Patent US5459127A successfully refreshed from Google Patents.
US5459127A
Expired / Lapsed
US
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Cationic lipids for intracellular delivery of biologically active molecules
Assignee: Fresh Tracks Therapeutics Inc
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Filing: 1993-09-16
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Granted: 1995-10-17
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Expires: 2013-09-16
Abstract
Abstract Disclosed are cationic lipids capable of facilitating transport of biologically active agents into cells, including the transfection of cells by therapeutic polynucleotides, the delivery of antiviral drugs, and the introduction of immunogenic peptides. The cationic lipids, comprising an ammonium group, have the general structure <IMAGE> (I) Also disclosed are adducts of these compounds comprising additional cationic sites that enhance the transfective or transport activity. Structure-activity correlations provide for the selection of preferred compounds to be synthesized for this purpose. Compositions disclosed for use of these cationic lipid include formulations for in vitro transfection and pharmaceutical formulations for parenteral and topical administration of therapeutic agents.
Inventors
- 👤 Philip L. Felgner
- 👤 Raj Kumar
- 👤 Channa Basava
- 👤 Richard C. Border
- 👤 Jiin-Yu Hwang-Felgner
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-06 19:53
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/87; C12N15/88; A; A61; A61K; A61K47/00; A61K47/06; A61K47/16; A61K47/18; A61K47/186; A61K47/50; A61K47/51; A61K47/54; A61K47/543; A61K47/544; A61K47/62; A61K47/64; A61K47/645; A61K9/00; A61K9/0012; A61K9/0014; A61K9/0019; A61K9/10; A61K9/127; A61K9/1271; A61K9/1272; A61P; A61P31/00; A61P31/12
Claims Analysis
Total Claims: 56
Claim 1
Independent Claim
52 words
A formulation for transfection of polynucleotides into cells, comprising a cationic lipid and an effective transfection-promoting amount of a lysophosphatide, having the structure ##STR20## wherein Y is selected from the group consisting of --O--CH2-- and --O--C(O)--; R is C 10 to C 23 alkyl or alkenyl; and Z is a phospholipid headgroup.
Claim 2
Dependent Claim
52 words
A formulation for transfection of polynucleotides into cells, comprising a cationic lipid and an effective transfection-promoting amount of a lysophosphatide, having the structure ##STR20## wherein Y is selected from the group consisting of --O--CH2-- and --O--C(O)--; R is C 10 to C 23 alkyl or alkenyl; and Z is a phospholipid headgroup.
Claim 3
Dependent Claim
260 words
A formulation for transfection of polynucleotides and peptides into cells, comprising a compound having the structure ##STR21## wherein Y 1 and Y 2 are the same or different and are --O--C(O)-- or --O--; R 1 is H, or C 1 to C 24 alkyl or alkenyl; R 2 is C 1 to C 24 alkyl or alkenyl; R 3 and R 4 are independently C 1 to C 24 alkyl or H; R 5 is C 1 to C 24 alkyl straight chain or branched chain; R 6 is --C(O)--(CH 2 ) m --NH--, a diaminocarboxylate ester group which is alkyl, aryl, or aralkyl, or --C(O)--(CH 2 ) m --NH-- linked to said diaminocarboxylate ester group, or is absent; R 7 is H, spermine, spermidine, a histone, or a protein with DNA-binding specificity, or the same groups wherein the amine functionalities of the R 7 moiety are quaternized with R 3 , R 4 , or R 5 groups; or R 7 is an L- or D-alpha amino acid having a positively charged group on the side chain, said amino acids comprising arginine, histidine, lysine or ornithine or analogues thereof, or wherein the amine of the R 7 moiety is quaternized with R 3 , R 4 or R 5 groups; or R 7 is a polypeptide selected from the group consisting of L- or D-alpha amino acids, wherein at least one of the amino acids residues comprises arginine, histidine, lysine, ornithine, or analogues thereof; n is 1 to 8; m is 1 to 18; and X is non-toxic anion.
Claim 4
Dependent Claim
260 words
A formulation for transfection of polynucleotides and peptides into cells, comprising a compound having the structure ##STR21## wherein Y 1 and Y 2 are the same or different and are --O--C(O)-- or --O--; R 1 is H, or C 1 to C 24 alkyl or alkenyl; R 2 is C 1 to C 24 alkyl or alkenyl; R 3 and R 4 are independently C 1 to C 24 alkyl or H; R 5 is C 1 to C 24 alkyl straight chain or branched chain; R 6 is --C(O)--(CH 2 ) m --NH--, a diaminocarboxylate ester group which is alkyl, aryl, or aralkyl, or --C(O)--(CH 2 ) m --NH-- linked to said diaminocarboxylate ester group, or is absent; R 7 is H, spermine, spermidine, a histone, or a protein with DNA-binding specificity, or the same groups wherein the amine functionalities of the R 7 moiety are quaternized with R 3 , R 4 , or R 5 groups; or R 7 is an L- or D-alpha amino acid having a positively charged group on the side chain, said amino acids comprising arginine, histidine, lysine or ornithine or analogues thereof, or wherein the amine of the R 7 moiety is quaternized with R 3 , R 4 or R 5 groups; or R 7 is a polypeptide selected from the group consisting of L- or D-alpha amino acids, wherein at least one of the amino acids residues comprises arginine, histidine, lysine, ornithine, or analogues thereof; n is 1 to 8; m is 1 to 18; and X is non-toxic anion.
Claim 5
Dependent Claim
123 words
A formulation for transfection of polynucleotides and peptides into cells, comprising a compound having the structure ##STR22## or an optical isomer thereof, wherein Y 1 and Y 2 are different and are either --O--C(O)-- or --O--; R 1 is C 1 to C 24 alkyl or alkenyl, or H; R 2 is C 1 to C 24 alkyl or alkenyl; R 3 , R 4 and R 5 are independently H, C 1 to C 14 alkyl, C 7 to C 11 aryl or alkaryl, or at least two of R 3 , R 4 and R 5 are taken together to form quinuclidino, piperidino, pyrrolidino, or morpholino; and X is a non-toxic anion; together with an effective transfection-promoting amount of a lysophosphatide.
Claim 6
Dependent Claim
123 words
A formulation for transfection of polynucleotides and peptides into cells, comprising a compound having the structure ##STR22## or an optical isomer thereof, wherein Y 1 and Y 2 are different and are either --O--C(O)-- or --O--; R 1 is C 1 to C 24 alkyl or alkenyl, or H; R 2 is C 1 to C 24 alkyl or alkenyl; R 3 , R 4 and R 5 are independently H, C 1 to C 14 alkyl, C 7 to C 11 aryl or alkaryl, or at least two of R 3 , R 4 and R 5 are taken together to form quinuclidino, piperidino, pyrrolidino, or morpholino; and X is a non-toxic anion; together with an effective transfection-promoting amount of a lysophosphatide.
Claim 7
Dependent Claim
32 words
A formulation for transfection of polynucleotides and peptides into cells, comprising a compound selected from the group consisting of 3,5-(N,N-dilysyl)-diaminobenxzoyl-3-(DL-1,2-dioleoyldimethylaminopropyl-.beta.-hydroxyethylamine; 3,5-(N,N-dilysyl)diaminobenxzoylglycyl-3-(DL-1,2-dioleoyl-dimethylaminopropyl-β-hydroxyethylamine); and L-spermine-5-carboxyl-3-(DL-1,2-dioleoyldimethylaminopropyl-β-hydroxyethylamine) together with an effective transfection-promoting amount of a lysophosphatide.
Claim 8
Dependent Claim
32 words
A formulation for transfection of polynucleotides and peptides into cells, comprising a compound selected from the group consisting of 3,5-(N,N-dilysyl)-diaminobenxzoyl-3-(DL-1,2-dioleoyldimethylaminopropyl-.beta.-hydroxyethylamine; 3,5-(N,N-dilysyl)diaminobenxzoylglycyl-3-(DL-1,2-dioleoyl-dimethylaminopropyl-β-hydroxyethylamine); and L-spermine-5-carboxyl-3-(DL-1,2-dioleoyldimethylaminopropyl-β-hydroxyethylamine) together with an effective transfection-promoting amount of a lysophosphatide.
Claim 9
Dependent Claim
21 words
A formulation according to claims 2 or 3, wherein said lysophosphatide is selected from the group consisting of lysophosphatidylcholine and lysophosphatidylethanolamine.
Claim 10
Dependent Claim
13 words
A formulation according to claim 5, wherein said lysophosphatide is a mono-oleoyl lysophosphatidylcholine.
Claim 11
Dependent Claim
16 words
A formulation according to claim 2 or 3, wherein said lysophosphatide has a negatively charged headgroup.
Claim 12
Dependent Claim
16 words
A formulation according to claim 2 or 3, wherein said lysophosphatide has a neutrally charged headgroup.
Claim 13
Dependent Claim
23 words
A formulation according to claim 2 or 3, wherein the molar ratio of said lysophosphatide to cationic lipid is less than about 0.50.
Claim 14
Dependent Claim
34 words
A formulation comprising a cationic lipid and a lysophosphatide, wherein said cationic lipid is selected from the group consisting of DOTMA, DOTAP, and compounds having a structure set forth in claim 2 or 3.
Claim 15
Dependent Claim
86 words
A lipid formulation for the transfection of polynucleotides and peptides into cells, comprising a cationic lipid or combination of cationic lipids selected from the group consisting of DOTMA, DOTAP, or a structure according to claim 2 or 3, wherein an effective transfection-promoting amount of said cationic lipids up to about one-third of the total cationic lipids are selected from species wherein Y 1 and R 1 or Y 2 and R 2 of said structure, or moieties of DOTMA or DOTAP corresponding thereto, are hydroxyl groups.
Claim 16
Dependent Claim
36 words
A liposomal formulation, comprising a cationic lipid according to claim 2 or 3, or a mixture of said cationic lipids, said lipid or mixture of lipids being in the form of vesicles in an aqueous media.
Claim 17
Dependent Claim
24 words
A liposomal formulation according to claim 12, further comprising a neutral lipid species, selected from the group consisting of phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, or cholesterol.
Claim 18
Dependent Claim
27 words
A liposomal formulation according to claim 13 wherein the molar ratio of said cationic lipid species to said neutral lipid species is from about 9/1 to 1/9.
Claim 19
Dependent Claim
14 words
A liposomal formulation according to claim 13 wherein said molar ratio is about 5/5.
Claim 20
Dependent Claim
29 words
A liposomal formulation according to claim 13 further comprising a lyso lipid selected from the group consisting of lysophosphatidylcholine, lysophosphatidylethanolamine, or a lyso form of a cationic lipid species.
Claim 21
Dependent Claim
26 words
A pharmaceutical product comprising a cationic lipid having a structure set forth in claim 2 or 3, together with a pharmacologically effective amount of therapeutic agent.
Claim 22
Dependent Claim
19 words
A pharmaceutical product according to claim 17, wherein said therapeutic agent is a corticosteroid or a non-steroidal anti-inflammatory agent.
Claim 23
Dependent Claim
20 words
A pharmaceutical product according to claim 17, wherein said therapeutic agent is a therapeutically effective nucleoside analogue or nucleotide analogue.
Claim 24
Dependent Claim
22 words
A pharmaceutical product according to claim 18, wherein said therapeutic agent is a phosphatidyl derivative or diphosphate diglyceride derivative of said analogue.
Claim 25
Dependent Claim
26 words
A pharmaceutical product according to claim 17, wherein said analogue is dideoxynucleoside, a didehydronucleoside, a halogenated or azido derivative of a nucleoside, or an acyclic nucleoside.
Claim 26
Dependent Claim
28 words
A pharmaceutical product according to claim 19, wherein said analogue is an antiviral nucleoside selected from the group consisting of a 3'-azido-2',3'-dideoxypyrimidine, a 3'-halopyrimidine dideoxynucleoside, or a 2',3'-didehydro-2',3'-dideoxynucleoside.
Claim 27
Dependent Claim
18 words
A pharmaceutical product according to claim 22, wherein said analogue is an antiviral nucleoside which is 3'-azido-3'-deoxythymidine (AZT).
Claim 28
Dependent Claim
27 words
A pharmaceutical product according to claim 21, wherein said analogue is an antiviral nucleoside selected from the group consisting of acyclovir, gancyclovir, 1-(2-deoxy-2'-fluoro-1-β-D-arabinofuranosyl)-5-iodocytosine (FIAC) or 1(2'-deoxy-2'-fluoro-1-β-D-arabinofuranosyl)5-iodouracil (FIAU).
Claim 29
Dependent Claim
11 words
A pharmaceutical product according to claim 19, comprising a therapeutic polynucleotide.
Claim 30
Dependent Claim
20 words
A pharmaceutical product according to claim 25, wherein said therapeutic polynucleotide is a ribozyme or an antisense RNA or DNA.
Claim 31
Dependent Claim
19 words
A pharmaceutical preparation according to claim 26, wherein said ribozyme or antisense DNA or RNA is directed against HIV.
Claim 32
Dependent Claim
21 words
A pharmaceutical preparation according to claim 26, wherein said ribozyme or antisense DNA or RNA is directed against the rev transactivator.
Claim 33
Dependent Claim
17 words
A pharmaceutical preparation according to claim 25, wherein said therapeutic polynucleotide is a 28-mer phosphorothioate antisense polynucleotide.
Claim 34
Dependent Claim
16 words
A pharmaceutical preparation according to claim 25, wherein said therapeutic polynucleotide codes for a therapeutic polypeptide.
Claim 35
Dependent Claim
18 words
The pharmaceutical preparation of claim 30, wherein said therapeutic polypeptide is deficient or absent in a disease state.
Claim 36
Dependent Claim
19 words
The pharmaceutical preparation of claim 30, wherein said therapeutic polypeptide is a natural hormone or a synthetic analog thereof.
Claim 37
Dependent Claim
13 words
The pharmaceutical preparation of claim 30, wherein said therapeutic polypeptide is an immunogen.
Claim 38
Dependent Claim
15 words
The pharmaceutical preparation of claim 27, wherein said therapeutic agent is a protein or peptide.
Claim 39
Dependent Claim
33 words
A pharmaceutical preparation for topical use comprising a compound having the structure set forth in claim 2 or 3, and a pharmacologically effective amount of a therapeutic agent in a pharmaceutically acceptable vehicle.
Claim 40
Dependent Claim
28 words
The pharmaceutical preparation of claim 25, wherein said therapeutic agent is a corticosteroid, a nonsteroidal anti-inflammatory agent, an antibiotic, an antifungal agent, an oxidant, or an antiviral nucleoside.
Claim 41
Dependent Claim
19 words
The pharmaceutical preparation of claim 25, wherein said therapeutic agent is a protein, a polypeptide, or a therapeutic polynucleotide.
Claim 42
Dependent Claim
21 words
The pharmaceutical preparation of claim 25 comprising a therapeutic polynucleotide which is a ribozyme or an antisense RNA or DNA sequence.
Claim 43
Dependent Claim
24 words
The pharmaceutical preparation of claim 25 comprising a therapeutic polynucleotide coding for a gene product that is deficient or absent in a disease state.
Claim 44
Dependent Claim
26 words
The pharmaceutical preparation of claim 25 comprising a therapeutic polynucleotide coding for an immunogenic peptide, a natural hormone, or a synthetic analogue of a natural hormone.
Claim 45
Dependent Claim
44 words
A pharmaceutical preparation for topical use in the treatment of Herpes simplex, comprising a compound having the structure set forth in claim 2 or 3 together with a pharmacologically effective concentration of acyclovir, gancyclovir, 1-(2-deoxy-2'-fluoro-1-β-D-arabinofuranosyl)-5-iodocytosine (FIAC) or 1(2'-deoxy-2'-fluoro-1-β-D-arabinofuranosyl)5-iodouracil (FIAU) in a pharmaceutically acceptable vehicle.
Claim 46
Dependent Claim
60 words
A method for introducing a biologically active agent into a cell of a plant or animal, comprising: a. preparing lipid vesicles comprising a cationic lipid having a structure set forth in claim 2 or 3 and containing said biologically active agent; b. contacting said cell with said lipid vesicles whereby said biologically active agent is taken up into said cell.
Claim 47
Dependent Claim
58 words
A method for introducing a biologically active agent into a cell of a plant or animal, comprising: preparing lipid vesicles comprising a cationic lipid having a structure set forth in claim 2 or 3; contacting said cell with a bioactive agent in the presence of said lipid vesicles, whereby said bioactive agent is taken up into said cell.
Claim 48
Dependent Claim
13 words
A method according to claim 43, wherein said contacting step occurs in vitro.
Claim 49
Dependent Claim
69 words
A method of treating a disease in a vertebrate, comprising the step of administering a pharmaceutical preparation comprising a cationic lipid having a structure set forth in claim 2 or 3 and a pharmacologically effective amount of a therapeutic agent specific for the treatment of said disease, and permitting said therapeutic agent to be incorporated into at least one cell of said vertebrate, whereby said disease is effectively treated.
Claim 50
Dependent Claim
28 words
A method according to claim 45, comprising the administration of said preparation to the cells of said vertebrate in vitro, which cells are then returned to said vertebrate.
Claim 51
Dependent Claim
21 words
A method according to claim 45, comprising the topical application of said preparation to the skin or to a mucosal surface.
Claim 52
Dependent Claim
22 words
A method according to claim 45, comprising the injection of said preparation into body cavity or into the tissues of said vertebrate.
Claim 53
Dependent Claim
13 words
A method according to claim 45, comprising the oral administration of said preparation.
Claim 54
Dependent Claim
14 words
A method according to claim 45, wherein said biologically active agent is a polynucleotide.
Claim 55
Dependent Claim
36 words
A method according to claim 45, wherein said biologically active agent is DNA or mRNA coding for a polypeptide, and said polypeptide is expressed after said DNA or said mRNA is taken up into said cell.
Claim 56
Dependent Claim
14 words
A method according to claim 45 wherein said biologically active agent is a drug.
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