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 analogues, and / or lipid-conjugated polymers to form lipid nanoparticles for delivering therapeutic and / or prophylactic agents. In some examples, the lipid nanoparticles are used to deliver 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 isomer or N-oxide thereof, wherein R 1 as defined herein There is also provided a pharmaceutical composition comprising a compound of one or more of the foregoing structural formula (I) 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 structural formula (I) 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 polymer-conjugated lipids) 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 compositions of the present invention may also include one or more cationic and / or ionizable amino lipids, neutral lipids including polyunsaturated lipids, polymer-conjugated lipids, steroids, and / or therapeutic and / or prophylactic agents in specific ratios. 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-based nanoparticle compositions, liposomes, and lipoplexes can be used effectively as delivery vehicles to transport 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 (polymer-conjugated lipids). 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 for improved cationic lipids and lipid nanoparticles for delivering oligonucleotides. Preferably, these lipid nanoparticles would provide an optimized drug:lipid ratio, 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 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 is not associated with 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 or isomer or N-oxide thereof, wherein:

[0010] R 1 is C 7 or C 8 alkyl.

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

[0012] Table 1 Representative Compounds

[0013]

[0014] 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.

[0015] 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.

[0016] 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 neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0017] In some embodiments, the steroid is selected from cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, α-tocopherol, and mixtures thereof. In some embodiments, the steroid is cholesterol.

[0018] In some embodiments, the polyethylene glycolylated lipid is 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol (PEG-DMG)

[0019] 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 neutral lipid, about 10 to 55 mol% of steroid, and about 0 to 10 mol% of polymer-conjugated lipid.

[0020] 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.

[0021] 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.

[0022] 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 a crude chemical, or can be formulated into a pharmaceutical composition. 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 readily determine the appropriate concentrations and dosages.

[0023] The administration of the composition of the present invention can be carried out by any acceptable mode of administration for a reagent of similar utility. The pharmaceutical composition 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 composition of the present invention is 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 a subject 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 the compound in the form of an aerosol of the present invention can contain multiple dosage units. 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 administered will contain a therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof to treat a related disease or condition in accordance with the teachings of the present invention.

[0024] The pharmaceutical composition of the present invention can be in solid or liquid form. In one aspect, the carrier is a microparticle such that the composition is in the form of a tablet or powder. The carrier can be a liquid, in which case the composition is an oral syrup or an injectable liquid or an aerosol suitable for inhalational administration.

[0025] When intended for oral administration, the pharmaceutical composition is preferably in solid or liquid form, where forms considered to be solid or liquid herein include semi-solid, semi-liquid, suspension and gel forms.

[0026] 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.

[0027] 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.

[0028] 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 injectable delivery. When intended for oral administration, the preferred composition contains, in addition to the compound of the present invention, one or more of sweeteners, preservatives, coloring agents and flavor enhancers. In the composition for administration by injection, one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizers and isotonic agents can be included.

[0029] The liquid pharmaceutical composition of the present invention, whether it is a solution, suspension or other similar form, 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, polyethylene glycol, glycerol, propylene glycol or other solvents that can be used as solvents or suspension 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. 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.

[0030] The liquid pharmaceutical composition of the present invention intended for parenteral or oral administration should contain an amount of the compound of the present invention that can obtain a suitable dose.

[0031] The pharmaceutical composition of the present invention can be intended for topical administration, in which case the carrier can suitably contain a solution matrix, an emulsion matrix, an ointment matrix or a gel matrix. The matrix can contain one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifying agents and stabilizers. Thickeners can be present in the pharmaceutical composition for topical administration. If intended for transdermal administration, the composition can include a transdermal patch or an iontophoresis device.

[0032] The pharmaceutical composition of the present invention can be intended for rectal administration, in the form of a suppository, which dissolves in the rectum and releases the drug. The composition for rectal administration can contain an oily matrix as a suitable non-irritating excipient. Such matrices include but are not limited to lanolin, cocoa butter and polyethylene glycol.

[0033] The pharmaceutical composition of the present invention may include various materials that modify the physical form of solid or liquid dosage units. The composition may include materials that form a coating shell around the active ingredient. The materials forming the coating shell are generally inert and may be sugars, shellac, and other enteric coating reagents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule.

[0034] The pharmaceutical composition of the present invention in solid or liquid form may include reagents that bind to the compounds of the present invention and thus assist in the delivery of the compounds. Suitable reagents that can act in this capacity include monoclonal or polyclonal antibodies or proteins.

[0035] The pharmaceutical composition of the present invention may consist of dosage units that can be administered as an aerosol. The term aerosol is used to denote a variety of 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 compounds 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.

[0036] The pharmaceutical composition of the present invention can be prepared by methods well known in the pharmaceutical art. The pharmaceutical composition intended to be administered 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. Surfactants can be added to facilitate the formation of a uniform solution or suspension. A surfactant is a compound that interacts non-covalently with the compounds of the present invention in order to facilitate the dissolution or uniform suspension of the compounds in an aqueous delivery system.

[0037] The composition of the present invention or its pharmaceutically acceptable salts are 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, gender and diet of the subject; the mode and time of administration; the excretion rate; drug combinations; the severity of the specific case, etc.

[0038] The compositions 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 respective separate pharmaceutical dosage formulations. For example, the composition of the present invention and the other active agents can be administered to a subject together in a single oral dosage composition (such as a tablet or capsule), or each agent can be administered in separate oral dosage formulations. 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 is understood that combination therapies include all such dosing regimens.

[0039] Methods for preparing the above compounds and compositions are described below and / or are known in the art.

[0040] 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 alkyl, aryl, or aralkyl esters. The protecting groups can be added or removed according to standard techniques that are known to those skilled in the art and described herein.

[0041] Those skilled in the art will also recognize that although such protected derivatives of the compounds of the present invention may not thereby have pharmaceutical activity, they can be administered to a mammal and then metabolized in vivo to form the pharmaceutically active compounds of the present invention. Such derivatives can therefore be described as "prodrugs". Accordingly, prodrugs of the compounds of the present invention are included within the scope of the present invention.

[0042] In addition, all compounds of the present invention in the form of the free base or free acid can be converted to 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 to their free base or acid forms by standard techniques.

[0043] The following examples are provided for purposes of illustration and not limitation.

[0044] In the following examples, unless otherwise indicated, all solvents and reagents used are commercially available and used as received.

[0045] The procedures described below can be used to synthesize Compounds A and B.

[0046] The following abbreviations are used herein:

[0047] EDC.HCL: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0048] DCM: Dichloromethane

[0049] DMAP: 4-Dimethylaminopyridine

[0050] DIEA: N,N-Diisopropylethylamine Detailed implementation mode

[0051] Example 1:

[0052] Representative route

[0053] Synthesis of Compound A: 2-Octyldecyl 8-((2-hydroxyethyl)(6-(undecyloxy)oxohexyl)amino)octanoate

[0054]

[0055] 1) Synthesis of Compound A-1

[0056]

[0057] Chemical formula: C 26 H 51 BrO 2

[0058] Molecular weight: 475.60

[0059] EDC.HCl (1.1 g, 5.8 mmol), DIEA (3.3 ml, 18.7 mmol), and DMAP (114 mg, 0.9 mmol) were successively added to a DCM mixture of 8-bromooctanoic acid (1.0 g, 4.5 mmol) and 2-octyldecanol (1.6 g, 5.8 mmol). The reaction was carried out at room temperature for 24 h, and then the system was diluted with DCM. It was successively washed with saturated aqueous sodium bicarbonate and dilute aqueous hydrochloric acid, dried over magnesium sulfate, filtered and concentrated. The obtained residue was purified by a silica gel column (0 - 15% ethyl acetate / hexane). Compound A-1 (951 mg, 2.0 mmol, 44%) was obtained.

[0060] 2) Synthesis of Compound A-2

[0061]

[0062] Chemical formula: C 28 H 57 NO 3

[0063] Molecular weight: 455.77

[0064] Compound A-1 (2.9 g, 6.0 mmol) was mixed with 3 mL of ethanol, 2-aminoethanol (15 mL, 248 mmol) was added, and the mixture was heated to reflux for 2 hours. The reaction system was concentrated under vacuum. Ethyl acetate and water were added to the residue, and the organic phase was separated and dried over anhydrous sodium sulfate. After concentration under vacuum, the residue was purified by silica gel column (0 - 100% (mixture of 1% NH 4 OH, 20% MeOH in dichloromethane) dichloromethane). Compound A-2 (2.4 g, 5.3 mmol, 89%) was obtained.

[0065] 3) Synthesis of Compound A-3

[0066]

[0067] Chemical formula: C 17 H 33 BrO 2

[0068] Molecular weight: 349.35

[0069] Compound A-3 can be prepared by referring to the synthesis method of A-1.

[0070] 4) Synthesis of Compound A

[0071]

[0072] Chemical formula: C 45 H 89 NO 5

[0073] Molecular weight: 724.21

[0074] DIEA (414 mg, 3.2 mmol) was added successively to the ethanol mixture of compound A-2 (1.0 g, 2.2 mmol) and A-3 (1.1 g, 3.2 mmol), and the temperature was raised to 65 °C and stirred for 24 h. Then the temperature was lowered and the solvent was concentrated under reduced pressure. The residue was dissolved in ethyl acetate, and the organic phase was washed with aqueous sodium bicarbonate solution. After drying over anhydrous sodium sulfate, it was concentrated under vacuum. The residue was purified by silica gel column (0 - 100% (mixture of 1% NH 4 OH, 20% MeOH in dichloromethane) dichloromethane) to obtain compound A (797 mg, 1.1 mmol, 50%).

[0075] C 45 H 89 NO 5 , Ms m / z: [M + H + 724.7; 1 H-NMR (300 MHz, CDCl 3) δ: ppm 4.10 (2H, t), 3.97 (2H, d), 3.67 - 3.47 (m, 2H), 2.76 - 2.36 (m, 5H), 2.28 (m, 4H), 1.88 - 1.41 (m, 15H), 1.38 - 1.17 (50H, m), 0.87 (9H, m).

[0076] Example 2:

[0077] Compound B: Synthesis of 2 - heptyldecyl 8 - ((2 - hydroxyethyl)(6 - (undecyloxy) - oxohexyl)amino)octanoate

[0078]

[0079] Chemical formula: C 44 H 87 NO 5

[0080] Molecular weight: 710.18

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

[0082] C 44 H 87 NO 5 , Ms m / z: [M + H + 711.0; 1 H - NMR (300 MHz, CDCl 3 ) δ: ppm 4.11 (2H, t), 3.98 (2H, d), 3.68 - 3.47 (m, 2H), 2.76 - 2.35 (m, 5H), 2.27 (m, 4H), 1.88 - 1.40 (m, 15H), 1.37 - 1.17 (48H, m), 0.88 (9H, m).

[0083] Example 3

[0084] In vivo evaluation of luciferase mRNA using lipid nanoparticle compositions

[0085] Dissolve cationic lipid, DSPC, cholesterol and PEG-lipid in ethanol at a molar ratio of 50:10:37:2 or 48:10:42:2. 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). 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 15 mL / min. Then remove 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 um. 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.

[0086] Conduct studies on female mice at 6 - 8 weeks of age and 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 freeze rapidly in liquid nitrogen, and store at -80 °C until used for analysis.

[0087] 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 Centro XS3LB 960 photometer (Berthold Technologies, Germany). The amount of protein assayed was determined by using a BCA protein assay kit (Pierce, Rockford IL). The relative light units (RLU) were then normalized to the total μg of protein assayed. To convert RLU to ng of luciferase, a standard curve was generated using QuantiLum recombinant luciferase (Promega).

[0088] The 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.

[0089] Example 4

[0090] Determination of the pKa of the formulated lipid

[0091] 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 3, an ordered method is used to prepare lipid nanoparticles containing cationic lipid / DSPC / cholesterol / PEG lipid (50 / 10 / 40 / 1.5 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 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 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 produces half-maximal fluorescence intensity.

[0092] Example 5

[0093] The efficacy of lipid nanoparticle formulations containing various cationic lipids was determined using a rodent model of in vivo luciferase mRNA expression.

[0094] The cationic lipids shown in Table 2 were previously tested with nucleic acids. For comparison purposes, these lipids were also used to formulate lipid nanoparticles containing FLuc mRNA (L-6107) using an in-line mixing method as described in Example 3. Lipid nanoparticles were formulated using the following molar ratios: 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 40% cholesterol / 1.5% PEG lipid ("PEG-DMG", i.e., (1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol, average PEG molecular weight of 2000). As described in Example 1, 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 3.

[0095] Table 2 Comparison of lipids showing activity with mRNA

[0096]

[0097] 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 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 falling within the scope described in this specification.

[0098] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof 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 A: Compound B:

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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