Methods for delivering compounds into a cell

• US 6,743,779 B1
• Filed: 04/29/1997
• Issued: 06/01/2004
• Est. Priority Date: 11/29/1994
• Status: Expired due to Fees

• Current Assignee: Cerevast Therapeutics, Inc.
• Original Assignee: Imarx Pharmaceutical Corp. (Sycamore Entertainment Group, Inc.)
• Inventors: McCreery, Thomas, Unger, Evan C.
• Primary Examiner(s): WILSON, MICHAEL C
• Application Number:
  US08/841,169
• Time in Patent Office: 2,590 Days
• Field of Search: 514/44, 435/325
• US Class Current: 514/44.00R
• CPC Class Codes: A61K 41/0028 Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61K 9/127 Liposomes
• CPC Class Codes: A61K 9/1272 with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids with cholesterol as the only non-phosphatidyl surfactant A61K9/127; lipids as modifying agent A61K47/543
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12N 15/88 using microencapsulation, e.g. using amphiphile liposome vesicle

Abstract

The present invention is directed, inter alia, to a method for delivering a compound into a cell comprising administering to the cell the compound to be delivered, an organic halide, and/or a carrier. Ultrasound may also be applied, if desired.

Claims

1. A method for delivering nucleic acid into a cell in vitro or in vivo, comprising:

(a) administering to the cell a composition comprising;

a nucleic acid sequence selected from the group consisting of a RNA sequence, and a DNA sequence;

a fluorinated compound selected from the group consisting of selenium hexafluoride, sulfur hexafluoride, 1-bromo-nonafluorobutane, perfluorooctyliodide, perfluorooctylbromide, 1-chloro-1-fluoro-1-bromomethane, 1,1,1-trichloro-2,2,2-trifluoroethane, 1,2-dichloro-2,2-difluoroethane, 1,1-dichloro-1,2-difluoroethane, 1,2-dichloro-1,1,3-trifluoropropane, 1-bromoperfluorobutane, 1-bromo-2,4-difluorobenzene, 2-iodo-1,1,1-trifluoroethane, 1,3-dichlorotetrafluoroacetone, bromine pentafluoride, 1-bromo-1,1,2,3,3,3-hexafluoropropane, 2-chloro 1,1,1,4,4,4-hexafluoro-2-butene, 2-chloropentafluoro-1,3-butadiene, iodotrifluoroethylene, 1,1,2-trifluoro-2-chloroethane, 1,2-difluorochloroethane, 1,1-difluoro-2-chloroethane, 1,1-dichlorofluoroethane, heptafluoro-2-iodopropane, bromotrifluoroethane, chlorotrifluoromethane, dichlorodifluoromethane, dibromofluoromethane, chloropentafluoroethane, bromochlorodifluoromethane, dichloro-1,1,2,2-tetrafluoroethane, 1,1,1,3,3-pentafluoropentane, perfluorotributylamine, perfluorotripropylamine, 3-fluorobenzaldehyde, 2-fluoro-5-nitrotoluene, 3-fluorostyrene, 3,5-difluoroaniline, 2,2,2-trifluoroeihylacrylate, 3-(trifluoromethoxy)-acetophenone, 1,1,2,2,3,3,4,4-octafluorobutane, 1,1,1,3,3-pentafluorobutane, 1-fluorobutane, 1,1,2,2,3,3,4,4-octafluorobutane, 1,1,1,3,3-pentafluorobutane, perfluoro-4 methylquinolizidine, perfluoro-N-meihyl-decahydroquinone, perfluoro-N-methyl-decahydroisoquinone, perfluoro-N-cyclohexyl-pyrrolidine, tetradecaperfluoroheptane, dodecaperfluorocyclohexane, perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluorononane, perfluorodecane, perfluoro-2-methyl-2-pentene, perfluorocyclohexane, perfluorodecalin, perfluorododecalin, perfluoropropylene, perfluorocyclobutane, perfluoro-2-butyne, perfluoro-2-butene, perfluorobuta-1,3-diene, perfluorobutylethyl ether, bis(perfluoroisopropyl) ether, bis(perfluoropropyl) ether, perfluorotetrahydropyran, perfluoromethyl tetrahydrofuran, perfluoro t-butyl methyl ether, perfluoro isobutyl methyl ether, perfluoro n-butyl methyl ether, perfluoro isopropyl ethyl ether, perfluoro n-propyl ethyl ether, perfluoro cyclobutyl methyl ether, perfluoro cyclopropyl ethyl ether, perfluoro isopropyl methyl ether, perfluoro n-propyl methyl ether, perflouro diethyl ether, perfluoro cyclopropyl methyl ether, perfluoro methyl ethyl ether, and perfluoro dimethyl ether; and

a lipid carrier.

2. The method of claim 1 wherein said lipid carrier comprises an ester linked fatty acid.

3. The method of claim 1 wherein said lipid carrier comprises phosphatidylcholine, or phosphatidylethanolamine.

4. The method of claim 1 wherein said fluorinated compound is selected from the group consisting of perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluorononane, perfluorodecane, perfluoro-2-methyl-2-pentene, perfluorocyclohexane, perfluorodecalin, perfluorododecalin, tetradecaperfluorohexane, and dodecaperfluorocyclohexane.

5. The method of claim 1 wherein said fluorinated compound is selected from the group consisting of perfluorobutylethyl ether, bis(perfluoroisopropyl)ether, bis(perfluoropropyl)ether, perfluorotetrahydropyran, perfluoromethyl tetrahydrofuran, perfluoro t-butyl methyl ether, perfluoro isobutyl methyl ether, perfluoro n-butyl methyl ether, perfluoro isopropyl ethyl ether, perfluoro n-propyl ethyl ether, perfluoro cyclobutyl methyl ether, perfluoro cyclopropyl ethyl ether, perfluoro isopropyl methyl ether, perfluoro n-propyl methyl ether, perflouro diethyl ether, perfluoro cyclopropyl methyl ether, perfluoro methyl ethyl ether, and perfluoro dimethyl ether.

6. The method of claim 1 wherein said fluorinated compound is selected from the group consisting of perfluorohexane and perfluorocyclohexane.

7. The method of claim 1 wherein said lipid carrier is a cationic lipid.

8. The method of claim 7 wherein said cationic lipid is N-[1(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride.

9. The method of claim 1 wherein said lipid carrier is selected from the group consisting of Lipofectamine, Transfectace, Transfectam, Cytofectin, DMRIE, DLRIE, GAP-DLRIE, DOTAP, DOPE, DMEAP, DODMP, DOPC, DDAB, DOSPA, EDLPC, EDMPC, DPH, TMADPH, CTAB, lysyl-PE, DC-Cho, -alanyl cholesterol;

DCGS, DPPES, DCPE, DMAP, DMPE, DOGS, DOHME, DPEPC, Pluronic, Tween, BRIJ, plasmalogen, phosphatidylethanolamine, phosphatidylcholine, glycerol-3-ethylphosphatidylcholine, dimethyl ammonium propane, trimethyl ammonium propane, diethylammonium propane, triethylammonium propane, dimethyldioctadecylammonium bromide, a sphingolipid, sphingomyelin, a lysolipid, a glycolipid, a sulfatide, a glycosphingolipid, cholesterol, cholesterol ester, cholesterol salt, oil, N-succinyldioleoylphosphatidylethanolamine, 1,2-dioleoyl-sn-glycerol, 1,3-dipalmitoyl-2-succinylglycerol, 1,2-dipalmitoyl-sn-3-succinylglycerol, 1-hexadecyl-2-palmitoylglycerophosphatidylethanolamine, palmitoylhomocystiene, N,N′

-Bis (dodecyaminocarbonylmethylene)-N,N′

-bis((-N,N,N-trimethylammoniumethyl-aminocarbonylmethylene)ethylenediamine tetraiodide;

N,N″

-Bis(hexadecylaminocarbonylmethylene)-N,N′

, N″

-tris((-N,N,N-trimethylammonium-ethylaminocarbonylmethylenediethylenetriamine hexaiodide;

N,N′

-Bis(dodecylaminocarbonylmethylene)-N,N″

-bis((-N,N,N-trimethylammoniumethylaminocarbonylmethylene)cyclohexylene-1,4-diamine tetraiodide;

1,7,7-tetra-((-N,N,N,N-tetramethylammoniumethylamino-carbonylmethylene)-3-hexadecylaminocarbonyl-methylene-1,3,7-triaazaheptane heptaiodide; and

N,N,N′

,N′

-tetra((-N,N,N-trimethylammonium-ethylaminocarbonylmethylene)-N′

-(1,2-dioleoylglycero-3-phosphoethanolaminocarbonylmethylene)diethylenetriamine tetraiodide.

10. The method of claim 1 wherein said lipid carrier comprises at least one of a member selected from the group consisting of dioleoylphosphatidylethanolamine, a fatty acid, a lysolipid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, a sphingolipid, a glycolipid, a glucolipid, a sulfatide, a glycosphingolipid, phosphatidic acid, palmitic acid, stearic acid, arachidonic acid, oleic acid, a lipid bearing a polymer, a lipid bearing a sulfonated saccharide, cholesterol, tocopherol hemisuccinate, a lipid with an ether-linked fatty acid, a lipid with an ester-linked fatty acid, a polymerized lipid, diacetyl phosphate, stearylamine, cardiolipin, a phospholipid with a fatty acid of 6-8 carbons in length, a phospholipid with asymmetric acyl chains, 6-(5-cholesten-3b-yloxy)-1-thio-b-D-galactopyranoside, digalactosyldiglyceride, 6-(5-cholesten-3b-yloxy)hexyl-6-amino-6-deoxy-1-thio-b-D-galactopyranoside, 6-(5-cholesten-3b-yloxy)hexyl-6-amino-6-deoxyl-1-thio-a-D-mannopyranoside, 12-(((7′

-diethylamino-coumarin-3-yl)carbonyl)methylamino)-octadecanoic acid;

N-[12-(((7′

-diethylaminocoumarin-3-yl)carbonyl)methyl-amino) octadecanoyl]-2-aminopalmitic acid;

cholesteryl)4′

-trimethyl-ammonio)butanoate;

N-succinyldioleoyl-phosphatidylethanolamine;

1,2-dioleoyl-sn-glycerol;

1,2-dipalmitoyl-sn-3-succinyl-glycerol;

1,3-dipalmitoyl-2-succinylglycerol, 1-hexadecyl-2-palmitoylglycero-phosphoethanolamine, and palmitoylhomocysteine.

11. The method of claim 10 wherein said lipid carrier comprises a phosphatidylcholine selected from the group consisting of dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine.

12. The method of claim 10 wherein said lipid carrier comprises dioloeylphosphatidylethanolamine.

13. The method of claim 10 wherein said sphingolipid is sphingomyelin.

14. The method of claim 10 wherein said lipid carrier comprises a glycolipid selected from ganglioside GM1 and ganglioside GM2.

15. The method of claim 10 wherein said lipid carrier comprises a lipid bearing a sulfonated saccharide.

16. The method of claim 10 wherein said lipid carrier comprises a cholesterol selected from the group consisting of cholesterol sulfate and cholesterol hemisuccinate.

17. The method of claim 10 wherein said lipid carrier comprises a phopholipid comprising asymmetric acyl chains comprising one acyl chain of about 6 carbons in length and another acyl chain of about 12 carbons in length.

18. The method of claim 1 wherein said lipid carrier comprises a lipid bearing polymer selected from the group consisting of polyethylene glycol, chitin, hyaluronic acid, and polyvinylpyrrolidone.

19. The method of claim 18 wherein said polymer is a polyethylethylene glycol having a molecular weight selected from the group consisting of about 2000, 5000, and 8000.

20. The method of claim 1 wherein said lipid carrier comprises about 82 mole percent dipalmitoylphosphatidylcholine, about 8 mole percent dipalmitoylphosphatidylethanolamine-polyethyleneglycol 5000 and about 10 mole percent dipalmitoylphosphatideic acid.

21. The method of claim 11 wherein said nucleic acid and said fluorinated carbon-based organic compound other than a lipid are encapsulated by said lipid carrier.

22. The method of claim 1 wherein said cell is a mammalian cell.

23. The method of claim 22 wherein said mammalian cell is a human cell.

24. The method of claim 1 wherein said nucleic acid sequence is a DNA sequence.

25. The method of claim 1 wherein said DNA sequence is a sequence that encodes a protein.

26. The method of claim 25 wherein said protein is vascular endothelial growth factor.

27. The method of claim 1 wherein said nucleic acid sequence is an RNA sequence.

28. The method of claim 1, wherein said method further comprises the step of:

(b) applying ultrasound to said cell.

29. The method of claim 28 wherein said ultrasound is applied to said cell after said administering step (a).

30. The method of claim 29 wherein said ultrasound is applied at a frequency from about 40 kilohertz to about 25 megahertz, and at an energy level of from about 500 milliwatts/cm² to about 10 watts/cm².

31. The method of claim 29 wherein said ultrasound is applied at a frequency from about 500 kilohertz to about 3 megahertz, and at an energy level of from about 500 milliwatts/cm² to about 2 watts/cm².

32. The method of claim 29 wherein said ultrasound is applied at a frequency from about 20 megahertz to about 1 megahertz, and at an energy level of from about 1 watt/cm² to about 2 watts/cm².

33. The method of claim 32 wherein said ultrasound is applied at a duty cycle from about 1% to about 100%.

34. A method of expressing in vitro or in vivo a nucleotide sequence in a mammalian cell comprising:

(a) administering to said mammalian cell a composition comprising;

a nucleic acid sequence selected from the group consisting of a RNA sequence, and a DNA sequence;

a fluorinated compound selected from the group consisting of selenium hexafluoride, sulfur hexafluoride, 1-bromo-nonafluorobutane, perfluorooctyliodide, perfluorooctylbromide, 1-chloro-1-fluoro-1-bromomethane, 1,1,1-trichloro-2,2,2-trifluoroethane, 1,2-dichloro-2,2-difluoroethane, 1,1-dichloro- 1,2-difluoroethane, 1,2-dichloro- 1,1,3-trifluoropropane, 1-bromoperfluorobutane, 1-bromo-2,4-difluorobenzene, 2-iodo-1,1,1-trifluoroethane, 1,3-dichlorotetrafluoroacetone, bromine pentafluoride, 1-bromo-1,1,2,3,3,3-hexafluoropropane, 2-chloro 1,1,1,4,4,4-hexafluoro-2-butene, 2-chloropentafluoro-1,3-butadiene, iodotrifluoroethylene, 1,1,2-trifluoro-2-chloroethane, 1,2-difluorochloroethane, 1,1-difluoro-2-chloroethane, 1,1-dichlorofluoroethane, heptafluoro-2-iodopropane, bromotrifluoroethane, chlorotrifluoromethane, dichlorodifluoromethane, dibromofluoromethane, chloropentafluoroethane, bromochlorodifluoromethane, dichloro-1,1,2,2-tetrafluoroethane, 1,1,1,3,3-pentafluoropentane, perfluorotributylamine, perfluorotripropylamine, 3-fluorobenzaldehyde, 2-fluoro-5-nitrotoluene, 3-fluorostyrene, 3,5-difluoroaniline, 2,2,2-trifluoroethylacrylate, 3-(trifluoromethoxy)-acetophenone, 1,1,2,2,3,3,4,4-octafluorobutane, 1,1,1,3 ,3-pentafluorobutane, 1-fluorobutane, 1,1,2,2,3,3,4,4-octafluorobutane, 1,1,1,3,3-pentafluorobutane, perfluoro-4 methylquinolizidine, perfluoro-N-methyl-decahydroquinone, perfluoro-N-methyl-decahydroisoquinone, perfluoro-N-cyclohexyl-pyrrolidine, tetradecaperfluoroheptane, dodecaperfluorocyclohexane, perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluorononane, perfluorodecane, perfluoro-2-methyl-2-pentene, perfluorocyclohexane, perfluorodecalin, perfluorododecalin, perfluoropropylene, perfluorocyclobutane, perfluoro-2-butyne, perfluoro-2-butene, perfluorobuta-1,3-diene, perfluorobutylethyl ether, bis(perfluoroisopropyl)ether, bis(perfluoropropyl) ether, perfluorotetrahydropyran, perfluoromethyl tetrahydrofuran, perfluoro t-butyl methyl ether, perfluoro isobutyl methyl ether, perfluoro n-butyl methyl ether, perfluoro isopropyl ethyl ether, perfluoro n-propyl ethyl ether, perfluoro cyclobutyl methyl ether, perfluoro cyclopropyl ethyl ether, perfluoro isopropyl methyl ether, perfluoro n-propyl methyl ether, perflouro diethyl ether, perfluoro cyclopropyl methyl ether, perfluoro methyl ethyl ether, and perfluoro dimethyl ether; and

a lipid carrier.

35. The method of claim 34 wherein said cell is a human cell.