CN117164468B - Ionizable lipid compound and application thereof - Google Patents
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Abstract
The invention discloses an ionizable lipid compound and application thereof. An ionizable lipid compound having a structure represented by formula (I), wherein the ionizable lipid compound of the invention has a polyoxastructure incorporated in the head thereof and is coordinated with a hydroxyl group; so that the lipid compound has good biocompatibility and excellent in vivo mRNA delivery efficiency. The LNP composition has novel structural design, proper proportion of each component of the matched LNP formulation, and animal experiments prove that the delivery effect reaches the international advanced level of commercial ionizable lipid, and the safety is good.
Description
Technical Field
The invention belongs to the field of new preparation materials, and relates to an ionizable lipid compound and application thereof.
Background
Compared with the conventional vaccine, the mRNA vaccine has the advantages of low cost, high production efficiency and high safety, and has the potential of synthesizing any protein. Therefore, the method has great application potential for the novel infectious viruses which cannot be handled by the traditional vaccine. However, the use of mRNA vaccines has been limited due to instability of the mRNA molecules, susceptibility to degradation by rnases, and low in vivo delivery efficiency. To achieve widespread use of mRNA vaccines, the delivery technology needs to be addressed with emphasis. The mRNA vaccine needs to have a proper delivery carrier to deliver the mRNA vaccine into the body so as to have better immune effect, so that the development of a high-efficiency nontoxic delivery system is key to the success of the mRNA vaccine.
Advanced delivery vehicles for mRNA are lipid nanoparticles (Lipid Nanoparticle, LNP), typically comprising at least ionizable lipids, neutral helper lipids, and phospholipid polyethylene glycol derivatives. At present, most of domestic mRNA vaccines are developed, ionized lipids which are invented by foreign companies and protected by patent restrictions are used, and commercial use is limited; the stability and overall safety assessment of LNP formulations has not been studied in depth.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide an ionizable lipid compound, and a preparation method and application thereof. mRNA-LNP prepared by the ionizable lipid compound with the brand new structure has the advantages of good biocompatibility, high in vivo delivery efficiency, high stability and good biosafety.
The aim of the invention can be achieved by the following technical scheme:
an ionizable lipid compound having a structure according to formula (i):
；
wherein m is selected from positive integers of 3-7; r is R 1 、R 2 、R 3 、R 4 The radicals are each independently-OH;
R 5 、R 6 、R 7 、R 8 the radicals being independently of one anotherAnd is a straight or branched C8-20 alkyl group, a straight or branched C8-20 alkenyl group, a straight or branched C8-20 alkynyl group, or at least 1C atom of said alkyl, alkenyl or alkynyl group is optionally replaced by a heteroatom independently selected from O, S or N.
As a preferred aspect of the present invention, m is 3, 4, 5, 6, 7; r is R 1 、R 2 、R 3 、R 4 The radicals are each independently-OH, R 5 、R 6 、R 7 、R 8 The radicals are each independently linear C12, C13, C14 alkyl radicals.
An ionizable lipid compound selected from any one of the following:
。
as a preferred aspect of the present invention, the ionizable lipid compound is selected from any one of the following:
。
the invention relates to an application of an ionizable lipid compound in preparing a nano lipid preparation.
A nanolipid particle composition comprising an ionizable lipid compound according to the invention.
Preferably, the nanoparticle composition further comprises one or more of an ionizable lipid and a neutral lipid, and a phospholipid polyethylene glycol derivative.
Preferably, the neutral lipid is selected from any one or both of DOPE and cholesterol.
Preferably, the phospholipid polyethylene glycol derivative is selected from phospholipid polyethylene glycol derivative DMG-PEG 2000.
As a preferred aspect of the present invention, the nanoparticle composition consists of the ionizable lipid compound of the present invention, cholesterol and phospholipid polyethylene glycol derivative DMG-PEG 2000.
As a further preferred aspect of the present invention, the composition of the nano-lipid particles comprises an ionizable lipid compound: cholesterol: the mole ratio of the phospholipid polyethylene glycol derivative DMG-PEG 2000 is 20-100:15-50:1-3, preferably 40-60:30-50:1-2. In one embodiment of the invention the ionizable lipid compounds of the invention: cholesterol: the molar ratio of the phospholipid polyethylene glycol derivative DMG-PEG 2000 is 50:38.5:1.5.
The nanometer lipid particle composition is applied to the preparation of mRNA drugs or vaccines.
The beneficial effects are that:
the ionizable lipid compound of the invention introduces a polyoxastructure and hydroxyl coordination at the head, so that the ionizable lipid compound has good biocompatibility and excellent in vivo mRNA delivery efficiency. The LNP dosage form has novel structural design, proper proportion of each component of the LNP dosage form, and animal experiments prove that the delivery effect reaches or even exceeds that of commercial lipid, and is in an international leading position.
The invention has high synthesis efficiency and low cost of the ionizable lipid compound, and is convenient for commercial application of large-scale production.
The proportion of the 3-component formula corresponding to LNP of the invention is different from the range of the 4-component formula of US8058069 patent in Arbunus name, and the invention is not limited.
The ionizable lipid compounds of the invention deliver equivalent levels of LNP formulations, in part exceeding SM-102, to SM-102.
The ionizable lipid compound has good stability corresponding to the LNP formula, the accelerated stability at 40 ℃ is 1 month, the detection shows that the particle size is slightly increased, the integral structure is maintained, and mRNA can be well encapsulated in LNP without leakage.
The safety of the ionized lipid compound corresponding to the LNP formulation is obviously better than that of the formulation of the Morgana SM-102, the ultra-large dose intravenous injection is carried out, most mice have no obvious discomfort, the discomfort of some mice of the examples can be relieved quickly, and the mice injected with the formulation of the control group SM-102 die. The ionizable lipid compounds and the corresponding LNP formulations of the invention are demonstrated to be excellent in safety.
Drawings
FIG. 1 HNMR spectrum (CDCl) of ionizable lipid compound H-1 3 Solution of
FIG. 2 HNMR spectra (CDCl) of ionizable lipid Compound H-2 3 Solution of
FIG. 3 HNMR spectrum (CDCl) of ionizable lipid compound H-3 3 Solution of
FIG. 4 HNMR spectrum (CDCl) of ionizable lipid compound H-4 3 Solution of
FIG. 5 HNMR spectrum (CDCl) of ionizable lipid compound H-5 3 Solution of
FIG. 6 HNMR spectrum (CDCl) of ionizable lipid compound H-6 3 Solution of
FIG. 7 HNMR spectrum (CDCl) of ionizable lipid compound H-7 3 Solution of
FIG. 8 HNMR spectrum (CDCl) of ionizable lipid compound H-8 3 Solution of
FIG. 9 HNMR spectrum (CDCl) of ionizable lipid compound H-9 3 Solution of
FIG. 10 HNMR spectra (CDCl) of ionizable lipid compound H-10 3 Solution of
FIG. 11 animal imaging apparatus photographs of mRNA lipid nanoparticles No. 1-10 and control samples prepared in example 11 after intramuscular injection of mice
Detailed Description
Example 1 synthesis of ionizable lipid compound H-1:
10mmol of 3,6, 9-trioxaundecane-1, 11-diamine, 50mmol of 1, 2-epoxytetradecane and 30mL of dichloromethane are sequentially added into a 50mL reaction bottle filled with magneton, the reaction tube is heated and stirred at 85 ℃ for reaction for 72 hours, after TLC monitoring that the reaction is complete, the reaction is cooled to room temperature, a rotary evaporator is used for removing the solvent, and the product is separated by a thin layer chromatography column (a silica gel column, eluent is dichloromethane: methanol: triethylamine volume ratio=93:6:1), so that a pale yellow oily product H-1 is obtained, and the yield reaches 89%. The specific reaction formula is as follows:
。
example 2 synthesis of ionizable lipid compound H-2:
10mmol of 3,6, 9-trioxaundecane-1, 11-diamine, 50mmol of 1, 2-epoxy hexadecane and 30mL of dichloromethane are sequentially added into a 50mL reaction bottle filled with magneton, the reaction tube is heated and stirred for reaction for 72 hours at the temperature of 85 ℃, after TLC monitoring that the reaction is complete, the reaction is cooled to room temperature, a rotary evaporator is used for removing the solvent, and the product is separated by a thin layer chromatography column (silica gel column, eluent is dichloromethane: methanol: triethylamine volume ratio=93:6:1), so that a milky white oily product H-2 is obtained, and the yield reaches 87%. The specific reaction formula is as follows:
the H-2 hydrogen spectrum of the ionizable lipid compound is shown in FIG. 2.
Example 3 synthesis of ionizable lipid compounds:
10mmol of 3,6,9, 12-tetraoxatetradecane-1, 14-diamine, 50mmol of 1, 2-epoxytetradecane and 30mL of dichloromethane are sequentially added into a 50mL reaction bottle filled with magneton, the reaction tube is heated and stirred at 90 ℃ for 72 hours, after TLC monitoring the reaction to be complete, the reaction is cooled to room temperature, a rotary evaporator is used for removing the solvent, and the product is separated by a thin layer chromatographic column (silica gel column, eluent is dichloromethane: methanol: triethylamine volume ratio=93:6:1), so that a pale yellow oily product H-3 is obtained, and the yield reaches 87%. The specific reaction formula is as follows:
the H-3 hydrogen spectrum of the ionizable lipid compound is shown in FIG. 3.
Example 4 synthesis of ionizable lipid compounds:
10mmol of 3,6,9, 12-tetraoxatetradecane-1, 14-diamine, 50mmol of 1, 2-epoxy hexadecane and 30mL of dichloromethane are sequentially added into a 50mL reaction bottle filled with magneton, the reaction tube is heated and stirred at 95 ℃ for 84 hours, after TLC monitoring the reaction is completed, the reaction is cooled to room temperature, a rotary evaporator is used for removing the solvent, and the product is separated by a thin layer chromatography column (a silica gel column, eluent is dichloromethane: methanol: triethylamine volume ratio=93:6:1), so that a pale yellow oily product H-4 is obtained, and the yield reaches 84%. The specific reaction formula is as follows:
the H-4 hydrogen spectrum of the ionizable lipid compound is shown in FIG. 4.
Example 5: synthesis of ionizable lipid compound H-5:
10mmol 3,6,9,12,15-pentaoxaheptadecane-1, 17-diamine, 50mmol of 1, 2-epoxytetradecane and 30mL of absolute ethyl alcohol are sequentially added into a 50mL reaction bottle filled with magneton, the reaction tube is heated and stirred at 90 ℃ for reaction for 72 hours, after TLC monitoring reaction is completed, the reaction is cooled to room temperature, a rotary evaporator is used for removing solvent, and the product is separated by a thin-layer chromatographic column (a silica gel column, eluent is dichloromethane: methanol: triethylamine volume ratio=93:6:1), so that a milky oily product H-5 is obtained, and the yield reaches 88%. The specific reaction formula is as follows:
the H-5 hydrogen spectrum of the ionizable lipid compound is shown in FIG. 5.
Example 6:
synthesis of Compound H-6: 10mmol 3,6,9,12,15-pentaoxaheptadecane-1, 17-diamine, 50mmol of 1, 2-epoxyhexadecane and 30mL of absolute ethyl alcohol are sequentially added into a 50mL reaction bottle filled with magneton, the reaction tube is heated and stirred at 90 ℃ for reaction for 72 hours, after TLC monitoring reaction is completed, the reaction is cooled to room temperature, a rotary evaporator is used for removing solvent, and the product is separated by a thin-layer chromatographic column (a silica gel column, eluent is dichloromethane: methanol: triethylamine volume ratio=93:6:1), so that a milky oily product H-6 is obtained, and the yield reaches 84%. The specific reaction formula is as follows:
the H-6 hydrogen spectrum of the ionizable lipid compound is shown in FIG. 6.
EXAMPLE 7 Synthesis of ionizable lipid Compound H-7
10mmol 3,6,9,12,15,18-hexaoxaeicosane-1, 20 diamine, 50mmol of 1, 2-epoxytetradecane and 30mL of absolute ethyl alcohol are sequentially added into a 50mL reaction bottle filled with magneton, the reaction tube is heated and stirred at 90 ℃ for reaction for 72 hours, after TLC monitoring reaction is completed, the reaction is cooled to room temperature, a rotary evaporator is used for removing the solvent, and the product is separated by a thin layer chromatographic column (a silica gel column, eluent is dichloromethane: methanol: triethylamine volume ratio=93:6:1), so that a milky oily product H-7 is obtained, and the yield reaches 86%. The specific reaction formula is as follows:
the H-7 hydrogen spectrum of the ionizable lipid compound is shown in FIG. 7.
Example 8 synthesis of ionizable lipid compound H-8:
10mmol 3,6,9,12,15,18-hexaoxaeicosane-1, 20-diamine, 50mmol of 1, 2-epoxy hexadecane and 30mL of absolute ethyl alcohol are sequentially added into a 50mL reaction bottle filled with magneton, a reaction tube is heated and stirred for reaction for 72 hours at 90 ℃, after TLC monitoring reaction is completed, the reaction is cooled to room temperature, a rotary evaporator is used for removing solvent, and a product is separated by a thin-layer chromatographic column (a silica gel column, eluent is dichloromethane: methanol: triethylamine volume ratio=93:6:1), so that a milky oily product H-8 is obtained, and the yield reaches 83%. The specific reaction formula is as follows:
the H-8 hydrogen spectrum of the ionizable lipid compound is shown in FIG. 8.
Example 9 synthesis of ionizable lipid compound H-9:
10mmol 3,6,9,12,15,18,21-heptaoxaditridecane-1, 23-diamine, 50mmol of 1, 2-epoxytetradecane and 30mL of absolute ethyl alcohol are sequentially added into a 50mL reaction bottle filled with magneton, the reaction tube is heated and stirred for reaction for 72 hours at 90 ℃, after the reaction is monitored by TLC, the reaction is cooled to room temperature, a rotary evaporator is used for removing the solvent, the product is separated by a thin layer chromatographic column (a silica gel column, eluent is dichloromethane: methanol: triethylamine volume ratio=93:6:1), and a milky oily product H-9 is obtained, and the yield reaches 86%. The specific reaction formula is as follows:
the H-9 hydrogen spectrum of the ionizable lipid compound is shown in FIG. 9.
Example 10 synthesis of ionizable lipid compound H-10:
10mmol 3,6,9,12,15,18,21-heptaoxaditridecane-1, 23-diamine, 50mmol of 1, 2-epoxy hexadecane and 30mL of absolute ethyl alcohol are sequentially added into a 50mL reaction bottle filled with magneton, a reaction tube is heated and stirred for reaction for 72 hours at 90 ℃, after the reaction is monitored by TLC, the reaction is cooled to room temperature, a rotary evaporator is used for removing the solvent, and the product is separated by a thin-layer chromatographic column (a silica gel column, eluent is dichloromethane: methanol: triethylamine volume ratio=93:6:1), so that a milky oily product H-10 is obtained, and the yield reaches 82%. The specific reaction formula is as follows:
the H-10 hydrogen spectrum of the ionizable lipid compound is shown in FIG. 10.
Example 11: preparation and detection of nanolipid particles (LNP formulations)
S1, preparing 5mM citric acid buffer solution with pH3.0 containing firefly luciferase (fluc) mRNA as an aqueous phase.
S2, preparing absolute ethanol solutions of the ionizable lipid compounds of examples 1-10, namely lipid, cholesterol and phospholipid polyethylene glycol derivative DMG-PEG 2000 as mother solutions, wherein the concentrations are respectively 10mg/ml, 10mg/ml and 2mg/ml. According to the ionizable lipid compound lipids: cholesterol: the molar ratio of DMG-PEG 2000 is 50:38.5:1.5, and a lipid mixed solution is prepared as an organic phase.
S3, mixing the two solutions in the microfluidic chip through the microfluidic device, wherein the aqueous phase and the organic phase solution respectively enter from two sides and a middle flow path of the microfluidic chip, the total flow rate of the aqueous phase is controlled at 3ml/min, the flow rate of the organic phase is controlled at 1ml/min, and the aqueous phase: organic phase flow speed ratio 3:1, mixing the two-phase flow paths after converging in a chip, wherein the weight ratio of total lipid to mRNA is about 15-30: 1, mRNA lipid nanoparticles (mRNA-LNP) were prepared by binding positively charged lipids to negatively charged mRNA.
S4, diluting the obtained mRNA-LNP 10 times by using 10mM PBS buffer solution with pH7.0, and purifying by ultrafiltration concentration through an ultrafiltration tube with a pore diameter of a 100KD filter membrane. The ultrafiltration mode is a 30-degree fixed angle rotor, centrifugal force is 2000g, and the concentration is carried out at room temperature of 25 ℃ to 1/10 volume.
S5, mRNA-LNP is filtered through a 0.22 μm filter and stored at 2-8 ℃.
S6, measuring the average particle size and the polydispersity index PDI of the mRNA-LNP sample by using a dynamic light scattering nano particle size analyzer, and the test results are shown in Table 1.
S7, according to the manufacturer' S instructions, using Quant-it Ribogreen RNA quantitative determination kit (Thermo
Fisher Scientific, UK) determines the encapsulation efficiency of lipid nanoparticles. The specific operation is as follows:
(1) The prepared mRNA-LNP suspension and PBS (negative control, equal volume of TE buffer) were diluted to 4 ng/. Mu.l with TE buffer in the kit to obtain mRNA-LNP working solution.
(2) The mRNA-LNP working solution was further diluted with TE buffer (or TE buffer containing 2% Triton-X100) and left to stand at 37℃for 10min after mixing (TE buffer without Triton-X100 was used to determine unencapsulated free mRNA, while TE buffer containing 2% Triton-X100 was used to determine Total mRNA in the mRNA-LNP working solution, including free mRNA and mRNA encapsulated in lipid nanoparticles).
(3) After calibrating the fluorescence intensity with the standard: after the standard curve of the concentration, a proper amount of Quant-it ™ RiboGreen RNA reagent nucleic acid dye is absorbed according to the instruction of the kit and added into each group of samples, the samples are dyed for 5min, each group of dyed samples are transferred into an enzyme-labeling instrument for detection, and the mRNA in the samples is accurately quantified by using software.
(4) mRNA encapsulation efficiency in lipid nanoparticles was calculated using the following formula:
encapsulation efficiency = [1-m (free mRNA): m (total mRNA) ]. Times.100% ], test results are shown in Table 1.
S8, LNP was prepared according to the 4-component formulation of the morganin coronal vaccine using the same preparation method using the commercial lipid SM-102 as control group 1, i.e. SM102: DSPC: cholesterol: the mole ratio of the phospholipid polyethylene glycol derivative DMG-PEG 2000 is 50:10:38.5:1.5.
S9, LNP was prepared according to a 3-component formulation without phospholipids, using the same preparation method, using the commercial lipid SM-102 as control group 2, i.e. SM102: cholesterol: the mole ratio of the phospholipid polyethylene glycol derivative DMG-PEG 2000 is 50:38.5:1.5.
TABLE 1
| mRNA lipid nanoparticle (mRNA-LNP) | Ionizable lipid compounds used | Particle size (nm) | Polydispersity index (PDI) | Encapsulation efficiency (%) |
| 1 | H-1 | 156.06 | 0.187 | 88 |
| 2 | H-2 | 157.56 | 0.138 | 89 |
| 3 | H-3 | 106.20 | 0.245 | 93 |
| 4 | H-4 | 107.37 | 0.233 | 93 |
| 5 | H-5 | 93.17 | 0.213 | 95 |
| 6 | H-6 | 95.57 | 0.226 | 96 |
| 7 | H-7 | 88.65 | 0.171 | 95 |
| 8 | H-8 | 75.03 | 0.075 | 94 |
| 9 | H-9 | 68.85 | 0.127 | 97 |
| 10 | H-10 | 67.40 | 0.133 | 95 |
| Control group 1 | SM-102 | 100.59 | 0.112 | 95 |
| Control group 2 | SM-102 | 117.56 | 0.170 | 94 |
Example 12 verification of delivery System Effect by in vivo animal experiments
The mRNA lipid nanoparticles 1 to 10 prepared in example 11 and the control sample were simultaneously injected into leg muscle of the mice at a dose of 5 μg mRNA/mouse, and the delivery effect was detected by calculating the fluorescence intensity of the injection site by photographing with an animal imaging apparatus after 24 hours. The results of the measurements are shown in Table 2 and FIG. 11.
TABLE 2
| mRNA lipid nanoparticle (mRNA-LNP) | Ionizable lipid compounds used | mRNA dose μg | Observation time point h | Total photon count p/s |
| 1 | H-1 | 5 | 24 | 1.907e+07 |
| 2 | H-2 | 5 | 24 | 2.922e+07 |
| 3 | H-3 | 5 | 24 | 8.659e+07 |
| 4 | H-4 | 5 | 24 | 9.894e+07 |
| 5 | H-5 | 5 | 24 | 2.208e+08 |
| 6 | H-6 | 5 | 24 | 2.890e+08 |
| 7 | H-7 | 5 | 24 | 3.489e+08 |
| 8 | H-8 | 5 | 24 | 3.747e+08 |
| 9 | H-9 | 5 | 24 | 4.104e+08 |
| 10 | H-10 | 5 | 24 | 5.843e+08 |
| Control group 1 | SM-102 | 5 | 24 | 5.424e+07 |
| Control group 2 | SM-102 | 5 | 24 | 8.928e+07 |
Example 13 accelerated stability test at 40℃
The mRNA lipid nanoparticles 1 to 10 prepared in example 11 and the mRNA lipid nanoparticles of the control sample were simultaneously placed in a drug stability test box at a set temperature of 40 ℃ and a humidity of 75%, and were taken out after being placed for 1 month. The average particle size and polydispersity index PDI of mRNA-LNP samples were determined using a dynamic light scattering nanoparticle size analyzer, and the encapsulation efficiency of lipid nanoparticles was determined using a Quant-it Ribogreen RNA quantification kit (ThermoFisher Scientific, UK). The results are shown in Table 3.
TABLE 3 Table 3
| mRNA lipid nanoparticle (mRNA-LNP) | Ionizable lipid compounds used | Size（nm） | Polydispersity index PDI | Encapsulation efficiency (%) |
| 1 | H-1 | 208.50 | 0.354 | 91 |
| 2 | H-2 | 204.14 | 0.275 | 91 |
| 3 | H-3 | 116.34 | 0.110 | 93 |
| 4 | H-4 | 116.23 | 0.118 | 95 |
| 5 | H-5 | 111.13 | 0.252 | 95 |
| 6 | H-6 | 105.44 | 0.275 | 97 |
| 7 | H-7 | 98.77 | 0.176 | 96 |
| 8 | H-8 | 99.77 | 0.267 | 99 |
| 9 | H-9 | 86.01 | 0.097 | 99 |
| 10 | H-10 | 73.84 | 0.107 | 99 |
| ControlGroup 1 | SM-102 | 199.43 | 0.391 | 90 |
| Control group 2 | SM-102 | 236.77 | 0.373 | 89 |
Example 14 in vivo animal safety test
The mRNA lipid nanoparticles 1 to 10 prepared in example 11 and the mRNA lipid nanoparticle of the control sample were simultaneously injected into the tail of the mice at a dose of 20 μg mRNA/mouse, and the clinical manifestations of the mice were observed, and physiological saline and formulation Buffer (PBS) were used as controls, and the results are shown in table 4.
TABLE 4 Table 4
| mRNA lipid nanoparticle Particle (mRNA-LNP) | The ionisable used Lipid compounds | mRNA administration Dose of mu g | Drug administration body Mu.l product | Clinical manifestations |
| 1 | H-1 | 20 | 200 | No obvious abnormality |
| 2 | H-2 | 20 | 200 | No obvious abnormality |
| 3 | H-3 | 20 | 200 | No obvious abnormality |
| 4 | H-4 | 20 | 200 | No obvious abnormality |
| 5 | H-5 | 20 | 200 | No obvious abnormality |
| 6 | H-6 | 20 | 200 | No obvious abnormality |
| 7 | H-7 | 20 | 200 | No obvious abnormality |
| 8 | H-8 | 20 | 200 | No obvious abnormality |
| 9 | H-9 | 20 | 200 | Activity was reduced 10min after the drug and recovery was achieved 30min after the drug |
| 10 | H-10 | 20 | 200 | Activity is reduced 10min after the medicine, and recovery is achieved 30min after the medicine |
| Control group 1 | SM-102 | 20 | 200 | Moderate paralysis, tetany, no creeping, body temperature lowering and death after the medicine is taken for 35min after the medicine is taken for 17min |
| Control group 2 | SM-102 | 20 | 200 | Moderate paralysis falls within 15min after the medicine, is loved to lie, has reduced body temperature and can not creep; urinary incontinence occurs in the middle course, and the limbs are twitched and shorted breath after the medicine is applied for 37minAnd die out |
| Preparation buffer | N/A | N/A | 200 | No obvious abnormality |
| Physiological saline | N/A | N/A | 200 | No obvious abnormality |
As can be seen from examples 11-14, the compounds of the present invention have the following advantages:
1. the preparation of mRNA-LNP with a 3-component formulation is simpler and less costly than the 4-component formulation of SM-102 dosage form of the Morgana novel crown vaccine, is convenient for production scale-up and is not limited by the 4-component formulation range of US8058069 patent in Arbutus.
2. According to the data in table 2, compounds H1 and H2, prepared mRAN-LNP, delivered in vivo in animals with the 4-component and 3-component formulation LNP delivery effect of the mordner SM-102 ionizable lipid compound were on the same order of magnitude, and compounds H3 and H4 delivered more than SM-102. The delivery effect of compound H5-H10 is an order of magnitude more advantageous than that of SM-102.
3. According to the data in Table 3, the 3-component formula mRAN-LNP prepared by the compounds H1 to H10 has good physical and chemical properties, and the accelerated stability at 40 ℃ is tested for 1 month, and the detection shows that the particle size is slightly increased, the whole structure is maintained, and mRNA can be well encapsulated in the LNP without leakage. After one month of standing at 40 ℃, the LNP prepared by the compounds H1-H10 has better overall encapsulation efficiency than the LNP prepared by SM-102.
4. Conventional mRNA vaccines are intramuscular injections, and some of the leading applications such as tumor immunomodulation treatment require intravenous administration of mRNA-LNP, which is a challenge to the safety of mRNA-LNP. According to the data in Table 4, the compounds H1 to H10 according to the invention, the 3-component formulation mRAN-LNP prepared, have very high safety. Large doses of intravenous injection (mice were injected with 20. Mu.g mRNA/mouse by hand, calculated according to a table in the pharmaceutical Experimental methodology on "equivalent dose ratio between human and animal in terms of body surface area", equivalent to 100ug/kg of human administration, which is far greater than 100 ug/human of the new crown vaccine, sufficient to cover the clinically maximum administered dose), no significant discomfort in compound H1-H8 mice, rapid relief of discomfort in compound H9 and H10 mice, and death in mice injected with the 3-and 4-component formulations of the SM-102 compound of the control group. The ionizable lipid compounds and the corresponding LNP formulations of the invention are shown to be excellent in safety far better than SM-102 compounds.
Claims (10)
1. An ionizable lipid compound having a structure according to formula (i):
m is 4, 5 and 6; r is R 1 、R 2 、R 3 、R 4 The radicals are each independently-OH; r is R 5 、R 6 、R 7 、R 8 The radicals are each independently linear C12, C13 or C14 alkyl radicals.
2. An ionizable lipid compound selected from any one of the following:
3. the ionizable lipid compound according to claim 2, characterized by being selected from any one of the following:
4. use of an ionizable lipid compound according to any one of claims 1-3 for the preparation of a nano-lipid formulation.
5. A nanolipid particle composition comprising the ionizable lipid compound of any one of claims 1-3.
6. The nanolipid particle composition according to claim 5, further comprising one or more of neutral lipids, phospholipid polyethylene glycol derivatives.
7. The nanolipid particle composition according to claim 6, wherein the neutral lipid is selected from any one or both of DOPE and cholesterol.
8. The nanoparticle composition of claim 6, wherein the phospholipid polyethylene glycol derivative is selected from the group consisting of phospholipid polyethylene glycol derivative DMG-PEG 2000.
9. The nanolipid particle composition according to claim 5, characterized in that it consists of an ionizable lipid compound according to any one of claims 1-3, cholesterol and a phospholipid polyethylene glycol derivative DMG-PEG 2000.
10. Use of the nanolipid particle composition of claim 5 in the preparation of an mRNA drug or vaccine.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102245559A (en) * | 2008-11-07 | 2011-11-16 | 麻省理工学院 | Aminoalcohol lipidoids and uses thereof |
| CA3206923A1 (en) * | 2021-02-05 | 2022-08-11 | Zihao Wang | Ionizable lipid molecule, preparation method therefor, and application thereof in preparation of lipid nanoparticle |
| CN114957164A (en) * | 2022-06-10 | 2022-08-30 | 华南理工大学 | Lipid compound and preparation method and application thereof |
| CN115154439A (en) * | 2022-09-08 | 2022-10-11 | 南京澄实生物科技有限公司 | mRNA lipid nanoparticle delivery system and preparation method and application thereof |
| CN115403474A (en) * | 2021-05-28 | 2022-11-29 | 北京启辰生生物科技有限公司 | Lipid compounds and their use in nucleic acid delivery |
| WO2023136688A1 (en) * | 2022-01-17 | 2023-07-20 | 에스티팜 주식회사 | Ionizable lipid containing biodegradable disulfide bond and lipid nanoparticles comprising same |
| CN116574070A (en) * | 2023-04-07 | 2023-08-11 | 华南理工大学 | Multi-tail type ionizable lipid, and preparation method and application thereof |
-
2023
- 2023-10-26 CN CN202311395529.3A patent/CN117164468B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102245559A (en) * | 2008-11-07 | 2011-11-16 | 麻省理工学院 | Aminoalcohol lipidoids and uses thereof |
| CA3206923A1 (en) * | 2021-02-05 | 2022-08-11 | Zihao Wang | Ionizable lipid molecule, preparation method therefor, and application thereof in preparation of lipid nanoparticle |
| CN115403474A (en) * | 2021-05-28 | 2022-11-29 | 北京启辰生生物科技有限公司 | Lipid compounds and their use in nucleic acid delivery |
| WO2023136688A1 (en) * | 2022-01-17 | 2023-07-20 | 에스티팜 주식회사 | Ionizable lipid containing biodegradable disulfide bond and lipid nanoparticles comprising same |
| CN114957164A (en) * | 2022-06-10 | 2022-08-30 | 华南理工大学 | Lipid compound and preparation method and application thereof |
| CN115154439A (en) * | 2022-09-08 | 2022-10-11 | 南京澄实生物科技有限公司 | mRNA lipid nanoparticle delivery system and preparation method and application thereof |
| CN116574070A (en) * | 2023-04-07 | 2023-08-11 | 华南理工大学 | Multi-tail type ionizable lipid, and preparation method and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| From lonic Liquid Epoxy Monomer to Tunable Epoxy-Amine Network: Reaction Mechanism and Final Properties;Livi Sebastien等;《ACS Sustainable Chemistry & Engineering》;第7卷(第3期);第3602-3613页 * |
| Kevin T. Love等.Lipid-like materials for low-dose,in vivo gene silencing.《PNAS》.2010,第107卷(第5期),第1864-1869页. * |
| RNA药物递送载体的研究进展;娄瑞等;《生命科学》;第35卷(第08期);第1012-1022页 * |
| 体外转录合成mRNA递送材料的研究进展;韩语诚等;《现代医药卫生》;第39卷(第16期);第2789-2794页 * |
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