TRANSFECTION REAGENTS

• US 20100159593A1
• Filed: 12/15/2009
• Published: 06/24/2010
• Est. Priority Date: 11/12/1998
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: CHU, Yongliang, Gebeyehu, Gulilat, Masoud, Malek
• Granted Patent: US 7,915,450 B2
• Time in Patent Office: Days
• US Class Current: 435/375
• 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)l, 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 anyone 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 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

1-20. -20. (canceled)

21. A compound or polycation having the formula:

wherein

m, n, q, r, s, u, and y are 1;

v and w are 0;

Q is N;

L is (CH₂)₁ wherein l is an integer from 1 to 4;

R₁ and R₄ are H;

R₂, R₃, R₅ and R₆, independently of one another, are H, or an alkyl or alkyl ether optionally substituted by one or more of an alcohol, an amino alcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, an alkylthio, a urea, a thiourea, a guanidyl, and a carbamoyl group;

at least one of R₃ and R₆ is a straight-chain alkyl group; and

R₃ and R₆ are covalently linked with each other to form a cyclic moiety;

X is a physiologically acceptable anion; and

a is the number of positive charges divided by the valence of the anion.

22. The compound or polycation of claim 21 wherein R₂ and R₅, independently of one another, are H, or an alkyl or alkyl ether substituted by one or more of an amide and an ester.

23. The compound or polycation of claim 21 wherein R₂ and R₅, independently of one another, are an alkyl or alkyl ether substituted by one or more of an amide and an ester.

24. The compound or polycation of claim 21 wherein R₂ and R₅, independently of one another, are H, or an alkyl or alkyl ether substituted by one or more of an amide.

25. The compound or polycation of claim 21 wherein R₂ and R₅, independently of one another, are H, or an alkyl or alkyl ether substituted by one or more of an ester.

26. A compound or polycation having the formula:

wherein

m, n, q, r, s, u, and y are 1;

v and w are 0;

Q is N;

L is (CH₂)₁ wherein l is an integer from 1 to 4;

R₁ and R₄ are H;

R₂ and R₅ are alkyl substituted by an ester;

R₃ and R₆ are covalently linked with each other to form a cyclic alkyl moiety;

X is a physiologically acceptable anion; and

a is the number of positive charges divided by the valence of the anion.

32. A composition for transfecting a cell comprising one or more compounds or polycations of claim 26 and a neutral lipid.

37. A method for introducing a polyanion into a eukaryotic cell, wherein the method comprises:

providing a liposome comprising one or more compounds or polycations of claim 26;

contacting the liposome with the polyanion to form a polyanion-liposome complex; and

incubating the complex with the cell to thereby introduce the polyanion into the cell.

27. A compound or polycation having the formula:

wherein

m, n, q, r, s, u, and y are 1;

v and w are 0;

Q is N;

L is (CH₂)₁ wherein l is an integer from 1 to 4;

R₁ and R₄ are H;

R₂ and R₅ are alkyl substituted by an amide;

R₃ and R₆ are covalently linked with each other to form a cyclic alkyl moiety;

X is a physiologically acceptable anion; and

a is the number of positive charges divided by the valence of the anion.

33. A composition for transfecting a cell comprising one or more compounds or polycations of claim 27 and a neutral lipid.

38. A method for introducing a polyanion into a eukaryotic cell, wherein the method comprises:

providing a liposome comprising one or more compounds or polycations of claim 27;

contacting the liposome with the polyanion to form a polyanion-liposome complex; and

incubating the complex with the cell to thereby introduce the polyanion into the cell.

28. A compound or polycation having the formula:

wherein

m, n, q, r, s, u, and y are 1;

v and w are 0;

Q is N;

L is (CH₂)₁ wherein l is an integer from 1 to 4;

R₁ and R₄ are H;

R₂ and R₅ are alkyl substituted by an ether;

R₃ and R₆ are covalently linked with each other to form a cyclic alkyl moiety;

X is a physiologically acceptable anion; and

a is the number of positive charges divided by the valence of the anion.

34. A composition for transfecting a cell comprising one or more compounds or polycations of claim 28 and a neutral lipid.

39. A method for introducing a polyanion into a eukaryotic cell, wherein the method comprises:

providing a liposome comprising one or more compounds or polycations of claim 28;

contacting the liposome with the polyanion to form a polyanion-liposome complex; and

incubating the complex with the cell to thereby introduce the polyanion into the cell.