Amide ester compound, drug carrier constructed by amide ester compound and application of amide ester compound

Abstract

The invention discloses an ionizable amide ester compound, a drug carrier constructed by the same and application of the ionizable amide ester compound. The structure of the compound is shown as a formula (I), and the compound mainly comprises cholesterol and analogues thereof, biodegradable ester bonds and a head of an amine structure. The invention relates to the field of nucleic acid delivery, in particular to a novel nucleic acid delivery system which is extremely low in toxicity, good in biodegradability and high in stability, and lipid nanoparticles further constructed by the novel nucleic acid delivery system have excellent liver, spleen and lung targeting performance, efficient in-vivo drug delivery efficiency and in-vitro transfection efficiency when applied to delivery of nucleic acid drugs, can efficiently deliver the nucleic acid drugs in a targeted mode and have important significance on prevention or treatment of diseases. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to an amide ester compound and a drug carrier constructed therefrom and applications thereof, and in particular to an amide ester compound capable of efficiently and targetedly delivering a nucleic acid drug and a drug carrier constructed therefrom and applications thereof. Background Art

[0002] Nucleic acid drugs have important clinical value in the prevention and treatment of infectious diseases, genetic diseases, cardiovascular diseases, tumors, etc. Since nucleic acid drugs such as mRNA are easily degraded by enzymes in the body, their effective delivery into the body is the key to the development of nucleic acid drugs. Although lipid nanoparticles (LNPs) based on ionizable lipids can deliver nucleic acids into the body, their delivery lacks targeting and has obvious off-target effects.

[0003] At present, the efficiency of targeted delivery is mainly enhanced by surface modification of LNP (such as binding peptides, antibodies, hyaluronic acid, mannose). For example, by binding CD5 antibodies to LNP, LNP can be targeted and delivered to T cells to obtain anti-fibrosis chimeric antigen receptor (CAR) T cells, further killing activated fibroblasts, and providing a new treatment method for patients with heart failure. Another example is the introduction of mannose groups on the surface of LNP. The modified LNP can specifically bind to the mannose receptors on the surface of dendritic cells (DC) and enhance the uptake of LNP by DC cells. Although the above strategies can improve the targeting of delivery, the structure of the modified LNP becomes very complex, making the production process cumbersome and complicated, and difficult to industrialize and clinically transform. Summary of the invention

[0004] Objectives of the invention: The first objective of the present invention is to provide an ionizable amide ester compound or a pharmaceutically acceptable salt thereof; the second objective is to provide a drug carrier constructed from the compound or a pharmaceutically acceptable salt thereof; the third objective is to provide a pharmaceutical composition constructed from the drug carrier and a nucleic acid drug; the fourth objective is to provide a pharmaceutical application of the compound and the drug carrier and pharmaceutical composition constructed therefrom.

[0005] Technical solution: The structure of the amide ester compound or a pharmaceutically acceptable salt thereof of the present invention is shown in (I),

[0006]

[0007] wherein R is selected from the structural fragments other than the hydroxyl hydrogen in the chemical structure of cholesterol, 7-dehydrocholesterol, campesterol, stigmasterol, brassicasterol, sitosterol, ergosterol, non-sterols, corticosteroids, ursolic acid or α-tocopherol;

[0008] X is selected from any of the following structures:

[0009]

[0010] Preferably, in the structure, R is selected from the structural fragments other than the hydroxyl hydrogen in the cholesterol chemical structure; X is selected from -N(CH 3 ) 2 .

[0011] Preferably, the amide ester compound or a pharmaceutically acceptable salt thereof of the present invention is selected from any one of the following compounds:

[0012]

[0013]

[0014] The present invention designs a class of ionizable amide ester compounds, which are mainly composed of cholesterol and its analogs, biodegradable ester bonds, and the head of an amine structure. They have extremely low toxicity, good biodegradability, and high stability. They can efficiently deliver nucleic acids to targeted organs, thereby reducing off-target effects and improving therapeutic effects, providing more options for the targeted delivery of nucleic acid drugs.

[0015] Preferably, the pharmaceutically acceptable salt of the present invention is a salt formed by the amide ester compound of the present invention and any one of the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid or ferulic acid.

[0016] The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention, prepared from compounds having specific substituents discovered by the present invention with relatively non-toxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the free form of such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts or similar salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the free form of such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid (forming carbonates or bicarbonates), phosphoric acid (forming phosphates, monohydrogen phosphates, dihydrogen phosphates, sulfuric acid (forming sulfates or bisulfates), hydroiodic acid, phosphorous acid, and the like; and organic acid salts such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, Citric acid, tartaric acid and methanesulfonic acid and similar acids; organic acid salts also include amino acids (such as arginine, etc.), glucuronic acid and other organic acid salts. Certain specific compounds of the present invention contain basic and acidic functional groups, and can be converted into any base or acid addition salt. Preferably, the salt is contacted with a base or acid in a conventional manner, and the parent compound is separated, thereby regenerating the free form of the compound. The free form of the compound differs from its various salt forms in certain physical properties, such as different solubility in polar solvents.

[0017] The "pharmaceutically acceptable salts" of the present invention can be synthesized from parent compounds containing acid radicals or bases by conventional chemical methods. In general, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of the two. In general, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred.

[0018] The drug carrier described in the present invention comprises the amide ester compound described in the present invention or a pharmaceutically acceptable salt thereof, a structural lipid, a neutral lipid, a polymer conjugated lipid and an ionizable lipid, and the molar ratio between them is (15-60):(15-45):(1-20):(0.5-5):(15-60), and more preferably (20-35):(20-35):(1-10):(0.5-1.5):(20-35).

[0019] Preferably, the structural lipid is selected from one or more of cholesterol, dehydrocholesterol, campesterol, stigmasterol, brassicasterol, sitosterol, ergosterol, non-sterol, corticosteroid, ursolic acid, α-tocopherol. The structural lipid can stabilize the structure of the carrier well.

[0020] More preferably, the molar ratio of the amide ester compound or a pharmaceutically acceptable salt thereof to the structural lipid is (1-10):1.

[0021] More preferably, in the chemical structure of the amide ester compound or a pharmaceutically acceptable salt thereof, RH is identical to the chemical structure of the structural lipid.

[0022] Preferably, the neutral lipid compound is selected from any lipid molecule that exists in an uncharged form or a neutral zwitterionic form at a selected pH value or range.

[0023] More preferably, the neutral lipid is selected from one or more of ceramide, sphingomyelin, phosphatidylcholine and phosphatidylethanolamine.

[0024] More preferably, the neutral lipid is selected from one or more of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 2-(((2,3-bis(oleoyloxy)propyl))dimethylammonium phosphate)ethyl hydrogen (DOCP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), sphingomyelin (SM), ceramide, and sterol.

[0025] Still further preferably, the neutral lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0026] More preferably, the molar ratio of the amide ester compound or a pharmaceutically acceptable salt thereof to the neutral lipid is 1:2 to 20:1.

[0027] Preferably, the polymer conjugated lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified ceramide, PEG-modified diacylglycerol, PEG-modified phosphatidic acid, PEG-modified dialkylamine, and PEG-modified dialkylglycerol. The polymer conjugated lipid can improve the stability of lipid nanoparticles and reduce protein absorption of lipids.

[0028] Further preferably, the polymer conjugated lipid is selected from PEG-modified phosphatidylethanolamine (PEG-DMG), PEG-modified dimyristoyl phosphatidylethanolamine (PEG-DMPE), PEG-modified dipalmitoyl phosphatidylcholine (PEG-DPPC), PEG-modified dilauroyl phosphatidylethanolamine (PEG-DLPE), PEG-modified distearoyl phosphatidylethanolamine (PEG-DSPE), PEG-modified cholesterol (Chol-PEG), and PEG-modified ceramide (Ceramide-PEG).

[0029] Still further preferably, the polymer conjugated lipid is selected from PEG-DMPE or PEG-DMG.

[0030] Still more preferably, the relative molecular mass of the PEG is 2000.

[0031] Most preferably, the polymer conjugated lipid is selected from DMG-PEG2000.

[0032] More preferably, the molar ratio of the amide ester compound or a pharmaceutically acceptable salt thereof to the polymer conjugated lipid is 1:10 to 200:1.

[0033] Preferably, the ionizable lipid is selected from 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioloxy-N,N-dimethylaminopropane (DODMA), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP) 、1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (MOEPC), (R)-5-(dimethylamino)pentane-1,2-diyl dioleate hydrochloride (DODAPen-Cl), (R)-5-guanidinopentane-1,2-diyl dioleate hydrochloride (DOPen-G), (R)-N,N,N-trimethyl-4,5-bis(oleoyloxy)pentan-1-ammonium chloride (DOTAPen), O,O'-bis(1-octylnonyl)-7,7'-((2-(bis(2-hydroxyethyl)amino)ethyl)azadiyl)diheptanoate (U-101), O,O'-bis(2-hexyldecyl)-7,7'-((2-(bis(2-hydroxyethyl)amino)ethyl) 6,6'-((3-(bis(2-hydroxyethyl)amino)propyl)azadiyl)dihexanoate (U-102), O-2-hexyldecyl-O'-dodecyl-6,6'-((3-(bis(2-hydroxyethyl)amino)propyl)azadiyl)dihexanoate (U-103), O-2-hexyldecyl-O'-dodecyl-6,6'-((3-(bis(2-hydroxyethyl)amino)ethyl)azadiyl)dihexanoate (U-104), O,O'-bis(1-octylnonyl)-6,6'-((3-(bis(2-hydroxyethyl)amino)propyl)azadiyl)dihexanoate (U-105), O,O'-bis(2-hexyldecyl)-6,6'-((3-(bis(2-hydroxyethyl)amino)propyl)azadiyl)dihexanoate (U-106), O-1-Octylnonyl-O'-dodecyl-6,6'-((3-(bis(2-hydroxyethyl)amino)propyl)azadiyl)dihexanoate (U-107), O-1-Octylnonyl-O'-dodecyl-7,7'-((2-(bis(2-hydroxyethyl)amino)ethyl)azadiyl)diheptanoate (U-108), O,O'-bis(1-heptyloctyl)-6,6'-((3-(bis(2-hydroxyethyl)amino)propyl)azadiyl)dihexanoate (U-109), O,O'-bis(1-heptyloctyl)-7,7'-((2-(bis(2-hydroxyethyl)amino)ethyl)azadiyl)diheptanoate (U-110), O-1-heptyloctyl-O'-dodecyl-6,One or more of 6'-((3-(bis(2-hydroxyethyl)amino)propyl)azadiyl)dihexanoate (U-111), O-1-heptyloctyl-O'-dodecyl-7,7'-((2-(bis(2-hydroxyethyl)amino)ethyl)azadiyl)diheptanoate (U-112), 6-((2-hexyldecanoyl)oxy)-N-6-(((2-ethyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1-amine (ALC-0315), and heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102). ,

[0034] More preferably, the ionizable lipid is selected from ALC-0315 or SM-102.

[0035] Further preferably, the molar ratio of the amide ester compound or a pharmaceutically acceptable salt thereof to the ionizable lipid is 1:10 to 10:1, further preferably (10 to 100):1, further preferably (15 to 50):1, and further preferably (20 to 30):1.

[0036] Preferably, the drug carrier comprises an amide ester compound or a pharmaceutically acceptable salt thereof, any one of cholesterol / β-sitosterol / dehydrocholesterol / ergosterol, DSPC, DMG-PEG2000 and any one of ALC-0315 / SM-102, and the molar ratio thereof is (3.85-19.25):(19.25-38.5):10:1.5:50.

[0037] Further preferably, the drug carrier comprises ergosterol amide ester compounds or pharmaceutically acceptable salts thereof, ergosterol, DSPC, DMG-PEG2000 and ALC-0315, and the molar ratio between them is 15.4:23.1:10:1.5:50, wherein the percentage of ergosterol amide ester compounds or pharmaceutically acceptable salts thereof / ergosterol amide ester compounds or pharmaceutically acceptable salts thereof plus ergosterol is 40%; or the drug carrier comprises β-sitosterol amide ester compounds or pharmaceutically acceptable salts thereof, β-sitosterol, DSPC, DMG-PEG2000 and SM-102, and the molar ratio between them is 7.7:30.8:10:1.5:50, wherein the percentage of β-sitosterol amide ester compounds or pharmaceutically acceptable salts thereof / β-sitosterol amide ester compounds or pharmaceutically acceptable salts thereof plus β-sitosterol is 20%.

[0038] The pharmaceutical composition of the present invention comprises the drug carrier of the present invention and one or more selected from single-stranded DNA, double-stranded DNA, siRNA, shRNA, saRNA, miRNA, mRNA, circRNA, dsRNA, tRNA, locked nucleic acid, and peptide nucleic acid drugs, wherein the mass ratio of the drug carrier to the drug is (1 to 100):1.

[0039] Preferably, the drug is an mRNA or circRNA drug.

[0040] Preferably, the drug is encapsulated in or adsorbed to a drug carrier.

[0041] More preferably, the mass ratio of the drug carrier to the drug is preferably (5-60):1, more preferably (8-40):1, and even more preferably (10-30):1.

[0042] Preferably, the pharmaceutical composition of the present invention is a lipid nanoparticle with an average particle size of 80 to 150 nm and a polydispersity index of less than 0.3.

[0043] Preferably, the pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable excipient.

[0044] The pharmaceutically acceptable excipients can be excipients widely used in the field of drug production. Excipients are mainly used to provide a safe, stable and functional pharmaceutical composition, and can also provide a method to dissolve the active ingredient at a desired rate after the subject receives the administration, or promote the effective absorption of the active ingredient after the subject receives the composition. The pharmaceutical excipients can be inert fillers, or provide a certain function, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. The pharmaceutical excipients can include one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents and sweeteners.

[0045] The pharmaceutical composition of the present invention can be prepared according to the disclosed content using any method known to those skilled in the art, such as conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding or lyophilizing processes.

[0046] The pharmaceutical composition of the present invention can be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid preparations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intraarterial, intramuscular) administration. The pharmaceutical composition of the present invention can also be a controlled release or sustained release dosage form (e.g., liposomes or microspheres). Examples of solid oral preparations include, but are not limited to, powders, capsules, caplets, soft capsules, and tablets. Examples of liquid preparations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical preparations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of preparations for parenteral administration include, but are not limited to, solutions for injection, dry powder preparations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories and lozenges.

[0047] The amide ester compound or its pharmaceutically acceptable salt, or the drug carrier constructed therefrom, or the drug composition constructed therefrom of the present invention is used in the preparation of nucleic acid drugs.

[0048] Preferably, the nucleic acid drug is an mRNA or circRNA drug.

[0049] Preferably, the drug is a drug targeting the liver, spleen, or lungs.

[0050] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0051] The present invention designs a new ionizable amide ester compound with extremely low toxicity, good biodegradability and high stability. When the lipid nanoparticles constructed by the invention are used to deliver nucleic acid drugs, they have excellent liver, spleen and lung targeting, high in vivo drug delivery efficiency and in vitro transfection efficiency, enrich the types of ionizable lipids, and can efficiently deliver nucleic acid drugs in a targeted manner, which is of great significance for the prevention or treatment of diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 is the NMR spectrum of ionizable cholesterol in Example 1;

[0053] Figure 2 is the mass spectrum of ionizable cholesterol in Example 1;

[0054] Figure 3 is the NMR spectrum of ionizable sitosterol in Example 1;

[0055] Figure 4is the mass spectrum of ionizable sitosterol in Example 1;

[0056] Figure 5 is the NMR spectrum of ionizable dehydrocholesterol in Example 1;

[0057] Figure 6 is the mass spectrum of ionizable dehydrocholesterol in Example 1;

[0058] Figure 7 is the NMR spectrum of ionizable ergosterol in Example 1;

[0059] Figure 8 is the mass spectrum of ionizable ergosterol in Example 1;

[0060] Fig. 9 TEM images of 0315-Chol-0, 0315-Chol-10, 0315-Chol-30 and 0315-Chol-50 in Example 2;

[0061] Fig.10 These are the results of in vivo delivery in mice in Example 5. Each figure contains three rows. The first number is the ratio of the ionizable cholesterol analog as the fifth component. The second row is the fluorescence imaging of mice under different ratios. The third row is the fluorescence imaging of mouse organs under different ratios. DETAILED DESCRIPTION

[0062] The technical solution of the present invention is further described below in conjunction with embodiments.

[0063] The meanings of the abbreviations are as follows:

[0064] Cholesterol: Chol, β-sitosterol: Sito, dehydrocholesterol: Dehy, ergosterol: Ergo, ALC-0315: 0315, SM-102: 102.

[0065] Example 1: Synthesis of ionizable cholesterol and its analogs

[0066] The compounds were synthesized according to the following route:

[0067]

[0068] Wherein R is selected from the following structural fragments other than the hydroxyl hydrogen in the chemical structure of cholesterol analogs:

[0069]

[0070] 1. Synthesis of Intermediate 1

[0071] 20mM cholesterol analog (cholesterol / β-sitosterol / dehydrocholesterol / ergosterol) was dissolved in 60mL dry dichloromethane solvent and stirred at room temperature for 30 minutes. Then 6.7mmol triphosgene and a few drops of pyridine were added under 0℃ ice bath conditions. The reaction mixture was stirred at 0℃ for 1 hour and then continued to stir at room temperature for 3 hours. After the reaction was completed, the solvent was removed by rotary evaporation to obtain intermediate 1 which was directly used in the next chemical reaction.

[0072] 2. Synthesis of Intermediate 2

[0073] 20 mM diethanolamine and 30 mM sodium carbonate were dissolved in a mixture of deionized water and tetrahydrofuran (50 mL: 25 mL) and stirred for 15 to 30 minutes under ice bath conditions. 20 mM intermediate 1 was added to the reaction mixture and reacted for 1 to 2 hours. Then the reaction was allowed to proceed at room temperature for about 14 hours. Finally, 200 mL of ethyl acetate was added to the reaction mixture, the organic layer was separated and washed with saturated sodium chloride aqueous solution, the aqueous layer was extracted twice with ethyl acetate, the organic layers were combined and washed with Na 2 SO 4 After drying, the solvent was removed by rotary evaporation to obtain intermediate 2 which was directly used in the next chemical reaction.

[0074] 3. Synthesis of the final product

[0075] The intermediate 2 was dissolved in dichloromethane, followed by the addition of 60 mM 3-(dimethylamino) propionic acid hydrochloride and 120 mM N, N-diisopropylethylamine (DIPEA), and then 44 mM 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and 4 mM 4-dimethylaminopyridine (DMAP) in batches, and the reaction was continued at room temperature. After the reaction was completed, it was diluted with dichloromethane, washed with saturated sodium bicarbonate solution and saturated brine, and the organic layers were combined and washed with anhydrous Na 2 SO 4 The final product was purified by silica gel column chromatography (methanol / dichloromethane = 1 / 20) and characterized by nuclear magnetic resonance spectrometry and mass spectrometry. Figures 1 to 8 .

[0076] Example 2: Preparation and characterization of lipid nanoparticles

[0077] 1. Preparation of lipid nanoparticles

[0078] Preparation of ethanol phase: The ethanol phase was prepared according to the specific formula ratios in Tables 1 and 2, 20 mM ALC-0315 or SM102, 4 mM 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 0.6 mM DMG-PEG2000, cholesterol analogs and ionizable cholesterol analogs totaling 15.4 mM, and the above substances were dissolved in 1 mL of anhydrous ethanol and vortexed to dissolve to obtain the ethanol phase. The ratio of cholesterol analogs and ionizable cholesterol analogs was changed according to Tables 1 and 2 to prepare different LNPs.

[0079] Table 1 Specific molar ratios of ethanol phase of 24 samples prepared based on ionizable lipid ALC-0315

[0080]

[0081]

[0082] Table 2 Specific molar ratios of ethanol phase of 24 samples prepared based on ionizable lipid SM-102

[0083]

[0084]

[0085] Preparation of the aqueous phase: A 200 μL solution of mRNA encoding firefly luciferase (Fluc mRNA) (concentration of 0.5 μg / μL) was added to 700 μL of a citric acid buffer solution at pH=4 as the aqueous phase.

[0086] The above aqueous phase and ethanol phase were blended by microfluidics to obtain a blended solution (the aqueous phase flow rate was 9 mL / min, and the ethanol phase flow rate was 3 mL / min). The prepared blended solution was quickly added to 10 volumes of PBS (pH = 7.4), and then the solution was transferred to an ultrafiltration tube and centrifuged at 5000 r / min for 30 min to remove ethanol and citrate buffer. Finally, a 220 nm filter membrane was used to filter the final sample. Each sample was named according to the abbreviation of ionizable lipids (e.g., 0315: ALC-0315 or 102: SM-102) and the name of the cholesterol analog (cholesterol: Chol, β-sitosterol: Sito, dehydrocholesterol: Dehy, ergosterol: Ergo) and the ratio of ionizable cholesterol analogs to total cholesterol analogs (the sum of ionizable cholesterol analogs and ionizable cholesterol analogs). For example, 0315-Ergo-40 means that the ionizable lipid is ALC-0315, ergosterol is used as the structural lipid, and the percentage of ionizable ergosterol / (ergosterol+ionizable ergosterol) is 40%. 102-Sito-20 means that the ionizable lipid is SM-102, β-sitosterol is used as the structural lipid, and the percentage of ionizable β-sitosterol / (β-sitosterol+ionizable β-sitosterol) is 20%. The obtained lipid nanoparticle preparation solution is calculated according to 5% sucrose, and sucrose is added for standby use.

[0087] 2. Characterization of lipid nanoparticles

[0088] The particle size and polydispersity index (PDI) of the lipid nanoparticles obtained above were measured using a Malvern particle size analyzer (Malvern UK). The results are shown in Tables 3 and 4.

[0089] RiboGreen reagent (Thermo Fisher Scientific, Cat No.5, R11491) was used to detect the mRNA encapsulation efficiency (EE%). First, in order to determine the free mRNA in the lipid nanoparticle preparation, 10 μL of the above-mentioned lipid nanoparticle preparation solution containing 5% sucrose was taken in a centrifuge tube, 990 μL of 1×TE buffer (Nanjing Novozyme Biotechnology Co., Ltd.) was added to dilute, and 100 μL of the diluted solution was added to a 96-well plate. Then, 100 μL of RiboGreen dye was added to the plate wells and incubated for 5 min. The free mRNA concentration value was determined using a spectrophotometer (Thermo Fisher Scientific, USA), with an excitation wavelength of 485 nm and an emission wavelength of 528 nm. In order to determine the total mRNA content in the lipid nanoparticle preparation, 10 μL of the above lipid nanoparticle preparation solution containing 5% sucrose was taken in a centrifuge tube, 990 μL of 2% TE-Triton buffer (Nanjing Novozyme Biotechnology Co., Ltd.) was added for dilution, and 100 μL of the diluted solution was added to a 96-well plate. Then, 100 μL of RiboGreen dye was added to the plate wells and incubated for 5 min. The total mRNA concentration value was determined using a spectrophotometer (ThermoFisher Scientific, USA), with an excitation wavelength of 485 nm and an emission wavelength of 528 nm. The encapsulation efficiency was calculated as follows:

[0090] Encapsulation efficiency = (total mRNA concentration - free mRNA concentration) / total mRNA concentration × 100%

[0091] The results are shown in Tables 3 and 4.

[0092] Table 3 Physicochemical properties of 24 samples based on ALC-0315 ionizable lipids

[0093] sample Particle size (nm) PDI Encapsulation rate (%) 0315-Chol-0 110 0.108 91.55 0315-Chol-10 98 0.132 90.43 0315-Chol-20 115 0.098 89.31 0315-Chol-30 112 0.086 90.25 0315-Chol-40 120 0.125 91.35 0315-Chol-50 180 0.085 87.25 0315-Sito-0 122 0.147 91.72 0315-Sito-10 154 0.085 91.02 0315-Sito-20 158 0.077 89.91 0315-Sito-30 162 0.096 88.47 0315-Sito-40 148 0.105 91.10 0315-Sito-50 143 0.098 88.51 0315-Dehy-0 123 0.142 92.02 0315-Dehy-10 103 0.138 91.35 0315-Dehy-20 105 0.154 89.21 0315-Dehy-30 133 0.125 90.89 0315-Dehy-40 145 0.147 88.95 0315-Dehy-50 165 0.168 91.15 0315-Ergo-0 115 0.143 91.35 0315-Ergo-10 151 0.151 90.88 0315-Ergo-20 158 0.156 89.95 0315-Ergo-30 153 0.129 90.34 0315-Ergo-40 150 0.136 88.86 0315-Ergo-50 156 0.141 90.10

[0094] Table 4 Physicochemical properties of 24 samples based on SM-102 ionizable lipids

[0095]

[0096]

[0097] As can be seen from Tables 3 and 4, the particle size range of the lipid nanoparticles prepared by the present invention is 95 to 170 nm, and the PDI value is less than 0.2. This shows that all 48 lipid nanoparticles have excellent physicochemical properties. At the same time, the lipid nanoparticles prepared by the present invention have a high mRNA encapsulation rate. As the proportion of ionizable cholesterol analogs increases, there is no obvious change in the physicochemical properties of the samples.

[0098] The surface morphology of lipid nanoparticles was observed by transmission electron microscopy (TEM, JEOL, Japan); four samples of lipid nanoparticles prepared above, 0315-Chol-0, 0315-Chol-10, 0315-Chol-30 and 0315-Chol-50, were selected for testing; lipid nanoparticles were dropped on different 200-mesh carbon film copper grids; after natural air drying, 2% (w / v) phosphotungstic acid was dropped on the copper grids for about 1 to 2 minutes; the treated samples were photographed at an accelerating voltage of 200 kV; the results are shown in FIG. Fig. 9 The transmission electron microscopy images of the four samples 0315-Chol-0, 0315-Chol-10, 0315-Chol-30 and 0315-Chol-50 in Example 2 show that the lipid nanoparticles prepared in this example are all spherical in size and are consistent with the test results of the Malvern particle size analyzer, all between 100 and 150 nm.

[0099] Example 3: Cell transfection experiment

[0100] Enhanced green fluorescent protein mRNA (eGFP mRNA) was encapsulated in lipid nanoparticles to obtain a lipid nanoparticle preparation solution encapsulating eGFP mRNA. The preparation process was the same as in Example 2. HEK293 cells or DC 2.4 cells (2×10 4 Cells / well, cells from the ATCC Biological Standard Resource Center in the United States) were inoculated in a 6-well plate, and the cells were cultured with 2mL DMEM medium until 80% confluence was reached; then, the cells were washed 3 times with PBS standard solution, and the medium was replaced with 2mL Opti-MEM serum-free medium, and the prepared 0315-Chol-0, 0315-Chol-10, 0315-Chol-30 and 0315-Chol-50 lipid nanoparticle preparation solutions containing 5% sucrose were added respectively (mRNA concentration was 1.5μg / mL). After incubation for 24h, the cells were collected and the positive rate of cell eGFP protein was detected by flow cytometry (Accui C6, USA), and the results are shown in Table 5.

[0101] Table 5 In vitro transfection data of 0315-Chol-0, 0315-Chol-10, 0315-Chol-30 and 0315-Chol-50 lipid nanoparticles

[0102]

[0103] As can be seen from Table 5, 0315-Chol-0, 0315-Chol-10, 0315-Chol-30 and 0315-Chol-50 lipid nanoparticle formulations can be effectively transfected in HEK293 cells or DC2.4 cells.

[0104] Example 4: Cytotoxicity Experiment

[0105] HEK293 cells were seeded in 96-well plates (2 × 10 3 cells / well) and cultured overnight; after the cells adhered, the lipid nanoparticle preparation solution containing 5% sucrose prepared in Example 2 was added to the 96-well plate to control the mRNA concentration to 2 μg / mL; after 24 hours of incubation, the cytotoxicity was evaluated using Cell Counting Kit-8 (CCK-8, APExBIO, USA); the OD value at 450nm was measured by a microplate reader. The cell survival rate was calculated based on the OD value, and the results are shown in Table 6.

[0106] Table 6 Cytotoxicity test results of lipid nanoparticles

[0107]

[0108]

[0109] As can be seen from Table 6, the introduction of ionizable cholesterol analogs into LNPs does not affect the cytotoxicity of LNPs.

[0110] Example 5: In vivo fluorescence detection

[0111] The 48 lipid nanoparticle samples encapsulating Fluc mRNA prepared in Example 2 were validated in vivo, and three mice were used for each sample. The steps are as follows: the sample prepared in Example 2 was injected into the mouse via the tail vein (dose of 0.5 mg / kg), and then a 20 mg / mL fluorescein potassium salt solution was prepared. After 3 hours, 100 μL of the fluorescein potassium salt solution was injected into the mouse via intraperitoneal injection. The bioluminescent signal image of the mouse was then detected by a mouse imager (PerkinElmer). The results are shown in Fig.10 .

[0112] Depend on Fig.10 It can be seen that all 48 lipid nanoparticle samples can effectively deliver mRNA and express proteins in mice. At the same time, ionizable cholesterol analogs can promote LNP delivery to the spleen under low addition conditions, and can promote most LNP targeted delivery to the lungs under high addition conditions. That is, selective targeted delivery of mRNA can be achieved by simply adjusting the ratio of ionizable cholesterol analogs in LNP.

[0113] After completing the in vivo fluorescence detection of the mice, the mice were dissected and the heart, liver, spleen, lungs, and kidneys were taken out and placed in a culture dish. The mouse imaging instrument was used again to detect the fluorescence of the mice's organs and perform quantitative analysis. At the same time, the percentage of the fluorescence intensity of the liver, spleen, and lungs was calculated based on the fluorescence detection results of the mice's organs. The results are shown in Tables 7 and 8.

[0114] Table 7 Organ fluorescence intensity values ​​of 24 samples based on ALC-0315 ionizable lipids

[0115]

[0116]

[0117] Table 8 Organ fluorescence intensity values ​​of 24 samples based on SM-102 ionizable lipids

[0118]

[0119]

[0120] As can be seen from Tables 7 and 8, ionizable cholesterol analogs can promote LNP delivery to the spleen under low addition conditions and can promote LNP delivery to the lungs under high addition conditions. The percentage of fluorescence intensity of the liver, spleen and lungs was analyzed based on the fluorescence detection results of mouse organs. Among them, the targeted delivery efficiency of the 0315-Ergo-40 sample to the spleen was as high as 94% (Table 7), and the targeted delivery efficiency of the 102-Sito-40 sample to the lungs was as high as 78% (Table 8).

Claims

1. An amide ester compound or a pharmaceutically acceptable salt thereof, characterized in that: The structure is shown in (I), wherein R is selected from the structural fragments other than the hydroxyl hydrogen in the chemical structure of cholesterol, 7-dehydrocholesterol, campesterol, stigmasterol, brassicasterol, sitosterol, ergosterol, non-sterols, corticosteroids, ursolic acid or α-tocopherol; X is selected from any of the following structures:

2. The amide ester compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Any one of the following compounds:

3. A drug carrier, characterized in that It comprises the amide ester compound or a pharmaceutically acceptable salt thereof according to claim 1, a structural lipid, a neutral lipid, a polymer conjugated lipid and an ionizable lipid, wherein the molar ratio between them is (15-60):(15-45):(1-20):(0.5-5):(15-60).

4. The drug carrier according to claim 3, characterized in that The structural lipid is selected from one or more of cholesterol, dehydrocholesterol, campesterol, stigmasterol, brassicasterol, sitosterol, ergosterol, non-sterol, corticosteroid, ursolic acid, α-tocopherol.

5. The drug carrier according to claim 3, characterized in that The neutral lipid is selected from one or more of ceramide, sphingomyelin, phosphatidylcholine and phosphatidylethanolamine.

6. The drug carrier according to claim 3, characterized in that The polymer conjugated lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified ceramide, PEG-modified diacylglycerol, PEG-modified phosphatidic acid, PEG-modified dialkylamine, and PEG-modified dialkylglycerol.

7. The drug carrier according to claim 3, characterized in that The ionizable lipid is selected from 1,2-dilinoleoyloxy-N,N-dimethylaminopropane, 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane, 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane, 1,2-dioloxy-N,N-dimethylaminopropane, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride, 1,2-dimyristoyl-sn-glyceryl-3-ethylcholine phosphate, (R )-5-(dimethylamino)pentane-1,2-diyl dioleate hydrochloride, (R)-5-guanidinopentane-1,2-diyl dioleate hydrochloride, (R)-N,N,N-trimethyl-4,5-bis(oleoyloxy)pentan-1-ammonium chloride, O,O'-bis(1-octylnonyl)-7,7'-((2-(bis(2-hydroxyethyl)amino)ethyl)azadiyl)diheptanoate, O,O'-bis(2-hexyldecyl)-7,7'-((2-(bis(2-hydroxyethyl)amino)ethyl)azadiyl)diheptanoate, O-2-hexyldecyl-O'-dodecyl-6,6'-((3-(bis(2-hydroxyethyl)amino)ethyl)azadiyl)diheptanoate )amino)propyl)azadiyl)dihexanoate, O-2-hexyldecyl-O'-dodecyl-6,6'-((3-(bis(2-hydroxyethyl)amino)ethyl)azadiyl)diheptanoate, O,O'-bis(1-octylnonyl)-6,6'-((3-(bis(2-hydroxyethyl)amino)propyl)azadiyl)dihexanoate, O,O'-bis(2-hexyldecyl)-6,6'-((3-(bis(2-hydroxyethyl)amino)propyl)azadiyl)dihexanoate, O-1-octylnonyl-O'-dodecyl-6,6'-((3-(bis(2-hydroxyethyl)amino)propyl)azadiyl)dihexanoate, O -1-octylnonyl-O'-dodecyl-7,7'-((2-(bis(2-hydroxyethyl)amino)ethyl)azadiyl)diheptanoate, O,O'-bis(1-heptyloctyl)-6,6'-((3-(bis(2-hydroxyethyl)amino)propyl)azadiyl)dihexanoate, O,O'-bis(1-heptyloctyl)-7,7'-((2-(bis(2-hydroxyethyl)amino)ethyl)azadiyl)diheptanoate, O-1-heptyloctyl-O'-dodecyl-6,6'-((3-(bis(2-hydroxyethyl)amino)propyl)azadiyl)dihexanoate, O-1-heptyloctyl-O'-dodecyl-7,One or more of 7'-((2-(bis(2-hydroxyethyl)amino)ethyl)azepinediyl)diheptanoate, 6-((2-hexyldecanoyl)oxy)-N-6-(((2-ethyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1-amine, and heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate.

8. A pharmaceutical composition, characterized in that The method comprises the drug carrier of claim 3 and one or more selected from single-stranded DNA, double-stranded DNA, siRNA, shRNA, saRNA, miRNA, mRNA, circRNA, dsRNA, tRNA, locked nucleic acid, and peptide nucleic acid drugs, wherein the mass ratio of the drug carrier to the drug is (1 to 100):

1.

9. The pharmaceutical composition according to claim 8, characterized in that The average particle size is 80-150 nm, and the polydispersity index is less than 0.

3.

10. Use of the amide ester compound or a pharmaceutically acceptable salt thereof according to claim 1, the drug carrier according to claim 3 or the pharmaceutical composition according to claim 8 in the preparation of a nucleic acid drug.

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