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• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
  • C07C219/02—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
  • C07C219/04—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
  • C07C219/08—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having at least one of the hydroxy groups esterified by a carboxylic acid having the esterifying carboxyl group bound to an acyclic carbon atom of an acyclic unsaturated carbon skeleton
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
  • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
  • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Liposomes
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
  • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
  • C07C217/02—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
  • C07C217/04—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
  • C07C217/28—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having one amino group and at least two singly-bound oxygen atoms, with at least one being part of an etherified hydroxy group, bound to the carbon skeleton, e.g. ethers of polyhydroxy amines
  • C07C217/40—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having one amino group and at least two singly-bound oxygen atoms, with at least one being part of an etherified hydroxy group, bound to the carbon skeleton, e.g. ethers of polyhydroxy amines having at least two singly-bound oxygen atoms, with at least one being part of an etherified hydroxy group, bound to the same carbon atom of the carbon skeleton, e.g. amino-ketals, ortho esters
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
  • C07C219/02—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
  • C07C219/04—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
  • C07C219/06—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having the hydroxy groups esterified by carboxylic acids having the esterifying carboxyl groups bound to hydrogen atoms or to acyclic carbon atoms of an acyclic saturated carbon skeleton
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
  • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

• cationic lipids that bind and transport polynucleotides, polypeptides, pharmaceutical substances and other biologically active species through membrane barriers. More specifically, symmetrical diamine cationic lipids are disclosed that complex with selected molecular species and facilitate delivery of those selected species into and through membranes and comparable boundary structures.
• LIPOFECTINTM is composed of a 1:1 formulation of the quaternary ammonium containing compound DOTMA and dioleoylphosphatidylethanolamine sonicated into small unilamellar vesicles in water.
• One problem with LIPOFECTINTM is that it contains non-metabolizable ether bonds.
• Other problems with LIPOFECTINTM are an inhibition of protein kinase C activity and direct cytotoxicity. In response to these problems, a number of other related compounds have been developed.
• the diamine compounds of the subject invention improve upon the capabilities of existing cationic transporters and serve as very efficient delivery means for biologically active chemicals.
• lipopolyamines and their use for transfecting eukaryotic cells.
• a polynucleotide is mixed with the subject lipopolyamine and contacted with the cells to be treated.
• U.S. Pat. Nos. 5,186,923 and 5,277,897 relate an enhancement of cellular accumulation of lipophilic cationic organometallic compounds by reduction of the intramembrane potential.
• Technetium containing compounds are disclosed.
• Lipophilic cationic compounds are presented in U.S. Pat. No. 5,208,036. Asymmetrical amine compounds are synthesized and employed in a method for DNA transfection.
• U.S. Pat. No. 5,264,618 discloses cationic lipids for intracellular delivery of biologically active molecules. Asymmetric ammonium containing cationic lipids are presented for transporting molecules into membranes enclosed systems.
• An object of the present invention is to disclose a category of diamines that greatly facilitate the delivery of biologically active compounds through membrane structures.
• Another object of the present invention is to present a group of symmetrical diamine cationic compounds that assist in the transport of selected macromolecules and other substances into and past membrane barriers.
• a further object of the present invention is to relate a collection of biologically active molecule transporters having easily variable lipid components linked to a symmetrical polyhydroxyl containing diamine core structure.
• novel diamine cationic transporter molecules that facilitate the delivery of such compounds as polynucleotides, polypeptides, and the like into and beyond membrane walls.
• symmetrically structured cationic diamines either polyhydroxylated or otherwise quaternized, having at least a pair of identical lipoyl moieties selected from a group consisting of an alkyl chain, an alkenyl chain, and an alkyl or alkenyl containing acyl chain.
• a preferred composition is N,N,N',N'-tetramethyl-N,N'-bis(2-hydroxyethyl)-2,3-di(oleoyloxy)-1,4-butanediaminium iodide, given the nickname PolyGum in view of its binding affinity.
• the length and double bond characteristics of the R 2 group (as is detailed below) and the presence or absence of a carbonyl in the R 2 group is variable.
• FIG. 1 is a graph that demonstrates the effectiveness of a 50:50 mixture of DOPE:PolyGum with DNA, compared with DNA only, in transfecting NIH 3T3 cells in a serum-free environment.
• FIG. 2 is a graph that illustrates that liposome structure (SV versus MLV forms) influences the efficiency of transfecting NIH 3T3 cells.
• FIG. 3 is a graph showing the effects of serum on PolyGum (MLV) mediated transfection of NIH 3T3 cells.
• FIG. 4 is a graph depicting the influence of phosphatidylethanolamine (PE) side chain structure on transfection of NIH 3T3 cells using a 50:50 mixture of the PE derivatives and PolyGum in the presence of 10% calf serum and without serum.
• PE phosphatidylethanolamine
• FIG. 5 is a graph showing the optimization of the mole ratio of DOPE to PolyGum (SV) in serum-free transfection of NIH 3T3 cells.
• FIG. 6 is a graph illustrating the optimization of the charge ratio of PolyGum (SV) to DNA in serum-free transfection of NIH 3T3 cells.
• FIG. 7 is a graph portraying an efficiency comparison of utilized cationic lipids in the serum-free transfection of DU-145 cells.
• FIG. 8 is a graph showing an efficiency comparison of utilized cationic lipids in transfection of DU-145 cells in the presence of 2% FBS.
• a symmetrical cationic diamine having at least a pair of identical lipoyl moieties selected from a group consisting of an alkyl chain, an alkenyl chain, and an alkyl or alkenyl containing acyl chain.
• R 1 is a hydrogen, an alkyl group, an alkenyl group, or a hydroxylated alkyl or alkenyl group
• R 2 is an alkyl group, an alkenyl group, or an alkyl or alkenyl
• n 0-10, usually between 0 and 3, preferably 1;
• R 1 is a hydrogen, an alkyl group, an alkenyl group, or a hydroxylatedalkyl or alkenyl group, generally having from 1 to 10 carbons, preferably 1carbon;
• R 2 is an alkyl group, an alkenyl group, or an alkyl or alkenylcontaining acyl group;
• R 3 is a hydrogen, an alkyl group, an alkenyl group, or a hydroxylated alkyl or alkenyl group, often an alkyl group of from 1 to 10 carbons, preferably a methyl group;
• X - is an anion, usually a halide, and preferably iodide.
• n 1-10;
• R 1 is a hydrogen, an alkyl group, an alkenyl group, or a hydroxylated alkyl or alkenyl group;
• R 2 is an alkyl group, an alkenyl group, or an alkyl or alkenyl containing acyl group;
• R 3 is a hydrogen, an alkyl group, an alkenyl group, or a hydroxylated alkyl or alkenyl group; and
• X is a halide.
• the first step involves reacting a tert-butyidiphenylsilyloxy derivatized starting material with 1,3-butane diepoxide in the presence of lithium perchlorate in absolute ethanol.
• the second step is a reaction with an alkyl or alkenyl halide or an alkyl or alkenyl containing acyl halide.
• the third step is tetrabutylammonium fluoride and THF initiated removal of the tert-butyldiphenylsilyloxy protection groups to produce the general precursor compound.
• the general precursor compound is then allowed to react with a selected alkyl, alkenyl, or hydroxylated alkyl or alkenyl halide.
• the subject composition has the structure: ##STR6##wherein R is an alkyl or alkenyl group, preferably --CH 2 (CH 2 ) 6 CH ⁇ CH(CH 2 ) 7 CH 3 and X - is an anion, preferably a halide such as iodide.
• R is an alkyl or alkenyl group, preferably --CH 2 (CH 2 ) 6 CH ⁇ CH(CH 2 ) 7 CH 3 and X - is an anion, preferably a halide such as iodide.
• a preferred species of the subject invention having the name N,N,N',N'-tetramethyl-N,N'-bis(2-hydroxyethyl)-2,3-di(oleoyloxy)-1,4-butanediaminium iodide, has the following structure: ##STR7##
• the long lipid tails are both oleoyl groups, however, other lipid tails are acceptable and within the realm of this disclosure.
• a study (Balasubramaniam, R. P., Bennett, M. J.,Gruenert, D., Malone, R. W., and Nantz, M. H., "Hydrophobic Domain of Cationic Lipids Influence Respiratory Epithelial Cell DNA Transfection" manuscript in preparation, which is herein incorporated by reference) involving cationic lipids, which contain a N,N-dimethyl-N-(2-hydroxyethyl)ammonium group (CH 3 ) 2 (HOCH 2 CH 2 --)N + --R!
• hydrophilic domain polar headgroup component present in PolyGum
• hydrophobic domain polar headgroup component present in PolyGum
• the cationic lipids showed activity as agents for polynucleotide transfection. Therefore, the various combinations of fatty acid side chains represent only analogous changes in the overall structure of the cationic lipid, andin each case the cationic lipid is apt to demonstrate transfection activity.
• DOHME a cationic asymmetric, lipid containing a mono-ammonium head group
• Transfection or membrane penetration is demonstrated by incorporating the subject diamines into various liposome/DNA complexes and exposing cells under desired conditions to initiate transfection.
• luciferase light emissions the luciferase plasmid pCMVL, see below, was utilized in a standard manner to detect transfection levels
• DOPE PolyGum SV very efficiently, as compared with DNA only, mediated DNA transfection of NIH 3T3 cells.
• FIG. 2 clearly indicates that with a 50:50 DOPE:PolyGum formulation, the SVs are much more capable transfection carriers.
• FIG. 3 The effects of serum on PolyGum (MLV) mediated transfection of NIH 3T3 cells is illustrated in FIG. 3. Under these transfection conditions, greatly increased transfection is found with serum-free conditions than inthe presence of 10% calf serum. Under other transfection conditions, littlechange is noted with serum transfection.
• MLV serum on PolyGum
• FIG. 4 plainly demonstrates how transfection of cells is influenced, with and without serum, by the side chain characteristics of the phosphatidylethanolamine utilized to generate the vesicles.
• the 50:50 PolyGum:DOPE formulation is superior for serum-free transfection, while the 50:50 PolyGum:DMPE formulation prevails in the serum added case.
• FIG. 6 shows the DNA charge ratio optimization data. For the ranges presented, clearly, a 2:1 PolyGum:DNA phosphate ratiomaximizes transfection.
• the presence of 2% FBS alters, as compared with serum-free conditions, the transfection levels for different formulations.
• the 50:50 DOPE:PolyGum (SV) formulation is most efficient for transfection.
• the 50:50 DOPE:POHME (MLV) formulation is most efficient, however, the 50:50 DOPE:PolyGum species is second highest in efficiency of transfection.
• the 50:50 DOPE:PolyGum (SV) formulation is once again the most efficient transfection agent, with the MLV 50:50 DOPE:PolyGum composition in second.Distinctly, the PolyGum reagent is a useful agent for transfecting cells.
• Toxicity of the subject compounds was evaluated by application of the standard Alamar Blue toxicity procedure. The results indicate less toxicity for both PolyGum MLV and SV formulations than for LIPOFECTINTM and several other amine containing vesicles.
• Dioleoylphosphatidylethanolamine was purchased from Avanti Polar Lipids Inc. (Birmingham, Ala.). Lipofectamine was obtained from Life Technologies. A liposome preparation containing DOTAP was obtained from Boehringer Mannheim. Cholesterol was purchased from Sigma Chemical Company(St. Louis, Mo.). Alamar blue was obtained from Alamar Biosciences (Sacramento, Calif.).
• reaction solution was allowed to cool to room temperature and then transferred to a separatory funnel containing Et 2 O (75 mL).
• Et 2 O 75 mL
• the resultant mixture was washed with saturated aqueous NaHCO 3 .
• the organic layer was separated and subsequently washed with H 2 O and brine, and then dried (Na 2 SO 4 ).
• the drying agent was filtered and the filtrate was concentrated by rotary evaporation to give the crude product as a yellow oil. Purification was accomplished by SiO 2 column chromatography (3% MeOH in CH 2 Cl 2 ) to afford 6.96 g (81%) of compound 3 (in above Specific Scheme) as an oil.
• Theorganic layer was dried (Na 2 SO 4 ), filtered, and the filtrate solvent removed in vacuo.
• the crude product so obtained was purified by SiO 2 column chromatography (1% MeOH in CH 2 Cl 2 ) to yield 4.21 g (57%) of compound 4 as an oil.
• NIH 3T3 cells were grown in Dulbecco's modified Eagles medium (DMEM)+10% fetal calf serum.
• DMEM Dulbecco's modified Eagles medium
• the luciferase reporterplasmid pCMVL was prepared at UC Davis, and consists of the P. pyralis luciferase cDNA subcloned into the plasmid pRc/CMV (Invitrogen).
• Multilamellar and small sonicated vesicles were prepared by addition of thecationic lipid DOHME together with DOPE, both as solutions in chloroform, to a 5 ml sample vial.
• the chloroform was removed via rotary evaporation with the water bath set at a constant temperature of 37° C.
• the resulting thin lipid films were placed under high vacuum overnight to insure that all traces of solvent had been removed.
• the lipid mixture was resuspended using distilled water (2 mmole total lipid/1 ml water) and vortex mixed to give a suspension of MLVs. This cloudy suspension was sonicated for fifteen minutes using a bath sonicator until a clear suspension containing SVs was obtained.
• Luciferase assays were performed with a luciferase assay kit (purchased from Analytical Luminescence Laboratories) using a Monolight 2010 luminometer (Analytical Luminescence Laboratories, San Diego, Calif.) according to themanufacturer's instructions. The media was removed from the transfected cells via aspiration. 0.5 ml of luciferase buffer/well was added, and the cells were placed on ice for 15 min. Luciferase light emissions from 100 ⁇ l of the lysate were measured using the luminometer.
• Alamar blue (Alamar Biosciences, Sacramento, Calif.), was diluted in cell culture media to 10%, added to cells, and incubated for up to two hours. Reduced dye was quantitated using a CytoFluor 2300 fluorescence plate reader with a 530 nm excitation filter and a 590 nm emission filter. Values expressed represent fluorescence less background. The same transfected cells can subsequently be assayed for reporter protein activity after Alamar Blue analysis.

Abstract

Description

______________________________________                                    
DC Cholesterol                                                            
            3β- N-(N',N'-dimethylaminoethane)-carbamoyl!             
            cholesterol                                                   
DCPE        Dicaproylphosphatidylethanolamine                             
DMAP        4-Dimethylaminopyridine                                       
DMEM        Dulbecco's modified Eagles medium                             
DMPE        Dimyristoylphosphatidylethanolamine                           
DOGS        Dioctadecylamidoglycyl spermidine                             
DOHME       N- 1-(2,3-dioleoyloxy)propyl!-N- 1-(2-                        
            hydroxyethyl)!-N,N-dimethylammonium iodide                    
DOPE        Dioleoylphosphatidylethanolamine                              
DOSPA       2,3-Dioleoyloxy-N- 2-                                         
            (sperminecarboxamido)ethyl!-N,N-dimethyl-1-                   
            propanaminium trifluoroacetate                                
DOTAP       N- 1-(2,3-dioleoyloxy)propyl!-N,N,N-                          
            trimethylammonium iodide  DIESTER!                            
            (Boehringer Mannheim GmbH)                                    
DOTMA       N- 1-(2,3-dioleyloxy)propyl!-N,N,N-                           
            trimethylammonium bromide  DIETHER!                           
DSPE        Distearoylphosphatidylethanolamine                            
DU-145      Human prostatic carcinoma cells for a                         
            representative human tumor cell line                          
FBS         Fetal Bovine Serum                                            
Lipofectamine                                                             
            DOSPA + DOPE                                                  
Lipofectin Reagent                                                        
            DOTMA + DOPE (Vical Inc.)                                     
NIH 3T3     Murine fibroblast cells for a representative                  
            human cell line                                               
MLV         Multilamellar vesicles                                        
PE          Phosphatidylethanolamine                                      
PolyGum     N,N,N',N'-tetramethyl-N,N'-bis(2-hydroxyethyl)-               
            2,3-di(oleoyloxy)-1,4-butanediaminium iodide                  
SV          Sonicated or extruded vesicles                                
Transfectam Reagent                                                       
            DOGS                                                          
______________________________________                                    

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Citations (9)

• 1994
  • 1994-09-30 US US08/316,719 patent/US5527928A/en not_active Expired - Lifetime
• 1995
  • 1995-09-21 WO PCT/US1995/012056 patent/WO1996010555A1/en active IP Right Grant
  • 1995-09-21 CA CA2200695A patent/CA2200695C/en not_active Expired - Fee Related
• 1996
  • 1996-05-16 US US08/648,543 patent/US5744625A/en not_active Expired - Lifetime

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