Galnac-lipid containing compounds and uses thereof

Abstract

A composition comprising a compound having the structure (I), (II), (III), or (IV) is provided: wherein: each R1 is independently selected from aliphatic alkyl C4-C100 groups optionally substituted with one or more of alkenyl, alkynyl, hydroxyl, amide, ester, and / or ether groups; R2 is selected from [-OCH2CH2-]p, [-OCH2CH2-]p-O-, or; R3 is selected from Formula (V), (VI), (VII), (VIII); R4 is an alkyl chain selected from [-CH2-]t; and m, p, s and t are independently selected from integers from 1 to 120. The compound is useful as a liver asialoglycoprotein receptor-targeted therapeutic agent in the form of a lipid nanoparticle, a liposome or a micelle including a drug or nucleic acid.

Description

[0001] GALNAC-LIPID CONTAINING COMPOUNDS AND USES THEREOF

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 776,541, filed March 24, 2025, the disclosure of which is incorporated by reference in its entirety for all purposes.

[0004] FIELD OF THE INVENTION

[0005] The invention relates to lipids comprising an N-acetylgalactosamine that targets liver asialoglycoprotein receptors.

[0006] BACKGROUND OF THE INVENTION

[0007] Compounds containing N-acetylgalactosamine moieties are known to bind to the liver asialoglycoprotein receptor (ASGR). However, these compounds are typically fairly hydrophobic and therefore less effective for drug or nucleic acid delivery to a person in need thereof. As such, there exists a need to provide more effective means for utilizing such compounds containing N-acetylgalactosamine moieties for drug or nucleic acid delivery to a person in need thereof.

[0008] H. Cui et al.; ACS Omega 2021 , 6, 16259-16265 provides a review of liver-targeted delivery of oligonucleotides with N-acetylgalactosamine conjugation.

[0009] K. Godbout et al.; Pharmaceutics 2022, 14, 2129 provides an overview of delivery of RNAs to specific organs by lipid nanoparticles for gene therapy.

[0010] L. N. Kasiewicz et al.; Nature Communications, (2023) 14:2776 discloses GalNAc-lipid nanoparticles to enable non-LDLR (low density lipoprotein receptor) dependent hepatic delivery of a CRISPR base editing therapy.

[0011] Y. Zhou et al. ACS Cent. Sci. 2021, 7, 499-506; discloses triantennary

[0012] N-acetylgalactosamine conjugates as degraders for extracellular proteins.

[0013] CA 02930393 A1 discloses carbohydrate conjugates as delivery agents for oligonucleotides.

[0014] The present disclosure provides liver asialoglycoprotein receptor targeted lipid compounds that overcome certain drawbacks limiting utility of compounds containing N-acetylgalactosamine moieties. These compounds provide a method of delivering therapeutic drugs or nucleic acids to the liver via the liver asialoglycoprotein receptors. Although ionically charged liver asialoglycoprotein receptor targeted lipid compoundscontaining hydrophilic linker moieties compounds have previously been described in PCT application no. PCT / US2024 / 050364, neutral compounds are now described; such neutral compounds have utility in applications where charged forms of material are not desirable, for example, when charged forms of material are not compatible with a particular therapeutic agent to be delivered, or the holistic drug formulation.

[0015] SUMMARY OF THE INVENTION

[0016] The present disclosure is directed to compounds incorporating lipids that are bound to two or more N-acetylgalactosamine units. The compounds target the liver asialoglycoprotein receptor and are useful for delivery of therapeutic agents, such as drugs or nucleic acids, to a person in need thereof. According to a particular embodiment, the compounds are used to form lipid nanoparticles that deliver the therapeutic agents, such as drugs or nucleic acids, to a person in need thereof. According to another embodiment, the compounds are used to form liposomes that deliver the therapeutic agents, such as drugs or nucleic acids, to a person in need thereof. According to another embodiment, the compounds are used to form micelles that deliver the therapeutic agents, such as drugs or nucleic acids, to a person in need thereof.

[0017] According to an embodiment, there is provided a composition comprising a compound having the structure (I), (II), (III), or (IV), or a combination of any two or more thereof.

[0018] AcHN

[0019]

[0020]

[0021] In these structures, (I), (II), (III), and (IV), each R1is independently selected from aliphatic alkyl C4-C100 groups optionally substituted with one or more of alkenyl, alkynyl, hydroxyl, amide, ester, and / or ether groups;

[0022] CH3

[0023] R2is selected from [-

[0024]

[0025] OCH2CH2-]P, [-OCH2CH2-]P-O- , or0R3is selected from Formula (V), Formula (VI), Formula (VII) and Formula (VIII):

[0026]

[0027] O'

[0028]

[0029] m, p, and s are independently selected from integers from 1 to 120.

[0030] According to an embodiment, the compounds having the structure (I), (II), (III), or (IV) do not have more than 3, 5, 7, 9, 11, 13, or 15 moieties of structure (VI). In other words, the compounds having the structure (I), (II), (III), or (IV) do not have more than 3, 5, 7, 9, 11, 13, or 15 N-acetylgalactosamine moieties per molecule.

[0031] According to an alternative embodiment a liver asialoglycoprotein receptor-targeted therapeutic agent comprising a drug or nucleic acid in a lipid nanoparticle, a liposome or a micelle comprising a compound having the structure (I), (II), (III), or (IV), or a combination of any two or more thereof is provided.

[0032] According to a further alternative embodiment a method of treatment, comprising administering the liver asialoglycoprotein receptor-targeted therapeutic agent to a patient in need thereof is also provided.

[0033] BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 shows a high-performance liquid chromatogram (HPLC) of a compound according to an embodiment of the invention, as further described in Example 1.

[0035] DETAILED DESCRIPTION OF THE INVENTION

[0036] DefinitionsAs used herein, the terms “prevention”, “prevent”, “preventing”, “suppression”, “suppress” and “suppressing” as used herein refer to a course of action (such as administering a compound or pharmaceutical composition) initiated prior to the onset of a symptom, aspect, or characteristics of a disease or condition so as to prevent or reduce such symptom, aspect, or characteristics. Such preventing and suppressing need not be absolute to be useful.

[0037] As used herein, the terms “treatment”, “treat” and “treating” as used herein refers a course of action (such as administering a compound or pharmaceutical composition) initiated after the onset of a symptom, aspect, or characteristics of a disease or condition so as to eliminate or reduce such symptom, aspect, or characteristics. Such treating need not be absolute to be useful.

[0038] As used herein, the term “in need of treatment” as used herein refers to a judgment made by a caregiver that a patient requires or will benefit from treatment.

[0039] This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that includes the knowledge that the patient is ill, or will be ill, as the result of a disease or condition that is treatable by a method or compound of the disclosure.

[0040] As used herein, the term “in need of prevention” as used herein refers to a judgment made by a caregiver that a patient requires or will benefit from prevention. This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that includes the knowledge that the patient will be ill or may become ill, as the result of a disease or condition that is preventable by a method or compound of the disclosure.

[0041] As used herein, the terms “individual”, “subject” or “patient” as used herein refers to any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and humans. The term may specify male orfemale or both or exclude male orfemale.

[0042] As used herein, the term “therapeutically effective amount” as used herein refers to an amount of a compound, either alone or as a part of a pharmaceutical composition, that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease or condition. Such effect need not be absolute to be beneficial.

[0043] As used herein, the term “alkyl”, whether used alone or as part of a substituent or linking group, includes straight hydrocarbon groups comprising from one to 100 carbon atoms. Thus, the phrase “alkyl” includes straight chain alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl and the like. Thephrase also includes branched chain isomers of straight chain alkyl groups, including but not limited to, the following which are provided by way of example: — CH(CH3)2,

[0044] — CH(CH3)(CH2CH3), — CH(CH2CH3)2, — C(CH3), — C(CH2CH3)3, — CH2CH(CH3)2, — CH2CH(CH3)(CH2CH3), — CH2CH(CH2CH3)2, — CHC(CH3)3, — CH2C(CH2CH3)3,

[0045] — CH(CH3)CH(CH3)(CH2CH3), — CH2CH2CH(CH3)2, — CH2CH2CH(CH3)(CH2CH3),

[0046] — CH2CH2CH(CH2CH3)2, — CH2CH2C(CH3)3, — CH2CH2C(CH2CH3)3,

[0047] — CH(CH3)CH2CH(CH3)2, — CH(CH3)CH(CH3)CH(CH3)CH(CH3)2,

[0048] — CH(CH2CH3)CH(CH3)CH(CH3)(CH2CH3) and others. The phrase also includes cyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl and such rings substituted with straight and branched chain alkyl groups as defined above. The phrase also includes polycyclic alkyl groups such as, but not limited to, adamantyl norbornyl, and bicyclo[2.2.2]octyl and such rings substituted with straight and branched chain alkyl groups as defined above.

[0049] As used herein, the term “alkylene”, whether used alone or as part of a substituent group, includes any group obtained by removing a hydrogen atom from an alkyl group; an alkylene group forms two bonds with other groups.

[0050] As used herein, the term “alkenyl”, whether used alone or as part of a substituent group, includes an alkyl group having at least one double bond between any two adjacent carbon atoms.

[0051] As used herein the term “alkynyl”, whether used alone or as part of a substituent group, includes an alkyl group having at least one triple bond between any two adjacent carbon atoms.

[0052] As used herein, the terms “unsubstituted alkyl,” “unsubstituted alkenyl” and “unsubstituted alkenyl” refer to alkyl, alkenyl and alkynyl groups that do not contain heteroatoms.

[0053] The phrases “substituted alkyl,” “substituted alkenyl,” and “substituted alkynyl” refer to alkyl, alkenyl, and alkynyl groups as defined above in which one or more bonds to a carbon(s) or hydrogen(s) are replaced by a bond to non-hydrogen or non-carbon atoms such as, but not limited to, a halogen atom in halides such as F, Cl, Br, and I; and oxygen atom in groups such as carbonyl, carboxyl, hydroxyl groups, alkoxy groups, aryloxy groups, and ester groups; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfone groups, sulfonyl groups, and sulfoxide groups; a nitrogen atom in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, enamines imines, oximes, hydrazones, and nitriles; a silicon atom in groups such as in trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilylgroups; and other heteroatoms in various other groups. Other alkyl groups include those in which one or more bonds to a carbon or hydrogen atom is replaced by a bond to an oxygen atom such that the substituted alkyl group contains a hydroxyl, alkoxy, aryloxy group, or heterocyclyloxy group. Still other alkyl groups include alkyl groups that have an amine, alkylamine, dialkylamine, arylamine, (alkyl)(aryl)amine, diarylamine, heterocyclylamine, (alkyl)(heterocyclyl)-mine, (aryl)(heterocyclyl)amine, or diheterocyclylamine group.

[0054] As used herein the term “alkynyl”, whether used alone or as part of a substituent group, includes an alkyl group having at least one triple bond between any two adjacent carbon atoms.

[0055] Compounds

[0056] According to an embodiment, a composition comprising a compound having a structure (I), (II), (III), or (IV), or a combination of any two or more thereof is provided.

[0057]

[0058]

[0059] In these structures, (I), (II), (III), and (IV), each R1is independently selected from aliphatic alkyl C4-C100 groups optionally substituted with one or more of alkenyl, alkynyl, hydroxyl, amide, ester, and / or ether groups;

[0060] R2is selected from [-

[0061]

[0062] OCH2CH2-]P, [-OCH2CH2-]P-O-, or R3is selected from Formula (V), (VI), (VII) or (VIII):

[0063]

[0064] 0 XN H

[0065] N

[0066] H

[0067]

[0068] R4is an alkyl chain selected from [-CH2-]t;

[0069] and m, p, s and t are independently selected from integers from 1 to 120.

[0070] According to some embodiments, the compounds do not have more than 3, 5, 7, 9, , 13, or 15 N-acetylgalactosamine moieties per molecule.

[0071] According to an embodiment, the compound (II) has structure 1:

[0072]

[0073] According to another embodiment the compound (II) has structure 2:

[0074]

[0075] According to another embodiment, the compound (II) has structure 3:

[0076]

[0077] According to some embodiments, R1is a substituted or an unsubstituted, straight chained or branched aliphatic or cyclic alkyl C4-C100 group, preferably C10-C80 group, more preferably C10-C50 group, most preferably an C12-C24 group. According to some other embodiments, R1may be substituted with at least one alkenyl group. According to another embodiment, R1may be substituted with at least one alkynyl group. According to an embodiment, R1may be substituted with at least hydroxyl group. According to an embodiment, R1may be substituted with at least one amide group. According to an embodiment, R1may be substituted with at least one ester group. Non-limiting examples of suitable R1moieties are C12-C24 straight chained alkyl groups. The C12-C24 groups may be branched. The C12-C24 groups may be cyclic.

[0078] According to an embodiment, R2is [-OCH2CH2-]Por [-OCH2CH2-]P-O-. Preferably R2is [-OCH2CH2-]P-O-.

[0079] CH3

[0080] According to another embodiment, R2is

[0081]

[0082] According to an embodiment, R2is [-OCH2CH2-]Por [-OCH2CH2-]P-O-, and p is an integer from 1 to 120, from 6 to 120, or from 10 to 80, particularly from 20 to 70, more particularly from 30 to 60, most particularly from 40 to 50. According to an embodiment, p is 43. According to an embodiment, p is 43 and R2is [-OCH2CH2-]Por [-OCH2CH2-]P-O-, and preferably p is 43 and R2is [-OCH2CH2-]P-O-.

[0083] CH3

[0084] According to another embodiment, R2is

[0085]

[0086] u sand s is an integer from 1 to 120, from 6 to 120, or from 10 to 80, particularly from 20 to 70, more particularly from 30 to60, most particularly from 40 to 50. According to an embodiment, s is 43 and R2is

[0087] CH3

[0088]

[0089] O Js

[0090] According to some embodiments, m is an integer selected from 1 to 50, particularly from 1 to 40, more particularly from 1 to 30, more particularly from 3 to 25, most particularly from 2 to 5.

[0091] According to an embodiment R4is an alkyl chain selected from [-CH2-]t, -, and t is an integer from 1 to 120, suitably from 1 to 60, or more suitably from 1 to 45, particularly from 1 to 20, more particularly from 1 to 10, most particularly from 1 to 5. Furthermore, preferably R4is the alkyl chain -CH2-, or -CH2CH2-. According to one embodiment, t is 1.According to alternative embodiment, t is 2.

[0092] Additionally, or alternatively, in some embodiments the compound having a structure (I), (II), (III), or (IV), or a combination of any two or more thereof further comprises a spacer or extender group (E). As such, in some embodiments the compound can be understood to have a structure (IE), (HE), (IIIE) or (IVE), or a combination of any two or more thereof. As such, it should be understood that in some embodiments the compound has a structure (I), (II), (III), (IV), (IE), (HE), (IIIE) or (IVE), or a combination of any two or more thereof. Without wishing to be bound by theory, the spacer or extender group (E) is believed to ease problems associated with steric hindrance which may be experienced during manufacture of a compound having a structure (I), (II), (III), or (IV), leading to improved yields of product, but without losing the beneficial properties of the compounds when in use.

[0093] AcHN .O"V" OH O r / - >N-E+O0

[0094] OH

[0095] AcHN HXO^V^ N

[0096] ■R2 mO' o x HR1\ X H R3

[0097]

[0098]

[0099] Additionally, or alternatively, R3, as described above, may also further comprise a spacer or extender group (E), such that R3is selected from Formula (VE), (VIE), (VI IE) or (VINE):

[0100]

[0101] Without wishing to be bound by theory, the spacer or extender group (E) is believed to ease problems associated with steric hindrance during manufacture of the compound having a structure (V) or (VI), leading to improved yields of product, but without losing the beneficial properties of the compositions containing said compounds when in use.

[0102] Preferably, in accordance with some embodiments of the present invention, when the spacer or extender group (E) is present, E is:

[0103]

[0104] wherein, n is an integer between the value 0 and 6, and X is an optional alkyl chain of between 0 and 4 carbons in length. Without wishing to be bound by theory, it is believed that overall shorter-chain versus longer-chain spacer or extender groups are preferable since this will result in a limited flexible conformation distribution in the final compound. As such, preferably n is 0, 1 or 2, more preferably 1 or 2. Furthermore, preferably X is the alkyl chain - CH2-, or -CH2CH2-.

[0105] Suitably, where the compound has a structure (IE), (HE), (HIE), or (IVE), such that the compound comprises the optional spacer or extender group (E), the Ragroup of the structure also comprises the spacer or extender group (E).

[0106] According to a further embodiment, the compound (i.e. further comprising the spacer or extender group (E)) has the structure:

[0107]

[0108] Reference to the structure (I), (II), (III), or (IV) throughout the description below also encompass the embodiments wherein said structure or a combination of any two or more thereof further comprises the spacer or extender group (E) in accordance with structures (IE), (HE), (HIE), or (IVE) and / or where R3 further comprises the spacer or extender group (E) in accordance with structures (VE), (VIE), (VI IE) or (VI 11 E), as described above.

[0109] Liver Asialoglycoprotein Receptor-Targeted Therapeutic Agent

[0110] As is known in the art, the N-acetylgalactosamine moieties present in the structures of compounds (I), (II), (III), or (IV) bond to liver asialoglycoprotein receptors and thus the liver uptakes the drug or nucleic acid in a lipid nanoparticle, liposome or micelle which includes the compound of the structure (I), (II), (III), or (IV), or a combination of any two or more thereof via the asialoglycoprotein receptor (ASGPR) pathway.According to some embodiments, RNA or DNA may be delivered to the liver in lipid nanoparticles (LNP). The lipid chains present in the compounds having structures (I), (II), (III), or (IV) described above are part of the lipids in the formulation of LNP. Preferably, the outer layer of LNP will contain DG-PEG2K-triGalNAc with triantennary GalNAc (e.g.

[0111] Example 1 , below) targeting groups projecting out of the LNP. LNP with GalNAc will accumulate more on hepatic cell surfaces and achieve the targeting delivery effect.

[0112] According to an embodiment, a liver asialoglycoprotein receptor-targeted therapeutic agent comprising a drug or nucleic acid conjugated with and / or covalently bound to a compound having the structure (I), (II), (III), or (IV), or a combination of any two or more thereof is provided.

[0113] According to some embodiments, the compound having the structure (I), (II), (III), or (IV), or a combination of any two or more thereof may be in the form of a liposome.

[0114] It may be particularly preferable to form LNP and liposomes with the compounds having the structure (I), (II), (III), and / or (IV) (which are neutral form materials), as in these cases utilizing a molecular entity with a neutral charge is important to prevent interfering with the desired complexation between an ionizable or charged lipid and the payload; maintenance of an overall neutral charge is particularly desirable in the case of liposomes.

[0115] According to some embodiments, the compound having the structure (I), (II), (III), or (IV), or a combination of any two or more thereof may be in the form of a micelle.

[0116] According to some embodiments, the present compounds having structure (I), (II), (III), or (IV) are useful when used as part of liver asialoglycoprotein receptor-targeted therapeutic agents that are in the form of lipid nanoparticles (LNP) that are designed to specifically target the liver. As in known in the art, these compounds (I), (II), (III), or (IV) may be used in conjunction with other lipids to form the LNP that encapsulates a therapeutic agent that targets the liver. Non-limiting examples of other lipids that may be used in conjunction with compounds having structure (I), (II), (III), or (IV) are ionizable charged lipids, permanently charged lipids, neutral lipids, polyethylene glycol (PEG) lipids, and / or sterols.

[0117] Since the liver is able to synthesize proteins, delivery of various drugs or nucleic acids via these LNPs that include compounds having the structure (I), (II), (III), or (IV) are especially attractive, since the present compounds target the liver asialoglycoprotein receptor. Thus, these compounds can be useful to deliver gene therapy to a patient in need thereof.

[0118] Suitable drugs may be antibodies, enzymes, proteins, small molecules, nucleotides, and especially drugs that are used to treat diseases of the liver. These include but are notlimited to treatments for hepatitis A, B or C, medications for bile acid synthesis disorders, medications for cholestatic pruritus in alagille syndrome, medications for hepatic tumors, medications for hepatic coma, medications for hyperbilirubinemia, medications for variable causes of liver cirrhosis, medications for liver metastasis in adenocarcinoma, medications for metabolic dysfunction-associated steatotic liver disease, medications for pancreatic exocrine dysfunction, medications for portal hypertension, medications to treat glycogen storage disorders, or medications for progressive familial intrahepatic cholestasis, medications for liver cancer. Specific examples may include cholic acid, maralixibat, odevixibat, sorafenib, phenobarbital, ursodiol, colchicine, azathioprine, obeticholic acid, floxuridine, pioglitazone, orlistat, betaine, rosiglitazone, rosiglitazone, pancrelipase, propranolol, nadolol, odevixibat, doxorubicin. For example, many types of active pharmaceutical ingredient(s) (APIs) can be included in a lipid particle, but the drug properties will dictate different composition and processing conditions. Hydrophilic drugs can be loaded into a liposome’s aqueous core. Lipophilic drugs can be loaded into the liposomal bilayer or sequestered in a micelle. Anionic drugs (nucleic acids or proteins, for example) can be loaded into lipid nanoparticle. Both hydrophilic and hydrophobic drugs can be loaded into emulsion preparations that include the subject compounds. The present compounds, which are in a neutral from, may provide advantages over e.g. ionically charged lipids when utilized in LNPs which are loaded with nucleic acids or peptides that are charge sensitive, or they may be advantageously used in providing liposomes encapsulating small molecules which desirably maintain a neutral charge.

[0119] Different size nucleic acids coding for expression or suppression of proteins, or editing of bases, codons, etc. through variable actions may be included in therapeutic agents that utilize the subject compounds. For example, some suitable nucleic acids are ABE (adenine base editor), miRNA inhibitors, antisense nucleic acids, ribozymes, DNAzymes, plasmids, immune stimulating nucleic acids, antagomir, antimir, mimic, supermir, and aptamers, allele-specific oligonucleotide, antisense oligonucleotide, ribonucleoproteins (RNP), mRNA, siRNA, sgRNA, gRNA, ss-siRNA, amongst others.

[0120] According to some embodiments, CRISPR (clustered regularly interspaced short palindromic repeats) base editing therapy can be delivered to the liver via utilization of the N-acetylgalactosamine moieties present in the compounds of structures (I), (II), (III), or (IV).

[0121] The therapeutic agents may be formulated into a composition for administration to a patient in need thereof. A formulated composition can assume a variety of states. In some examples, the composition may be at least partially crystalline, uniformly crystalline, and / or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In another example, the formulation is in an aqueous phase, e.g., in a solution that includes water.The aqueous phase or the crystalline compositions can, e.g., be incorporated into a delivery vehicle, e.g., a lipid nanoparticle. Generally, the composition is formulated in a manner that is compatible with the intended method of administration.

[0122] In some embodiments, the liver asialoglycoprotein receptor-targeted therapeutic agent comprising a drug or nucleic acid in the form of a lipid nanoparticle, and / or in the form of a liposome including a compound having the structure (I), (II), (III), or (IV), or a combination of any two or more thereof may be prepared by at least one of the following methods: spray drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques; or sonication with a lipid, freeze-drying, condensation and other self-assembly.

[0123] The liver asialoglycoprotein receptor-targeted therapeutic agent comprising a drug or nucleic acid in the form of a lipid nanoparticle including a compound having the structure (I), (II), (III), or (IV); or a combination of any two or more thereof may be formulated into pharmaceutical compositions suitable for administration. The liver asialoglycoprotein receptor-targeted therapeutic agent comprising a drug or nucleic acid and the compound having the structure (I), (II), (III), or (IV) may also be in the form of a lipid nanoparticle or may be in the form of a liposome. As is known in the art, liposomes have one or more rings of lipid bilayer surrounding an aqueous pocket, but lipid nanoparticles usually only have a phospholipid outer layer that encapsulates a solid interior, which may be non-aqueous.

[0124] The therapeutic agent, e.g. drug and / or nucleic acid preparation can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar, multilamellar, and multivesicular vesicles that have a membrane formed from a lipophilic material comprising a compound having structure (I), (II), (III), or (IV), or a combination of any two or more thereof and an aqueous interior.

[0125] The interior aqueous or non-aqueous portion contains the therapeutic agent composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the therapeutic agent composition, although in some examples, it may. Liposomes, micelles and lipid nanoparticles are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal or lipid nanoparticle or micelle membrane is structurally similar to biological membranes, when liposomes, lipid nanoparticles or micelles are applied to a tissue, the exterior layer of the lipid particle fuses with bilayer of the cellular membranes. As the merging of the liposome, lipid nanoparticle, or micelle and cell progresses, the internal contents that include the therapeutic agentcomposition are delivered into the cell. As discussed herein, the compound having structure (I), (II), (III), or (IV) target the liver asialoglycoprotein receptor.

[0126] "Micelles" are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic in which inverted micelles are formed.

[0127] A micelle containing a drug can be prepared by a variety of methods. In one example, the lipid component is dissolved in a detergent, emulsifier, and / or surfactant so that micelles are formed with the lipid component. For example, the lipid component can include the present compound having structure (I), (II), (III), or (IV) or combination of any two or more thereof. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents, emulsifier, and / or surfactants include cholate, CHAPS (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate), octylglucoside, deoxycholate, and lauroyl sarcosine. The drug preparation is then added to the micelles that include the lipid component, to yield a micelle preparation of a drug.

[0128] A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the nucleic acid or drug composition, an alkali metal C8 to C22 alkyl sulphate, and micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof.

[0129] The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients, but vigorous mixing will provide smaller size micelles.

[0130] In one method a first micellar composition is prepared which contains the nucleic acid or drug composition and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the nucleic acid or drug composition, the alkali metal alkyl sulphate and at least one of the micellesforming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.

[0131] Phenol and / or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and / or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.

[0132] For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e., there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g., through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray. Propellants may include hydrogencontaining chlorofluorocarbons, hydrogen containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1, 1, 1,2 tetrafluoroethane) may be used.

[0133] The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.

[0134] Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al. Biochim. Bio phys. Acta 858: 161, 1986). Microfluidization, high pressure extrusion, high pressure homogenization or solvent injection can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired.

[0135] A lipid nanoparticle (LNP) is a vesicle formed of multiple lipid components and a nucleic acid, where a complex interaction forms between the nucleic acid and one or more of the lipidic components to create particle, which may have a solid core. Lipid nanoparticles may include up to four or more types of lipids. These may include neutral phospholipids (helper lipids), charged lipids, sterol, and polymeric lipids. Lipid nanoparticles may be prepared by mixing solvent / lipid mixture including the present compounds (I), (II), (III), or (IV) with an aqueous buffer solution containing protein(s), small molecules(s), enzyme(s) nucleic acid(s) and / or drug(s). A 1:3 ratio of lipid mixture to aqueous buffer is generally used. The general method is to combine the lipid elements with the therapeutic agent (nucleic acids, proteins, enzymes, or other drugs) under controlled conditions to precipitate lipid nanoparticles. Non-limiting examples of suitable solvents are those that are miscible withwater, including C1-C4 alcohols, methanol, ethanol, propanols, butanols, for example. Suitable aqueous buffers are those that maintain a pH in the correct buffering range for the lipids and cargo present.

[0136] Such compositions include the liver asialoglycoprotein receptor-targeted therapeutic agent comprising a drug or nucleic acid in the form of a lipid nanoparticle including a compound having the structure (I), (II), (III), or (IV), or as a liposome, or as a micelle and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

[0137] The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated.

[0138] Methods of T reatment

[0139] According to an embodiment, a method of treatment is provided. The method comprising administering the liver asialoglycoprotein receptor-targeted therapeutic agent to a patient in need thereof. Suitable administration to a subject in need thereof includes a variety of routes. Non-limiting examples include: parenteral, intravenous, topical, rectal, anal, vaginal, nasal, pulmonary, or ocular.

[0140] The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. The target of the present liver asialoglycoprotein receptor-targeted therapeutic agent is the liver, and the administration method should therefore be chosen to enhance uptake by the liver. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.

[0141] Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.

[0142] Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0143] Compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, elixirs or non-aqueous media, tablets, capsules, lozenges, or troches. In the case of tablets, carriers that can be used include lactose, sodium citrate andsalts of phosphoric acid. Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the liver asialoglycoprotein receptor-targeted therapeutic agent comprising a drug or nucleic acid in the form of a lipid nanoparticle, liposome or micelle including a compound having the

[0144] structure (I), (II), (III), or (IV) can be combined with emulsifying and suspending agents. If desired, certain sweetening and / or flavoring agents can be added.

[0145] According to some embodiments, compositions for intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers,

[0146] diluents and other suitable additives.

[0147] According to some embodiments, the administration of the pharmaceutical composition is parenteral, e.g., intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser which delivers a metered dose. Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers,

[0148] diluents and other suitable additives. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. For intravenous use, the total concentration of solutes may be controlled to render the preparation isotonic.

[0149] According to some embodiments the administration of the pharmaceutical composition is intramuscular.

[0150] The invention will now be described by reference to non-limiting examples.

[0151] EXAMPLES

[0152] Example 1: Preparation of a compound of structure 1 (C18:0 DG-PEG2K-triGalNAc)

[0153]

[0154] Experimental procedure:

[0155] Scheme 1 - Synthesis of C18 DG PEG2K-NHS

[0156] Et3N, DMAP •o OH 'O OMs CH2CI2O°C- rt, 1 h NaN3, DMF. 70°C 24 h

[0157]

[0158] 10 Synthesis of (R)-2,3-bis(octadecyloxy)propyl methanesulfonate (5):

[0159] To a solution of (S)-2,3-bis(octadecyloxy)propan-1-ol (C18 DG) 4 (25 g, 41.9 mmol) in dichloromethane (200mL) at 0°C under nitrogen was added triethyl amine (TEA) (23.34 ml, 167 mmol) and 4-dimethylaminopyridine (DMAP) (0.512 g, 4.19 mmol). To this solution was added a solution of methane sulfonyl chloride (6.48 ml, 84 mmol) in dichloromethane (DCM) (40 mL)) and the stirring was continued at 0°C for 10 minutes. The mixture was allowed to warm to room temperature and stirred for 1 hour. The contents of the flask were diluted with cold water (20 mL). The organic phase was separated, washed with 1 N HCI (10 mL), water, brine, dried over anhydrous sodium sulphate (Na2SO4> and evaporated to obtain mesylate (5) quantitatively as a white powder. This material (5) was pure enough to use for the next step without further purification. 1 H NMR (400 MHz, CDCI3) 54.39 (dd, J = 3.6 Hz, 1H), 4.28 (dd, J = 5.7 Hz, 1H), 3.71-3.66 (m, 1H), 3.58 (t, J = 6.7 Hz, 2H), 3.56-3.48 (m, 2H), 3.47-3.43 (m, 2H), 3.06 (s, 3H), 1.62-1.53 (m, 4H), 1.28 (s, 60H), 0.9 (t, J = 13.7 Hz, 6H);

[0160] Synthesis of (S)-1-(3-azido-2-(octadecyloxy) propoxy) octadecane (6):

[0161] Mesylate (5) (28g, 41.5 mmol) and sodium azide (4.04g, 62.2 mmol) were dissolved in anhydrous dimethylformamide (DMF) (940 mL) and the resulting suspension heated to 70°C with continued stirring under nitrogen. Heating was continued at 70°C for 24 hours.

[0162] Subsequently, the reaction mixture was allowed to cool and diluted with diethyl ether andwashed with water (4x200 mL); the combined organic phase was then washed with brine, dried over anhydrous Na2SC , and filtered. The resultant filtrate was evaporated and dried over pump to obtain corresponding azide (6) quantitatively as a white powder. This material (6) was pure enough to use for the next step without further purification. 1 H NMR (400 MHz, CDCI3) 53.63-3.50 (m, 4H), 3.47-3.43 (m, 3H), 3.4-3.31 (m, 2H), 1.63-1.53 (4H), 1.27 (s 60 H), 0.90 (t, J = 13.7 Hz, 6H).

[0163] Synthesis of (S)-2,3-bis(octadecyloxy)propan-1-amine (7):

[0164] The azide (6) (26g, 41.8 mmol) was dissolved in a 4:1 mixture of tetrahydrofuran THF:HOAc (100 mL) and the reaction mixture cooled to 0°C before the addition of zinc (32.8g, 502 mmol) portion wise. The mixture was stirred vigorously at room temperature (RT) for 4 to 6 hours. The progress of the reaction was checked by thin layer chromatography (TLC) (chloroform: methanol 8:2, Rf 0.6). Upon completion, the reaction mixture was diluted with chloroform (200 mL) and filtered through Celite™(ex. Imerys S.A., Paris, France) and washed with chloroform (2x100 mL) and methanol (2x100 mL). The resulting amine containing filtrate was concentrated and dried over pump. Subsequently the crude amine was purified over silica gel column chromatography using chloroform and methanol as eluents to obtain the amine (7) (21g, 84% yield). 1 H NMR (400 MHz, CDCI3) 5 3.66-3.60 (m, 1H), 3.52-3.48 (m, 2H), 3.46-3.38 (m, 4H), 2.90 (dd, J - 3.9 Hz, 1H), 2.78 (dd, J = 6.4 Hz, 1H), 1.59-1.54 (m, 4H), 1.27 (s, 60H), 0.9 (t, J =13.7 Hz, 6H).

[0165] Synthesis of (S)-55-(octadecyloxy)-52-oxo-49l5,50l5-3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 57-pentatetracontaoxa-53-azapentaheptacont-49-enoic acid-ethene (1 / 41) (9):

[0166] PEG2K-Diacid (8) (85g, 41.9 mmol) was dissolved in toluene heated to 70°C under a nitrogen atmosphere. When dissolved completely, carbonyl diimidazole CDI (6.8 g, 41.9 mmol) was added in portion wise and stirred for 10 minutes. Subsequently amine (7) as described above (21g, 35.6 mmol) was added and stirred at 70°C for 24 hours. The progress of the reaction was checked by thin layer chromatography (TLC) (chloroform: methanol 8:2, Rf 0.6). Upon completion, solvent was removed and partition with ethyl acetate and 1N HCI (100 mL), washed with water, dried over anhydrous NazSC and filtered. The filtrate was evaporated and dried over pump. Subsequently, the crude product was purified over reverse phase silica gel column chromatography using methanol and water as eluents to obtain (9) (44g, 40%) as a white solid. 1H NMR (400 MHz, CD3OD) 57.87 (t, J = 5.3 Hz, 1H, NH), 4.12 (s, 2H), 4.01 (s, 2H), 3.84-3.82 (m, 1H), 3.70-3.65 (m, 174 H), 3.51-3.46 (m, 6H), 1.61-1.56 (m, 4H), 1.31 (s, 60H), 0.9 (t, J = 13.7 Hz, 6H).Synthesis of 2,5-dioxopyrrolidin-1-yl (S)-55-(octadecyloxy)-52-oxo-4915,5015-3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 57-pentatetracontaoxa-53-azapentaheptacont-49-enoate-ethene (1 / 41) (10):

[0167] C18 DG-PEG2K-acid (9) (15g, 5.75 mmol) was dissolved in 300 ml anhydrous dichloromethane / acetonitrile (DCM / CAN) (8:2) and added DMAP (0.141g, 1.151 mmol), followed by N, N'-disuccinimidyl carbonate (DSC) (1.92 g, 7.48 mmol), and stirred at room temperature (RT) for 24 hours. The progress of the reaction was monitored by NMR. In doing so, a little aliquot was dried and run 1H-NMR in anhydrous Deuterated chloroform (CDCI3). The level of activation was measured by integrating the peak at -4.5 ppm (S, 2H). If less than 90% conversion, an additional amount of DSC (10%) and DMAP (10%) were added and stirred for 1h. The progress of the reaction was checked by thin layer chromatography (TLC) (chloroform: methanol 8:2, Rf 0.6). Upon completion, the solvent was removed and redissolved in DCM (300 mL) and washed with saturated brine (50 mL), dried over anhydrous Na2SO4 and filtered. The filtrate was evaporated and cycle with toluene (3 x 100 mL) and dried over pump to obtain (10) (14.4 g, 90%) as white powder. Stored under nitrogen at-20°C and checked NMR purity (85-90%) before using the next step. 1H NMR (400 MHz, CDCI3) 54.55 (s, 2H), 4.02 (s, 2H), 3.85-3.81 (m, 3H), 3.73-3.72 (m, 3H), 3.68-3.66 (m, 169 H), 3.52-3.41 (m, 6H), 2.88 (s, 4H), 1.60-1.53 (m, 4H), 1.27 (s, 60 H), 0.90 (t, J = 13.7 Hz, 6H).

[0168] Scheme 2. Synthesis of Z-Lys-tri-GalNAc

[0169] Scandium triflate, 1 ,2-Dichloroethane, TEA

[0170]

[0171] 90 °C, 3 hrs 11 13

[0172] 3% KOH in Methanol, 25 °C drop pH to 75 with acidic resin

[0173]

[0174]

[0175] Synthesis of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(2-(2-(2-azido-ethoxy)ethoxy)ethoxy)tetrahvdro-2H-pyran-3,4-diyl diacetate (13):

[0176] Tetraacetate GalNAc (11) (60.0 g, 154 mmol) and azido-Gly-alcohol (12) (29.7g, 170 mmol) were suspended in 1,2-dichloroethane (1.7 L) in a dry 3 litter round bottom flask and scandium triflate (4.55 g, 9.25 mmol) was added to the reaction flask. The reaction flask was warmed to 90° C with stirring for 3 hours under nitrogen and checked the progress by TLC (chloroform: methanol, 93:7, Rf 0.5). The reaction mixture was cooled to 10°C and quenched with TEA (86 mL, 616 mol) and concentrated. The crude product was purified via silica flash column chromatography to obtain product (13) Azido-Gly-GalNAc (54.4 g, 70%) as a paleyellow powder. 1H NMR (400 MHz, CDCI3) 52.01 (s, 3H), 2.03 (s, 3H), 2.07 (s, 3H), 2.18 (s, 3H), 3.49 (t, J = 4.8 Hz, 2H), 3.65-3.75 (m, 9H), 3.85-3.95 (m, 3H), 4.13-4.29 (m, 3H), 4.81 (d, J = 8.5 Hz, 1H), 5,08 (dd, J = 3.4, 3.4 Hz, 1H), 5.34 (s,1H, NH).

[0177] Synthesis of N-((2R,3R,4R,5R,6R)-2-(2-(2-(2-azidoethoxy) ethoxy )ethoxy)-4,5-dihydroxy-6-(hvdroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (14):

[0178] Azido-Gly-GalNAc (13) (10.0 g, 19.8 mmol) was dissolved in 3% potassium hydroxide solution in methanol (methanolic KOH solution) (4.45 g, 79.0 mmol) and stirred for 3 hours and checked the status of the progress via TLC (chloroform: methanol: ammonium hydroxide: 65:30:6, Rf 0.3). Then the reaction mixture was neutralized with strong acidic resin to pH 7.5. The resin was filtered off, washed with methanol, and filtrate was concentrated in rotary evaporator for the subsequent column chromatography. The crude product was then purified via silica flash column chromatography to obtain Azido-GalNAc (14) (6.0 g, 80%) as a colorless gel. 1 H NMR (400 MHz, CD3OD) 52.00 (s, 3H), 3.40 (t, J = 5.1 Hz, 2H), 3.51 (t, J = 5.6 Hz, 1H), 3.60 (dd, J = 3.3, 3.4 Hz, 1H), 3.65-3.71 (m, 9H), 3.72-3.82 (m, 3H), 3.84 (d, J = 3.0 Hz, 1H), 3.90-4.00 (m, 2H); MS (ESI) m / z calculated for C20H34N2O11 (M+H)+ : 479.50; found 478.0.

[0179] Synthesis of N-((2R,3R,4R,5R,6R) -2-(2-(2-(2-aminoethoxy) ethoxy)ethoxy)-4,5-dihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-3-yl) acetamide (15):

[0180] In a 400 ml parr glass vessel, Azido-GalNAc (14) (5.20 g, 13.4 mmol) was dissolved in ethanol / HzO (4:1, 100 mL) and purged with nitrogen, subsequently 10% palladium on carbon (360 mg) was added to the vessel. The product was then hydrogenated for 12 hours at room temperature (RT). The mixture was filtered through the bed of Celite and concentrated. The crude was used for the next step without further any purification, yield (15) (4.8 g, quantitative) as a colorless gel. 1H NMR (400 MHz, D2O) 5 1.98 (s, 3H), 2.56-2.57 (m,3H), 3.52-3.66 (m, 12H), 3.67-3.90 (m, 3H), 4.33 (d, J = 4 Hz, 1H).Synthesis of Benzyl (1-(((2S,3S,4S,5S,6S)-3-acetamido-4,5-dihvdroxy-6-(hvdroxymethyl) -tetrahydro-2H-pyran-2-yl)oxy)-12-(2-((2-(2-(2-(((2S,3S,4S,5S, 6S)-3-acetamido-4,5-dihvdroxy-6-(hvdroxymethyl)tetrahvdro-2H-pyran-2-yl)-oxy)ethoxy)ethoxy) ethvDamino)- 2-oxoethyl)-13-((2-(2-(2-(((2S,3S,4S,5S,6S)-3-acetamido-4,5-dihvdroxy-6-(hvdroxym ethyl) tetrahvdro-2H-pyran-2-yl)oxy)-ethoxy)ethoxy)ethyl)carbamoyl)-10-oxo-3,6-dioxa-9, 12-diazaheptadecan-17-yl)carbamate (17):

[0181] Cbz-Lysine-Triacid (2,2'-((5-(((benzyloxy)carbonyl) amino)-1-carboxypentyl)azanediyl)diacetic acid) (16) (1.4 g, 3.5 mmol) was dissolved in dry DMF and N-hydroxy succinimide (NHS) (1.6 g, 14.1 mmol) was added to the solution and stirred for 10 minutes, then N,N'-dicyclohexylcarbodiimide (DCC) (2.33 g, 11.3 mmol) was added to the mixture and stirred for 12 to 24 hours at 50°C. Amine-GalNAc (15) (4.1 g, 11.6 mmol) and TEA (2 mL, 14.0 mmol) were added and stirred the reaction mixture at 50°C for 24 hours. The progress of the reaction was checked by TLC (chloroform: methanol: water: 50:40:10, Rf 0.3) and solvent was removed under reduced pressure. The crude product was purified by silica flash column chromatography to obtain Z-Lys-tri-GalNAc (17) (1.25 g, 51%) as a waxy white solid.1H NMR (400 MHz, CDCI3:CD3OD, 8:2) 5 1.23-1.43 (m, 2H), 1.42-1.51 (m, 2H), 1.52-1.70 (m, 2H), 2.01 (s, 9H), 3.07-3.16 (m, 3H), 3.21-3.28 (m, 1H)3.36-3.43 (m, 6H), 3.44-3.70 (m, 36H), 3.71-3.80 (m,12H), 3.81-3.95 (m, 3H) 4.42-4.50 (m, 3H), 5.03 (s, 2H), 7.31 (s, 5H).

[0182] Scheme 3. Synthesis of C18:0 DG-PEG2K-triGalNAc

[0183]

[0184] Synthesis of 2,2'-((1-((2-(2-(2-(((2S,3S,4S,5S,6S)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)amino)-6-amino-1-oxohexan-2-yl)azanediyl)bis(N-(2-(2-(2-(((2S,3S,4S,5S,6S)-3-acetamido-4,5-dihydroxy-6-(hvdroxymethyl)tetrahvdro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)acetamide) (18):

[0185] In a 400 ml parr glass vessel, Z-Lys-tri-GalNAc (17) (2.00 g, 1.43 mmol) was dissolved in ethanol / H2O (4:1, 60 mL) and purged with nitrogen, 10% palladium hydroxide on carbon (100 mg) was added to the vessel. The mixture was hydrogenated for 12 hours at room temperature (RT). The mixture was filtered through the bed of Celite and concentrated. The crude was used for the next step without any purification, yield (18) (1.75 g, quantitative) as a semi solid. 1 H NMR (400 MHz, CDCI3:CD3OD, 8:2) 5 1.30-1.61 (m, 3H), 1.63-1.79 (m, 3H), 2.01 (s, 9H), 2.94-3.01 (m, 1H), 3.23-3.37 (m, 2H), 3.38-3.47 (m, 6H), 3.48-3.81 (m, 44H), 3.83-3.88 (m, 3H), 3.91-4.01 (m, 5H), 4.39-4.48 (m, 3H).TOF m / z calculated for C52H96N8O27 (M+H)+ : 1265.64; found 1265.6448.

[0186] Synthesis of 2,2'-(((70S)-1-(((2S,3S,4S,5S,6S)-3-acetamido-4,5-dihydroxy-6-(hvdroxymethyl)tetrahvdro-2H-pyran-2-yl)oxy)-70-(octadecyloxy)-10,17,21 ,67-tetraoxo-3, 6, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 65, 72-hexatetracontaoxa-9,16,68-triazanonacont-19-en-11-yl)azanediyl) -bis(N-(2-(2-(2-(((2S,3S,4S,5S,6S)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)acetamide)-ethene (1 / 41) (17):

[0187] C18 DG PEG2K-NHS (10) (2.65g, 2.1 mmol) and NH2-Lysine-tri-GalNAc (18) (8.2 g, 2.72 mmol) in DMF (50 mL) under nitrogen was added TEA (0.636 g, 6.28 mmol) and stirred at RT for 24 hours. The progress of the reaction was monitored by TLC (65:35:8 -chloroform: methanol: water). Solvent was evaporated and dried. The crude product was purified by three consecutive columns (Normal Phase / Reverse Phase / Normal Phase) to obtain the pure product C18:0 DG-PEG2K-triGalNAc (19) (2.7 g, 34%) as a white powder.

[0188] 1 H NMR (400 MHz, CD3OD) 54.46 (d, J - 8.43 Hz, 3H), 4.01 (s, 2H), 40 (s, 2H), 3.97-3.92 (m, 6H), 3.85 (d, J = 3.08 Hz, 3H), 3.84-3.81 (m, 2H), 3.79-3.76 (m, 5H), 3.73-3.57 (m, 202H), 3.54-3.50 (m, 6H), 3.49-3.46 (m, 6H), 3.45-3.43 (m, 6H), 3.34-3.22 (m, 6H), 2.01 (s, 9H), 1.70-1.54 (m, 8H), 1.31 (brs, 62H), 0.92 (t, J = 13.7 Hz, 6H); ESI-MS: m / z 3829.572 (PD 1.002).

[0189] The final product C18:0 DG-PEG2K-triGalNAc (19) was freeze-dried from tBuOH / Water (97:3) to obtain fine white powder. The HPLC purity of this final product was 99.82% and the HPLC chromatogram is shown in Figure 1.

[0190] HPLC conditions:Chromatographic conditions of C18:0-DG-PEG2K-TriGalNAc (19): Detector: evaporative light scattering detector (ELSD), concentration: 1.Omg / mL (delusion with methanol), gradient: solvent A (80:20:0.5:0.5 Methanol:Water:Ammonium Acetate:Acetic Acid) with solvent B (100:0.5 MethanokAmmonium Hydroxide), injection volume = 10 pL. Flow rate = 1.0 mL / min. Column = Phenomenex Luna-C18, 5 pm 4.6 x 250 mm, column temperature: 60 °C and detector temperature: 50°C.

[0191] Example 2 Preparation of Lipid Nanoparticles:

[0192] 100 mg 18:1 1 ,2-dioleoyl-3-trimethylammonium-propane (TAP), 23.7 mg 18:0 PC, 46.5 mg cholesterol, and 30 mg of the Example 1 compound (19) are mixed together in ethanol at 25 mg / mL. cDNA is dissolved in RNAse free phosphate-buffered saline (PBS) (pH=7.4). The two solutions are mixed together using microfluidics to produce lipid nanoparticles. Ethanol is removed by solvent exchange with RNAse free buffer (pH=7.4).The resultant lipid nanoparticles are 70-1 OOnm in diameter.

[0193] Example 3 Preparation of Lipid Nanoparticles:

[0194] 141.5 mg 1,2-dioleyloxy-3-dimethylaminopropane(DODMA), 72.8 mg cholesterol, 37.2 mg 18:0 1,2-distearoyl-sn-glycero-3-phosphocholine (PC), and 28.4 mg of the Example 1 compound (19) are dissolved in ethanol at 20 mg / mL. mRNA is dissolved in 100 mM citrate buffer, pH=4. The two solutions are combined under controlled mixing using microfluidics. Ethanol is removed and buffer pH is adjusted by dialysis.

[0195] Example 4 Preparation of Liposomes:

[0196] 5.4 grams HSPC L-alpha-phosphatidylcholine, hydrogenated (HSPC), 1.8 grams cholesterol, 1.6 grams of DSPE-PEG2000, and 0.2 grams of Example 1 compound (19) are dissolved in chloroform. Chloroform is removed by rotary evaporation and further removed under vacuum to create a thin film. Lipid film is hydrated with 250 mM ammonium sulfate buffer (pH=5.4) at 25 mg / mL at 60°C. Hydrated lipids are extruded to a final particle size 80-120nm. Buffer is exchanged for phosphate buffered saline (pH=7.4) by tangential flow filtration. Separately, doxorubicin hydrochloride salt is dissolved in PBS (phosphate buffered saline) at elevated temperature, then combined with liposome solution and incubated at 60°C. Unencapsulated doxorubicin is removed, and buffer exchanged for 10 mM histidine with 10% sucrose.

[0197] Example 5 Preparation of Liposomes:

[0198] 29.8 mg 18:0 PC , 11 mg cholesterol, 10 mg of the Example 1 compound (1) and 10 mg of a hydrophobic API (active pharmaceutical ingredient) are mixed together inchloroform. Chloroform is removed under nitrogen to create a thin film. Lipid / API film is hydrated in phosphate-buffered saline (pH=7.4) and extruded to a size 115 nm.

[0199] Unencapsulated API is removed by size exclusion.

[0200] Example 7 Preparation of Micelles:

[0201] 60 mg of soy phosphotidylcholine, 40 mg of the Example 1 compound (19), and 10 mg doxorubicin hydrochloride are dissolved in chloroform. Solvent is removed under nitrogen. Lipids are hydrated with phosphate-buffered saline and sonicated to achieve micelles. Micelles are prepared of between 15 and 35 nm in diameter.

[0202] Within this specification embodiments have been described in a way which enables a clear and concise specification to be written, but it is intended and will be appreciated that embodiments may be variously combined or separated without departing from the invention. For example, it will be appreciated that all preferred features described herein are applicable to all aspects of the invention described herein.

[0203] The foregoing description of various forms of the invention has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Numerous modifications or variations are possible in light of the above teachings. The forms discussed were chosen and described to provide the best illustration of the principles of the invention and its practical application to thereby enable one of ordinary skill in the art to utilize the invention in various forms and with various modifications as are suited to the particular use contemplated. All such modifications and variations are within the scope of the invention as determined by the appended claims when interpreted in accordance with the breadth to which they are fairly, legally, and equitably entitled.

Claims

What is claimed is:

1. A composition comprising a compound having the structure (I), (II), (III), or (IV); or a combination of any two or more thereof:AcHNwherein:each R1is independently selected from aliphatic alkyl C4-C100 groups optionally substituted with one or more of alkenyl, alkynyl, hydroxyl, amide, ester, and / or ether groups;R2is selected from [-OCH2CH2-]P, [-OCH2CH2-]P-O-, oru J s; R3is selected from Formula (V), (VI), (VII), (VIII), (VE), (VIE), (VIIE), and (VINE):HN(VII)(VIII)R4is an alkyl chain selected from [-CH2-]t;m, p, s and t are independently selected from integers from 1 to 120, and wherein E is an optional spacer or extender group.

2. The compound according to claim 1, wherein the compound includes at most 15 N-acetylgalactosamine moieties per molecule.

3. The compound according to claim 1 or claim 2, wherein R1is an unsubstituted aliphatic alkyl C4-C100 group.

4. The compound according to any of claims 1-3, wherein R2is [-OCH2CH2-]P, [- OCH2CH2-]P-O-, or [-OCH2CH2-]P-O-.

5. The compound according to any of claims 1-3, wherein R2iss6. The compound according to any of claims 1-5, wherein p is an integer selected from 10 to 80, preferably from 20 to 70, more preferably from 30 to 60, most preferably from 40 to 50.

7. The compound according to any of claims 1-5, wherein s is an integer selected from 10 to 80, preferably from 20 to 70, more preferably from 30 to 60, most preferably from 40 to 50.

8. The compound according to any of claims 1-7, wherein m is an integer selected from 1 to 50, preferably from 1 to 20, more preferably from 1 to 10, most preferably from 2 to 5.

9. The compound according to any of claims 1-8, wherein t is an integer from 1 to 120, preferably 1 to 45, even more preferably 1 to 20, and most preferably 1 to 5.

10. The compound according to any of claims 1-9, wherein the structure (I), (II), (III), or (IV) further comprising a spacer or extender group (E), where E has the structure:oand wherein, n is an integer between the value 0 and 6, and X is an alkyl chain of between 0 and 4 carbons in length.

11. The compound according to claim 10, comprising the structure (IE), (HE), (HIE), or (IVE); or a combination of any two or more thereof:AcHN12. The compound according to claim 10 or 11 , wherein R3according to claim 1 necessarily comprises a spacer or extender group (E), such that R3is selected from Formula (VE), (VIE), (VIIE), and (VI HE),and wherein E is:owherein, n is an integer between the value 0 and 6, and X is an optional alkyl chain of between 0 and 4 carbons in length.

13. A pharmaceutical composition comprising the composition of any one of claims 1 to 12, an active pharmaceutical ingredient and pharmaceutically acceptable carrier.

14. A liver asialoglycoprotein receptor-targeted therapeutic agent comprising a drug nucleic acid, antibody, enzyme, protein, small molecule, or nucleotide, in a form of a lipid nanoparticle, or in a form of a liposome, or in a form of a micelle comprising a composition in accordance with any one of claims 1 to 12.

15. The liver asialoglycoprotein receptor-targeted therapeutic agent according claim 14, wherein the drug comprises at least one of medications for bile acid synthesis disorders, medications for cholestatic pruritus in alagille syndrome, medications for hepatic tumors, medications for hepatic coma, medications for hyperbilirubinemia, medications for liver cirrhosis, medications for liver metastasis in adenocarcinoma, medications for metabolic dysfunction-associated steatotic liver disease, medications for pancreatic exocrine dysfunction, medications for portal hypertension, or medications for progressive familial intrahepatic cholestasis.

16. The liver asialoglycoprotein receptor-targeted therapeutic agent according to claim 14 or 15, wherein the nucleic acid comprises at least one of ABE (adenine base editor), miRNA inhibitors, antisense nucleic acids, ribozymes, DNAzymes, plasmids, immune stimulating nucleic acids, antagomir, antimir, mimic, supermir, and aptamers, allele-specific oligonucleotide, antisense oligonucleotide, ribonucleoproteins (RNP), mRNA, siRNA, sgRNA, gRNA, ss-siRNA, or combinations thereof.

17. A pharmaceutical composition comprising the liver asialoglycoprotein receptor- targeted therapeutic agent according to any of claims 14 to 16 and a pharmaceutically acceptable carrier.

18. A method of treatment, comprising administering the liver asialoglycoprotein receptor-targeted therapeutic agent or pharmaceutical composition according any of claims 13 to 16 to a patient in need thereof.

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