Transfection reagents

• US 9,358,300 B2
• Filed: 07/21/2014
• Issued: 06/07/2016
• Est. Priority Date: 11/12/1998
• Status: Expired due to Fees

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Chu, Yongliang, Masoud, Malek, Gebeyehu, Gulilat
• Primary Examiner(s): Davis, Brian J
• Application Number:
  US14/336,887
• Publication Number:
  US 20150190522A1
• Time in Patent Office: 687 Days
• Field of Search: None
• US Class Current: 1/1
• 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. A transfection complex formed in an aqueous medium, said transfection complex comprising:

an expressible nucleic acid;

a compound having the structure;

2. The transfection complex according to claim 1, wherein the protein or peptide transfection enhancer enhances cell-targeting, uptake, internalization, nuclear targeting or expression of the expressible nucleic acid.

3. The transfection complex according to claim 1, wherein the protein or peptide transfection enhances uptake of the expressible nucleic acid.

4. The transfection complex according to claim 1, wherein R₅ is an alkyl group substituted with one amine and one or more alcohols.

5. The transfection complex according to claim 1, wherein the amine substituent of the R₅ alkyl group comprises an aminoethanol, an aminopropanol or an aminobutanol group.

6. The transfection complex according to claim 5, wherein the amine substituent of the R₅ alkyl group has the structure:

7. The transfection complex according to claim 1, wherein R₅ is an alkyl group having 3-30 carbon atoms and is substituted by one amine and one or more alcohols.

8. The transfection complex according to claim 7, wherein the amine substituent of the R₅ alkyl group comprises an aminoethanol, an aminopropanol or an aminobutanol group.

9. The transfection complex according to claim 7, wherein the amine substituent of the R₅ alkyl group has the structure:

10. The transfection complex according to claim 2, wherein the protein or peptide transfection enhancer enhances uptake, internalization or nuclear targeting of the expressible nucleic acid.

11. The transfection complex according to claim 10, wherein the amine substituent of the R₅ alkyl group has the structure:

12. The transfection complex according to claim 1, wherein R₇ is H.

13. The transfection complex according to claim 12, wherein R₄ is a straight-chain alkyl group having 8 to 24 carbon atoms.

14. The transfection complex according to claim 13, wherein R₅ is an alkyl group having 3-30 carbon atoms and is substituted with one amine and one or more alcohols.

15. The transfection complex according to claim 14, wherein the amine substituent of the R₅ alkyl group comprises an aminoethanol, an aminopropanol or an aminobutanol group.

16. The transfection complex according to claim 14, wherein the amine substituent of the R₅ alkyl group has the structure:

17. The transfection complex according to claim 14, wherein R₅ is a propyl or a butyl group which is substituted by one amine and one or more alcohols.

18. The transfection complex according to claim 17, wherein the amine substituent of the R₅ alkyl group comprises an aminoethanol, an aminopropanol or an aminobutanol group.

19. The transfection complex according to claim 18, wherein the amine substituent of the R₅ alkyl group has the structure:

20. The transfection complex according to claim 1, wherein R₄ is a Straight-chain alkyl group having 8 to 24 carbon atoms, R₅ is a butyl group substituted with one amine and one or more alcohols, and R₇ is H.

21. The transfection complex according to claim 20, wherein the amine substituent of the R₅ alkyl group comprises an aminoethanol, an aminopropanol or an aminobutanol group.

22. The transfection complex according to claim 21, wherein the amine substituent of the R₅ alkyl group has the structure:

23. The transfection complex according to claim 1, wherein R₄ is a straight-chain alkyl group having 8 to 24 carbon atoms, R₅ is a propyl group substituted with one amine and one alcohol, and R₇ is H.

24. The transfection complex according to claim 23, wherein the amine substituent of the R₅ alkyl group comprises an aminoethanol, an aminopropanol or an aminobutanol group.

25. The transfection complex according to claim 24, wherein the amine substituent of the R₅ alkyl group has the structure:

26. The transfection complex according to claim 1, wherein the transfection complex further comprises one or more neutral lipids.

27. The transfection complex according to claim 26, wherein the neutral lipid is DOPE, DOPC or cholesterol.

28. The transfection complex according to claim 1, wherein the transfection complex comprises a liposome.