CN117946201A

CN117946201A - Lipid compounds and lipid nanoparticle compositions

Info

Publication number: CN117946201A
Application number: CN202410070063.8A
Authority: CN
Inventors: 董翊洁; 吕凯
Original assignee: Renjing International Hong Kong Ltd; Renjing Suzhou Biotechnology Co ltd
Current assignee: Renjing International Hong Kong Ltd; Renjing Suzhou Biotechnology Co ltd
Priority date: 2023-01-18
Filing date: 2024-01-17
Publication date: 2024-04-30

Abstract

The application relates to a lipid compound and a lipid nanoparticle composition, in particular to a lipid compound, the structural formula of which is shown in a formula (I), and salts and isomers thereof, and a nanoparticle composition containing the compound or the salts or the isomers thereof.

Description

Lipid compounds and lipid nanoparticle compositions

Technical Field

The application relates to the field of biotechnology, in particular to a lipid compound and a lipid nanoparticle composition.

Background

Efficient targeted delivery of biologically active substances such as small molecule drugs, proteins and nucleic acids is a continuing medical challenge. The key to success of gene therapy is whether the therapeutic drug can be delivered safely and effectively into the target cells via the vector in vivo. Delivery of nucleic acids to cells is made difficult by the relative instability of the nucleic acids and the low cell permeability of such substances. Accordingly, there is a need to develop methods and compositions to facilitate the delivery of therapeutic and/or prophylactic agents, such as nucleic acids, to cells. Gene therapy vectors are classified into viral vectors and nonviral vectors. Although viral vectors are used as efficient delivery systems for gene transfection and therapy purposes, the viral vectors are widely focused on the basis of their advantages of good in vitro stability, in vivo degradation, safety and reliability, and are largely used in gene therapy research of congenital and acquired genetic defects due to the fact that they contain immunogenic viral proteins, limited gene load capacity, high price, and the like.

Lipid-containing nanoparticles or lipid nanoparticles, liposomes and lipid complexes have proven to be effective transport vehicles for bioactive substances such as small molecule drugs, proteins and nucleic acids into and/or within cells. LNPs refers to vesicles formed from one or more lipid components that can efficiently compress and deliver a variety of nucleic acid molecules, from DNA, RNA, to chromosomes, and even cells; LNPs is favorable for large-scale production due to the characteristics of the determined construction scheme, easy modification of the targeting ligand and the like.

LNPs typically comprise one or more cationic lipids and/or amino (ionizable) lipids, phospholipids containing polyunsaturated lipids, structural lipids (e.g., sterols), and/or lipids containing polyethylene glycol (PEG lipids). Cationic and/or ionizable lipids include, for example, amine-containing lipids, which can be readily protonated.

Lipid Nanoparticle (LNP) formulations represent a revolution in the field of nucleic acid delivery. Onpattro ^TM is an early example of a lipid nanoparticle product approved for clinical use. Onpattro ^TM is a lipid nanoparticle-based short interfering RNA (siRNA) pharmaceutical formulation for the treatment of multiple neuropathy caused by hereditary transthyretin amyloidosis. The success of this LNP delivery system paves the way for the leading clinical development of LNP-based COVID-19mRNA vaccines.

Onpattro ^TM LNP formulation consists of four major lipid components, namely the ionizable amino lipid MC3, distearoyl phosphatidylcholine (DSPC), cholesterol and polyethylene glycol conjugated lipid (PEG-lipid), in molar amounts of 50/10/38.5/1.5, respectively. Onpattro ^TM is still considered the gold standard for comparison of LNP studies.

Although studies relating to LNP mediated nucleic acid delivery have advanced significantly, it is well known that Onpattro ^TM formulations accumulate primarily in liver tissue. The ability of LNP to accumulate in organs and tissues other than the liver will greatly expand the clinical utility of these delivery systems.

Disclosure of Invention

Based on the above, the application discloses a lipid compound and a lipid nanoparticle composition containing the same, wherein the lipid has the advantages of high encapsulation efficiency, high expression, spleen targeting and the like, and has the characteristic of low toxicity, in particular low hepatotoxicity.

Specifically, the application adopts the following technical scheme

1. A compound of formula (I), or a salt or isomer thereof,

X ₁、X₂ is independently selected from c=o or O, Y ₁、Y₂ is independently selected from c=o or O, provided that X ₁ and Y ₁、X₂ and Y ₂ are not both c=o or O; r ₁ is R; r ₂、R₃ is independently selected from H, C-C14 alkyl, R, provided that R ₂、R₃ is not H at the same time;

The R is

Ra, rb, rc, rd, re is independently selected from H, C1-C6 alkyl,

R' is selected from C1-C10 alkyl, C1-C10 alkenyl;

R ₄、R₅、R₆、R₇ is independently selected from H, C1-C6 alkyl;

The dotted line represents a single bond or a double bond;

n is selected from 0,1,2,3,4,5,6;

o, p are independently selected from 1,2,3,4,5,6,7,8,9, 10.

2. The compound of item 1, R ₂、R₃ is independently selected from H, C1-C14 alkyl, provided that R ₂、R₃ is not simultaneously H.

3. A compound according to item 2, R ₂、R₃ is a C6-C11 linear or branched alkyl group.

4. The compound of item 3, wherein R ₂、R₃ is C8 straight or branched alkyl.

5. The compound of item 2, R ₂ is H, R ₃ is a C10-C12 linear or branched alkyl.

6. The compound of item 5, R ₂ is a linear or branched alkyl group having H, R ₃ as C11.

7. The compound of item 1, wherein R ₂ is H, R ₃ is R.

8. The compound of clauses 1-7, wherein R' is C8 alkyl or C8 alkenyl.

9. The compound of item 8, wherein R' is

The dotted line represents a single bond or a double bond.

10. The compound of any one of claims 1-9, wherein R is: Preference/>

11. The compound of any one of claims 1-10, wherein o is selected from 1,2,3,4,5,6,7.

12. The compound according to item 11, wherein o is 5.

13. The compound of any one of claims 1-12, wherein p is selected from 1,2,3,4,5,6,7.

14. The compound according to item 13, wherein p is 7.

15. The compound of any one of claims 1-14, wherein n is 2.

16. The compound of any one of claims 1-14, wherein n is 6.

17. The compound of any one of claims 1-16, wherein X ₁、X₂ is c=o and Y ₁、Y₂ is O.

18. The compound of any one of claims 1-16, wherein X ₁ is O, Y ₁ is c=o, X ₂ is c=o, and Y ₂ is O.

19. The compound of any one of claims 1-16, wherein X ₁ is c=o, Y ₁ is O, X ₂ is O, and Y ₂ is c=o.

20. The compound of any one of claims 1-16, wherein X ₁,X₂ is O and Y ₁,Y₂ is c=o.

21. The compound of any one of claims 1-20, which is a compound of formula (II):

22. a compound or a salt or isomer thereof, wherein the compound is selected from the group consisting of

Compound 1

Compound 2

Compound 3

Compound 4

Compound 5

Compound 6

Compound 7

Compound 8

Compound 9

Compound 10

Compound 11

Compound 12

Compound 13

Compound 14

Compound 15

Compound 16

23. The compound according to item 22, or a salt or isomer thereof, wherein the compound is selected from the group consisting of

Compound 1

Compound 2

24. A nanoparticle composition comprising a lipid component comprising a compound of any one of claims 1-23.

25. The nanoparticle composition of item 24, wherein the lipid component further comprises a phospholipid.

26. The nanoparticle composition according to item 25, wherein the phospholipid is selected from one or more of the following compounds: dilauroyl Lecithin (DLPC), dimyristoyl phosphatidylcholine (DMPC), dioleoyl lecithin (DOPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DUPC), palmitoyl Oleoyl Phosphatidylcholine (POPC), 1, 2-di-O-octadecyl-SN-glycero-3-phosphocholine (18:0 DietherPC), 1-oleoyl-2-cholesterol dimethyl succinic acid-SN-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-SN-glycero-3-phosphocholine (C16 Lyso PC), 1, 2-divinyl-SN-glycero-3-phosphocholine, 1, 2-diaryl acyl-SN-glycero-3-phosphoethanolamine (DOPE), 1, 2-dihydrotin oxide-SN-glycero-3-phosphoethanolamine (16.0), 1, 2-oleoyl-SN-glycero-3-phosphoethanolamine (2, 2-hexadecyl-SN-glycero-3-phosphoethanolamine (C16 Lyso PC), 1, 2-divinyl-SN-glycero-3-phosphoethanolamine, 1, 2-diacyl-phosphatidylethanolamine, 2-diacylglycerol-SN-3-phosphoethanolamine, 1, 2-dithiohexaenoic acid-sn-glycerol-3-phosphate ethanolamine, 1, 2-diol-sn-glycerol-3-phosphate- (1-glycerol) sodium salt (DOPG), or sphingomyelin.

27. The nanoparticle composition of item 25, wherein the phospholipid is DOPE.

28. The nanoparticle composition of item 25, wherein the phospholipid is DSPC.

29. The nanoparticle composition of any one of claims 24-28, wherein the lipid component further comprises a structural lipid.

30. The nanoparticle composition of claim 29, wherein the structural lipid is selected from one or more of cholesterol, stigmasterol, sitosterol, ergosterol, stigmasterol.

31. The nanoparticle composition of item 29, wherein the structural lipid is cholesterol.

32. The nanoparticle composition of any one of claims 24-31, wherein the lipid component further comprises a PEG lipid.

33. The nanoparticle composition of any one of claims 32, wherein the PEG lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, or PEG-modified dialkylglycerol.

34. The nanoparticle composition of any one of claims 24-33, wherein the lipid component further comprises a cationic and/or ionizable lipid.

35. The nanoparticle composition according to any one of claims 24 to 34, further comprising a therapeutic and/or prophylactic agent selected from a vaccine or a compound capable of eliciting an immune response, a nucleic acid, preferably RNA, selected from one or more of siRNA, aiRNA, miRNA, dsRNA, shRNA or mRNA.

36. The nanoparticle composition according to any one of claims 24 to 35, wherein the encapsulation efficiency of the therapeutic and/or prophylactic agent is greater than or equal to 50%; or more than or equal to 80 percent; or more than or equal to 90 percent.

37. The nanoparticle composition of any one of claims 24-36, wherein the nanoparticle composition has an average particle size of 50nm-110nm.

38. The nanoparticle composition of any one of claims 24-37, wherein the nanoparticle composition has a dispersibility index of 0.006-0.20.

39. Use of a compound of any one of claims 1-23 in the preparation of a lipid nanoparticle composition.

40. A pharmaceutical composition comprising the nanoparticle composition of any one of claims 24-38 and a pharmaceutically acceptable carrier.

41. A method of delivering a therapeutic and/or prophylactic agent to a mammalian cell, the method comprising administering to a subject the nanoparticle composition of any one of claims 24-38 or the pharmaceutical composition of claim 40, the administering comprising contacting a cell with the nanoparticle composition or the pharmaceutical composition to deliver the therapeutic and/or prophylactic agent to the cell.

42. The method of item 41, wherein the mammalian cell is in a mammal.

43. The method of clause 41 or 42, wherein the mammal is a human.

44. The method of any one of items 41-43, wherein the nanoparticle composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

45. A method of producing a polypeptide of interest in a mammalian cell, the method comprising contacting the cell with the nanoparticle composition of any one of claims 24-38 or the pharmaceutical composition of claim 40 to deliver a therapeutic and/or prophylactic agent to the cell, wherein the therapeutic and/or prophylactic agent is an mRNA encoding the polypeptide of interest, whereby the mRNA is capable of translation in the cell to produce the polypeptide of interest.

46. The method of item 45, wherein the mammalian cell is in a mammal.

47. The method of any one of items 45 or 46, wherein the mammal is a human.

48. The method of any one of claims 45-47, wherein the nanoparticle composition or pharmaceutical composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

49. A method of treating a disease or disorder in a mammal, the method comprising administering to the mammal a therapeutically effective amount of the nanoparticle composition of any one of claims 24-38 or the pharmaceutical composition of claim 40.

50. The method of item 49, wherein the disease or disorder is characterized by dysfunctional or abnormal protein or polypeptide activity.

51. The method of clause 49 or 50, wherein the disease or disorder is selected from infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, renal vascular diseases, or metabolic diseases.

52. The method of any one of items 49-51, wherein the mammal is a human.

53. The method of any one of claims 49-52, wherein the nanoparticle composition or pharmaceutical composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

54. A method of specifically delivering a therapeutic and/or prophylactic agent to an organ of a mammal, the method comprising administering to the mammal the nanoparticle composition of any one of claims 24-38 or the pharmaceutical composition of claim 40, the administering comprising contacting the organ of the mammal with the nanoparticle composition, thereby delivering the therapeutic and/or prophylactic agent to the organ.

55. The method of item 54, wherein the mammal is a human.

56. The method of item 54 or 55, wherein the nanoparticle composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

57. The method of any one of claims 54-56, wherein the mammal is pretreated 24 hours or less prior to the contacting or administering step.

58. The method of any one of claims 54-57, wherein the mammal is pretreated about one hour prior to the contacting or administering step.

59. Use of a nanoparticle composition of any one of items 24-38 or a pharmaceutical composition of item 40 in the manufacture of a medicament for the treatment of a disease or disorder in a mammal.

60. Use of the nanoparticle composition of any one of claims 24-38 or the pharmaceutical composition of claim 40 in the treatment of a disease or disorder in a mammal.

The application provides a compound of formula (I), or a pharmaceutically acceptable salt or stereoisomer thereof,

X ₁、X₂ is independently selected from c=o or O, Y ₁、Y₂ is independently selected from c=o or O, provided that X ₁ and Y ₁、X₂ and Y ₂ are not both c=o or O;

r ₁ is R, and the total number of the components is R,

R ₂、R₃ is independently selected from H, C-C14 alkyl, R, provided that R ₂、R₃ is not H at the same time;

The R is

Ra, rb, rc, rd, re is independently selected from H, C1-C6 alkyl,

R' is selected from C1-C10 alkyl, C1-C10 alkenyl,

The dotted line represents a single bond or a double bond;

R ₄、R₅、R₆、R₇ is independently selected from H, C1-C6 alkyl;

n is selected from 0,1,2,3,4,5,6;

o, p are independently selected from 1,2,3,4,5,6,7,8,9, 10.

In some embodiments, R ₂、R₃ is independently selected from H, C1-C14 alkyl, provided that R ₂、R₃ is not both H.

In some embodiments, R ₂、R₃ is a C6-C11 linear or branched alkyl group.

In some embodiments, R ₂、R₃ is a C8 linear or branched alkyl group.

In some embodiments, R ₂ is H, R ₃ is a C10-C12 linear or branched alkyl group.

In some embodiments, R ₂ is a linear or branched alkyl group H, R ₃ that is C11.

In some embodiments, R ₂ is H, R ₃ is R.

In some embodiments, the R' is C8 alkyl or C8 alkenyl.

In some embodiments, the R' isThe dotted line represents a single bond or a double bond.

In some embodiments, the R is: Preferably

In some embodiments, the o is selected from 1,2,3,4,5,6,7.

In some embodiments, the o is 5.

In some embodiments, the p is selected from 1,2,3,4,5,6,7.

In some embodiments, p is 7.

In some embodiments, n is 2.

In some embodiments, n is 6.

In some embodiments, the X ₁、X₂ is c=o and the Y ₁、Y₂ is O.

In some embodiments, the X ₁ is O, Y ₁ is c=o, the X ₂ is c=o, and Y ₂ is O.

In some embodiments, the X ₁ is c=o, Y ₁ is O, the X ₂ is O, and Y ₂ is c=o.

In some embodiments, the X ₁,X₂ is O and the Y ₁,Y₂ is c=o.

In some embodiments, R ₂、R₃ is a C6-C11 linear or branched alkyl group; x ₁、X₂ is independently selected from c=o or O, Y ₁、Y₂ is independently selected from c=o or O, provided that X ₁ and Y ₁、X₂ and Y ₂ are not both c=o or O; r ₁ is R; r ₄、R₅、R₆、R₇ is independently selected from H, C1-C6 alkyl; the dotted line represents a single bond or a double bond; n is selected from 0,1,2,3,4,5,6; said p is selected from 1,2,3,4,5,6,7; and o is 5.

In some embodiments, R ₂、R₃ is a C8 linear or branched alkyl; x ₁、X₂ is independently selected from c=o or O, Y ₁、Y₂ is independently selected from c=o or O, provided that X ₁ and Y ₁、X₂ and Y ₂ are not both c=o or O; r ₁ is R; r ₄、R₅、R₆、R₇ is independently selected from H, C1-C6 alkyl; the dotted line represents a single bond or a double bond; n is selected from 0,1,2,3,4,5,6; said p is selected from 1,2,3,4,5,6,7; and o is 5.

In some embodiments, R ₂、R₃ is a C6-C11 linear or branched alkyl group; x ₁、X₂ is independently selected from c=o or O, Y ₁、Y₂ is independently selected from c=o or O, provided that X ₁ and Y ₁、X₂ and Y ₂ are not both c=o or O; r ₁ is R; r ₄、R₅、R₆、R₇ is independently selected from H, C1-C6 alkyl; the dotted line represents a single bond or a double bond; n is selected from 0,1,2,3,4,5,6; p is 7; and o is 5.

In some embodiments, R ₂、R₃ is a C8 linear or branched alkyl; x ₁、X₂ is independently selected from c=o or O, Y ₁、Y₂ is independently selected from c=o or O, provided that X ₁ and Y ₁、X₂ and Y ₂ are not both c=o or O; r ₁ is R; r ₄、R₅、R₆、R₇ is independently selected from H, C1-C6 alkyl; the dotted line represents a single bond or a double bond; n is selected from 0,1,2,3,4,5,6; p is 7; and o is 5.

In some embodiments, R ₂、R₃ is a C6-C11 linear or branched alkyl group; x ₁、X₂ is independently selected from c=o or O, Y ₁、Y₂ is independently selected from c=o or O, provided that X ₁ and Y ₁、X₂ and Y ₂ are not both c=o or O; r ₁ is R; r ₄、R₅、R₆、R₇ is independently selected from H, C1-C6 alkyl; the dotted line represents a single bond or a double bond; n is 2; p is 7; and o is 5.

In some embodiments, R ₂、R₃ is a C8 linear or branched alkyl; x ₁、X₂ is independently selected from c=o or O, Y ₁、Y₂ is independently selected from c=o or O, provided that X ₁ and Y ₁、X₂ and Y ₂ are not both c=o or O; r ₁ is R; r ₄、R₅、R₆、R₇ is independently selected from H, C1-C6 alkyl; the dotted line represents a single bond or a double bond; n is 2; p is 7; and o is 5.

In some embodiments, R ₂、R₃ is a C6-C11 linear or branched alkyl group; x ₁、X₂ is independently selected from c=o or O, Y ₁、Y₂ is independently selected from c=o or O, provided that X ₁ and Y ₁、X₂ and Y ₂ are not both c=o or O; r ₁ is R; r ₄、R₅、R₆、R₇ is independently selected from H, C1-C6 alkyl; the dotted line represents a single bond or a double bond; n is 6; p is 7; and o is 5.

In some embodiments, R ₂、R₃ is a C8 linear or branched alkyl; x ₁、X₂ is independently selected from c=o or O, Y ₁、Y₂ is independently selected from c=o or O, provided that X ₁ and Y ₁、X₂ and Y ₂ are not both c=o or O; r ₁ is R; r ₄、R₅、R₆、R₇ is independently selected from H, C1-C6 alkyl; the dotted line represents a single bond or a double bond; n is 6; p is 7; and o is 5.

In some embodiments, R ₂、R₃ is a C6-C11 linear or branched alkyl group; x ₁、X₂ is c=o, Y ₁、Y₂ is O; r ₁ is R; r ₄、R₅、R₆、R₇ is independently selected from H, C1-C6 alkyl; the dotted line represents a single bond or a double bond; n is 2; p is 7; and o is 5.

In some embodiments, R ₂、R₃ is a C8 linear or branched alkyl; x ₁、X₂ is c=o, Y ₁、Y₂ is O; r ₁ is R; r ₄、R₅、R₆、R₇ is independently selected from H, C1-C6 alkyl; the dotted line represents a single bond or a double bond; n is 2; p is 7; and o is 5.

In some embodiments, R ₂、R₃ is a C6-C11 linear or branched alkyl group; x ₁、X₂ is c=o, Y ₁、Y₂ is O; r ₁ is R; r ₄、R₅、R₆、R₇ is independently selected from H, C1-C6 alkyl; the dotted line represents a single bond or a double bond; n is 6; p is 7; and o is 5.

In some embodiments, R ₂、R₃ is a C8 linear or branched alkyl; x ₁、X₂ is c=o, Y ₁、Y₂ is O; r ₁ is R; r ₄、R₅、R₆、R₇ is independently selected from H, C1-C6 alkyl; the dotted line represents a single bond or a double bond; n is 6; p is 7; and o is 5.

In some embodiments, R ₂、R₃ is a C6-C11 linear or branched alkyl group; x ₁ is O, X ₂ is c=o, Y ₁ is c= O, Y ₂ is O; r ₁ is R; r ₄、R₅、R₆、R₇ is independently selected from H, C1-C6 alkyl; the dotted line represents a single bond or a double bond; n is 2; p is 7; and o is 5.

In some embodiments, R ₂、R₃ is a C8 linear or branched alkyl; x ₁ is O, X ₂ is c=o, Y ₁ is c= O, Y ₂ is O; r ₁ is R; r ₄、R₅、R₆、R₇ is independently selected from H, C1-C6 alkyl; the dotted line represents a single bond or a double bond; n is 2; p is 7; and o is 5.

In some embodiments, R ₂、R₃ is a C6-C11 linear or branched alkyl group; x ₁ is O, X ₂ is c=o, Y ₁ is c= O, Y ₂ is O; r ₁ is R; r ₄、R₅、R₆、R₇ is independently selected from H, C1-C6 alkyl; the dotted line represents a single bond or a double bond; n is 6; p is 7; and o is 5.

In some embodiments, R ₂、R₃ is a C8 linear or branched alkyl; x ₁ is O, X ₂ is c=o, Y ₁ is c= O, Y ₂ is O; r ₁ is R; r ₄、R₅、R₆、R₇ is independently selected from H, C1-C6 alkyl; the dotted line represents a single bond or a double bond; n is 6; p is 7; and o is 5.

In some embodiments, R ₂、R₃ is a C6-C11 linear or branched alkyl group; x ₁ is c=o, X ₂ is O, Y ₁ is O, Y ₂ is c=o; r ₁ is R; r ₄、R₅、R₆、R₇ is independently selected from H, C1-C6 alkyl; the dotted line represents a single bond or a double bond; n is 2; p is 7; and o is 5.

In some embodiments, R ₂、R₃ is a C8 linear or branched alkyl; x ₁ is c=o, X ₂ is O, Y ₁ is O, Y ₂ is c=o; r ₁ is R; r ₄、R₅、R₆、R₇ is independently selected from H, C1-C6 alkyl; the dotted line represents a single bond or a double bond; n is 2; p is 7; and o is 5.

In some embodiments, R ₂、R₃ is a C6-C11 linear or branched alkyl group; x ₁ is c=o, X ₂ is O, Y ₁ is O, Y ₂ is c=o; r ₁ is R; r ₄、R₅、R₆、R₇ is independently selected from H, C1-C6 alkyl; the dotted line represents a single bond or a double bond; n is 6; p is 7; and o is 5.

In some embodiments, R ₂、R₃ is a C8 linear or branched alkyl; x ₁ is c=o, X ₂ is O, Y ₁ is O, Y ₂ is c=o; r ₁ is R; r ₄、R₅、R₆、R₇ is independently selected from H, C1-C6 alkyl; the dotted line represents a single bond or a double bond; n is 6; p is 7; and o is 5.

In some embodiments, R ₂、R₃ is a C6-C11 linear or branched alkyl group; x ₁、X₂ is O, Y ₁、Y₂ is c=o; r ₁ is R; r ₄、R₅、R₆、R₇ is independently selected from H, C1-C6 alkyl; the dotted line represents a single bond or a double bond; n is 2; p is 7; and o is 5.

In some embodiments, R ₂、R₃ is a C8 linear or branched alkyl; x ₁、X₂ is O, Y ₁、Y₂ is c=o; r ₁ is R; r ₄、R₅、R₆、R₇ is independently selected from H, C1-C6 alkyl; the dotted line represents a single bond or a double bond; n is 2; p is 7; and o is 5.

In some embodiments, R ₂、R₃ is a C6-C11 linear or branched alkyl group; x ₁、X₂ is O, Y ₁、Y₂ is c=o; r ₁ is R; r ₄、R₅、R₆、R₇ is independently selected from H, C1-C6 alkyl; the dotted line represents a single bond or a double bond; n is 6; p is 7; and o is 5.

In some embodiments, R ₂、R₃ is a C8 linear or branched alkyl; x ₁、X₂ is O, Y ₁、Y₂ is c=o; r ₁ is R; r ₄、R₅、R₆、R₇ is independently selected from H, C1-C6 alkyl; the dotted line represents a single bond or a double bond; n is 6; p is 7; and o is 5.

In some embodiments, it is a compound of formula (II):

In some embodiments, it is a compound of formula (II) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein n is selected from 2,3,4,5,6; o is selected from 3,4,5; p is selected from 7 or 8; r1 is R2 and R3 are each independently selected from C7-C9 linear alkyl.

In some embodiments, it is a compound of formula (II) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein n is selected from 2,3,4,5,6; o is 5; p is 7; r1 isR2 and R3 are each independently selected from C7-C9 linear alkyl.

In some embodiments, it is a compound of formula (II) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein n is selected from 2,3,4,5,6; o is 5; p is 7; r1 isR2 and R3 are each independently C8 linear alkyl.

In some embodiments, it is a compound of formula (II) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein n is selected from 2 or 6; o is 5; p is 7; r1 isR2 and R3 are each independently selected from C8 linear alkyl.

In some embodiments, the compound is selected from

Compound 1

Compound 2

Compound 3

Compound 4

Compound 5/>

Compound 6

Compound 7

Compound 8

Compound 9

Compound 10

Compound 11

Compound 12

Compound 13

Compound 14/>

Compound 15

Compound 16

In some embodiments, the compound is selected from

Compound 1

Compound 2

In some embodiments, compound 1 and stereoisomers thereof are selected from

Compound 1aOr (b)

Compound 1bOr racemates of compound 1a and compound 1 b.

In some embodiments, compound 2 and stereoisomers thereof are selected from

Compound 2aOr/>

Compound 2bOr racemates of compound 2a and compound 2 b.

Effects of the invention

The compound can be used for preparing lipid nano-particles, the nano-particle composition containing the compound can realize encapsulation and delivery of a therapeutic agent/preventive agent, safely deliver the therapeutic agent/preventive agent to a target position, realize high expression, exert the effect of the therapeutic agent/preventive agent, and have the characteristic of low toxicity, particularly low hepatotoxicity.

The lipid nanoparticle prepared by the application has small average particle size and high encapsulation efficiency, and has wide application prospect in the field of drug delivery.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

Fig. 1 shows the fluorescence intensity of luciferases shown by different LNP formulations 6 hours after intravenous injection.

Fig. 2 shows the Luciferase fluorescence intensity displayed in the spleen 24 hours after intravenous injection for the different LNP formulations.

Fig. 3 shows the Luciferase fluorescence intensity shown by the different LNP formulations 6 hours after intramuscular injection.

Detailed Description

Exemplary embodiments of the application are described below, including various details of embodiments of the application to facilitate understanding, which should be considered as merely exemplary. Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope and spirit of the application. Also, descriptions of well-known functions and constructions are omitted in the following description for clarity and conciseness.

Terminology and definitions

As used herein, the term "alkyl" refers to a compound containing one or more carbon atoms (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more carbon atoms) that is optionally substituted. The term "C1-C14 alkyl" refers to an optionally substituted straight or branched chain saturated hydrocarbon comprising 1 to 14 carbon atoms, the term "C1-C10 alkyl" refers to an optionally substituted straight or branched chain saturated hydrocarbon comprising 1 to 10 carbon atoms, and the term "C1-C6 alkyl" refers to an optionally substituted straight or branched chain saturated hydrocarbon comprising 1 to 6 carbon atoms. Unless otherwise indicated, alkyl groups described herein refer to unsubstituted and substituted alkyl groups.

As used herein, the term "alkenyl" refers to a group comprising one or more carbon atoms (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more carbon atoms) that is optionally substituted. The term "C1-C14 alkenyl" refers to an optionally substituted straight or branched chain unsaturated hydrocarbon comprising 1 to 14 carbon atoms, the term "C1-C10 alkenyl" refers to an optionally substituted straight or branched chain unsaturated hydrocarbon comprising 1 to 10 carbon atoms, and the term "C1-C6 alkenyl" refers to an optionally substituted straight or branched chain unsaturated hydrocarbon comprising 1 to 6 carbon atoms. Unless otherwise indicated, alkenyl groups described herein refer to both unsubstituted and substituted alkenyl groups.

Unless otherwise indicated, alkyl groups may be optionally substituted. The optional substituents may be selected from, but are not limited to, halogen atoms (e.g., chlorine, bromine, fluorine, OR iodine), carboxylic acids (e.g., -C (O) OH), alcohols (e.g., -OH), esters (e.g., -C (O) OR OR-OC (O) R), aldehydes (e.g., -C (O) H), carbonyl groups (e.g., -C (O) R, OR represented by C=O), acyl halides (e.g., -C (O) X, where X is a halide selected from bromide, fluoride, chloride, and iodide), carbonates (e.g., -OC (O) OR), alkoxy (e.g., -OR), acetal, phosphate, thiols (e.g., -SH), sulfoxides (e.g., -S (O) R), sulfurous acids (e.g., -S (O) OH), sulfonic acids (e.g., -S (O) ₂ OH), thiols (e.g., -C (S) H), sulfates, sulfonyl groups (e.g., -S (O) ₂ -), amides (e.g., -C (O) NR ₂ OR-N (R) C (O) R), azido (e.g., -nitro) (e.g., -32) N), e.g., -cyano (e.g., -34) cyano-), acyloxy (e.g., -OC (O) R), amino (e.g., -NR ₂, -NRH or-NH ₂), carbamoyl (e.g., -OC (O) NR ₂, -OC (O) NRH or-OC (O) NH ₂), sulfonamide, alkyl, alkenyl, and cyclic groups (e.g., carbocyclyl or heterocyclyl). In any of the foregoing, R is alkyl or alkenyl as defined herein. In some embodiments, the substituents themselves may be further substituted with, for example, one, two, three, four, five or six substituents as defined herein. For example, a C _1-6 alkyl group may be further substituted with 1,2, 3, 4, 5, or 6 substituents as described herein.

As used herein, the term "compound" is meant to include all isomers and isotopes of the described structures. "isotope" refers to atoms having the same atomic number, but different mass numbers due to different numbers of neutrons in the nuclei. Isotopes of hydrogen include, for example, tritium and deuterium. In addition, the compounds, salts or complexes of the application may be prepared by conventional methods in combination with solvents or water molecules to form sols and hydrates.

As used herein, the term "contacting" refers to establishing a physical connection between two or more entities. For example, contacting a mammalian cell with a nanoparticle composition means that the mammalian cell and the nanoparticle share a physical connection. Methods for contacting cells with external entities in vivo and ex vivo are well known in the biological arts. For example, nanoparticle compositions can be contacted with mammalian cells disposed within a mammal by a variety of routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous), and can involve a variety of amounts of nanoparticle compositions. Furthermore, the nanoparticle composition may be contacted with more than one mammalian cell.

As used herein, the term "delivery" refers to the direction of a destination providing entity. For example, delivering a therapeutic and/or prophylactic agent to a subject can include administering to the subject a nanoparticle composition that includes the therapeutic and/or prophylactic agent (e.g., via an intravenous, intramuscular, intradermal, or subcutaneous route). Administration of the nanoparticle composition to a mammal or mammalian cell may involve contacting one or more cells with the nanoparticle composition.

As used herein, the term "enhanced delivery" refers to the delivery of more (e.g., at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold) of a therapeutic and/or prophylactic drug to a target tissue (e.g., mammalian liver) by nanoparticles as compared to the level of therapeutic and/or prophylactic drug delivered to the target tissue (e.g., MC3, KC2, or DLinDMA) by control nanoparticles. The amount of therapeutic and/or prophylactic agent in the tissue may be compared to the weight of the tissue, the amount of protein produced in the tissue may be compared to the amount of total protein in the tissue, or the amount of therapeutic and/or prophylactic agent in the tissue may be compared to the amount of total therapeutic and/or prophylactic agent in the tissue. It will be appreciated that enhanced delivery of nanoparticles to target tissue need not be determined in the subject being treated, but may be determined in an alternative such as an animal model (e.g., a rat model). In certain embodiments, the nanoparticle compositions comprise compounds of formulas (I), (II) with substantially the same level of enhancement of delivery, regardless of the route of administration.

As used herein, the term "specific delivery" or "specific delivery" refers to the delivery of more (e.g., at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold) of a therapeutic and/or prophylactic drug to a target tissue of interest (e.g., mammalian liver) by nanoparticles than to non-target tissue. The level of nanoparticle delivery to a particular tissue can be measured by comparing the weight of protein produced in the tissue to the weight of the tissue, comparing the amount of treatment and/or prevention in the tissue to the weight of the tissue, comparing the weight of protein produced in the tissue to the weight of total protein in the tissue, or comparing the amount of treatment and/or prevention in the tissue to the amount of total treatment and/or prevention in the tissue.

As used herein, "encapsulation efficiency" refers to the amount of therapeutic and/or prophylactic agent that becomes part of the nanoparticle composition relative to the total amount of therapeutic or prophylactic agent used in preparing the nanoparticle composition. For example, if 97mg of therapeutic and/or prophylactic agent is encapsulated in the nanoparticle composition in a total of 100mg of therapeutic and/or prophylactic agent initially provided to the composition, the encapsulation efficiency may be 97%. As used herein, "encapsulating" may refer to completely, substantially, or partially encapsulating, enclosing, or enveloping.

As used herein, "expression" of a nucleic acid sequence refers to translation of mRNA into a polypeptide or protein and/or post-translational modification of a polypeptide or protein.

As used herein, the term "in vitro" refers to an event that occurs in an artificial environment, such as in a test tube or reaction vessel, in a cell culture, in a petri dish, etc., rather than in an organism. (e.g., animal, plant or microorganism).

As used herein, the term "in vivo" refers to an event that occurs within an organism (e.g., an animal, plant or microorganism or a cell or tissue thereof).

As used herein, the term "ex vivo" refers to an event that occurs outside an organism (e.g., an animal, plant or microorganism or cell or tissue thereof). An ex vivo event may occur in an environment that minimally alters from the natural (e.g., in vivo) environment.

As used herein, the term "isomer" refers to any geometric isomer, tautomer, zwitterionic, stereoisomer, enantiomer or diastereomer of a compound. The compounds may contain one or more chiral centers and/or double bonds and thus may exist as stereoisomers, such as double bond isomers (i.e., geometric E/Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis-trans isomers. The present application encompasses any and all isomers of the compounds described herein. The enantiomers and mixtures of stereoisomers of a compound and methods for resolving them into their component enantiomers or stereoisomers are well known.

As used herein, a "lipid component" is a component of a nanoparticle composition that comprises one or more lipids. For example, the lipid component may include one or more cationic/ionizable lipids, pegylated lipids, structural lipids, or other lipids, such as phospholipids.

As used herein, a "linker" is a moiety that connects two moieties, e.g., a connection between two nucleosides of a cap species. The linker may include one or more groups including, but not limited to, phosphate groups (e.g., phosphates, borophosphates, thiophosphates, selenophosphates, and phosphates), alkyl groups, amides, or glycerols. For example, two nucleosides of a cap analogue can be linked at their 5' positions by a triphosphate group or by a chain comprising two phosphate moieties and a borophosphate moiety.

As used herein, "method of administration" may include intravenous, intramuscular, intradermal, subcutaneous, or other methods of delivering the composition to a subject. Any of the methods of administration may be selected to target delivery (e.g., specific delivery) to a particular region or system of the body.

As used herein, "modified" refers to non-natural. For example, the RNA may be modified RNA. That is, the RNA can include one or more non-naturally occurring nucleobases, nucleosides, nucleotides, or linkers. "modified" materials may also be referred to herein as "engineered" materials. The substance may be chemically, structurally or functionally modified or engineered. For example, the modified nucleobase species can include one or more non-naturally occurring substitutions.

As used herein, a "nanoparticle composition" is a composition that comprises one or more lipids. The particle size of the nanoparticle composition is typically on the order of microns or less and may include a lipid bilayer. Nanoparticle compositions include Lipid Nanoparticles (LNPs), liposomes (e.g., lipid vesicles) and lipid complexes. For example, the nanoparticle composition may be a liposome having a lipid bilayer with a diameter of 500nm or less.

As used herein, "naturally occurring" refers to occurring naturally without artificial assistance.

As used herein, "patient" refers to a subject who may seek or need treatment, is receiving treatment, will receive treatment, or is being treated by a trained professional for a particular disease.

As used herein, "PEG lipid" or "pegylated lipid" refers to a lipid comprising a polyethylene glycol component.

The term "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The nanoparticle compositions of the present application may also comprise salts of one or more compounds. The salt may be a pharmaceutically acceptable salt. As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds wherein the parent compound is altered by converting the existing acid or base moiety to its salt form (e.g., by reacting the free base with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; acidic residues such as bases or organic salts of carboxylic acids, and the like. Representative acid addition salts include acetates, adipates, alginates, ascorbates, aspartate, benzenesulfonates, benzoate, bisulfate, borate, butyrate, camphoride, camphorsulfonate, citrate, cyclopentane propionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, hydrobromide, hydrochloride, hydroiodite, 2-hydroxy-ethanesulfonate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pectate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, tosylate, undecanoate, valerate salts, and the like.

Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium and amine cations including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Pharmaceutically acceptable salts of the application include, for example, conventional non-toxic salts of the parent compound formed from non-toxic inorganic or organic acids. Pharmaceutically acceptable salts of the application can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. In general, these salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of both. In general, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred.

As used herein, a "phospholipid" is a lipid that includes a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains. The phospholipid may comprise one or more (e.g. double or triple) bonds (e.g. one or more unsaturated bonds). Specific phospholipids may promote fusion with the membrane. For example, a cationic phospholipid may interact with one or more negatively charged phospholipids of a membrane (e.g., a cell membrane or an intracellular membrane). The fusion of the phospholipid with the membrane may allow one or more elements of the lipid-containing composition to pass through the membrane, thereby allowing, for example, delivery of the one or more elements to the cell.

As used herein, a "polydispersity index" is a ratio that describes the uniformity of the particle size distribution of a system. Smaller values, for example less than 0.3, represent a narrow particle size distribution.

As used herein, the term "polypeptide" or "polypeptide of interest" refers to a polymer of amino acid residues that are typically linked by peptide bonds, which may be produced naturally (e.g., isolated or purified) or synthetically.

As used herein, "RNA" refers to ribonucleic acid that may be naturally or non-naturally occurring. For example, RNA can include modified and/or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. The RNA may include cap structures, chain terminating nucleosides, stem loops, polyA sequences and/or polyadenylation signals. The RNA may have a nucleotide sequence encoding a polypeptide of interest. For example, the RNA may be messenger RNA (mRNA). Translation of an mRNA encoding a particular polypeptide, e.g., in vivo translation of an mRNA inside a mammalian cell, can produce the encoded polypeptide. The RNA may be selected from a non-limiting group including small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), double-stranded RNA (dsRNA), small hairpin RNA (shRNA), mRNA and mixtures thereof.

As used herein, a "single unit dose" is a dose of any therapeutic agent administered in one dose/single route/single point of contact, i.e., a single administration.

As used herein, a "split dose" is a single unit dose or total daily dose divided into two or more doses.

As used herein, a "total daily dose" is a given or prescribed amount within 24 hours. It can be administered in a single unit dose.

As used herein, in the context of a nanoparticle composition, "particle size" or "average particle size" refers to the average diameter of the nanoparticle composition.

As used herein, the term "subject" or "patient" refers to any organism to which a composition according to the application may be administered, e.g., for experimental, diagnostic, prophylactic and/or therapeutic purposes. Typical subjects include animals (e.g., mammals, such as mice, rats, rabbits, non-human primates, and humans) and/or plants.

As used herein, "targeted cells" refers to any one or more cells of interest. Cells can be found in vitro, in vivo, in situ, or in a tissue or organ of an organism. The organism may be an animal, preferably a mammal, more preferably a human, most preferably a patient.

As used herein, "target tissue" refers to any one or more target tissue types, wherein therapeutic and/or prophylactic delivery will result in a desired biological and/or pharmacological effect. Examples of target tissues include specific tissues, organs, and systems or groups thereof. In particular applications, the target tissue may be kidney, lung, spleen, vascular endothelium in the kidney (e.g., intracoronary or intrafemoral) or in a blood vessel (e.g., by intratumoral injection). "non-target tissue" refers to any tissue type or types in which expression of the encoded protein does not result in a desired biological and/or pharmacological effect. In particular applications, non-target tissue may include liver and spleen.

The term "therapeutic agent" or "prophylactic agent" refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or causes a desired biological and/or pharmacological effect. Therapeutic agents are also referred to as "active agents" or "active ingredients. Such substances include, but are not limited to, cytotoxins, radioions, chemotherapeutics, small molecule drugs, proteins, and nucleic acids.

As used herein, the term "therapeutically effective amount" refers to an amount of an agent (e.g., nucleic acid, drug, composition, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient to be delivered when: a subject suffering from or susceptible to an infection, disease, disorder and/or condition is administered to treat, ameliorate symptoms thereof, diagnose, prevent and/or delay the onset of the infection, disease, disorder and/or condition.

As used herein, "transfection" refers to the introduction of a species (e.g., RNA) into a cell. Transfection may be performed, for example, in vitro, ex vivo or in vivo.

As used herein, the term "treatment" refers to partial or complete relief, alleviation, amelioration, remission, delay of onset thereof, inhibition of progression thereof, reduction of severity thereof and/or reduction of incidence of one or more symptoms or features thereof. Specific infections, diseases, disorders and/or conditions. For example, "treating" cancer may refer to inhibiting the survival, growth, and/or spread of a tumor. To reduce the risk, subjects that do not exhibit a disease, disorder, and/or condition and/or subjects that exhibit only early signs of a disease, disorder, and/or condition may be treated. A pathologically developed condition associated with a disease, disorder and/or condition.

In a preferred embodiment, in the compounds of the invention, R ₂ is selected from C1-C14 alkyl, for example R ₂ is selected from C2-C14 alkyl, R ₂ is selected from C3-C14 alkyl, R ₂ is selected from C4-C14 alkyl, R ₂ is selected from C5-C14 alkyl, R ₂ is selected from C6-C14 alkyl, R ₂ is selected from C7-C14 alkyl, R ₂ is selected from C8-C14 alkyl, R ₂ is selected from C9-C14 alkyl, R ₂ is selected from C10-C14 alkyl, C10-C13 alkyl, C10-C12 alkyl, C10-C11 alkyl, for example R ₂ is C1 alkyl, R ₂ is C2 alkyl, R ₂ is C3 alkyl, R ₂ is C4 alkyl, R ₂ is C5 alkyl, R ₂ is C6 alkyl, R ₂ is C7 alkyl, R ₂ is C8 alkyl, R ₂ is C9 alkyl, R8 is C10 alkyl, R11 is C12 alkyl, R7248 is C12 alkyl, R3275 is C13 alkyl. In a further preferred embodiment, R ₂ is C10 alkyl, and in a further preferred embodiment, R ₂ is C11 alkyl.

In a preferred embodiment, the R ₂ is selected from C1-C14 linear alkyl, for example, R ₂ is selected from C2-C14 linear alkyl, R ₂ is selected from C3-C14 linear alkyl, R ₂ is selected from C4-C14 linear alkyl, R ₂ is selected from C5-C14 linear alkyl, R ₂ is selected from C6-C14 linear alkyl, R ₂ is selected from C7-C14 linear alkyl, R ₂ is selected from C8-C14 linear alkyl, R ₂ is selected from C9-C14 linear alkyl, R ₂ is selected from C10-C14 linear alkyl, C10-C13 linear alkyl, C10-C12 linear alkyl, C10-C11 linear alkyl, for example, R ₂ is C1 alkyl, R ₂ is C2 alkyl, R ₂ is C3 linear alkyl, R ₂ is C4 linear alkyl, R ₂ is linear C5 alkyl, R ₂ is C6 linear alkyl, R ₂ is C7 linear alkyl, R ₂ is C8 linear alkyl, R ₂ is C9 linear alkyl, R728 linear C12 linear alkyl, R7248 is linear C12 linear alkyl. In a further preferred embodiment, R ₂ is a C10 linear alkyl group, and in a further preferred embodiment, R ₂ is a C11 linear alkyl group.

In a preferred embodiment, in the compounds of the invention, R ₃ is selected from C1-C14 alkyl, for example, R ₃ is selected from C2-C14 alkyl, R ₃ is selected from C3-C14 alkyl, R ₃ is selected from C4-C14 alkyl, R ₃ is selected from C5-C14 alkyl, R ₃ is selected from C6-C14 alkyl, R ₃ is selected from C7-C14 alkyl, R ₃ is selected from C8-C14 alkyl, R ₃ is selected from C8-C13 alkyl, R ₃ is selected from C8-C12 alkyl, R ₃ is selected from C8-C11 alkyl, R ₃ is selected from C8-C10 alkyl, R ₃ is selected from C8-C9 alkyl, for example, R ₃ is C1 alkyl, R ₃ is C2 alkyl, R ₃ is C3 alkyl, R ₃ is C4 alkyl, R ₃ is C5 alkyl, R2 is C6 alkyl, R2 is C7 alkyl, R2 is C8 alkyl, R9 is C8-C11 alkyl, R ₃ is C2 alkyl, R2 is R39312 is C12 alkyl, R39312 is R39312 alkyl. In a preferred embodiment, R ₃ is C8 alkyl.

In a preferred embodiment, the R ₃ is selected from C1-C14 linear alkyl, e.g., R ₃ is selected from C2-C14 linear alkyl, R ₃ is selected from C3-C14 linear alkyl, R ₃ is selected from C4-C14 linear alkyl, R ₃ is selected from C5-C14 linear alkyl, R ₃ is selected from C6-C14 linear alkyl, R ₃ is selected from C7-C14 linear alkyl, R ₃ is selected from C8-C14 linear alkyl, R ₃ is selected from C8-C13 linear alkyl, R ₃ is selected from C8-C12 linear alkyl, R ₃ is selected from C8-C11 linear alkyl, R ₃ is selected from C8-C10 linear alkyl, R ₃ is selected from C8-C9 linear alkyl, e.g., R ₃ is C1 alkyl, R ₃ is C2 alkyl, R ₃ is C3 linear alkyl, R ₃ is C4 linear alkyl, R ₃ is linear C5 alkyl, R2 is C6 linear alkyl, R ₃ is C7 linear alkyl, R2 is C8-C11 linear alkyl, R ₃ is C2 linear alkyl, R ₃ is C10 linear alkyl. In a preferred embodiment, R ₃ is a C8 straight chain alkyl.

The present application further provides nanoparticle compositions comprising a lipid component comprising a compound provided by the present application or a pharmaceutically acceptable salt or stereoisomer thereof.

In some embodiments, the nanoparticle composition has an average particle size of 50nm to 110nm. For example, it may be 51nm、52nm、53nm、54nm、55nm、56nm、57nm、58nm、59nm、60nm、61nm、62nm、63nm、64nm、65nm、66nm、67nm、68nm、69nm、70nm、71nm、72nm、73nm、74nm、75nm、76nm、77nm、78nm、79nm、80nm、81nm、82nm、83nm、84nm、85nm、86nm、87nm、88nm、89nm、90nm、91nm、92nm、93nm、94nm、95nm、96nm、97nm、98nm、99nm、100nm、101nm、102nm、103nm、104nm、105nm、106nm、107nm、108nm、109nm.

The nanoparticle composition may include, for example, lipid Nanoparticles (LNP), liposomes, lipid vesicles, and lipid complexes. In some embodiments, the nanoparticle composition is a vesicle comprising one or more lipid bilayers. In some embodiments, the nanoparticle composition comprises two or more concentric bilayers separated by an aqueous compartment. Lipid bilayers can be functionalized and/or crosslinked to each other. The lipid bilayer may include one or more ligands, proteins or channels.

The nanoparticle composition according to the present application comprises a lipid component comprising at least one compound according to formula (I) or (II). For example, the lipid component of the nanoparticle composition may include one or more of the compounds of the present application. The nanoparticle composition may also include a variety of other components. For example, the lipid component of the nanoparticle composition may include one or more other lipids in addition to the compound according to formula (I).

The lipid component of the nanoparticle composition may include one or more PEG or PEG-modified lipids. Such materials may alternatively be referred to as pegylated lipids. PEG lipids are lipids modified with polyethylene glycol. The PEG lipid may be selected from the non-limiting group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, the PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC or PEG-DSPE lipid.

The lipid component of the nanoparticle composition may include one or more structural lipids. The structural lipid may be selected from, but is not limited to, cholesterol, stigmasterol, sitosterol, ergosterol, stigmasterol, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipids include cholesterol and corticosteroids (e.g., prednisolone, dexamethasone, prednisone, and hydrocortisone), or combinations thereof.

The lipid component of the nanoparticle composition may include one or more phospholipids, and the phospholipids used in the nanoparticle compositions and methods may be selected from the group consisting of

Dilauroyl Lecithin (DLPC),

Dimyristoyl phosphatidylcholine (DMPC),

Di-oleoyl lecithin (DOPC),

Dipalmitoyl phosphatidylcholine (DPPC),

Distearoyl phosphatidylcholine (DSPC),

Di-oleoyl phosphatidylcholine (DUPC),

Palmitoyl Oleoyl Phosphatidylcholine (POPC),

1, 2-Di-O-octadecyl-sn-glycero-3-phosphorylcholine (18:0 Dither PC),

1-Oleoyl-2-cholesteryl dimethyl succinic acid-sn-glycero-3-phosphorylcholine (OChemsPC),

L-hexadecyl-sn-glycero-3-phosphorylcholine (C16 Lyso PC),

1, 2-Divinyl-sn-glycero-3-phosphorylcholine,

1, 2-Diaryl-acyl-sn-glycero-3-phosphorylcholine,

1, 2-Dioleoyl-SN-glycerol-3-phosphorylethanolamine (DOPE),

1, 2-Tin dihydroxide-sn-glycerol-3-phosphate ethanolamine (ME 16.0 PE),

1, 2-Distearoyl-sn-glycero-3-phosphoethanolamine,

1, 2-Divinyl-sn-glycero-3-phosphoethanolamine,

1, 2-Divinyl-sn-glycerol-3-phosphato ethanolamine,

1, 2-Diaryl-sn-glycero-3-phosphato ethanolamine,

1, 2-Dithiohexaenoic acid-sn-glycero-3-phosphoethanolamine,

1, 2-Diol-sn-glycerol-3-phosphate- (1-glycerol) sodium salt (DOPG) or sphingomyelin.

In some embodiments, the nanoparticle composition comprises DSPC. In some embodiments, the nanoparticle composition comprises DOPE. In some embodiments, the nanoparticle composition comprises DSPC and DOPE.

In some embodiments, nanoparticle compositions comprising one or more lipids described herein may further comprise one or more adjuvants, such as, for example, glucopyranosyl Lipid Adjuvants (GLA), cpG oligodeoxynucleotides (e.g., class a or class B), poly (I: C), aluminum hydroxide, and Pam3CSK4.

The nanoparticle composition may comprise one or more therapeutic and/or prophylactic agents.

The present application provides methods of delivering therapeutic and/or prophylactic agents to mammalian cells or organs, producing a polypeptide of interest in mammalian cells, and treating a disease or disorder in a mammal in need thereof, comprising administering to the mammal and/or contacting the mammalian cells with a nanoparticle composition comprising a therapeutic and/or prophylactic agent.

The therapeutic and/or prophylactic agent includes a biologically active substance, and may alternatively be referred to as an "active agent". The therapeutic and/or prophylactic agent can be a substance that, once delivered to a cell or organ, produces a desired change in the cell, organ or other body tissue or system. Such substances may be used to treat one or more diseases, disorders or conditions. In some embodiments, the therapeutic and/or prophylactic agent is a small molecule drug that can be used to treat a particular disease, disorder, or condition. Examples of drugs that may be used in the nanoparticle compositions include, but are not limited to, antineoplastic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecine, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), antineoplastic agents (e.g., dactinomycin, vincristine), vinblastine, cystine arabinoside, anthracyclines, alkylating agents, platinum compounds, antimetabolites and nucleoside analogs (e.g., methotrexate, purine and pyrimidine analogs), antiinfectives, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic blockers (e.g., propranolol, timolol, and labetalol), antihypertensives (e.g., clonidine and hydralazine), antidepressants (e.g., imipramine, amitriptyline, and doxepin), anti-converting agents (e.g., phenytoin), antihistamines, chlorphenamine, and promethazine, antibiotics/antimicrobials (e.g., gentamicin, ciprofloxacin, and ciprofloxacin), antagonists, fluconazole, and antimuscarin, fluconazole, 25, and the antagonists, such as well as the antagonists, fluconazole, and the antagonists, such as fluconazole, and the antimuscarines.

In some embodiments, the therapeutic and/or prophylactic is a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that elicits an immune response, and/or another therapeutic and/or prophylactic. Cytotoxins or cytotoxic agents include any agent that may be detrimental to cells. Examples include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, ketomilsone, 1-nor testosterone, aspergillus oryzae, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, e.g., maytansinol, rapamycin (CC-1065), and analogs or homologs thereof. Radioions include, but are not limited to, iodine (e.g., iodine 125 or iodine 131), strontium 89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium 90, sa 153, and. Vaccines include compounds and formulations capable of providing immunity to one or more conditions associated with infectious diseases (e.g., influenza, measles, human Papilloma Virus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis, and tuberculosis), including mRNA encoding infectious disease-derived antigens and/or epitopes. Vaccines also include compounds and formulations that direct immune responses against cancer cells, and may include mRNA encoding tumor cell-derived antigens, epitopes, and/or neoepitopes. Compounds that elicit an immune response may include vaccines, corticosteroids (e.g., dexamethasone) and other species.

Other therapeutic and/or prophylactic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarboxylase), alkylating agents (e.g., meclothiazine, thiopatadine-chloramphenicol, rapamycin (CC-1065), melphalan, carmustine (BSNU)), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cisplatin (DDP), anthracyclines (e.g., daunorubicin (formerly daunorubicin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, milteframycin, and Anthracycline (AMC)), and antimitotics (e.g., vincristine, vinblastine, taxol, and zein)

In some embodiments, vaccines and/or compounds capable of eliciting an immune response are administered intramuscularly by a composition comprising compounds of formula (I), (II), e.g., one or more of compounds 1-14. Other therapeutic and/or prophylactic measures include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarboxylazine), alkylating agents (e.g., methoxyethylamine, thiamine chlorobutyrate, triptomycin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cisplatin (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., actinomycin, melamycin, doxorubicin, actinomycin), anthracycline (AMC)) and antimitotics (e.g., vincristine, vinblastine, paclitaxel and maytansinoids).

In other embodiments, the therapeutic and/or prophylactic agent is a protein. Therapeutic proteins useful in the nanoparticle compositions of the present application include, but are not limited to, insulin, erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), factor VIR, luteinizing hormone-releasing hormone (LHRH) analogs, interferons, heparin, hepatitis b surface antigen, typhoid vaccines and cholera vaccines.

In some embodiments, the therapeutic agent is a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The term "polynucleotide" in its broadest sense includes any compound and/or substance that binds or can be bound into an oligonucleotide chain. Exemplary polynucleotides for use in accordance with the present invention include, but are not limited to, one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) including messenger mRNA (mRNA), hybrids thereof, RNAi-inducing agents, RN-Ai reagents, siRNA, shRNA, miRNA, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, aptamers, vectors, and the like. In some embodiments, the therapeutic and/or prophylactic is RNA. RNA useful in the compositions and methods described herein can be selected from, but is not limited to, a short polymer, an anti-gametophyte, an antisense, a ribozyme, a small interfering RNA (siRNA), an asymmetric interfering RNA (aiRNA), microRNA (miRNA), a double-stranded RNA (dsRNA), a small hairpin RNA (shRNA), a transfer RNA (tRNA), a messenger RNA (mRNA), and mixtures thereof. In certain embodiments, the RNA is mRNA.

In certain embodiments, the therapeutic and/or prophylactic agent is mRNA. The mRNA may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA may be of any size and may have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA may have a therapeutic effect when expressed in a cell.

In other embodiments, the therapeutic and/or prophylactic agent is an siRNA. The siRNA may be capable of selectively knocking down or down-regulating expression of the gene of interest. For example, the siRNA can be selected to silence a gene associated with a particular disease, disorder, or condition upon administration of a nanoparticle composition comprising the siRNA to a subject in need thereof. The siRNA may comprise a sequence complementary to an mRNA sequence encoding a gene or protein of interest. In some embodiments, the siRNA can be an immunomodulatory siRNA.

In some embodiments, the therapeutic and/or prophylactic agent is a shRNA or a vector or plasmid encoding the shRNA. After delivery of the appropriate construct to the nucleus, shRNA can be produced inside the target cell. Constructs and mechanisms related to shRNA are well known in the relevant arts.

The nanoparticle compositions of the present application may also comprise one or more components in addition to those described in the preceding section. For example, the nanoparticle composition may comprise one or more small hydrophobic molecules, such as vitamins (e.g., vitamin a or vitamin E) or sterols.

In addition to the above components, the nanoparticle composition may include any substance useful in pharmaceutical compositions. For example, nanoparticle compositions may comprise one or more pharmaceutically acceptable excipients or co-ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersing aids, suspending agents, granulating aids, disintegrants, fillers, glidants, liquid carriers, binders, surfactants, isotonic agents, thickening or emulsifying agents, buffers, lubricants, oils, preservatives and other substances. Excipients, for example waxes, butter, colorants, coatings, flavors and perfuming agents, may also be included. Pharmaceutically acceptable excipients are well known in the art.

Nanoparticle compositions of the present application may comprise a lipid component and one or more other components, such as therapeutic and/or prophylactic agents. Nanoparticle compositions can be designed for one or more specific applications or targets. The elements of the nanoparticle composition may be selected based on the particular application or goal and/or based on the efficacy, toxicity, cost, ease of use, availability or other characteristics of one or more of the elements. Similarly, a particular formulation of the nanoparticle composition may be selected for a particular application or target based on, for example, the efficacy and toxicity of a particular combination of elements.

The lipid component of the nanoparticle composition may include, for example, compounds according to formula (I), phospholipids (e.g., unsaturated lipids such as DOPE or DSPC), PEG lipids, and structural lipids.

The nanoparticle composition may be designed for one or more specific applications or targets. For example, nanoparticle compositions can be designed to deliver therapeutic and/or prophylactic agents such as RNA to a particular cell, tissue, organ, or system or group thereof in a mammal. The physicochemical properties of the nanoparticle composition can be altered to increase selectivity for a particular bodily target. For example, the particle size may be adjusted based on the fenestration size of the different organs. The therapeutic and/or prophylactic agents included in the nanoparticle compositions can also be selected based on the desired delivery target or targets. For example, therapeutic and/or prophylactic agents may be selected for a particular indication, condition, disease or disorder and/or delivery (e.g., local or specific delivery) to a particular cell, tissue, organ, or system or group thereof. In certain embodiments, nanoparticle compositions can include mRNA encoding a polypeptide of interest that can be translated in a cell to produce the polypeptide of interest. Such compositions may be designed to be specifically delivered to a particular organ. In some embodiments, the composition may be designed for specific delivery to the liver of a mammal.

The amount of therapeutic and/or prophylactic agent in the nanoparticle composition can depend on the size, composition, desired target and/or application or other property and the nature of the therapeutic and/or prophylactic agent, e.g., the amount of RNA that can be used in the nanoparticle composition can depend on the size, sequence and other characteristics of the RNA. The relative amounts of therapeutic and/or prophylactic and other elements (e.g., lipids) in the nanoparticle composition can also vary.

The characteristics of the nanoparticle composition may depend on its components. For example, nanoparticle compositions comprising cholesterol as a structural lipid may have different characteristics than nanoparticle compositions comprising a different structural lipid. Similarly, the characteristics of a nanoparticle composition may depend on the absolute or relative amounts of its components. For example, nanoparticle compositions comprising a higher mole fraction of phospholipids may have different characteristics than nanoparticle compositions comprising a lower mole fraction of phospholipids. The characteristics may also vary depending on the method and conditions of preparation of the nanoparticle composition.

The nanoparticle composition can be characterized by a variety of methods. For example, a microscope (e.g., a transmission electron microscope or a scanning electron microscope) may be used to examine the morphology and size distribution of the nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometry) can be used to measure zeta potential. Dynamic light scattering can also be used to determine particle size.

The nanoparticle composition has an average particle size of 50nm to 110nm.

The nanoparticle composition may be relatively uniform. The polydispersity index may be used to indicate the uniformity of the nanoparticle composition, e.g., the particle size distribution of the nanoparticle composition. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. The nanoparticle composition has a polydispersity index of 0.04 to 0.20. For example, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19 are possible.

Encapsulation efficiency of a therapeutic and/or prophylactic agent describes the amount of therapeutic and/or prophylactic agent that is encapsulated or otherwise associated with the nanoparticle composition after preparation relative to the initial amount provided. The higher the encapsulation efficiency is expected to be, the better (e.g., near 100%). Encapsulation efficiency may be measured, for example, by comparing the amount of therapeutic and/or prophylactic agent of a solution containing the nanoparticle composition before and after decomposing the nanoparticle composition with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic and/or prophylactic (e.g., RNA) in solution. For nanoparticle compositions described herein, the therapeutic and/or prophylactic agent can have an encapsulation efficiency of at least 50%, e.g., 50%,55%,60%,65%,70%,75%,80%,85%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In certain embodiments, the encapsulation efficiency may be at least 90%.

The nanoparticle composition may optionally comprise one or more coatings. For example, the nanoparticle composition can be formulated as a capsule, film or tablet with a coating. Capsules, films or tablets comprising the compositions described herein may be of any useful size, tensile strength, hardness or density.

The nanoparticle compositions of the present application may be formulated in whole or in part as pharmaceutical compositions. The pharmaceutical composition may comprise one or more nanoparticle ingredients. For example, the pharmaceutical composition may include one or more nanoparticle compositions comprising one or more different therapeutic and/or prophylactic agents. The pharmaceutical composition may further comprise one or more pharmaceutically acceptable excipients or adjunct ingredients, such as those described herein, unless any conventional excipient or adjunct ingredient may be incompatible with one or more components of the nanoparticle composition. If the excipient or adjunct ingredient is incompatible with the components of the nanoparticle composition, its combination with the component may result in any undesirable biological or other deleterious effects.

In some embodiments, one or more excipients or adjunct ingredients can comprise greater than 50% of the total mass or volume of the pharmaceutical composition including the nanoparticle composition. For example, one or more excipients or auxiliary ingredients may constitute 50%,60%,70%,80%,90% or more of the pharmaceutical convention. In some embodiments, the pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% pure. In some embodiments, the excipient is approved for human and veterinary use.

The relative amounts of the nanoparticle composition(s), the pharmaceutically acceptable excipient(s) and/or any other ingredients in the pharmaceutical composition according to the present application will vary depending on the identity, size and/or disease condition of the subject being treated, and further depending on the route of administration of the composition. For example, the pharmaceutical composition may comprise from 0.1% to 100% (wt/wt) of one or more nanoparticle compositions.

The nanoparticle composition and/or pharmaceutical composition comprising one or more of the nanoparticle compositions may be administered to any patient or subject, including those patients or subjects who may benefit from the therapeutic effects provided by delivery of therapeutic and/or prophylactic agents to one or more specific cells, tissues, organs, or systems or groups thereof (e.g., the renal system). Although the description of nanoparticle compositions and pharmaceutical compositions comprising nanoparticle compositions provided herein is primarily directed to compositions suitable for administration to humans, those skilled in the art will appreciate that such compositions are generally suitable for administration to any other mammal. Modifications to compositions suitable for human administration in order to adapt the compositions to various animal administration are well known, and generally skilled veterinary pharmacologists may merely design and/or make such modifications through ordinary (if any) experimentation, including but not limited to humans, other primates and other man-made animals, including commercially relevant mammals such as cows, pigs, hoses, sheep, cats, dogs, mice and/or rats.

Pharmaceutical compositions comprising one or more nanoparticle compositions may be prepared by any method known or later developed in the pharmacological arts. Typically, such preparation involves combining the active ingredient with excipients and/or one or more other auxiliary ingredients, and then, if desired or necessary, dividing, shaping and/or packaging the product into the desired single or multiple dose units.

Pharmaceutical compositions according to the present application may be prepared, packaged and/or sold in batches in single unit doses and/or in multiple single unit doses. As used herein, a "unit dose" is a precise amount of a pharmaceutical composition that is small, comprising a predetermined amount of an active ingredient (e.g., a nanoparticle composition). The amount of active ingredient is typically equal to the dose of active ingredient to be administered to the subject and/or a convenient portion of the dose, e.g., half or one third of the dose.

Pharmaceutical compositions may be prepared in a variety of forms suitable for a variety of routes and methods of administration. For example, pharmaceutical compositions may be formulated in liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups and medicaments), injectable forms, solid dosage forms (e.g., capsules, tablets, etc.) to prepare slave liquids, powders and granules), dosage forms for topical and/or transdermal administration (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and patches), suspensions, powders and other forms. Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups and/or medicaments.

In addition to the active ingredient, the liquid dosage forms may include inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitol and mixtures thereof. In addition to inert diluents, the oral compositions can also include other therapeutic and/or prophylactic agents, other agents, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and/or perfuming agents. In certain embodiments for parenteral administration, the composition is mixed with a solubilizing agent (e.g., alcohol, oil, modified oil, glycol, polysorbate, cyclodextrin, polymer, and/or combinations thereof).

Injectable formulations, such as sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing, wetting and/or suspending agents. The sterile injectable preparation may be a sterile injectable solution, suspension and/or emulsion in a non-toxic parenterally acceptable diluent and/or solvent, for example, as a solution in 1, 3-butanediol. Acceptable vehicles and solvents that may be used include water. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids such as oleic acid find use in the preparation of injectables.

The injectable formulations may be sterilized, for example, by filtration through a bacterial-retaining filter and/or by incorporating sterilizing agents in the form of sterile solid compositions which may be employed prior to dissolution or dispersion in sterile water or other sterile injectable medium.

In order to prolong the action of the active ingredient, it is generally desirable to slow down the absorption of the active ingredient from subcutaneous or intramuscular injection. This can be achieved by using liquid suspensions of crystalline or amorphous materials that are poorly water soluble. The rate of absorption of a drug then depends on its rate of dissolution, which in turn depends on the crystal size and crystal form. Or by dissolving or suspending the drug in an oil carrier. The injectable depot forms are made by forming a matrix of microcapsules of the drug in a biodegradable polymer such as polylactic acid-polyethylene glycol. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (ortho esters) and poly (anhydrides). Stock injectable formulations are prepared by embedding the drug in liposomes or microemulsions that are compatible with body tissues.

Compositions for rectal or vaginal administration are generally suppositories which can be prepared by mixing the composition with suitable non-irritating excipients such as cocoa butter, polyethylene glycols or suppository waxes, which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.

Solid dosage forms for oral administration include capsules, tablets, pills, films, powders and granules. In such solid dosage forms, the active ingredient is admixed with at least one inert pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate and/or fillers or extenders (e.g., starches, lactose, sucrose, glucose, mannitol, and silicic acid), binders (e.g., carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia), humectants (e.g., glycerin), disintegrants (e.g., agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate), slow solvents (e.g., paraffin), absorption accelerators (e.g., quaternary ammonium compounds), wetting agents (e.g., cetyl alcohol and glycerol monostearate), absorbents (e.g., kaolin and bentonite, silicates) and lubricants (e.g., talc), calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate), and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may comprise buffering agents.

Solid compositions of a similar type may be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols and the like. Solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may have a composition that only releases the active ingredient, or preferably in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes. Solid compositions of a similar type can be used as lactose and excipients such as lactose and high molecular weight polyethylene glycols and the like as fillers in soft and hard-filled gelatin capsules.

Dosage forms for topical and/or transdermal administration of the composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and/or patches. Typically, the active ingredient is combined under sterile conditions with pharmaceutically acceptable excipients and/or any preservatives and/or buffers that may be required. In addition, the present application contemplates the use of transdermal patches, which generally have the additional advantage of providing controlled delivery of the compound to the body. Such dosage forms may be prepared, for example, by dissolving and/or partitioning the compound in a suitable medium. Alternatively or additionally, the rate may be controlled by any of the following to provide a rate controlling membrane and/or by dispersing the compound in the polymer matrix and/or gel.

Formulations suitable for topical administration include, but are not limited to, liquid and/or semi-liquid formulations such as liniments, lotions, oil-in-water and/or water-in-oil emulsions such as creams, ointments and/or pastes and/or solutions and/or suspensions, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the other ingredients described herein.

The pharmaceutical composition may be prepared, packaged and/or sold in a formulation suitable for pulmonary administration via the oral cavity. Such formulations may comprise dry particles of the active ingredient. Such compositions are conveniently in dry powder form for administration using a device comprising a dry powder reservoir and/or using a self-propelled solvent/powder dispensing container (e.g. a device containing the active ingredient dissolved and/or suspended in a low boiling point propellant in a sealed container), and may comprise a solid fine powder diluent, such as a sugar, conveniently provided in unit dosage form.

Low boiling point propellants typically include liquid propellants having a boiling point below 65°f at atmospheric pressure. Typically, the propellant may comprise 50% to 99.9% (wt/wt) of the composition, while the active ingredient may comprise 0.1% to 20% (wt/wt) of the composition. The propellant may further comprise other ingredients such as liquid nonionic and/or solid anionic surfactants and/or solid diluents (which may have particle sizes of the same order of magnitude as the particles comprising the active ingredient).

Pharmaceutical compositions formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and/or suspension. Such formulations may be prepared, packaged and/or sold as aqueous and/or diluted alcoholic solutions and/or suspensions, optionally sterile, containing the active ingredient, and may be conveniently administered using any of the nebulization and/or nebulization devices. Such formulations may further comprise one or more other ingredients including, but not limited to, flavoring agents such as sodium saccharin, volatile oils, buffers, surfactants, and/or preservatives such as methyl hydroxybenzoate. The average diameter of the droplets provided by this route of administration may be in the range of 1nm to 200 nm.

Formulations described herein that can be used for pulmonary delivery can be used for intranasal delivery of pharmaceutical compositions. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle size of from 0.2 μm to 500 μm. Such formulations are administered by way of snuff administration by rapid inhalation through the nasal passages from a powder container held near the nose.

Formulations suitable for nasal administration may, for example, comprise from 0.1% (wt/wt) to 100% (wt/wt) of the active ingredient, and may comprise one or more other active ingredients as described herein. The pharmaceutical compositions may be prepared, packaged and/or sold in formulations suitable for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges prepared using conventional methods, and may, for example, be from 0.1% to 20% (wt/wt) of the active ingredient, the balance comprising the orally dissolvable and/or degradable composition, and optionally one or more other ingredients described herein. Or formulations suitable for oral administration may comprise powders and/or nebulized and/or sprayed solutions and/or suspensions containing the active ingredient. Such powdered, aerosolized and/or aerosolized formulations may have an average particle and/or droplet size in the range of 0.1nm to 200nm when dispersed, and may further comprise one or more other ingredients described herein.

The pharmaceutical compositions may be prepared, packaged and/or sold in a form suitable for ophthalmic administration. Such formulations may, for example, be in the form of drops, including, for example, 0.1/1.0% (wt/wt) solutions and/or suspensions of the active ingredient in aqueous or oily liquid vehicles. Such drops may further comprise buffers, salts, and/or one or more of any of the other additive ingredients described herein. Other formulations useful for ophthalmic administration include formulations comprising the active ingredient in microcrystalline form and/or in liposomal formulations. Methods of producing polypeptides in cells within the scope of the present disclosure are believed to be ear drops and/or eye drops

The application further provides methods of producing a polypeptide of interest in a mammalian cell. The method of producing a polypeptide comprises contacting a cell with a nanoparticle composition comprising an mRNA encoding the polypeptide of interest. After contacting the cell with the nanoparticle composition, the mRNA can be taken up and translated in the cell to produce the polypeptide of interest.

In general, the step of contacting the mammalian cells with a nanoparticle composition comprising mRNA encoding the polypeptide of interest can be performed in vivo, ex vivo, in culture, or in vitro. The amount of nanoparticle composition contacted with the cells and/or the amount of mRNA therein can depend on the type of cell or tissue being contacted, the mode of administration, the physicochemical properties of the nanoparticle composition and the mRNA (e.g., size, charge and chemical composition), among other factors. In general, an effective amount of the nanoparticle composition will allow for efficient production of the polypeptide in the cell. Metrics for efficiency may include polypeptide translation (expressed by polypeptide expression), mRNA degradation levels, and immune response indicators.

The step of contacting the nanoparticle composition comprising mRNA with the cell may involve or cause transfection. The phospholipids comprised in the lipid component of the nanoparticle composition may facilitate transfection and/or increase transfection efficiency, e.g., by interacting and/or fusing with cells or cell membranes. Transfection may allow translation of intracellular mRNA.

In some embodiments, nanoparticle compositions described herein can be used in therapy. For example, an mRNA included in a nanoparticle composition can encode a therapeutic polypeptide (e.g., in a translatable region) and produce the therapeutic polypeptide upon contact with and/or entry (e.g., transfection) into a cell, and in other embodiments, an mRNA included in a nanoparticle composition can encode a polypeptide that can improve or increase immunity in a subject. For example, the mRNA may encode granulocyte colony stimulating factor or trastuzumab.

In certain embodiments, the mRNA included in the nanoparticle composition can encode a recombinant polypeptide that can replace one or more polypeptides that may be substantially absent from cells contacted with the nanoparticle composition. One or more polypeptides that are substantially absent may be absent due to genetic mutations of the encoding gene or its regulatory pathways. Alternatively, recombinant polypeptides produced by translation of mRNA can antagonize the activity of endogenous proteins present in, on the surface of, or secreted from a cell. Resistant recombinant polypeptides may be desirable to combat deleterious effects caused by the activity of endogenous proteins, such as activity changes or mutation-induced localization. In another alternative, the recombinant polypeptide produced by translation of the mRNA may indirectly or directly antagonize the activity of a biological moiety present in the cell, on the cell surface, or secreted from the cell. Antagonistic biological moieties can include, but are not limited to, lipids (e.g., cholesterol), lipoproteins (e.g., low density lipoproteins), nucleic acids, carbohydrates and small molecule toxins. Recombinant polypeptides produced by mRNA translation may be engineered to be located within a cell, for example within a specific compartment such as the nucleus, or may be engineered to be secreted or transported from a cell to the plasma membrane of a cell.

In some embodiments, contacting the cell with a nanoparticle composition comprising mRNA can reduce the innate immune response of the cell to exogenous nucleic acids. The cell may be contacted with a first nanoparticle composition comprising a first amount of a first exogenous mRNA, the first exogenous mRNA comprising a translatable region, and the level of the cell's innate immune response to the first exogenous mRNA may be determined. Subsequently, the cell can be contacted with a second composition comprising a second amount of the first exogenous mRNA, the second amount being a small amount of the first exogenous mRNA compared to the first amount. Or the second composition may comprise a first amount of a second exogenous mRNA different from the first exogenous mRNA. The step of contacting the cells with the first and second compositions may be repeated one or more times. In addition, the efficiency of polypeptide production (e.g., translation) in the cell can optionally be determined, and the cell can be repeatedly contacted again with the first and/or second composition until the target protein production efficiency is reached.

The application further provides a method of treating a disease or disorder in a mammal, the method comprising administering to the mammal a therapeutically effective amount of a nanoparticle composition of any of the foregoing, in particular, the nanoparticle composition is useful for treating a disease, disorder or condition characterized by a loss or abnormality in protein or polypeptide activity. For example, nanoparticle compositions comprising mRNA encoding a deleted or aberrant polypeptide may be administered or delivered to cells. Subsequent translation of the mRNA may produce the polypeptide, thereby reducing or eliminating problems due to lack or abnormal activity caused by the polypeptide. Because translation may occur rapidly, these methods and compositions are useful in treating acute diseases, disorders or conditions such as sepsis, stroke, and myocardial infarction. The therapeutic and/or prophylactic agents included in the nanoparticle compositions may also be capable of altering the transcription rate of a given species, thereby affecting gene expression.

Diseases, disorders and/or conditions characterized by dysfunctional or aberrant protein or polypeptide activity include, but are not limited to, rare diseases, infectious diseases (e.g., vaccines and therapeutics), cancer and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

The present application provides methods involving administration of nanoparticle compositions comprising one or more therapeutic and/or prophylactic agents and pharmaceutical compositions comprising nanoparticle compositions. The terms therapeutic and prophylactic may be used interchangeably herein with respect to the features and embodiments of the present application. The therapeutic composition or imaging, diagnostic or prophylactic composition thereof can be administered to a subject using any reasonable amount and any route of administration effective to prevent, treat, diagnose or image a disease, disorder and/or condition and/or any other purpose. The particular amount of a particular subject administered may vary depending on the type, age, and general condition of the subject; management purposes; specific components; a management mode; etc. The compositions according to the application may be formulated in unit dosage form for ease of administration and uniformity of dosage. However, it will be appreciated that the total daily dosage of the compositions of the present application will be determined by the attending physician within the scope of sound medical judgment. The particular therapeutically effective, prophylactically effective, or other appropriate dosage level (e.g., for imaging) for any particular patient will depend on a variety of factors including the severity and identity (if any) of the disease being treated; one or more therapeutic and/or prophylactic agents used; the specific components are used; age, weight, general health, sex and diet of the patient; the time of administration, route of administration and rate of excretion of the particular pharmaceutical component employed; duration of treatment; a medicament for use in combination or simultaneously with the particular pharmaceutical ingredient being used; factors well known in the medical community.

The application further provides a method of specifically delivering a therapeutic and/or prophylactic agent to an organ of a mammal, the method comprising administering to the mammal a nanoparticle composition of any of the foregoing, the administering comprising contacting the organ of the mammal with the nanoparticle composition, thereby delivering the therapeutic and/or prophylactic agent to the organ. Therapeutic and/or prophylactic agents, e.g., proteins, cytotoxic agents, radioactive ions, chemotherapeutic agents, or nucleic acids (e.g., RNA, e.g., mRNA) may be delivered to a cell or organ. Where the therapeutic and/or prophylactic is an mRNA, the translatable mRNA can be translated in the cell to produce the polypeptide of interest when the cell is contacted with the nanoparticle composition. However, substantially nontranslatable mRNAs may also be delivered to the cells. The substantially nontranslatable mRNA can be used as a vaccine and/or can sequester the translated components of the cell to reduce expression of other species in the cell.

In some embodiments, the nanoparticle composition can target a particular type or class of cells (e.g., cells of a particular organ or system thereof). For example, nanoparticle compositions comprising a target treatment and/or prophylaxis may be specifically delivered to the liver, kidney, spleen, femur or lung of a mammal. Specific delivery to a particular class of cells, organs, or systems or groups thereof means that a higher proportion of nanoparticle composition including therapeutic and/or prophylactic agent(s) is delivered to the target destination (e.g., tissue) relative to other destinations, e.g., upon administration of the nanoparticle composition to a mammal. In some embodiments, the target tissue is selected from liver, kidney, lung, spleen, femur, ocular tissue (e.g., by intraocular, subretinal, or intravitreal injection), vascular endothelium in blood vessels (e.g., intracoronary or intrafemoral), or kidney, and tumor tissue (e.g., by intratumoral injection).

As another example of targeted or specific delivery, mRNA encoding a protein binding partner (e.g., an antibody or functional fragment thereof, a scaffold protein, or peptide) or receptor on the cell surface may be included in the nanoparticle composition. mRNA may be used to increase the rate of synthesis and extracellular localization of lipids, carbohydrates or other biological moieties alternatively or in the alternative. Alternatively, other therapeutic and/or prophylactic agents or elements (e.g., lipids or ligands) of the nanoparticle composition may be selected based on their affinity for a particular receptor (e.g., a low density lipoprotein receptor) such that the nanoparticle composition may more readily interact with a target cell population that includes the receptor.

In some embodiments, the ligand may be a surface-bound antibody, which may allow for modulation of cell targeting specificity. This is particularly useful because highly specific antibodies can be raised against the target epitope of the desired targeting site. In one embodiment, multiple antibodies are expressed on the cell surface, and each antibody may have a different specificity for a desired target. This approach can increase the affinity and specificity of the targeted interactions.

The targeted cells may include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells.

In some embodiments, the nanoparticle composition may target hepatocytes.

The nanoparticle compositions of the present application may be administered by any route. In some embodiments, compositions comprising a prophylactic, diagnostic or imaging composition are administered by one or more of a variety of routes, including oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal or intradermal, rectal, intravaginal, intraperitoneal, intraocular, subretinal, intravitreal, topical (e.g., by powders, ointments, creams, gels, lotions and/or drops), mucosal, nasal, buccal, intestinal, vitreous, intratumoral, sublingual, intranasal; by intratracheal instillation, bronchial instillation and/or inhalation; as an oral spray and/or powder, nasal spray and/or aerosol, and/or via a portal vein catheter. In some embodiments, the composition may be administered intravenously, intramuscularly, intradermally, intraarterially, intratumorally, subcutaneously, intraocularly, subretinally, intravitreally, or by inhalation. However, in view of the possible advances in drug delivery science, the present application contemplates delivery or description of the compositions of the present application by any suitable route. Generally, the most suitable route of administration depends on a variety of factors, including the nature of the nanoparticle composition, including one or more therapeutic and/or prophylactic measures (e.g., its stability in various bodily environments (e.g., blood and gastrointestinal tract)), the condition of the patient (e.g., whether the patient is able to tolerate a particular route), and the like.

In certain embodiments, wherein a therapeutic and/or prophylactic agent is administered to a mammal at a dose of 0.0001mg/kg to 10 mg/kg. For example, it may be 0.0001 to 10mg/kg,0.001 to 10mg/kg,0.005 to 10mg/kg,0.01 to 10mg/kg,0.05 to 10mg/kg,0.1 to 10mg/kg,0.0001 to 5mg/kg,0.001 to 5mg/kg,0.005 to 5mg/kg,0.01 to 5mg/kg,0.05 to 5mg/kg,0.1 to 5mg/kg,1 to 5mg/kg,2mg to 5mg/kg,0.0001 to 2.5mg/kg,0.0001 to 5mg/kg,0.01 to 5mg to 25mg/kg,0.01 to 5mg/kg, 0.25mg to 0.25mg/kg,0.01 to 5mg/kg, 0.25mg to 0.005mg to 5mg/kg,0.01 to 25mg/kg. In some embodiments, a therapeutic and/or prophylactic (e.g., mRNA) dose of about 0.001mg/kg to about 10mg/kg of the nanoparticle composition may be administered. In other embodiments, therapeutic and/or prophylactic doses of about 0.005mg/kg to about 2.5mg/kg may be administered. In certain embodiments, a dose of about 0.1mg/kg to about 1mg/kg may be administered. In other embodiments, a dose of about 0.05mg/kg to about 0.25mg/kg may be administered. The doses may be administered one or more times per day in the same or different amounts to obtain the desired level of mRNA expression and/or therapeutic, diagnostic, prophylactic or imaging effect. The desired dose may be delivered, for example, three times per day, twice per day, once per day, every other day, every third day, weekly, biweekly, every third week, or every fourth week. In certain embodiments, multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, twelve, thirteen, ten or more administrations) may be used to deliver the desired dose. In some embodiments, a single dose may be administered, for example, before or after surgery, or in the case of an acute disease, disorder, or condition.

Nanoparticle compositions comprising one or more therapeutic and/or prophylactic agents may be used in combination with one or more other therapeutic, prophylactic, diagnostic or imaging agents. "in combination with … …" does not mean that the agents must be administered and/or formulated simultaneously for delivery together, although such delivery methods are within the scope of the application. For example, one or more nanoparticle compositions comprising one or more different therapeutic and/or prophylactic agents may be administered in combination. The composition may be administered simultaneously, before or after one or more other desired therapeutic or medical procedures. Typically, each agent will be administered at a dosage and/or schedule determined for that agent. In some embodiments, the application includes delivering the composition or an imaging, diagnostic or prophylactic composition thereof in combination with an agent that increases its bioavailability, reduces and/or alters its metabolism, inhibits its excretion and/or modulates contractions in the body.

It will be appreciated that the therapeutic, prophylactic, diagnostic or imaging agents used in combination may be administered together in a single composition or separately in different compositions. In general, it is desirable that the levels of agents used in combination do not exceed the levels at which they are used alone. In some embodiments, the levels used in combination may be lower than the levels used alone.

The particular combination of treatments (treatments or procedures) used in the combination regimen will take into account the compatibility of the desired treatment and/or procedure and the desired therapeutic effect to be achieved. It will also be appreciated that the treatments employed may achieve a desired effect on the same condition (e.g., the composition used to treat the cancer may be administered concurrently with the chemotherapeutic agent), or they may achieve a different effect (e.g., control of any side effects, such as infusion-related reactions).

In some embodiments, a method of treating a subject in need thereof or delivering a therapeutic and/or prophylactic agent to a subject (e.g., mammal) may comprise pre-treating the subject with one or more agents prior to administration of the nanoparticle composition. For example, the subject may be pretreated with an effective amount (e.g., 10mg, 20mg, 30mg, 40mg, 50mg, 60mg, 70mg, 80mg, 90mg, 100mg, or any other effective amount) of dexamethasone, methotrexate, acetaminophen, hl receptor blocker, or H2 receptor blocker. Pretreatment may occur 24 hours or less (e.g., 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 4 hours, 2 hours, 1 hour, 50 minutes, 40 minutes, 30 minutes, 20 minutes, or 10 minutes) prior to administration of the nanoparticle composition, and may occur once, twice, or more, e.g., with increasing doses.

Examples

The present invention is illustrated below in conjunction with specific examples which are not intended to limit the scope of the invention, but rather to provide guidance to those skilled in the art in making and using the compounds, compositions of the present invention.

The synthetic routes for the compounds of this example section are as follows:

Example 1

The synthetic route is as follows:

To a solution of compound a-1 (570 mg,2.92 mmol) in DCM (6 mL) was added B (640 mg,3.49 mmol) and DCC (910 mg,4.41 mmol), the reaction was stirred for 15 hours at 25 ℃, monitored by TLC, filtered after completion of the reaction, concentrated, and chromatographed on silica gel (PE: ea=50:1) to give compound C-1 (1510 mg, 92% yield).

To a solution of compound E-1 (1000 mg,2.17 mmol) in ethanol (3 mL) was added 10-fold equivalent of compound D-1 (2540 mg,21.67 mmol), DIEA (840 mg,6.50 mmol), and the reaction was heated at 75℃for 24 hours, followed by TLC monitoring the reaction, and after completion of the reaction, it was cooled to room temperature. The reaction mixture was diluted with water, extracted with ethyl acetate (50 mL. Times.3), concentrated, and chromatographed on silica gel column (PE: EA=5:1) to give oily compound F-1 (659 mg, yield 61%)

To a mixed solvent of acetonitrile and cyclopentyl methyl ether (cyclopentyl methyl ether: acetonitrile=1:1, 4 ml) of compound C-1 (300 mg,0.53 mmol) were added 1.0-fold equivalent of compound F-1 (270 mg,0.53 mmol), K2CO3 (220 mg,1.59 mmol) and KI (200 mg,1.20 mmol), and the reaction was heated at 75 ℃ for 24 hours, followed by cooling to room temperature after completion of the reaction by TLC monitoring. The reaction solution was diluted with water, extracted with ethyl acetate (30 ml×3), concentrated, and chromatographed on silica gel (dichloromethane: methanol=20:1) to give compound 1 (234 mg, yield 45％),¹H NMR(400MHz,CDCl3)δ5.42(d,J＝5.0Hz,1H),4.91(p,J＝6.3Hz,1H),4.65(ddt,J＝11.5,8.0,4.3Hz,1H),3.69(q,J＝6.0Hz,2H),2.56(s,6H),2.37–2.30(m,6H),2.09–1.99(m,2H),1.89(ddt,J＝13.9,9.3,4.4Hz,4H),1.76–1.49(m,11H),1.33(d,J＝24.4Hz,54H),1.17(qd,J＝10.8,4.4Hz,6H),1.06(s,4H),0.98–0.85(m,15H),0.72(s,3H).MS-ESI(m/z):981(M+H)⁺.

Example 2

The preparation method is the same as that of the compound 1, and 6-bromohexanoic acid, ethanolamine, cholesterol and heptadec-9-yl 8-bromooctanoate are used as raw materials to prepare oily compound 2,¹H NMR(400MHz,CDCl3)δ5.41(d,J＝4.9Hz,1H),4.90(p,J＝6.2Hz,1H),4.65(ddt,J＝11.5,8.2,4.3Hz,1H),3.69(s,2H),2.70(d,J＝48.8Hz,6H),2.40–2.25(m,6H),2.13–1.96(m,2H),1.89(ddt,J＝14.2,9.7,4.7Hz,4H),1.74–1.46(m,9H),1.33(d,J＝24.3Hz,48H),1.22–1.10(m,6H),1.06(s,4H),0.98–0.86(m,15H),0.72(s,3H).MS-ESI(m/z):925.2(M+H)⁺.

Example 3

The preparation method is the same as that of the compound 1, and 6-bromohexanoic acid, ethanolamine, cholesterol and 6-bromohexanoic acid undecyl ester are used as raw materials to prepare oily compound 3,¹H NMR(400MHz,CDCl3)δ5.46–5.38(m,1H),4.65(dtd,J＝11.6,8.6,4.3Hz,1H),4.10(t,J＝6.8Hz,2H),3.70(s,2H),2.72(d,J＝43.8Hz,6H),2.40–2.27(m,6H),2.10–1.97(m,2H),1.89(ddt,J＝14.4,9.7,4.6Hz,4H),1.75–1.56(m,11H),1.46–1.24(m,32H),1.16(dd,J＝9.9,4.4Hz,6H),1.06(s,4H),0.97–0.87(m,12H),0.72(s,3H).MS-ESI(m/z):813.0(M+H)⁺.

Example 4

The preparation method is the same as that of the compound 1, and 6-bromohexanoic acid, 6-amino-1-hexanol, cholesterol and 6-bromohexanoic acid undecyl ester are used as raw materials to prepare oily compounds 4,¹H NMR(400MHz,CDCl3)δ5.41(d,J＝5.1Hz,1H),4.65(dtd,J＝11.6,8.6,4.3Hz,1H),4.09(t,J＝6.8Hz,2H),3.68(t,J＝6.4Hz,2H),2.61(s,6H),2.39–2.27(m,6H),2.09–1.96(m,2H),1.88(ddt,J＝14.1,9.5,4.6Hz,4H),1.74–1.48(m,13H),1.33(d,J＝26.3Hz,38H),1.16(tt,J＝14.1,7.2Hz,6H),1.06(s,4H),0.97–0.86(m,12H),0.71(s,3H).MS-ESI(m/z):869.0(M+H)⁺.

Example 5

The preparation method is the same as that of the compound 1, and 8-bromooctanoic acid, 6-amino-1-hexanol, cholesterol and 6-bromohexanoic acid undecyl ester are used as raw materials to prepare oily compounds 5,¹H NMR(400MHz,CDCl3)δ5.42(d,J＝5.0Hz,1H),4.73–4.57(m,1H),4.10(t,J＝6.8Hz,2H),3.70(t,J＝6.2Hz,2H),3.02(d,J＝9.4Hz,6H),2.44–2.26(m,6H),2.04(t,J＝15.3Hz,2H),1.94–1.77(m,4H),1.76–1.58(m,13H),1.57–1.26(m,42H),1.17(tt,J＝13.4,7.0Hz,6H),1.07(d,J＝2.3Hz,4H),0.99–0.87(m,12H),0.72(s,3H).MS-ESI(m/z):897.1(M+H)⁺.

Example 6

The preparation method is the same as that of the compound 1, and 8-bromooctanoic acid, 6-amino-1-hexanol, cholesterol and heptadec-9-yl 8-bromooctanoate are used as raw materials to prepare oily compound 6,¹H NMR(400MHz,CDCl3)δ5.41(d,J＝5.0Hz,1H),4.90(t,J＝6.2Hz,1H),4.71–4.59(m,1H),3.68(t,J＝6.6Hz,2H),2.52(s,6H),2.39–2.27(m,6H),2.10–1.97(m,2H),1.88(ddd,J＝13.8,9.3,4.9Hz,4H),1.69–1.47(m,11H),1.32(d,J＝23.3Hz,58H),1.16(dd,J＝9.9,4.4Hz,6H),1.06(s,4H),0.96–0.86(m,15H),0.72(s,3H).MS-ESI(m/z):1009.2(M+H)⁺.

Example 7

The preparation method is the same as that of the compound 1, and 8-bromooctanoic acid, ethanolamine, cholesterol and heptadec-9-yl 8-bromooctanoate are used as raw materials to prepare oily compound 7,¹H NMR(400MHz,CDCl3)δ5.45–5.38(m,1H),4.90(p,J＝6.3Hz,1H),4.65(ddt,J＝11.4,8.1,4.3Hz,1H),3.67(t,J＝5.1Hz,2H),2.75(d,J＝6.5Hz,2H),2.63(d,J＝8.0Hz,4H),2.40–2.26(m,6H),2.10–1.96(m,2H),1.90(d,J＝13.6Hz,4H),1.72–1.48(m,9H),1.33(d,J＝24.7Hz,52H),1.22–1.10(m,6H),1.06(s,4H),0.97–0.88(m,15H),0.72(s,3H).MS-ESI(m/z):953.2(M+H)+.

Example 8

The preparation method is the same as that of the compound 1, and 8-bromooctanoic acid, ethanolamine, cholesterol and 6-bromohexanoic acid undecyl ester are used as raw materials to prepare oily compound 8,¹H NMR(400MHz,CDCl3)δ5.44–5.38(m,1H),4.65(ddt,J＝11.4,8.0,4.2Hz,1H),4.09(t,J＝6.8Hz,2H),3.64(t,J＝5.2Hz,2H),2.71(t,J＝5.2Hz,2H),2.58(p,J＝4.9Hz,4H),2.39–2.27(m,6H),2.09–1.96(m,2H),1.88(ddd,J＝14.0,9.4,4.9Hz,4H),1.73–1.46(m,11H),1.43–1.25(m,36H),1.17(ddd,J＝19.5,9.7,5.9Hz,6H),1.06(s,4H),0.95–0.88(m,12H),0.71(s,3H).MS-ESI(m/z):841.1(M+H)⁺.

Example 9

The preparation method is the same as that of the compound 1, and 4-bromobutyric acid, 6-amino-1-hexanol, cholesterol and heptadec-9-yl 8-bromooctanoate are used as raw materials to prepare oily compound 9,¹H NMR(400MHz,CDCl3)δ5.41(d,J＝4.9Hz,1H),4.90(t,J＝6.2Hz,1H),4.65(dtd,J＝11.6,8.5,4.2Hz,1H),3.69(t,J＝6.5Hz,2H),2.50(s,6H),2.34(dt,J＝17.5,6.9Hz,6H),2.12–1.96(m,2H),1.87(tdd,J＝13.1,9.4,4.9Hz,4H),1.71–1.47(m,11H),1.46–1.24(m,50H),1.22–1.11(m,6H),1.06(s,4H),0.98–0.87(m,15H),0.72(s,3H).MS-ESI(m/z):953.3(M+H)⁺.

Example 10

The preparation method is the same as that of the compound 1, and 4-bromobutyric acid, ethanolamine, cholesterol and heptadec-9-yl 8-bromocaprylate are used as raw materials to prepare oily compound 10,¹H NMR(400MHz,CDCl3)δ5.41(dd,J＝4.9,1.8Hz,1H),4.90(p,J＝6.3Hz,1H),4.66(dtd,J＝11.5,8.4,4.2Hz,1H),3.60(t,J＝5.2Hz,2H),2.65(t,J＝5.2Hz,2H),2.53(dt,J＝19.5,6.7Hz,4H),2.33(dt,J＝11.4,7.6Hz,6H),2.10–1.96(m,2H),1.95–1.76(m,4H),1.71–1.44(m,9H),1.43–1.23(m,44H),1.16(dd,J＝10.0,4.3Hz,6H),1.06(s,4H),0.97–0.85(m,15H),0.72(s,3H).MS-ESI(m/z):897.1(M+H)⁺.

Example 11

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To a solution of Compound C-3 (500 mg,0.93 mmol) in acetonitrile (4 mL) was added 0.45-fold equivalent of 6-amino-1-hexanol (50 mg,0.42 mmol), K2CO3 (180 mg,1.30 mmol) and KI (160 mg,0.96 mmol), and the reaction was heated at 75deg.C for 24 hours, monitored by TLC, and after completion of the reaction, cooled to room temperature. The reaction solution was diluted with water, extracted with ethyl acetate (30 ml×3), concentrated, and chromatographed on silica gel (dichloromethane: methanol=30:1) to give compound 11 (215 mg, yield 50％)¹H NMR(400MHz,CDCl3)δ5.45–5.38(m,2H),4.65(dt,J＝8.1,3.2Hz,2H),3.68(t,J＝6.5Hz,2H),2.47(s,4H),2.34(dd,J＝7.4,3.5Hz,8H),2.09–1.96(m,4H),1.94–1.74(m,10H),1.70–1.46(m,16H),1.45–1.24(m,22H),1.16(ddd,J＝18.9,11.0,4.2Hz,12H),1.06(s,8H),0.95(d,J＝6.5Hz,6H),0.90(dd,J＝6.6,1.9Hz,12H),0.72(s,6H).MS-ESI(m/z):1027.2(M+H)⁺.

Example 12

The preparation method is the same as that of the compound 11, and 4-bromobutyric acid, ethanolamine and cholesterol are used as raw materials to prepare a white solid compound 12,¹H NMR(400MHz,CDCl3)δ5.48–5.39(m,2H),4.67(dt,J＝7.9,3.5Hz,2H),3.65(s,2H),2.66(d,J＝39.2Hz,4H),2.37(dd,J＝7.8,5.2Hz,8H),2.15–1.98(m,4H),1.91(ddd,J＝13.4,9.0,4.8Hz,10H),1.70–1.48(m,14H),1.40(d,J＝8.2Hz,16H),1.18(ddd,J＝19.0,11.1,4.3Hz,12H),1.08(s,8H),0.98(d,J＝6.4Hz,6H),0.93(dd,J＝6.6,1.8Hz,12H),0.74(s,6H).MS-ESI(m/z):971.2(M+H)⁺.

Example 13

The preparation method is similar to the previous compound.

Example 14

The preparation method is similar to the previous compound.

Example 15

The preparation method is the same as that of the compound 1, and 4-bromobutyric acid, ethanolamine, cholesterol and 6-bromohexanoic acid undecyl ester are used as raw materials to prepare oily compound 15,¹H NMR(400MHz,CDCl₃)δ4.08(t,J＝6.8Hz,2H),3.58(t,J＝5.3Hz,2H),2.62(t,J＝5.3Hz,2H),2.51(dt,J＝14.4,7.3Hz,5H),2.33(t,J＝7.4Hz,6H),2.02(ddt,J＝20.5,16.9,4.3Hz,2H),1.94–1.75(m,5H),1.73–1.44(m,14H),1.43–1.24(m,25H),1.23–0.98(m,8H),0.97–0.82(m,14H),0.71(s,3H).MS-ESI(m/z):785.0(M+H)⁺.

Example 16

The preparation method is the same as that of the compound 1, and 4-bromobutyric acid, 6-amino-1-hexanol, cholesterol and 6-bromohexanoic acid undecyl ester are used as raw materials to prepare oily compounds 15,¹H NMR(400MHz,CDCl₃)δ4.07(t,J＝6.8Hz,2H),3.65(t,J＝6.6Hz,2H),2.46(dt,J＝11.7,7.2Hz,6H),2.32(td,J＝7.6,1.8Hz,6H),2.08–1.93(m,3H),1.92–1.71(m,6H),1.71–1.46(m,16H),1.46–1.26(m,28H),1.26–0.85(m,27H),0.70(s,3H).MS-ESI(m/z):841.1(M+H)⁺.

Test examples

Test example 1 mRNA-LNP entrapment and Performance test

MRNA stock solution was dispersed in 20mM acetic acid solution (pH 5.0) to give a final concentration of 200. Mu.g/mL (aqueous phase). The compounds according to the examples: cholesterol: DSPC: DMG-pe2000=50: 38.5:10: the mixture was mixed to a mixed fat (oil phase) at a molar ratio of 1.5. The flow rates of the water phase and the oil phase are controlled through a T mixed flow mode, and mRNA is mixed with the lipid mixture to obtain LNP-entrapped mRNA. The entrapped LNP was diluted 10-fold with buffer, then concentrated by ultrafiltration and the dilution was replaced, and finally the LNP was concentrated to mRNA to 200ug/mL while the LNP pH was adjusted to around 7-8. Finally, using Ribogreen kit and 10% OTG as demulsifier to detect total mRNA content and free content in LNP, and calculating the encapsulation efficiency of LNP. The LNP final product was diluted with the diluent, added to 1ml of the particle size pool, and placed on a Markov Zetasizer instrument to examine the particle size of the LNP, and the results are shown in Table 1.

Particle size, PDI and encapsulation efficiency are all important quality attributes of lipid nanoparticles. As can be seen from the following table, the test compounds all have good encapsulation efficiency, particle size suitable for mRNA delivery, and narrow PDI. LNP based on compound 3, compound 4, compound 5 and compound 8 has precipitation phenomenon in the preparation process, has poor drug formation and is not studied later.

Table 1: LNP characterization data for the Compounds of the examples

Numbering of compounds	Encapsulation efficiency/%	Particle size/nm	PDI
				1	97.72	78.76	0.04752
2	97.85	78.35	0.02884
				3	98.1	96.34	0.1799
4	63.9	149.8	0.1513
				5	89.3	128.9	0.006391
6	85.5	91.39	0.07442
				7	79.4	80.68	0.04916
8	92.9	107	0.1525
				9	93.6	82.38	0.06221
15	98.9	81.65	0.1541
				16	94.1	118.2	0.2082

Test example 2 test of intravenous delivery Effect

1. The mRNA expressing luciferases was entrapped in LNP formulations of compound MC3, compound 1, compound 2, compound 9, compound 15 and compound 16, and the LNP formulation preparation method and the mRNA entrapment method were as described in test example 1;

2. The entrapped LNP formulation was intravenously injected into Balb/c mice at a dose of 1mg/kg, 5 per group.

3. The fluorescence expression intensity of luciferases was measured at 6 hours/24 hours for each mouse.

4. Fluorescence expression intensity detection: each mouse was intraperitoneally injected with D-fluorescein sodium salt (dose: 150 mg/kg) 10 minutes before detection, and then the mice were anesthetized with isoflurane and placed in an IVIS apparatus, and bioluminescence was selected for detection.

5. The delivery effect of the compound was judged as a fluorescence intensity result.

The Luciferase assay is the main method for examining mRNA expression in vivo by fluorescence intensity, and it is understood from the results of FIG. 1 that compound 15 and compound 16 groups generate substantially no fluorescence after 6 hours of intravenous injection, while compound 1, compound 2 and compound 9 groups generate fluorescence. Compared with the positive control MC3, the expression of the compound 9 is slightly lower, the expression of the compound 2 is equivalent to the expression of the compound 1, and the expression of the compound 1 is obviously higher, so that the compound has excellent in-vivo expression effect.

As can be seen from fig. 2, compound 1, compound 2, and compound 9 had significantly higher expression in the spleen compared to the positive control group MC3 after intravenous injection for 24 hours, and in particular, the advantage of compound 1 was particularly apparent, showing that it has excellent spleen targeted delivery effect.

Test example 3 test of intramuscular delivery effect

1. The mRNA expressing luciferases was entrapped into LNP formulations of compound MC3, compound SM102, compound 1, compound 2, compound 9, LNP formulation preparation method and mRNA entrapment method were as described in test example 1;

2. The entrapped LNP formulation was intramuscularly injected into Balb/c mice at a dose of 5 μg/dose, 5 per group.

3. The fluorescence expression intensity of luciferases was measured at 6 hours for each mouse.

4. Fluorescence expression intensity detection: each mouse was intraperitoneally injected with D-fluorescein sodium salt (dose: 150 mg/kg) 10 minutes before detection, and then the mouse was anesthetized with isoflurane and then placed in an IVIS apparatus, bioluminescence was selected for detection, and after detection was completed, the fluorescence intensity at the intramuscular injection site was analyzed.

As can be seen from fig. 3, compound 1, compound 2 and compound 9 also produced fluorescence by intramuscular administration, wherein both compound 1 and compound 2 were significantly better than MC3, compound 9 was comparable to MC3, and compound 1 was better than positive control SM102, and compound 2 was only slightly lower than SM102, reflecting that the above compounds also had excellent delivery effect by intramuscular administration.

Although the embodiments of the present application have been described above in connection with the above, the present application is not limited to the above-described specific embodiments and fields of application, which are merely illustrative, instructive, and not restrictive. Those skilled in the art, having the benefit of this disclosure, may effect numerous forms of the application without departing from the scope of the application as claimed.

Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt or stereoisomer thereof,

r ₁ is R;

The R is

Ra, rb, rc, rd, re is independently selected from H, C1-C6 alkyl,

R' is selected from C1-C10 alkyl, C1-C10 alkenyl;

R ₄、R₅、R₆、R₇ is independently selected from H, C1-C6 alkyl;

The dotted line represents a single bond or a double bond;

n is selected from 0,1,2,3,4,5,6;

o, p are independently selected from 1,2,3,4,5,6,7,8,9, 10.

2. A compound according to claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, R ₂、R₃ is independently selected from H, C1-C14 alkyl, provided that R ₂、R₃ is not simultaneously H, preferably R ₂、R₃ is C6-C11 linear or branched alkyl, more preferably R ₂、R₃ is C8 linear or branched alkyl.

3. A compound according to claim 1 or 2, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R 'is C8 alkyl or C8 alkenyl, preferably wherein R' is

The dotted line represents a single bond or a double bond, more preferably, the R is: most preferably, the R is: /(I)

4. A compound according to any one of claims 1-3, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein o is selected from 1,2,3,4,5,6,7, preferably wherein o is 5.

5. A compound according to any one of claims 1-4, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein p is selected from 1,2,3,4,5,6,7, preferably wherein p is 7.

6. The compound according to any one of claims 1-5, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein n is selected from 2,3,4,5,6; preferably n is 2 or 6.

7. The compound of any one of claims 1-6, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein X ₁、X₂ is c=o, Y ₁、Y₂ is O, or X ₁ is O, Y ₁ is c=o, X ₂ is c=o, Y ₂ is O, or X ₁ is c=o, Y ₁ is O, X ₂ is O, Y ₂ is c=o, or X ₁,X₂ is O, and Y ₁,Y₂ is c=o.

8. The compound according to any one of claims 1-7, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, which is a compound of formula (II):

9. a compound or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound is selected from the group consisting of

Compound 1

Compound 2

Compound 3

Compound 4

Compound 5

Compound 6

Compound 7

Compound 8

Compound 9

Compound 10

Compound 11

Compound 12

Compound 13

Compound 14

Compound 15

Compound 16

10. A nanoparticle composition comprising a lipid component comprising a compound according to any one of claims 1-9, or a pharmaceutically acceptable salt or stereoisomer thereof.

11. Use of a compound according to any one of claims 1-9, or a pharmaceutically acceptable salt or stereoisomer thereof, for the preparation of a lipid nanoparticle composition.

12. A pharmaceutical composition comprising the nanoparticle composition of any one of claims 10 and a pharmaceutically acceptable carrier.

13. A method of producing a polypeptide of interest in a mammalian cell, the method comprising contacting the cell with the nanoparticle composition of any one of claims 10 or the pharmaceutical composition of claim 12 to deliver a therapeutic and/or prophylactic agent to the cell, wherein the therapeutic and/or prophylactic agent is an mRNA encoding the polypeptide of interest, whereby the mRNA is capable of translation in the cell to produce the polypeptide of interest.

14. A method of treating a disease or disorder in a mammal, the method comprising administering to the mammal a therapeutically effective amount of the nanoparticle composition of any one of claims 10 or the pharmaceutical composition of claim 12.

15. A method of specifically delivering a therapeutic and/or prophylactic agent to an organ of a mammal, the method comprising administering to the mammal the nanoparticle composition of any one of claims 10 or the pharmaceutical composition of claim 12, the administering comprising contacting the organ of the mammal with the nanoparticle composition, thereby delivering the therapeutic and/or prophylactic agent to the organ.