WO2023029928A1 - Aminolipide et son application - Google Patents

Aminolipide et son application Download PDF

Info

Publication number
WO2023029928A1
WO2023029928A1 PCT/CN2022/111656 CN2022111656W WO2023029928A1 WO 2023029928 A1 WO2023029928 A1 WO 2023029928A1 CN 2022111656 W CN2022111656 W CN 2022111656W WO 2023029928 A1 WO2023029928 A1 WO 2023029928A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino lipid
cancer
preparation
lipid
amino
Prior art date
Application number
PCT/CN2022/111656
Other languages
English (en)
Chinese (zh)
Inventor
查高峰
彭星华
夏恒
Original Assignee
深圳虹信生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳虹信生物科技有限公司 filed Critical 深圳虹信生物科技有限公司
Publication of WO2023029928A1 publication Critical patent/WO2023029928A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/30—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/31—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atoms of the sulfonamide groups bound to acyclic carbon atoms
    • C07C311/32—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atoms of the sulfonamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C303/00—Preparation of esters or amides of sulfuric acids; Preparation of sulfonic acids or of their esters, halides, anhydrides or amides
    • C07C303/02—Preparation of esters or amides of sulfuric acids; Preparation of sulfonic acids or of their esters, halides, anhydrides or amides of sulfonic acids or halides thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C303/00—Preparation of esters or amides of sulfuric acids; Preparation of sulfonic acids or of their esters, halides, anhydrides or amides
    • C07C303/36—Preparation of esters or amides of sulfuric acids; Preparation of sulfonic acids or of their esters, halides, anhydrides or amides of amides of sulfonic acids
    • C07C303/38—Preparation of esters or amides of sulfuric acids; Preparation of sulfonic acids or of their esters, halides, anhydrides or amides of amides of sulfonic acids by reaction of ammonia or amines with sulfonic acids, or with esters, anhydrides, or halides thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/12—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms
    • C07D295/125—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/13—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C07D309/08—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D309/14—Nitrogen atoms not forming part of a nitro radical
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
    • C07C2601/14—The ring being saturated

Definitions

  • the invention relates to the technical field of medicinal chemistry, in particular to an amino lipid and its application.
  • Gene medicine refers to the introduction of exogenous normal genes into target cells to correct or compensate diseases caused by gene defects and abnormalities to achieve therapeutic purposes; or the genes are expressed to produce corresponding antigens, thereby inducing memory immune responses.
  • gene medicine has pushed the research from preclinical to clinical in many disease treatment fields, and has irreplaceable advantages for diseases caused by genetic abnormalities that are difficult to solve in the medical field, such as tumors.
  • Common gene medicines include plasmid DNA (plasmid DNA, pDNA), antisense oligonucleotide (antisense ODN), small interfering RNA (siRNA) and messenger RNA (mRNA).
  • the gene carrier has to go through multiple complex processes when transporting the gene: reaching the target cell through blood circulation, cell uptake, escape of endosomes, intracellular movement, and release of gene material by the carrier. Its main barriers are mainly extracellular barriers in the complex blood environment and intracellular barriers in the degradation of lysosomal enzymes. Therefore, it is an urgent problem for gene carrier researchers to find a good gene carrier to make the target gene reach the target site and play a role.
  • viral vector system a natural carrier resource.
  • Viral genome has simple structure, high transfection efficiency, and strong target cell specificity.
  • its disadvantages such as poor orientation, low carrying capacity, and immunogenicity limit its use. Therefore, the non-viral vector system with diversity, non-immunogenicity and easy-to-control production has attracted much attention in recent years and has been applied in many therapeutic fields.
  • Commonly used non-viral vector systems are mainly cationic lipids vectors.
  • Cationic lipids have three important structural regions: a positively charged hydrophilic polar head gene, a connecting chain in the middle responsible for connecting the polar and nonpolar ends, and a hydrophobic lipid chain.
  • the polar head containing amine groups plays a role in the interaction between liposome and RNA, liposome/RNA complex and cell membrane, affects the charge of lipid, and plays a major role in the process of lysosome escape.
  • Linking chains determine the chemical and biological stability of cationic liposomes, especially the resulting cytotoxicity.
  • the hydrophobic region can be in the form of a carbon chain or a variety of structures such as steroids, and the length of the carbon chain, whether it is saturated or not, and the specific type will affect the behavior of the lipid, which not only provides sufficient fluidity for the lipid bilayer, but also promotes the formation of cationic liposomes. Lipid fusion in vivo.
  • Cationic liposomes form liposome/gene complexes through electrostatic interactions with negatively charged groups.
  • the complex is positively charged due to the excess of cationic liposomes, and the positively charged liposome/gene complex is adsorbed on the negatively charged cell surface due to electrostatic interaction. It then enters the cell by fusion with the cell membrane or by endocytosis.
  • the main feature of cationic lipids for gene therapy is charge-influenced membrane fusion during endosomal escape. But at the same time, the excess positive charge of the cationic lipid/gene complex and the refractory characteristics of some cationic lipids also lead to cytotoxicity. Therefore lower transfection efficiency and cytotoxicity are the main disadvantages limiting the application of cationic lipids.
  • the present invention attempts to design ionizable cationic lipids to solve the above problems, so as to achieve better gene therapy effect,
  • the invention provides an amino lipid and its application.
  • a kind of amino lipid, its structure is as shown in formula (I):
  • L is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene;
  • R2, R3, R4 and R5 are the same or different from each other, and each independently selected from H, C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl; said C1-C24 alkyl, C2-C24 alkenyl Base, C2-C24 alkynyl can be optionally substituted by C1-C6 hydrocarbon group;
  • R2 and R3 are connected to form a 4-10-membered heterocycle, wherein the multi-membered heterocycle contains 1-6 heteroatoms, and the heteroatoms are selected from nitrogen, sulfur or oxygen.
  • said R2 is selected from C6-C24 alkyl, C6-C24 alkenyl, C6-C24 alkynyl; said C6-C24 alkyl, C6-C24 alkenyl, C6-C24 alkynyl can be optionally Substituted by C1-C6 hydrocarbyl.
  • L and R1 are connected to form NH2-L-R1 selected from A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14 , A15, A16, A17, A18, A19, A20, A21, A22, A23, A24, A25, A26, A27, A28, A29, A30, A31, A32, A33, A34, A35, A36, A37, A38, A39 , A40 in one.
  • L and R1 are linked to N atom after linking.
  • the position substituted by NH2 in the above NH2-L-R1 is the free radical position connected to the compound of formula (I).
  • R2, R3 and adjacent N atoms form R2R3-NH, wherein H is the position of a free radical;
  • R2R3-NH is selected from N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, One of N11, N12, N13, N14, N15, N16, N17, N18, N19, N20, N21, N22, N23.
  • R2 and R3 are connected to the same N atom, and the N atom is connected to the S atom on the sulfonyl group.
  • the H atom in the above R2R3-NH is the position connected to the S atom in the formula (I).
  • the preparation method of described amino lipid comprises the following steps:
  • step S2 Add R2R3NH to the reaction system in step S1, and heat the reaction in the presence of a base.
  • the method comprises the steps of:
  • the aminolipid compound of formula I is obtained.
  • the heating temperature in the above step S2 is 50-120°C.
  • the base used in the above preparation method is an organic base or an inorganic base, such as: triethylamine, DIPEA, pyridine, DMAP, sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate and the like.
  • amino lipid and pharmaceutically acceptable salts, prodrugs or stereoisomers thereof in the preparation of medicaments for gene therapy, gene vaccination, antisense therapy or therapy by interfering RNA.
  • the above application is an application in the preparation of therapeutic drugs for cancer or genetic diseases.
  • the above-mentioned application is the application in the preparation of medicines for lung cancer, gastric cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphatic cancer, blood cancer or prostate cancer, and the genetic disease is hemophilia, Mediterranean One or more of anemia, Gaucher's disease.
  • the above application is the application in the preparation of medicines for treating cancer, allergy, toxicity and pathogenic infection.
  • the above application is the application in the preparation of medicines for nucleic acid transfer.
  • the nucleic acid is RNA, messenger RNA (mRNA), antisense oligonucleotide, DNA, plasmid, ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small inhibitory RNA ( siRNA) and small nuclear RNA (snRNA).
  • mRNA messenger RNA
  • rRNA ribosomal RNA
  • miRNA microRNA
  • tRNA transfer RNA
  • siRNA small inhibitory RNA
  • snRNA small nuclear RNA
  • An aminolipid compound disclosed by the present invention uses vinyl sulfonyl fluoride (ESF), a bifunctional electrophile, to construct an amino head group and a hydrophobic chain into the aminolipid, making full use of the advantages of ESF Click chemistry reaction characteristics, mild reaction conditions in the process of constructing amino lipids, no need for protection and deprotection, high atom economy. In in vitro and in vivo delivery studies, it shows excellent ability to deliver nucleic acid to cells.
  • the above-mentioned amino lipid compound has two sulfonamides, and the introduction of this group significantly enhances the stability of the lipid nanoparticle, improves the circulation time in vivo, and thus improves the delivery efficiency in vivo.
  • the preparation method of the amino lipid compound has the advantages of readily available raw materials, mild reaction conditions, good reaction selectivity, high reaction yield, low requirements for instruments and equipment and simple operation.
  • OVA mRNA stimulates BMDC in Fig. 1 embodiment 10, differentiate into the ratio figure of the cell population that presents OVA antigen;
  • test methods used in the following experimental examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available reagents and materials unless otherwise specified.
  • PEG2000-DMG (1-(Monomethoxypolyethylene glycol)-2,3 Dimyristoylglycerol
  • Embodiment 1 Parallel synthesis and characterization of A1Ny series amino amino lipid compound library
  • Step I reaction solution In a 250mL reaction flask, add n-hexylamine (25.3mg, 0.25mmol), vinylsulfonyl fluoride (55mg, 0.5mmol), anhydrous tetrahydrofuran 2.5mL, and stir at room temperature for 5min to obtain the Step I reaction solution (2.5 mL, 0.1M).
  • Table 1 MW/z values of A1Ny series amino amino lipid compound library
  • HeLa cell line HeLa cell line (ATCC)
  • DMEM Human fetal bovine serum
  • Detection Percentage of GFP fluorescent cell number relative to total cell number (determined using nuclear dye Hoechst - see Figure 2). Lipofectamine 2000 (Invitrogen) was used as a positive control group according to the manufacturer's instructions.
  • Method Use an 8-channel pipette to add samples. Amounts shown are for a single well of a 96-well plate.
  • the amino lipid compound described in Example 1 is mixed with dioleoylphosphatidylethanolamine (DOPE), cholesterol, and the molar ratio of PEG2000-DMG is 45:10:42.5:2.5 and dissolved in absolute ethanol ;
  • the mass ratio of aminolipid compound to green fluorescent protein mRNA (EGFP mRNA) is about 8:1, and the amount of mRNA per hole is 100ng.
  • lipid nanoparticle solution was incubated at room temperature for 30 min, 90 ⁇ L of freshly resuspended HeLa cells (3-5 ⁇ 10 4 cells) were added and mixed with a pipette. Transfer 100 ⁇ L of cells+lipid nanoparticles to separate wells of a 96-well culture plate and place in an incubator at 37 °C with 5% CO2 .
  • Hoechst33258 (Invitrogen) was added to the cells at a final concentration of 0.2 ⁇ g/ml, and incubated at 37° C. for 15 minutes in the dark. Then the cells were washed once with PBS solution, and culture medium was added for 20 to 24 hours.
  • Embodiment 8 Transfection of lipid nanoparticles prepared by aminolipid compounds on BMDC primary cells
  • Animal preparation 6-week-old female C57BL/6 mice were selected, weighing about 20 g, and the feeding environment was an SPF-grade breeding room. Animal experiments were carried out in strict accordance with the guidelines of the national health agency and animal ethics requirements.
  • lipid nanoparticles encapsulating luciferase mRNA to the 96-well all-white ELISA plate covered with primary cells, and control the amount of luciferase mRNA lipid nanoparticles added in each well to 3ug . Then place it in an incubator at 37° C. with 5% CO 2 concentration for 12 hours to fully express luciferase mRNA.
  • Detection of transfection efficiency Add 10ul of 10mg/ml D-luciferin potassium salt to each well of a 96-well all-white microplate plate, and immediately place it in a microplate reader to detect the luminescence intensity.
  • the expression intensity of Fluc mRNA transfected by representative aminolipid compounds on BMDC is shown in Table 3.
  • DLin-MC3 was used as a control, and the expression intensity of many amino lipids was similar to that of MC3, and some of them were significantly better than the positive control.
  • Example 9 In vivo delivery performance evaluation of luciferase mRNA of lipid nanoparticles prepared from amino lipid compounds
  • the amino lipid compound of the present invention is mixed with DOPE, cholesterol, (1-(monomethoxypolyethylene glycol)-2,3 dimyrisyl glycerol (PEG2000-DMG) according to the ratio of 45:10:42.5:2.5
  • the molar ratio is mixed and dissolved in absolute ethanol, so that the molar concentration of the amino lipid compound is 0.001-0.01mmol/L.
  • TriLink FLuc-mRNA
  • the particle size and PDI of the prepared lipid nanoparticles were measured by Nano-ZSZEN3600 (Malvern). Take 40uL of LNP solution for particle size measurement, and cycle three times, each cycle 30s.
  • Encapsulation rate detection use The RNA HS Assay kit was used to detect the concentration of LNP RNA. Theoretical RNA concentration is the amount of total RNA input divided by the total volume of the final solution.
  • the preparation method is the same as the preparation method 1, except that amino lipid compound, DSPC, cholesterol and PEG2000-DMG are used in a molar ratio of 50:10:38.5:1.5.
  • the resulting lipid nanoparticle (LNP) solution was administered to the test animals by tail vein and intramuscular injection.
  • In vivo delivery 9 C57BL/6 mice were randomly selected in each group, and the dosage of 0.5mg/kg mRNA was injected into the lipid nanoparticle solution by subcutaneous, intramuscular and tail vein injection respectively (each administration method 3 mice). After 12 hours, 200 ⁇ L of 10 mg/mL D-luciferin potassium salt was injected into each mouse through the tail vein, and after 10 minutes, the mouse was placed under an in vivo imaging system (IVIS-200, Xenogen), and each mouse was observed. The total fluorescence intensity of each mouse was recorded by taking pictures. The expression intensities of Fluc mRNA delivered by representative amino lipid compounds through the three administration methods are shown in Table 6-8. DLin-MC3 served as a control.
  • Table 8 Expression intensity of Fluc mRNA delivered by tail vein administration of representative aminolipid compounds
  • Preparation method mix and dissolve the amino lipid compound described in the present invention with DOPE, cholesterol, and PEG2000-DMG in a molar ratio of 45:10:42.5:2.5 in absolute ethanol.
  • Use two micro-injection pumps, control the ratio of ethanol solution to sodium acetate solution (50mM, pH 4.0) to be 1:3, prepare a crude solution of lipid nanoparticles in the micro-channel chip, and then use Dialysis cassettes (Fisher, MWCO 20,000) were dialyzed in 1X PBS at a temperature controlled temperature of 4°C for 6 h, and filtered through a 0.22 ⁇ m microporous membrane before use.
  • the mass ratio of amino lipid compound to ovalbumin mRNA (OVA mRNA) is about 8:1.
  • Animal preparation 6-week-old female C57BL/6 mice were selected, weighing about 20 g, and the feeding environment was an SPF-grade breeding room. Animal experiments were carried out in strict accordance with the guidelines of the national health agency and animal ethics requirements.
  • Activation of immune cells add 1ug of ovalbumin mRNA lipid nanoparticles to each well of a 12-well plate, and place in a 37°C, 5% CO 2 incubator for 24 hours.
  • the cells were blown down with PBS solution, washed and centrifuged (100g, 5 minutes) with PBS three times, and then incubated with CD11c-APC antibody and SIINFEKL-H-2Kb-PE antibody, CD11c-APC antibody and MHC-II-PE antibody for 30 Minutes, then washed with PBS and centrifuged (100g, 5 minutes) once to remove unbound antibodies, and then detected by flow cytometry (Beckman cytoflex LX).
  • CD11 is a marker of BMDC
  • the CD11c-APC antibody is used for the labeling of the DC population
  • the SIINFEKL-H-2Kb-PE antibody is used for labeling the cell population presenting the OVA antigen in the cell population
  • the MHC-II-PE antibody is used for labeling Mature DC population.
  • Example 11 Evaluation of in vivo delivery performance of luciferase mRNA of lipid nanoparticles prepared from aminolipid compounds
  • Preparation method mix and dissolve the amino lipid compound described in the present invention with DOPE, cholesterol, and PEG2000-DMG in a molar ratio of 45:10:42.5:2.5 in absolute ethanol.
  • Animal preparation 6-week-old female C57BL/6 mice were selected, weighing about 20 g, and the feeding environment was an SPF-grade breeding room. Animal experiments were carried out in strict accordance with the guidelines of the national health agency and animal ethics requirements.
  • mice were randomly selected in each group, and lipid nanoparticles were injected subcutaneously at a dose of 0.5 mg/kg. Six hours later, each mouse was injected with 200 ⁇ L of 10 mg/mL D-luciferin potassium salt through the tail vein, and after 10 minutes, the mice were placed under an in vivo imaging system (IVIS-200, Xenogen) to observe The total fluorescence intensity of each mouse was taken and recorded.
  • IVIS-200, Xenogen in vivo imaging system
  • the expression intensity of Fluc mRNA delivered by representative aminolipid compounds is shown in Table 10, and DLin-MC3 was used as a control.
  • the expression intensity of the multiple amino lipids is similar to that of Dlin-MC3, and some of them are significantly better than the positive control.
  • Example 12 In vivo delivery of ovalbumin mRNA and immune performance evaluation of lipid nanoparticles prepared from amino lipid compounds
  • Preparation method mix and dissolve the amino lipid compound described in the present invention with DOPE, cholesterol, and PEG2000-DMG in a molar ratio of 45:10:42.5:2.5 in absolute ethanol.
  • Use two micro-injection pumps, control the ratio of ethanol solution to sodium acetate solution (50mM, pH 4.0) to be 1:3, prepare a crude solution of lipid nanoparticles in the micro-channel chip, and then use Dialysis cassettes (Fisher, MWCO 20,000) were dialyzed in 1X PBS at a temperature controlled temperature of 4°C for 6 h, and filtered through a 0.22 ⁇ m microporous membrane before use.
  • the mass ratio of amino lipid compound to ovalbumin mRNA (OVA mRNA) is about 8:1.
  • Animal preparation 6-week-old female C57BL/6 mice were selected, weighing about 20 g, and the feeding environment was an SPF-grade breeding room. Animal experiments were carried out in strict accordance with the guidelines of the national health agency and animal ethics requirements.
  • mice were randomly selected in each group, and lipid nanoparticles were injected subcutaneously at a dose of 0.5 mg/kg (Day 0). After 7 days, use the same amount for another booster (Day 7). On the 21st day, blood was collected from the tail vein for serological analysis, and DLin-MC3 was used as a control.
  • Enzyme-linked immunosorbent assay (ELISA): flat-bottomed 96-well plates (Nunc) were pre-coated in 50 mM carbonate buffer, the concentration of OVA protein was 0.5 ⁇ g protein per well (pH 9.6) overnight at 4 ° C, and then used 5% glycine blocked. The sera of the immunized animals were diluted from 10 ⁇ 2 to 10 ⁇ 6 using PBS-0.05% Tween (PBS-T, pH 7.4), added to the wells and incubated at 37° C. for 1 hour. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG was labeled at a dilution of 1:10,000 in PBS-T-1% BSA.
  • HRP horseradish peroxidase
  • the absorbance at 450 nm was detected in an ELISA plate reader (Bio-Rad) to determine the optical density at one wavelength.
  • the IgG antibody titers produced by A14N13 and MC3 were comparable, while the IgG antibody titers of A32N22, A34N22, and A35N20 were significantly better than the MC3 control group.
  • Example 13 In vivo immune and tumor therapeutic effect evaluation of lipid nanoparticles prepared from aminolipid compounds
  • Preparation method mix and dissolve the amino lipid compound described in the present invention with DOPE, cholesterol, and PEG2000-DMG in a molar ratio of 45:10:42.5:2.5 in absolute ethanol.
  • Use two micro-injection pumps, control the ratio of ethanol solution to sodium acetate solution (50mM, pH 4.0) to be 1:3, prepare a crude solution of lipid nanoparticles in the micro-channel chip, and then use Dialysis cassettes (Fisher, MWCO 20,000) were dialyzed in 1X PBS at a temperature controlled temperature of 4°C for 6 h, and filtered through a 0.22 ⁇ m microporous membrane before use.
  • the mass ratio of amino lipid compound to ovalbumin mRNA (OVA mRNA) is about 8:1.
  • Animal preparation 6-week-old female C57BL/6 mice were selected, weighing about 20g, and the feeding environment was an SPF-grade breeding room. Animal experiments were carried out in strict accordance with the guidelines of the national health agency and animal ethics requirements.
  • B16-OVA melanoma cells (1.5 x 105 ) were injected subcutaneously into the right side of 4-6 week old mice. Vaccination was started when the tumor size was less than 50 mm3 (approximately on day 4 or day 5 after tumor inoculation). Animals were immunized by intramuscular injection of LNP preparations containing 15 ⁇ g of OVA-mRNA. Tumor growth was measured 3 times a week using a digital caliper, and the calculation formula was 0.5 ⁇ length ⁇ width. Euthanize the mice when the tumor volume reaches 2,000 mm. Tumor suppression was compared to mice bearing freshly inoculated tumors. The median survival period of the non-vaccinated group was 29 weeks. After vaccination, the corresponding median survival periods were: 38 weeks (MC3), 49 weeks (A32N22), and 52 weeks (A35N20), as shown in Figure 4.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Health & Medical Sciences (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Oncology (AREA)
  • Biotechnology (AREA)
  • Plant Pathology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Communicable Diseases (AREA)
  • Molecular Biology (AREA)
  • Toxicology (AREA)
  • Pulmonology (AREA)
  • Immunology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

L'invention concerne un aminolipide et son application, à l'aide de fluorure d'éthènesulfonyle (ESF), un électrophile à double fonctionnalité, pour construire un groupe de tête amine et une chaîne hydrophobe dans un aminolipide, utilisant complètement une réaction de chimie clic de l'ESF ; dans le procédé de construction de l'aminolipide, les conditions de réaction sont ménagées, une protection et une déprotection ne sont pas nécessaires et l'économie en atomes est élevée. Des études d'administration in vitro et in vivo présentent une excellente capacité à introduire un acide nucléique dans les cellules. Le composé aminolipide est pourvu de deux sulfonamides, et l'introduction du groupe amplifie significativement la stabilité des nanoparticules lipidiques et améliore le temps de cycle in vivo, ce qui améliore l'efficacité de l'administration in vivo. Le procédé de préparation du composé aminolipide présente les avantages de matières premières aisément disponibles, de conditions de réaction ménagées, d'une bonne sélectivité de la réaction, d'un rendement élevé de la réaction, de faibles besoins en instruments et équipement, et d'opérations simples.
PCT/CN2022/111656 2021-09-01 2022-08-11 Aminolipide et son application WO2023029928A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111017566.1 2021-09-01
CN202111017566.1A CN113461577B (zh) 2021-09-01 2021-09-01 一种氨基脂质及其应用

Publications (1)

Publication Number Publication Date
WO2023029928A1 true WO2023029928A1 (fr) 2023-03-09

Family

ID=77867037

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/111656 WO2023029928A1 (fr) 2021-09-01 2022-08-11 Aminolipide et son application

Country Status (2)

Country Link
CN (1) CN113461577B (fr)
WO (1) WO2023029928A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113461577B (zh) * 2021-09-01 2021-12-14 中山大学附属第七医院(深圳) 一种氨基脂质及其应用
CN113880735A (zh) * 2021-11-08 2022-01-04 湖北九宁化学科技有限公司 一种双氟磺酰二乙胺锂的制备方法
WO2023125184A1 (fr) * 2021-12-29 2023-07-06 华南理工大学 Composé lipidique d'amine tertiaire et son utilisation

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1041361A (zh) * 1988-09-06 1990-04-18 邻位药品公司 乙磺酰胺衍生物的制备
CN101346468A (zh) * 2005-06-15 2009-01-14 麻省理工学院 含胺脂质和其用途
CN109414408A (zh) * 2016-05-16 2019-03-01 得克萨斯州大学系统董事会 阳离子磺酰胺氨基脂质和两亲性两性离子氨基脂质
WO2021055849A1 (fr) * 2019-09-19 2021-03-25 Modernatx, Inc. Composés lipidiques à têtes polaires et compositions pour administration intracellulaire d'agents thérapeutiques
CN113461577A (zh) * 2021-09-01 2021-10-01 中山大学附属第七医院(深圳) 一种氨基脂质及其应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004292331A (ja) * 2003-03-26 2004-10-21 Sankyo Co Ltd クロマン類縁化合物
CN100588401C (zh) * 2004-06-28 2010-02-10 沈阳药科大学 磺胺类化合物脂质体的制备方法及其制剂
US20080187998A1 (en) * 2007-02-01 2008-08-07 University Of Utah Research Foundation Sulfonamide-based oligomers and polymers for destabilization of biological membranes
CA2984026C (fr) * 2008-10-09 2020-02-11 Arbutus Biopharma Corporation Lipides amines ameliores et procedes d'administration d'acides nucleiqu s

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1041361A (zh) * 1988-09-06 1990-04-18 邻位药品公司 乙磺酰胺衍生物的制备
CN101346468A (zh) * 2005-06-15 2009-01-14 麻省理工学院 含胺脂质和其用途
CN109414408A (zh) * 2016-05-16 2019-03-01 得克萨斯州大学系统董事会 阳离子磺酰胺氨基脂质和两亲性两性离子氨基脂质
WO2021055849A1 (fr) * 2019-09-19 2021-03-25 Modernatx, Inc. Composés lipidiques à têtes polaires et compositions pour administration intracellulaire d'agents thérapeutiques
CN113461577A (zh) * 2021-09-01 2021-10-01 中山大学附属第七医院(深圳) 一种氨基脂质及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GAN LIN, OLSON JENNIFER L., RAGSDALE CLIFTON W., YU LUPING: "Poly(β-aminosulfonamides) as gene delivery vectors: synthesis and in vitro screening", CHEMICAL COMMUNICATIONS, ROYAL SOCIETY OF CHEMISTRY, UK, no. 5, 13 September 2007 (2007-09-13), UK , pages 573 - 575, XP093042483, ISSN: 1359-7345, DOI: 10.1039/B714278A *

Also Published As

Publication number Publication date
CN113461577A (zh) 2021-10-01
CN113461577B (zh) 2021-12-14

Similar Documents

Publication Publication Date Title
WO2023029928A1 (fr) Aminolipide et son application
US9511024B2 (en) Amino lipids, their synthesis and uses thereof
AU2004246904B2 (en) Sphingolipids polyalkylamine conjugates for use in transfection
EP4282855A1 (fr) Molécule lipidique ionisable, son procédé de préparation et son utilisation dans la préparation de nanoparticules lipidiques
WO2023236976A1 (fr) Composé lipidique et procédé de préparation s'y rapportant, et son utilisation
WO2023142600A1 (fr) Lipide aminé, son procédé de préparation et son utilisation
WO2021170034A1 (fr) Composé aminolipide, son procédé de préparation et son application
CN113968968B (zh) 氨基脂质化合物、其制备方法和应用
KR102560772B1 (ko) 신규한 이온화지질 및 이를 이용한 지질나노입자 조성물
CN116574070A (zh) 一种多尾型可电离脂质及其制备方法与应用
AU737579B2 (en) New class of cationic transfecting agents for nucleic acids
CN115073316B (zh) 长链烷基酯胺类脂质化合物及其制备方法和在核酸递送方面的应用
CN115521220B (zh) 长链烷基酯胺类化合物及其制备方法和在核酸递送方面的应用
Soltan et al. Design and Synthesis of N 4, N 9-Disubstituted Spermines for Non-viral siRNA Delivery–Structure-Activity Relationship Studies of siFection Efficiency Versus Toxicity
CN113214171B (zh) 两亲性树形分子、合成及其作为药物递送系统的应用
US6812218B2 (en) Lipid derivatives of polythiourea
EP3141582B1 (fr) Synthèse et utilisation de polyalkylamines
Wu et al. Evaluation of pentaerythritol-based and trimethylolpropane-based cationic lipidic materials for gene delivery
CN114874106B (zh) 一种氨基脂质及其制备方法和应用
CN117257965B (zh) 一种核酸递送载体组合物及其应用
WO2023222081A1 (fr) Composé lipidique d'amine d'ester alkylique à longue chaîne, son procédé de préparation et son utilisation dans l'administration d'acide nucléique
AU759301B2 (en) New agents for transferring nucleic acids, compositions containing them and their uses
JP2005530771A (ja) 生物学的活性物質の細胞への運搬に適した複合体
CN114805410A (zh) 一类两亲性树形分子、合成及其在核酸递送方面的应用
WO2023190166A1 (fr) Lipide cationique ayant une liaison disulfure, structure de membrane lipidique le comprenant, agent d'introduction d'acide nucléique et composition pharmaceutique contenant l'un quelconque de ceux-ci, procédé d'introduction d'acide nucléique dans une cellule ou une cellule cible, et procédé de production d'un produit pharmaceutique cellulaire

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22863069

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE