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  • C07C275/04—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms
  • C07C275/06—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms of an acyclic and saturated carbon skeleton
  • C07C275/14—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms of an acyclic and saturated carbon skeleton being further substituted by nitrogen atoms not being part of nitro or nitroso groups
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  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
  • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
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  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
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  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
  • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
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  • A61K9/51—Nanocapsules; Nanoparticles
  • A61K9/5107—Excipients; Inactive ingredients
  • A61K9/513—Organic macromolecular compounds; Dendrimers
  • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
• • A—HUMAN NECESSITIES
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  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • 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
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/53—DNA (RNA) vaccination
• • A—HUMAN NECESSITIES
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
  • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
  • A61K2039/6018—Lipids, e.g. in lipopeptides
• • C—CHEMISTRY; METALLURGY
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  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/20011—Coronaviridae
  • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• the present invention provides a use of the aforementioned compound in preparing liposomes, drug carriers or complexes.
• the aforementioned compound has an ionizable property and can be used to prepare liposomes, and the prepared liposomes can be used as drug carriers to form nucleic acid drug-liposome complexes.
• the present invention provides a drug carrier.
• the drug carrier includes the aforementioned compound or the aforementioned liposome.
• the aforementioned compound is an ionizable lipid
• the drug carrier is an ionizable carrier
• the aforementioned compound or liposome can be used to load the drug and deliver the drug into the cell.
• FIG8 is the H1-NMR spectrum of compound 10 of the present invention.
• FIG9 is the H1-NMR spectrum of compound 11 of the present invention.
• FIG12 is the H1-NMR spectrum of compound 15 of the present invention.
• Figure 16 is the H1-NMR spectrum of compound 19 of the present invention.
• the compounds of the present invention comprising the aforementioned isotopes and/or other isotopes of other atoms and pharmaceutically acceptable salts of the compounds are all included within the scope of the present invention.
• Isotope-labeled compounds of the present invention such as radioactive isotopes, such as 3 H and 14 C are incorporated into the compounds of the present invention and can be used for drug and/or substrate tissue distribution analysis. Due to ease of preparation and detection, tritiated, i.e. 3 H, and carbon-14, i.e. 14 C, isotopes are particularly preferred.
• substitution with heavy isotopes, such as deuterium, i.e. 2 H can provide some therapeutic advantages derived from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements. Therefore, it may be preferred in some cases.
• tautomer or “tautomeric form” refers to structural isomers of different energies that are interconvertible via a low energy barrier. If tautomerism is possible (such as in solution), a chemical equilibrium of the tautomers can be achieved.
• proton tautomers also known as prototropic tautomers
• Valence tautomers include interconversions that occur via reorganization of some of the bonding electrons.
• keto-enol tautomerism is the interconversion of pentane-2,4-dione and 4-hydroxypent-3-en-2-one tautomers.
• Another example of tautomerism is phenol-keto tautomerism.
• a specific example of phenol-keto tautomerism is the interconversion of pyridine-4-ol and pyridine-4(1H)-one tautomers. Unless otherwise indicated, all tautomeric forms of the compounds of the invention are within the scope of the invention.
• solvate refers to an association formed by one or more solvent molecules and the compounds of the present invention.
• Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, aminoethanol.
• hydrate refers to an association formed by a solvent molecule that is water.
• pharmaceutically acceptable salt refers to organic and inorganic salts of the compounds of the present invention.
• Pharmaceutically acceptable salts are well known in the art, as described in the literature: SM Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19.
• salts formed by non-toxic acids include, but are not limited to, inorganic acid salts (such as hydrochlorides, hydrobromides, phosphates, sulfates, perchlorates) formed by reaction with amino groups, and organic acid salts (such as acetates, oxalates, maleates, tartrates, citrates, succinates, malonates), or other methods described in books and literature, such as ion exchange methods, to obtain these salts.
• inorganic acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, perchlorates
• organic acid salts such as acetates, oxalates, maleates, tartrates, citrates, succinates, malonates
• salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionat
• Pharmaceutically acceptable salts further include appropriate, non-toxic ammonium, quaternary ammonium salts and amine cations formed by counter ions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, C1 - C8 sulfonates and aromatic sulfonates.
• the terms “optionally”, “optional” or “optionally” generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
• one or more means “one, two, three, four or five, especially one, two, three or four, more especially one, two or three, even more especially one or two”.
• halogen refers to a fluorine, chlorine, bromine or iodine atom.
• the minimum and maximum values of the carbon atom content in the hydrocarbon group are indicated by prefixes, for example, the prefix Ca - Cb refers to "a” to "b” carbon atoms.
• the prefix Ca - Cb refers to "a" to "b” carbon atoms.
• C1 - Cn refers to a straight or branched saturated/unsaturated carbon chain containing 1, 2, 3, 4, 5, ... or n carbon atoms; it is further understood that “ C1 - Cn " should be interpreted as any sub-range included therein, for example, C1 - C40 , C2 - C40 , C1 - C24 , C3 - C24 , C1 - C11 , C4 - C10 , C4- C8 , C1 - C3 .
• C 1 -C 40 alkyl refers to a linear or branched saturated monovalent hydrocarbon group having 1, 2, 3, 4, 5, ... or 40 carbon atoms, for example, C 2 -C 40 alkyl, C 2 -C 24 alkyl, C 3 -C 24 alkyl, C 3 -C 11 alkyl, C 4 -C 10 alkyl, C 4 -C 8 alkyl.
• n-propyl n - Pr, -CH2CH2CH3
• isopropyl i-Pr, -CH( CH3 ) 2 )
• n - butyl n-Bu, -CH2CH2CH2CH3
• isobutyl i-Bu, -CH2CH ( CH3 ) 2
• sec - butyl s-Bu, -CH( CH3 ) CH2CH3 )
• tert-butyl t-Bu, -C( CH3 ) 3
• n - pentyl -CH2CH2CH2CH2CH3
• 2-pentyl -CH( CH3 ) CH2CH2CH3
• 3 - pentyl -CH( CH2CH3 ) 2 )
• 2 - methyl-2-butyl -C( CH3 ) 2CH2CH3 )
• C2 - C40 alkynyl refers to a linear or branched monovalent hydrocarbon group having 2, 3, 4, 5, ... or 40 carbon atoms, wherein at least one position CC is in an unsaturated state of sp triple bond, wherein the alkynyl group may be independently unsubstituted or substituted with one or more substituents described in the present invention, and specific examples include but are not limited to alkynylethyl ( -C ⁇ CH2 ), propargyl ( -CH2C ⁇ CH ), 1-propynyl (-C ⁇ C- CH3 ), etc.
• heteroalkyl means that heteroatoms such as oxygen, sulfur, phosphorus, and nitrogen (in the form of a tertiary amine moiety) may be present in an alkyl group to provide a heteroalkyl group (e.g., an alkyl group containing one or more ether, thioether, or amino bonds).
• heteroalkenyl means that heteroatoms such as oxygen, sulfur, phosphorus, and nitrogen (in the form of a tertiary amine moiety) may be present in an alkenyl group to provide a heteroalkenyl group (e.g., an alkenyl group containing one or more ether, thioether, or amino bonds).
• heteroalkynyl means that heteroatoms such as oxygen, sulfur, phosphorus, and nitrogen (in the form of a tertiary amine moiety) may be present in an alkynyl group to provide a heteroalkynyl group (e.g., an alkynyl group containing one or more ether, thioether, or amino bonds).
• the term "pharmaceutically acceptable excipients” includes any solvent, dispersion medium, coating material, surfactant, antioxidant, preservative (e.g., antibacterial agent, antifungal agent), isotonic agent, salt, drug stabilizer, binder, excipient, dispersant, lubricant, sweetener, flavoring agent, coloring agent, or combination thereof, which are known to those skilled in the art (such as Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Except for the case where any conventional carrier is incompatible with the active ingredient, its use in treatment or pharmaceutical composition is covered.
• treatment refers to the use of drugs to obtain the desired pharmacological and/or physiological effects.
• the effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and/or may be therapeutic in terms of partially or completely curing a disease and/or the adverse effects caused by the disease.
• Treatment covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease.
• the present invention provides a compound and its use, a liposome, a drug carrier, a complex, a drug composition and its pharmaceutical use, which will be described in detail below.
• the compound of the present invention has low cytotoxicity and can show good biocompatibility; the compound has a strong delivery ability and can be used as a delivery carrier to deliver nucleic acid drugs and the like into an animal body, for example, to the heart, liver, spleen, lungs and kidneys, especially to the spleen, which can effectively activate the body's immunity and increase the level of specific antibodies in the animal body.
• X 1 and X 2 are each independently C 4 -C 12 alkylene.
• R1 and R2 are the following structures:
• R 1 and R 2 each independently have a structure represented by formula (III):
• R7 and R8 are each independently H, optionally substituted C1 - C20 alkyl, optionally substituted C1-C20 heteroalkyl, optionally substituted C2 - C20 alkenyl, optionally substituted C2- C20 heteroalkenyl, optionally substituted C2-C20 alkynyl , or optionally substituted C2 - C20 heteroalkynyl, wherein the substituted groups are each independently selected from one or more halogen, -OH, -SH, -NH2 , -NO2 , cyano or C1 - C3 alkyl .
• the compounds of the present invention or their salts may also be obtained in the form of their hydrates, or include other solvents for their crystallization.
• the compounds of the present invention may inherently or by design form solvates with pharmaceutically acceptable solvents (including water); therefore, the present invention is intended to include solvated and unsolvated forms.
• the present invention provides a liposome.
• the liposome comprises: the aforementioned compound.
• the liposome according to the embodiment of the present invention has the advantages of low cytotoxicity, good biocompatibility and strong delivery ability, and has a good immune activation effect.
• the molar ratio of compound:neutral lipid:steroid:PEG-lipid is (20-80):(5-50):(10-60):(0.01-10).
• the molar ratio of compound:neutral lipid:steroid:PEG-lipid is (40-60):(5-10):(30-50):(0.5-5).
• the present invention proposes a pharmaceutical composition.
• the pharmaceutical composition comprises: the aforementioned compound, the aforementioned liposome, the aforementioned drug carrier or the aforementioned complex.
• the aforementioned compound, liposome, drug carrier and complex have the advantages of low cytotoxicity, good biocompatibility, strong delivery ability and good immune activation effect.
• the aforementioned compound, liposome or drug carrier is loaded with biologically active ingredients, and the biologically active ingredients can be delivered into the body, which is beneficial for the loaded biologically active ingredients to exert their efficacy and be used for the treatment of diseases.
• the pharmaceutical composition includes: a pharmaceutically acceptable excipient.
• the present invention proposes a use of the aforementioned compound, the aforementioned liposome, the aforementioned drug carrier, the aforementioned complex or the aforementioned pharmaceutical composition in the preparation of a drug, wherein the drug is used to target at least one of the heart, liver, spleen, lungs and kidneys.
• the aforementioned compound, liposome, drug carrier and complex have the advantages of low cytotoxicity, good biocompatibility, strong delivery ability and good immune activation effect.
• the aforementioned compound, liposome or drug carrier is loaded with bioactive ingredients, and the bioactive ingredients can be delivered to the heart, liver, spleen, lungs and kidneys, which is conducive to the loaded bioactive ingredients to exert their efficacy for the treatment of diseases.
• the medicament is used to target the spleen.
• targeting means that the bioactive component loaded by liposomes can be delivered to a predetermined target, so that the bioactive component is present in large quantities in the predetermined target, and the targeting is non-specific.
• targeting spleen means that the bioactive component is present in large quantities in the spleen, and does not exclude the presence of the bioactive component in other parts of the body.
• the virus-related diseases include but are not limited to the diseases caused by the new coronavirus.
• Compound 2a was synthesized according to the method described in Example 1.
• the encapsulation efficiency test results show that the encapsulation efficiency of LNPs@mRNA prepared by different ionizable lipids prepared in Example 1 and Example 2 of the invention and different ionizable lipids available on the market are all above 80%, indicating that the ionizable lipids provided in the present invention have good protection for mRNA in different preparation methods.
• LNPs@mRNA prepared by the compounds of the present invention to deliver mRNA in vivo was investigated.
• the inventors selected firefly luciferase as a reporter gene marker mRNA, namely Fluc mRNA, and then prepared LNPs@Fluc mRNA using different ionizable lipids (compound 8, MC3, ALC-0315 or SM-102) according to the method of Example 3, respectively, which were called LNP@Fluc mRNA preparations, MC3@Fluc mRNA, ALC-0315@Fluc mRNA and SM-102@Fluc mRNA, respectively, and investigated the ability of LNPs@mRNA preparation systems prepared by the compounds of the present invention to express mRNA in vivo and the distribution of expression through two administration routes, intravenous injection (step 2.1 of this embodiment) and intramuscular injection (step 2.2 of this embodiment).
• each preparation was adjusted to 0.1 mg/mL using PBS solution, and the osmotic pressure of the preparation was adjusted to isotonic.
• Each BALB/c mouse was injected with 200 ⁇ L of hind leg muscle, i.e., 20 ⁇ g FLuc mRNA/mouse, 3 mice per group, and PBS was used as a negative control (Control).
• the mice were kept on a normal diet after administration. 8 hours after administration, 200 ⁇ L of substrate solution (15 mg/mL, fluorescein potassium salt) was injected intraperitoneally, i.e., 3 mg/mouse. The timing started after the substrate was injected, and the mice were placed in the gas anesthesia device 10 minutes later.
• the results of the in vivo expression investigation experiment by intramuscular injection showed that the ionizable lipid of compound 8 had stronger mRNA expression ability than the three positive control materials in terms of the total luminescence in vivo by intramuscular injection.
• the LNPs@mRNA prepared by the lipid of the present invention had a certain expression level in the spleen, which is beneficial to the development of tumor vaccines by intramuscular injection.
• LNPs@Delta S-2P mRNA was prepared using Compound 8 and SM-102 as ionizable lipids, respectively, and the lipid SM-102 of the marketed product was used as a positive control.
• Delta S-2P mRNA encodes the Delta S-2P protein as shown in the amino acid sequence of SEQ ID NO: 1, and Delta S-2P is a full-length protein of the S protein of the Delta mutant strain B.1.617.2 with K986P and V987P mutations.
• the amino acid sequence is as follows:
• the results showed that the mRNA vaccine prepared with compound 8 could effectively activate the body's immunity and produce specific antibodies, with a binding antibody titer of up to 10 6 .
• the mRNA vaccine prepared with compound 8 had a significant advantage in immune activation, with a GMT increase of more than 2 times and up to 14 times.
• mice 6-8 week old Balb/c male mice were taken and immunized by intramuscular injection. A total of two doses were administered, each dose was 1 ⁇ g, and the interval between the two doses was 14 days.
• Mouse serum was collected 28 days after the first administration, and the antibody titer of the RBD of the S protein of the wild Delta strain in the serum was detected by ELISA, and the lipid SM-102 of the marketed product was used as a positive control.
• the geometric mean titer (GMT) of each compound was counted, and the experimental results are shown in Table 3.
• Example 3 the drug selection used the compounds prepared in Example 1 and Example 2 as lipids to prepare LNPs@Delta S-2P mRNA, which are respectively referred to as Compound 7-Compound 13, Compound 15-Compound 19 and Compound 20.
• the specific preparation method is shown in Example 3.
• Delta S-2P mRNA encodes the Delta S-2P protein shown in the amino acid sequence of SEQ ID NO: 1.

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