WO2023029593A1

WO2023029593A1 - Novel cationic lipid compound

Info

Publication number: WO2023029593A1
Application number: PCT/CN2022/093432
Authority: WO
Inventors: 黄才古; 谭俊荣; 黄铁强
Original assignee: 广州谷森制药有限公司
Priority date: 2021-09-03
Filing date: 2022-05-18
Publication date: 2023-03-09
Also published as: CN115745819A

Abstract

The present invention relates to a lipid compound, which can be used alone or in combination with other lipid components such as a neutral lipid, a charged lipid, a steroid and/or an analog thereof, and/or a polymer-conjugated lipid for forming a lipid nanoparticle for the delivery of a therapeutic and/or prophylactic agent. In some examples, the lipid nanoparticle is used to deliver a nucleic acid, such as a messenger RNA and/or antisense RNA. Further provided is a method for treating and/or preventing various diseases by means of using such a lipid nanoparticle. In one embodiment, provided is a compound having a structure as represented by following formula (I), or a salt, isomer or N-oxide thereof, wherein R is as defined herein, and further provided is a pharmaceutical composition comprising one or more of the above-mentioned compounds as represented by structural formula (I) and the therapeutic and/or prophylactic agent. In some embodiments, the pharmaceutical composition further comprises one or more components selected from a neutral lipid, a charged lipid, a steroid and a polymer-conjugated lipid. Such a composition is useful for forming a lipid nanoparticle for delivering the therapeutic and/or prophylactic agent. In the other embodiments, provided in the present invention is a method for administering the therapeutic and/or prophylactic agent to a subject in need thereof, and the method comprises preparing the pharmaceutical composition of lipid nanoparticles containing the compound as shown in the structural formula (I) and the therapeutic and/or prophylactic agent, and delivering the composition to the subject.

Description

Novel Cationic Lipid Compounds

technical field

The present invention provides novel cationic lipids that 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 combining a A method of delivering one or more therapeutic and/or prophylactic agents to a mammalian cell or organ and/or producing a polypeptide in a mammalian cell or organ. In addition to the novel lipids, the lipid nanoparticle compositions of the invention may also include, in specified proportions, one or more cationic and/or ionizable amino lipids, neutral lipids including polyunsaturated lipids, Lipids, polymer-conjugated lipids, steroids, and/or therapeutic and/or prophylactic agents.

Background technique

Efficient targeted delivery of bioactive substances such as small molecule drugs, proteins and nucleic acids presents a persistent medical problem. Specifically, delivery of nucleic acids to cells is made difficult by the relative instability and low cell permeability of these species. Accordingly, 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.

Studies have proved that using lipid-containing nanoparticle compositions, liposomes and liposome complexes as transport media can effectively deliver biologically active substances such as small molecule drugs, proteins and nucleic acids to cells and/or intracellularly in the compartment. These compositions generally comprise one or more "cationic" lipids, neutral lipids including polyunsaturated lipids (such as phospholipids), structured lipids (such as steroids) and/or polyethylene glycol-containing Lipids of alcohols (polymer-conjugated lipids). Cationic lipids include, for example, amine-containing lipids that can be readily protonated.

However, the use of oligonucleotides in a therapeutic setting currently faces two problems. First, free RNA is readily digested by nucleases in plasma. Second, cell-free RNA has limited access to intracellular compartments where associated translation machinery exists. Lipid nanoparticles formed from cationic lipids with other lipid components such as neutral lipids, cholesterol, PEG, PEGylated lipids, and oligonucleotides have been used to prevent RNA degradation in plasma and promote Cellular uptake of oligonucleotides.

There remains a need for improved cationic lipids and lipid nanoparticles for the delivery of 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. In addition, these preferred lipid-nucleic acid particles should be well tolerated and provide a sufficient therapeutic index such that treatment of the patient at effective doses of the nucleic acid does not create unacceptable toxicity and/or risk to the patient. The present invention provides these and related advantages.

Contents of the invention

The present invention provides the following novel compounds and methods involving these compounds:

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

or its salt or its isomer or its N-oxide, wherein:

R can be alkyl, alkenyl or hydroxyalkyl

In various embodiments, the compound has one of the structures shown in Table 1 below

Table 1 Representative compounds

In some embodiments, there is provided a composition comprising any one or more of the compounds of the above formulas and a therapeutic and/or prophylactic agent.

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

In some embodiments, the neutral lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine Alkaline (DPPC), 1,2-Dimyristyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palm Acyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM) or Various. In some embodiments, the preferred neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

In some embodiments, the steroid is selected from one of cholesterol, fecal sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, α-tocopherol one or more species. In some embodiments, the preferred steroid is cholesterol.

In some embodiments, the pegylated lipid is 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG)

In some embodiments, the composition ratio ranges from about 10 to 60 mol% of the compound, about 0 to 30 mol% of neutral lipids, about 10 to 55 mol% of steroids, and about 0 to 10 mol% of polymer-conjugated lipids. quality.

In embodiments of some 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 following composition: siRNA, aiRNA, miRNA, dsRNA, shRNA, mRNA and mixtures thereof. In some embodiments, the RNA is selected from mRNA.

In other various embodiments, the present invention relates to methods of administering therapeutic and/or prophylactic agents to a subject in need thereof, the method comprising preparing or providing any of the compositions described above and administering to the subject combination.

For purposes of use, the compounds of the present invention may be used as drug substances, or may be formulated as pharmaceutical compositions (usually in the form of lipid nanoparticles in combination with therapeutic and/or prophylactic agents). The pharmaceutical composition of the present invention comprises a compound of structure (I) and one or more pharmaceutically acceptable carriers, diluents or excipients. Compounds of structure (I) are effective to form lipid nanoparticles and deliver therapeutic and/or prophylactic agents. Appropriate concentrations and dosages can be readily determined by those skilled in the art.

The use of the compositions of the present invention may be by any of the acceptable modes of use for agents of similar utility. The pharmaceutical composition of the present invention can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microgels, etc. Balls and Aerosols. Typical routes of use 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 injections, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques. The pharmaceutical compositions of the present invention are formulated so that the active ingredients therein are bioavailable in the subject. The composition to be administered to a subject or patient may be in one or more dosage forms where a tablet may be a single dosage unit and a container of the compound of the invention in aerosol form may hold multiple dosage units. Current methods for the preparation of such 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 a compound of the invention, or a pharmaceutically acceptable salt thereof, for treating the associated disease or condition in accordance with the teachings of the invention.

The pharmaceutical compositions of the present invention may be in solid or liquid form. In one aspect, the carrier can be particulate, such that the composition is in tablet or powder form. 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.

When intended for oral use, the pharmaceutical composition is preferably in solid or liquid form, where solid or liquid forms are considered herein to include semi-solids, semi-liquids, suspensions and gels.

As a solid composition for oral use, the pharmaceutical composition can be formulated in the form of powder, granule, tablet, pill, capsule, chewing gum, flake and the like. Such solid compositions will generally contain one or more inert diluents or edible carriers. In addition, there may be one or more of the following: 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; and coloring agents.

When the pharmaceutical composition is in capsule form, it may contain liquid carriers other than materials of the above type, such as polyethylene glycol or oil.

Pharmaceutical compositions may be in liquid form, such as syrups, solutions, emulsions or suspensions. Liquids may be for oral use or for injectable delivery, as two examples. When intended for oral use, preferred compositions contain, in addition to the compounds of the present invention, one or more of sweetening agents, preservatives, dyeing/coloring agents and flavor enhancers. In compositions for use by injection, one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers and isotonic agents may be included.

The liquid pharmaceutical composition of the present invention, no matter it is a solution, a suspension or other similar forms, may include one or more of the following auxiliary materials: sterile diluents, such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride; fixed oils such as synthetic mono- or diglycerides, polyethylene glycol, glycerol, propylene glycol, or other solvents that can be used as a solvent or suspending medium; antibacterial antioxidants, such as methylparaben, etc.; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetate, citrate, or phosphate; Reagents for tonicity, such as sodium chloride or glucose; reagents used as cryoprotectants, such as sucrose or trehalose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is the preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.

The pharmaceutical composition of the present invention may be intended for topical use, in which case the carrier may suitably comprise a solution base, emulsion base, ointment base or gel base. The base may contain one or more of petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in pharmaceutical compositions for topical use. If intended for transdermal use, the composition may include a transdermal patch or iontophoretic device.

The pharmaceutical compositions of the invention can include various materials which modify the physical form of solid or liquid dosage forms. The composition may include materials which form a coating shell around the active ingredient. The materials forming the coating shell are generally inert and can be sugar, shellac and other enteric coating agents. Alternatively the active ingredient may be enclosed in a gelatin capsule.

Pharmaceutical compositions of the invention in solid or liquid form may include vehicles which facilitate delivery of the compound in combination with the compound of the invention. Such vectors include monoclonal or polyclonal antibodies or proteins.

The pharmaceutical compositions of the invention may consist of formulations available as aerosols. The term aerosol denotes systems comprising colloidal properties and systems consisting of pressurized packs. Delivery may be by liquefied or compressed gas, or by a suitable pump system which dispenses the active ingredient. Aerosols of the compounds of the invention may be delivered as single-phase, bi-phasic or triphasic systems to deliver the active ingredient. Aerosol delivery includes the necessary containers, activators, valves, sub-containers, etc., which together may form a drug delivery device. Preferred aerosols can be determined by those skilled in the art without undue experimentation.

The pharmaceutical compositions of the present invention can be prepared by methods well known in the field of pharmacy. The pharmaceutical composition for injection can be prepared by combining the lipid nanoparticles of the present invention with sterile distilled water or other carriers into a solution. Surfactants can be added to promote the formation of a uniform solution or suspension. Surfactants interact non-covalently with the compounds of the invention thereby facilitating dissolution or homogeneous suspension of said compounds in aqueous media.

The compositions of the present invention, or pharmaceutically acceptable salts thereof, are used in therapeutically effective amounts, which will vary depending on a number of factors, including the activity of the particular therapeutic agent used; the metabolic stability and duration of action of the therapeutic agent; The patient's age, weight, general health, sex, and diet; mode and duration of use; rate of excretion; drug combination; severity of the specific case, etc.

Compositions of the invention may also be administered concurrently with, prior to, or subsequent to administration of one or more other therapeutic agents. Such therapeutic combinations include formulations using the compositions of the invention alone as well as combinations using the compositions of the invention and one or more other active ingredients. For example, the compositions of the present invention and other active ingredients may be administered to a subject together in a single oral dosage formulation such as a tablet or capsule, or each active ingredient may be administered in separate oral dosage formulations. When different dosage formulations are used, the compound of the invention and one or more additional active ingredients may be administered at the same time, or sequentially at times staggered from each other; it is understood that combination therapy includes all such dosing regimens.

The structural modification and design of the above-mentioned novel deuterated cationic lipid compounds have achieved more advantageous physical and chemical properties, including more suitable pKa and better chemical stability, and are used in mRNA nanoliposome compositions, which can realize anti-ion Nucleic acid-like drugs are more effectively combined and delivered, and at the same time, their chemical structure is more stable, which is convenient for synthesis and beneficial for development as pharmaceutical excipients.

Methods for the preparation of the above compounds and compositions are described below, and/or are known in the art.

Those skilled in the art will recognize that in the methods described herein, 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 hydroxy include trialkylsilyl or diarylalkylsilyl, tetrahydrofuranyl, benzyl, and the like. Suitable protecting groups for amino include t-butoxycarbonyl, benzyloxycarbonyl and the like. Suitable protecting groups for carboxylic acids include hydroxy, aryl or aryl esters. Protecting groups can be added or removed according to standard techniques, known to those skilled in the art and described herein.

Those skilled in the art will also recognize that while such protected derivatives of compounds of the invention may not thereby be pharmaceutically active, they may be administered to a mammal and then metabolized in vivo to form pharmacologically active compounds of the invention. Such derivatives can thus be described as "prodrugs". Prodrugs of the compounds of the present invention are therefore included within the scope of the present invention.

In addition, all compounds of the present invention that exist in free base or free acid form can be converted to their pharmaceutically acceptable salts by treatment with an appropriate inorganic or organic base or acid according to methods known to those skilled in the art. Salts of compounds of the present invention may be converted to their free base or acid forms by standard techniques.

The following examples are offered for purposes of illustration and not limitation.

In the following examples, unless otherwise indicated, all solvents and reagents used were obtained commercially and used as received.

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

This article uses the following abbreviations:

EDC.HCl: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride

DCM: dichloromethane

DMAP: 4-Dimethylaminopyridine

DIEA: N, N-Diisopropylethylamine

THF: Tetrahydrofuran

Detailed ways

Example 1:

representative route

Synthesis of compound 5

1) Synthesis of Compound A

Chemical formula: C ₃₇ H ₆₈ O

Molecular weight: 528.95

Under the protection of nitrogen, magnesium chips (1.1g, 46mmol) were added to THF (6mL), and a solution of bromolinolene (14g, 42.5mmol) in THF (15mL) was added dropwise, and the temperature was controlled at 45°C for 4 hours. Cool down to 0°C, add a THF solution of ethyl formate (3.3g, 44.6mmol) dropwise under temperature control, return to room temperature and stir for 18 hours, cool down to -10°C and slowly add dilute hydrochloric acid and water, extract with n-hexane, and the organic phase Concentrate in vacuo. Add ethanol (25mL), water (5mL) and potassium hydroxide (1.7g) in sequence, stir at room temperature for 2 hours, concentrate in vacuo to remove ethanol, add hydrochloric acid to acidify, extract with n-hexane, wash the organic phase with water, and dry with anhydrous sodium sulfate , concentrated, and column purified (ethyl acetate/n-hexane) to obtain compound A (8.7 g, 16.4 mmol).

2) Synthesis of Compound B

Chemical formula: C ₄₁ H ₇₃ BrO ₂

Molecular weight: 677.94

Add EDC.HCl (3.1g, 16.2mmol), DMAP (0.4g, 3.3 mmol) and DIEA (8.3 g, 64.2 mmol) were reacted at room temperature for 24 hours. It was washed successively with saturated aqueous sodium bicarbonate solution and dilute aqueous hydrochloric acid solution, the organic phase was separated, dried over anhydrous sodium sulfate, concentrated in vacuo, and the residue was purified by silica gel column (ethyl acetate/n-hexane). Compound B (7.0 g, 10.3 mmol) was obtained.

3) Synthesis of compound C

Chemical formula: C ₄₃ H ₇₉ NO ₃

Molecular weight: 658.11

Compound B (5.0 g, 7.6 mmol) was mixed with ethanol (5 mL), 2-aminoethanol (13.8 g, 230.0 mmol) was added, and the mixture was reacted at 60° C. for 32 hours. The system was concentrated in vacuo, ethyl acetate and water were added to the residue, the organic phase was separated and dried over anhydrous sodium sulfate, concentrated in vacuo, and the residue was purified by silica gel column (ammonia/methanol/DCM). Compound C (2.9 g, 4.4 mmol) was obtained.

4) Synthesis of Compound 5

Chemical formula: C ₆₂ H ₁₁₃ NO ₃

Molecular weight: 920.59

Compound C (2.0g, 3.0mmol), bromolinolene (1.2g, 3.5mmol) and DIEA (0.5g, 3.8mmol) were sequentially added to ethanol (5mL), and the temperature was raised to 60°C for 24 hours. The system was concentrated in vacuo, ethyl acetate and water were added to the residue, the organic phase was separated and dried over anhydrous sodium sulfate, concentrated in vacuo, and the residue was purified by silica gel column (ammonia/methanol/DCM). Compound 5 (1.3 g, 1.4 mmol) was obtained.

C ₆₂ H ₁₁₃ NO ₃ , Ms m/z: [M+H ⁺ ]921; ¹ H-NMR (300MHz, CDCl ₃ ) δ: ppm 5.50-5.10 (12H, m), 4.47 (1H, m), 3.43 (2H,t), 3.00(2H,t), 2.80(6H,m), 2.60-2.30(6H,m), 2.10(12H,m), 1.84(2H,m)1.64～1.05(60H,m) , 0.89 (9H, m).

Example 2:

Compound 1:

Molecular weight: 672.14

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

C ₄₄ H ₈₁ NO ₃ , Ms m/z: [M+H ⁺ ]673; ¹ H-NMR (300MHz, CDCl ₃ ) δ: ppm 5.50-5.10(8H,m), 4.47(1H,m), 3.43 (2H, t), 2.80 (4H, m), 2.60-2.30 (6H, m), 2.15-2.00 (11H, m), 1.84 (2H, m), 1.64～1.05 (40H, m), 0.89 (6H, m).

Example 3:

Compound 2:

Chemical formula: C ₄₅ H ₈₃ NO ₃

Molecular weight: 686.16

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

C ₄₅ H ₈₃ NO ₃ , Ms m/z: [M+H ⁺ ]687; ¹ H-NMR (300MHz, CDCl ₃ ) δ: ppm 5.50-5.10(8H,m), 4.47(1H,m), 3.43 (2H, t), 3.00 (2H, m), 2.80 (4H, m), 2.60-2.30 (6H, m), 2.15-2.00 (8H, m), 1.84 (2H, m) 1.64 ~ 1.05 (43H, m), 0.89 (6H, m).

Example 4:

Compound 3:

Chemical formula: C ₄₆ H ₈₅ NO ₃

Molecular weight: 700.19

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

C ₄₆ H ₈₅ NO ₃ , Ms m/z: [M+H ⁺ ]701; ¹ H-NMR (300MHz, CDCl ₃ ) δ: ppm 5.50-5.10 (8H, m), 4.47 (1H, m), 3.43 (2H, t), 2.80 (4H, m), 2.60-2.30 (8H, m), 2.15-2.00 (8H, m), 1.84 (2H, m), 1.64～1.05 (42H, m), 0.89 (9H, m).

Example 5:

Compound 4:

Chemical formula: C ₆₂ H ₁₁₇ NO ₃

Molecular weight: 924.62

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

C ₆₂ H ₁₁₇ NO ₃ , Ms m/z: [M+H ⁺ ]925; ¹ H-NMR (300MHz, CDCl ₃ ) δ: ppm 5.50-5.10 (8H, m), 4.47 (1H, m), 3.43 (2H, t), 3.00 (2H, t), 2.80 (4H, m), 2.60-2.30 (6H, m), 2.15-2.00 (8H, m), 1.84 (2H, m) 1.64～1.05 (74H, m), 0.89(9H, m).

Embodiment 6:

Compound 6:

Chemical formula: C ₆₂ H ₁₁₇ NO ₄

Molecular weight: 940.62

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

C ₆₂ H ₁₁₇ NO ₄ , Ms m/z: [M+H ⁺ ]941; ¹ H-NMR (300MHz, CDCl ₃ ) δ: ppm 5.50-5.10(8H,m), 4.47(1H,m), 3.43 (3H, m), 2.80 (4H, m), 2.60-2.30 (8H, m), 2.15-2.00 (8H, m), 1.84 (2H, m), 1.64～1.05 (72H, m), 0.89 (9H, m).

Embodiment 7:

Compound 7:

Chemical formula: C ₄₅ H ₈₃ NO ₄

Molecular weight: 702.16

Compound 7 can be synthesized according to the representative route described in Example 1.

C ₄₅ H ₈₃ NO ₄ , Ms m/z: [M+H ⁺ ] 703; ¹ H-NMR (300MHz, CDCl ₃ ) δ: ppm 5.50-5.10(8H,m), 4.47(1H,m), 3.43 (4H, m), 2.80 (4H, m), 2.60-2.30 (8H, m), 2.15-2.00 (8H, m), 1.84 (2H, m), 1.64～1.05 (40H, m), 0.89 (6H, m).

Example 8

In vivo evaluation of luciferase mRNA using a lipid nanoparticle composition

Cationic lipids, DSPC, cholesterol and PEG-lipids were dissolved in ethanol at a molar ratio of 50:10:38:2 or 48:10:40:2. Lipid nanoparticles (LNPs) were prepared at a weight ratio of total lipid to mRNA of about 10:1 to 30:1. Briefly, mRNA was diluted to 0.15 mg/mL in 10 ml to 50 ml citrate buffer (pH=4). Using a syringe pump, mix the ethanolic lipid solution with the aqueous mRNA solution at a ratio of about 1:5 to 1:3 (vol/vol) at a total flow rate of 10 ml/min or more. Ethanol was then removed and the external buffer was replaced by 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, as 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.

The study was performed on 6-8 week old female C57BL/6 mice, 8-10 week old CD-1 mice according to the guidelines established by the National Science and Technology Commission. Different doses of mRNA lipid nanoparticles were administered systemically via tail vein injection, and animals were euthanized at specific time points (eg, 5 hours) after administration. Livers and spleens were collected in pre-weighed tubes, weight determined, immediately snap frozen in liquid nitrogen, and stored at -80°C until analysis.

For liver, approximately 50 mg was cut for analysis in 2 mL FastPrep tubes (MP Biomedicals, Solon OH). 1/4" ceramic balls (MP Biomedicals) were 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. Using the FastPrep24 instrument (MP Biomedicals), the Liver tissue was homogenized at 2x6.0m/s for 15 s. The homogenate was incubated at room temperature for 5 min, then diluted 1:4 in GLB and evaluated using the SteadyGlo luciferase assay system (Promega). Specifically , 50uL of diluted tissue homogenate was reacted with 50μL of SteadyGlo substrate, shaken for 10 seconds, then incubated for 5 minutes, and then quantified using a SpectraMAX_L chemiluminescent microplate reader (Megu Molecular Instruments (Shanghai) Co., Ltd.). Use the BCA protein quantification kit (Shanghai Yise Medical Technology Co., Ltd.) to determine the amount of assayed protein. The relative luminescence units (RLU) are then normalized to the total μg of assayed protein. In order to convert RLU into μg fluorescein Enzymes, standard curves were generated using QuantiL μM recombinant luciferase (Promega).

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 culture to measure gene expression and cell viability. It emits bioluminescence in the presence of the substrate luciferin. This capped and polyadenylated mRNA is completely replaced by 5-methylcytidine and pseudouridine.

Example 9

Determination of the pKa of the prepared lipid

The pKa of the formulated cationic lipid correlates with the effectiveness of the LNP for delivery of nucleic acids. The preferred pKa range is 5-7. The pKa of each cationic lipid was determined in lipid nanoparticles using an assay based on the fluorescence of 2-(p-toluidino)-6-phthanesulfonic acid (TNS). As described in Example 8, a sequenced method was used to prepare cationic lipids/DSPC/cholesterol/PEG lipids (50/10/38/2 mol%) at a concentration of 0.4 mM total lipids in PBS. Lipid nanoparticles. TNS was prepared as a 100 μM stock solution in distilled water. The vesicles were diluted to contain 24 μM lipid in 2 mL of a buffer solution containing 10 mM HEPES, 10 mM MES, 10 mM sodium acetate, 130 mM NaCl, wherein the pH value was 2.5-11. Aliquots of TNS solution were added to give a final concentration of 1 μM, and after vortex mixing, fluorescence intensity was measured in a SLM Aminco Series 2 Luminescence Spectrophotometer at room temperature using excitation and emission wavelengths of 321 nm and 445 nm. Sigmoid best-fit analysis was applied to the fluorescence data, and pKa was measured as the pH that yielded half the maximum fluorescence intensity.

Example 10

Determination of the Potency of Lipid Nanoparticle Formulations Containing Various Cationic Lipids Using a Rodent Model of In Vivo Luciferase mRNA Expression

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 8. 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, the average PEG molecular weight is 2000). As described in Example 8, 5 hours after administration via tail vein injection, by Relative activity was determined by measuring luciferase expression in the liver. The activity was compared at doses of 0.3 and 1.0 mg mRNA/kg and expressed as ng luciferase measured 5 hours after administration as described in Example 8 /g liver. Embodiment 8 and 9 results are shown in table 2.

Table 2 Comparison of lipids exhibiting activity with mRNA

The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.

The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the scope of the disclosed patents. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims

Have following structure (I) compound:

or its salt or its isomer or its N-oxide, wherein:

R can be alkyl, alkenyl or hydroxyalkyl.
The compound of claim 1, wherein R has a straight carbon chain.
The compound as claimed in claim 1, wherein the carbon number of the straight chain carbon chain is not more than 20.
A composition comprising a compound according to any one of claims 1 to 3 and a therapeutic agent and/or a preventive agent.
The composition of claim 4, further comprising one or more excipients selected from neutral lipids, steroids, and polymer-conjugated lipids.
The composition as claimed in claim 5, wherein the neutral lipid in the component is selected from one or more of the following mixtures: 1,2-distearoyl-sn-glycero-3-phosphate Choline (DSPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Dimyristyl-sn-glycerol-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).
The composition of claim 6, wherein the neutral lipid is DSPC.
The composition as claimed in claims 5-7, wherein the steroid in the component is selected from one or more of the following mixtures: cholesterol, fecal sterol, sitosterol, ergosterol, campesterol, Stigmasterol, brassicasterol, tomatine, ursolic acid, alpha-tocopherol.
A composition as claimed in claim 8, wherein said steroid is cholesterol.
The composition of the preceding claims 5-9, wherein the polymer-conjugated lipid in the component is a pegylated lipid.
The composition of claim 10, wherein the pegylated lipid is 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG).
The composition of any one of the preceding claims, wherein the therapeutic and/or prophylactic agent is a vaccine or compound capable of eliciting an immune response, including nucleic acids.
The composition of claim 12, wherein the nucleic acid is RNA selected from the group consisting of siRNA, aiRNA, miRNA, dsRNA, shRNA, mRNA and mixtures thereof.
The composition of claim 13, wherein said RNA is mRNA.
A method of administering a therapeutic and/or prophylactic agent to a subject in need thereof, the method comprising preparing or providing a composition according to any one of the preceding claims, and administering the composition to the subject.
The subject of any one of the preceding claims is a mammal or a human.