Amine-Containing Transfection Reagents and methods for making and using same

• US 20120136073A1
• Filed: 11/15/2011
• Published: 05/31/2012
• Est. Priority Date: 11/15/2010
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Yang, Zhiwei, Angrish, Parul, de Mollerat du Jeu, Xavier, Wiederholt, Kristin
• Application Number:
  US13/297,231
• Publication Number:
  US 20120136073A1
• Time in Patent Office: Days
• US Class Current: 514/785
• CPC Class Codes: A61K 47/54 the modifying agent being an organic compound
• CPC Class Codes: A61K 47/543 Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
• CPC Class Codes: A61K 48/0008 characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
• CPC Class Codes: C07C 227/06 by addition or substitution reactions, without increasing the number of carbon atoms in the carbon skeleton of the acid
• CPC Class Codes: C07C 229/08 the nitrogen atom of the amino group being further bound to hydrogen atoms
• CPC Class Codes: C07C 229/26 having more than one amino group bound to the carbon skeleton, e.g. lysine
• CPC Class Codes: C07D 211/28 to which a second hetero atom is attached
• CPC Class Codes: C07D 211/58 attached in position 4
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 15/88 using microencapsulation, e.g. using amphiphile liposome vesicle
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/141 MicroRNAs, miRNAs

Abstract

There are provided for herein novel amine-containing transfection compounds and methods for making and using same. The compounds are generally obtained by reacting a primary amine with an unsaturated compound. Transfection complexes made using the amine-containing transfection compounds in combination with additional compounds to encapsulate biologically active agents such as nucleic acids are also provided for herein. Methods of using the transfection complexes for the in vivo or in vitro delivery of biologically active agents are also described. The transfection complexes of the present invention are highly potent, thereby allowing effective modulation of a biological activity at relatively low doses compared to analogous transfection compounds known in the art.

Claims

1. A compound having the general structure I, or pharmaceutically acceptable salts thereof:

2. The compound of claim 1, or pharmaceutically acceptable salts thereof, wherein each of R₁ and R₂ is independently selected from the group consisting of substituted or unsubstituted, branched or unbranched alkyl or alkenyl groups having between 3 and about 20 carbon atoms, and between 0 and 4 double bonds.

3. The compound of claim 2, or pharmaceutically acceptable salts thereof, wherein each of R₁ and R₂ is independently selected from the group consisting of substituted or unsubstituted, unbranched alkyl or alkenyl groups having between 8 and about 18 carbon atoms, and between 0 and 2 double bonds.

4. The compound of claim 1, or pharmaceutically acceptable salts thereof, wherein each of n and m independently has the value of 1 or 3, and R₃ is selected from the group consisting of

5. The compound of claim 1, or pharmaceutically acceptable salts thereof, wherein x has the value between 1 and 6.

6. The compound of claim 1 having the general structure II or pharmaceutically acceptable salts thereof:

7. The compound of claim 6, or pharmaceutically acceptable salts thereof, wherein at east one X₁ is NH.

8. The compound of claim 6, or pharmaceutically acceptable salts thereof, wherein at least one X₁ is O.

9. The compound of claim 6, or pharmaceutically acceptable salts thereof, wherein each R₁ is independently selected from the group consisting of substituted or unsubstituted, branched or unbranched alkyl or alkenyl groups having between 3 and about 20 carbon atoms, and between 0 and 4 double bonds.

10. The compound of claim 9, or pharmaceutically acceptable salts thereof, wherein each of R₁ is independently selected from the group consisting of substituted or unsubstituted, unbranched alkyl or alkenyl groups having between 8 and about 18 carbon atoms, and between 0 and 2 double bonds.

11. The compound of claim 6, or pharmaceutically acceptable salts thereof, wherein each of n and m independently has the value of 1 or 3, and R₃ is selected from the group consisting of

12. The compound of claim 6, or pharmaceutically acceptable salts thereof, wherein x has the value between 1 and 6.

13. The compound of claim 1, or pharmaceutically acceptable salts thereof, the compound being selected from the group consisting of compounds 1-87 or isomers thereof:

14. A method for synthesizing a compound having the structure I, or pharmaceutically acceptable salts thereof,

15. The method of claim 14, wherein each of R₁ and R₂ is independently selected from the group consisting of substituted or unsubstituted, branched or unbranched alkyl or alkenyl groups having between 3 and about 20 carbon atoms, and between 0 and 4 double bonds.

16. The method of claim 14, wherein each of R₁ and R₂ is independently selected from the group consisting of substituted or unsubstituted, branched or unbranched alkyl or alkenyl groups having between 3 and about 20 carbon atoms, and between 0 and 4 double bonds.

17. The method of claim 16, wherein each of R₁ and R₂ is independently selected from the group consisting of substituted or unsubstituted, unbranched alkyl or alkenyl groups having between 8 and about 18 carbon atoms, and between 0 and 2 double bonds.

18. The method of claim 14, wherein each of n and m independently has the value of 1 or 3, and the primary amine NH₂—

R₃ is selected from the group consisting of

19. The method of claim 14, wherein the amino component is a primary amine NH₂—

R₃, and the molar ratio between the unsaturated component and the primary amine is between about 1;

1 and about 6;

1.

20. The method of claim 14, wherein each of R₄, R₅, R₆ and R₇ is hydrogen and each of Y and Z is C═

O.

21. The method of claim 20, or pharmaceutically acceptable salts thereof, wherein each R₁ is independently selected from the group consisting of substituted or unsubstituted, branched or unbranched alkyl or alkenyl groups having between 3 and about 20 carbon atoms, and between 0 and 4 double bonds.

22. The method of claim 21, or pharmaceutically acceptable salts thereof, wherein each of R₁ is independently selected from the group consisting of substituted or unsubstituted, unbranched alkyl or alkenyl groups having between 8 and about 18 carbon atoms, and between 0 and 2 double bonds.

23. A transfection complex comprising an amine-containing transfection reagent having the general structure I, or pharmaceutically acceptable salts thereof:

24. The transfection complex of claim 23, or pharmaceutically acceptable salts thereof, wherein each of R₁ and R₂ is independently selected from the group consisting of substituted or unsubstituted, branched or unbranched alkyl or alkenyl groups having between 3 and about 20 carbon atoms, and between 0 and 4 double bonds.

25. The transfection complex of claim 24, or pharmaceutically acceptable salts thereof, wherein each of R₁ and R₂ is independently selected from the group consisting of substituted or unsubstituted, unbranched alkyl or alkenyl groups having between 8 and about 18 carbon atoms, and between 0 and 2 double bonds.

26. The transfection complex of claim 23, or pharmaceutically acceptable salts thereof, wherein each of n and m independently has the value of 1 or 3, and R₃ is selected from the group consisting of

27. The transfection complex of claim 23, or pharmaceutically acceptable salts thereof, wherein x has the value between 1 and 6.

28. The transfection complex of claim 23, wherein the amine-containing transfection reagent has the general structure II, or pharmaceutically acceptable salts thereof:

29. The transfection complex of claim 28, or pharmaceutically acceptable salts thereof, wherein at east one X₁ is NH.

30. The transfection complex of claim 28, or pharmaceutically acceptable salts thereof, wherein at least one X₁ is O.

31. The transfection complex of claim 28, or pharmaceutically acceptable salts thereof, wherein each R₁ is independently selected from the group consisting of substituted or unsubstituted, branched or unbranched alkyl or alkenyl groups having between 3 and about 20 carbon atoms, and between 0 and 4 double bonds.

32. The transfection complex of claim 31, or pharmaceutically acceptable salts thereof, wherein each of R₁ is independently selected from the group consisting of substituted or unsubstituted, unbranched alkyl or alkenyl groups having between 8 and about 18 carbon atoms, and between 0 and 2 double bonds.

33. The transfection complex of claim 28, or pharmaceutically acceptable salts thereof, wherein each of n and m independently has the value of 1 or 3, and R₃ is selected from the group consisting of

34. The transfection complex of claim 28, or pharmaceutically acceptable salts thereof, wherein x has the value between 1 and 6.

35. The transfection complex of claim 23, or pharmaceutically acceptable salts thereof, the compound being selected from the group consisting of compounds 1-87 or isomers thereof:

36. The transfection complex of claim 23, further comprising at least one helper lipid.

37. The transfection complex of claim 36, wherein the helper lipid is selected from the list consisting of BMOP (N-(2-bromoethyl)-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-propana minimun bromide), DDPES (Dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide), DSPC, CTAB:

DOPE (formulation of cetyltrimethylammonium bromide (CATB) and DOPE), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPE (dioleoylphosphatidylethanolamine), DMG, DMAP (4-dimethylaminopyridine), DMPE (Dimyristoylphospatidylethanolamine), DOMG, DMA, DOPC (Dioleoylphosphatidylcholine), DMPC (dimyristoylphosphatidylcholine), DPEPC (Dipalmitoylethylphosphatidylcholine), DODAC (dioleoydimethylammonium chloride), DOSPER (1,3-Di-Oleoyloxy-2-(6-Carboxyspermyl)-Propylamid), DOTMA (N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammoniumchloride), DDAB (didoceyl methylammonium bromide), DOTAP(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate), DOTAP.Cl, DC-chol (3,13-N,(N′

,N′

-dimethylaminoethane)-carbamoyl]cholesterol), DOSPA (2-(sperminecarboxamido)ethyl)-N,N-dimethyl-lammonium trifluoroacetate), DC-6-14 (O,O′

-Ditetradecanoyl-N-(alphatrimethylammonioacetyl) diethanolamine chloride), DCPE (Dicaproylphosphtidylethanolamine), DLRIE (dilauryl oxypropyl-3-dimethylhydroxy ethylammonium bromide), DODAP (1,2-Dioleoyl-3-dimethylammonium-propane), Ethyl-PC, DOSPA (2,3-dioleoyloxy-N-[2-(sperminecarboxamidoethyl]-N,N-di-met-hyl-1-propanaminium trifluoroacetate), DOGS (dioctadecylamidoglycyl carboxyspermine), DMRIE (N-[1-(2,3 dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl) ammonium bromide), DOEPC (Dioleoylethyl-phosphocholine), DOHME (N-[1-(2,3-dioleoyloxy)propyl]-N-[1-(2-hydroxyethyl)]-N,Ndimethylammonium iodide), GAP-DLRIE;

DOPE (N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propaniminium bromide/dioleyl phosphatidylethanolamine), DPPC (Dipalmitoylphosphatidylcholine), DOPG (1,2-dioleoyl-sn-glycero-3-[phospho-rac-(3-lysyl(1-glycerol)).Cl), N-lauroylsarcosine, (R)-(+)-limonene, lecithins (and derivatives thereof);

phosphotidylethanolamine (and derivatives thereof);

phosphatidylethanolamines, dioleoylphosphatidylethanolamine), DPhPE (diphytanoylphosphatidylethanolamine), DPPE dipalmitoylphosphatidylethanolamine), dipalmiteoylphosphatidylethanolamine, O-Chol (3 beta[1-ornithinamidecarbamoyl]cholesterol), POPE (palmitoyloleoylphosphatidylethanolamine) and distearoylphosphatidylethanolamine;

phosphotidylcholine;

phosphatidylcholines, DPPC (dipalmitoylphosphatidylcholine) POPC (palmitoyloleoylphosphatidylcholine) and distearoylphosphatidylcholine;

phosphatidylglycerol;

piperazine-based cationic lipids, phosphatidylglycerols, such as DOPG (dioleoylphosphatidylglycerol), DPPG (dipalmitoylphosphatidylglycerol), and distearoylphosphatidylglycerol;

phosphatidylserine (and derivatives thereof);

phosphatidylserines, such as dioleoyl- or dipalmitoylphosphatidylserine;

diquaternary ammonium salts such as N,N′

-dioleyl-N,N,N′

,N′

-tetramethyl-1,2-ethanediamine (TmedEce), N,N′

-dioleyl-N,N,N′

,N′

-tetramethyl-1,3-propanediamine (PropEce), N,N′

-dioleyl-N,N,N′

,N′

-tetramethyl-1,6-hexanediamine (HexEce), and their corresponding N,N′

-dicetyl saturated analogues (TmedAce, PropAce and HexAce), diphosphatidylglycerols;

fatty acid esters;

monocationic transfection lipids such as 1-deoxy-1-[dihexadecyl(methyl)ammonio]-D-xylitol;

1-deoxy-1-[methyl(ditetradecyl)ammonio]-Darabinitol;

1-deoxy-1-[dihexadecyl(methyl)ammonio]-D-arabinitol;

1-deoxy-1-[methyl(dioctadecyl)ammonio]-Darabinitol, glycerol esters;

sphingolipids;

cardolipin;

cerebrosides; and

ceramides; and

mixtures thereof. Neutral lipids also include cholesterol and other 3β

OH-sterols as well as derivatives thereof phosphatidyl choline or commercially available cationic lipid mixtures such as, for example, LIPOFECTIN®

CELLFECTIN®

(1;

1.5 (M/M) formulation of N, NI,NII, NIII-tetramethyl-N, NI, NII, NIII-tetrapalmitylspermine (TMTPS) and dioleoyl phosphatidylethanolamine (DOPE), LIPOFECTACE®

, GS 2888 CYTOFECTIN®

, FUGENE 6®

, EFFECTENE®

, and LIPOFECTAMINE®

, LIPOFECTAMINE 2000®

, LIPOFECTAMINE PLUS®

, LIPOTAXI®

, POLYECT®

, SUPERFECT®

, TFXN™

, TRANSFAST™

, TRANSFECTAM®

, TRANSMESSENGER®

, vectamidine (3-tetradecylamino-N-tert-butyl-N′

-tetradecylpropionamidine (diC14-amidine), OLIGOFECTAMINE®

.

38. The transfection complex of claim 23, further comprising at least one pegylated lipid.

39. The transfection complex of claim 23, further comprising at least one bioactive agent.

40. The transfection complex of claim 39, wherein the bioactive agent is a DNA molecule, and RNA molecule, a protein of a drug.

41. The transfection complex of claim 40, wherein the RNA molecule is an siRNA, an shRNA, an miRNA and stRNA or an mRNA.

42. The transfection complex of claim 39, wherein the bioactive agent is an siRNA molecule.

43. The transfection complex of claim 39, wherein the bioactive agent is an mRNA molecule.

44. The transfection complex of claim 39, wherein the bioactive agent is an DNA molecule.

45. The transfection complex of claim 23, further comprising a targeting moiety.

46. The transfection complex of claim 23, further comprising a targeting moiety selected from the list consisting of peptide, a modified peptide, an antibody, a modified antibody, a receptor molecule, a modified receptor molecule, a single or a double stranded nucleic acid molecule, a modified single or double stranded nucleic acid molecule, a peptide or nucleic acid aptamer, a modified peptide or nucleic acid aptamer, an organic molecule, a polysaccharide.