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 instances, the lipid nanoparticles are used to deliver nucleic acids such as messenger RNA and / or antisense RNA. Also provided are methods of using such lipid nanoparticles to treat and / or prevent various diseases. In one embodiment, compounds having the structures of formula (I) and formula (II) are provided: or a salt or an isomer or an N-oxide thereof, wherein R 1 , R 2 and R 3 are as defined herein. Also provided are pharmaceutical compositions comprising a compound of one or more of the foregoing structural formulas and a therapeutic and / or prophylactic agent. In some embodiments, the pharmaceutical composition further comprises one or more components selected from neutral lipids, charged lipids, steroids and lipid-conjugated polymers. Such compositions are useful for forming lipid nanoparticles for delivering therapeutic and / or prophylactic agents. In other embodiments, the present invention provides a method of administering a therapeutic and / or prophylactic agent to a subject in need thereof, the method comprising preparing a pharmaceutical combination of a lipid nanoparticle comprising a compound of the foregoing structural formula and a therapeutic and / or prophylactic agent, and delivering the composition to the subject.

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 the lipids, the lipid nanoparticle compositions of the present invention may further include 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 cellular 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 include one or more "cationic" lipids, neutral lipids (such as phospholipids) including polyunsaturated lipids, structural lipids (such as steroids), and / or lipids containing polyethylene glycol (lipid-polymer conjugates). Cationic lipids include 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 prone to being digested by nucleases in plasma. Second, the ability of free RNA to enter intracellular compartments where the relevant translation machinery exists 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 with 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 are alkyl or hydrogen, but not both hydrogen.

[0011] In another aspect, the present invention relates to compounds of the following structural formula (II):

[0012]

[0013] or a salt thereof or an isomer thereof or an N-oxide thereof, wherein:

[0014] R 3 is methyl or hydrogen.

[0015] In various different embodiments, the compound has one of the structures shown in Table 1 below

[0016] Table 1 Representative Compounds

[0017]

[0018]

[0019] In some embodiments, there is provided a composition comprising any one or more of the compounds of the said structural formula and a therapeutic agent and / or a prophylactic agent.

[0020] 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. In some embodiments, the composition comprises any one of the compounds of structural formula (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.

[0021] In some embodiments, the neutral lipid is selected from one or more of 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), and sphingomyelin (SM). In some embodiments, the preferred neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0022] In some embodiments, the steroid is selected from one or more of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, and α-tocopherol. In some embodiments, the preferred steroid is cholesterol.

[0023] In some embodiments, the polyethylene glycolated lipid is 1,2-dimyristoyl-sn-glycero-methoxypolyethylene glycol (PEG-DMG)

[0024] In some embodiments, the composition ratios are in the following ranges: about 10 to 60 mol% of the compound, about 0 to 30 mol% of the neutral lipid, about 10 to 55 mol% of the steroid, and about 0 to 10 mol% of the polymer-conjugated lipid.

[0025] In some embodiments of the foregoing compositions, the therapeutic and / or prophylactic agent comprises a nucleic acid. 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.

[0026] In other different embodiments, the present invention relates to a method of using a therapeutic and / or prophylactic agent in a subject in need thereof, the method comprising preparing or providing any one of the above compositions and administering the composition to the subject.

[0027] For use purposes, the compounds of the present invention can be used as the active ingredient, or can be formulated into a pharmaceutical composition (usually in the form of a lipid nanoparticle combined with a therapeutic and / or prophylactic agent). The pharmaceutical composition of the present invention comprises a compound of structure (I) and one or more pharmaceutically acceptable carriers, diluents, or excipients. The compound of structure (I) is effective to form lipid nanoparticles and deliver the therapeutic and / or prophylactic agent. Those skilled in the art can easily determine the appropriate concentration and dosage.

[0028] The use of the compositions of the present invention can be carried out by any acceptable use mode of reagents for similar utilities. 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 using 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 make the active ingredients therein bioavailable in a subject. The form of the composition to be used for an object or patient can be one or more dosage forms, wherein the tablet can be a single-dose unit, and the container of the compound in the form of aerosol of the present invention can contain multiple dose units. The current methods for preparing these dosage forms are known or will be apparent to those skilled in the art. In any case, the composition to be used will contain a therapeutically effective amount of the compound of the present invention or its pharmaceutically acceptable salt so as to treat related diseases or conditions according to the teachings of the present invention.

[0029] The pharmaceutical compositions of the present invention can be in solid or liquid forms. On the one hand, the carrier can be granules such that the composition is in the form of a tablet or powder. The carrier can also be a liquid, in which case the composition is an oral syrup or an injectable liquid or an aerosol suitable for inhalation use.

[0030] When to be used for oral use, the pharmaceutical composition is preferably in solid or liquid forms, wherein the solid or liquid forms herein are considered to include semi-solids, semi-liquids, suspensions and gels.

[0031] As a solid composition for oral use, the pharmaceutical composition can be formulated into forms such as powders, granules, tablets, pills, capsules, chewing gums, wafers, etc. Such solid compositions generally will contain one or more inert diluents or edible carriers. Additionally, one or more of the following can 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 mint, etc.; and coloring agents.

[0032] When the pharmaceutical composition is in the form of a capsule, it can contain a liquid carrier in addition to the materials of the above types, such as polyethylene glycol or oil.

[0033] 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 use or for injection delivery. When intended for oral use, the preferred compositions contain one or more of a sweetening agent, a preservative, a coloring agent and a flavoring agent in addition to the compounds of the present invention. In the compositions for use 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.

[0034] The liquid pharmaceutical compositions of the present invention, whether in solution, suspension or other similar forms, can include one or more of the following excipients: 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, polyethylene glycol, glycerol, propylene glycol or other solvents that can be used as solvents or suspending media; 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. Parenteral preparations can be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic. Physiological saline is the preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.

[0035] The pharmaceutical compositions of the present invention can be used for topical use, in which case the carrier can suitably comprise a solution matrix, an emulsion matrix, an ointment matrix or a gel matrix. The matrix can comprise one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifying and stabilizing agents. Thickeners can be present in the pharmaceutical compositions for topical use. If intended for transdermal use, the composition can include a transdermal patch or an iontophoresis device.

[0036] The pharmaceutical compositions of the present invention can include various materials that modify the physical form of the solid or liquid dosage forms. The composition can include materials that form a coating shell around the active ingredient. The materials forming the coating shell are usually inert and can be sugars, shellac and other enteric coating reagents. Alternatively, the active ingredient can be encapsulated in a gelatin capsule.

[0037] The pharmaceutical compositions in solid or liquid form of the present invention can include carriers that bind to the compounds of the present invention and facilitate the delivery of the compounds. Such carriers include monoclonal or polyclonal antibodies or proteins.

[0038] The pharmaceutical composition of the present invention may consist of a formulation that can be used as an aerosol. The term "aerosol" refers to a system having colloidal properties and a system consisting of a pressurized package. It can be delivered by a liquefied gas or a compressed gas, or by a suitable pump system for dispersing the active ingredient. The aerosol of the compound of the present invention can be delivered in a single-phase, two-phase or three-phase system for delivering the active ingredient. The delivery of the aerosol includes the necessary container, activator, valve, sub-container, etc., which together can form a drug delivery device. A person skilled in the art can determine the preferred aerosol without additional experiments.

[0039] The pharmaceutical composition of the present invention can be prepared by methods well known in the pharmaceutical field. The pharmaceutical composition for 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 promote the formation of a uniform solution or suspension. The surfactant interacts non-covalently with the compound of the present invention, thereby promoting the dissolution or uniform suspension of the compound in a water-soluble medium.

[0040] The composition of the present invention or a pharmaceutically acceptable salt thereof is used in a therapeutically effective amount, and this amount will vary according to various 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 status, gender and diet of the subject; the mode and time of use; the excretion rate; drug combinations; the severity of the specific case, etc.

[0041] The composition of the present invention can also be used simultaneously with, before or after the use of one or more other therapeutic agents. Such therapeutic combinations include the use of the formulation of the composition of the present invention alone and the combination of the composition of the present invention and one or more other active ingredients. For example, the composition of the present invention and other active ingredients can be used together in a single oral dosage form (such as a tablet or a capsule) for the subject, or each active ingredient can be used in different oral dosage forms. When using different dosage forms, the compound of the present invention and one or more additional active ingredients can be used at the same time, or used sequentially at staggered times; it should be understood that combination therapy includes all these dosing regimens.

[0042] The structural modification and design of the above deuterated cationic lipid compound have achieved more advantageous physical and chemical properties, including a more suitable pKa and better chemical stability, for mRNA nanoliposome compositions, which can achieve more effective binding and delivery of ionic nucleic acid drugs. At the same time, its chemical structure is more stable, facilitating synthesis and being favorable for development as a pharmaceutical excipient.

[0043] The preparation methods of the above compounds and compositions are described below and / or are known in the art.

[0044] Those skilled in the art will recognize that in the methods described herein, the functional groups of the 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 hydroxyl, aryl or aralkyl esters. The protecting groups can be added or removed according to standard techniques, which are known to those skilled in the art and described herein.

[0045] 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.

[0046] In addition, all compounds of the present invention in the form of free bases or free acids 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.

[0047] The following examples are provided for the purpose of illustration only, not limitation.

[0048] In the following examples, unless otherwise indicated, all solvents and reagents used are commercially available and used as received.

[0049] The procedures described below can be used to synthesize related compounds.

[0050] The following abbreviations are used herein:

[0051] EDC.HCl: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0052] DCM: Dichloromethane

[0053] DMAP: 4-Dimethylaminopyridine

[0054] DIEA: N,N-Diisopropylethylamine

[0055] THF: Tetrahydrofuran

[0056] PCC: Pyridinium chlorochromate Detailed Description

[0057] Example 1:

[0058] Representative Route 1: Synthesis of Compound 1

[0059]

[0060] 1) Compound A

[0061]

[0062] Chemical formula: C 9 H 19 NO 2

[0063] Molecular weight: 173.26

[0064] 4-Bromomethyl-2,2,5-trimethyl-1,3-dioxolane (10.5 g, 0.05 mol) and a tetrahydrofuran solution of dimethylamine (2 M, 1250 mL) were successively added to a reaction flask. After stirring at room temperature for 2 days, it was concentrated under vacuum to obtain a crude product of Compound A (9.2 g).

[0065] 2) Compound B

[0066]

[0067] Chemical formula: C 6 H 15 NO 2

[0068] Molecular weight: 133.19

[0069] The crude product of Compound A (9.2 g) and water (10 mL) were added to a reaction flask. The temperature was controlled at 25 °C, and the pH of the system was adjusted to 4 with 5 M hydrochloric acid. After stirring for 3 hours, it was concentrated under vacuum. The residue was dissolved in DCM, sodium bicarbonate and anhydrous sodium sulfate were successively added. After filtration, it was concentrated under vacuum. The residue was purified by column chromatography to obtain Compound B (4.7 g, 0.03 mol).

[0070] 3) Compound 1

[0071]

[0072] Chemical formula: C 42 H 79 NO 2

[0073] Molecular weight: 630.10

[0074] Compound B (1.3 g, 0.01 mol), linoleyl methanesulfonate (6.9 g, 0.02 mol), tetrabutylammonium hydrogen sulfate (1.7 g, 5 mmol) and toluene (50 ml) were successively added to a reaction flask. 50 ml of 40% aqueous sodium hydroxide solution was added dropwise at 25 °C. The mixture was stirred at room temperature for 3 days, diluted with water, and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated in vacuo, and the residue was purified by column chromatography to obtain Compound 1 (2.5 g, 4 mmol).

[0075] C 42 H 79 NO 2 , Ms m / z: [M+H + 631; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 5.40 - 5.30 (4H, m), 5.30 - 5.00 (4H, m), 3.60 - 3.30 (6H, m), 2.76 (4H, m), 2.53 (2H, m), 2.31 - 2.07 (14H, m), 1.49 - 1.26 (36H, m), 1.10 (3H, s), 0.88 (6H, m).

[0076] Example 2:

[0077] Compound 2

[0078]

[0079] Chemical formula: C 42 H 79 NO 2

[0080] Molecular weight: 630.10

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

[0082] C 42 H 79 NO 2 , Ms m / z: [M+H + 631; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 5.50 - 5.35 (4H, m), 5.30 - 5.10 (4H, m), 3.60 - 3.30 (6H, m), 2.76 (4H, m), 2.38 - 2.05 (16H, m), 1.49 - 1.16 (39H, m), 0.88 (6H, m).

[0083] Example 3:

[0084] Compound 3

[0085]

[0086] Chemical formula: C 43 H 81 NO 2

[0087] Molecular weight: 644.13

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

[0089] C 43 H 81 NO 2 , Ms m / z: [M+H + 645; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 5.50 - 5.35 (4H, m), 5.30 - 5.10 (4H, m), 3.50 - 3.20 (6H, m), 2.80 (4H, m), 2.50 - 2.20 (8H, m), 2.18 - 2.01 (8H, m), 1.49 - 1.16 (38H, m), 0.88 (9H, m).

[0090] Example 4:

[0091] Representative Route 2: Synthesis of Compound 4

[0092]

[0093] 1) Compound C

[0094]

[0095] Chemical formula: C 37 H 68 O

[0096] Molecular weight: 528.95

[0097] Under nitrogen protection, magnesium chips (1.1 g, 46 mmol), THF (6 mL) and several iodine particles were added to the reaction flask. A solution of linolenyl bromide (14 g, 42.5 mmol) in THF (15 mL) was added dropwise to the system in batches. After the system was kept at 40 °C for 5 hours, the temperature was lowered to 0 °C, and then a THF solution of ethyl formate (3.3 g, 44.6 mmol) was added dropwise. The reaction was carried out at room temperature for 18 hours. The temperature was lowered to -10 °C, and 2N hydrochloric acid was added dropwise for acidification. Extraction was carried out with n-hexane, followed by washing with saturated brine and concentration. The residue was dissolved in ethanol (25 mL), a solution of potassium hydroxide (1.7 g) in water (5 mL) was added, and the mixture was stirred for 3 hours. Ethanol was removed by vacuum concentration, neutralized with 2N hydrochloric acid, extracted with n-hexane, vacuum concentrated, and the residue was purified by column chromatography to obtain compound C (8.7 g, 16.4 mmol).

[0098] 2) Compound D

[0099]

[0100] Chemical formula: C 37 H 66 O

[0101] Molecular weight: 526.93

[0102] Under nitrogen protection, PCC (8.0 g, 37.1 mmol) and sodium carbonate (0.8 g) were successively added to a solution of compound C (8.0 g, 15.1 mmol) in dichloromethane (100 mL). The mixture was stirred at room temperature for 3 hours, filtered, concentrated, and the residue was purified by column chromatography to obtain compound D (6.4 g, 12.1 mmol).

[0103] 3) Compound E

[0104]

[0105] Chemical formula: C 41 H 70 O 5

[0106] Molecular weight: 643.01

[0107] While controlling the temperature at 0 °C, compound D (6.0 g, 11.4 mmol) and boron trifluoride diethyl etherate (1.1 g, 7.7 mmol) were added to a solution of malic acid (762 mg, 5.7 mmol) in THF (15 mL) in batches. After stirring at room temperature for 2 days, the mixture was vacuum concentrated. The residue was dissolved in DCM, washed with saturated brine, dried over anhydrous sodium sulfate, vacuum concentrated, and purified by column chromatography to obtain compound E (1.8 g, 2.8 mmol).

[0108] 4) Compound F

[0109]

[0110] Chemical formula: C 41 H 72 O 4

[0111] Molecular weight: 629.02

[0112] Compound E (1.5 g, 2.3 mmol) and THF (10 mL) were successively added to the reaction flask. Sodium borohydride (105 mg, 2.8 mmol) was added in batches. The temperature was lowered to 0 °C. After injecting boron trifluoride diethyl etherate (392 mg, 2.8 mmol), the temperature was raised to 50 °C and the reaction was carried out for 5 hours. The reaction was quenched by adding an aqueous sodium bicarbonate solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated in vacuo, and the residue was purified by column chromatography to obtain compound F (1.2 g, 1.9 mmol).

[0113] 5) Compound G

[0114]

[0115] Chemical formula: C 42 H 74 O 6 S

[0116] Molecular weight: 707.11

[0117] Compound F (1.0 g, 1.6 mmol), DCM (10 mL), and triethylamine (240 mg, 2.4 mmol) were successively added to the reaction flask. The temperature was cooled to 0 °C, and a solution of methanesulfonyl chloride (230 mg, 2.0 mmol) in dichloromethane (5 mL) was added dropwise. The temperature was returned to room temperature and the reaction was carried out for 8 hours. The reaction system was diluted with DCM and washed successively with water, an aqueous sodium bicarbonate solution, and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated. The residue was purified by column chromatography to obtain compound G (1.0 g, 1.4 mmol).

[0118] 6) Compound 4

[0119]

[0120] Chemical formula: C 43 H 77 NO 3

[0121] Molecular weight: 656.09

[0122] Under nitrogen protection, compound G (1.0 g, 1.4 mmol) and a THF solution of dimethylamine (2 M, 35 mL) were successively added to the reaction flask. The mixture was stirred at room temperature for 5 days, concentrated in vacuo, and the residue was purified by column chromatography. Compound 4 (0.6 g, 0.9 mmol) was obtained

[0123] C 43 H 77 NO 3 ,Ms m / z:[M+H + 657; 1 H-NMR(300MHz,CDCl 3 )δ:ppm 5.50 - 5.10(8H, m), 4.00(1H, t), 2.80(4H, m), 2.60 - 2.50(2H, m), 2.40(6H, m) 2.20 - 2.00(8H, m), 1.83(2H, m), 1.63(4H, m), 1.49 - 1.16(36H, m), 0.88(6H, m).

[0124] Example 5:

[0125] Compound 5:

[0126]

[0127] Chemical formula: C 44 H 79 NO 3

[0128] Molecular weight: 670.12

[0129] Compound 5 can be synthesized according to the representative Route 2 described in Example 4.

[0130] C 44 H 79 NO 3 ,Ms m / z:[M+H + 671; 1 H-NMR(300MHz,CDCl 3 )δ:ppm 5.50 - 5.10(8H, m), 2.80(4H, m), 2.60 - 2.50(2H, m), 2.40 - 2.10(14H, m), 1.83(2H, m), 1.63(4H, m), 1.49 - 1.16(39H, m), 0.88(6H, m).

[0131] Example 6

[0132] In vivo evaluation of luciferase mRNA using lipid nanoparticle compositions

[0133] 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 (LNPs) at a total lipid to mRNA weight ratio of approximately 10:1 to 30:1. Briefly, dilute mRNA to 0.15 mg / mL in 10 mL to 50 mL of citrate buffer (pH = 4.0). Using a syringe pump, mix the ethanol solution of lipid with the aqueous solution of mRNA 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.

[0134] 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.

[0135] For the liver, approximately 50 mg was cut for analysis in a 2 mL FastPrep tube (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 × 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 luciferase, a standard curve was generated using QuantiL μM recombinant luciferase (Promega).

[0136] 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.

[0137] Example 7

[0138] The pKa of the formulated cationic lipid is related to the efficacy of the LNP used 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 6, 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, with a pH value ranging 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 at room temperature in an SLM Aminco Series 2 luminescence spectrophotometer using excitation and emission wavelengths of 321 nm and 445 nm. 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.

[0139] Example 8

[0140] The efficacy of lipid nanoparticle formulations containing various cationic lipids was determined using a rodent model of in vivo luciferase mRNA expression.

[0141] For comparison purposes, these lipids were also used to formulate lipid nanoparticles containing FLuc mRNA (L-6107) using the ordered mixing method as described in Example 6. 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 6, 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 6. The results of Examples 6 and 7 are shown in Table 2.

[0142] Table 2 Comparison of lipids showing activity with mRNA

[0143]

[0144]

[0145] 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-described 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 recorded in this specification.

[0146] The above-described embodiments only express several implementation manners of the present invention, and the description thereof is relatively specific and detailed. However, 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 the present 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 4: Compound 5:

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 a mixture of one or more of the following substances: 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 a mixture of one or more of the following substances: 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 a mixture of one or more of the following substances: siRNA, aiRNA, miRNA, dsRNA, shRNA, mRNA.

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

Images