 3- LC-MS rt(min):0.59 3

[0222] Intermediate 26 (Figure 22): (Blocked DBCO)-TCO-Trivalent GalNAc A solution of 1-azido-3,6,9-trioxaundecane-11-ol (2.17 mg, 9.88 μmol) in DMF (0.50 mL) was added to DBCO-TCO-trivalent GalNAc (14.6 mg, 4.94 μmol). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative MP-LC.1C The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (10.00 mg, 64%) as a white solid. Purity was 98% based on LC-MS. LRMS (m / z): 1750 [fragment] LC-MS rt(min):2.33 1B

[0223] Ls-(blocked DBCO)-BNA oligo-trivalent GalNAc (Figure 23) (Blocked DBCO)-TCO-trivalent GalNAc (10.0 mg, 3.15 μmol) and methyltetrazine-BNA oligo (10.0 mg, 1.83 μmol) were dissolved in a 20 mM ammonium bicarbonate / acetonitrile solution (3:1, v / v, 1.00 mL). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 3 hours, the reaction mixture was frozen and freeze-dried overnight. The crude composition was dissolved in 20 mM ammonium bicarbonate (1 mL), and the resulting solution was subjected to preparative LC-MS. 1B The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (GalNAc)3-Ls-BNA) (11.3 mg, 72%) as a white, cottony solid. Purity was 95% based on LC-MS (multiple (broad) peaks due to positional isomers). LRMS(m / z):2149 [M-4] 4- , 2866 [M-3] 3- LC-MS rt(min):2.92 1A

[0224] Synthesis of anti-CD71-saporin We had a custom CD71 mab-saporin conjugate generated and purchased it from Advanced Targeting Systems (San Diego, CA). The CD71 antibody (anti-CD71 clone OKT-9, InVivoMab) was purchased from BioXCell.

[0225] HSP27BNA, ApoB and ApoB scrambledOligo sequences HSP27(5'-GGCacagccagtgGCG-3')[SEQ ID NO: 1] (Antisense BNA (HSP27) is a BNA having the oligonucleotide sequence 5'-GGCacagccagtgGCG-3', as defined by Zhang et al. (2011) [Y Zhang, Z Qu, S Kim, V Shi, B Liao1, P Kraft, R Bandaru, Y Wu, LM Greenberger and ID Horak, Down-modulation of cancer targets using locked nucleic acid (LNA)-based antisense oligonucleotides without transfection, Gene Therapy (2011) 18, 326-333], more specifically BNA NC (was), ApoB (also called ApoB#01) (5'-GCCTCagtctgcttcGCACC-3') [SEQ ID NO: 2] and ApoB scrambled (5'-GGCCTctctacaccgCTCGT-3')[SEQ ID NO: 3]BNA oligonucleotide, and apoB#02(5'-GCattggtatTCA-3')[SEQ ID NO: 12]BNA oligonucleotide compatible with mouse and human sequences. NC Oligonucleotides were ordered from Bio-Synthesis Inc. (Lewisville, Texas) with a 5'-thiol C6 linker, BNA bases capitalized, and a complete phosphorothioate backbone.

[0226] RNA isolation and gene expression analysis of cell lines RNA was isolated from cells and analyzed according to a standard procedure (Biorad). The qPCR primers used are shown in Table A2.

[0227] [Table 6]

[0228] Cell viability assay After treatment, cells were incubated at 37°C for 72 hours, and then cell viability was measured by MTS assay, performed according to the manufacturer's instructions (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega). In summary, the MTS solution was diluted 20-fold with phenol red-free DMEM (PAN-Biotech GmbH) supplemented with 10% FBS. Cells were washed once with 200 μL / PBS well, and then 100 μL of diluted MTS solution was added to each well. The plates were incubated at 37°C for approximately 20-30 minutes. Subsequently, the OD at 492 nm was measured using a Thermo Scientific Multiskan FC plate reader (Thermo Scientific). For quantification, the background signal of the "medium only" well was subtracted from all other wells, and then the percentage of treated / untreated cell viability was calculated by dividing the background-corrected signal of the treated well by the background-corrected signal of the untreated well (×100).

[0229] FACS analysis Cells were seeded at appropriate densities for each cell line in DMEM (PAN-Biotech GmbH) supplemented with 10% fetal bovine serum (PAN-Biotech GmbH) and 1% penicillin / streptomycin (PAN-Biotech GmbH) in T75 flasks, and incubated for 72-96 hours until confluence reached 90% (5% CO2, 37°C). Next, the cells were trypsinized (TryplE Express, Gibco Thermo Scientific) to isolate single cells, transferred to 15 mL Falcon tubes, and centrifuged (1,400 rpm, 3 mins). The supernatant was discarded while the cell pellet remained immersed. 500,000 cells were transferred to round-bottom FACS tubes and 3 mL of cold DPBS (Mg 2+ and Ca 2+ Washed with free, 2% FBS. Centrifuged at 1800 rpm for 3 minutes at 4°C and rinsed in 200 μL of cold DPBS (Mg 2+ and Ca 2+Free, 2% FBS) or 195 μL of cold DPBS (Mg 2+ and Ca 2+ Cells were resuspended in 200 μL of antibody solution containing 5 μL of antibody in free (2% FBS). PE anti-human CD71 (#334106, Biolegend) was used to stain the transferrin receptor, with PE mouse IgG2a, κ isotype control FC (#400212, Biolegend) as a matching isotype control. PE anti-human ASGPR1 (#130-122-963, Miltenyi) was used to stain the ASGPR1 receptor, with PE mouse IgG1, isotype control (#130-113-762, Miltenyi) as a matching isotype control. Samples were incubated at 4°C for 30 minutes. Cells were then chilled in cold DPBS (Mg 2+ and Ca 2+ Wash twice with free, 2% FBS, and DPBS (Mg 2+ and Ca 2+ Cells were fixed at room temperature for 20 minutes using a 2% PFA solution in free (2% FBS). The cells were washed once with cold DPBS and resuspended in 1000 μL of cold DPBS for FACS analysis. Samples were analyzed using a Sysmex Cube 8 flow cytometry system (Sysmex) and FCS Express 7 Research edition software. The FACS analysis results are summarized in Table A3.

[0230] [Table 7]

[0231] RNA isolation and gene expression analysis from primary mouse and human hepatocytes RNA was isolated from cells using TRI Reagent® solution (Thermo Scientific) according to the manufacturer's instructions. Conversion to cDNA was performed using the iScript® cDNA synthesis kit (Biorad) following standard procedures. ApoB expression levels and specific hepatocyte housekeeping gene levels were measured using quantitative real-time PCR assays (qRT-PCR) with iTaq® Universal SYBR® Green Supermix (BioRad) and Light Cycler 480 (Roche Diagnostics, Rotkreuz, Switzerland), including the specific DNA primers listed in Table A4 (mouse) and Table A2 (human). ApoB expression was analyzed by the ΔCt method to compare it with two hepatocyte-specific housekeeping control mRNAs. Each analytical reaction was performed three times.

[0232] [Table 8]

[0233] Treatment of primary mouse hepatocytes Frozen primary mouse hepatocytes (PRIMACYT Cell Culture Technology GmbH, Germany) were thawed in hepatocyte thawing medium (HTM, PRIMACYT Cell Culture Technology GmbH, Germany) and washed once with hepatocyte washing medium (HWM, PRIMACYT Cell Culture Technology GmbH, Germany). The cells were then placed in hepatocyte plating medium (HPM Cryo, PRIMACYT Cell Culture Technology GmbH, Germany) at a density of approximately 0.275 × 10⁶. 6The cells were resuspended at a density of cells / ml. Cells were seeded onto type I collagen-coated plates at a density of 88,000 cells / well or 26,400 cells / well for 48 or 96-well plates (Greiner BioOne). Before initiating treatment, cells were pre-cultured at 37°C for 4–6 hours to adhere to the cell culture plate. The plating medium was replaced with 315 μl or 108 μl of assay medium (MHM, PRIMACYT Cell Culture Technology GmbH, Germany), and then the conjugate was added from a 10-fold stock solution in PBS. Plates were incubated at 37°C for 72 hours and harvested for analysis of gene expression and cell viability.

[0234] Treatment of primary human hepatocytes Cryopreserved primary human hepatocytes (Cytes Biotechnologies SL, Spain) were thawed in hepatocyte thawing medium (Cytes Biotechnologies SL, Spain). The cells were resuspended in hepatocyte plating medium (Cytes Biotechnologies SL, Spain). Cells were seeded into type I collagen-coated plates at densities of 215,600 cells / well or 66,600 cells / well in 48 or 96-well plates (Greiner BioOne). Before initiating treatment, the cells were pre-cultured at 37°C for 4–6 hours to adhere to the cell culture plate. The plating medium was replaced with 315 μl or 108 μl of maintenance medium (Cytes Biotechnologies SL, Spain), and then a conjugate from a 10-fold concentration of stock solution in PBS was added. The plates were incubated at 37°C for 72 hours and harvested for analysis of gene expression and cell viability.

Claims

1. A pharmaceutical compound, - A conjugate of an effector molecule and a ligand for the asialocrycoprotein receptor (ASGPR), wherein the ligand for ASGPR comprises at least one N-acetylgalactosamine (GalNAc) moiety, and the effector molecule comprises or comprises an oligonucleotide; - A saponin which is a monodesmoside triterpene glycoside or a videsmoside triterpene glycoside, selected from chiric acid and gypsogenin, or C of the chiric acid or gypsogenin. ２３ It comprises a saponin containing an aglycone core structure, selected from chiral acid and gypsogenin, in which the aldehyde group at the position is derivatized. A pharmaceutical formulation in which the aldehyde group at the C23 position of the chiric acid or gypsogenin is derivatized by reduction to an alcohol or by the formation of a covalent bond with another molecule at the C23 position of the aglycone core structure.

2. The pharmaceutical formulation according to claim 1, wherein the ligand of ASGPR comprises three or four GalNAc moieties, or consists of a single GalNAc moiety.

3. A pharmaceutical formulation according to claim 1 or 2, comprising a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable diluent.

4. The pharmaceutical formulation according to any one of claims 1 to 3, wherein the ligand of ASGPR and the effector molecule are conjugated via a covalent bond.

5. The pharmaceutical formulation according to any one of claims 1 to 4, wherein the ligand of ASGPR and the effector molecule are conjugated via at least one linker.

6. The pharmaceutical formulation according to any one of claims 1 to 5, wherein the aldehyde group is derivatized by conversion to a hydrazone bond, thereby forming a covalent bond between the aglycone core and the other molecule.

7. The saponin is conjugated with the ligand of ASGPR via the C23 position of the chiric acid or gypsogenin, The pharmaceutical formulation according to any one of claims 1 to 6, wherein the ligand of ASGPR comprises at least one GalNAc moiety.

8. The pharmaceutical formulation according to claim 7, wherein the ligand of ASGPR conjugated to the saponin comprises or consists of (GalNAc)3-tris or monovalent GalNAc.

9. The aforementioned effector molecules include small interfering RNA (siRNA), small hairpin RNA (shRNA), anti-hairpin microRNA (miRNA), single-stranded RNA, aptamer RNA, double-stranded RNA (dsRNA), antimicroRNA (anti-miRNA, anti-miR), antisense oligonucleotide (ASO), mRNA, DNA, antisense DNA, locked nucleic acid (LNA), cross-linked nucleic acid (BNA), and 2'-O,4'-aminoethylene cross-linked nucleic acid (BNA). ＮＣ The pharmaceutical formulation according to any one of claims 1 to 8, wherein the oligonucleotide is selected from one or more of the following: BNA-based siRNA and BNA-based antisense oligonucleotide (BNA-AON).

10. The pharmaceutical formulation according to any one of claims 1 to 9, wherein the effector molecule is an oligonucleotide selected from chemically modified siRNA, metabolically stable siRNA, and chemically modified and metabolically stable siRNA.

11. The pharmaceutical formulation according to any one of claims 1 to 10, wherein the effector molecule is an oligonucleotide capable of silencing any one of the following genes: HSP27, apolipoprotein B (ApoB), transthyretin (TTR), the proprotein convertase subtilisin / kexin type 9 (PCSK9), δ-aminolevulinic acid synthase 1 (ALAS1), antithrombin 3 (AT3), glycolate oxidase (GO), complement component C5 (CC5), the X gene of hepatitis B virus (HBV), the S gene of HBV, α1-antitrypsin (AAT), and lactate dehydrogenase (LDH), and / or targeting abnormal miRNA.

12. The pharmaceutical formulation according to any one of claims 1 to 11, wherein the effector molecule, when present inside a mammalian cell, can target mRNA involved in the expression of any one of the following proteins: HSP27, ApoB, TTR, PCSK9, ALAS1, AT3, GO, CC5, the expression product of the HBV X gene, the expression product of the HBV S gene, AAT, and LDH, or is an oligonucleotide that can weaken or restore miRNA function, such as inhibiting oncogenic miRNA (onco-miR) or suppressing the expression of onco-miR.

13. The pharmaceutical formulation according to any one of claims 1 to 12, wherein the aglycone core structure of the saponin is the chiric acid.

14. A pharmaceutical formulation according to any one of claims 1 to 13, - The aforementioned saponins belong to group A: GlucA-, Glc-, Gal-, Rha-(1→2)-Ara-, Gal-(1→2)-[Xyl-(1→3)]-GlcA-, Glc-(1→2)-[Glc-(1→4)]-GlcA-, Glc-(1→2)-Ara-(1→3)-[Gal-(1→2)]-GlcA-, Xyl-(1→2)-Ara-(1→3)-[Gal-(1→2)]-GlcA-, Glc-(1→3)-Gal-(1→2)-[Xyl-(1→3)]-Glc-(1→4)-Gal-, Rha-(1→2)-Gal-(1→3)-[Glc-(1→2)]-GlcA-, Ara-(1→4)-Rha-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-, Ara-(1→4)-Fuc-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-, Ara-(1→4)-Rha-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-, Ara-(1→4)-Fuc-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-, Ara-(1→4)-Rha-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-, Ara-(1→4)-Fuc-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-, Ara-(1→4)-Rha-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-, Ara-(1→4)-Fuc-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-, Xyl-(1→4)-Rha-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-, Xyl-(1→4)-Fuc-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-, Xyl-(1→4)-Rha-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-, Xyl-(1→4)-Fuc-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-, Xyl-(1→4)-Rha-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-, Xyl-(1→4)-Fuc-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-, Xyl-(1→4)-Rha-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-, and Xyl-(1→4)-Fuc-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA- A selection from which a sugar chain is attached to the aglycone core structure is included, or - The aforementioned saponins belong to group B: Glc-, Gal-, Rha-(1→2)-[Xyl-(1→4)]-Rha-, Rha-(1→2)-[Ara-(1→3)-Xyl-(1→4)]-Rha-, Ara-, Xyl-, Xyl-(1→4)-Rha-(1→2)-[R1-(→4)]-Fuc-(wherein R1 is 4E-methoxycinnamate), Xyl-(1→4)-Rha-(1→2)-[R2-(→4)]-Fuc-(wherein R2 is 4Z-methoxycinnamate), Xyl-(1→4)-[Gal-(1→3)]-Rha-(1→2)-4-OAc-Fuc-, Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-3,4-di-OAc-Fuc-, Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R3-(→4)]-3-OAc-Fuc-(wherein R3 is 4E-methoxycinnamate), Glc-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-4-OAc-Fuc-, Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-4-OAc-Fuc-, (Ara- or Xyl-)(1→3)-(Ara- or Xyl-)(1→4)-(Rha- or Fuc-)(1→2)-[4-OAc-(Rha- or Fuc-)(1→4)]-(Rha- or Fuc-), Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Qui-(1→4)]-Fuc-, Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-Fuc-, Xyl-(1→4)-[Gal-(1→3)]-Rha-(1→2)-Fuc-, Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-Fuc-, Ara / Xyl-(1→4)-Rha / Fuc-(1→4)-[Glc / Gal-(1→2)]-Fuc-, Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R4-(→4)]-Fuc-(wherein R4 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R5-(→4)]-Fuc-(wherein R5 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Rha-(1→3)]-4-OAc-Fuc-, Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4-OAc-Fuc-, 6-OAc-Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3-OAc-Rha-(1→3)]-Fuc-, Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3-OAc--Rha-(1→3)]-Fuc-, Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Qui-(1→4)]-Fuc-, Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-[Qui-(1→4)]-Fuc-, Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc-, Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3,4-di-OAc-Qui-(1→4)]-Fuc-, Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-Fuc-, 6-OAc-Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-Fuc-, Glc-(1→3)-[Xyl-(1→3)-Xyl-(1→4)]-Rha-(1→2)-Fuc-, Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc-, Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4OAc-Fuc-, Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4OAc-Fuc-, Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R6-(→4)]-Fuc-(wherein R6 is 5-O-[5-O-Rha-(1→2)-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid) Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R7-(→4)]-Fuc-(wherein R7 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid) Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R8-(→4)]-Fuc-(wherein R8 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R9-(→4)]-Fuc- (wherein R9 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R10-(→4)]-Fuc- (wherein R10 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R11-(→3)]-Fuc-(wherein R11 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R12-(→3)]-Fuc-(wherein R12 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), and Glc-(1→3)-[Glc-(1→6)]-Gal- A selection from which a sugar chain is attached to the aglycone core structure is included, or - A pharmaceutical formulation in which the saponin is a videsmoside triterpene glycoside comprising a first sugar chain selected from group A bonded to the aglycone core structure and a second sugar chain selected from group B bonded to the aglycone core structure.

15. The aforementioned saponins are Quillaja bark saponins, NP-017777, NP-017778, NP-017774, NP-018110, NP-017772, NP-018109, NP-017888, NP-017889, NP-018108, SA1641, AE X55, NP-017674, NP-017810, AG1, NP-003881, NP-017676, NP-017677, NP-017706, NP-017705, NP-017773, NP-017 775, SA1657, AG2, SO1861, GE1741, SO1542, SO1584, SO1658, SO1674, SO1832, SO1862, SO1904, QS-7, QS1861, QS-7 api, QS1862, QS-17, QS-18, QS-21 A-apio, QS-21 A-xylo, QS-21 A pharmaceutical formulation according to any one of claims 1 to 14, selected from the group consisting of B-apio, QS-21 B-xylo, stereoisomers thereof, and combinations thereof.

16. The pharmaceutical formulation according to any one of claims 1 to 15, wherein the saponin is selected from the group consisting of QS-21, SO1861, SA1641, GE1741, and combinations thereof.

17. The pharmaceutical formulation according to any one of claims 1 to 16, wherein the saponin is selected from the group consisting of QS-21 saponin, SO1861 saponin, and combinations thereof.

18. The pharmaceutical formulation according to any one of claims 1 to 17, wherein the saponin is SO1861 saponin.

19. The saponin is one of the following saponins: i. The saponin contains an aglycone core structure that includes an aldehyde group derivatized by reduction to an alcohol or conversion to a hydrazone bond that forms a covalent bond between the aglycone core and another molecule; or ii. The saponin comprises a sugar chain containing a carboxyl group that is derivatized by conversion to an amide bond that forms a covalent bond with another molecule; or iii. The saponin contains a sugar chain containing an acetoxy (Me(CO)O-) group which has been derivatized by conversion to a hydroxyl group (HO-) by deacetylation; or iv. A pharmaceutical formulation according to any one of claims 1 to 18, wherein any combination of derivatization i., ii., and iii. exists.

20. The aforementioned saponins include SO1861, SA1657, GE1741, SA1641, QS-21, QS-21A, QS-21A-api, QS-21A-xyl, QS-21B, QS-21B-api, QS-21B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862, Quillaja saponin, Saponinum album. A pharmaceutical formulation according to any one of claims 1 to 19, selected from the group consisting of album), QS-18, Quil-A, Gyp1, dypsoside A, AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, SO1904, their stereoisomers, and combinations thereof.

21. The aforementioned saponin has a molecule of 1: 【Chemistry 1】 [In the formula, A １ represents hydrogen, monosaccharide, or linear or branched oligosaccharide; A ２ represents hydrogen, monosaccharide, or linear or branched oligosaccharide; Furthermore, R is represented as either a hydrogen atom in gypsogenin or a hydroxyl atom in chiral acid; The derivative of the saponin corresponds to the saponin represented by molecule 1, and is derivatized as follows: i. C of the chiric acid or gypsogenin ２３ The aldehyde group at the position is derivatized by reduction to an alcohol or conversion to a hydrazone bond that forms a covalent bond between the aglycone core and another molecule; ii. A １ A represents a sugar chain selected from group A as described in claim 14, and A １ If it contains or consists of a glucuronic acid portion, A １ The carboxyl group of the glucuronic acid portion is derivatized by conversion to an amide bond that forms a covalent bond with another molecule; and iii. A ２ represents a sugar chain selected from Group B according to claim 14, and A ２ when contains at least one acetoxy group, A ２ at least one of the one sugar moiety or one or more of the two or more sugar moieties of A is derivatized by conversion of one or more acetoxy groups to hydroxyl groups (HO-) by deacetylation, A pharmaceutical formulation according to any one of claims 1 to 20.

22. A １ A represents a sugar chain selected from group A as described in claim 14, and includes or consists of a glucuronic acid portion. １ The glucuronic acid portion of is derivatized by conversion of the carboxyl group to an amide bond that forms a covalent bond with another molecule, and / or A ２ A represents a sugar chain selected from group B as described in claim 14, and A ２ It contains at least one acetoxy group, A ２ The pharmaceutical formulation according to claim 21, wherein at least one acetoxy group is derivatized by conversion to a hydroxyl group (HO-) by deacetylation.

23. The saponin derivative corresponds to the saponin represented by molecule 1, and the following derivatization is performed: i. C of the chiric acid or gypsogenin ２３ The aldehyde group at position - Reduction to alcohol; or - Conversion to a hydrazone bond by reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), thereby resulting in saponin-Ald-EMCH; or - Conversion to a hydrazone bond by reaction with N-[β-maleimidopropionic acid]hydrazide (BMPH); or - Conversion to a hydrazone bond through reaction with N-[κ-maleimidoundecanoic acid]hydrazide (KMUH) It is derivatized by; ii. A １ A represents a sugar chain selected from group A as described in claim 14, and A １ If it contains or consists of a glucuronic acid portion, A １ The carboxyl group of the glucuronic acid portion is derivatized by conversion to an amide bond through reaction with 2-amino-2-methyl-1,3-propanediol (AMPD) or N-(2-aminoethyl)maleimide (AEM), thereby yielding saponin-Glu-AMPD; iii. A ２ A represents a sugar chain selected from group B as described in claim 14, and A ２ If it contains at least one acetoxy group, A ２ One or more acetoxy groups of one or more sugar moieties are derivatized by conversion to a hydroxyl group (HO-) through deacetylation. A pharmaceutical compound according to claim 21 or 22, wherein at least one of the following is present.

24. A １ However, Gal-(1→2)-[Xyl-(1→3)]-GlcA and / or A ２ The pharmaceutical compound according to any one of claims 21 to 23, wherein the compound is Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc.

25. The saponin is a saponin derivative, i. The saponin derivative is - Reduction to alcohol; or - Conversion to a hydrazone bond by reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), thereby resulting in saponin-Ald-EMCH; or - Conversion to a hydrazone bond by reaction with N-[β-maleimidopropionic acid]hydrazide (BMPH); or - Conversion to a hydrazone bond through reaction with N-[κ-maleimidoundecanoic acid]hydrazide (KMUH) It comprises an aglycone core structure containing an aldehyde group derivatized by; or ii. The saponin derivative comprises a sugar chain containing a carboxyl group that has been derivatized by conversion to an amide bond through reaction with 2-amino-2-methyl-1,3-propanediol (AMPD) or N-(2-aminoethyl)maleimide (AEM); or iii. The saponin derivative contains a sugar chain containing an acetoxy (Me(CO)O-) group that has been derivatized by conversion to a hydroxyl group (HO-) by deacetylation; or iv. The saponin derivative includes any combination of derivatizations i., ii., and iii.; A pharmaceutical compound according to any one of claims 19 to 24.

26. The saponin is a saponin derivative, i. The saponin derivative comprises an aglycone core structure containing an aldehyde group that has been derivatized by conversion to a hydrazone bond through reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), thereby yielding saponin-Ald-EMCH; or ii. The saponin comprises a sugar chain containing a carboxyl group that has been derivatized by conversion to an amide bond through reaction with N-(2-aminoethyl)maleimide (AEM); or iii. The pharmaceutical formulation according to claim 25, wherein the saponin derivative comprises a combination of derivatization i. and ii.

27. The aforementioned saponin has two molecules: 【Chemistry 2】 It is a saponin derivative represented by, Alternatively, the saponin derivative is molecular 3: 【Transformation 3】 A pharmaceutical formulation according to any one of claims 1 to 25, which is a saponin derivative represented by .

28. A pharmaceutical compound according to any one of claims 1 to 27, for use as a pharmaceutical agent.

29. A pharmaceutical formulation according to any one of claims 1 to 27, for the treatment of a disease or health problem caused by the expression of one or more of the following genes: ApoB, HSP27, TTR, PCSK9, ALAS1, AT3, GO, CC5, the HBV X gene, the HBV S gene, AAT, and LDH, wherein the effector molecule, when present inside a mammalian cell, can target mRNA involved in the expression of one of the following proteins: HSP27, ApoB, TTR, PCSK9, ALAS1, AT3, GO, CC5, the expression product of the HBV X gene, the expression product of the HBV S gene, AAT, and LDH, or can weaken or restore miRNA function such as inhibiting oncogenic miRNA (onco-miR) or suppressing the expression of onco-miR.

30. A pharmaceutical formulation according to any one of claims 1 to 27 for the treatment or prevention of cancer, infectious diseases, viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, AAT-related liver disease, acute hepatic porphyria, TTR-mediated amyloidosis, hereditary TTR amyloidosis (hATTR), complement-mediated diseases, hepatitis B infection, diseases or disorders related to HSP27 expression, or autoimmune diseases.

31. An in vitro or ex vivo method for transferring an effector molecule according to any one of claims 1 to 12 from outside a cell to inside the cell, a) A step of providing cells that express ASGPR on their surface; b) A step of providing a conjugate according to any one of claims 1 to 27, comprising the effector molecule to be migrated; c) A step of providing a saponin according to any one of claims 1 to 27; d) A method comprising the step of contacting the cells of step a) with the conjugate of step b) and the saponin of step c) in vitro or ex vivo, thereby causing the transfer of the conjugate containing the effector molecule from outside the cell to inside the cell, and causing the transfer of the conjugate to cause the transfer of the effector molecule from outside the cell to inside the cell.

32. An in vitro or ex vivo method for transferring a conjugate according to any one of claims 1 to 27 from outside a cell to inside the cell, a) A step of providing cells that express ASGPR on their surface; b) A step of providing a conjugate according to any one of claims 1 to 27; c) A step of providing a saponin according to any one of claims 1 to 27; d) A method comprising the step of contacting the cells of step a) with the conjugate of step b) and the saponin of step c) in vitro or ex vivo, thereby causing the conjugate to move from outside the cell to inside the cell.

33. When the aforementioned effector molecules are present inside mammalian cells, the following genes may be affected: apolipoprotein B (ApoB), HSP27, transthyretin (TTR), the proprotein convertase subtilisin / kexin type 9 (PCSK9), δ-aminolevulinic acid synthase 1 (ALAS1), antithrombin 3 (AT3), glycolate oxidase (GO), complement component C5 (CC5), the X gene of hepatitis B virus (HBV), the S gene of HBV, α1-antitrypsin (AAT), and lactate dehydrogenase (L). The method according to claim 31 or 32, wherein the oligonucleotide can silence any one of DH) and / or target abnormal miRNAs and / or target mRNAs involved in the expression of any one of the following proteins: HSP27, ApoB, TTR, PCSK9, ALAS1, AT3, GO, CC5, the expression product of the HBV X gene, the expression product of the HBV S gene, AAT, and LDH, or weakens or restores miRNA function.

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