Transfection reagents

• US 7,166,745 B1
• Filed: 11/12/1999
• Issued: 01/23/2007
• Est. Priority Date: 11/12/1998
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Gebeyehu, Gulilat, Chu, Yongliang, Masoud, Malek
• Primary Examiner(s): DAVIS, BRIAN J
• Application Number:
  US09/438,365
• Time in Patent Office: 2,629 Days
• Field of Search: 435/458, 435/238, 424/450, 514/7, 514/2, 514/44, 530/358, 540/467, 544/357, 546/186, 546/264, 564/155, 564/159, 564/158, 564/236, 564/290, 564/292, 564/295, 564/346, 564/355, 564/372, 564/503, 564/504, 564/506, 564/507, 564/509, 564/511, 564/512
• US Class Current: 564/155
• CPC Class Codes: A61K 31/14 Quaternary ammonium compounds, e.g. edrophonium, choline betaines A61K31/205
• CPC Class Codes: A61K 38/28 Insulins
• CPC Class Codes: A61K 38/40 Transferrins, e.g. lactoferrins, ovotransferrins
• CPC Class Codes: A61K 47/543 Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
• CPC Class Codes: A61K 9/16 Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction A61K9/20 takes precedence if the final form is a tablet; microspheres with drug-free outer coating, microcapsules A61K9/50; mixture of different granules, microcapsules, (coated) microparticles A61K9/5084; nanoparticles A61K9/51
• CPC Class Codes: C07C 211/21 Monoamines
• CPC Class Codes: C07C 211/63 having quaternised nitrogen atoms bound to acyclic carbon atoms
• CPC Class Codes: C07C 211/64 having quaternised nitrogen atoms bound to carbon atoms of six-membered aromatic rings
• CPC Class Codes: C07C 229/26 having more than one amino group bound to the carbon skeleton, e.g. lysine
• CPC Class Codes: C07D 209/48 with oxygen atoms in positions 1 and 3, e.g. phthalimide
• CPC Class Codes: C07D 241/12 with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
• CPC Class Codes: C12N 15/88 using microencapsulation, e.g. using amphiphile liposome vesicle
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

Disclosed are compounds capable of facilitating transport of biologically active agents or substances into cells having the general structure:

wherein

• • Q is selected from the group consisting of N, O and S; L is any bivalent organic radical capable of linking each Q, such as C, CH, (CH₂)₁, or {(CH₂)_i—Y—(CH₂)_j}_k, wherein Y is selected from the group consisting of CH₂, an ether, a polyether, an amide, a polyamide, an ester, a sulfide, a urea, a thiourea, a guanidyl, a carbamoyl, a carbonate, a phosphate, a sulfate, a sulfoxide, an imine, a carbonyl, and a secondary amino group and wherein Y is optionally substituted by —X₁—L′—X₂—Z or —Z; R₁–R₆, independently of one another, are selected from the group consisting of H, —CH₂)_p—D—Z, an alkyl, an alkenyl, an aryl, and an alkyl or alkyl ether optionally substituted by one or more of an alcohol, an aminoalcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, an alkylthio, a urea, a thiourea, a guanidyl, or a carbamoyl group, and wherein at least one of R₁, R₃, R₄ and R₆ is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group; and any one of R₁, R₃, R₄ and/or R₆ may optionally be covalently linked with each other, with Y or with L when L is C or CH to form a cyclic moiety; Z is selected from the group consisting of amine, spermiyl, carboxyspermiyl, guanidyl, spermidinyl, putricinyl, diaminoalkyl, pyridyl, piperidinyl, pyrrolidinyl, polyamine, amino acid, peptide, and protein; X₁ and X₂, independently of one another, are selected from the group consisting of NH, O, S, alkylene, and arylene; L′ is selected from the group consisting of alkylene, alkenylene, alkynylene, arylene, alkylene ether, and polyether; D is Q or a bond; A₁ and A₂, independently of one another, are selected from the group consisting of CH₂O, CH₂S, CH₂NH, C(O), C(NH), C(S) and (CH₂)_t; X is a physiologically acceptable anion; m, n, r, s, u, v, w and y are 0 or 1, with the proviso that when both m and n are 0 at least one of r, s, u and y is other than 0; i, j, k, l, p and t are integers from 0 to about 100; q is an integer from 1 to about 1000; and a is the number of positive charge divided by the valence of the anion.

Claims

1. A compound having the formula:

79. A method for introducing a biologically active substance into a cell, said method comprising forming a lipid aggregate of a compound of any one of claims 1, 2, 5, 9, 17, 20, 21, 23, 25, 27, 30, 32, 38, 46, 52, 58, 65, or 73 and a biologically active substance and incubating the lipid aggregate with a cell or cell culture.

2. A compound having the formula:

3. The compound as claimed in claim 2, which is:

4. The compound as claimed in claim 3, wherein R₇ and R₈ are H.

96. The compound of claim 2 wherein R₁, R₃, R₄, and R₆, independently of one another, are selected from the group consisting of H, and an alkyl group and at least two of R₁, R₃, R₄, and R₆, are straight-chain alkyl groups having from 8 to 24 carbon atoms attached to each N.

97. The compound of claim 96 wherein at least two of R₁, R₃, R₄, and R₆, are straight-chain alkyl groups having 14 or 16 carbon atoms attached to each N.

5. A compound having the formula:

6. The compound as claimed in claim 5, which is:

7. The compound which is:

8. The compound which is:

9. A compound having the formula:

10. The compound as claimed in claim 9 wherein R₇ and R₈ are H.

11. The compound as claimed in claim 9 which is:

12. The compound according to claim 11, wherein R₇ and R₈ are H.

13. The compound which is

14. The compound as claimed in claim 13, wherein R₇ and R₈ are H.

15. The compound which is

16. The compound as claimed in claim 15, wherein R₇ and R₈ are H.

17. A compound having the formula:

18. The compound as claimed in claim 17, which is:

19. The compound as claimed in claim 17, which is:

74. A composition comprising one or more compounds of any one of claims 2, 5, 9, 17, 20, 21, 23, 25, 27, 30, 32, 38, 46, 52, 58, 65, or 73.

75. A composition comprising one or more compounds of any one of claims 2, 5, 9, 17, 20, 21, 23, 25, 27, 30, 32, 38, 46, 52, 58, 65, or 73 and at least one additional component selected from the group consisting of a cell, cells, a cell culture, a cell culture media, a neutral lipid, a nucleic acid, and a transfection enhancer.

76. A lipid aggregate comprising one or more compounds of any one of claims 2, 5, 9, 17, 20, 21, 23, 25, 27, 30, 32, 38, 46, 52, 58, 65, or 73.

77. A kit comprising one or more compounds of any one of claims 2, 5, 9, 17, 20, 21, 23, 25, 27, 30, 32, 38, 46, 52, 58, 65, or 73 and at least one additional component selected from the group consisting of a cell, cells, a cell culture medium, a nucleic acid, a transfection, enhancer and instructions for transfecting a cell or cells.

78. A method for introducing a polyanion into a cell or cells, said method comprising forming a lipid aggregate from a positively charged compound of any one of claims 2, 5, 9, 17, 20, 21, 23, 25, 27, 30, 32, 38, 46, 52, 58, 65, or 73, contacting the lipid aggregate with a polyanion to form a positively-charged polyanion-lipid aggregate complex and incubating the complex with a cell or cells.

82. A composition comprising one or more compounds of any one of claims 2, 5, 9, 17, 20, 21, 23, 25, 27, 30, 32, 38, 46, 52, 58, 65, or 73 and at least one additional lipid aggregating compound.

83. A composition comprising one or more compounds of any one of claims 2, 5, 9, 17, 20, 21, 23, 25, 27, 30, 32, 38, 46, 52, 58, 65, or 73 and at least one additional lipid aggregating compound selected from DOPE, DOPC or cholesterol.

84. A composition comprising one or more compounds of any one of claims 2, 5, 9, 17, 20, 21, 23, 25, 27, 30, 32, 38, 46, 52, 58, 65, or 73 and at least one neutral lipid or at least one other cationic lipid.

85. A composition comprising one or more compounds of any one of claims 2, 5, 9, 17, 20, 21, 23, 25, 27, 30, 32, 38, 46, 52, 58, 65, or 73 and at least one cationic lipid selected from the group consisting of DOSPA, DOTMA, DMRIE, DOTAP, DOGS and TM-TPS.

86. A kit comprising one or more compounds of any one of claims 2, 5, 9, 17, 20, 21, 25, 27, 30, 32, 38, 46, 52, 58, 65, or 73.

87. A kit comprising one or more compounds of any one of claims 2, 5, 9, 17, 20, 21, 23, 25, 27, 30, 32, 38, 46, 52, 58, 65, or 73 and at least one additional lipid aggregating forming compound.

88. A kit comprising one or more compounds of any one of claims 2, 5, 9, 17, 20, 21, 23, 25, 27, 30, 32, 38, 46, 52, 58, 65, or 73 and at least one additional lipid aggregating forming compound selected from DOPE, DOPC or cholesterol.

89. A kit comprising one or more compounds of any one of claims 2, 5, 9, 17, 20, 21, 23, 25, 27, 30, 32, 38, 46, 52, 58, 65, or 73 and at least one neutral lipid or at least one other cationic lipid.

90. A kit comprising one or more compounds of any one of claims 2, 5, 9, 17, 20, 21, 23, 25, 27, 30, 32, 38, 46, 52, 58, 65, or 73 and at least one other cationic lipid selected from the group consisting of DOSPA, DOTMA, DMRIE, DOTAP, DOGS and TM-TPS.

91. A lipid aggregate comprising one or more compounds of any one of claims 2, 5, 9, 17, 20, 21, 23, 25, 27, 30, 32, 38, 46, 52, 58, 65, or 73 and at least one additional lipid aggregating forming compound.

92. A lipid aggregate comprising one or more compounds of any one of claims 2, 5, 9, 17, 20, 21, 23, 25, 27, 30, 32, 38, 46, 52, 58, 65, or 73 and at least one additional lipid aggregating forming compound selected from DOPE, DOPC or cholesterol.

93. A lipid aggregate comprising one or more compounds of any one of claims 2, 5, 9, 17, 20, 21, 23, 25, 27, 30, 32, 38, 46, 52, 58, 65, 73 and at least one neutral lipid or at least one other cationic lipid.

94. A lipid aggregate comprising one or more compounds of any one of claims 2, 5, 9, 17, 20, 21, 23, 25, 27, 30, 32, 38, 46, 52, 58, 65 or 73 and at least one other cationic lipid selected from the group consisting of DOSPA, DOTMA, DMRIE, DOTAP, DOGS and TM-TPS.

95. A kit comprising a lipid aggregate comprising one or more compounds of any one of claims 2, 5, 9, 17, 20, 21, 23, 25, 27, 30, 32, 38, 46, 52, 58, 65, and 73 and at least one neutral lipid or at least one other cationic lipid.

20. A compound having the formula:

21. The compound as claimed in claim 20, which is:

22. The compound as claimed in claim 21, wherein R₇ and R₈ are H.

23. The compound as claimed in claim 20, which is:

24. The compound as claimed in claim 23, wherein R₇ and R₈ are H.

25. A compound having the formula:

26. The compound as claimed in claim 25, wherein R₇ and R₈ are both H.

27. A compound having the formula:

28. A compound having the formula:

29. A compound having the formula:

30. A compound having the formula:

31. The compound which is:

32. A compound having the formula:

33. The compound which is:

34. A compound having the formula:

35. The compound as claimed in claim 34, wherein R₇ and R₈ are H.

36. A compound having the formula:

37. A compound having the formula:

38. A compound having the formula:

39. The compound as claimed in claim 38 wherein R₁ and R₄, independently of one another, are straight-chain, branched, or cyclic alkyl, alkenyl, alkynyl or aryl groups having from about 8 to about 24 carbon atoms.

40. A compound having the formula:

41. A compound having the formula:

42. The compound as claimed in claim 41, wherein R₇ and R₈ are H.

43. A compound having the formula:

44. A compound having the formula:

45. The compound as claimed in claim 44, wherein R₇ and R₈ are H.

46. A compound having the formula:

47. The compound which is:

48. A compound having the formula:

49. The compound as claimed in claim 48, wherein R₇ and R₈ are H.

50. A compound having the formula:

51. The compound as claimed in claim 50, wherein R₇ and R₈ are H.

52. A compound having the formula:

53. The compound which is:

54. A compound having the formula:

55. The compound according to claim 54, wherein R₇ and R₈ are H.

56. A compound having the formula:

57. The compound as claimed in claim 56, wherein R₇ and R₈ are H.

58. A compound having the formula:

59. A compound having the formula:

60. A compound having the formula:

61. The compound as claimed in claim 60, wherein R₇ and R₈ are H.

62. A compound having the formula:

63. The compound as claimed in claim 62, wherein R₇ and R₈ are H.

64. A compound having the formula:

65. A compound having the formula:

66. The compound as claimed in claim 65, wherein at least one of R₁ and R₄ is a straight-chain, branched, or cyclic alkyl, alkenyl, alkynyl or aryl group having from about 8 to about 24 carbon atoms.

67. The compound as claimed in claim 65, wherein the alkyl ether optionally substituted by one or more alcohol groups is a carbohydrate.

68. The compound as claimed in claim 67, wherein the carbohydrate is selected from the group consisting of galactose, fructose, glucose, maltose, sucrose, cellobiose, lactose, mannose, glucopyranose, mannopyranose and galactopyranose.

69. The compound as claimed in claim 65, wherein Y is selected from the group consisting of CH₂, an ether, a polyether, an amide, a polyamide, an ester, a sulfide, a urea, a thiourea, a guanidyl, a carbamoyl, a carbonate, and a secondary amino group.

70. The compound as claimed in claim 69, wherein at least one of R₁ and R₄ is a straight-chain, branched, or cyclic alkyl, alkenyl, alkynyl or aryl group having from about 8 to about 24 carbon atoms.

71. The compound as claimed in claim 69, wherein the alkyl ether optionally substituted by one or more alcohol groups is a carbohydrate.

72. The compound as claimed in claim 71, wherein the carbohydrate is selected from the group consisting of galactose, fructose, glucose, maltose, sucrose, cellobiose, lactose, mannose, glucopyranose, mannopyranose and galactopyranose.

73. A compound having the formula:

98. The compound of claim 73 wherein R₁, R₃, R₄, and R₆, independently of one another, are selected from the group consisting of alkyl group, an alkenyl group, or an alkynyl group.

99. The compound of claim 73 wherein R₁, R₃, R₄, and R₆, independently of one another, are selected from the group consisting of H, and an alkyl group.

100. The compound of claim 73 wherein R₇ and R₈ are H.

101. The compound of claim 73 wherein two of R₁, R₃, R₄, and R₆ are alkyl groups having 14 or 16 carbons.

80. A compound which is:

N¹,N⁴-dipalmitolyl-N¹,N⁴-di-[2-hydroxy-3-(N-aminopropyl)]-diaminobutane;

N¹,N⁴-distearyl-N¹,N⁴-di-[2-hydroxy-3-(N-aminopropyl)]-diaminobutane;

N¹,N⁴-dilauryl-N¹,N⁴-di-[2-hydroxy-3-(N-aminopropyl)]-diaminobutane;

N¹,N²-dimyristyl-N¹,N²-di-[2-hydroxy-3-(N-aminopropyl)]-diaminoethane;

N¹,N²-dipalmity-N¹,N²-di-[2-hydroxy-3-(N-aminopropyl)]-diaminoethane;

N¹,N²-dipalmitolyl-N¹,N²-di-[2-hydroxy-3-(N-aminopropyl)]-diaminoethane;

N¹,N²-distearyl-N¹,N²-di-[2-hydroxy-3-(N-aminopropyl)]-diaminoethane;

N¹,N²-dilauryl-N¹, N²-di-[2-hydroxy-3-(N-aminopropyl)]-diaminoethane;

N¹,N⁸-dimyristyl-N¹,N⁸-di-[2-hydroxy-3-(N-aminopropyl)]-Jeffamine;

N¹,N⁸-dipalmityl-N¹,N⁸-di-[2-hydroxy-3-(N-aminopropyl)]-Jeffamine;

N¹,N⁸-dipalmitolyl-N¹,N⁸-di-[2-hydroxy-3-(N-aminopropyl)]-Jeffamine;

N¹,N⁸-distearyl-N¹,N⁸-di-[2-hydroxy-3-(N-aminopropyl)]-Jeffamine;

N¹,N⁸-dilauryl-N¹,N⁸-di-[2-hydroxy-3-(N-aminopropyl)]-Jeffamine;

N¹,N⁸-dioleyl-N¹,N⁸-di-[2-hydroxy-3-(N-aminopropyl)]-Jeffamine;

N¹,N⁴-dimyristyl-N¹,N⁴-di-[2-hydroxy-3-(N-sperminecarboxamido)aminopropyl]-diaminobutane;

N¹,N⁴-dipalmityl-N¹,N⁴-di-[2-hydroxy-3-(N-sperminecarboxamido)aminopropyl]-diaminobutane;

N¹,N⁴-dipalmitolyl-N¹,N⁴-di-[2-hydroxy-3-(N-sperminecarboxamido)aminopropyl]-diaminobutane;

N¹,N⁴-distearyl-N¹,N⁴-di-[2-hydroxy-3-(N-sperminecarboxamido)aminopropyl]-diaminobutane;

N¹,N⁴-dilauryl-N¹,N⁴-di-[2-hydroxy-3-(N-sperminecarboxamido)aminopropyl]-diaminobutane;

N¹,N⁸-dimyristyl-N¹,N⁸-di-[2-hydroxy-3-(N-sperminecarboxamido)aminopropyl]-Jeffamine;

N¹,N⁸-dipalmityl-N¹,N⁸-di-[2-hydroxy-3-(N-sperminecarboxamido)aminopropyl]-Jeffamine;

N¹,N⁸-dipalmitolyl-N¹,N⁸-di-[2-hydroxy-3-(N-sperminecarboxamido)aminopropyl]-Jeffamine;

N¹,N⁸-distearyl-N¹,N⁸-di-[2-hydroxy-3-(N-sperminecarboxamido)aminopropyl]-Jeffamine;

N¹,N⁸-dilauryl-N¹,N⁸-di-[2-hydroxy-3-(N-sperminecarboxamido)aminopropyl]-Jeffamine;

N¹,N⁸-dioleyl-N¹,N⁸-di-[2-hydroxy-3-(N-sperminecarboxamido)aminopropyl]-Jeffamine;

N¹,N²-dimyristyl-N¹,N²-di-[2-hydroxy-3-(N-sperminecarboxamido)aminopropyl]-diaminoethane;

N¹,N²-dipalmityl-N¹,N²-di-[2-hydroxy-3-(N-sperminecarboxamido)aminopropyl]-diaminoethane;

N¹,N²-dipalmitolyl-N¹,N²-di-[2-hydroxy-3-(N-sperminecarboxamido)aminopropyl]-diaminoethane;

N¹,N²-distearyl-N¹,N²-di-[2-hydroxy-3-(N-sperminecarboxamido)aminopropyl]-diaminoethane;

or

N¹,N²-dilauryl-N¹,N²-di-[2-hydroxy-3-(N-sperminecarboxamido)aminopropyl]-diaminoethane.

81. A compound which is: