Cationic lipid compound

Abstract

The present invention relates to lipid compounds which can be used alone or in combination with other lipid components such as neutral lipids, charged lipids, steroids and / or their analogs, and / or lipid-conjugated polymers to form lipid nanoparticles for delivering therapeutic and / or prophylactic agents. In some examples, the lipid nanoparticles are used for delivering nucleic acids such as messenger RNA and / or antisense RNA. Methods of using such lipid nanoparticles to treat and / or prevent various diseases are also provided. In one embodiment, a compound having the structure of formula (I) is provided: or a salt or an isomer or an N-oxide thereof, wherein R 1 is as defined in the text. 2 is as defined in the text.

Description

Technical Field

[0001] The present invention provides cationic lipids which can be used in combination with other lipid components (such as neutral lipids, steroids, and lipid-polymer conjugates) to form a nucleic acid mRNA lipid nanoparticle composition for delivering one or more therapeutic and / or prophylactic agents to mammalian cells or organs and / or for producing polypeptides in mammalian cells or organs. In addition to lipids, the lipid nanoparticle composition of the present invention may further comprise one or more cationic and / or ionizable amino lipids, neutral lipids including polyunsaturated lipids, lipid-polymer conjugates, steroids, and / or therapeutic and / or prophylactic agents in specific proportions. Background Art

[0002] The effective targeted delivery of bioactive substances such as small molecule drugs, proteins, and nucleic acids poses a persistent medical challenge. Specifically, delivering nucleic acids to cells is difficult due to the relative instability and low cell permeability of these species. Therefore, there is a need to develop methods and compositions that facilitate the delivery of therapeutic and / or prophylactic agents such as nucleic acids to cells.

[0003] It has been demonstrated that lipid-containing nanoparticle compositions, liposomes, and lipoplexes can be used as delivery vehicles to effectively deliver bioactive substances such as small molecule drugs, proteins, and nucleic acids to cells and / or intracellular compartments. These compositions generally comprise one or more "cationic" lipids, neutral lipids including polyunsaturated lipids (such as phospholipids), structural lipids (such as steroids), and / or lipids containing polyethylene glycol (lipid-polymer conjugates). Cationic lipids include, for example, amine-containing lipids that can be readily protonated.

[0004] However, the use of oligonucleotides in a therapeutic setting currently faces two problems. First, free RNA is susceptible to nuclease digestion in plasma. Second, the ability of free RNA to enter intracellular compartments where the relevant translation machinery is present is limited. Lipid nanoparticles formed from cationic lipids and other lipid components (such as neutral lipids, cholesterol, PEG, PEGylated lipids, and oligonucleotides) have been used to prevent the degradation of RNA in plasma and to promote the cellular uptake of oligonucleotides.

[0005] There is still a need to improve cationic lipids and lipid nanoparticles for delivering oligonucleotides. Improved lipid nanoparticles would provide optimized drug delivery, protect nucleic acids from degradation and clearance in serum, be suitable for systemic or local delivery, and provide intracellular delivery of nucleic acids. Additionally, these preferred lipid-nucleic acid particles should be well-tolerated and provide a sufficient therapeutic index such that patient treatment at an effective dose of nucleic acid does not cause unacceptable toxicity and / or risk to the patient. The present invention provides these and related advantages. Summary of the Invention

[0006] The present invention provides the following compounds and methods involving these compounds:

[0007] In a first aspect, the present invention relates to compounds of the following structural formula (I):

[0008]

[0009] or a salt thereof or an isomer thereof or an N-oxide thereof, wherein:

[0010] R 1 、R 2 is C 1-3 alkyl or hydrogen, but not both hydrogen.

[0011] In various different embodiments, the compound has one of the structures shown in Table 1 below

[0012] Table 1 Representative Compounds

[0013]

[0014]

[0015]

[0016] In some embodiments, there is provided a composition comprising any one or more of the compounds of structural formula (I) and a therapeutic agent and / or a prophylactic agent.

[0017] In some embodiments, there is provided a composition comprising any one or more of the compounds of structure (I) and a therapeutic agent and / or a prophylactic agent. In some embodiments, the composition comprises any one of the compounds of structure (I) and a therapeutic agent and / or a prophylactic agent and one or more excipients selected from neutral lipids, steroids, and polymer-conjugated lipids. Other pharmaceutically acceptable excipients and / or carriers are also included in various embodiments of the composition.

[0018] In some embodiments, the neutral lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM), and mixtures thereof. In some embodiments, the preferred neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0019] In some embodiments, the steroid is selected from cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, and mixtures thereof. In some embodiments, the preferred steroid is cholesterol.

[0020] In some embodiments, the polyethylene glycolylated lipid is 1,2-dimyristoyl-sn-glycero-methoxypolyethylene glycol (PEG-DMG)

[0021] In some embodiments, the composition ratios are in the following ranges: about 10-60 mol% of the compound, about 0-30 mol% of neutral lipid, about 10-55 mol% of steroid, and about 0-10 mol% of polymer-conjugated lipid.

[0022] In some embodiments of the foregoing compositions, the therapeutic and / or prophylactic agent comprises a nucleic acid. Wherein the nucleic acid is RNA, which is selected from the group consisting of siRNA, aiRNA, miRNA, dsRNA, shRNA, mRNA, and mixtures thereof. In some embodiments, the RNA is selected from mRNA.

[0023] In other different embodiments, the present invention relates to a method of administering a therapeutic and / or prophylactic agent to a subject in need thereof, the method comprising preparing or providing any one of the above compositions and administering the composition to the subject.

[0024] For the purpose of administration, the compounds of the present invention (usually in the form of lipid nanoparticles combined with a therapeutic and / or prophylactic agent) can be administered as the active pharmaceutical ingredient, or can be formulated into a pharmaceutical composition. The pharmaceutical composition of the present invention comprises a compound of formula (I) and one or more pharmaceutically acceptable carriers, diluents or excipients. The compound of formula (I) is effective to form lipid nanoparticles and deliver the therapeutic and / or prophylactic agent. Those skilled in the art can readily determine the appropriate concentrations and dosages.

[0025] The administration of the compositions of the present invention can be carried out by any acceptable mode of administration for a reagent of similar utility. The pharmaceutical compositions of the present invention can be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal and intranasal routes. The term parenteral as used herein includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques. The pharmaceutical compositions of the present invention are formulated to allow the active ingredient contained therein to be bioavailable after administration of the composition to a subject. The composition to be administered to an object or patient is in the form of one or more dosage units, wherein a tablet can be a single dosage unit, and a container of a compound in aerosol form of the present invention can contain multiple dosage units. Current methods of preparing these dosage forms are known or will be apparent to those skilled in the art. In any case, the composition to be administered will contain a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof to treat a relevant disease or condition according to the teachings of the present invention.

[0026] The pharmaceutical compositions of the present invention can be in solid or liquid form. In one aspect, the carrier is particulate such that the composition is in the form of a tablet or powder. The carrier can be liquid, in which case the composition is an oral syrup or an injectable liquid or an aerosol suitable for inhalational administration.

[0027] When intended for oral administration, the pharmaceutical composition is preferably in solid or liquid form, where forms considered solid or liquid herein include semi-solid, semi-liquid, suspension and gel forms.

[0028] As a solid composition for oral administration, the pharmaceutical composition can be formulated into forms such as powders, granules, compressed tablets, pills, capsules, chewing gums, wafers, etc. Such solid compositions will generally contain one or more inert diluents or edible carriers. Additionally, one or more of the following may be present: binders such as gelatin, cellulose, etc.; excipients such as lactose, etc.; disintegrants such as alginic acid, etc.; lubricants such as magnesium stearate, etc.; glidants such as silica gel, etc.; sweeteners such as sucrose or saccharin; flavoring agents such as peppermint, etc.; and coloring agents.

[0029] When the pharmaceutical composition is in capsule form, it can contain a liquid carrier in addition to materials of the above types, such as polyethylene glycol or oil.

[0030] The pharmaceutical composition can be in liquid form, such as syrup, solution, emulsion or suspension. As two examples, the liquid can be used for oral administration or for injection delivery. When intended for oral administration, the preferred composition contains, in addition to the compound of the present invention, one or more of a sweetening agent, a preservative, a coloring agent, and a flavor enhancer. In the composition for administration by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffering agent, a stabilizing agent, and an isotonic agent can be included.

[0031] The liquid pharmaceutical composition of the present invention, whether it is a solution, a suspension or other similar forms, can include one or more of the following adjuvants: sterile diluents, such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride; non-volatile oils, such as synthetic monoglycerides or diglycerides that can be used as solvents or suspending media, polyethylene glycol, glycerol, propylene glycol or other solvents; antibacterial agents, such as methylparaben, etc.; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffering agents, such as acetate, citrate or phosphate; and reagents for adjusting tonicity, such as sodium chloride or glucose; reagents used as cryoprotectants, such as sucrose or trehalose. The parenteral preparation can be encapsulated in ampoules, disposable syringes or multi-dose bottles made of glass or plastic. Physiological saline is the preferred adjuvant. The injectable pharmaceutical composition is preferably sterile.

[0032] The pharmaceutical composition of the present invention can consist of dosage units that can be administered as an aerosol. The term aerosol is used to denote various systems ranging from colloidal systems to systems consisting of pressurized packages. It can be delivered by liquefied gas or compressed gas, or by a suitable pump system that disperses the active ingredient. The aerosol of the compound of the present invention can be delivered in a single-phase, two-phase system or a three-phase system in order to deliver the active ingredient. The delivery of the aerosol includes the necessary containers, activators, valves, sub-containers, etc., which together can form a kit. A person skilled in the art can determine the preferred aerosol without undue experimentation.

[0033] The pharmaceutical composition of the present invention can be prepared by methods well known in the pharmaceutical field. The pharmaceutical composition intended for administration by injection can be prepared by combining the lipid nanoparticles of the present invention with sterile distilled water or other carriers to form a solution. A surfactant can be added to facilitate the formation of a uniform solution or suspension. A surfactant is a compound that interacts non-covalently with the compound of the present invention in order to facilitate the dissolution or uniform suspension of the compound in an aqueous delivery system.

[0034] The composition of the present invention or a pharmaceutically acceptable salt thereof is administered in a therapeutically effective amount, which will vary depending on a variety of factors, including the activity of the specific therapeutic agent used; the metabolic stability and duration of action of the therapeutic agent; the age, weight, general health, sex and diet of the subject; the mode and time of administration; the rate of excretion; the drug combination; the severity of the specific case, etc.

[0035] The composition of the present invention can also be administered simultaneously with, before or after the administration of one or more other therapeutic agents. Such combination therapies include single pharmaceutical dosage formulations that administer the composition of the present invention and one or more additional active agents, as well as the administration of the composition of the present invention and the active agents in their own separate pharmaceutical dosage formulations. For example, the composition of the present invention and other active agents can be administered to a subject together in a single oral dosage composition (such as a tablet or capsule), or each reagent can be administered in a different oral dosage formulation. When different dosage formulations are used, the compounds of the present invention and one or more additional active agents can be administered at substantially the same time, or sequentially at staggered times; it should be understood that combination therapies include all of these dosing regimens.

[0036] The structural modification and design of the above-mentioned cationic lipid compounds have achieved more advantageous physicochemical properties, including more suitable pKa and better chemical stability, for mRNA nanoliposome compositions, enabling more effective binding and delivery of ionic nucleic acid drugs. At the same time, its chemical structure is more stable, facilitating synthesis and being conducive to development as a pharmaceutical excipient.

[0037] The preparation methods of the above-mentioned compounds and compositions are described below and / or are known in the art.

[0038] Those skilled in the art will recognize that in the methods described herein, the functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxyl, amino and carboxylic acid. Suitable protecting groups for hydroxyl include trialkylsilyl or diarylalkylsilyl, tetrahydrofuranyl, benzyl, etc. Suitable protecting groups for amino include tert-butoxycarbonyl, benzyloxycarbonyl, etc. Suitable protecting groups for carboxylic acid include alkyl, aryl or aryl ester. The protecting groups can be added or removed according to standard techniques that are known to those skilled in the art and described herein.

[0039] Those skilled in the art will also recognize that although such protected derivatives of the compounds of the present invention may not thereby have pharmacological activity, they can be administered to mammals and then metabolized in vivo to form the pharmacologically active compounds of the present invention. Such derivatives can therefore be described as "prodrugs". Therefore, prodrugs of the compounds of the present invention are included within the scope of the present invention.

[0040] In addition, all compounds of the present invention in the form of free base or free acid can be converted into their pharmaceutically acceptable salts by treatment with appropriate inorganic or organic bases or acids according to methods known to those skilled in the art. The salts of the compounds of the present invention can be converted into their free base or acid forms by standard techniques.

[0041] The following examples are provided for illustrative purposes only and are not intended to be limiting.

[0042] In the following examples, unless otherwise indicated, all solvents and reagents used are commercially available and used as received.

[0043] The procedures described below can be used to synthesize related compounds.

[0044] The following abbreviations are used herein:

[0045] EDC.HCl: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0046] DCM: Dichloromethane

[0047] DMAP: 4-Dimethylaminopyridine

[0048] DIEA: N,N-Diisopropylethylamine

[0049] THF: Tetrahydrofuran Detailed Description of the Invention

[0050] Example 1:

[0051] Representative Route

[0052] Synthesis of Compound 7

[0053]

[0054] 1) Synthesis of Compound A-2

[0055]

[0056] Chemical Formula: C 18 H 36 O 2

[0057] Molecular Weight: 284.48

[0058] Dissolve diisopropylamine (3 mL, 21.4 mmol) in 10 mL of THF, cool to -78 °C, and dropwise add n-butyllithium solution (2.5 M in hexane, 7.7 mL, 19.3 mmol). Slowly warm to 0 °C and dropwise add a 20 mL THF mixture of 2-hexyldecanoic acid (4.49 g, 17.5 mmol). Stir at room temperature for 30 minutes, then add bromoethane (2.10 g, 19.3 mmol), warm to 30 °C and react for 8 hours. Quench the reaction with dilute hydrochloric acid, extract with ethyl acetate, dry the organic phase over anhydrous sodium sulfate, and concentrate in vacuo. Purify the residue by silica gel column (ethyl acetate / hexane). Obtain compound A-2 (1.5 g, 5.3 mmol, 30%).

[0059] 2) Synthesis of compound B-2

[0060]

[0061] Chemical formula: C 24 H 47 BrO 2

[0062] Molecular weight: 447.54

[0063] To a 20 mL DCM mixture of compound A-2 (1.50 g, 5.3 mmol) and 6-bromohexanol (1.20 g, 6.6 mmol), sequentially add EDC·HCl (1.26 g, 6.6 mmol), DMAP (0.06 g, 0.5 mmol), and DIEA (2.05 g, 15.9 mmol), and react at room temperature for 24 hours. Wash sequentially with saturated aqueous sodium bicarbonate and dilute aqueous hydrochloric acid, separate the organic phase, dry over anhydrous sodium sulfate, concentrate in vacuo, and purify the residue by silica gel column (ethyl acetate / hexane). Obtain compound B-2 (1.5 g, 3.4 mmol, 64%).

[0064] 3) Synthesis of compound 7

[0065]

[0066] Chemical formula: C 52 H 103 NO 5

[0067] Molecular weight: 822.40

[0068] Compound B-2 (1.50 g, 3.4 mmol) was mixed with ethanol, 4-amino-1-butanol (0.13 g, 1.5 mmol) and DIEA (1.03 g, 10.2 mmol) were added, and the reaction was carried out at 60 °C for 32 hours. The reaction system was concentrated in vacuo. Ethyl acetate and water were added to the residue, and the organic phase was separated and dried over anhydrous sodium sulfate. After concentration in vacuo, the residue was purified by silica gel column (ammonia water / methanol / DCM). Compound 7 (0.9 g, 1.1 mmol, 73%) was obtained.

[0069] C 52 H 103 NO 5 ,Ms m / z:[M+H + 823; 1 H-NMR(300MHz,CDCl 3 )δ:ppm 4.10(4H,t), 3.49(2H,t), 3.05(6H, t), 1.76~1.41(18H, m), 1.38~1.17(54H, m),0.87(18H, m).

[0070] Example 2:

[0071] Compound 1:

[0072]

[0073] Chemical formula: C 49 H 97 NO 5

[0074] Molecular weight: 780.32

[0075] Compound 1 can be synthesized according to the representative route described in Example 1.

[0076] C 49 H 97 NO 5 ,Ms m / z:[M+H + 781; 1 H-NMR(300MHz,CDCl 3 )δ:ppm 4.08(4H,t), 3.47(2H,t), 3.03(6H, t), 2.11(1H, m), 1.74~1.48(14H, m), 1.43~1.15(57H, m),0.88(12H, m).

[0077] Example 3:

[0078] Compound 2:

[0079]

[0080] Molecular weight: 794.34

[0081] Compound 2 can be synthesized according to the representative route described in Example 1.

[0082] C 50 H 99 NO 5 , Ms m / z: [M+H + 795; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.09 (4H, t), 3.48 (2H, t), 3.04 (6H, t), 2.11 (1H, m), 1.74~1.48 (16H, m), 1.43~1.15 (54H, m), 0.88 (15H, m).

[0083] Example 4:

[0084] Compound 3:

[0085]

[0086] Chemical formula: C 51 H 101 NO 5

[0087] Molecular weight: 808.37

[0088] Compound 3 can be synthesized according to the representative route described in Example 1.

[0089] C 51 H 101 NO 5 , Ms m / z: [M+H + 809; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.06 (4H, t), 3.46 (2H, t), 3.02 (6H, t), 2.11 (1H, m), 1.78~1.45 (16H, m), 1.43~1.15 (56H, m), 0.86 (15H, m).

[0090] Example 5:

[0091] Compound 4:

[0092]

[0093] Chemical formula: C 50 H 99 NO 5

[0094] Molecular weight: 794.34

[0095] Compound 4 can be synthesized according to the representative route described in Example 1.

[0096] C 50 H 99 NO 5 , Ms m / z: [M+H + 795; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.11 (4H, t), 3.46 (2H, t), 3.02 (6H, t), 1.70~1.50 (14H, m), 1.48~1.13 (60H, m), 0.88 (12H, m).

[0097] Example 6:

[0098] Compound 5:

[0099]

[0100] Chemical formula: C 51 H 101 NO 5

[0101] Molecular weight: 808.37

[0102] Compound 5 can be synthesized according to the representative route described in Example 1.

[0103] C 51 H 101 NO 5 , Ms m / z: [M+H + 809; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.11 (4H, t), 3.46 (2H, t), 3.02 (6H, t), 1.70~1.48 (16H, m), 1.47~1.13 (57H, m), 0.88 (15H, m).

[0104] Example 7:

[0105] Compound 6:

[0106]

[0107] Chemical formula: C 52 H 103 NO 5

[0108] Molecular weight: 822.40

[0109] Compound 6 can be synthesized according to the representative route described in Example 1.

[0110] C 52 H 103 NO 5 , Ms m / z: [M+H + 823; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.10 (4H, t), 3.45 (2H, t), 3.03 (6H, t), 1.73~1.48 (16H, m), 1.47~1.15 (59H, m), 0.88 (15H, m).

[0111] Example 8:

[0112] Compound 8:

[0113]

[0114] Chemical formula: C 53 H 105 NO 5

[0115] Molecular weight: 836.43

[0116] Compound 8 can be synthesized according to the representative route described in Example 1.

[0117] C 53 H 105 NO 5 , Ms m / z: [M+H + 837; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.07 (4H, t), 3.47 (2H, t), 3.01 (6H, t), 1.69~1.47 (18H, m), 1.45~1.13 (56H, m), 0.87 (18H, m).

[0118] Example 9:

[0119] Compound 9:

[0120]

[0121] Chemical formula: C 54 H 107 NO 5

[0122] Molecular weight: 850.45

[0123] Compound 9 can be synthesized according to the representative route described in Example 1.

[0124] C 54 H 107 NO 5 , Ms m / z: [M+H + 851; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.06 (4H, t), 3.47 (2H, t), 3.01 (6H, t), 1.65~1.48 (18H, m), 1.47~1.15 (58H, m), 0.88 (18H, m).

[0125] Example 10

[0126] In vivo evaluation of luciferase mRNA using lipid nanoparticle compositions

[0127] Dissolve cationic lipid, DSPC, cholesterol, and PEG-lipid in ethanol at a molar ratio of 50:10:38:2 or 48:10:40:2. Prepare lipid nanoparticles (LNP) at a total lipid to mRNA weight ratio of approximately 10:1 to 30:1. Briefly, dilute the mRNA to 0.15 mg / mL in 10 mL to 50 mL of citrate buffer (pH = 4). Using a syringe pump, mix the ethanol solution of the lipid with the aqueous mRNA solution at a ratio of approximately 1:5 to 1:3 (volume / volume) with a total flow rate of more than 10 mL / min. Then remove the ethanol and replace the external buffer with PBS by dialysis. Finally, filter the lipid nanoparticles through a sterile filter with a pore size of 0.2 μm. The particle size of the lipid nanoparticles determined by quasi-elastic light scattering using a Malvern Zetasizer Nano ZS is approximately 65 - 105 nm in diameter, and in some cases, approximately 75 - 100 nm in diameter.

[0128] Conduct studies on female C57BL / 6 mice at 6 - 8 weeks of age and CD-1 mice at 8 - 10 weeks of age according to the guidelines established by the National Science and Technology Council. Systemically administer different doses of mRNA lipid nanoparticles by tail vein injection and euthanize the animals at specific time points after administration (e.g., 5 hours). Collect the liver and spleen in pre-weighed tubes, determine the weight, immediately snap-freeze in liquid nitrogen, and store at -80 °C until used for analysis.

[0129] For the liver, approximately 50 mg was cut for analysis in 2 mL FastPrep tubes (MP Biomedicals, Solon OH). A 1 / 4" ceramic bead (MP Biomedicals) was added to each tube, and 500 μL of Glo lysis buffer - GLB (Promega, Madison WI) equilibrated to room temperature was added to the liver tissue. The liver tissue was homogenized at 2 x 6.0 m / s for 15 seconds using a FastPrep24 instrument (MP Biomedicals). The homogenate was incubated for 5 minutes at room temperature, then diluted 1:4 in GLB and evaluated using the SteadyGlo luciferase assay system (Promega). Specifically, 50 μL of the diluted tissue homogenate was reacted with 50 μL of the SteadyGlo substrate, shaken for 10 seconds, then incubated for 5 minutes, and then quantified using a SpectraMAX_L chemiluminescent microplate reader (Molecular Devices (Shanghai) Co., Ltd.). The amount of protein assayed was determined by using a BCA protein quantification kit (Shanghai Yise Medical Technology Co., Ltd.). The relative light units (RLU) were then normalized to the total μg of protein assayed. To convert RLU to μg of luciferase, a standard curve was generated using QuantiLum recombinant luciferase (Promega).

[0130] FLuc mRNA (L - 6107) from Trilink Biotechnologies will express the luciferase protein, which was originally isolated from the firefly (Photinus pyralis). Fluc is commonly used in mammalian cell cultures to measure gene expression and cell viability. It emits bioluminescence in the presence of the substrate luciferin. This capped and polyadenylated mRNA is completely substituted with 5 - methylcytidine and pseudouridine.

[0131] Example 11

[0132] Determination of the pKa of the formulated lipids

[0133] The pKa of the formulated cationic lipid is related to the efficacy of the LNP for nucleic acid delivery. The preferred pKa range is 5 - 7. The pKa of each cationic lipid is determined in lipid nanoparticles using an assay based on the fluorescence of 2-(p-toluidino)-6-naphthalene sulfonic acid (TNS). As described in Example 3, an ordered method is used to prepare lipid nanoparticles containing cationic lipid / DSPC / cholesterol / PEG lipid (50 / 10 / 38 / 2 mol%) at a total lipid concentration of 0.4 mM in PBS. TNS is prepared as a 100 μM stock solution in distilled water. The vesicles are diluted to contain 24 μM lipid in 2 mL of buffer solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl, where the pH ranges from 2.5 to 11. Equal aliquots of the TNS solution are added to give a final concentration of 1 μM, and after vortex mixing, the fluorescence intensity is measured in an SLM Aminco Series 2 luminescence spectrophotometer at excitation and emission wavelengths of 321 nm and 445 nm at room temperature. A sigmoidal best-fit analysis is applied to the fluorescence data, and the pKa is measured as the pH that gives half-maximal fluorescence intensity.

[0134] Example 12

[0135] The efficacy of lipid nanoparticle formulations containing various cationic lipids was determined using a rodent model of in vivo luciferase mRNA expression.

[0136] For comparison purposes, these lipids were also used to formulate lipid nanoparticles containing FLuc mRNA (L-6107) using an ordered mixing method as described in Example 10. Lipid nanoparticles were formulated using the following molar ratios: 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38% cholesterol / 2% PEG lipid ("PEG-DMG", i.e., (1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol, average PEG molecular weight of 2000). As described in Example 10, the relative activity was determined by measuring luciferase expression in the liver 5 hours after administration via tail vein injection. The activity was compared at doses of 0.3 and 1.0 mg mRNA / kg and expressed as ng luciferase / g liver measured 5 hours after administration as described in Example 10. The results of Examples 10 and 11 are shown in Table 2.

[0137] Table 2 Comparison of lipids showing activity with mRNA

[0138]

[0139]

[0140] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the various technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0141] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation to the scope of the disclosed patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of this invention patent shall be subject to the appended claims.

Claims

1. A cationic lipid compound, which is a compound having one of the following structures: Compound 1: Compound 2: Compound 5: Compound 6:

2. A composition comprising the compound according to claim 1 and a therapeutic agent and / or a prophylactic agent.

3. The composition according to claim 2, which further comprises one or more excipients selected from neutral lipids, steroids, and polymer-conjugated lipids.

4. The composition according to claim 3, wherein the neutral lipid is selected from one or more of the following mixtures: 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and sphingomyelin.

5. The composition according to claim 4, wherein the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine.

6. The composition according to claim 3, wherein the steroid is selected from one or more of the following mixtures: cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol.

7. The composition according to claim 6, wherein the steroid is cholesterol.

8. The composition according to claim 3, wherein the polymer-conjugated lipid is a polyethylene glycolated lipid.

9. The composition according to claim 8, wherein the polyethylene glycolated lipid is 1,2-dimyristoyl-sn-glycero-methoxypolyethylene glycol.

10. The composition according to any one of claims 2-9, wherein the therapeutic agent and / or the prophylactic agent is a vaccine or a compound capable of eliciting an immune response.

11. The composition according to claim 10, wherein the therapeutic agent and / or the prophylactic agent is a nucleic acid.

12. The composition according to claim 11, wherein the nucleic acid is selected from one or more of the following mixtures: siRNA, aiRNA, miRNA, dsRNA, shRNA, mRNA.

13. The composition according to claim 12, wherein the nucleic acid is mRNA.

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