Piperazine based divalent ionizable lipids and their uses

Abstract

A compound having the structure (I) is provided. The compound is useful as a therapeutic agent in the form of a lipoplex, or in a form of a polyplex, in a form of a lipid nanoparticle, or in a form of a liposome, or in a form of a micelle, or in a form of an emulsion including a drug, vaccine, oligonucleotide, antibody, enzyme, protein, small molecule, or nucleotide.

Description

PIPERAZINE BASED DIVALENT IONIZABLE LIPIDS AND THEIR USESCROSS REFERENCE TO PRIOR APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 663,524 filed June 24, 2024, the contents of which are incorporated by reference herein in their entirety for all purposes.FIELD OF THE INVENTION

[0002] The invention relates to ionizable lipids useful for forming lipid nanoparticles, emulsions, liposomes, lipoplexes, and polyplexes.BACKGROUND

[0003] Lipid compounds are used to prepare delivery systems for drug, oligonucleotide, antibody, enzyme, protein, small molecule, or nucleotide. The delivery systems may encompass lipid nanoparticles, lipoplex, or in a form of a polyplex, in a form of a lipid nanoparticle, or in a form of a liposome, or in a form of a micelle. The delivery system together with the drug, oligonucleotide, antibody, enzyme, protein, small molecule, or nucleotide thus form a therapeutic agent. It is desirable for such therapeutic agents to be non-toxic, while at the same time effectively delivering the drug, oligonucleotide, antibody, enzyme, protein, small molecule, or nucleotide in order to achieve the desired therapeutic effect. In case of oligonucleotides and nucleotides, for example, the objective is to induce a cell to express a desired protein, without causing harm to the target cell or other cells in the organism.

[0004] Other lipids that may be used in such delivery systems are those referred to as ALC-0315 lipids (e.g. [(4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate)),which are described in WO 2018 / 08081480 A1. These lipids suffer from higher than desirable cell toxicity and lower encapsulation efficiency of the therapeutic agents, as well as a reduced protein expression from targeted cells, if used to deliver a nucleotide or oligonucleotide to the cells. Accordingly, there remains a need for alternative lipids that simultaneously provide low cell toxicity and high encapsulation efficiency of the drug, oligonucleotide, antibody, enzyme, protein, small molecule, or nucleotide. SUMMARY

[0005] The present disclosure is directed to lipid compounds, especially ionizable cationic lipid compounds that are low toxicity and according to an embodiment may provide increased protein expression in the target cells when used as part of a composition to deliver nucleic acids to a cell. The lipid compounds may also provide reduced toxicity and improved encapsulation efficiency. The improvements may be due to spatial geometry of the lipid head group and fatty acid chains for improved delivery of nucleic acids, drugs, oligonucleotides, antibodies, enzymes, proteins, or small molecules. The lipid compounds are thus useful for delivery of therapeutic agents comprising a drug, oligonucleotide, antibody, enzyme, protein, small molecule, or nucleotide, to a person in need thereof.

[0006] It has been found that in lipid nanoparticle (LNP) formulations, cationic ionizable lipids may complex with negatively charged DNA or RNA. Aside from playing a major role in the LNP formulation itself, cationic lipids also serve a major role in the biological delivery of DNA or RNA to target cells.

[0007] Cationic lipids are able to form aggregate complexes with anionic genetic materials such as DNA or RNA. When in aqueous solution, these aggregated liposomal structures have a positive surface charge, mediating the interaction of RNA or DNA and the cell membrane. The positive charge of the liposome thus prevents the cell from distinguishing it from the cell membrane, enabling introduction of DNA or RNA to the cell and thereby improving the expression of protein from the call.

[0008] According to some embodiments, the inclusion of the lipids disclosed herein may be used in a human or veterinary therapeutic agent, such as a vaccine, drug, oligonucleotide, antibody, enzyme, protein, small molecule, or nucleotide.

[0009] The inclusion of the lipids disclosed herein may also be used for agriculture applications, for delivery of sensitive compounds before release. For example, liposomes, lipid nanoparticles, micelles, lipoplexes polyplexes, or emulsions may be used to slow, delay, or meter delivery of seed enhancement or pest control agents, for example.

[0010] According to a particular embodiment, the lipid compounds may be used to form lipid nanoparticles that are the therapeutic agent that delivers a drug, vaccine, oligonucleotide, antibody, enzyme, protein, small molecule, or nucleotide, to a person in need thereof. According to another embodiment, the lipid compounds may be used to form liposomes that deliver the drug, vaccine, oligonucleotide, antibody, enzyme, protein, small molecule, or nucleotide, to a person in need thereof. According to another embodiment, the lipid compounds may be used to form micelles that deliver the drug, vaccine, oligonucleotide, antibody, enzyme, protein, small molecule, or nucleotide, to a person in need thereof. According to other embodiments, the lipid compounds may be used to form lipoplexes that deliver the drug, vaccine, oligonucleotide, antibody, enzyme, protein, small molecule, or nucleotide, to a person in need thereof. According to other embodiments, the lipid compounds may be used to form polyplexes that deliver the drug, vaccine, oligonucleotide, antibody, enzyme, protein, small molecule, or nucleotide to a person in need thereof. According to other embodiments, the lipid compounds may be used to form emulsions that deliver the drug, vaccine, oligonucleotide, antibody, enzyme, protein, small molecule, or nucleotide, to a person in need thereof.

[0011] According to an embodiment, the compounds have the structure (I) or stereoisomers thereof or a salt or ion thereof, or a combination of any two or more thereof.where: each R1is CH3 or together are -[CH2]2-; R2is selected from -[CH2]-, -S-(C=O)-, and -S-(C=S)-; R3, R4, R5, and R6are each independently selected from -O-(C=O)-, -(C=O)-O-, -NH-(C=O)-, and -C(=O)-NH-; X is selected from OH, -CH3, NH2, and SH; V, W, Y, and Z are independently selected from H, aliphatic alkyl C4-C100 groups optionally substituted with one or more of alkenyl, alkynyl, hydroxyl, amide, ester, and / or ether groups, with the proviso that V, W, Y, and Z are not all H; n, p, and r are independently selected integers from 1 to 8; and m, q, and s are independently selected integers from 0 to 8.

[0012] A therapeutic agent comprising a drug or oligonucleotide in a lipid nanoparticle, a liposome or a micelle including a compound having the structure (I) or stereoisomers thereof or a salt or ion thereof, or a combination of any two or more thereof is provided.

[0013] A method of treatment, comprising administering the therapeutic agent to a patient in need thereof is also provided.DETAILED DESCRIPTION Definitions

[0014] As 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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 or female or both, or exclude male or female.

[0020] 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.

[0021] 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 includes straight chain alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl and the like. The phrase 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, —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, —CH(CH3)CH(CH3)(CH2CH3), —CH2CH2CH(CH3)2, — CH2CH2CH(CH3)(CH2CH3)—, CH2CH2CH(CH2CH3)2, —CH2CH2C(CH3)3, — CH2CH2C(CH2CH3)3, —CH(CH3)CH2CH(CH3)2, —CH(CH3)CH(CH3)CH(CH3)CH(CH3)2, — 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] As used herein, the terms “unsubstituted alkyl,” “unsubstituted alkenyl” and “unsubstituted alkenyl” refer to alkyl, alkenyl and alkynyl groups that do not contain heteroatoms.

[0026] 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 triarylsilyl groups; 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. Compounds

[0027] According to an embodiment, a compound having a structure (I) or stereoisomers thereof or a salt or ion thereof, or a combination of any two or more thereof is provided.CDA-501WO Patent (I)

[0028] In this structure (I), each R1is CH3 or together are -[CH2]2-; R2is selected from - [CH2]-, -S-(C=O)-, and -S-(C=S)-; R3, R4, R5, and R6are each independently selected from -O- (C=O)-, -(C=O)-O-, -NH-(C=O)-, and -C(=O)-NH-; X is selected from OH, -CH3, NH2, and SH; V, W, Y, and Z are independently selected from H, aliphatic alkyl C4-C100 groups optionally substituted with one or more of alkenyl, alkynyl, hydroxyl, amide, ester, and / or ether groups, with the proviso that V, W, Y, and Z are not all H; n, p, and r are independently selected integers from 1 to 8; and m, q, and s are independently selected integers from 0 to 8.

[0029] This structure covers any chirality of the asymmetric carbons or mixtures thereof.

[0030] According to some embodiments, each R1together may be -[CH2]2-; i.e., they form a ring. According to some embodiments, each R1may be -CH3.

[0031] According to some embodiments, X may be -OH. According to some embodiments X may be -NH2. According to some embodiments, X may be -SH. According to some embodiments, X may be -CH3.

[0032] According to some embodiments, R2may be -CH2-. According to some embodiments, R2may be –S-(C=O)-. According to some embodiments, R2may be -S-(C=S)-.

[0033] According to some embodiments, V W, Y, and Z may be independently selected from H and aliphatic alkyl C4-C100 groups optionally substituted with one or more of alkenyl, alkynyl, hydroxyl, amide, ester, and / or ether groups, with the proviso that V, W, Y, and Z are notall H. According to some embodiments, V W, Y, and Z may be independently selected from H and aliphatic alkyl C4-C75 groups optionally substituted with one or more of alkenyl, alkynyl, hydroxyl, amide, ester, and / or ether groups, with the proviso that V, W, Y, and Z are not all H. According to some embodiments, V W, Y, and Z may be independently selected from H and aliphatic alkyl C4-C100 groups optionally substituted with one or more of alkenyl, alkynyl, hydroxyl, amide, ester, and / or ether groups, with the proviso that V, W, Y, and Z are not all H. According to some embodiments, V W, Y, and Z may be independently selected from H and aliphatic alkyl C4-C50 groups optionally substituted with one or more of alkenyl, alkynyl, hydroxyl, amide, ester, and / or ether groups, with the proviso that V, W, Y, and Z are not all H. According to some embodiments, V W, Y, and Z may be independently selected from H and aliphatic alkyl C4-C100 groups optionally substituted with one or more of alkenyl, alkynyl, hydroxyl, amide, ester, and / or ether groups, with the proviso that V, W, Y, and Z are not all H. According to some embodiments, V W, Y, and Z may be independently selected from H and aliphatic alkyl C4-C24 groups optionally substituted with one or more of alkenyl, alkynyl, hydroxyl, amide, ester, and / or ether groups, with the proviso that V, W, Y, and Z are not all H.

[0034] According to some embodiments, V W, Y, and Z may be independently selected from H and aliphatic alkyl C4-C100 groups optionally substituted with one or more alkenyl groups, with the proviso that V, W, Y, and Z are not all H. According to some embodiments, V W, Y, and Z may be independently selected from H and aliphatic alkyl C4-C75 groups optionally substituted with one or more alkenyl groups, with the proviso that V, W, Y, and Z are not all H. According to some embodiments, V W, Y, and Z may be independently selected from H and aliphatic alkyl C4-C100 groups optionally substituted with one or more alkenyl groups, with the proviso that V, W, Y, and Z are not all H. According to some embodiments, V W, Y, and Z may be independently selected from H and aliphatic alkyl C4-C50 groups optionally substituted with one or more alkenyl groups, with the proviso that V, W, Y, and Z are not all H. According to some embodiments, V W, Y, and Z may be independently selected from H and aliphatic alkyl C4-C100 groups optionally substituted with one or more alkenyl groups, with the proviso that V, W, Y, and Z are not all H. According to some embodiments, V W, Y, and Z may be independently selected from H and aliphatic alkyl C4-C24 groups optionally substituted with one or more alkenyl groups, with the proviso that V, W, Y, and Z are not all H.

[0035] According to some embodiments, n may be an integer selected from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 3. According to some embodiments, n may be 1, 2, 3, 4, 5, 6, 7, or 8. According to some embodiments, n may be an integer selected from 2 to 8, from 2 to 7, from 2 to 6, from 2 to 5, from 2 to 4. According to some embodiments, n may be an integer selected from 3 to 8, from 3 to 7, from 3 to 6, from 3 to 5. According to some embodiments, n may be an integer selected from 4 to 8, from 4 to 7, from 4 to 6. According to some embodiments, n may be an integer selected from 5 to 8, from 5 to 7. According to some embodiments, n may be an integer selected from 6 to 8.

[0036] According to some embodiments, m may be an integer selected from 0 to 8, from 0 to 7, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, or from 0 to 2. According to some embodiments, m may be an integer selected from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 3. According to some embodiments, m may be an integer selected from 2 to 8, from 2 to 7, from 2 to 6, from 2 to 5, from 2 to 4. According to some embodiments, m may be an integer selected from 3 to 8, from 3 to 7, from 3 to 6, from 3 to 5. According to some embodiments, m may be an integer selected from 4 to 8, from 4 to 7, from 4 to 6. According to some embodiments, m may be an integer selected from 5 to 8, from 5 to 7. According to some embodiments, m may be an integer selected from 6 to 8. According to some embodiments, m may be 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0037] According to some embodiments, r may be an integer selected from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 3. According to some embodiments, r may be an integer selected from 2 to 8, from 2 to 7, from 2 to 6, from 2 to 5, from 2 to 4. According to some embodiments, r may be an integer selected from 3 to 8, from 3 to 7, from 3 to 6, from 3 to 5. According to some embodiments, r may be an integer selected from 4 to 8, from 4 to 7, from 4 to 6. According to some embodiments, r may be an integer selected from 5 to 8, from 5 to 7. According to some embodiments, r may be an integer selected from 6 to 8. According to some embodiments, r may be 1, 2, 3, 4, 5, 6, 7, or 8.

[0038] According to some embodiments, p may be an integer selected from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 3. According to some embodiments, p may be an integer selected from 2 to 8, from 2 to 7 from 2 to 6 from 2 to 5, from 2 to 4. According to someembodiments, p may be an integer selected from 3 to 8, from 3 to 7, from 3 to 6, from 3 to 5. According to some embodiments, p may be an integer selected from 4 to 8, from 4 to 7, from 4 to 6. According to some embodiments, p may be an integer selected from 5 to 8, from 5 to 7. According to some embodiments, p may be an integer selected from 6 to 8. According to some embodiments, p may be 1, 2, 3, 4, 5, 6, 7, or 8.

[0039] According to some embodiments, s may be an integer selected from 0 to 8, from 0 to 7, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, or from 0 to 2. According to some embodiments, s may be an integer selected from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 3. According to some embodiments, s may be an integer selected from 2 to 8, from 2 to 7, from 2 to 6, from 2 to 5, from 2 to 4. According to some embodiments, s may be an integer selected from 3 to 8, from 3 to 7, from 3 to 6, from 3 to 5. According to some embodiments, s may be an integer selected from 4 to 8, from 4 to 7, from 4 to 6. According to some embodiments, s may be an integer selected from 5 to 8, from 5 to 7. According to some embodiments, s may be an integer selected from 6 to 8. According to some embodiments, s may be 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0040] According to some embodiments, q may be an integer selected from 0 to 8, from 0 to 7, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, or from 0 to 2. According to some embodiments, q may be an integer selected from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 3. According to some embodiments, q may be an integer selected from 2 to 8, from 2 to 7, from 2 to 6, from 2 to 5, from 2 to 4. According to some embodiments, q may be an integer selected from 3 to 8, from 3 to 7, from 3 to 6, from 3 to 5. According to some embodiments, q may be an integer selected from 4 to 8, from 4 to 7, from 4 to 6. According to some embodiments, q may be an integer selected from 5 to 8, from 5 to 7. According to some embodiments, q may be an integer selected from 6 to 8. According to some embodiments, q may be 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0041] According to an embodiment, the compound may have structure (II) or stereoisomers thereof or a salt or ion thereof:

[0042] According to an embodiment, the compound may have structure (III) or stereoisomers thereof or a salt or ion thereof:

[0043] According to an embodiment, the compound may have structure (IV) or stereoisomers thereof or a salt or ion thereof:

[0044] According to an embodiment, the compound may have structure (V) or stereoisomers thereof or a salt or ion thereof:

[0045] According to an embodiment, the compound may have structure (VI) or stereoisomers thereof or a salt or ion thereof:

[0046] According to an embodiment, the compound may have structure (VII) or stereoisomers thereof or a salt or ion thereof:(VII).

[0047] According to an embodiment, the compound may have structure (VIII) or stereoisomers thereof or a salt or ion thereof:

[0048] According to an embodiment, the compound may have structure (IX) or stereoisomers thereof or a salt or io

[0049] According to some embodiments, the compound may be structure (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), or stereoisomers thereof or a salt of any thereof or an ion or any thereof, or a combination of any two or more thereof.

[0050] According to some embodiments, the structure (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX) may be an ion. According to some embodiments, the structure (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX) may be a cation. If the structure (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX) is a cation, the counterion may be a halide, such as C1-, Br-, F-, or I- or a triflate ion ( OSO2CF3).Therapeutic Agent

[0051] The immune system of higher organisms has been characterized as distinguishing foreign agents (or “non-self ’) agents from familiar or “self’ components, such that foreign agents elicit immune responses while “self’ components are ignored or tolerated. Immune responses have traditionally been characterized as either humoral responses, in which antibodies specific for antigens are produced by differentiated B lymphocytes known as plasma cells, or cell mediated responses, in which various types of T lymphocytes act to eliminate antigens by a number of mechanisms. For example, CD4+ helper T cells that are capable of recognizing specific antigens may respond by releasing soluble mediators such as cytokines to recruit additional cells of the immune system to participate in an immune response. Also, CD8+ cytotoxic T cells that are also capable of specific antigen recognition may respond by binding to and destroying or damaging an antigen-bearing cell or particle. It is known in the immunological arts to provide certain vaccines according to a variety of formulations, usually for the purpose of inducing a desired immune response in a host.

[0052] Several strategies for eliciting specific immune responses through the administration of a vaccine to a host include immunization with heat-killed or with live, attenuated infectious pathogens such as viruses, bacteria or certain eukaryotic pathogens; immunization with a non-virulent infective agent capable of directing the expression of genetic material encoding the antigen(s) to which an immune response is desired; and immunization with subunit vaccines that contain isolated immunogens (such as proteins) from a particular pathogen in order to induce immunity against the pathogen. (See, e.g., Liu, 1998 Nature Medicine 4(5 suppl. ): 515.) For certain antigens there may be one or more types of desirable immunity for which none of these approaches has been particularly effective, including the development of vaccines that are effective in protecting a host immunologically against human immunodeficiency viruses or other infectious pathogens, cancer, autoimmune disease, or other clinical conditions.

[0053] In other embodiments there is provided a vaccine composition comprising (a) an antigen; the compound having structures (I) - (IX) or stereoisomers thereof or salts or ions thereof or a mixture of two or more thereof; and a toll-like receptor (TLR) agonist, wherein in certain further embodiments the TLR agonist may be selected from lipopolysaccharide, peptidoglycan, polyLC, CpG, 3M003, flagellin, Leishmania homolog of eukaryotic ribosomal elongation and initiation factor 4a (LelF) and at least one hepatitis C antigen. In another embodiment there is provided a vaccine composition comprising: an antigen; the compound having structures (I) - (IX) or stereoisomers thereof or salts or ions thereof or a combination of any two or more thereof; and at least one co-adjuvant that is selected from saponins and saponin mimetics. In another embodiment there is provided a vaccine composition comprising an antigen; the compound having structures (I) - (IX) or stereoisomers thereof or salts or ions thereof or a combination of any two or more thereof; and a carrier that comprises at least one of an oil and ISCOMATRIX™. In another embodiment there is provided a vaccine composition comprising an antigen; the compound having structures (I) - (IX) or stereoisomers thereof or salts or ions thereof or a combination of any two or more thereof; and one or more of: (i) at least one co-adjuvant, (ii) at least one TLR agonist, (iii) at least one imidazoquinoline immune response modifier, and (iv) at least one double stem loop immune modifier (dSLIM). In certain further embodiments (i) the co-adjuvant, when present, may be selected from alum, a plant alkaloid and a detergent, wherein the plant alkaloid may be selected from tomatine and thedetergent may be selected from saponin, Polysorbate 80, Span 85 and Stearyl tyrosine, (ii) the TLR agonist, when present, may be selected from lipopolysaccharide, peptidoglycan, polyI:C, CpG, 3M003, flagellin, Leishmania homolog of eukaryotic ribosomal elongation and initiation factor 4a (LeIF) and at least one hepatitis C antigen, and (iii) the imidazoquinoline immune response modifier, when present, may be selected from resiquimod (R848), imiquimod and gardiquimod. In another embodiment there is provided a vaccine composition comprising: an antigen; the compound having structures (I) – (IX) or stereoisomers thereof or salts or ions thereof or a combination of any two or more thereof; and at least one of a co-adjuvant and a pharmaceutically acceptable carrier, wherein: the co-adjuvant is selected from a cytokine, a detergent, and a block copolymer or biodegradable polymer, and the pharmaceutically acceptable carrier comprises a carrier that is selected from calcium phosphate, an oil-in-water emulsion, a water-in-oil emulsion, a liposome, a novosome, a non-ionic surfactant vesicle (e.g., niosome) and a microparticle. In a particular embodiment, where a liposome or similar carrier is used, a compound having structures (I) – (IX) or stereoisomers thereof or salts or ions thereof or a combination of any two or more thereof is in the laminar structure of the liposome or is encapsulated. In another particular embodiment, where a microparticle is used, the microparticle is one that is based on or comprises polymer fat lipids.

[0054] In certain further embodiments the cytokine is selected from GM-CSF, IL-2, IL-7, IL-12, TNF-α and IFN-gamma, the block copolymer or biodegradable polymer may be selected from Pluronic L121, CRL1005, PLGA, PLA, PLG, and polyI:C, and the detergent may be selected from the group consisting of saponin, Polysorbate 80, Span 85 and Stearyl tyrosine.

[0055] In other embodiments there is provided a vaccine composition comprising: at least one recombinant expression construct which comprises a promoter operably linked to a nucleic acid sequence encoding an antigen and the compound having structures (I) – (IX) or salts or ions thereof or a combination of any two or more thereof. In one embodiment the recombinant expression construct may be present in a viral vector, which in certain further embodiments is present in a virus that is selected from an adenovirus, an adeno-associated virus, a herpesvirus, a lentivirus, a poxvirus, and a retrovirus.

[0056] According to certain of any of the above-described embodiments that include a TLR agonist, the TLR agonist may be capable of delivering a biological signal by interacting with at least one TLR that may be selected from TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8 and TLR-9. In certain further embodiments the TLR agonist may be selected from lipopolysaccharide, peptidoglycan, polyI:C, CpG, 3M003, flagellin, Leishmania homolog of eukaryotic ribosomal elongation and initiation factor 4a (LeIF) and at least one hepatitis C antigen. In a particular embodiment, where a TLR-7 and / or TLR-8 agonist is used, the TLR-7 and / or TLR-8 agonist may be entrapped within a vesicle.

[0057] According to certain of any of the above-described embodiments, the antigen may be derived from at least one infectious pathogen that may be selected from a bacterium, a virus, and a fungus.

[0058] In certain further embodiments the bacterium may be an Actinobacterium, and in certain still further embodiments the Actinobacterium is a Mycobacterium. In certain other related embodiments, the Mycobacterium may be selected from M. tuberculosis and M. leprae. In certain other related embodiments, the bacterium may be selected from Salmonella, Neisseria, Borrelia, Chlamydia and Bordetella.

[0059] In certain other related embodiments, the virus may be selected from a herpes simplex virus, a human immunodeficiency virus (HIV), a feline immunodeficiency virus (FIV), cytomegalovirus, Varicella Zoster Virus, hepatitis virus, Epstein Barr Virus (EBV), respiratory syncytial virus, human papilloma virus (HPV) and a cytomegalovirus. According to certain of any of the above-described embodiments, the antigen may be derived from a human immunodeficiency virus, which in certain further embodiments is selected from HIV-1 and HIV- 2.

[0060] In certain other related embodiments, the fungus may be selected from Aspergillus, Blastomyces, Coccidioides and Pneumocystis. In certain other related embodiments, the fungus may be a yeast, which in certain further embodiments may be a Candida, wherein in certain still further embodiments the Candida may be selected from C. albicans, C. glabrata, C. krusei, C. lusitaniae, C. tropicalis and C. parapsilosis.

[0061] According to certain of any of the above-described embodiments, the antigen may be derived from a parasite, which in certain further embodiments may be a protozoan, which in certain further embodiments may be a Plasmodium, which in certain still further embodiments may be selected from P. falciparum, P. vivax, P. malariae and P. ovale. In certain other embodiments the parasite may be selected from Acanthamoeba, Entamoeba histolytica, Angiostrongylus, Schistosoma mansonii, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mekongi, Cryptosporidium, Ancylostoma, Entamoeba histolytica, Entamoeba coli, Entamoeba dispar, Entamoeba hartmanni, Entamoeba polecki, Wuchereria bancrofti, Giardia, Leishmania, Enterobius vermicularis, Ascaris lumbricoides, Trichuris trichiura, Necator americanus, Ancylostoma duodenale, Brugia malayi, Onchocerca volvulus, Dracanculus medinensis, Trichinella spiralis, Strongyloides stercoralis, Opisthorchis sinensis, Paragonimus sp, Fasciola hepatica, Fasciola magna, Fasciola gigantica, Taenia saginata and Taenia solium.

[0062] According to certain of any of the above-described embodiments, the antigen may be derived from at least one cancer cell. In certain further embodiments the cancer cell may originate in a primary solid tumor, and in certain other embodiments the cancer cell may originate in a cancer that is a metastatic or secondary solid tumor, and in certain other embodiments the cancer cell originates in a cancer that may be a circulating tumor or an ascites tumor. In certain related embodiments the cancer cell may originate in a cancer that may be selected from cervical cancer, ovarian cancer, breast cancer, prostate cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, pseudomyxoma petitonei, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma and Wilms' tumor. In certain other related embodiments, the cancer cell originates in a cancer that is selected from testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oliodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia and heavy chain disease.

[0063] According to certain of any of the above-described embodiments, the antigen may be derived from, or may be immunologically cross-reactive with, at least one epitope, biomolecule, cell or tissue that is associated with an autoimmune disease. In certain further embodiments the epitope, biomolecule, cell or tissue that is associated with an autoimmune disease may be selected from snRNP when the autoimmune disease is systemic lupus erythematosus, at least one of thyroglobulin, thyrotropin receptor and a thyroid epithelial cell when the autoimmune disease is Graves' disease, a platelet when the autoimmune disease is thrombocytopenic purpura, at least one of pemphigus antigen, desmoglein-3, desmoplakin, envoplakin and bullous pemphigoid antigen 1 when the autoimmune disease is pemphigus, myelin basic protein when the autoimmune disease is multiple sclerosis, a pancreatic islet beta cell when the autoimmune disease is type 1 diabetes, and an acetylcholine receptor when the autoimmune disease is myasthenia gravis.

[0064] In another embodiment there is provided a pharmaceutical composition for inducing or enhancing an immune response, comprising a compound having structures (I) - (IX) or stereoisomers thereof or salts or ions thereof or mixture of any two or more thereof; and a pharmaceutically acceptable carrier or excipient. In another embodiment there is provided a pharmaceutical composition for inducing or enhancing an immune response comprising an antigen and a compound having structures (I) - (IX) or stereoisomers thereof or salts or ions thereof or mixture of any two or more thereof; and a pharmaceutically acceptable carrier or excipient. In another embodiment there is provided a pharmaceutical composition for inducing or enhancing an immune response comprising an antigen; a compound having structures (I) - (IX) or stereoisomers thereof or salts or ions thereof or mixture of any two or more thereof; a toll-like receptor (TLR) agonist; and a pharmaceutically acceptable carrier or excipient. In a further embodiment the TLR agonist is selected from lipopolysaccharide, peptidoglycan, polykC, CpG, 3M003, flagellin, Leishmania homolog of eukaryotic ribosomal elongation and initiation factor 4a (LelF) and at least one hepatitis C antigen. In another embodiment there is provided a pharmaceutical composition for inducing or enhancing an immune response comprising: an antigen; a compound having structures (I) - (IX) or stereoisomers thereof or salts or ions thereofor mixture of any two or more thereof; at least one adjuvant; and a pharmaceutically acceptable carrier or excipient. In another embodiment there is provided a pharmaceutical composition for inducing or enhancing an immune response comprising antigen; a compound having structures (I) – (IX) or stereoisomers thereof or salts or ions thereof or mixture of any two or more thereof; and a pharmaceutically acceptable carrier that comprises at least one of an oil and ISCOMATRIX™. In another embodiment there is provided a pharmaceutical composition for inducing or enhancing an immune response comprising: (a) an antigen; a compound having structures (I) – (IX) or stereoisomers thereof or salts or ions thereof or mixture of any two or more thereof; (c) one or more of: (i) at least one adjuvant, (ii) at least one TLR agonist, (iii) at least one imidazoquinoline immune response modifier, and (iv) at least one double stem loop immune modifier (dSLIM); and (d) a pharmaceutically acceptable carrier or excipient. In certain further embodiments (i) the adjuvant, when present, may be selected from alum, a plant alkaloid and a detergent, wherein the plant alkaloid may be tomatine and the detergent may be selected from Polysorbate 80, Span 85 and Stearyl tyrosine, (ii) the TLR agonist, when present, may be selected from lipopolysaccharide, peptidoglycan, polyI:C, CpG, 3M003, flagellin, Leishmania homolog of eukaryotic ribosomal elongation and initiation factor 4a (LeIF) and at least one hepatitis C antigen, and (iii) the imidazoquinoline immune response modifier, when present, is selected from resiquimod (R848), imiquimod and gardiquimod.

[0065] In another embodiment there is provided a pharmaceutical composition comprising: at least one recombinant expression construct which comprises a promoter operably linked to a nucleic acid sequence encoding an antigen; a compound having structures (I) – (IX) or stereoisomers thereof or salts or ions thereof or mixture of any two or more thereof; and a pharmaceutically acceptable carrier or excipient. In certain further embodiments the recombinant expression construct may be present in a viral vector, which in certain further embodiments is present in a virus that is selected from an adenovirus, an adeno-associated virus, a herpesvirus, a lentivirus, a poxvirus, and a retrovirus.

[0066] According to some embodiments, RNA or DNA may be delivered in lipid nanoparticles (LNP). The present lipid compounds having structures (I) – (IX) or stereoisomers thereof or salts or ions thereof or mixture of any two or more thereof are part of the lipids in theformulation of LNP. The outer layer of LNP could contain targeting groups projecting out of the LNP.

[0067] According to an embodiment, a therapeutic agent comprising a drug or oligonucleotide conjugated with and / or covalently bound to the compound having structures (I) – (IX) or stereoisomers thereof or salts or ions thereof, or a combination of any two or more thereof is provided.

[0068] According to some embodiments, the compound having structures (I) – (IX) or stereoisomers thereof or salts or ions thereof, or a combination of any two or more thereof may be in the form of a liposome.

[0069] According to some embodiments, the compound having structures (I) – (IX) or stereoisomers thereof or salts or ions thereof, or a combination of any two or more thereof may be in the form of a micelle.

[0070] According to some embodiments, the compound having structures (I) – (IX) or stereoisomers thereof or salts or ions thereof, or a combination of any two or more thereof may be in the form of a polyplex.

[0071] According to some embodiments, the compound having structures (I) – (IX) or stereoisomers thereof or salts or ions thereof, or a combination of any two or more thereof may be in the form of a lipoplex.

[0072] According to some embodiments, the compound having structures (I) – (IX) or stereoisomers thereof or salts or ions thereof, or a combination of any two or more thereof may be in the form of an emulsion.

[0073] As in known in the art, these compounds of structures (I) – (IX) or stereoisomers thereof or salts or ions thereof or mixtures of any two or more thereof may be used in conjunction with other lipids to form a LNP that encapsulates a therapeutic agent. Non-limiting examples of other lipids that may be used in conjunction with compounds having structures (I) – (IX) or stereoisomers thereof or salts or ions thereof or mixtures of any two or more thereof areother ionizable cationic lipids, permanently charged cationic lipids, polyethylene glycol (PEG) lipids, and / or cholesterol.

[0074] The lipids of structures (I) – (IX) or stereoisomers thereof or salts or ions thereof as described herein may be used to provide adjuvant systems. Such systems may include three or four or more components used together including at least one of the lipids disclosed herein. These adjuvants may be provided for example in the form of liposomes, emulsions, lipid nanoparticles, lipoplexes, or polyplexes. Non-limiting examples of additional components that may be used with the present lipids of structures (I) – (IX) or stereoisomers thereof or salts or ions thereof or mixtures of any two or more thereof are squalene (and / or derivative thereof such as squalane), such as the sustainably sourced squalene (Croda). Other such adjuvant lipids that may be used in combination with the lipids of structures (I) – (IX) or stereoisomers thereof or salts or ions thereof disclosed herein to provide pharmaceutical compositions include the family of the PHAD® adjuvants (Croda) which are synthetic structural analogs of monophosphoryl Lipid A (MPLA). Non-limiting examples include the following. PHAD®-504 a synthetic structural analog of detoxified MPLA derived from E. coli lipopolysaccharide (LPS) is suitable optional adjuvant. It is structurally similar to PHAD®, differing only in the length of a single fatty acid chain.3D-PHAD® which is a homogeneous synthetic equivalent for the 3-deacylated MPLA derived from bacterial LPS is also a suitable adjuvant that may be used in combination with the lipids disclosed herein. The MPLA structural analog, 3D(6-acyl)-PHAD® is also suitable to be used in conjunction with the lipids of structures (I) – (IX) or stereoisomers thereof or salts or ions thereof disclosed here. Other adjuvants that may be used to prepare liposomes in combination with the present lipids include sterols. Suitable sterols may include but are not limited to s-sitosterol, beta-sitosterol, campesterol, stigmasterol, ergosterol, ergocalciferol and cholesterol. Sterols, such as cholesterol may also be used to prepare lipid nanoparticles. The sterol works as a stability enhancer by filling gaps in the lipid layer and assists in the transfection of the pharmaceutical ingredient, such as RNA. Structural analogs of cholesterol, such as β- sitosterol, can also be used to create LNPs with a comparable particle size (diameter ~100 nm) and encapsulation efficiency (~95% using 200 ng mRNA) as cholesterol. β-sitosterol-substituted LNPs may improve transfection efficiency when compared to cholesterol containing LNPs.

[0075] Neutral lipids may also be used with the present lipids of structures (I) – (IX) or stereoisomers thereof or salts or ions thereof. A neutral lipid, for example phosphatidylcholine, which may be non-crystalline at room temperature. Eggyolk phosphatidylcholine, dioleoyl phosphatidylcholine, dioleoyl ethanolamine and dilauryl phosphatidylcholine are non-limiting examples.

[0076] Delivery of various drugs, vaccines, or oligonucleotides, antibodies, enzymes, proteins, small molecules, or nucleotides via these LNPs that include compounds of structures (I) – (IX) or stereoisomers thereof or salts or ions thereof are especially attractive, since the present compounds may be in a form of a cation. Thus, these compounds can be useful to deliver gene therapy to a patient in need thereof.

[0077] Suitable drugs are antibodies, enzymes, and especially drugs that are used to treat diseases. Other suitable pharmaceuticals are anti-tumor antigens that may be useful for immunotherapeutically treating cancers.

[0078] Suitable oligonucleotides are ABE (adenine base editor), miRNA inhibitors, antisense oligonucleotides, ribozymes, DNAzymes, plasmids, immune stimulating nucleic acids, antagomir, antimir, mimic, supermir, and aptamers, allele-specific oligonucleotide, antisense oligonucleotide, ribonucleoproteins (RNP), saRNA, siRNA, dsRN ,,mRNA, siRNA, sgRNA, gRNA, ss-siRNA, cDNA, and cRNA where the c = circular.

[0079] Vaccines and adjuvants for vaccines may be delivered by the use of the lipids of structures (I) – (IX) or stereoisomers thereof or ions or salts or mixtures of any two or more thereof disclosed herein. For example, optional inclusion of adjuvants (particular for vaccines) may include immunologically active saponin fractions having adjuvant activity, typically derived from the bark of the South American tree Quillaja Saponaria Molina, including for example QS21 as a preferred material which may also be derived more sustainably from plant tissue culture such as QS21 (Croda). The lipids disclosed herein may also be used with aluminum hydrogels available as Alhydrogel™ (Croda). QS21 may be used in combination with Alhydrogel as a useful adjuvant together with the lipid disclosed herein.

[0080] According to some embodiments, CRISPR (clustered regularly interspaced short palindromic repeats) base editing therapy can be delivered using the present structures of formulas (I)-(IX) or stereoisomers thereof or ions or salts thereof or mixture of any two or more thereof.

[0081] 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 10wt% water). In another example, the formulation is in an aqueous phase, e.g., in a solution that includes water.

[0082] 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.

[0083] In some embodiments, the therapeutic agent comprising a drug or oligonucleotide in the form of a lipid nanoparticle, and / or in the form of a liposome including a compound having the structure of formulas (I)-(IX) or stereoisomers thereof or ions or salts thereof, or a combination of any two or more thereof may be prepared by at least one of the following methods: microfluidics, impinging jet mixing, lipid film hydration, high shear mixing, high pressure homogenization, high pressure extrusion, 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.

[0084] The therapeutic agent comprising a drug or oligonucleotide in the form of a lipid nanoparticle including a compound having a structure (I) - (IX) or stereoisomers thereof or ions or salts or mixtures of any two or more thereof may be formulated into pharmaceutical compositions suitable for administration. The therapeutic agent comprising a drug or oligonucleotide and the compound having a structure (I) - (IX) or stereoisomers thereof or ions or salts or mixtures of any two or more thereof 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 single phospholipid outer layer that encapsulates the interior, which may be non-aqueous.

[0085] The therapeutic agent, e.g. drug and / or oligonucleotide 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 multivesicular liposomes (MVLs), unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material comprising a compound having structure (I) – (IX) or stereoisomers thereof or ions or salts or mixtures of any two or more thereof or a combination of any two or more thereof and an aqueous interior.

[0086] A liposome containing an oligonucleotide or a drug can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can include the present compound having structure (I) – (IX) or stereoisomers thereof or ions or salts or mixtures of any two or more thereof. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate), octylglucoside, deoxycholate, and lauroyl sarcosine. The oligonucleotide or a drug preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the oligonucleotide or drug and condense around the oligonucleotide or a drug to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of an oligonucleotide or a drug.

[0087] 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 or micelle membrane is structurally similar to biological membranes, when liposomes, lipid nanoparticles or micelles are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the lipid nanoparticle and cell progresses, the internal contents that include the therapeutic agent composition are delivered into the cell.

[0088] "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.

[0089] A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the oligonucleotide or drug composition, an alkali metal C8 to C22 alkyl sulphate, and a 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, chenodeoxy cholate, deoxycholate, and mixtures thereof.

[0090] 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.

[0091] In one method a first micellar composition is prepared which contains the oligonucleotide 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 oligonucleotide or drug composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.

[0092] 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 fol mation of the mixed micellar composition.

[0093] 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 hydrogen-containing chlorofluorocarbons, hydrogen containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1, 1, 1,2 tetrafluoroethane) may be used.

[0094] 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.

[0095] 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 can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired.

[0096] Lipid nanoparticles may include four types of lipids. These are phospholipids (helper lipids), ionizable cationic lipids, cholesterol, and polyethylene glycol (PEG) lipids. The present compounds (I) – (IX) or stereoisomers thereof or ions or salts or mixtures of any two or more thereof may be used as the ionizable cationic lipids. The purpose of the compounds having structure (I) – (IX) or stereoisomers thereof or ions or salts or mixtures of any two or more thereof is thus to improve the stability of the nanoparticle. Lipid nanoparticles may be prepared by mixing an ethanolic lipid mixture including the present compounds (I) – (IX) or stereoisomers thereof or ions or salts or mixtures of any two or more thereof with an acidic aqueous solution containing oligonucleotides and / or a drug. A 1:3 ratio of ethanolic lipid mixture to aqueous buffer is generally used. The general method is to combine the lipid elements with the ethanol, the therapeutic agent (oligonucleotides or drug) in aqueous, and rapidly mix them to form the lipid nanoparticles.

[0097] Such compositions include the therapeutic agent comprising a drug or oligonucleotide in the form of a lipid nanoparticle including a compound having the structure (I)- (IX) or stereoisomers thereof or salts or ions thereof or mixture of any two or more thereof, 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.

[0098] The compounds of structures (I)-(IX) or stereoisomers thereof or salts or ions thereof or mixture of any two or more thereof, may be used to form polyplexes. The polyplexes may be formed by the combination of cationic polymers (e.g. poly-L-Lysine (PLL), poly-L- ornithine (POL), polyethyleneimine (PEI), polyamidoamine (PAM) or combinations thereof) together with the lipids of structures (I)-(IX) or stereoisomers thereof or salts or ions thereof or mixture of any two or more thereof, helper lipids and a nucleic acid (e.g. DNA, RNA, etc.) These may be mixed together and will self-assemble via electrostatic interactions into the polyplexes. Non-limiting methods of combining include microfluidics, sonication, and extrusion for example.

[0099] The compounds of structures (I)-(IX) or stereoisomers thereof or salts or ions thereof or mixture of any two or more thereof, may be used to form lipoplexes. The lipoplexes may be formed by combining the present cationic lipids of structures (I)-(IX) or stereoisomers thereof or salts or ions thereof or mixture of any two or more thereof, optionally with neutral and / or amphoteric lipids and a therapeutic agent, such as anionic charged polynucleic acids (e.g. DNA, RNA, etc.). Typically lipoplexes are formed as suspensions in water, and so the lipid and therapeutic agent may suitably be provided in an aqueous environment; each may be provided as an aqueous solution and subsequently the two aqueous solutions mixed together, alternatively the lipid may be provided as an aqueous solution and the therapeutic agent subsequently added to this or conversely the therapeutic agent may be provided as an aqueous solution and the lipid subsequently added. Desirably, the lipid may be provided as a liposome or lipid nanoparticle prior to subsequent introduction of the therapeutic agent. Lipoplexes utilizing cationic lipids are known to be readily formed with negatively charged DNA or RNA where spontaneous selfassembly occurs from simple mixing to provide the nano-sized lipid based lipoplex materials.Like polyplexes, lipoplexes may be formed by mixing together the components which may self- assemble via electrostatic interaction into lipoplexes. Non-limiting methods of combining include microfluidics, sonication, and extrusion for example.

[0100] The compounds of structures (I)-(IX) or stereoisomers thereof or salts or ions thereof or mixture of any two or more thereof, may be formed into emulsions to deliver therapeutic agent by combining the compounds of structures (I)-(IX) or stereo isomers thereof or salts or ions thereof or mixture of any two or more thereof, with the therapeutic agent and an optional emulsifier. Such emulsions may be prepared using standard techniques. Suitable emulsions may be oil-in-water (o / w) or water-in-oil (w / o) emulsions. The water-in-oil emulsions may also be processed into a water-in-oil-in-water emulsion (also called "double emulsions"), where the internal and external aqueous phases are separated by an oil phase.

[0101] In general, the aqueous phase, the oil phase comprising the compounds of structures (I)-(IX) or stereoisomers thereof or salts or ions thereof or mixture of any two or more thereof, optionally any emulsifiers and / or surfactants and therapeutic agent(s) are brought together and emulsified until a stable emulsion having the desired viscosity is obtained (the desired viscosity is application-dependent. Suitable viscosities would be known to the relevant skilled person). Compounds of structures (I)-(IX) or stereoisomers thereof or salts or ions thereof or mixture of any two or more thereof may be present in the oil phase. Water-in-oil (w / o) emulsions are two phase systems including an oil phase (continuous phase) comprising the compounds of structures (I)-(IX) or stereoisomers thereof or salts or ions thereof or mixture of any two or more thereof and an aqueous emulsions phase (discontinuous phase). The aqueous phase may be dispersed as small droplets in the oil phase, and the emulsion may contain one or more surfactants and / or emulsifiers. Water-in-oil emulsions are generally used in pharmaceutical formulations as a vehicle for delivery of therapeutic agents, especially in case of water-insoluble or water-sensitive active agents. In vaccinations, water-in-oil emulsions are commonly used as an adjuvant to stimulate the immune response against target antigens derived from one or more infectious agents. These water-in-oil emulsion vaccines may be delivered to a patient in need thereof via injection.

[0102] When emulsions are prepared, energy must be expended to form an interface between the oil phase and aqueous phase. Therefore, emulsification equipment suitable to prepare emulsions includes a wide variety of agitators, homogenizers, colloid mills, jet mixers and ultrasonic devices for example. Production-size agitators can be propeller shaped or paddle shaped stirring systems, with rotation speed usually up to 2,000 rpm, and are considered as low shear mixing procedures. Another type of production-site agitator is the colloid mill. The principle of operation of the colloid mill is the passage of the mixed phases of an emulsion formula between a stator and a high-speed rotor revolving at speeds of 2,000 to 18,000 rpm that is considered as a high shear mixing procedure.

[0103] Optionally, once the emulsion has been prepared a drying step may be utilized to provide a dried powder product comprising the compounds of structures (I)-(IX) or stereoisomers thereof or salts or ions thereof or mixture of any two or more thereof and therapeutic agent. In this case the therapeutic agent may be encapsulated in the compounds of structures (I)-(IX) or stereoisomers thereof or salts or ions thereof or mixture of any two or more thereof and optionally other lipids as well. Such a drying step may advantageously utilize a spray drier, although other drying methods are known in the art. 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. Methods of Treatment

[0104] According to an embodiment, a method of treatment is provided. The method comprising administering the 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.

[0105] 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. 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.

[0106] Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.

[0107] Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0108] 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 and salts 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 therapeutic agent comprising a drug or oligonucleotide in the form of a lipid nanoparticle, liposome or micelle including a compound having the structure of any (I)-(IX) or salts or ions thereof or mixtures of any two or more of any thereof, can be combined with emulsifying and suspending agents. If desired, certain sweetening and / or flavoring agents can be added.

[0109] According to some embodiments, compositions for intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.

[0110] 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, intratumor, 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, diluents and other suitableadditives. 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.

[0111] According to some embodiments the administration of the pharmaceutical composition is intramuscular. EXAMPLES

[0112] EDC is N-(3-Dimethylaminopropyl)-N ′-ethylcarbodiimide hydrochloride or 1- Ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0113] DCM is dichloromethane.

[0114] DMAP is 4-(Dimethylamino)pyridine.

[0115] DMF is dimethylformamide.

[0116] TBAI is Tetra-n-butylammonium iodide.

[0117] DIPEA is N,N-Diisopropylethylamine.

[0118] DCM is Dichloromethane.

[0119] TFA is trifluoroacetic acid.

[0120] Boc is tert-butyloxycarbonyl.

[0121] ACN is Acetonitrile.

[0122] TBSCI is tert-Butyldimethylsilyl chloride.

[0123] TBS is tert-Butyldimethylsilyl.

[0124] TBAF is Tetrabutylammonium fluoride.

[0125] THF is tetrahydrofuran.

[0126] NMR conditions:1H NMR is performed with 400 MHz instrument, using CD3OD standard.

[0127] Example 1 : Preparation of Compound (III):

[0128] Compound (III) was prepared according to the following synthetic scheme.Synthesis of Intermediate la:

[0129] 2-Hexyldecan-l-ol (20 g, 82 mmol) and glutaric anhydride (9.41 g, 82 mmol) were dissolved in THF anhydrous (103 ml). DMAP (1.0 g, 8.25 mmol) and TFA (18.4 ml, 132 mmol) were then added. The solution was heated to reflux for 3 hours and then cooled to roomtemperature. 160 mL 1 N HC1 was then added to the mixture with stirring, extracted with ether 160 ml three times. The organic phase was combined, and washed with water two times, brined and then dried with Na2SC>4. The organic phase was concentrated under reduced pressure and the resulting crude material was purified by flash column chromatography using ethyl acetate and hexane to afford Intermediate la as clear oil (19.2 g, yield 65%).JH NMR (400 MHz, CDC13) 6 3.98 (d, J = 5.6 Hz, 2 H), 2.46-2.39 (m, 4H), 2.00-1.93 (m, 2H), 1.30-1.22 (m, 25H), 0.88 (t, J= 6.8 Hz, 6H).Synthesis of Intermediate 2a:

[0130] Intermediate la (18.9 g, 52.9 mmol), DMAP (0.63 g, 5.16 mmol) and 3- bromopropane-l,2-diol (4.0 g, 25.8 mmol) were added to a dried round flask. The flask was filled with N2, DCM (103 ml) anhydrous was then added, and the mixture cooled to 0 °C. After EDC (12.4 g, 64.5 mmol) was added in three portions, the mixture was stirred at 0 °C for 1 hour, then at room temperature for another 1 hour. 100 mL ice-water and 500 mL hexane were added. The organic phase was washed with citric acid (aqueous), water, NaHCCh, and brine. The organic phase was concentrated under reduced pressure and the resulting crude material was purified by flash column chromatography using ethyl acetate / hexane to afford Intermediate 2 as clear oil (18.5 g, yield 86%).JH NMR (400 MHz, CDC13) 5 ’H NMR (400 MHz, CDC13) 8 5.21 (m, 1H), 4.36 (dd, J= 12.0, 4.0 Hz, 1H), 4.24 (dd, J= 12.0, 5.6 Hz 1H), 3,98 (d, J= 5.6 Hz, 4 H), 3.55-3.45 (m, 2H), 2.44-2.37 (m, 8H), 1.99-1.93 (m, 4H), 1.65-1.55 (m, 2H), 135-1.20 (m, 50H), 0.88 (t, .7= 6.8 Hz, 12H).Synthesis of compound (III):

[0131] Intermediate 2a (14 g, 16.83 mmol), TBAI (1.243 g, 3.37 mmol), 3 -(piperazin- 1- yl)propan-l-ol (2.91 g, 20.19 mmol) and DIPEA (3.53 ml, 20.19 mmol) were dissolved in DMF (42.1 ml). After purging with N2, the solution was stirred at 50 °C overnight. The mixture was allowed to cool to room temperature, and then diluted with 400 mL ether. The organic phase was washed with water 200 mL three times, and then brine 100 mL. The organic phase was concentrated under reduced pressure and the resulting crude material was purified by flash column chromatography using hexane / chloroform and chloroform / methanol to afford compound (III) as clear oil (9.1 g, yield 60%). MS (ESI, m / z) [M+H]+895.7 ;XH NMR (400 MHz, CDC13)5 5.24-5.19 (m, 1H), 4.36 (dd, J= 12.0, 4.0 Hz, 1H), 4.08 (dd, J = 12.0, 4.0 Hz 1H), 3.98 (dd, J = 7.2, 4.0 Hz, 4 H), 3.79 (t, J= 4.0 Hz, 2H), 2.61-2.37 (m, 20H), 1.98-1.90 (m, 4H), 1.72-1.68 (m, 2H), 1.32-1.26 (m, 50H), 0.88 (t, J= 6.8 Hz, 12H).

[0132] Example 2: Preparation of Compound (IV):

[0133] Compound (IV) was prepared according to the following synthetic scheme.Synthesis of intermediate 2b:

[0134] Intermediate lb was prepared according to the synthesis of intermediate la (above). Intermediate lb (17.65 g, 47.6 mmol) and 3-bromopropane-l,2-diol (3.6 g, 23.23 mmol) were added to a dried round flask, charged with N2. Then DCM (93 ml) anhydrous, and DMAP (0.568 g, 4.65 mmol) were added. The mixture was cooled to 0 °C under N2. EDCI (11.13 g, 58.1 mmol) was added in three portions, then stirred at 0 °C for another hour, then stirred atroom temperature (about 25°) for 1 hour then cooled to 0 °C and quenched with water. Solvent was removed and partitioned with ethyl acetate and washed with citric acid, water, NaHCO3, and brine, then dried over Na2SO4and filtered. The solvent was removed and dried. The crude product was purified over silica gel column chromatography using hexane / ethyl acetate as eluents to obtain the Intermediate 2b (19.3 g, 22.44 mmol) in 97% yield.1H NMR (400 MHz, CDCl3) δ 5.26-5.20 (m, 1H), 4.92-4.86 (m, 2H), 4.38 (dd, J = 11.9, 4.4 Hz, 1H), 4.26 (dd, J = 11.9, 5.5 Hz, 1H), 3.47 (ddd, J = 15.8, 5.5 Hz, 2H), 2.4-2.35 (m, 8H), 2.02-1.95 (m, 4H), 1.53- 1.52 (m, 8H), 1.28 (br s, 48H), 0.89 (t, J = 13.7 Hz, 12H). Synthesis of Compound (IV)

[0135] Intermediate 2b (19.3 g, 22.44 mmol), 3-(piperazin-1-yl)propan-1-ol (3.88 g, 26.9 mmol),TBAI (1.658 g, 4.49 mmol), and DIPEA (4.70 ml, 26.9 mmol) were combined in anhydrous DMF and stirred at 50 °C under N2 till the bromo compound consumption was at a maximum (24h to 48 h). The mixture was then diluted with ethyl acetate, and washed with water and brine. The mixture was then dried over anhydrous Na2SO4 and filtered. The solvent was removed and dried. The crude product was purified over silica gel column using dichloromethane and methanol as eluant to provide the Compound (IV) (11.25g, 12.18 mmol) in 54% yield.1H NMR (400 MHz, CDCl3) δ 5.22 (tt, J = 6.5, 3.2 Hz, 1H), 4.91-4.84 (m, 2H), 4.36 (dd, J = 11.9, 3.1 Hz, 1H), 4.10 (d, 6.5 Hz, 1H), 3.80 (t, J = 10.4 Hz, 2H), 2.60 (t, J = 11.4 Hz, 2H), 2.53-2.42 (m, 8H), 2.40-2.35 (m, 8H), 1.99-1.90 (m, 8H), 1.73- 1.68 (m, 2H), 1.52-1.50 (m, 8H), 1.27 (s, 48 H), 0.889 (t, J = 13.7 Hz, 12 H);13C NMR (100 MHz, CDCl3) δ 172.7, 172.6, 172.3, 74.5, 74.4, 68.7, 64.7, 64.4, 64.1, 58.8, 58.0, 53.6, 53.3, 34.1, 34.0, 33.6, 33.5, 33.4, 33.1, 31.8, 29.5, 29.4, 29.2, 26.9, 25.3, 25.33, 25.32, 22.7, 20.3, 20.2, 14.1.

[0136] Compound (IV) structure was confirmed with1H and13C NMR.

[0137] Example 3: Preparation of Compound (V):

[0138] Compound (V) was prepared according to the following synthetic scheme. B TFA, DCM OHSynthesis of compound 53:

[0140] A suspension of compound 51 (4g, 21.25 mmol) and 2-bromoethan-1-ol (2.92 g, 23.37 mmol) and K2CO3(15 mmol) in dry acetonitrile (40 ml) was stirred at 90 °C in a closed vessel for 20 h. The solid was filtered off and washed with acetonitrile. The solvent was removed in vacuo and purified by flash chromatography on silica gel by using 2% methanol in dichloromethane as a mobile phase to obtain compound 52 (4g, 17.22 mmol) in 81% yield.1H NMR (400 MHz, CDCl3) δ 3.57 (t, J = 10.6 Hz, 2H), 3.31 (br s, 2H), 2.86 (s, 3H), 2.56 (q, J = 5.4 Hz, 4H), 2.30 (s, 3H), 1.45 (s, 9H);13C NMR (100 MHz, CDCl3) δ 156.05, 79.5, 59.1, 58.5, 55.1, 46.9, 41.9, 34.6, 28.4. Compound 52 (4g, 12.91 mmol) was dissolved in dichloromethane (20 mL) and a solution of 4N HCl in Dioxane (20 mL) was added dropwise and stirred at roomtemperature for 2 h. The solvent was removed via rotovap. The product was redissolved in DCM and treated with 4N NaOH solution 5 mL and Na2SO4 was added and washed with DCM. The solid was filtered off and solvent was removed to obtain the compound 53 (2 g, yield 88%).1H NMR (400 MHz, 8:2 CDCl3:CD3OD) δ 3.59 (t, J = 6.1 Hz, 2H), 2.62 (t, J = 6.1 Hz, 2H), 2.51 (t, J = 5.62 Hz, 4H), 2.38 (s, 3H), 2.24 (s, 3H);13C NMR (100 MHz, 8:2 CDCl3:CD3OD) δ 59.43, 59.0, 56.4, 48.60, 42.1, 35.6. Synthesis of Compound (V)

[0141] Intermediate 2a, compound 53 (0.763 g, 5.77 mmol) and TBAI (0.355 g, 0.961 mmol) and DIPEA (4.20 ml, 24.04 mmol) (4g, 4.81 mmol),were dissolved in DMF (12.02 ml) under N2, and stirred at 50 °C under N2for 24-72 h. The mixture was then diluted with ethyl acetate, washed with water and brine. The crude product was purified by silica gel column chromatography using hexane / ethyl acetate as eluents.1H NMR (400 MHz, CDCl3) δ 4.22 (t, J = 5.9 Hz, 2H), 4.14 (dd, J = 11.3, 4.2 Hz, 1H), 4.02 (dd, J = 11.3, 6.0 Hz, 1H), 3.98 (d, J = 5.8 Hz, 4H), 3.89-3.84 (m, 1H), 2.78-2.78 (m, 7H), 2.54-2.39 (m, 9H), 2.37 (s, 3H), 2.32 (s, 3H), 2.01- 1.92 (m, 4H), 1.62 (br s, 2H), 1.28 (br s, 48H), 0.89 (t, J = 6.0 Hz, 12H);13C NMR (100 MHz, CDCl3) δ 173.1, 172.9, 172.8, 67.3, 66.6, 66.5, 61.7, 59.1, 55.5, 55.4, 54.7, 43.7, 42.2, 37.2, 33.4, 33.3, 33.2, 31.9, 31.8, 31.2, 29.5, 29.6, 29.5, 29.3, 26.7, 26.6, 22.7, 22.6, 20.14, 20.12, 14.11, 14.10.

[0142] Example 4: Preparation of Compound (VI):

[0143] Compound (VI) was prepared according to the following synthetic scheme.

[0144] Experimental procedure:Synthesis of compound 63:

[0145] To compound 61 (5 g, 34.7 mmol) in dichloromethane (25 mL) was added tertbutylchlorodimethylsilane (6.27 g, 41.6 mmol). The reaction mixture was stirred at roomtemperature. The reaction mixture was stirred overnight, then diluted one time with CH2Cl2 and extracted against 0.5 N NaOH (100 mL). The aqueous mixture was extracted further with 10% methanol in DCM (three times at 100 mL). The combined organics were dried with Na2SO4and concentrated in vacuo. The crude product was purified over silica gel chromatography using DCM and methanol N and 1% NH4OH additive to provide compound 61 quantitatively.1H NMR (400 MHz, 8:2 CDCl3:CD3OD) δ 3.67 (t, J = 9.8 Hz, 2H), 2.91 (t, J = 9.8 Hz, 4H), 2.43- 2.39 (m, 6H), 1.76-1.69 (m, 2H), 0.91 (s, 9H), 0.67 (s, 6H);13C NMR (100 MHz, 8:2 CDCl3:CD3OD) δ 61.33, 55.88, 53.89, 45.09, 29.3, 25.74, 18.2, -5.59. Compound 62 (5g, 19.34 mmol) was dissolved in ethyl acetate (130 ml). NaOH (4.64 g, 34.8 mmol) was added to the reaction mixture while maintaining the temperature at 5 °C. Subsequently, CS2(2.099 ml, 34.8 mmol) in ethyl acetate (30 ml) was added drop-wise to the reaction mixture with stirring at 5 °C. The mixture was stirred for 4h until a solid formed. The solid was filtered and washed (three times in 100 mL) with ethyl acetate to afford compound 63.1H NMR (400 MHz, 8:2 CDCl3:CD3OD) δ 4.43 (t, J = 10.0 Hz, 3H), 3.66-3.61 (m, 2H), 2.91 (t, J = 9.98 Hz, 1H), 2.48 (t, J = 10.28 Hz, 4H), 2.45-2.39 (m, 2H), 1.75-1.65 (m, 2H); 0.86 (s, 9H), 0.027 (s, 6H);13C NMR (100 MHz, 8:2 CDCl3:CD3OD) δ 210.9, 61.3, 54.9, 52.9, 50.2, 29.6, 25.7, 18.2, -5.55. Synthesis of intermediate 3a

[0146] Compound 63 (2 g, 5.61 mmol) and intermediate 2a (3.89 g, 4.67 mmol) in acrylonitrile 50 mL was stirred at room temperature overnight. The mixture was concentrated under reduced pressure, and the crude product was treated with water and extracted with ethyl acetate, washed with water, brine, and dried over Na2SO4 and filtered. Filtrate was put into a rotovap and dried. Finally, the crude product was purified over silica gel column chromatography using hexane / ethyl acetate to obtain the desired product (3.6 g, 3.32 mmols) in 71% yield.1H NMR (400 MHz, CDCl3) δ 5.37-5.3 (m, 1H), 4.42 (dd, J = 12.0, 3.6 Hz, 1H), 4.34 (br s, 2H), 4.19 (dd, J = 12.0, 5.4 Hz, 1H), 3.99 (d, J = 5.77 Hz, 4H), 3.9 (br s, 2H), 3.81 (dd, J = 14.3, 4.9 Hz, 1H), 3.68 (t, J = 6.2 Hz, 2H), 3.53 (dd, J = 14.2, 7.4 Hz, 1H), 2.53 (br s, 4H), 2.49- 2.4 (m, 2H), 2.42-2.37 (m, 8H), 2.0-1.93 (m, 4H), 1.75-1.68 (m, 2H), 1.63 (br s, 2H), 1.28 (br s, 48H), 0.91 (s, 9H) 0.89 (t, J = 6.8 Hz, 12H), 0.06 (s, 6H);13C NMR (100 MHz, CDCl3) δ 195.0, 173.0, 172.5, 171.9, 70.2, 67.3, 63.9, 61.0, 54.5, 52.5, 37.3, 37.2, 33.3, 33.2, 30.1, 31.9, 31.8, 31.2, 29.9, 29.6, 29.5, 29.3, 26.7, 266 259 227 226 20.1, 20.0, 18.3, 14.1, -5.3.Synthesis of compound (VI)

[0147] A room temperature solution of the intermediate 3a (3.6 g, 3.32 mmol) in THF (100 mL) was treated with a solution of TBAF (4.97 ml, 4.97 mmol). After stirring for 4 hours, the reaction mixture was quenched with water and extracted with dichloromethane. The combined dichloromethane layers were dried (Na2SO4), filtered and concentrated to afford the desired product. The crude product was purified over silica gel column chromatography using hexane / ethyl acetate as eluent to obtain the compound (VI) (2.7g, 2.78 mmols) in 84% yield.1H NMR (400 MHz, CDCl3)δ 5.36-5.31 (m, 1H), 4.41 (dd, J = 12.0, 3.6 Hz, 1H), 4.34 (br s, 2H), 4.18 (dd, J = 12.0, 5.5 Hz, 1H), 3.99 (d, J = 5.4 Hz, 4H), 3.98 (br s, 2H), 3.84-3.78 (m, 3H), 3.55-3.49 (m, 1H), 2.67-2.61 (6H), 2.42-2.37 (m, 8H), 1.99-1.92 (m, 4H), 1.80-1.74 ( m, 2H), 1.63 (br s, 2H), 1.28 (br s, 48 H), 0.89 (t, J = 6.8 Hz, 12H);13C NMR (100 MHz, CDCl3) δ 195.4, 173.0, 172.4, 171.9, 70.1, 67.3, 64.1, 63.9, 60.4, 57.9, 52.6, 37.4, 37.2, 33.3, 33.1, 31.9, 31.8, 31.2, 29.9, 29.6, 29.5, 29.3, 27.4, 26.7, 26.6, 22.6, 22.6, 21.0, 20.1, 20.2, 14.2, 14.1.

[0148] Compound (VI) structure was confirmed with1H and13CNMR

[0149] Example 5: Preparation of Compound (VII):

[0150] Compound (VII) is prepared according to the following synthetic scheme.

[0151] Example 6: Preparation of Compound (VIII):

[0152] Experimental procedure: Synthesis of compound (VIII)

[0153] Intermediate 2a (5 g, 6.01 mmol) was dissolved in DMF (15 mL) under nitrogen, followed by the addition of TBAI (0.444 g, 1.202 mmol), 2-(piperazin-1-yl)ethan-1-ol (0.939 g, 7.21 mmol) and DIPEA (1.259 mL, 7.21 mmol). Mixture was stirred at 50 °C for 2 days. Thin layer chromatography (hexane:ethyl acetate 8:2, staining method: iodine and char) was used to follow the reaction. Once complete, the reaction was diluted with ethyl acetate and partitioned with water and brine. The organic layer was dried over anhydrous sodium sulfate and solvent was removed. Purification was done by DCM and methanol in a column). Compound (VIII) was isolated as a light yellow oil (2.67 g, 50%). HNMR (CDCl3, ppm): 5.22 (m, 1H, NCH2CH), 4.35 (dd, J = 11.9, 3.1 Hz, 1H, NCH2CH), 4.09 (m, 1H, NCH2CH), 3.97 (dd, J = 7.0, 4.6 Hz, 2H, COO-CHCH2), 3.59 (t, J = 6.8 Hz, 2H, CH2OH), 2.53-2.35 (m, 20H, glutarate and piperazine CH2), 1.95 (m, 4H, CH2), 1.58 (m, 10H, CH2), 1.26 (m, 48H, aliphatic chain), 0.88 (t, J = 6.8 Hz, 12H, CH3).

[0154] Compound (VIII) structure was confirmed with1H NMR.

[0155] Example 7 Preparation of Compound (IX):

[0156] Compound (IX) was prepared according to the following synthetic scheme. O O

[0157] Experimental procedure: Synthesis of Compound (IX)

[0158] Intermediate 2a (5 g, 6.01 mmol) was dissolved in DMF (15 mL) under nitrogen, followed by the addition of TBAI (0.444 g, 1.202 mmol), 1-methylpiperazine (0.800 mL, 7.21 mmol) and DIPEA (1.259 ml, 7.21 mmol). Mixture was stirred at 50 °C for 2 days. TLC (hexane:ethyl acetate 8:2, staining method: iodine and char) was used to follow the reaction. Once complete, the reaction was diluted with ethyl acetate and partitioned with water and brine. Organic layer was dried over anhydrous sodium sulfate and solvent was removed. Purification was done by DCM:methanol:acetone column). Compound (IX) was isolated as a light yellow oil (2.92 g, 57%). HNMR (CDCl3, ppm): 5.22 (m, 1H, NCH2CH), 4.35 (dd, J = 11.9, 3.1 Hz, 1H, NCH2CH), 4.09 (m, 1H, NCH2CH), 3.97 (dd, J = 7.0, 4.6 Hz, 2H, COO-CHCH2), 3.59 (t, J = 6.8 Hz, 2H, CH2OH), 2.53-2.35 (m, 20H, glutarate and piperazine CH2), 1.95 (m, 4H, CH2), 1.58 (m,10H, CH2), 1.26 (m, 48H, aliphatic chain), 0.88 (t, J = 6.8 Hz, 12H, CH3). MS (ESI, m / z) [M+H]+852.70.

[0159] Compound (IX) structure was confirmed with1H NMR as well as mass spectrometry.

[0160] Example 8 Preparation of Lipid Nanoparticles:

[0161] 20mg of Structure (III) (Example 2), 6.5 mg cholesterol, 3.5 mg Distearoylphosphatidylcholine (DSPC), and 1.7 mg 1,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG2000) were mixed together in ethanol at 50 mg / mL. This was combined with mRNA under controlled conditions with microfluidics to produce lipid nanoparticles and then dialyzed against PBS (Phosphate-buffered saline) to remove ethanol. Particles produced were 60-150 nm.

[0162] Example 9 Preparation of Lipoplex:

[0163] A lipid film composed of 40 mg Structure (III) (Example 2) and 10 mg 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was prepared and hydrated with an aqueous solution containing DNA then sonicated to form a lipoplex.

[0164] Example 10 Preparation of Lipoplex:

[0165] Structure (III) (Example 2) was hydrated with an aqueous solution containing DNA at a 4:1 ratio lipid:DNA. The lipoplex was sonicated and extruded to reduce size.

[0166] 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.

[0167] 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

AMENDED CLAIMS received by the International Bureau on 06 January 2026 (06.01.2026)Claims

1. A compound having the structure (I), or stereoisomers thereof, or a salt or ion thereof, or a combination of any two or more thereof:where: each R1is CH3or together are -[CH2]2-;R2is selected from -[CHJ-, -S-(C=O)-, and -S-(C=S)-;R3, R4, R5, and R6are each independently selected from -O- (C=O)-, -(C=O)-O-, -NH-(C=O)-, and -C(=O)-NH-;X is selected from OH, -CH3, NH2, and SH;V, W, Y, and Z are independently selected from H, aliphatic alkyl C4-C100 groups optionally substituted with one or more of alkenyl, alkynyl, hydroxyl, amide, ester, and / or ether groups, with the proviso that V, W, Y, and Z are not all H; n, p, and r are independently selected integers from 1 to 8; and m, q, and s are independently selected integers from 0 to 8.

2. The compound of claim 1, wherein each R1together are -[CH2]2-.

3. The compound of claim 1 or claim 2, wherein each R1is -CH3.

4. The compound of any of claims 1-3, wherein X is -OH.

5. The compound of any of claims 1-3, wherein X is -CH3.

6. The compound of any of claims 1-3, wherein X is -SH.

7. The compound of any of claims 1-3, wherein X is -NH2.

8. The compound of any of claims 1-7, wherein R2is -CH2-.

9. The compound of any of claims 1-7, wherein R2is -S-(C=O)-.

10. The compound of any of claims 1-7, wherein R2is -S-(C=S)-.53

11. The compound of any of claims 1-10, wherein V, W, Y, and Z are independently selected from H and aliphatic alkyl C4-C100 groups optionally substituted with one or more alkenyl groups, with the proviso that V, W, Y, and Z are not all H.

12. The compound of any of claims 1-10, wherein V, W, Y, and Z are independently selected from H, aliphatic alkyl C4-C24 groups optionally substituted with one or more of alkenyl, alkynyl, hydroxyl, amide, ester, and / or ether groups, with the proviso that V, W, Y, and Z are not all H.

13. The compound of any of claims 1-12, wherein n is an integer selected from 1 to 3.

14. The compound of any of claims 1-13, wherein m is an integer selected from 0 to 3.

15. The compound of any of claims 1-14, wherein r is an integer selected from 1 to 3.

16. The compound of any of claims 1-14, wherein p is an integer selected from 1 to 3.

17. The compound of any of claims 1-16, wherein s is an integer selected from 0 to 3.

18. The compound of any of claims 1-17, wherein q is an integer selected from 0 to 3.

19. The compound of claim 1 wherein the compound comprises the structure (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), or stereoisomers thereof, or a salt or ion of any thereof, or a mixture of two or more thereof:5455(IX).

20. A therapeutic agent comprising a drug, vaccine, oligonucleotide, antibody, enzyme, protein, small molecule, or nucleotide, the agent being in a form of a lipoplex, or in a form of a polyplex, in a form of a lipid nanoparticle, or in a form of a liposome, or in a form of a micelle, or in a form of an emulsion, comprising a compound having a structure of any of claims 1-19.

21. A pharmaceutical composition comprising the therapeutic agent of claim 20 and a pharmaceutically acceptable carrier.

22. A method of treatment, comprising administering the therapeutic agent of claim 20 or the pharmaceutical composition of claim 21 to a patient in need thereof.56

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