ionizable lipid compound, composition, drug carrier and pharmaceutical formulation

专利摘要:
IONIZABLE LIPIDE COMPOUND, COMPOSITIONS, DRUG VEHICLE AND PHARMACEUTICAL FORMULATION. The description is directed to ionizable lipids useful to enhance the delivery of therapeutic agents to liposomes.
公开号:BR112014030714B1
申请号:R112014030714-8
申请日:2013-06-07
公开日:2020-12-22
发明作者:Joseph E. Payne；John A. Gaudette；Jun Zhang；Lei Yu；Mohammad Ahmadian；Neda Safarzadeh；Priya Karmali；Richard P. Witte；Victor Knopov；Violetta Akopian；Wenbin Ying；Zheng Hou
申请人:Nitto Denko Corporation；
IPC主号:

专利说明:

REMISSIVE REFERENCE TO RELATED ORDERS
[001] This application claims the benefit of Provisional Patent Application No. US 61 / 657,480 filed on June 8, 2012, which is hereby incorporated by reference in its entirety. TECHNICAL FIELD
[002] The description is directed to ionizable lipids to intensify the delivery of therapeutic agents. BACKGROUND
[003] Numerous techniques are available for delivering a therapeutic agent, for example, siRNA, nucleic acids, etc., to a cell. These techniques include viral and non-viral transfection systems. Non-viral transfection systems can include, for example, polymers, lipids, liposomes, micelles, dendrimers and nanomaterials. Polymers that have been studied for cell transfection include cationic polymers such as, for example, poly (L-lysine) ("PLL"), polyethyleneimine ("PEI"), chitosan and poly (2-dimethylamino) ethyl methacrylate ("pDMAEMA ").
[004] However, viral and non-viral transfection techniques have disadvantages. For example, viral systems can yield high transfection effectiveness, but they may not be completely safe. In addition, viral systems can be complicated and / or expensive to prepare.
[005] Non-viral transfection systems, for example, those employing cationic polymers, have been reported to transfer plasmid DNA into cells. However, cationic polymers can be unstable and can be toxic to cells.
[006] As such, there is a need for new compounds, compositions and methods to use a cationic composition to enhance the delivery of therapeutic agents to cells, tissues and organisms. SHORT DESCRIPTION
[007] The present description is directed to Formula I ionizable lipid compounds:
where neither are independently 1, 2, 3 or 4; R1 and R2 are independently C10-18 alkyl or C12-18 alkenyl; x is -CH2-, S, O, N or absent; L is C1-4 alkylene; -S-C1-4 alkylene; -O-C 1-4 alkylene; -OC (O) -1- C1-4 alkylene alkylene; -S (O) 2-C 1-4 alkylene;
or a pharmaceutically acceptable salt form thereof. Also described are the compositions, pharmaceutical formulations, drug carriers and methods of using the compounds of Formula I. BRIEF DESCRIPTION OF THE FIGURES
[008] Figure 1 shows the in vitro synergistic efficacy of ionizable lipid realizations: ionizable lipid of the present description Figure 2 shows the in vitro synergistic efficacy of ionizable lipid realizations: ionizable lipid of the present description Figure 3 shows the synergistic efficacy in vitro of ionizable lipid realizations: cationic lipid of the present description Figure 4 shows the in vivo efficacy of ionizable lipid realizations: cationic lipid of the present description DETAILED DESCRIPTION OF THE ILLUSTRATIVE ACHIEVEMENTS
[009] The present description is directed to compounds of ionizable lipid, as well as their uses in the delivery of therapeutic agents to cells, tissues and organisms.
[0010] Formula I ionizable lipid compounds are within the scope of the description:
where neither are independently 1, 2, 3 or 4; R1 and R2 are independently C10-18 alkyl or C12-18 alkenyl; X is -CH2-, S, O, N or absent; L is C1-4 alkylene; -S-C1-4 alkylene; -O-C 1-4 alkylene; -OC (O) - xj n — A «CM alkylene / NJ1>" V ^ N C 1-4 alkylene; -S (O) 2-C 1-4 alkylene;
or a pharmaceutically acceptable salt form thereof.
[0011] Within the scope of the description, n and m can be the same or different. In preferred embodiments, n and m are equal. Particularly preferred are those embodiments in which n and m are 1 or n and m are 2.
[0012] In some embodiments of the description, X is a link. In other embodiments, X is -CH2-. In still other embodiments, X is S. Also preferred are those in which X is O. Those in which X is N are also within the scope of the description.
[0013] In some embodiments of the description, L is C1-4 alkylene. In other embodiments, L is -S-C1-4 alkylene. In still other embodiments, L is - O-C 1-4 alkylene. In still other embodiments, L is -OC (O) -C1-4 alkylene. Alternatively, L is -S (O) 2-C 1-4 alkylene. Also within the scope of the i-lalkylene CH description are the realizations in which L is "

[0014] In those embodiments where X is a bond, L is preferably C 1-4 alkylene. Such examples of L include -CH2-, -CH2-CH2-, - CH2CH2CH2- and -CH2CH2CH2CH2-. In other realizations where X is a
connection, L is, for example,

[0015] In those embodiments where X is -CH2-, L is preferably -S-C1-4 alkylene. Such examples of L include -S-CH2-, -S-CH2- CH2-, -S-CH2CH2CH2- and -S-CH2CH2CH2CH2-.
[0016] In other embodiments where X is -CH2-, L is preferably -S (O) 2-C1-4 alkylene. Such examples of L include -S (O) 2-CH2, - S (O) 2-CH2-CH2, -S (O) 2-CH2-CH2-CH2-, and S (O) 2-CH2-CH2- CH2-CH2-.
[0017] In still other embodiments where X is -CH2-, L is -O- C1-4 alkylene. Such examples of L include -O-CH2-, -O-CH2-CH2-, -O- CH2CH2CH2- and -O-CH2CH2CH2CH2-.
[0018] In those embodiments of the description where X is S, L is preferably C1-4 alkylene. Such examples of L include -CH2-, -CH2-CH2-, - CH2CH2CH2-, and -CH2CH2CH2CH2-.
[0019] In those embodiments where X is O, L is preferably C1-4 alkylene. Such examples of L include -CH2-, -CH2-CH2-, -CH2CH2CH2- and - CH2CH2CH2CH2-.
[0020] Within the scope of the description, R1 and R2 can be the same or different. Preferably, R1 and R2 are the same. Preferably, R1 and R2 are C10-18 alkyl. Also preferred are embodiments in which the C10-18 alkyl is straight chain C10-18 alkyl. More preferred are embodiments in which R1 and R2 are C12-18 alkyl. Also preferred are embodiments in which R1 and R2 are C12-15 alkyl. In most preferred embodiments, R1 and R2 are both C13 alkyl.
[0021] In other embodiments of the description, R1 and R2 are C12-18 alkenyl. Preferably, R1 and R2 are C13-17 alkenyl. More preferably, R1 and R2 are each oleyl:

[0022] In other embodiments of the description, R1 and R2 are C12-18 alkenyl. Preferably, R1 and R2 are C13-17 alkenyl. More preferably, R1 and R2 are each linoleoyl.

[0023] Compositions comprising a compound of Formula I in a liposome, wherein the liposome comprises a bilayer of lipid molecules, are also within the scope of the description. Although the Formula I compound can comprise any molecular percentage of the lipid molecules in such compositions, it is preferred that the Formula I compound has about 5 to about 50 mol% of the lipid molecules of the compositions of the description.
[0024] The compositions of the description which comprise a compound of Formula I in a liposome may contain more than one compound of Formula I. In preferred embodiments, such compositions of the description include two compounds of Formula I. In those embodiments, it is preferred that the molar ratio of the two Formula I compounds is from about 10:30 to about 30:10.
[0025] The compositions of the description which comprise a compound of Formula I in a liposome may further comprise a cationic lipid. In such embodiments, the cationic lipid has about 5 to about 40 mol% of the lipid molecules in the composition. In addition, in those embodiments that comprise a Formula I compound in a liposome with a cationic lipid, the molar ratio between the Formula I compound and the cationic lipid is from about 5:35 to about 35: 5. More preferably, the ratio is from about 10:30 to about 30:10.
[0026] Within the scope of the description, any composition of the description may additionally comprise a liquid medium. Preferably, the liquid medium is suitable for injection into a living organism. In some embodiments, the liquid medium comprises an organic solvent. Alternatively, the liquid medium used in certain embodiments of the description comprises water and an organic solvent. In other embodiments of the description, the liquid medium may additionally comprise a non-aqueous medium.
[0027] Any composition of the description that may additionally comprise at least one phospholipid is also within the scope of the description.
[0028] In other embodiments of the description, any composition of the description may additionally comprise at least one PEG-conjugated lipid.
[0029] Star-cell specific drug vehicles are also within the scope of the description. These embodiments of the description include any of the compositions mentioned above, as well as a specific amount of stellate cell of a targeting molecule consisting of an (retinoid) n-ligand- (retinoid) n, where n = 0, 1, 2 or 3; and wherein the linker comprises a polyethylene glycol (PEG) or a PEG-like molecules.
[0030] In preferred embodiments, the drug carriers of the description will additionally comprise a siRNA molecule.
[0031] Pharmaceutical formulations are also within the scope of the description. Pharmaceutical formulations within the scope of the description include any of the drug carrier mentioned above in the description and a pharmaceutically acceptable carrier or diluent. It is preferred that in such formulations, the siRNA is encapsulated by the liposome of the compositions of the description.
[0032] Methods of delivering a drug to a patient in need of treatment are also within the scope of the description. These methods comprise providing a pharmaceutical formulation within the scope of the description and administering the pharmaceutical formulation to the patient. DEFINITIONS
[0033] The following terms are used throughout this specification.
[0034] As used herein, "cationic lipid" refers to a compound that includes at least a chemical portion of lipid and a positively charged quaternary nitrogen associated with a counterion. In the art, "lipids" are understood to comprise a chemical moiety of hydrophobic alkyl or alkenyl and a chemical moiety of carboxylic acid or ester. The preferred cationic lipids for use in the present


[0035] As used herein, "ionizable lipid" refers to a compound of Formula I within the scope of the description. These compounds have the ability to form charged species when they come in contact with an appropriate counter-ion species, for example, a species that includes an ionizable hydrogen atom.
[0036] As used herein, the term "alkyl" refers to a straight or branched, fully saturated (without double or triple bond) hydrocarbon group, for example, a group that has the general formula -CnH2n + 1. The alkyl group can have 1 to 50 carbon atoms (whenever it appears in this document, a numerical range like "1 to 50" refers to each integer in the given range; for example, the expression "1 to 50 carbon atoms ”Means that the alkyl group can consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 50 carbon atoms, although this definition also covers the occurrence of the term“ alkyl ”where it does not no numeric range is designated). The alkyl group can also be a medium-sized alkyl having 1 to 30 carbon atoms. The alkyl group could also be a lower alkyl that has 1 to 5 carbon atoms. The alkyl group of the compounds can be referred to as "C1-4alkyl" or similar designations. Just as an example, “C1-4 alkyl” indicates that there are one to four carbon atoms in the alkyl chain, that is, the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl and the like.
[0037] As used herein, "alkylene" refers to an alkanediyl functional group, for example, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2- and the like.
[0038] As used herein, "alkenyl" refers to an alkyl group that contains, in the linear or branched hydrocarbon chain, one or more double bonds. An alkenyl group can be unsubstituted or substituted. When substituted, the substituent (s) can be selected from the same groups disclosed above in relation to the alkyl group substitution unless otherwise indicated. Oleyl is an example of such an alkenyl group.
[0039] As used herein, the term "pharmaceutical carrier" refers to a pharmaceutical compound that facilitates the incorporation of a compound into cells or tissues. For example, dimethyl sulfoxide (DMSO) is a commonly used vehicle that facilitates the absorption of many organic compounds in the cells or tissues of an organism.
[0040] As used herein, the term "diluent" refers to pharmaceutical compounds diluted in water that will dissolve the formulation of interest (for example, the formulation that may include a compound, a retinoid, a second lipid, an agent stabilizing agent and / or a therapeutic agent) as well as stabilizing the biologically active form of the formulation. Salts dissolved in buffered solutions are used as diluents in the art. A commonly used buffered solution is phosphate buffered saline due to the fact that it simulates the salt conditions of human blood. Since buffer salts can control the pH of a solution in low concentrations, a buffered diluent rarely modifies the biological activity of the formulation. As used herein, an "excipient" refers to an inert substance that is added to a formulation to provide, without limitation, volume, consistency, stability, binding capacity, lubrication, disintegration capacity, etc., for the composition . A "thinner" is a type of excipient.
[0041] "Organic solvents" used within the scope of the description are known in the art alone and include, for example, C1-4alkyl alcohols, dimethyl sulfoxide ("DMSO") and the like.
[0042] As used herein, the term "therapeutic agent" refers to a compound that, upon administration to a mammal in a therapeutically effective amount, provides a therapeutic benefit to the mammal. A therapeutic agent can be referred to herein as a drug. Those skilled in the art will find that the term "therapeutic agent" is not limited to drugs that have received regulatory approval. A "therapeutic agent" can be operatively associated with a compound as described herein, a retinoid and / or a second lipid. For example, a second lipid as described herein can form a liposome and the therapeutic agent can be operatively associated with the liposome, for example, as described herein.
[0043] As used herein, a “retinoid” is a member of the class of compounds consisting of four isoprenoid units joined head to tail, see GP Moss, “Biochemical Nomenclature and Related Documents,” 2nd Ed. Portland Press , pages 247 to 251 (1992). "Vitamin A" is the generic descriptor for retinoids that qualitatively exhibit the biological activity of retinol. As used herein, the term “retinoid” refers to natural and synthetic retinoids that include first generation, second generation and third generation retinoids. Examples of naturally occurring retinoids include, but are not limited to, (1) 11-cis-retinal, (2) fully trans retinol, (3) retinyl palmitate, (4) fully trans retinoic acid, and (5) 13 -retinoic cis-acids. In addition, the term “retinoid” includes retinois, retinal, retinoic acids, retinoids and derivatives thereof.
[0044] As used herein, the term "retinoid conjugate" refers to a molecule that includes at least one chemical portion of retinoid. In preferred embodiments of the description, the retinoid conjugate will be present at a concentration of about 0.3 to about 30% by weight, based on the total weight of the composition or formulation, which is equivalent to about 0.1 to about 10 mol%, which is equivalent to a molar ratio of about 0.1 to about 10. Preferably, the retinoid conjugate is a retinoid-ligand-lipid molecule or a retinoid-ligand-retinoid molecule .
[0045] An example of a retinoid conjugate includes those compounds of Formula II:
where q, r, es are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and enantiomers and diastereomers thereof.
Preferred Formula II compounds include those in which q, res are each independently 1, 2, 3, 4, 5, 6 or 7. More preferred are those Formula II compounds in which q, res are , each independently 3, 4 or 5. Even more preferred are those compounds of Formula II in which q is 3, r is 5 and s is 3. An example of a Formula II compound is

[0047] DiVA-PEG-DiVA includes stereocenters and all enantiomers and diastereomers are considered to be within the scope of the description.
[0048] As used herein, the term "retinoid-ligand-lipid molecule" refers to a molecule that includes at least one chemical portion of retinoid linked to at least one chemical portion of lipid through at least one ligand as , for example, a chemical portion of PEG.
[0049] As used herein, the term "ligand-retinoid molecule" refers to a molecule that includes at least one chemical portion of retinoid linked to at least one other chemical portion of retinoid (which may be the same or different) ) through at least one linker, such as a chemical PEG portion.
[0050] As used herein, the terms "lipid" and "lipophilic" are used in this document in their usual meanings as understood by those skilled in the art. Non-limiting examples of lipids and lipophilic groups include fatty acids, sterols, C2-C50 alkyl, C2-C50 heteroalkyl, C2-C50 alkenyl, C2-C50 heteroalkenyl, C2-C50 aryl, heteroaryl, C2-C50 alkynyl, C2-C50 heteroalkynyl , C2-C50 carboxyalkenyl and C2-C50 carboxyeroalkenyl. A fatty acid is a long-chain saturated or unsaturated monocarboxylic acid that contains, for example, 12 to 24 carbon atoms. A lipid is characterized by being essentially insoluble in water, presenting a water solubility of less than about 0.01% (based on weight). As used herein, the terms "chemical lipid moiety" and "lipophilic chemical moiety" refer to a lipid or moiety that has joined another group. For example, a lipid group can attach to another compound (for example, a monomer) through a chemical reaction between a functional group (such as a carboxylic acid group) on the lipid and an appropriate functional group on a monomer.
[0051] As used in this document, the term "star cell" refers to neurons with various dendrites radiating from the cell body. Examples of stellate cells are inhibitory interneurons.
[0052] As used in this document, the term "siRNA" refers to a small interference RNA, also known in the art as short interference RNA or silencing RNA. SiRNA is a class of double-branched RNA molecules that have a variety of effects known in the art, the most notable of which is interference with the expression of specific protein and gene expression.
[0053] The term "liposome" is used in this document in its usual meaning as understood by the person skilled in the art and refers to a lipid bilayer structure that contains lipids attached to polar hydrophilic groups that form a substantially closed structure in aqueous media . In some embodiments, the liposome may be operatively associated with one or more compounds, such as a therapeutic agent and a retinoid. A liposome can be comprised of a single lipid bilayer (i.e., unilamellar) or it can be comprised of two or more lipid bilayers (i.e., multilamellar). Although the interior of a liposome can consist of a variety of compounds, the exterior of the liposome is accessible to the aqueous formulation that comprises the liposome. A liposome can be approximately spherical or ellipsoidal in shape.
[0054] In some embodiments, siRNA will be encapsulated by the liposome so that siRNA is inaccessible to the aqueous medium. During siRNA encapsulation, the liposome will have a solid nucleus; such siRNA encapsulating liposomes and which have a solid nucleus are referred to herein as "lipid nanoparticles". In other embodiments, siRNA will not be encapsulated by the liposome. In such embodiments, siRNA can be complexed on the outer surface of the liposome by mixing preformed liposomes to RNA in an aqueous solution. In these embodiments, siRNA is accessible to the aqueous medium. Liposomes that have siRNA bound only on their outer surface are referred to in this document as "lipoplexes".
[0055] The formulations of the description can also include PEG-conjugated lipids. PEG-conjugated lipids within the scope of the description are known in the art alone. Suitable PEG-lipids include PEG-phospholipids and PEG-ceramides such as, for example, PEG2000-DSPE, PEG2000-DPPE, PEG2000-DMPE, PEG2000-DOPE, PEG1000-DSPE, PEG1000-DPPE, PEG1000-DMPE, PEG1000-DOPE, PEG550-DSPE, PEG550-DPPE, PEG-550DMPE, PEG-1000DOPE, PEG-BML, PEG-Cholesterol. PEG2000-Ceramide, PEG1000-Ceramide, PEG750-Ceramide, PEG550- Ceramide.
[0056] The foregoing compositions of the description can include one or more phospholipids such as 1,2-distearoil-sn-glycero-3-phosphocholine ("DSPC"), dipalmitoylphosphatidylcholine ("DPPC"), 1,2-dipalmitoyl -sn- glycero-3-phosphoethanolamine ("DPPE"), and 1,2-dioleoyl-sn-glycero-3 phosphoethanolamine ("DOPE"). Preferably, the assisting lipid is DOPE.
[0057] Pharmaceutical formulations that include any of the compositions mentioned above in addition to a pharmaceutically acceptable carrier or diluent are also within the scope of the description. The pharmaceutical formulations of the description will include at least one therapeutic agent. Preferably, the therapeutic agent is a siRNA. It is envisaged that any siRNA molecule can be used within the scope of the description. For example, siRNA can include: Senso (5 '-> 3') GGACAGGCCUCUACAACUATT (SEQ. ID. NO. 1) Antisense (3 '-> 5') TTCCUGUCCGGAGAUGUUGAU (SEQ. ID. NO. 2) and Senso (5 '-> 3') GGACAGGCCUGUACAACUATT (SEQ. ID. NO. 3) Antisense (3 '-> 5') TTCCUGUCCGGACAUGUUGAU (SEQ. ID. NO. 4)
[0058] In preferred formulations of the description that include siRNA, siRNA is encapsulated by the liposome. In other embodiments, siRNA may be outside the liposome. In those embodiments, siRNA can be complexed out of the liposome.
[0059] Methods of delivering a therapeutic agent to a patient are also within the scope of the description. Such methods comprise providing a pharmaceutical formulation that includes any of the foregoing compositions and a pharmaceutically acceptable carrier or diluent; and administering the pharmaceutical formulation to the patient.
[0060] In another aspect, the present description refers to a pharmaceutical formulation comprising one or more physiologically acceptable surface active agents, pharmaceutical vehicles, diluents, excipients and suspending agents, or a combination thereof; and a formulation (for example, a formulation that can include a compound, a retinoid, a second lipid, a stabilizing agent and / or a therapeutic agent) disclosed herein. pharmaceutical carriers or additional diluents acceptable for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA (1990), which is incorporated herein reference title in its entirety. Preservatives, stabilizers, dyes and the like can be provided in the pharmaceutical formulation. For example, sodium benzoate, ascorbic acid and esters of p-hydroxybenzoic acid can be added as preservatives. In addition, antioxidants and suspending agents can be used. In various embodiments, alcohols, esters, sulfated aliphatic alcohols and the like can be used as surface active agents; sucrose, glucose, lactose, starch, crystallized cellulose, mannitol, light anhydrous silicate, magnesium aluminate, magnesium aluminate metasilicate, synthetic aluminum silicate, calcium carbonate, sodium acid carbonate, calcium calcium phosphate, calcium carboxymethyl calcium cellulose and the like can be used as excipients; coconut oil, olive oil, sesame oil, peanut oil, soybean oil can be used as suspending agents or lubricants; cellulose acetate phthalate as a derivative of a carbohydrate such as cellulose or sugar, or methylacetate-methacrylate copolymer as a polyvinyl derivative can be used as suspending agents; and plasticizers such as phthalate esters and the like can be used as suspending agents.
[0061] The pharmaceutical formulations described in this document can be administered to a human patient alone or in pharmaceutical formulations in which they are mixed with other active ingredients, as in combination therapy, or suitable pharmaceutical carriers or excipient (s) . Techniques for the formulation and administration of the compounds of the present application can be found in "Remington’s Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, 18th edition, 1990.
[0062] Suitable routes of administration may include, for example, parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal or intraocular injections. The formulation (for example, the formulation which may include a compound, a retinoid, a second lipid, a stabilizing agent and / or a therapeutic agent) can also be administered in sustained or controlled release dosage forms, including injections of deposits , osmotic pumps and the like, for prolonged and / or timed pulsed administration at a predetermined rate. In addition, the administration route can be local or systematic.
[0063] Pharmaceutical formulations can be manufactured in a way that is known in itself, for example, by means of conventional mixing, dissolving, granulating, dragee, levigation, emulsification, encapsulation, trapping or tabletting processes.
[0064] Pharmaceutical formulations can be formulated in any conventional manner with the use of one or more physiologically acceptable pharmaceutical carriers comprising excipients and auxiliaries that facilitate the processing of active compounds to form preparations that can be used pharmaceutically. The appropriate formulation depends on the route of administration chosen. Any of the well-known techniques, pharmaceutical vehicles and excipients can be used as appropriate and as understood in the art; for example, in Remington’s Pharmaceutical Sciences, above.
[0065] Injectable products can be prepared in conventional ways, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid before injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride and the like. In addition, if necessary, injectable pharmaceutical formulations may contain small amounts of non-toxic auxiliary substances, such as wetting agents, pH buffering agents and the like. Physiologically compatible buffers include, but are not limited to, Hanks' solution, Ringer's solution or physiological saline buffer. If desired, absorption enhancing preparations can be used.
[0066] Pharmaceutical formulations for parenteral administration, for example, by bolus injection or continuous infusion, include aqueous solutions of the active formulation (for example, the formulation which may include a compound, a retinoid, a second lipid, an agent stabilizing agent and / or a therapeutic agent) in water-soluble form. In addition, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension can also contain stabilizers or suitable agents that increase the solubility of the compounds to allow the preparation of highly concentrated solutions. Injection formulations can be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative. The formulations can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles and can contain formulating agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, for example, water without sterile pyrogen, before use.
[0067] In addition to the preparations described above, the formulations can also be formulated as a depot preparation. Such long-acting formulations can be administered through intramuscular injection. Thus, for example, formulations (for example, the formulation which may include a compound, a retinoid, a second lipid, a stabilizing agent and / or a therapeutic agent) can be formulated with suitable polymeric or hydrophobic materials (for example , as an emulsion in an acceptable oil) or ion exchange resins, or as reduced soluble derivatives, for example, as a reduced soluble salt.
[0068] The compositions and formulations of the description can also be formulated for topical delivery and can be applied to the skin of an individual using any process suitable for the application of topical delivery vehicle. For example, the formulation can be applied manually, using an applicator or through a process that involves both. After application, the formulation can be slowly penetrated into an individual's skin, for example, by rubbing. The application can be carried out multiple times a day or once a day. For example, the formulation can be applied to an individual's skin once a day, twice a day or multiple times a day, or can be applied once every two days, once every three days or about once. per week, once every two weeks or once every few weeks.
[0069] Some achievements in this document are directed to a method of delivering a therapeutic agent to a cell. For example, some achievements are directed at a method of delivering a therapeutic agent like siRNA to a cell. Cells suitable for use according to the methods described herein include prokaryotic, yeast or higher eukaryotic cells, including cells of plant or animal origin (e.g., mammalian cells). In some embodiments, the cells may be human fibrosarcoma cells (for example, HT1080 cell line). In other embodiments, the cells may be hepatic stellate cells (cell line LX2). In other embodiments, the cells may be cancer cells. In still other embodiments, the cells can be stem cells (cell line pHSC). Cell lines that are model systems for cancer can be used, including, but not limited to, breast cancer (cell lines MCF-7, MDA-MB-438), glioblastoma cell line U87, B16F0 cells (melanoma), cells HeLa (cervical cancer), A549 cells (lung cancer), and GH3 and 9L mouse tumor cell lines. In these embodiments, the formulations described in this document can be used to transfect a cell. Such embodiments may include bringing the cell into contact with a formulation described herein that includes a therapeutic agent, thereby delivering a therapeutic agent to the cell.
[0070] Methods of treating a condition characterized by abnormal fibrosis, which may include administering a therapeutically effective amount of a formulation described in this document, are disclosed herein. Conditions characterized by abnormal fibrosis can include cancer and / or a fibrotic disease. The types of cancer that can be treated or improved by a formulation described in this document include, but are not limited to, lung cancer, pancreatic cancer, breast cancer, liver cancer, stomach cancer and colon cancer. In one embodiment, the cancer that can be treated or improved is pancreatic cancer. In another embodiment, the cancer that can be treated or improved is lung cancer. The types of fibrotic disease that can be treated or ameliorated by a formulation described in this document include, but are not limited to, liver fibrosis, liver cirrhosis, pancreatitis, pancreatic fibrosis, cystic fibrosis, vocal cord healing, vocal cord mucosal fibrosis , laryngeal fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, myelofibrosis, retroperitoneal fibrosis and nephrogenic systemic fibrosis. In one embodiment, the condition that can be treated or improved is liver fibrosis.
[0071] The pharmaceutical formulations or compositions described in this document can be administered to the individual through any suitable means. Non-limiting examples of methods of administration include, among others, (a) administration via injection, subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally, intraorbitally, intracapsularly, intraspinally, intrasternally or the like, including delivery by infusion pump; (b) local administration such as through direct injection into the renal or cardiac area, for example, through implantation of a deposit; as well as it is considered by those skilled in the art to put the active compound in contact with living tissue.
[0072] Pharmaceutical compositions suitable for administration include formulations (for example, the formulation which may include a compound, a retinoid, a second lipid, a stabilizing agent and / or a therapeutic agent) in which the active ingredients are contained in a effective amount to achieve its intended purpose. The therapeutically effective amount of the compounds disclosed herein required as a dose will depend on the route of administration, the type of animal, including humans, being treated and the physical characteristics of the specific animal under consideration. A dose can be customized to achieve a desired effect, but it will depend on such factors as weight, diet, concomitant mediation and other factors that those skilled in the medical art will recognize. More specifically, the term "a therapeutically effective amount" means an amount of compound effective to prevent, alleviate or ameliorate the symptoms of the disease or prolong the survival of the subject being treated. The determination of a therapeutically effective amount is well within the ability of those skilled in the art, especially in light of the detailed description provided in this document.
[0073] As will become apparent to the person skilled in the art, the useful in vivo dosage to be administered and the particular mode of administration will vary depending on the age, weight and species of mammal treated, the particular compounds employed and the specific use for which these compounds are used. The determination of effective dosage levels, that is, the dosage levels necessary to achieve the desired result, can be carried out by the person skilled in the art with the use of routine pharmacological methods. Typically, human clinical applications of products are initiated at lower dosage levels, where the dosage level is increased until the desired effect is achieved. Alternatively, acceptable in vitro studies can be used to establish useful doses and routes for administering the compositions identified by the present methods with the use of established pharmacological methods.
[0074] In studies of non-human animals, applications of potential products are initiated at higher dosage levels, where the dosage is decreased until the desired effect is no longer achieved or adverse side effects disappear. The dosage can be in a wide range, depending on the desired effects and the therapeutic indication. Typically, dosages can be from about 10 microgram / kg to about 100 mg / kg of body weight, preferably about 100 microgram / kg to about 10 mg / kg of body weight. Alternatively, dosages can be based and calculated on the patient's surface area, as understood by one skilled in the art.
[0075] The exact formulation, route of administration and dosage for pharmaceutical compositions can be chosen by the individual physician in the light of the patient's condition. (See, for example, Fingl et al. 1975, in “The Pharmacological Basis of Therapeutics”, which is hereby incorporated into this document as a reference in its entirety, with particular reference to Chap. 1, page 1 ). Typically, the dose setting of the composition administered to the patient can be from about 0.5 to about 1,000 mg / kg of the patient's body weight. The dosage can be single or a series of two or more given over the course of one or more days, as needed by the patient. In examples where human dosages for compounds have been established for at least some condition, dosages will be almost equal, or dosages that are from about 0.1% to about 500%, more preferably, about 25% at about 250% of the established human dosage. Where no human dosage is established, as will be the case for recently disclosed pharmaceutical compositions, a suitable human dosage can be deducted from ED50 or ID50 values, or from other appropriate values derived from in vitro or in vivo studies, as qualified by studies of toxicity and efficacy studies in animals.
[0076] It should be noted that the attending physician would know how and when to complete, interrupt or adjust the administration due to toxicity or organ dysfunction. Conversely, the attending physician would also know how to adjust treatment to higher levels if the clinical response is not adequate (excluding toxicity). The magnitude of a dose administered in the management of the disorder of interest will vary with the severity of the condition and the route of administration. The severity of the condition can, for example, be assessed, in part, using standard prognostic assessment methods. In addition, the dose, and perhaps the dose frequency, will also vary according to the age, body weight and response of the individual patient. A program comparable to the one discussed above can be used in veterinary medicine.
[0077] Although the exact dosage is determined on a drug-by-drug basis, in most cases, some generalizations related to the dosage can be made. The daily dosage regimen for an adult human patient can be, for example, a dose of about 0.1 mg to 2,000 mg of each active ingredient, preferably about 1 mg to about 500 mg, for example, 5 to 200 mg. In other embodiments, an intravenous, subcutaneous or intramuscular dose of each active ingredient of about 0.01 mg to about 100 mg, preferably about 0.1 mg to about 60 mg, for example, about 1 to about 40 mg. In cases of administration of a pharmaceutically acceptable salt, dosages can be calculated as the free base. In some embodiments, the formulation is administered 1 to 4 times a day. Alternatively, the formulations can be administered by continuous intravenous infusion, preferably at a dose of each active ingredient up to about 1,000 mg per day. As will be understood by the person skilled in the art, in certain situations, it may be necessary to administer the formulations disclosed in this document in amounts that exceed, or that greatly exceed, the preferential dosage range set out above in order to effectively and aggressively treat diseases or infections particularly aggressive. In some embodiments, the formulations will be administered over a period of continuous therapy, for example, for a week or more, or for months or years.
[0078] The dosage amount and interval can be individually adjusted to provide plasma levels of the active chemical portion that are sufficient to maintain modulation effects, or the minimum effective concentration (MEC). The MEC will vary for each compound, but can be estimated from in vitro data. The dosages required to achieve MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.
[0079] Dosage intervals can also be determined using an MEC value. The compositions should be administered using a regimen that maintains plasma levels above the ECM for 10 to 90% of the time, preferably between 30 to 90% and, more preferably, between 50 to 90%.
[0080] In cases of local administration or selective absorption, the effective local concentration of the drug may not be related to the plasma concentration.
[0081] The amount of formulation administered may depend on the individual being treated, the individual's weight, the severity of the affliction, the manner of administration and the prescribing physician's assessment.
[0082] The formulations disclosed in this document (for example, the formulation that may include a compound, a retinoid, a second lipid, a stabilizing agent and / or a therapeutic agent) can be evaluated for effectiveness and toxicity with the use of known methods. For example, the toxicology of a particular compound, or a subset of the compounds, sharing certain chemical moieties, can be established by determining toxicity in vitro towards a cell line, such as a mammalian cell line and, preferably, of a human being. The results of such studies are often predictive of toxicity in animals, such as mammals or, more specifically, humans. Alternatively, the toxicity of particular compounds in an animal model, such as mice, rats, rabbits or monkeys, can be determined using known methods. The effectiveness of a particular compound can be established using a number of recognized methods, such as in vitro methods, animal models or clinical experiments on humans. Recognized in vitro models exist for almost all classes of disease, including, but not limited to, cancer, cardiovascular disease and various autoimmune disorders. Similarly, acceptable animal models can be used to establish the effectiveness of chemicals to treat such conditions. When selecting a model to determine effectiveness, the subject can be guided by the state of the art to choose an appropriate model, dose and route of administration, and regimen. Of course, clinical experiments on humans can also be used to determine the effectiveness of a compound in humans.
[0083] The formulations can, if necessary, be presented in a package or dispenser device that can contain one or more unit dosage forms containing the active ingredient. The package may, for example, comprise a sheet of metal or plastic, such as a blister-type package. The package or dispensing device may be accompanied by instructions for administration. The package or dispenser may also be accompanied with a notice associated with the container in a form prescribed by a government agent that regulates the manufacture, use or marketing of pharmaceutical products, the notice of which reflects the approval by the agency in the form of the drug for administration in humans or veterinary. Such a warning, for example, may be labeling approved by the United States Food and Drug Administration to prescribe drugs, or the insertion of an approved product. Compositions comprising a compound formulated in a compatible pharmaceutical carrier can also be prepared, placed in an appropriate container and labeled for the treatment of an indicated condition.
[0084] It is understood that, in any compound described in this document that has one or more stereocenters, if an absolute stereochemistry is not expressly indicated, then each center can independently have an R configuration or an S configuration or a mixture thereof. Thus, the compounds provided in this document can be enantiomerically pure or stereoisomeric mixtures. Furthermore, it should be understood that, in any compound that has one or more double bonds that generate geometric isomers that can be defined as E or Z, each double bond can be independently E or Z or a mixture thereof. Likewise, all tautomeric forms are also intended to be included.
[0085] Preferred Formula I compounds within the scope of the description are shown in Table 1. In vitro and in vivo data (see below) are also shown in Table 1. TABLE 1




[0086] The description can be further exemplified by reference to the following examples. These examples are illustrative only and are not intended to limit the description. EXPERIMENTAL SECTION PREPARATION OF ((2- (DIMETHYLAMINE) ACETYL) AZANODHL) BIS (PROPANO-3,1-DHL) DITETRADECANOATE (I-PR-DC)
STEP 1: PREPARATION OF INTERMEDIATE 1: 3,3'-AZANODI-ILBIS (PROPAN-1-OL)

[0087] A mixture of 3-amino-1-propanol (14.5 ml, 19.0 mmoles), 1-chloro-3-hydroxy propane (8.00 ml, 95.6 mmoles) and H2O (~ 50 ml) was refluxed for 24 hours. Then, potassium hydroxide (5.40 g) was added. After dissolution, the entire water was evaporated to leave viscous oil and large amounts of potassium chloride. They were filtered and washed with dry acetone and dichloromethane. The organic phase was dried over Na2SO4, filtered and evaporated to leave an oil. Purification by silica gel chromatography eluting with a gradient from DCM / MeOH yielded 3,3'-azanedi-ylbis (propan-1-ol) (12.5 g). STEP 2: PREPARATION OF INTERMEDIATE 2: BIS (3-HYDROXYPROPYL) TERC-BUTYL CARBAMATE

[0088] 3,3'-azanedi-ylbis (propan-1-ol) (12.5 g, 95.4 mmoles) was diluted in DCM (25 ml). A solution of di-tert-butyl dicarbonate (26.0 g, 119 mmoles) in DCM (25 ml) was slowly added during stirring under an argon gas purge. The reaction was allowed to be stirred throughout the night. The reaction mixture was concentrated. Purification by silica gel chromatography eluting with a DCM / MeOH gradient yielded tert-butyl bis (3-hydroxypropyl) carbamate. STEP 3: PREPARATION OF INTERMEDIATE 3: ((TERC-BUTOXICARBONIL) AZANODI-

[0089] tert-Butyl bis (3-hydroxypropyl) carbamate (4.00 g, 17.3 mmoles), Et3N (4.8 ml, 34.6 mmoles) and DMAP (529 mg, 4.33 mmoles) were dissolved in chloroform (50 ml). While stirring in an ice bath, a solution of myristoyl chloride was added over 15 minutes. The addition was carried out so that the reaction temperature did not exceed 30 ° C. The reaction was allowed to stir at room temperature overnight. The next day, MeOH (50 ml) and 0.9% saline (50 ml) were added to abruptly cool the reaction. The organic layer was separated and washed with 1M NaHCO3. The solvent was dried over Na2SO4, filtered and concentrated in vacuo to yield ((tert-butoxycarbonyl) azanediol) bis (propane-3,1-diyl) dithetradecanoate as an oil which was passed on without further purification. STEP 4: PREPARATION OF INTERMEDIATE 4: AZANODI-ILBIS TFA SALT (PROPANO-3,1-DI-IL) DITETRADECANOATE

[0090] ((Terc-butoxycarbonyl) azanediyl) bis (propane-3,1-diyl) ditetradecanoate (11.3 g, 17.3 mmol) was dissolved in TFA / CHCl3 (1: 1, 20 ml ) and the mixture was allowed to stir at room temperature for 15 minutes. Then, the material was concentrated in vacuo. This was repeated a second time. Then, the material was dissolved in DCM and washed with H2O, dried with Na2SO4, concentrated in vacuo and dried completely overnight. The reaction mixture was concentrated. Purification by chromatography on silica gel eluting with a gradient of DCM / MeOH yielded TFA salt of azanediolis (propane-3,1-diyl) dithetradecanoate (7.5 g). STEP 5: PREPARATION OF I-PR-DC: ((2- (DIMETHYLAMINE) ACETYL) AZANODI-IL) BIS (PROPANO-3,1-DI-IL) DITETRADECANOATE

[0091] The TFA salt of azanedi-ylbis (propane-3,1-di-yl) dithetradecanoate (750 mg, 1.35 mmol) was diluted in DCM (5 ml) and added to a pre-activated mixture of N , N-dimethylglycine (154 mg, 1.49 mmol), HATU (616 mg, 1.62 mmol) and DIEA (495 µL, 2.84 mmol) in DCM (5 ml). The flask was drained with argon and allowed to stir at room temperature overnight. The reaction mixture was concentrated. Purification by silica gel chromatography eluting with a DCM / MeOH gradient yielded ((2- (dimethylamino) acetyl) azanediyl) bis (propane-3,1-diyl) dithetradecanoate (465 mg). QTOF MS ESI +: m / z 639.6 (M + H). PREPARATION OF (Z) - ((2- (DIMETHYLAMINE) ACETYL) AZANODHL) BIS (PROPANO-3,1-DHL) DIOLEATE (I-PR-DODC)
STEP 1: PREPARATION OF INTERMEDIATE 1: 3,3'-AZANODI-ILBIS (PROPAN-1-OL

[0092] A mixture of 3-amino-1-propanol (14.5 ml, 19.0 mmol), 1-chloro-3-hydroxy propane (8.00 ml, 95.6 mmol) and water (50 ml) was refluxed for 24 hours. Then, potassium hydroxide (5.40 g) was added. After dissolution, the entire water was evaporated to leave viscous oil and large amounts of potassium chloride. They were filtered and washed with dry acetone and dichloromethane. The organic phase was dried over Na2SO4, filtered and evaporated to leave an oil. Purification by silica gel chromatography eluting with a DCM / MeOH gradient yielded 3,3'-azanedi-ylbis (propan-1-ol) (12.5g). STEP 2: PREPARATION OF INTERMEDIATE 2: TERC-BUTIL BIS (3- HYDROXYPROPYL) CARBAMATE

[0093] 3,3'-azanedi-ylbis (propan-1-ol) (12.5 g, 95.4 mmoles) was diluted in DCM (25 ml). A solution of di-tert-butyl dicarbonate (26.0 g, 119 mmoles) in DCM (25 ml) was slowly added during stirring under an argon gas purge. The reaction was allowed to be stirred throughout the night. The reaction mixture was concentrated. Purification by silica gel chromatography eluting with a DCM / MeOH gradient yielded tert-butyl bis (3-hydroxypropyl) carbamate. STEP 3: PREPARATION OF INTERMEDIATE 3: (Z) - ((TERC-BUTOXICARBONIL) AZANODI-IL) BIS (PROPANO-3,1-DI-IL) DIOLEATO

[0094] Terc-butyl bis (3-hydroxypropyl) carbamate, triethylamine and DMAP were dissolved in chloroform. While stirring in an ice bath, a solution of oleyl chloride was added over 15 minutes. The addition was carried out so that the reaction temperature did not exceed 30 ° C. The reaction was allowed to stir at room temperature overnight. The next day, MeOH (50 ml) and 0.9% saline (50 ml) were added to abruptly cool the reaction. The organic layer was separated and washed with 1M NaHCO3. The solvent was dried with Na2SO4, filtered and concentrated in vacuo to yield (Z) - ((tert-butoxycarbonyl) azanediyl) bis (propane-3,1-diyl) dioleate as an oil which was passed on without purification additional. STEP 4: PREPARATION OF INTERMEDIATE 4: TFA SALT FROM (Z) -AZANODI- ILBIS (PROPANO-3,1-DI-IL) DIOLEATO

[0095] (Z) - ((tert-butoxycarbonyl) azanediyl) bis (propane-3,1-diyl) dioleate (13.2 g, 17.3 mmol) was dissolved in TFA / CHCl3 (1: 1, 20 ml) and the mixture was allowed to stir at room temperature for 15 minutes. Then, the material was concentrated in vacuo. This was repeated a second time. Then, the material was dissolved in DCM and washed with H2O, dried with Na2SO4 and concentrated in vacuo. Purification by silica gel chromatography eluting with a DCM / MeOH gradient yielded a TFA salt of (Z) -azanediolis (propane-3,1-diyl) dioleate. STEP 5: PREPARATION OF I-PR-DODC: (Z) - ((2- (DIMETHYLAMINE) ACETYL) AZANODI-IL) BIS (PROPANO-3,1-DI-IL) DIOLEATE

[0096] TFA salt of (Z) -azanodi-ylbis (propane-3,1-di-yl) dioleate (750 mg, 1.13 mmoles) was diluted in DCM (5 ml) and added to a pre-activated mixture of N, N-dimethylglycine (128mg, 1.24mmol), HATU (517mg, 1.36mmol) and DIEA (413uL, 2.37mmol) in DCM (5ml). The flask was drained with argon and allowed to stir at room temperature overnight. The reaction mixture was concentrated. Purification by silica gel chromatography eluting with a gradient from DCM / MeOH yielded (Z) - ((2- (dimethylamino) acetyl) azanediyl) bis (propane-3,1-diyl) dioleate (450 mg) . QTOF MS ESI +: m / z 747.7 (M + H). PREPARATION OF ((2- (DIMETHYLAMINE) ACETYL) AZANODI-IL) BIS (ETANO-2,1-DI-IL) DITETRADECANOATE, (I-DC)
STEP 1: PREPARATION OF INTERMEDIATE 1: ((TERC-BUTOXICARBONIL) AZANODI-IL) BIS (ETANO-2,1-DI-IL) DITETRADECANOATE

[0097] N-Boc diethanolamine (MW 205.25; 8.4 g, 0.041 mol), triethylamine (MW 101.19; 11.5 ml, 0.083 mol) and 4- (dimethylamino) pyridine (MW 122.17; 1.3 g, 0.011 mol) were dissolved in chloroform (170 ml). While stirring in an ice / water bath, a solution of myristoyl chloride (MW 246.82; 22 ml, 80.9 mmoles) in 100 ml of chloroform was added by dripping. The reaction mixture was then removed from the ice bath and stirring was continued at room temperature for 2 hours. A mixture of 200 ml of methanol and 200 ml of 0.9% saline solution was added to abruptly cool the reaction. Stirring was stopped and the organic layer was isolated. The solvent was removed by rotary evaporation to provide ((tert-butoxycarbonyl) azanediyl) bis (ethane-2,1-diyl) dithetradecanoate as a colorless oil (25.7 g) which was carried forward without purification additional. STEP 2: PREPARATION OF INTERMEDIATE 2: AZANEDI-ILBIS TFA SALT (ETHANE-2,1-DI-IL) DITETRADECANOATE

[0098] To a solution of ((tert-butoxycarbonyl) azanediol) bis (ethane-2,1-di-yl) ditetradecanoate (33.0 g, 0.053 mol) in 100 ml of chloroform was added trifluoroacetic acid (150 ml, 2.02 mol). The reaction mixture was stirred at room temperature overnight. After the solvent was removed by rotary evaporation, the resulting soft solid was recrystallized from 80 ml of methanol to yield TFA salt from azanedi-ilbis (ethane-2,1-di-yl) dithetradecanoate TFA salt (16 , 6 g) as a white solid. STEP 3: PREPARATION OF I-DC: ((2- (DIMETHYLAMINE) ACETYL) AZANEDI-IL) BIS (ETHANE-2,1-Di-iL) DiTETRADECANoATE

[0099] The TFA salt of azanedi-ilbis (ethane-2,1-di-yl) dithetradecanoate (10 g, 16 mmol) was diluted with dimethyl glycine (42.5 g, 25 mmol), DCC (4.7 g, 23 mmol) and DIEA (6.33 ml, 40 mmol) in Pyridine (20 ml). The round-bottom flask was drained with argon gas and the reaction mixture was heated to 55 ° C overnight. The next day, the reaction mixture was concentrated. After purification by silica gel chromatography eluting with a DCM / MeOH, the pooled fractions were concentrated to yield ((2- (dimethylamino) acetyl) azanedi-yl) bis (ethane-2,1-di-yl) ditetradecanoate . PREPARATION OF ((3- (DIMETHYLAMINE) PROPANOIL) AZANEDHL) BIS (ETANO-2,1-DHL) DITETRADECANOATE (I-ET-DC, ALSO CALLED IN THE PRESENT INVENTION OF ET104)
I-ET-DC PREPARATION: ((3- (DIMETHYLAMINE) PROPANOIL) AZANEDI-IL) BIS (ETANE-2,1-DI-IL) DITETRADECANOATE

[00100] TFA salt synthesis of azanedi-ilbis (ethane-2,1-di-yl) ditetradecanoate described above. The TFA salt of azanedi-ilbis (ethane-2,1-di-yl) dithetradecanoate (1.50 g, 2.85 mmol) was diluted with DCM (10 ml) and added to a pre-activated mixture of salt of 3- (dimethylamino) propionic acid HCl (482 mg, 3.14 mmol), HATU (1.30 g, 3.42 mmol) and DIEA (1.04 ml, 5.98 mmol) in DCM (10 ml) . The round-bottom flask was drained with argon gas and the reaction mixture was allowed to stir at room temperature overnight. The reaction mixture was concentrated. After purification by silica gel chromatography eluting with a DCM / MeOH, the pooled fractions were concentrated and stirred in DCM (20 ml) and 10% K2CO3 (20 ml) between 0 and 5 ° C for 30 min. The organic layer was isolated and the aqueous layer further extracted with DCM (2 x 10 ml). The combined organics were stirred with MgSO4 for 30 min. between 0 and 5 ° C, filtered, washed with DCM and concentrated to yield ((3- (dimethylamino) propanoyl) azanediol) bis (ethane-2,1-diyl) dithetradecanoate (1.01 g). QTOF MS ESI +: m / z 625.6 (M + H). PREPARATION OF (Z) - ((3- (DIMETHYLAMINE) PROPANOIL) AZANEDI-IL) BIS (ETANO-2,1-DI-IL) DIOLEATE I-ET-DODC
STEP 1: PREPARATION OF INTERMEDIATE 1: (Z) - ((TERC-BUTOXICARBONIL) AZANEDI-IL) BIS (ETANO-2,1-DI-IL) DIOLEATE

[00101] N-Boc diethanolamine (17.8 g, 0.087 mol), triethylamine (24.4 ml, 0.176 mol) and 4- (dimethylamino) pyridine (2.76 g, 0.023 mol) were dissolved in 350 ml of chloroform . During stirring, a solution of oleyl chloride (61.6 g, 0.174 mol) in 100 ml of chloroform was added for 10 min. (Alternatively, the N-Boc diethanolamine chloroform solution was immersed in an ice / water bath while the oleyl chloride was added). The addition was carried out in such a way that the temperature of the reaction mixture did not exceed 50 ° C. The reaction mixture was stirred at room temperature for 2 h. A mixture of 200 ml of methanol and 200 ml of 0.9% saline was added to cool the reaction. The organic layer was separated and washed with 2 x 100 ml of dilute aqueous sodium bicarbonate. The solvent was removed by rotary evaporation to provide (Z) - ((tert-butoxycarbonyl) azanediol) bis (ethane-2,1-diyl) dioleate as a pale yellow oil (59.5 g). This material was used for the next step without further purification. 1H NMR (400 MHz, CDCl3) 0.87 (t, 6H, CH3), 1.20-1.40 (m, 40H, CH2), 1.45 (s, 9H, tBu CH3), 1.59 (m, 4H, CH2CH2C (= O)), 2.00 (m, 8H, CH2CH = CH), 2.33 (t, 4H, CH2C (= O)), 3.48 (m, 4H, NCH2CH2O) , 4.18 (m, 4H, NCH2CH2O), 5.33 (m, 4H, CH = CH). STEP 2: PREPARATION OF INTERMEDIATE 2: TFA SALT FROM (Z) -AZANEDI- ILBIS (ETANO-2,1-DI-IL) DIOLEATE

[00102] (Z) - ((tert-butoxycarbonyl) azanedi-yl) bis (ethane-2,1-di-yl) dioleate (59.5 g, 0.081 mol) was treated twice with 100 ml of trifluoroacetic acid ( 100 ml, 1.35 mol) and 100 ml of chloroform. Each consisted of stirring at room temperature for 10 min., And the solvent was removed by rotary evaporation at the end of each treatment. The residue was dissolved in 200 ml of methylene chloride and the mixture was washed with 100 ml of water twice. The residue was purified by silica gel chromatography using a mixture of methanol and methylene chloride as the eluent to yield TFA salt of (Z) -azanedi-ilbis (ethane-2,1-di-yl) dioleate ( 44.0 g). 1H NMR (400 MHz, CDCl3) 0.87 (t, 6H, CH3), 1.20-1.40 (m, 40H, CH2), 1.59 (m, 4H, CH2CH2C (= O)), 2.00 (m, 8H, CH2CH = CH), 2.33 (t, 4H, CH2C (= O)), 3.31 (m, 4H, NCH2CH2O), 4.38 (m, 4H, NCH2CH2O), 5.33 (m, 4H, CH = CH). STEP 3: PREPARATION OF I-ET-DODC: (Z) - ((3- (DIMETHYLAMINE) PROPANOIL) AZANEDI-IL) BIS (ETHANE-2,1-DI-IL) DIOLEATE

[00103] TFA salt of (Z) -azanedi-ilbis (ethane-2,1-di-yl) dioleate (1.50 g, 2.37 mmol) was diluted with DCM (10 ml) and added to a pre-mix activated HCl salt of 3- (dimethylamino) propionic acid (383 mg, 2.49 mmol), HATU (1.03 g, 2.72 mmol) and DIEA (831 ul, 4.77 mmol) in DCM (10 ml). The round-bottom flask was drained with argon gas and the reaction mixture was allowed to stir at room temperature overnight. The reaction mixture was concentrated. After purification by silica gel chromatography eluting with a DCM / MeOH, the pooled fractions were concentrated and stirred in DCM (20 ml) and 10% K2CO3 (20 ml) between 0 and 5 ° C for 30 min. The organic layer was isolated and the aqueous layer further extracted with DCM (2 x 10 ml). The combined organics were stirred with MgSO4 for 30 min. between 0 and 5 ° C, filtered, washed with DCM and concentrated to yield (Z) - ((3- (dimethylamino) propanoyl) azanedi-yl) bis (ethane-2,1-di-yl) dioleate. QTOF MS ESI +: m / z 733.6 (M + H). PREPARATION OF ((4- (DIMETHYLAMINE) BUTANOIL) AZANEDI-IL) BIS (ETANO-2,1-DI-IL) DITETRADECANOATE [I-PROP-DC (ALSO CALLED IN THE PRESENT INVENTION OF PR104)]

[00104] TFA salt synthesis of azanedi-ilbis (ethane-2,1-di-yl) dithetradecanoate previously described. The TFA salt of azanedi-ilbis (ethane-2,1-di-yl) dithetradecanoate (1.00 g, 1.90 mmol) was diluted with DCM (5 ml) and added to a pre-activated mixture of salt of 4- (dimethylamino) butyric acid HCl (382 mg, 2.28 mmol), HATU (867 mg, 2.28 mmol) and DIEA (728 ul, 4.18 mmol) in DCM (5 ml). The round-bottom flask was drained with argon gas and the reaction mixture was allowed to stir at room temperature overnight. The reaction mixture was concentrated. After purification by silica gel chromatography eluting with a DCM / MeOH, the pooled fractions were concentrated and stirred in DCM (20 ml) and 10% K2CO3 (20 ml) between 0 and 5 ° C for 30 min. The organic layer was isolated and the aqueous layer further extracted with DCM (2 x 10 ml). The combined organics were stirred with MgSO4 for 30 min. between 0 and 5 ° C, filtered, washed with DCM and concentrated to yield ((4- (dimethylamino) butanoyl) azanediyl) bis (ethane-2,1-diyl) dithetradecanoate. LCMS ESI +: m / z 639.6 (M + H). PREPARATION OF (Z) - ((4- (DIMETHYLAMINE) BUTANOIL) AZANEDI-IL) BIS (ETANO-2,1-DI-IL) DIOLEATE [I-PROP-DODC (ALSO CALLED IN THE PRESENT INVENTION OF PR104- DO)]

[00105] TFA salt synthesis of (Z) -azanedi-ilbis (ethane-2,1-di-yl) dioleate previously described. The TFA salt of (Z) -azanedi-ilbis (ethane-2,1-diyl) dioleate (1.00 g, 1.58 mmol) was diluted with DCM (5 ml) and added to a pre-mix. activated HCl salt of 4- (dimethylamino) butyric acid (317 mg, 1.89 mmol), HATU (719 mg, 1.89 mmol) and DIEA (606 ul, 3.48 mmol) in DCM (5 ml) . The round-bottom flask was drained with argon gas and the reaction mixture was allowed to stir at room temperature overnight. The reaction mixture was concentrated. After purification by silica gel chromatography eluting with a DCM / MeOH, the pooled fractions were concentrated and stirred in DCM (20 ml) and 10% K2CO3 (20 ml) between 0 and 5 ° C for 30 min. The organic layer was isolated and the aqueous layer further extracted with DCM (2 x 10 ml). The combined organics were stirred with MgSO4 for 30 min. between 0 and 5 ° C, filtered, washed with DCM and concentrated to yield (Z) - ((4- (dimethylamino) butanoyl) azanedi-yl) bis (ethane-2,1-di-yl) dioleate. LCMS ESI +: m / z 747.7 (M + H). PREPARATION OF (Z) - ((2 - ((2- (DIMETHYLAMINE) ETHYL) TIO) ACETYL) AZANEDI-IL) BIS (ETHANE-2,1-DI-IL) DIOLEATE (S104-DO)

[00106] TFA salt synthesis of (Z) -azanedi-ilbis (ethane-2,1-di-yl) dioleate previously described. The TFA salt of (Z) -azanedi-ilbis (ethane-2,1-diyl) dioleate (4.06 g, 6.41 mmol) was stirred in DCM (60 ml) with 10% K2CO3 (30 ml) between 0 and 5 ° C. After 30 min., The organic phase is separated and the aqueous phase was further extracted with DCM (30 ml). The combined organic phases are stirred with MgSO4 for a period of 30 min. between 0 and 5 ° C, filtered and washed with DCM (~ 30 ml). To the combined filtrates were added 2 - ((2- (dimethylamino) ethyl) thio) acetic acid (1.26 g, 7.70 mmol), EDC HCl salt (1.84 g, 9.62 mmol), DMAP (78.3 mg, 0.64 mmol) and the fine suspension was stirred overnight at room temperature. The next day, H2O (60 ml) and MeOH (30 ml) are added and after stirring for 10 min., The clear organic phase was isolated. The cloudy aqueous phase was extracted with DCM. The combined organic extracts are concentrated. The crude material was filtered through a silica plug and absorbed in DCM (40 ml) and PBS (pH = 11.50 ml) was added. The mixture was stirred at room temperature for ~ 10 min. Subsequently, the organic phase was separated and the aqueous phase was extracted again with DCM (15 ml). The combined organics are dried (MgSO4) for 30 min., Filtered, washed with DCM and concentrated to yield (Z) - ((2 - ((2- (dimethylamino) ethyl) thio) acetyl) azanediol) bis (ethane -2,1-diyl) dioleate (3.44 g). LCMS ESI +: m / z 780.2 (M + H). PREPARATION OF ((5- (DIMETHYLAMINE) PENTANOIL) AZANEDHL) BIS (ETHANE-2,1-DHL) DITETRADECANOATE (C104)

[00107] TFA salt synthesis of azanedi-ilbis (ethane-2,1-di-yl) dithetradecanoate previously described. The TFA salt of azanedi-ilbis (ethane-2,1-di-yl) dithetradecanoate (730 mg, 1.14 mmol) was stirred in DCM (20 ml) with 10% K2CO3 (10 ml) between 0 and 5 ° C. After 30 min, the organic phase was separated and the aqueous phase was further extracted with DCM (10 ml). The combined organic phases are stirred with MgSO4 for a period of 30 minutes between 0 and 5 ° C, filtered and washed with DCM (10 ml). To the combined filtrates are added 5- (dimethylamino) pentanoic acid (248 mg, 1.37 mmol), EDC HCl salt (328 mg, 1.71 mmol), DMAP (14 mg, 0.114 mmol) and the fine suspension was stirred overnight at room temperature, after which the solution became clear. The next day, H2O (20 ml) and MeOH (10 ml) are added and after stirring for 10 min., The clear organic phase was isolated. The cloudy aqueous phase was extracted with DCM. The combined organic extracts are concentrated. After purification by silica gel chromatography eluting with 100% ethyl acetate followed by 10% MeOH / DCM, the purified residue was taken up in DCM (25 ml) and PBS (pH = 11.25 ml). The mixture was stirred at room temperature for 15 min. Subsequently, the organic phase was separated and the aqueous phase was extracted again with DCM (15 ml). The combined organics are dried (MgSO4) for 30 min., Filtered, washed with DCM and concentrated to yield ((5- (dimethylamino) pentanoyl) azanedi-yl) bis (ethane-2,1-di-yl) ditetradecanoate (405 mg). LCMS ESI +: m / z 654.1 (M + H). PREPARATION OF ((2 - ((2- (DIMETHYLAMINE) ETHYL) SULFONY) ACETYL) AZANEDI-IL) BIS (ETHANE-2,1-DI-IL) DITETRA-DECANOATE (SO2-S104)

[00108] Synthesis of ((2 - ((2- (dimethylamino) ethyl) thio) acetyl) azanediol) bis (ethane-2,1-diyl) dithetradecanoate, also known as S104, has been described. DCM (10) ((2 - ((2- (dimethylamino) ethyl) thio) acetyl) azanedi-yl) bis (ethane-2,1-di-yl) ditetradecanoate in a round-bottom flask drained with argon ml). The solution was cooled by an ice bath. To this, mCPBA (a DCM solution) was added slowly for 5 min. The ice bath was removed after the addition and the reaction was allowed to stir overnight at room temperature. After 3.5 hours, 2M DMA / THF (4.55 ml) was added slowly and the reaction mixture was allowed to stir overnight. The reaction mixture was then diluted with DCM to 75 ml., Washed with H2O (2 x 50 ml) and 10% K2CO3 (50 ml). All aqueous washes were back-extracted with DCM (40 ml). The combined organics were dried (MgSO4), filtered and concentrated to yield a colorless oil. The reaction mixture was concentrated. Purification by silica gel chromatography eluting with an ethyl acetate / MeOH gradient yielded ((2 - ((2- (dimethylamino) ethyl) sulfonyl) acetyl) azanedi-yl) bis (ethane-2,1-di -il) dithetradecanoate (540 mg). LCMS ESI +: m / z 704.0 (M + H). PREPARATION OF (((((2- (DIMETHYLAMINE) ETHYL) TIO) CARBONY) AZANEDI-IL) BIS (ETHANE-2,1-DI-IL) DITETRADECANOATE (TU104)
STEP 1: PREPARATION OF INTERMEDIATE 1: AZANEDI-ILBIS (ETANO-2,1-DI-IL) DITETRADECANOATE

[00109] TFA salt synthesis of azanedi-ilbis (ethane-2,1-di-yl) dithetradecanoate previously described. The TFA salt of azanedi-ylbis (ethane-2,1-diyl) dithetradecanoate was dissolved in DCM (50 ml) and PBS (pH = 11.50 ml) was added. The mixture was stirred at room temperature for 15 min. Subsequently, the organic phase was separated and the aqueous phase was extracted again with DCM (25 ml). The combined organics were dried (MgSO4) for 30 min., Filtered, washed with DCM and concentrated to yield azanediolis (ethane-2,1-diyl) dithetradecanoate as the free base. STEP 2: TU104 PREPARATION: (((((2- (DIMETHYLAMINE) ETHYL) UNCLE) CARBONY) AZANEDI-IL) BIS (ETHANE-2,1-DI-IL) DITETRA-DECANOATE

[00110] Trichloromethyl chloroformate (also known as diphosgene) (257 µl, 2.13 mmol) was added to a solution of HCl salt of 2- (dimethylamino) ethanethiol (302 mg, 2.13 mmol) in dry DCM ( 20 ml) and stirred under an argon purge at room temperature for 4 h. Subsequently, DCM and excess diphosgene were removed under vacuum. The free base of azanedi-ilbis (ethane-2,1-di-yl) dithetradecanoate (1,068 mg, 2.03 mmol), DCM (20 ml) and triethylamine (580 ul, 4.16 mmol) were then added . After 16 h at room temperature, the reaction mixture was diluted with DCM and washed with 1M HCl (75 ml), H2O (75 ml) and PBS (pH = 11.75 ml), dried (MgSO4), filtered and concentrated. Purification by silica gel chromatography eluting with ethyl acetate followed by a DCM / MeOH gradient yielded ((((2- (dimethylamino) ethyl) thio) carbonyl) azanedi-yl) bis (ethane-2,1- di-yl) dithetradecanoate (120 mg). LCMS ESI +: m / z 657.5 (M + H). PREPARATION OF ((2- (2- (DIMETHYLAMINE) ETOXI) ACETYL) AZANEDI-IL) BIS (ETANO-2,1-DI-IL) DITETRADECANOATE (O104)
STEP 1: PREPARATION OF INTERMEDIATE 1: ((2-BROMOACETYL) AZANEDIIL) BIS (ETANO-2,1-DI-IL) DITETRADECANOATE

[00111] TFA salt synthesis of azanedi-ilbis (ethane-2,1-di-yl) ditetradecanoate previously described. The TFA salt of azanedi-ilbis (ethane-2,1-di-yl) dithetradecanoate (1,500 mg, 2.34 mmol) was dissolved in DCM (20 ml) and placed in an ice bath. Bromoacetyl bromide (214 µl, 2.46 mmol) was added followed by triethylamine (685 µl, 4.91 mmol). The ice bath was removed and the reaction was allowed to stir overnight at room temperature under an inert gas purge. The next day, diluted with DCM to 100 ml. Washed with 1M HCl (75 ml), H2O (75 ml), saturated NaHCO3 solution (75 ml) and saturated brine solution (75 ml). All aqueous washes were back-extracted with DCM (25 ml). The organics were dried over MgSO4, filtered and concentrated in vacuo. Purified by silica gel chromatography eluting with 100% ethyl acetate. The fractions were pooled and concentrated to yield ((2-bromoacetyl) azanedi-yl) bis (ethane-2,1-di-yl) ditetradecanoate (1220 mg). STEP 2: O104 PREPARATION: ((2- (2- (DIMETHYLAMINE) ETOXI) ACETYL) AZANEDIIL) BIS (ETHANE-2,1-DI-IL) DITETRADECANOATE

[00112] To a round-bottom flask equipped with a stir bar, ((2-bromoacetyl) azanedi-yl) bis (ethane-2,1-di-yl) dithetradecanoate (1.22 g, 1.87 mmol) was added ), N, N-dimethylethanolamine (197 µl, 1.96 mmol), potassium iodide (6.2 mg, 0.0374 mmol) and dry THF (25 ml). The resulting solution was cooled to -40 ° C. DBU (588 µl, 3.93 mmol) was added dropwise over 5 min. and the reaction was heated to 0 ° C for 2 hours. The reaction mixture was concentrated. The residue was absorbed with DCM, 1M HCl (12 ml) was added and the biphasic mixture stirred for 15 min. Then, basified with the use of PBS (pH = 11). The organic layer was isolated, dried (MgSO4), filtered and concentrated. Purification by chromatography on silica gel eluting with 100% ethyl acetate followed by a DCM / MeOH gradient yielded ((2- (2- (dimethylamino) ethoxy) acetyl) azanedi-yl) bis (ethane-2, 1-di-yl) dithetradecanoate (53 mg). LCMS ESI +: m / z 655.6 (M + H). PREPARATION OF ((2 - ((4- (DIMETHYLAMINE) BUTANOIL) OXI) ACETYL) AZANEDI-IL) BIS (ETANO-2,1-DI-IL) DITETRA-DECANOATE (HEDC-M1)
STEP 1: PREPARATION OF INTERMEDIATE 1: ((2- (BENZILOXI) ACETYL) AZANEDI-IL) BIS (ETANO-2,1-DI-IL) DITETRADECANOATE

[00113] TFA salt synthesis of azanedi-ilbis (ethane-2,1-di-yl) dithetradecanoate previously described. The TFA salt of azanedi-ilbis (ethane-2,1-di-yl) dithetradecanoate was stirred in DCM (25 ml) with 10% K2CO3 (12.5 ml) between 0 and 5 ° C. After 30 min., The organic layer was isolated and the aqueous layer was further extracted with DCM (12 ml). The combined organic phases are stirred with MgSO4 for 30 min. between 0 and 5 ° C, filtered, washed with DCM (12 ml). To the combined filtrates are added benzyloxy acetic acid (402 µl, 2.81 mmol), EDC HCl salt (673 mg, 3.51 mmol) and DMAP (29 mg, 0.234 mmol). The suspension was allowed to stir at room temperature overnight. The next day, H2O (25 ml) and MeOH (12 ml) were added and after stirring for 10 min., The clear organic phase was isolated. The cloudy aqueous phase was extracted with DCM (25 ml). The combined organic extracts dried with MgSO4, filtered and concentrated. Purification by silica gel chromatography eluting with a gradient of hexanes / ethyl acetate yielded ((2- (benzyloxy) acetyl) azanedi-yl) bis (ethane-2,1-di-yl) dithetradecanoate (1.28 g). STEP 2: PREPARATION OF INTERMEDIATE 2: ((2-HYDROXYACETYL) AZANEDI-IL) BIS (ETANO-2,1-DI-IL) DITETRADECANOATE

[00114] ((2- (benzyloxy) acetyl) azanedi-yl) bis (ethane-2,1-di-yl) ditetradecanoate (1.28 g, 1.80 mmol) was dissolved in a round bottom flask with MeOH (20 ml). The flask was capped and drained with argon. 10% Pd / C (135 mg) was added and the flask was again drained with argon. All air was removed using a vacuum pump and then an 8-inch balloon filled with H2 gas was added. The reaction was allowed to stir vigorously at room temperature. After 30 min., The reaction mixture was filtered (celite), washed with methanol, concentrated to residue, absorbed in DCM (25 ml) and 10% K2CO3 (25 ml). The mixture was stirred for 15 min. and then the isolated organic layer. The aqueous wash was back-extracted with DCM (15 ml). The combined organics were dried over MgSO4, filtered and concentrated to yield ((2-hydroxyacetyl) azanedi-yl) bis (ethane-2,1-di-yl) dithetradecanoate (990 mg). STEP 3: PREPARATION OF HEDC-M1: ((2 - ((4- (DIMETHYLAMINE) BUTANOIL) OXI) ACETYL) AZANEDI-IL) BIS (ETHANE-2,1-DI-IL) DI- TETRADECANOATO

[00115] ((2-Hydroxyacetyl) azanedi-yl) bis (ethane-2,1-di-yl) dithetradecanoate (990 mg, 1.70 mmol) was stirred in DCM (20 ml) and 4-dimethylamino-butyric acid (268 mg), EDC HCl salt (487 mg) and DMAP (21 mg) were added. The suspension was allowed to stir at room temperature overnight. The next day, H2O (20 ml) and MeOH (10 ml) added and after stirring for 10 minutes, the clear organic phase was isolated. The cloudy aqueous phase was extracted with DCM (20 ml). The combined organic extracts dried with MgSO4, filtered and concentrated. The crude material was purified by silica gel chromatography which eluted with a DCM / MeOH gradient. The fractions were pooled and concentrated were concentrated and absorbed with DCM (25 ml) and PBS (pH = 11, 25 ml). The mixture was stirred at room temperature for 15 min. Subsequently, the organic phase was isolated and the aqueous phase was extracted again with DCM (25 ml). The combined organics are dried (MgSO4), filtered and concentrated to yield ((2 - ((4- (dimethylamino) butanoyl) oxy) acetyl) azanedi-yl) bis (ethane-2,1-di-yl) ditetradecanoate (672 mg). LCMS ESI +: m / z 697.6 (M + H). PREPARATION OF (Z) - ((5- (DIMETHYLAMINE) PENTANOIL) AZANEDI-IL) BIS (ETANO-2,1-DI-IL) DIOLEATE (C104-DO)


[00116] TFA salt synthesis of (Z) -azanedi-ilbis (ethane-2,1-di-yl) dioleate previously described. The TFA salt of (Z) -azanedi-ilbis (ethane-2,1-di-yl) dioleate (1.50 g, 2.37 mmol) was stirred in DCM (20 ml) with 10% K2CO3 (10 ml) between 0 and 5 ° C. After 30 min., The organic phase was isolated and the aqueous phase was further extracted with DCM (10 ml). The combined organic phases are stirred with MgSO4 for a period of 30 min. between 0 and 5 ° C, filtered and washed with DCM (15 ml). To the combined filtrates are added 5- (dimethylamino) pentanoic acid (516 mg, 2.84 mmol), EDC HCl salt (681 mg, 3.55 mmol), DMAP (29 mg, 0.237 mmol) and the suspension was stirred overnight at room temperature, after which the clear solution was formed. The next day, H2O (20 ml) and MeOH (10 ml) were added and, after stirring for 10 min., The clear organic phase was isolated. The cloudy aqueous phase was extracted with DCM. The combined organics are dried (MgSO4), filtered and concentrated. After purification by silica gel chromatography eluting with a DCM / MeOH gradient, the fractions that have been pooled and concentrated are absorbed with DCM (25 ml) and PBS (pH = 11, 25 ml). The mixture was stirred at room temperature for ~ 10 min. Subsequently, the organic phase was isolated and the aqueous phase was extracted again with DCM (15 ml). The combined organics are dried (MgSO4), filtered and concentrated to yield (Z) - ((5- (dimethylamino) pentanoyl) azanedi-yl) bis (ethane-2,1-di-yl) dioleate (1.10 g) . LCMS ESI +: m / z 761.7 (M + H). PREPARATION OF ((5 - ((DIMETHYLAMINE) METHIL) THIOPHENE-2-CARBONY) AZANEDI-IL) BIS (ETHANE-2,1-DI-IL) DI-TETRADECANOATE (T104)


[00117] TFA salt synthesis of azanedi-ilbis (ethane-2,1-di-yl) dithetradecanoate previously described. The TFA salt of azanediylbis (ethane-2,1-diyl) dithetradecanoate (1004 mg, 1.57 mmol) was stirred in DCM (20 ml) with 10% K2CO3 (20 ml) between 0 and 5 ° C. After 30 min., The organic phase was isolated and the aqueous phase was further extracted with DCM (10 ml). The combined organics are dried with MgSO4 for 30 min. between 0 and 5 ° C, filtered and washed with DCM (10 ml). To the combined filtrates were added ((dimethylamino) methyl) thiophene-2-carboxylic acid (350 mg, 1.89 mmol), EDC HCl salt (452 mg, 2.36 mmol) and DMAP (19.2 mg, 0.157 mmol). The suspension was allowed to stir at room temperature overnight. The following day, H2O (20 ml) and MeOH (10 ml) were added and after stirring for 10 min., The clear organic phase was isolated. The cloudy aqueous phase was extracted with DCM (25 ml). The combined organics were dried (MgSO4), filtered and concentrated. After purification by silica gel chromatography eluting with a hexanes / ethyl acetate gradient, the fractions that were pooled and concentrated were absorbed with DCM (20 ml) and PBS (pH = 11, 20 ml). The mixture was stirred at room temperature for ~ 10 min. Subsequently, the organic phase was isolated and the aqueous phase was extracted again with DCM (15 ml). The combined organics were dried (MgSO4) for a period of 30 min., Filtered, washed with DCM and concentrated to yield ((5- ((dimethylamino) methyl) thiophene-2-carbonyl) azanedi-yl) bis (ethane-2 , 1-diyl) dithetradecanoate (482 mg). LCMS ESI +: m / z 693.6 (M + H). PREPARATION OF (Z) - ((((2- (DIMETHYLAMINE) ETHYL) TIO) CARBONY) AZANEDI-IL) BIS (ETHANE-2,1-DI-IL) DIOLEATE (TU104-DO)

[00118] Synthesis of (Z) - ((tert-butoxycarbonyl) azanedi-yl) bis (ethane-2,1-di-yl) dioleate previously described. (Z) - ((tert-butoxycarbonyl) azanediol) bis (ethane-2,1-diyl) dioleate (4.20 g, 5.72 mmol) was dissolved in DCM (20 ml) and cooled to 0 ° C in an ice bath. TFA (20 ml) was added and the mixture was allowed to stir under an inert gas purge for 20 min. Subsequently, the reaction mixture was concentrated in vacuo. The residue was divided between 10% K2CO3 (20 ml) and DCM (20 ml) and stirred in an ice bath for 20 min. The organic layer was isolated, dried (MgSO4) and filtered. Diphosgene (1.38 ml, 11.4 mmol) was added to (Z) -azanediolis (ethane-2,1-diyl) dioleate material in DCM and stirred under an inert gas purge at room temperature . The next day, DCM and excess diphosgene were removed in vacuo. The HCl salt of 2- (dimethylamino) ethane thiol (4.05 g, 28.6 mmol) was absorbed in DCM (50 ml) and triethylamine (5.2 ml, 37.2 mmol) and added to a residue of ( Z) - ((chlorocarbonyl) azanedi-yl) bis (ethane-2,1-di-yl) dioleate. The material was allowed to stir overnight at room temperature. The next day, diluted with DCM and washed with 0.3M HCl (75 ml), H2O (75 ml) and 10% K2CO3 (75 ml). All aqueous washes were back-extracted with DCM (25 ml). The combined organics were dried over MgSO4, filtered and concentrated. Purification by silica gel chromatography eluting with a DCM / MeOH gradient yielded (Z) - (((((2- (dimethylamino) ethyl) thio) carbonyl) azanedi-yl) bis (ethane-2,1-di -yl) dioleate (1.90 g). LCMS ESI +: m / z 765.7 (M + H). PREPARATION OF ((((3- (DIMETHYLAMINE) PROPOXI) CARBONY) AZANEDI-IL) BIS (ETHANE-2,1-DI-IL) DITETRADECANOATE (CB104)

[00119] Synthesis of azanedi-ilbis (ethane-2,1-di-dithetradecanoate) previously described. Diphosgene (266 ul, 2.2 mmol) was added to dimethylaminopropanol (413 mg, 4.00 mmol) in DCM (10 ml) and stirred under an inert gas purge at room temperature for 4 h. DCM and excess diphosgene were removed in vacuo and azanedi-ilbis (ethane-2,1-di-yl) dithetradecanoate was added. The round bottom flask was drained with argon and DCM (10 ml) and triethylamine (859 µl, 6.16 mmol) was added. The material was allowed to stir overnight at room temperature. The reaction mixture was then diluted with DCM and washed with 0.3M HCl (75 ml), H2O (75 ml) and 10% K2CO3 (75 ml). All aqueous washes were back-extracted with DCM (25 ml). The combined organics were dried over MgSO4, filtered and concentrated. Purification by silica gel chromatography eluting with a DCM / MeOH gradient yielded (((3- (dimethylamino) propoxy) carbonyl) azanedi-yl) bis (ethane-2,1-di-yl) ditetradecanoate (87 mg) . LCMS ESI +: m / z 655.59 (M + H). PREPARATION OF (((2- (DIMETHYLAMINE) ETOXI) CARBONIL) AZANEDI-IL) BIS (ETANO-2,1-DI-IL) DITETRADECANOATE (CA104)
STEP 1: PREPARATION OF INTERMEDIATE 1: ((((4-NITROFENOXI) CARBONY) AZANEDI-IL) BIS (ETANO-2,1-DI-IL) DITETRADECANOATE

[00120] TFA salt synthesis of azanedi-ilbis (ethane-2,1-di-yl) dithetradecanoate previously described. The TFA salt of azanedi-ylbis (ethane-2,1-diyl) dithetradecanoate was dissolved in dry DCM (10 ml) and triethylamine (654 µl, 4.69 mmol) was added. The reaction vessel was drained with inert gas and 4-nitrophenyl chloroformate was added. The material was allowed to stir at room temperature overnight. The reaction mixture was cooled with water (50 ml) and DCM (50 ml). The organic layer was isolated and the aqueous layer was further extracted with DCM (2 x 50 ml). The combined organics were dried over MgSO4, filtered and concentrated. Purification by chromatography on silica gel eluting with a hexanes / ethyl acetate gradient yielded (((4-nitrophenoxy) carbonyl) azanedi-yl) bis (ethane-2,1-di-yl) dithetradecanoate. STEP 2: CA104 PREPARATION: ((((2- (DIMETHYLAMINE) ETOXI) CARBONY) AZANEDI-IL) BIS (ETHANE-2,1-DI-IL) DITETRA-DECANOATE

[00121] A (((4-nitrophenoxy) carbonyl) azanedi-yl) bis (ethane-2,1-di-yl) ditetradecanoate, 2-dimethylaminoethanol (2 ml) was added and heated to 140oC with a condensation column by 20 min Subsequently, the crude material was purified by silica gel chromatography eluting with a gradient from DCM / MeOH to yield (((2- (dimethylamino) ethoxy) carbonyl) azanediol) bis (ethane-2,1-di- il) dithetradecanoate (38 mg). LCMS ESI +: m / z 641.7 (M + H). PREPARATION OF ((2- (DIMETHYLAMINE) ACETYL) AZANEDHL) BIS (ETHANE-2,1-DHL) DITETRADECANOATE (INT-4)

[00122] Synthesis of (Z) - ((2- (dimethylamino) acetyl) azanediyl) bis (ethane-2,1-diyl) dioleate was prepared in a similar manner to i-Prop-DODC with the substitution of dimethyl glycine by 3- (dimethylamino) propionic acid. QTOF MS ESI +: m / z 720.1 (M + H). PREPARATION OF ((2 - ((2- (DIMETHYLAMINE) ETHYL) THIO) ACETYL) AZANEDI-IL) BIS (ETHANE-2,1-DI-IL) DITETRADECANOATE (S104)
STEP 1: PREPARATION OF INTERMEDIATE 1: CHLORHYDROIC ACID 2 - ((2- (DIMETHYLAMINE) ETHYL) TIO) ACETIC:

[00123] Ethanol (500 ml) was degassed by triple evacuation to 0.01 MPa (60 mBar) for 1 to 2 minutes and repressurization with nitrogen. Chloroacetic acid (36.9 g, 0.391 mol) was added and a clear solution was formed after being stirred for 5 minutes at 17 to 20 ° C. 2- (dimethylamino) -ethanethiol hydrochloride (52.7 g, 0.372 mol) was added and a clear solution was formed after being stirred for 20 minutes at 25 ° C. Solid sodium hydroxide (47.7 g, 1.19 mol) was added in portions over a period of 20 minutes with cooling in order to keep the temperature below 35 ° C - a short period at 44 ° C was observed. Almost immediate precipitation was observed. The reaction mixture was eventually heated and stirred at 40 ° C for a period of two hours, at which point the TLC indicated the completion of the reaction. Celite 545 (74 g) was added and the mixture was filtered through a plate sintered with G3 glass for 6 minutes, washing with ethanol (2 x 105 ml). The combined cloudy filtrates are evaporated from a 50 ° C bath to generate 110 g of white solid. The solid was dissolved in water (250 ml), then the pH was adjusted from 13.1 (temperature 30 ° C) to 10.5 (temperature 31 ° C), using concentrated HCl (5.5 ml) to generate a very pale yellow solution. The aqueous phase was washed with DCM (3x 100 ml) to remove the disulfide impurity (all 3 washes required). The concentrated HCl was added to the aqueous phase (pH 10.7, temperature 22 ° C) until the pH was 1.4 (57.5 ml added, temperature 35 ° C). The aqueous phase was washed with DCM (100 ml) and then concentrated to dryness (bath temperature 55 ° C). Toluene (250 ml) was added, the mixture was concentrated to dryness (bath temperature 55 ° C) and this was repeated once to generate a wet white solid (98 g). Acetonitrile (750 ml) was added to the solid, the mixture was stirred at 55 ° C for a period of 45 minutes and then filtered through a plate sintered with G3 glass. Acetonitrile (250 ml) was added to the filter cake, the mixture was stirred at 55 ° C for a period of 25 minutes and then filtered through a plate sintered with G3 glass, washing with acetonitrile (50 ml). The combined filtrates are concentrated to 300 ml, resulting in a heavy white precipitate. The mixture was cooled under nitrogen and stirred at 0 ° C for a period of 30 minutes. The precipitate was isolated by filtration through a plate sintered with a G3 filter and the filter cake was washed with cold acetonitrile (100 ml). Drying under reduced pressure for 3 days generates 47.0 g (63%) of 2 - ((2- (dimethylamino) ethyl) thio) acetic hydrochloric acid. STEP 2: PREPARATION OF S104: ((2 - ((2- (DIMETHYLAMINE) ETHYL) TIO) ACETYL) AZANEDI-IL) BIS (ETHANE-2,1-DI-IL) DITETRADECANOATE

[00124] TFA salt synthesis of azanedi-ilbis (ethane-2,1-di-yl) dithetradecanoate previously described. The TFA salt of azanedi-ilbis (ethane-2,1-di-yl) dithetradecanoate (152 g, 238 mmol) was stirred with DCM (2.3 l) and 10% potassium bicarbonate (1.15 l) between 0 and 5 ° C. The organic phase was separated and the aqueous phase was further extracted with DCM (1.15 l). The combined organic phases were stirred with magnesium sulfate hydrate (236 g) for a period of 30 minutes between 0 and 5 ° C, filtered and washed with DCM (1.15 l). To the combined filtrates, 2 - ((2- (dimethylamino) ethyl) thio) acetic acid (57.0 g, 285 mmol), EDC hydrochloride (68.4 g, 357 mmol) and DMAP (2.91 g) were added , 23.8 mmol), and the fine suspension was stirred overnight at room temperature, after which time a clear solution was formed. Water (2.3 l) and methanol (460 ml) are added and after being stirred for a period of 10 minutes, the clear organic phase was separated. The cloudy aqueous phase (pH 3.0) was extracted with DCM (575 ml). The combined organic extracts were concentrated yielding 143 g of crude material such as the hydrochloride salt. The crude material (142.6 g) was transferred to a distillation flask with DCM (500 ml) and ethyl acetate (1 l) was added. The solution was heated for distillation at atmospheric pressure, and the distillation was continued for a period of 70 minutes in order to obtain a residue temperature of 76 ° C. A total volume of 1.4 l was obtained by adding ethyl acetate (800 ml), and ethanol (70 ml) was added. The clear solution at 50 ° C was cooled to 37 ° C and the seed crystals were added. With the initiation of significant crystallization observed for a period of 10 minutes between 37 and 35 ° C, the suspension was cooled and stirred at 0 ° C overnight and the precipitate was isolated by filtration and washed with cold ethyl acetate (210 ml). Drying for dry weight at room temperature in an oil pump vacuum for a period of 4.5 hours generated 134 g of recrystallized material such as the hydrochloride salt, white crystalline solid.
[00125] Tripotassium phosphate (85 g, 0.40 mol) and dipotassium hydrogen phosphate (226 g, 1.30 mol) were added to the purified water (1.7 l), and the solution formed at pH 10, 9 was cooled to 18 to 20 ° C. DCM (1.3 l) and recrystallized hydrochloride S104 (133.0 g, 0.188 mol) are added, and the mixture was stirred for a period of 10 min. The clear organic phase was separated at a moderate rate (over a period of 35 minutes), and the cloudy aqueous phase was further extracted with DCM (650 ml). The combined organic phases were stirred with magnesium sulfate hydrate (65 g) for a period of 40 minutes and the mixture was filtered, washing with DCM (200 ml). The combined filtrates were evaporated from a 50 ° C water bath under reduced pressure (down to 0.2 KPa (20 mBar), which pressure evaporation was continued for one hour). Further evaporation from a 15 to 20 ° C water bath in an oil pump vacuum resulted in 126 g of partially solidified oil. The cooling in a cooling bath at -20 ° C generated complete solidification and, after drying at -20 ° C under vacuum for two days, 126 g of ((2 - ((2- (dimethylamino) ethyl) thio) were obtained acetyl) azanedi-yl) bis (ethane-2,1-di-yl) dithetradecanoate, also known as S104. HPLC indicates 98.1% purity. QTOF MS ESI +: m / z 671.6 (M + H). PREPARATION OF (9Z, 9'Z) - ((2 - ((2- (DIMETHYLAMINE) ETHYL) TIO) ACETYL) AZANEDIIL) BIS (ETANO-2,1-DI-IL) BIS (TETRADEC-9-ENOATO) ( S104-DMO)
STEP 1: PREPARATION OF INTERMEDIATE 1: (9Z, 9'Z) - ((TERCBUTOXICARBONIL) AZANEDI-IL) BIS (ETANO-2,1-DI-IL) BIS (TETRADEC-9-ENOATO)

[00126] N-Boc-diethanolamine (454 mg, 2.21 mmol), myristoleic acid (1,000 mg, 4.42 mmol) and DMAP (54 mg, 0.442 mmol) was dissolved in DCM (25 ml) and placed in a room temperature water bath in a round bottom flask was drained with inert gas. The EDC HCl salt (932 mg, 4.86 mmol) was added in 3 portions over 5 min. The reaction was allowed to stir overnight at room temperature under an inert gas purge. The next day, H2O (25 ml) was added and stirred for 10 min. The organic layer was isolated and the aqueous layer was further extracted with DCM (50 ml). The combined organics were dried (MgSO4) for 10 min., Filtered and concentrated. Purification by silica gel chromatography eluting with a hexane / ethyl acetate gradient yielded (9Z, 9'Z) - ((tert-butoxycarbonyl) azanediol) bis (ethane-2,1-di-yl) bis (tetradec-9-enoate) (1.10 g). STEP 2: PREPARATION OF INTERMEDIATE 2: (9Z, 9'Z) -AZANEDI-ILBIS (ETANO-2,1-DI-IL) BIS (TETRADEC-9-ENOATO)

[00127] A (9Z, 9'Z) - (((tert-butoxycarbonyl) azanedi-yl) bis (ethane-2,1-di-yl) bis (tetradec-9-enoate) (1,100 mg, 1.77 mmol ) in a round bottom flask DCM (10 ml) was added and placed in an ice bath. TFA (10 ml) was added and the mixture was allowed to stir for 20 min. The reaction mixture was then concentrated. Toluene was added to the residue to assist in azeotroping the excess TFA. The residue was placed back in the ice bath and PBS (pH = 11.25 ml) and DCM (25 ml) were added. The mixture was stirred for 15 min. and the organic layer was then isolated. The cloudy aqueous layer was extracted with DCM (10 ml). The combined organics were dried (MgSO4) at 0 ° C for 15 min., Filtered and concentrated to yield (9Z, 9'Z) -azanedi-ilbis (ethane-2,1-di-yl) bis (tetradec-9- enoate) (923 mg). STEP 3: PREPARATION OF S104-DMO: (9Z, 9'Z) - ((2 - ((2- (DIMETHYLAMINE) ETHYL) TIO) ACETYL) AZANEDI-IL) BIS (ETHANE-2,1-DI-IL) BIS (TETRADEC-9- ENOATO)

[00128] A mixture of (9Z, 9'Z) -azanedi-ilbis (ethane-2,1-di-yl) bis (tetradec-9-enoate) (923 mg, 1.77 mmol), 2- ( (2- (dimethylamino) ethyl) thio) acetic acid (346 mg, 2.12 mmol) and EDC HCl salt (509 mg, 2.66 mmol) suspended in DCM (10 ml). DMAP (21.6 mg, 0.177 mmol) was added and the mixture was allowed to stir at room temperature overnight. On the following day, H2O (10 ml) and MeOH (10 ml) were added and after stirring for 10 min., The clear organic phase was isolated. The cloudy aqueous phase was extracted with DCM (2 x 20 ml). The combined organic extracts are dried with MgSO4, filtered and concentrated. After purification by silica gel chromatography eluting with a DCM / MeOH, the fractions that were pooled and concentrated were absorbed in DCM (25 ml) and PBS (pH = 11.25 ml). The mixture was stirred at room temperature for ~ 10 min. Subsequently, the organic phase was isolated and the aqueous phase was extracted again with DCM (2 x 15 ml). The combined organic phase was dried (MgSO4), filtered and concentrated to yield (9Z, 9'Z) - ((2 - ((2- (dimethylamino) ethyl) thio) acetyl) azanedi-yl) bis (ethane-2 , 1-di-yl) bis (tetradec-9-enoate) (589 mg). LCMS ESI +: m / z 667.6 (M + H). PREPARATION OF (R) - ((1-METHYLPYRROLIDINE-2-CARBONY) AZANEDI-IL) BIS (ETANE-2,1-DI-IL) DITETRADECANOATE (PRO-DC)
STEP 1: PRO-DC PREPARATION: (R) - ((1-METHYLPYRROLIDINE-2-CARBONY) AZANEDI-IL) BIS (ETHANE-2,1-DI-IL) DITETRADECANOATE

[00129] TFA salt synthesis of azanedi-ilbis (ethane-2,1-di-yl) dithetradecanoate previously described. The TFA salt of azanedi-ilbis (ethane-2,1-di-yl) dithetradecanoate (1,000 mg, 1.56 mmol) was stirred with DCM (10 ml) and N-Methyl-L-Proline (228 mg, 1 , 77 mmol), HOBt.H2O (239 mg, 1.77 mmol) was added. NMM (365 µl, 3.32 mmol) was added and the solution became mostly clear. A suspension of EDC hydrochloride (518 mg, 2.70 mmol), NMM (257 µl, 2.34 mmol) and DMAP (19 mg, 0.156 mmol) in DCM (10 ml) was added and the mixture was stirred for about 12 hours at room temperature, after which time a clear solution was formed. Thereafter, the mixture was diluted with DCM (50 ml) and washed with 10% aqueous K2CO3 (60 ml). The organics were dried over MgSO4, filtered and concentrated. The resulting compound was crude purified by silica gel chromatography, which elutes with a methanol (0 to 10)% gradient in DCM to yield (R) - ((1-methylpyrrolidine-2-carbonyl) azanedi-yl) bis ( ethane-2,1-diyl) dithetradecanoate. LCMS ESI +: m / z 637.6 (M + H). PREPARATION OF (9Z, 9'Z, 12Z, 12'Z) - ((2 - ((2- (DIMETHYLAMINE) ETHYL) UNCLE) ACETYL) AZANEDI-IL) BIS (ETANO-2,1-DI-IL) BIS (OCTADECA- 9,12-DIENOATE) (S104-DLIN)
STEP 1: PREPARATION OF INTERMEDIATE 1: (9Z, 9'Z, 12Z, 12'Z) - ((TERC- BUTOXICARBONIL) AZANEDI-IL) BIS (ETANO-2,1-DI-IL) BIS (OCTADECA-9, 12-DIENOATE)

[00130] N-Boc-diethanolamine (5 g, 24.4 mmol), linoleic acid (14.4 g, 51.2 mmol) and was dissolved in DCM (100 ml). The EDC HCl salt (10.3 g, 53.7 mmol) was added followed by DMAP (596 mg, 4.88 mmol). The reaction was allowed to stir for about 12 hours at room temperature under an inert gas purge. Subsequently, 50 ml of water and 50 ml of methanol were added and the mixture was stirred for 10 min. The organic layer was isolated and the aqueous layer was further extracted with DCM (150 ml). The combined organics were dried (MgSO4) for 10 min., Filtered and concentrated. Purification by silica gel chromatography, which elutes with a hexane / ethyl acetate gradient yielded (9Z, 9'Z, 12Z, 12'Z) - (((tert-butoxycarbonyl) azanedi-yl) bis (ethane-2 , 1-di-yl) bis (octadeca-9,12-dienoate) (15.9 g). STEP 2: PREPARATION OF INTERMEDIATE 2: (9Z, 9'Z, 12Z, 12'Z) -AZANEDI- ILBIS (ETANO-2,1-DI-IL) BIS (OCTADECA-9,12-DIENOATO)

[00131] A (9Z, 9'Z, 12Z, 12'Z) - ((tert-butoxycarbonyl) azanediol) bis (ethane-2,1-diyl) bis (octadeca-9,12-dienoate) (5.33 g, 7.30 mmol) in a round bottom flask DCM (50 ml) was added and placed in an ice bath. TFA (50 ml) was added and the mixture was allowed to stir for 30 min. The reaction mixture was then concentrated. Toluene was added to the residue to assist in azeotroping the excess TFA. The residue was placed back in the ice bath and 10% K2CO3 (50 ml) and DCM (50 ml) were added. The mixture was stirred for 15 min. and the isolated organic layer. The cloudy aqueous layer was extracted with DCM (20 ml). The combined organics were dried (MgSO4), filtered and concentrated to yield (9Z, 9'Z, 12Z, 12'Z) - azanedi-ilbis (ethane-2,1-di-yl) bis (octadeca-9,12- dienoate) (Quantitative). STEP 3: PREPARATION OF S104-DLIN: (9Z, 9'Z, 12Z, 12'Z) - ((2 - ((2- (DIMETHYLAMINE) ETHYL) UNCLE) ACETYL) AZANEDI-IL) BIS (ETHANE-2, 1-DI-IL) BIS (OCTADECA- 9,12-DIENOATE)

[00132] A mixture of (9Z, 9'Z, 12Z, 12'Z) -azanedi-ilbis (ethane-2,1-di-yl) bis (octadeca-9,12-dienoate) (4.68 g, 7.30 mmol), 2 - ((2- (dimethylamino) ethyl) thio) acetic acid (1.43 g, 8.76 mmol) and EDC HCl salt (2.10 g, 10.95 mmol) were suspended in DCM (100 ml). DMAP (89 mg, 0.73 mmol) was added, stirred at room temperature for 12 hours. Fifty ml each of water and methanol were added and, after stirring for 10 minutes, the clear organic phase was isolated. The cloudy aqueous phase was extracted with DCM (2 x 20 ml) and the combined organic extracts washed with PBS (pH = 11, 100 ml). The product was dried over MgSO4, filtered and concentrated. Purification was by chromatography on silica gel eluting with a MeOH in DCM gradient. The fractions that were grouped and concentrated yielded (9Z, 9'Z, 12Z, 12'Z) - ((2- ((2- (dimethylamino) ethyl) thio) acetyl) azanedi-yl) bis (ethane-2,1 di-yl) bis (octadeca-9,12-dienoate) (4.3 g). LCMS ESI +: m / z 775.9 (M + H). PREPARATION OF (9Z, 9'Z, 12Z, 12'Z) - ((2 - ((2- (DIMETHYLAMINE) ETHYL) UNCLE) ACETYL) AZANEDI-IL) BIS (ETANO-2,1-DI-IL) BIS (OCTADECA- 9,12-DIENOATE) (TU104-DLIN)
STEP 1: TU104-DLIN PREPARATION: (9Z, 9'Z, 12Z, 12'Z) - ((2 - ((2- (DIMETHYLAMINE) ETHYL) TIO) ACETYL) AZANEDI-IL) BIS (ETHANE-2, 1-DI-IL) BIS (OCTADECA- 9,12-DIENOATE)

[00133] Synthesis of (9Z, 9'Z, 12Z, 12'Z) -azanedi-ilbis (ethane-2,1-di-yl) bis (octadeca-9,12-dienoate) described above. Trichloromethyl chloroformate (also known as diphosgene) (740 ul) was added to a solution (9Z, 9'Z, 12Z, 12'Z) -azanedi-ilbis (ethane-2,1-di-yl) bis (octadeca- 9.12- dienoate) (2.6 g) in dry DCM (40 ml) and stirred under an argon purge at room temperature for 12 hours. DCM and excess diphosgene were removed under vacuum. The HCl salt of 2- (dimethylamino) ethanethiol (2.9 g), DCM (40 ml) and triethylamine (3.7 ml) were then added. After 16 h at room temperature, the reaction mixture was diluted with DCM and washed with 10% K2CO3 (75 ml), dried (MgSO4), filtered and concentrated. Purification was purified by silica gel chromatography eluting with ethyl acetate followed by a gradient of DCM / MeOH to yield (9Z, 9'Z, 12Z, 12'Z) - ((2 - ((2- (dimethylamino ) ethyl) thio) acetyl) azanedi-yl) bis (ethane-2,1-di-yl) bis (octadeca-9,12-dienoate) (850 mg). LCMS ESI +: m / z 761.9 (M + H).
[00134] Formation of non-diVA siRNA containing liposomes. Ionizable lipid, DOPE, cholesterol and a lipid by PEG were solubilized in absolute EtOH (grade 200 proof) at a final weight concentration of ~ 4.4 mg / ml. The siRNA was solubilized in citrate buffer at a concentration of ~ 0.26 mg / ml and the temperature was adjusted to 35 to 40 ° C. The ethanol / lipid mixture was then added to the siRNA-containing buffer during agitation to spontaneously form liposomes loaded with siRNA. The lipids were combined with siRNA to achieve a final lipid to siRNA ratio of 7: 1 to 14: 1 (p: p). The liposomes loaded with siRNA were diafiltered in 10x volumes of PBS to remove ethanol and change the buffer. The final product was filtered through a 0.22 μm filter with a degree of sterilization to reduce bioburden. This process yielded liposomes with an average particle diameter of 40 to 100 nm, PDI <0.2, and> 85% efficiency of RNA encapsulation (entrapment).
[00135] Formation of siRNA which contains cosolubilized liposomes with diVA. The siRNA-diVA-liposome formulations were prepared using the method described above. diVA-PEG-diVA was cosolubilized in absolute ethanol with the other lipids (ionizable lipid, DOPE, cholesterol and lipids by PEG) before adding to the buffer containing siRNA. The molar content of diVA-PEG-diVA was in the range of 0.1 to 5 mol%. This process yielded liposomes with an average particle diameter of 40 to 100 nm, PDI <0.2, and> 85% of trapping efficiency.
[00136] Formation of siRNA containing liposomes with cationic and ionizable lipids. SiRNA-diVA-Liposome formulations and siRNA-Liposome formulations were prepared using the method described above. The cationic lipid was cosolubilized in absolute ethanol with the other lipids (ionizable lipid, DOPE, cholesterol, lipids by PEG and diVA-PEG-diVA) before adding to the buffer containing siRNA. The molar content of cationic lipid ranged from 5 to 40 mol%. This process yielded liposomes with an average particle diameter of 40 to 100 nm, PDI <0.2, and> 85% of trapping efficiency. EFFECTIVENESS IN VITRO (PHSC, GP46 KD @ 20 NM)
[00137] PHSCs in 96-well plate were incubated with the formulation composed of each of the formulation of ionizable lipid C104, the formulation of ionizable lipid Tu104 or the combination formulations with different ratios of ionizable lipids (C104: Tu104). After 30 minutes, the medium was replaced with fresh growth medium. Forty-eight hours later, cells were lysed and gp46 and GAPDH mRNA levels measured by quantitative RT-PCR assay (TaqMan®), and gp46 levels were normalized to GAPDH levels. The normalized gp46 levels were expressed as a percentage of simulated control cells. Error bars indicate standard deviations (n = 3). The results are revealed in Figure 1. As demonstrated by the results, the combination of two ionizable lipids from the description resulted in the synergistic reduction observed in gene expression.
[00138] Similar experiments were performed with other combinations of ionizable lipid: ionizable lipid and ionizable lipid: cationic lipid. The results for combinations S104-DO: Tu104-DO are revealed in Figure 2. The results for combinations of HEDODC: Tu104 are revealed in Figure 3. The combinations of both ionizable and ionizable lipids: cationic again resulted in synergistic reduction in expression genic. EFFECTIVENESS IN VIVO (DMNQ)
[00139] The in vivo activity of the target formulation was evaluated in the short-term liver damage model (called the Quick Model or DMNQ). In this model, short-term liver damage induced by treatment with a hepatotoxic agent such as dimethylnitrosamine (DMN) is accompanied by increased levels of gp46 mRNA. To induce these changes, male Sprague-Dawley rats were injected intraperitoneally with DMN for six consecutive days. At the end of the DMN treatment period, the animals were randomized into groups based on the individual animal's body weight. The formulations were administered as a single IV dose, one hour after the last injection of DMN. Twenty-four hours later, the liver lobes were excised and both gp46 and MRPL19 mRNA levels were determined by quantitative RT-PCR (TaqMan) assay. The mRNA levels for gp46 were normalized to the levels of MRPL19. The results are shown in Figure 4. Various combinations of ionizable lipids: cationic reached 50% reduction in gene expression for a single dose of encapsulated siRNA of 0.5 mg per kg of animal body weight. IN VITRO TOXICOLOGY DATA, IN VITRO CYTOTOXICITY (HEPG2 @ 200 NM)
[00140] The addition of 20 mol% of S104 in formulations of the description improved cell viability in an improved HepG2 cytotoxicity assay from 27% to 52%.
DESCRIPTION OF HEPG2 CITOTOXICITY TEST
[00141] HepG2 cells, an adherent cell line derived from human hepatocellular carcinoma, were grown in MEM / EBSS (Hyclone, Logan, Utah, USA, catalog number SH30024.01) supplemented with 10% FBS (Hyclone, Logan , Utah, USA, catalog number SH30910). HepG2 cells were seeded in black 96-well Optilux plates (BD Falcon, catalog number BD353220) in 5,000 cells / well overnight. The formulations were added to each well for the indicated final siRNA concentration (n = 3). Within 48 h after formulation addition, cell viability was determined using the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega, catalog number G7572) following the manufacturer's instructions. The chemiluminescent signal was measured on the Clarity Luminescence Microplate reader (502- Biotek, Winooski, Vermont, Canada). Viability was calculated based on% of chemiluminescent signal in treated cavity with standardized formulation in simulated treated cavities. IN VIVO TOXICOLOGY DATA
[00142] The HEDC: S104 (20:20) formulation of the present description is exceptionally well tolerated as shown in toxicity studies. No toxicity was observed when the formulation was injected intravenously into rats and monkeys at doses up to 25 mg / kg and 12 mg / kg, respectively, which is considered by the experts in the art to be superior. TRANSFECTION WITH DESCRIPTION FORMULATIONS
[00143] The transfection method is the same for LX-2 and pHSCs. The liposome formulations or lipoplex formulations of the description were mixed with growth medium in desired concentrations. 100 μl of the mixture was added to the cells in a 96-well plate and the cells were incubated for 30 min. at 37 ° C in the 5% CO2 incubator. After 30 min, the medium was replaced with fresh growth medium. After 48 h of transfection, the cells were processed using Cell-to-Ct lysis reagents (Applied Biosystems) according to the manufacturer's instructions. QUANTITATIVE RT-PCR (Q) TO MEASURE MRP EXPRESSION OF HSP47
[00144] The HSP47 and GAPDH TaqMan® and One-Step RT-PCR master mix assays were purchased from Applied Biosystems. Each PCR reaction contained the following composition: 5 μl One-Step RT-PCR mixture, 0.25 μl TaqMan® RT enzyme mixture, 0.25 μl TaqMan® (HSP47) gene expression probe, probe 0.5 μl TaqMan® gene expression assay (GAPDH), 3.25 μl RNase free water, 0.75 μl cell lysate, 10 μl total volume. GAPDH was used as an endogenous control for the relative quantification of HSP47 mRNA levels. Quantitative RT-PCR was performed on the ViiA 7 real-time PCR system (Applied Biosciences). All values were normalized to the mean HSP47 expression of the simulated transfected cells and expressed as a percentage of HSP47 expression compared to the simulation.

权利要求:
Claims (24)
[0001]
1. IONIZABLE LIPIDE COMPOUND, characterized by formula I:
[0002]
COMPOSITE according to claim 1, characterized in that n and m are 1 or 2.
[0003]
COMPOSITE according to claim 1, characterized in that X is absent.
[0004]
COMPOSITE according to claim 1, characterized in that X is -CH2-.
[0005]
COMPOSITE according to claim 4, characterized in that L is -S-C 1-4 alkylene.
[0006]
6. COMPOSITE according to claim 5, characterized in that L is -S-CH2- or -S-CH2-CH2-.
[0007]
7. COMPOUND according to claim 4, characterized in that L is -S (O) 2-C 1-4 alkylene, or -O-C (O) -C 1-4 alkylene.
[0008]
8. COMPOUND according to claim 1, characterized in that X is S.
[0009]
9. COMPOUND according to claim 1, characterized in that X is O.
[0010]
10. COMPOUND, as characterized by R1 and R2 each being C10-18 alkyl. claim 1,
[0011]
COMPOSITE according to claim 1, characterized in that R1 and R2 are each C12-18 alkenyl.
[0012]
12. COMPOUND according to claim 11, characterized in that R1 and R2 are each C13-17 alkenyl.
[0013]
13. COMPOSITION, characterized in that it comprises a unilamellar or multilamellar lipid bilayer comprising a compound, as defined in any one of claims 1 to 12.
[0014]
COMPOSITION according to claim 13, characterized in that the compound has 5 to 50 mol% of a unilamellar or multilamellar lipid bilayer.
[0015]
COMPOSITION according to claim 14, characterized in that it comprises two compounds of Formula I, as defined in any one of claims 1 to 12, and in which the molar ratio of the two compounds of Formula I is 10:30 to 30 : 10.
[0016]
16. COMPOSITION according to any one of claims 13 to 15, characterized in that it additionally comprises a
[0017]
17. COMPOSITION, according to claim 16, characterized in that the cationic lipid has 5 to 40 mol% of a unilamellar or multilamellar lipid bilayer.
[0018]
18. COMPOSITION, according to claim 17, characterized by the molar ratio of the compound, as defined in any one of claims 1 to 13, and the cationic lipid is from 5:35 to 35: 5.
[0019]
19. COMPOSITION according to any one of claims 14 to 18, characterized in that it further comprises a liquid medium, in which the liquid medium comprises an organic solvent or water and an organic solvent, in which the organic solvent is chosen from alcohols of C1-4 alkyl and dimethyl sulfoxide (DMSO).
[0020]
20. COMPOSITION according to any one of claims 13 to 19, characterized in that it further comprises: a phospholipid selected from the group 1,2-distearoil-sn-glycero-3-phosphocholine (“DSPC”), dipalmitoylphosphatidylcholine (“DPPC ”), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (“ DPPE ”), and 1,2-dioleoyl-sn-glycero-3 phosphoethanolamine (“ DOPE ”), and / or a PEG-conjugated lipid , wherein the PEG-conjugated lipids are PEG-phospholipids or PEG-ceramides, such as, for example, PEG2000-DSPE, PEG2000-DPPE, PEG2000-DMPE, PEG2000-DOPE, PEG1000-DSPE, PEG1000-DPPE, PEG1000-DMPE , PEG1000-DOPE, PEG550-DSPE, PEG550-DPPE, PEG-550DMPE, PEG-1000DOPE, PEG-BML, PEG-Cholesterol. PEG2000-Ceramide, PEG1000-Ceramide, PEG750-Ceramide, PEG550- Ceramide.
[0021]
21. PHARMACEUTICAL VEHICLE, characterized in that it comprises the composition, as defined in any one of claims 13 to 20, and a compound of the structure (retinoid) n-ligand- (retinoid) n, where n = 0, 1, 2 or 3; and wherein the binder comprises a polyethylene glycol (PEG).
[0022]
22. VEHICLE, according to claim 21, characterized in that it additionally comprises a small interference RNA (siRNA) molecule.
[0023]
23. PHARMACEUTICAL FORMULATION, characterized in that it comprises the drug carrier, as defined in any one of claims 21 to 22, and a pharmaceutically acceptable carrier or diluent.
[0024]
24. FORMULATION, according to claim 23, characterized in that the siRNA is encapsulated by a liposome.

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同族专利:

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公开号 | 公开日

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SI2858974T1|2019-01-31|

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AU2013270685B2|2017-11-30|

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BR112014030714A2|2017-06-27|

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ES2697680T3|2019-01-25|

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US10441659B2|2019-10-15|

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LT3489220T|2021-09-27|

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US20160074514A1|2016-03-17|

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WO2013185116A1|2013-12-12|

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LT2858974T|2018-12-10|

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KR102102801B1|2020-04-22|

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EP3489220A1|2019-05-29|

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CY1120929T1|2019-12-11|

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EP2858974A1|2015-04-15|

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SI3489220T1|2021-11-30|

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EP3489220B9|2021-11-10|

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TWI654997B|2019-04-01|

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RU2014153658A|2016-07-27|

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RS62288B1|2021-09-30|

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TR201816986T4|2019-01-21|

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RS58108B1|2019-02-28|

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US20130330401A1|2013-12-12|

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US11103583B2|2021-08-31|

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JP5873604B2|2016-03-01|

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US9308267B2|2016-04-12|

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HRP20181855T1|2018-12-28|

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HRP20211424T2|2022-02-18|

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CA2876148C|2021-01-12|

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HRP20211424T1|2022-02-04|

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EP3489220B1|2021-08-18|

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JP2015523990A|2015-08-20|

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CN104640841A|2015-05-20|

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RS62288B9|2021-12-31|

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PL3489220T3|2021-11-02|

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CA2876148A1|2013-12-12|

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PT3489220T|2021-10-22|

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HUE041882T2|2019-06-28|

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TW201402146A|2014-01-16|

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CN104640841B|2018-04-13|

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ES2897216T3|2022-02-28|

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RU2658007C2|2018-06-19|

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EP2858974B1|2018-10-17|

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DK3489220T3|2021-09-06|

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KR20150021566A|2015-03-02|

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PT2858974T|2018-11-29|

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AU2013270685A1|2015-01-29|

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US20200085957A1|2020-03-19|

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PL2858974T3|2019-03-29|

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DK2858974T3|2018-12-03|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

US5264618A|1990-04-19|1993-11-23|Vical, Inc.|Cationic lipids for intracellular delivery of biologically active molecules|

---

US5650386A|1995-03-31|1997-07-22|Emisphere Technologies, Inc.|Compositions for oral delivery of active agents|

---

US5994317A|1996-04-09|1999-11-30|Vical Incorporated|Quaternary cytofectins|

---

CN1277024A|1999-06-11|2000-12-20|克林诺斯生物药品工业有限公司|Application of cationic liposome and polydeoxyribonucleosides composite using as medicament|

---

CN101163796B|2004-06-07|2012-08-29|普洛体维生物治疗公司|Cationic lipids and methods of use|

---

AU2005320014B2|2004-12-22|2011-05-26|Nitto Denko Corporation|Drug carrier and drug carrier kit for inhibiting fibrosis|

---

JP2006254877A|2005-03-18|2006-09-28|Fukuoka Prefecture|Carrier containing glycolipid and method for transferring gene using the same|

---

WO2011136368A1|2010-04-28|2011-11-03|協和発酵キリン株式会社|Cationic lipid|

---

US9393315B2|2011-06-08|2016-07-19|Nitto Denko Corporation|Compounds for targeting drug delivery and enhancing siRNA activity|

---

US10196637B2|2011-06-08|2019-02-05|Nitto Denko Corporation|Retinoid-lipid drug carrier|

---

TWI658830B|2011-06-08|2019-05-11|日東電工股份有限公司|Retinoid-liposomes for enhancing modulation of hsp47 expression|

---

ES2743540T3|2011-06-08|2020-02-19|Nitto Denko Corp|Compounds to direct the administration of drugs and enhance the activity of siRNA|

---

US9011903B2|2011-06-08|2015-04-21|Nitto Denko Corporation|Cationic lipids for therapeutic agent delivery formulations|

---

CN103906504B|2011-11-04|2017-11-17|日东电工株式会社|The method for preparing the lipidic nanoparticles for medicine delivery|

---

US9579338B2|2011-11-04|2017-02-28|Nitto Denko Corporation|Method of producing lipid nanoparticles for drug delivery|

---

TWI554244B|2011-12-19|2016-10-21|愛爾康眼科手術激光股份有限公司|Image processor for intra-surgical optical coherence tomographic imaging of laser cataract procedures|

---

PL3489220T3|2012-06-08|2021-11-02|Nitto Denko Corporation|Lipids for therapeutic agent delivery formulations|US20120141576A1|2007-03-15|2012-06-07|Benjamin Johnson|Treatment of Dermatologic Skin Disorders|

---

US8822663B2|2010-08-06|2014-09-02|Moderna Therapeutics, Inc.|Engineered nucleic acids and methods of use thereof|

---

EP2625189B1|2010-10-01|2018-06-27|ModernaTX, Inc.|Engineered nucleic acids and methods of use thereof|

---

WO2012135805A2|2011-03-31|2012-10-04|modeRNA Therapeutics|Delivery and formulation of engineered nucleic acids|

---

US9464124B2|2011-09-12|2016-10-11|Moderna Therapeutics, Inc.|Engineered nucleic acids and methods of use thereof|

---

CN110511939A|2011-10-03|2019-11-29|现代泰克斯公司|Nucleosides, nucleotide and nucleic acid of modification and application thereof|

---

AU2012352180A1|2011-12-16|2014-07-31|Moderna Therapeutics, Inc.|Modified nucleoside, nucleotide, and nucleic acid compositions|

---

US9283287B2|2012-04-02|2016-03-15|Moderna Therapeutics, Inc.|Modified polynucleotides for the production of nuclear proteins|

---

AU2013243955B2|2012-04-02|2018-02-22|Modernatx, Inc.|Modified polynucleotides for the production of oncology-related proteins and peptides|

---

US9878056B2|2012-04-02|2018-01-30|Modernatx, Inc.|Modified polynucleotides for the production of cosmetic proteins and peptides|

---

US9572897B2|2012-04-02|2017-02-21|Modernatx, Inc.|Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins|

---

PL3489220T3|2012-06-08|2021-11-02|Nitto Denko Corporation|Lipids for therapeutic agent delivery formulations|

---

EP2922554B1|2012-11-26|2022-02-23|ModernaTX, Inc.|Terminally modified rna|

---

US10258698B2|2013-03-14|2019-04-16|Modernatx, Inc.|Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions|

---

US8980864B2|2013-03-15|2015-03-17|Moderna Therapeutics, Inc.|Compositions and methods of altering cholesterol levels|

---

WO2015048744A2|2013-09-30|2015-04-02|Moderna Therapeutics, Inc.|Polynucleotides encoding immune modulating polypeptides|

---

US10323076B2|2013-10-03|2019-06-18|Modernatx, Inc.|Polynucleotides encoding low density lipoprotein receptor|

---

US9365610B2|2013-11-18|2016-06-14|Arcturus Therapeutics, Inc.|Asymmetric ionizable cationic lipid for RNA delivery|

---

JP6637988B2|2014-11-18|2020-01-29|アークトゥルス セラピューティクス， インコーポレイテッド|Ionizable cationic lipids for RNA delivery|

---

EP3071547A1|2013-11-18|2016-09-28|Arcturus Therapeutics, Inc.|Ionizable cationic lipid for rna delivery|

---

WO2015152332A1|2014-04-02|2015-10-08|日東電工株式会社|Targeting molecule and utilization thereof|

---

BR112016024644A2|2014-04-23|2017-10-10|Modernatx Inc|nucleic acid vaccines|

---

US10792299B2|2014-12-26|2020-10-06|Nitto Denko Corporation|Methods and compositions for treating malignant tumors associated with kras mutation|

---

US10264976B2|2014-12-26|2019-04-23|The University Of Akron|Biocompatible flavonoid compounds for organelle and cell imaging|

---

TW201701887A|2015-02-19|2017-01-16|日東電工股份有限公司|RNA interference therapeutics against Ebola virus|

---

CN107708739A|2015-06-24|2018-02-16|日东电工株式会社|Ionizable compound, composition and their purposes|

---

US10221127B2|2015-06-29|2019-03-05|Acuitas Therapeutics, Inc.|Lipids and lipid nanoparticle formulations for delivery of nucleic acids|

---

PT3324932T|2015-07-22|2021-04-06|Nitto Denko Corp|Compositions and methods for nanoparticle lyophile forms|

---

CA2998810A1|2015-09-17|2017-03-23|Modernatx, Inc.|Compounds and compositions for intracellular delivery of therapeutic agents|

---

EP3386484A4|2015-12-10|2019-07-31|ModernaTX, Inc.|Compositions and methods for delivery of therapeutic agents|

---

JP2019508371A|2015-12-22|2019-03-28|モデルナティエックス インコーポレイテッドＭｏｄｅｒｎａＴＸ，Ｉｎｃ．|Compounds and compositions for intracellular delivery of drugs|

---

US10780174B2|2015-12-23|2020-09-22|The University Of British Columbia|Lipid-linked prodrugs|

---

US9834510B2|2015-12-30|2017-12-05|Arcturus Therapeutics, Inc.|Aromatic ionizable cationic lipid|

---

JP6833456B2|2016-11-02|2021-02-24|日東電工株式会社|Skin fibrosis treatment agent|

---

CA3045131A1|2016-12-14|2018-06-21|Ligandal, Inc.|Methods and compositions for nucleic acid and protein payload delivery|

---

US10383952B2|2016-12-21|2019-08-20|Arcturus Therapeutics, Inc.|Ionizable cationic lipid for RNA delivery|

---

US10526284B2|2016-12-21|2020-01-07|Arcturus Therapeutics, Inc.|Ionizable cationic lipid for RNA delivery|

---

CA3056132A1|2017-03-15|2018-09-20|Modernatx, Inc.|Compounds and compositions for intracellular delivery of therapeutic agents|

---

WO2018170322A1|2017-03-15|2018-09-20|Modernatx, Inc.|Crystal forms of amino lipids|

---

WO2019074071A1|2017-10-11|2019-04-18|日東電工株式会社|Regulation of nucleic acid molecule expression|

---

CN108148870B|2018-01-12|2021-09-21|中国人民解放军第四军医大学|Method for improving siRNA transferring efficiency in vitro of chitosan|

---

US11066355B2|2019-09-19|2021-07-20|Modernatx, Inc.|Branched tail lipid compounds and compositions for intracellular delivery of therapeutic agents|

---

WO2021262746A1|2020-06-24|2021-12-30|Bristol-Myers Squibb Company|Process for synthesizing lipids|

---

WO2021262745A1|2020-06-24|2021-12-30|Bristol-Myers Squibb Company|Process for synthesizing cationic lipids|

---

WO2022037652A1|2020-08-20|2022-02-24|Suzhou Abogen Biosciences Co., Ltd.|Lipid compounds and lipid nanoparticle compositions|

法律状态:
2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-12-04| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-04-24| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-08-13| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-03-03| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2020-10-27| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-12-22| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 07/06/2013, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201261657480P| true| 2012-06-08|2012-06-08|

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US61/657,480|2012-06-08|

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PCT/US2013/044849|WO2013185116A1|2012-06-08|2013-06-07|Lipids for therapeutic agent delivery formulations|

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