CA2260034A1 - Cationic amphiphile/dna complexes - Google Patents

Cationic amphiphile/dna complexes Download PDF

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Publication number
CA2260034A1
CA2260034A1 CA002260034A CA2260034A CA2260034A1 CA 2260034 A1 CA2260034 A1 CA 2260034A1 CA 002260034 A CA002260034 A CA 002260034A CA 2260034 A CA2260034 A CA 2260034A CA 2260034 A1 CA2260034 A1 CA 2260034A1
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amphiphiles
amphiphile
cationic
cells
group
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CA002260034A
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French (fr)
Inventor
Rebecca G. Bagley
Simon J. Eastman
Seng H. Cheng
Edward R. Lee
David J. Harris
Craig S. Siegel
John Marshall
Ronald K. Scheule
Chau-Dung Chang
Nelson S. Yew
C. Shirley Hubbard
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Genzyme Corp
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    • 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
    • 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
    • A61K47/51—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 non-active ingredient being a modifying agent
    • A61K47/54—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 non-active ingredient being a modifying agent the modifying agent being an organic compound
    • 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
    • A61K47/51—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 non-active ingredient being a modifying agent
    • A61K47/54—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 non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • 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
    • A61K47/51—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 non-active ingredient being a modifying agent
    • A61K47/54—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 non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • 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
    • A61K47/51—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 non-active ingredient being a modifying agent
    • A61K47/54—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 non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/554—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 non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0005—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring the nitrogen atom being directly linked to the cyclopenta(a)hydro phenanthrene skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
    • C07J41/0055—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
    • C07J41/0055—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives
    • C07J41/0061—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives one of the carbon atoms being part of an amide group
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J43/00—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C07J43/003—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton not condensed
    • 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

Abstract

Novel cationic amphiphiles are provided that facilitate transport of biologically active (therapeutic) molecules into cells. The amphiphiles contain lipophilic groups derived from steroids, from mono or dialkylamines, or from alkyl or acyl groups; and cationic groups, protonatable at physiological pH, derived from amines, alkylamines or polyalkylamines. There are provided also therapeutic compositions prepared typically by contacting a dispersion of one or more cationic amphiphiles with the therapeutic molecules. Therapeutic molecules that can be delivered into cells according to the practice of the invention include DNA, RNA, and polypeptides. Representative uses of the therapeutic compositions of the invention include providing gene therapy, and delivery of antisense polynucleotides or biologically active polypeptides to cells. With respect to therapeutic compositions for gene therapy, the DNA is provided typically in the form of a plasmid for complexing with the cationic amphiphile. Novel and highly effective plasmid constructs are also disclosed, including those that are particularly effective at providing gene therapy for clinical conditions complicated by inflammation. Additionally, targeting of organs for gene therapy by intravenous administration of therapeutic compositions is described.

Description

CA 02260034 l999-01-ll Cationic Amphiphile / DNA Complexes This application is a continuation-in-part of U.S. patent appLication 08/545,473 file~ October 19, 1995 and entitled " Organ-Specific Targeting of Cationic Arnph iphile /DNA Complexes for Gene Therapy", itself a continuation-in-part of U.S. patent application 08/ 540,867 filed October 11, 1995 and entitled "Cationic Arnp:~Liphiles Containing Steroid Lipophilic Groups for Intracellular Delivery of Therapeutic Molecules", itself a continuation-in-part of U.S.
application ~o. 08/352,479 entitled "Cationic Amphiphiles for Intracellular Delivery of Therapeutic Molecules", as filed on December 9, 1994. This application also claims the priority of (1) IJnited States provisional patent application identified as Express Mail Label TB79S223107 US, filed September 26,1995 and entitled "Molecular Model of Cationic Lipid/DNA Complexes", and (2) United States provisional patent application identified as Express Mail Label EF109437051 US filed on September 27, 199a and entitled "Intravenous Delivery of Therapeutic Gompositions for Gene Therapy" .
This application is also a continuation-in-part of U.S. patent application identified as Express Mail Label No. EM288778977 US filed June 20, 1996 and entitled " Orgasl-Specific Targeting of Cationic A~phiphile /DNA Complexes for Gene Therapy.

... . . .. ... .. ...

CA 02260034 l999-01-ll W O 98/02190 PCT~US97/12105 The complete text, claims and drawings of all of the above applications are incorporated herein by reference in their ~IL. . ~Ly.
Background of the Invention The present invention relates to novel cationic amphiphilic compounds 5 that facilitate the intracellular delivery of biologically active (therapeutic) molecules. The ~resellt invention relates also to pharmaceutical compositions that comprise such cationic amphiphiles, and that are useful to deliver into thecells of patients therapeutically effective amounts of biologically active molecules. The novel cationic amphiphilic compounds of the invention are 10 particularly useful in relation to gene therapy.
Effective therapeutic use of many types of biologically active molecules has not been achieved simply because methods are not available to cause delivery of therapeutically effective amounts of such substances into the particular cells of a patient for which treatment therewith would provide 15 therapeutic benefit. Efficient delivery of therapeutically sufficient a~nounts of such molecules into cells has often proved difficult, if not irnpossible, since, for example, the cell membrane presents a selectively-permeable barrier.
Additionally, even when biologically active molecules successfully enter targeted cells, they may be degraded directly in the cell cytoplasm or even transported to 20 structures in the the cell, such as lysosomal compa~ .ents, specialized for degradative processes. Thus both the nature of substances that are allowed to enter cells, and the amounts thereof that ultimately arrive at targeted locations within cells, at which they can provide therapeutic benefit, are strictly lirnited.
Although such selectivity is generally necessary in order that proper cell 25 function can be maintained, it comes with the disadvantage that many therapeutically valuable substances (or therapeutical}y effective amounts thereof) are excluded. Additionally, the complex structure, behavior, and envirorunent .. . . , . ... ~ . .. . .

CA 02260034 l999-01-ll presented by an intact tissue that is targeted for intracellular delivery of biologically active molecules ofte;n interfere substantially with such delivery, in comparison with the case presented by populations of cells cultured in zntro Examples of biologically active m~ lPs for which effective la~,elillg to a 5 patients' tissues is often not achieved: (1) numerous ~role, s including immunoglobin proteins, (2) polynucleotides such as genornic DNA, cDNA, or mRNA t3) antisense polynucleotides; and (4) many low molec~ weight compounds, whether synthetic or naturally occurring, such as the peptide hormones a nd a ntibiotics.
One of the fundamental challenges now facing medical practicioners is that although the defective genes that are associated with numerous inherited diseases (or that lepres~.t disease risk factors including for various cancers) have been isolated and c}narac~Pn~e-l, methods to correct the disease states thernselves by providing pa tients with normal copies of such genes (the technique of gene 15 therapy) are substantially }aclcing. Accordingly, the development of irnproved methods of intracellular delivery therefor is of great medical importance.
Examples of diseases that it is hoped can be treated by gene therapy include inherited disorders such as cys~c fibrosis, Gaucher's disease, Fabr,v s disease, and muscular dystrophy. R~esellLative of acquired disorders that can 20 be treated are: (1) for cancers--multiple myeloma, leukernias, melanomas, ovarian carcinon:~a and small cell lung cancer; (2) for cardiovascular conditions--progressive heart failure, restenosis, and hemophilias; and (3) for neurologicalconditions--traumatic brain injury.
Gene therapy requires successful transfection of target cells in a patient.
25 Transfection may generally be defined as the process of introducing an exylessible pol~ucleotide (for example a gene, a cDNA, or an mRNA pattemed thereon) into a cell. Successful expression of the encoding polynucleotide leads ll CA 02260034 l999-Ol-ll to production in the cells of a normal protein and leads to correction of the disease state associated with the abnormal gene. Therapies based on providing such proteins directly to target cells (protein replacement therapy) are often ineffective for the reasons mentioned above.
Cystic fibrosis, a common lethal genetic disorder, is a particular example of a disease that is a target for gene therapy. The disease is caused by the presence of one or more mutations in the gene that encodes a protein known as cystic fibrosis transmembrane conductance regulator ("CFIR"), and which regulates the movement of ions (and therefore fluid) across the cell membrane ofepithelial cells, including lung epithelial cells. Abnormnal ion transport in airway cells leads to abnormal mucous secretion, infla~ ion and infection, tisssue damage, and eventually death.
It is widely hoped that gene therapy will provide a long lasting and predictable form of therapy for certain disease states, and it is likely the only form of therapy suitable for many inhereted diseases. There remains however a critical need to develop compounds that faciliate entry of functional genes intocells, and whose activity in this regard is sufficient to provide for in vivo delivery of genes or other such biologically active therapeutic molecules in concentrations thereof that are sufficient for intracellular therapeutic effect.
Reported Developments In as much as compounds designed to facilitate intracellular delivery of biologically active molecules must interact with both non-polar and polar environments ( in or on, for example, the plasma membrane, tissue fluids, compartments within the cell, and the biologically active molecule itself ), such compounds are designed typically to contain both polar and non-polar domains.
Compounds having both such domains may be termed amphiphiles, and many lipids and synthetic lipids that have been disclosed for use in facilitating such CA 02260034 l999-01-ll intracellular deliver,v (whether for in ~itro or in viw application) meet thi s definition. One p~ rticularly important class of such arnphiphiles is the cationic amphiphiles. In general, cationic arnphiphiles have polar groups that are capable of being positively charged at or around physiological pH, and this property is understood in the art to be important in defining how the amphiphiles interact with the many types of biologically active (therapeutic) molenllpc induding, forexample, negatively dharged polynucleotides such as DNA.
Examples of cationic amphiphilic compounds that have both polar and non-polar domair~s and that are stated to be useful in relation to intracellulardelivery of biologically active molecl-l~s are found, for example, in the fo~lowing references, which contain also useful rlic~lccion of (1) the properties of such compounds that are understood in the art as making them suitable for such applications, and (2) the nature of structures, as understood in the art, that are formed by complexing of such amphiphiles with therapeutic molecules intended for intracellular delivery.
(1) Felgner, et al., Proc. Natl. Acad. Sci. USA, 84, 7413-7417 (1987) disclose use of positively-charp,ed synthetic cationic lipids including N-[1(2,3-dioleyloxy)propylJ-N,N,N-trimethylammoni-lm chloride ("DOTMA"), to form lipid/DNA complexes suitable for transfections. See also Felgner et al., The Toumal of ~3io~ogical Chemistry 269(4), 2550-2561 (1994).
(2) ~ehr et al., ]'roc. Natl. Acad. Sci. .USA, 86, 6982-6986 (1989) disclose numerous amphiphiles including dioctadecylamidologlycylspe~nine ("DOGS").
(3) U.S. Patent 5, 283,185 to Epand et al. des~ibes additional classes and species of amphiphiles including 3~ [N-(Nl,Nl - dimethylaminoethane)-carbamoyl] choles~:erol, termed "DC-chol".
(4) Additional compounds that facilitate transport of biologically active molecules into cel]s are disclosed in U.S. Patent No. 5,264,618 to Felgner et al. See CA 02260034 l999-01-ll WO 98/02190 PCT/US97/1210~i aLso Felgner et al., The Tournal Of Biological Chemistry, 269(4), pp. 255~ 2561 (1994? for disclosure therein of further compounds including "DMRIE" 1,2-dimyrLstyloxy~u~yl-3-dimethyl-hydroxyethyl ammonium bromide, which is ~iccl-cse-l below.
5 (S) Reference to amphiphiles suitable for intr~c~lll-l~r delivery of biologically active mo~ec~ s is alsû found in U.S. Patent No. 5,334,761 to Gebeyehu et al., and in Felgner et al., Methods (Methods in Enzymolûgy), 5, 67- 75 (1993).
Although the compounds mentioned in the above-identified references have been demonstrated to facilitate (although in many such cases only in ~itro ) 10 the entry of biologically active ~c~lecllt~ into cells, it is believed that the uptake efficiencies provided thereby are irl~ nt to support numerous therapeutic applications, particulary gene therapy. Additionally, since the above-identifiedcompounds are understood to have only modest activity, subsL~-Iial quantities thereof must be used leading to concerns about the toxicity of such compounds 1~ or of the metabolites thereof. Accordingly there LS a need to develop a "second generation" of cationic amphiphiles whose activity is so sufficient that successful therapies can be achieved therewith.

CA 02260034 l999-Ol-ll WO 98/02190 PCTtUS97tl2105 Summary of the Invention This invention provides for cationic arnphiphiles that are particularly effective to farilit~te transport of biologicaLLy active molecl-le~ into ceLLs. The 5 cationic amphiphiLes of the invention are divided into four (4) groups, aLthough it will be seen that there are certain structural and functional features that many of the amphiphiles share.
Accordingly, there are provided r~tic)nir amphiphiLes of Group I (see Figure 1, paneLs A, B, and C) capable of farilit~tirlg transport of biologiicaLLy 10 active molecuLes into ceLLs, said amphiphiiles having the structure (1), (R3) (R1) (~ (Y) (~

(R4) 'R2'' (I) wherein:
1o Z is a steroid;
X is a carbon atom or a nitrogen atom;
Y is a short linking group, or Y is absent;
R3 is H, or a saturated or unsaturated aliphatic group;
R1 is--NH--, an a].kylamine, or a polyalkylamine;
20 R4 is H, or a saturated or unsaturated aliphatic group;
R2 is -NH--, an alkylamine, or a polyalkylamine;
and wherein R1 is the same or is different from R2, except that both R1 and R2 cannot be --NH--.

CA 02260034 l999-01-ll W O 98/02190 PCT~US97112105 In one ~le~l~ed embodiment, the steroid component "Z" is s~l~cte~l from the group cc,l~isL-g of ~sterols, wherein said sterol nlolecl]lP is linked by the O- group thereof, or by N- in replacement thereof, to Y (or directly to X, if Y is absent ). According to this aspect of the invention, particularly efre~Lve 5 amphiphiles include, for example, spermidine cholesterol carbarnate ( N4 -spermidine cholesteryl carbamate, amphiphile No. 53), and spermine cholesterol carbamate ( Ngspermine cholesteryl carbamate, amphiphile No. 67), and amphiphiles patterned thereon.
In a further ~,efelled embodiment, the steroid group is linked to Y (or 10 directly to X, if Y is absent) from ring position 17 of the steroid nucleus (see Figures 1 and 22), or from the arm that normally extends from position 17 in many steroids( see the structure of cholesterol in Figure 1), or from any shortened form of said arm.
In other preferred embodiments, within linking group Y are contained no 15 more than about three or four atoms that themselves form a bridge of covalentbonds between X and Z. In a specific preferred embodiment of the invention, Y
is a linking group wherein no more than one atom of said group forms a bond with both X and Z, or Y is absent.

20 Representative amphiphiles provided according to Group I include:

0~ ~ ~ ~0~

No. ~3 NH2 No. 67 NH2 N4-spermidine cholesteryl N~-spermine cholesteryl carbamate carbamate CA 02260034 l999-01-ll H N~'N ~ NO ~~
N~H H2N~N No. 78 No. 75 l NH2 ~ NH2 N~ Bis (~ar ~ir~opropyl~ N4 - N(N4-~aminopluy~y~ e) 5p.om~irlin~ chole.;teryl c~l,~.ate ~hr~ yl carbamate Additionally there are provided cationic amphiphiles of Group II (see Figure 5) capable of farilitating transport of biologically active ~olr-clllr-S into 5 cells said amphiphiles having the structure (lI), (R3) (R1)\
\

(X) (Y) (~) //

(R4) (R2)/ (II) 10 wherein:
Z is a steroid;
X is a carbon atom or a nitrogen atom;
Y is a link~g group or Y is absent;
R3 is an amino acid, a derivatized am no acid, H or alkyl;
15 Rl is--NH--, an alkylarnine, or a polyalkylamine;
R4 is an amino acid, a derivatized amino acid, H or al~yl;
R2 is --NH--, an alkylamine, or a polyalkylamine;

CA 02260034 l999-Ol-ll and wherein Rl is the same or is different from R2, except that both R1 and R2 cannot be--NH--.

Representative amphiphiles provided according to Group ~ include:

H2N~ NH

H2N~ ~
N ~~--NH
HN ~ No. 95 N H
H2N ,N ~ H----N~2 HN~ NH2 N~ is(arginine ~I,o ~
No. 91 N4-spem~idine cholestery carbamate Additionally there are provided cationic amphiphiles of Group m (see 10 Figure 6) capable of facilitating transport of biologically active molecules into cells said amphiphiles having the structure (m), (R3~--(R1) (X) (~') (Z) (R ) (R2~ (~) 15 wherein:

CA 02260034 l999-Ol-ll Z is an aLlcylamine or a dialkylamine, linked by the N-atom thereof, to Y ( or directly to X, if Y is absent ), wherein if Z is a diaLl~ylamine, the aL~cyl groups thereof can be the same or different;
X is a carbon atom or a nitrogen atom;
5 Y is a short linking group, or Y is absent;
R3 is H, or a saturated or unsaturated aliphatic group;
R1 is--NH--, an aL~cylamine, or a polyallcylamine;
1i~4 is H, or a saturated or unsaturated aliphatic group;
R2 is--NH--, an alkylamine, or a polyaLIcylarnine;
10 and wherein R1 is the same or is different from R2, except that both Rl and R2 cannot be--NH-.
With respect to amphiphiles provided according to Structure (m), it is again ~ure~ d that within linking group Y there are contained no more than about three or fDur atorns that themselves form a bridge of covalent bonds 15 between X and Z. In a specific preferred embodiment of the invention, Y is a linking group, such as > C=O, wherein no more than one atom of said group forms a bond with both X and Z, or Y is absent.

Representative arnphiphiles provided according to Group m include: .

O [CH2],7CH3 H2N--~N~ [CH2]17CH3 NH2 ~--N~
2 HCl NH2 [CH2]17CH3 3HCl [CH2l17CH3 No. 43 Nl,Nl-d~ y~
N,N-dioctadecyll)~sineamide diHCl salt trialrunohexane tri HCI salt CA 02260034 l999-Ol-ll Additionally there are provided cationic amphiphiles of Group I~,7 (see Figure 7) capable of facilitating transport of biologically active molecules into cells said amphiphiles having the stmcture (IV), (A)~ ~(D) (E)~

(B) (R~) (R
(R5) (R6) (IV~

wherein:
A and B are independently 0, N or S;
R5 and R6 are independently aL~yl or acyl groups and may be saturated or 10 contain sites of unsaturation;
C is selected from the group consisting of--CH2--, >C=O, and >C=S;
E is a carbon atom or a nitrogen atom;
D is a lin~ing group such as -NH(C=O)- or -O(C=O)-, or D is absent;
R3 is H, or a saturated or unsaturated aliphatic group;
1~ Rl is--NH-, an alkylamine, or a polyalkylamine;
R4 is H, or a saturated or unsaturated aliphatic group;
R2 is--NH--, an alkylamine, or a polyalkylamine;
and wherein R1 is the same or is different from R2, except that both R1 and R2 cannot be -NH-.

CA 02260034 lsss-ol-ll wO 98/02190 PCT/USg7/12105 Representative amphiphiles of Group IV include:

~ ~ N ~

No. 102 N H
No. 89 NH ,,r ~ NH2 NH2 N4-spem~ine-2,~dilauryl-1 -(N~-spermine)-2,~dilaL~ry1-o~.u~ylamine glycerol carbanlate o The invention provides also for pharmaceutical compositions that 5 comprise one or more cationic amphiphiles, and one or more biologically activemolecules, wherein said compositions facilitate intracellular delivery in the tissues of patients of therapeutical1y effective amounts of the biologically active molecules. The pharma-~e~ r~l compositions of the invention may be formulated to contain one or more additional physiologically acceptable 10 substances that stabili~e the compositions for storage and/or contribute to th successful intracellular delivery of the biologically active molecules.
In a further aspect, the invention provides a method for facilitating the transfer of biologically active molecules into cells comprising the steps of:
preparing a dispersion of a cationic amphiphile of the invention; contacting said 15 dispersion with a biologically active molecule to form a complex between saidamphiphile and said molecule, and contacting cells with said complex thereby facilitating transfer of said biologically-active molecule into the cells.
For pharmaceutical use, the cationic amphiphile(s) of the invention may be formulated with one or more additional cationic amphiphiles including those 20 known in the art, or with neutral co-lipids such as dioleoylphosphatidyl-ethanolamine, (" DOPE"), to facilitate delivery to cells of the biologically active molecules. Additionally, compositions that comprise one or more cationic CA 02260034 l999-01-ll WO 98/021gO PCTIUS97/12105 amphiphiles of the invention can be used to introduce biologically active molecules into plant cells, such as plant cells in tissue culture.
Additionally, the present application provides for novel plasmids suitable for complexing with the amphiphiles of the invention in order to treat patients by gene therapy, so that a high level of e*,l~ssion of the ~ ro~ te therapeutic transgene can be achieved. Representative examples thereof include the plasrnid pCMV~ and pCFI. pCF1 plasrnid contains the enhancer/promoter region from the immediate early gene of cytomegalovirus. The plamid also contains a hybrid intron located between the promoter and the transgene cDNA. The polyadenylation signal of the bovine growth hormone gene was selected for placement downstream from the transgene. These and other features contribute substantially to the improved transgene e~ression possible with this plasrnid.
Further enhancements in plasrnid performance are made possible by the provision of replicating episomal pl~micl~ Additional therapeutic 1~ enhancements are made possible by providing plasrnids in which expression of the therapeutic transgene is placed under the control of a transcriptional promoter that is sensitive to the concentration of inflammation-related substances in the target tissue. Such plasmids are of particular use for the treatment of clinical cases in which inflammation is a major complication.
In a still further embodiment of the invention, particular organs or tissues may be targeted for gene therapy, by intravenous administration of amphiphile/transgene complexes, by adjusting the ratio of amphiphile to DNA
in such complexes, and by adjusting the apparent charge or zeta potential thereof.
Further additional and representative aspects of the invention are described according to the Detailed Description of the Invention which follows directly.

Brief Descriphon of the Drawir~gs FIGU~E1 depicts representative Group I cationic amphophiles.
FIGURE 2 depicts representative steroid lipophilic groups.
FTGURE 3 depicts representative steroid lipophilic groups.
5 FIGURE 4 depicts a transacylation reaction.
.. FIGURE 5 depicts represenative Group II cationic amphiphiles.
FIGURE 6 depicts represenative Group m cationic amphiphiles.
FIGURE 7 depicts representative Group IV cationic amphiphiles.
FIGURE 8 provides a route of synthesis for sperrr idine cholesterol carbamate.
10 FIGURE 9 provides a route of synthesis for sperrnine choiesterol carbamate FIGURE 10 provides a comparison of ;n vivo transfection efficiency for certain cationic amphiphiles under particular conditions.
FIGURE 11 is a depiction of in vivo transfection effeciency as a function of DNA
concentration for a particular cationic amphiphi~e.
15 FIGUI~E 12 is a depiction of in vivo transfection effeciency as a function of concentration of a par~i~lar cationic amphiphile.
FIGURE 13 provides relative transfection efficiencies for Group I amphiphiles.
FIGURE 14 provides relative transfection efficiencies for Group Il amphiphiles.
FIGURE 1~ provides relative transfection efficiencies for Group IV arnphiphiles.
20 FIGI JRE 16 provides a map of pCMVHI-CAT plasmid.
FIGU~E 17 shows the hybrid intron of pCMVHI-CAT.
FIGURE 18 (panel A) provides a map of pCF1 /CAT plasmid.
FIGURE 18 (panel ~3) provides a map of pCF2/CAT plasmid.
FIGURE l9 (panel ~'~) shows a plot of corrected chloride ion transport in pCMV-CFTR
25 transfected nasal polyp epithelial cells from a cystic fibrosis patient.
FIGURE 19 (panel I3) shows a plot of chloride ion transport using pCMV-~-galactosidase control.
FIGURE 20 provides a map of pMyc4-C~T~ plasmid.
FIGURE 2 l demonstralcs intravenous targeting of ~hc hcart ~nd lung.

. _.. ...

CA 02260034 l999-Ol-ll FIGURE 22 demonstrates expression of SEAP following intravenous administration in BALB/c rnice.

.. . .

Detailed Desaiption of the Invention Inforrnation Conceming the Structure of Cationic Amphiphiles of the Invention This inve'ntiion provides for cationic amphiphile compounds, and compositions containing them, that are useful to facilitate transport of 5 biologically active molec~ c into cells. The amphiphiles are particularly useful in facilitating the transport of biologically active polynucleotides into cells, and in particular to the cells of patients for the purpose of gene therapy.
Cationic amphiphiles according to the pràctice of the invention possess several novel features. These features may be seen in comparison with, for 10 example, cationic arnphiphile structures such as those rlicrlose~l in U.S. Patent No. 5, 2&3,185 to Epand et al., a le~v,es~-tative stmcture of which is is 3B [N-(N l,N 1 - dirnethylaminoethane)-carbamoyl] cholesterol, commonly known as "DC-chol", and to those disclosed by Behr et al. Proc. Natl. Acad. Sci., USA, 86, 6982- 6986 (1989), a representative structure of which is dioctadecylamidolo-15 glycylsperrnine ("DOGS").
Cationic amphiphiles of the present invention contain distinctivestructural featuIes: (1) the presence of a lipophilic group which is connected directly, or through a linking group, to two cationic groups (see below) that themselves comprise amino, alkylamine or polyalkylamine groups, there 20 resulting an overall and novel "T-shaped" structure; and (2) in many cases, and in comparison with numerous art-recognized amphiphiles, the use of a relatively short linking group to bring into close proximity the lipophilic and cationic regions of the arnphiphile. Without being limited as to theory, it is believed that these features contribute substantially to the transfection-enhancing capability of 25 these compouncls. As an example of this, Figure 10 below demonstrates the very substantial in vi vo transfection-enhancing capability of spermidine cholesterol .

carbamate (a novel amphiphile of the invention) in comparision to DC- chol and DMRE--two well recognized transfectants.
In connection with the practice of the present invention, it is noted that "cationic" means that the R groups, as defined herein, tend to have one or more 5 positive charges in a solution that is at or near physiological pH. Such cationic character may enhance interaction of the amphiphile with therapeutic rnolPc (such as nucleic acids) or with cell structures (such as plasma membrane glyco~lotei~ls) thereby contributing to successful entry of the therapeutic mole~ into cells, or processing within subcompartments (such as the nucleus) 10 thereof. In this regard, the reader is referred to the numerous theories in the art concerning transfection-enhancing function of cationic amphiphiles, none of which is to be taken as limiting on the practice of the present invention.
Biological molecules for which transport into cells can be facilitated according to the practice of the invention include, for example, genomic DNA, 15 cDNA, mRNA, antisense RNA or DNA, polypeptides and small molecular weight drugs or hormones. Representative examples thereof are mentioned below in connection with the description of therapeutic (pharmaceutical) compositions of the invention.
In an imporant embodiment of the invention the biologically active 20 molecule is an encoding polynucleotide that is expressed when placed in the cells of a patient leading to the correction of a metabolic defect. In a particularly important example, the polynucleotide encodes for a polypeptide having an amino acid sequence sufficiently duplicative of that of human cystic fibrosis transmembrane regulator ("CFI R") to allow possession of the biological 2~ property of epithelial celi anion channel regulation.
As aforementioned, characteristic and novel features of the amphiphiles of the invention include first, that the linking group that connects the two cationic W O 98/02190 PCTrUS97/12105 amine groups to the lipophilic group is very short, or absent entirely, and second, that the resultant linking of the the two cationic R groups to the lipophilic group forms a T-shaped structure when viewed from the position of atom "X" (a carbon or nitrogen atom) as depicted, for example, in Structures (I), (II), (m) and 5 (IV, see atom "E").
As examples of the cationic amphiphiles of the invention, both spermidine cholesterol carbamate ( N4 -sperrnidine cholesteryl carbamate) and spermine cholesterol carbamate ( N4 -spermine cholester,vl carbamate) have been determinedto be superior transfectants in vivo in comparison with non "T-10 shaped" amphiphiles having otherwise equivalent amounts of cationicalkylarnine stru-ture. Superior performance (see also Exarnple 3) has been determined for:

~Y
o H2N ~N
~--NH2 ( spermidine cholesterol carbamate ) 1~ in comparison with, for example, H2N~ 0 --~4 H (N1-spermidine cholesteryl carbamate).

CA 02260034 l999-Ol-ll Additionally, superior performance has been determined for H2N~'N~

H
NH2 ( spermine cholesterol carbamate ) 5 in comparison with, for example, 0~
H2N ~ N----N----N O
H H H (N l-~errnospermine cholesteryl carbamate), and \~
H O ~ 4 H2N N ~. N----N O
H (Nl-Spermine cholesteryl carbamate).

Applicants have also noted that numerous of the cationic amphiphiles of 15 the invention have structural features in common with naturally occurring polyamines such as spermine and spermidine (including N-atom spacing). In this regard, the structures of amphiphiles 53, 67, 7~, 90, and 91 are representative.
As can be seen by examination of the data in Figures 13, 14 and 15, the placement of the nitrogen atoms in the polar head groups of the amphiphiles such that they20 are separated by one or more combinations of 3 and 4 carbon atoms leads to high in vivo transfection efficiency for plasmid transgenes complexed therewith.
Applicants have also noted that these in-common structural features may have a ~ I r T

CA 02260034 lsss-ol-ll WO 98/02190 PCT/USg7/12105 useful effect upon the bind~ng of the amphiphiles to DNA, and on interaction with cell surface polyamine receptors. Interaction with cell polyarnine receptors may be particularly important with respect to the treatment of cancer cells by gene therapy, since the DNA replication requirements of such cells may lead to 5 high level ex~ression of such receptors.
- Group I Amphiphiles In connection with the design of the Group I amphiphiles of the invention, the following considerations are of note. Many of these design features are thendiscussed in co ;mection with the other amphiphiles of the invention, those 10 ~ 5ifie~1 under Groups II, II and IV.
Accordingly, there are provided cationic amphiphiles of Group I (see Fi~ure I, panels A, B, and C) capable of facilitating transport of biologically active molecules into cells, said amphiphiles having the structure (I), (R3) (Rl) (~ (Y) (~
/

(R4) (R2)/ (I) wherein:
Z is a steroid;
X is a carbon atom or a nitrogen atom;
20 Y is a short linking group, or Y is absent;
R3 is H, or a saturated or unsaturated aliphatic group;
R1 is --NH--, an alkylamine, or a polyalkylamine;
R4 is H, or a sa~urated or unsaturated aliphatic group;

CA 02260034 l999-01-ll WO 98102190 PCT/US97/1210~;

R2 is--NH--, an alkylamine, or a polyalkylamine;
and wherein R1 is the same or is different from R2, except that both R1 and R2 cannot be--NH--.
The Linking Group Preferably the linking group that connects the lipophilic group to the two cationic R groups is relatively short. It is ~lere~ d that within linking group Y
are contained no more than about three or four atorns that themselves form a bridge of covalent bonds between X and Z. Examples of Y groups include---(CH2)2-; -(CH2)3-; -(CH2)-(C=O)-; -(CH2)n-NH-(C=O)- where n is preferably 1~1 4 or less. Additional lir king groups useful in the practice of the invention are those pattemed on small amino acids such as glycinyl, alanyl, beta-alanyl, serinyl, and the like.
With respect to the above le~es~ atiorls, the left hand side thereof-as depicted- is intended to bond to atom "X", and the right hand side thereof to group "Z"( see structure I).
ln certain preferred embodiments of the invention, Y is a linking group wherein no more than one atom of this group forms a bond with both "X"and "Z". Examples of preferred linking groups include --C~2-, >C=S, and >C=O.
Altematively, the linking group "Y"may be absent entirely.
'70 As aforementioned (see Structure I, directly above), "X" forms a connecting point in the amphiphiles to which is also attached the two cationic Rgroups. As can be seen therein (see also Figure 1), the placement of the nitrogen atom that represents "X" clearly causes the molecule to assume a T-shape.
Steroid Lipophilic Groups Cationic amphiphiles according to the practice of the invention may include a variety of structures as lipophilic group. Steroids represent a preferred group of such structures.

With respect to the design and orientation of steroids as lipophilic groups according to the practice of the invention, the following considerations are of note. Steroids are widely distributed in the animal, microbial and plant kingdoms. They may be defined as solid alcohols that typically contain, as their5 basic skeleton, 17 carbon atoms arranged in the form of a perhydrocyclopenteno-. phenanthrene ring system. Accordingly, such compounds include bile acids, cholesterol and related substances, vitamin D, certain insect molting hormones, certain sex horrnones, corticoid horrnones, certain antibiotics, and derivatives of all of the above wherein additional rings are added or are deleted from the basic structure. [see Natural Products Chernist~y, K. Nakanashi et al. eds., AcadernicPress, Inc., New York (1974), volume 1, at Chapter 6 for a further discussion ofthe broad classes of molecules that are understood in the art to be steroids~.
Additionally, for the purposes of the invention, the term steroid is used broadly to include related molecules derived from multiple isoprenoid units, such as vitamin E. Steroids representative of those useful in the practice of the invention are shown in Fic~ures 1, 7, 3 and 5.
As elaborated below, certain preferred amphiphiles of the invention include a steroicl component "Z" that is selected from the group consisting of 3-sterols, wherein said sterol molecule is linked by the 3-O- group thereof, or byN- in replacement thereof, to Y (see Figure 1). Such structures include, for example, sperrrlidine cholesterol carbamate, spermine cholesterol carbamate, spermidine 7-dehydrocholesteryl carbamate, lysine 3-N-dihydrocholesteryl carbamate, spermidine cholestamine urea, and N-3-aminopropyl-N-4-aminobutylcholestamine.
In a further preferred embodiment, the steroid group is linked to Y (or directly to X if 'Y is absent) from ring position 17 of the steroid nucleus (see Figures 1 and 3), or from the arm that norma~ly extends from position 17 in many steroids (see Figures 1 and 3), or from any shortened form of said arm.
In connection with the selection of steroids for inclusion in the amphiphiles of the invention, it is yreft:~led that the molecules have structures 5 which can be metabolized by the body and are nontoxic at the doses thereof that are used. Preferred are steroids such as cholesterol and ergosterol that are substantially non toxic and which possess biologically normal stereospecificity in order to facilitate their safe metabolism in patients. Additional steroids useful in the practice of the invention include, for example, ergosterol B1, ergosterol B2, 10 ergosterol B3, androsterone, cholic acid, desoxycholic acid, chenodesoxycholic acid, lithocholic acid and, for example, various derivatives thereof as are shown in the panels of Figures 2 and 3.
With respect to the orientation of the steroid lipophilic group, that is, how the group is attached(with or without a linker) to the cationic (al}~yl) amine 15 groups of an amphiphile, the following further information is of note. Any ring position or substituent on the steroid can in general be used as point of attachment. It is preferred, however, to use a point of attachment that (1) rrLilnimi7es the complexity of chemical syntheses, and (2) is positioned near either "end" of the steroid molecule, for example, a position near ring position20 3, or near ring position 17( or the arm that typically extends therefrom). Such positions provide an orientation of the steroid with respect to the rest of the amphiphile structure that faciliates bilayer formation, and/or micelle formation, and/or stabilizes interaction with the biolog~ically active molecules to be carried into the target cells. Representative structures showing attachment of the 25 cationic (alkyl) amine groups to the steroid lipophilic group through the armextending from ring position 17 therof are shown in Figure 3 (panels A, B). Withrespect to this type of structure, it is further preferred that any polar groups on , . . . . ..

CA 02260034 lsss-ol-ll the steroid, such as may be attached to ring position 3, be either removed or capped (for exarnple, hydroxy as methoxy) to avoid potentially destabilizing bilayer or micelle structures.
The representation in Figure 3 of cationic amphiphiles in which the steroid 5 lipophilic group thereof is linked to the cationic aL~cylamine groups through steroid ring position 17 is but an example of the invention. Sirnilarly, the representation iIl Figures 1 to 4 of cationic amphiphiles in which the steroid lipophilic group thereof is linked to the cationic alkylamine groups through steroid ring position 3 is an example of the invention. As aforementioned, use of 10 any steroid ring position (or moiety or branch extending theLef~oL~-) as point of attachment is within the practice of the invention.
Preferred steroids for use as group "Z" according to the practice of the invention include:
3- sterols (derived from cholesterol~
~y o 15 --0~

3-N sten~l groups (pattemed on cholesterol) ~~y -- N J~----~J

ergostero] and derivatives ,~

HO~
Representative species of steroid that are patterened on ergosterol and that may be used to define the structure of cationic amphiphiles of the invention include: ergosterol (double bonds as shown); ergosterol B1 (A 8, 9; ~14,15; A '~2, 23); ergosterol B1 (~ 6, 7; ~ 8,14; ~ 22, 23); ergosterol B1 (~ 7, 8; ~14, 15; ~ 22, 23);
and lumisterol ( the 9b-H isomer of ergosterol).

cholic acid and derivatives ~,COOH
~, ,~

HOJ~ r1 - Representative species of steroid that are patterened on choiic acid and that may be used to define the structure of cationic amphiphiles of the invention include: cholic acid wherein r1 and r2 = OH; desoxycholic acid wherein r1 = H
1~ and r2 = OH; chenodesoxycholic acid wherein r1 = OH and r2 = H; and lithocholic acid wherein r1 and r2 = H.

~ . . ..

CA 02260034 l999-01-ll W O 98/02190 PCT~US97/12105 androsterone and derivatives thereof ~3~
HO~

Selection of Groups _~, R2, R3, and R4 For_3and R4:
According to the practice of the invention R3 and R4 are, independently, H, or saturated or unsaturated aliphatic groups. The aliphatic groups can be branched or unbranched. Representative groups include alkyl, alkenyl, and cycloall-~yl.
For Rl and R2:
R1 and R2 represent structures recognized in the art as being amine;
alkylamines (including primary, secondary, and tertiary amines), or extended 1~ versions thereof-herein termed "polyalkylamines". It is understood that both alkylamine and polyalkylamine groups as defined herein may include one or more carbon-carbon double bonds and the use of such alkenylamines is therefore within the praciice of the invention.
Representative alkylamines include: (a) -- NH-~CH2)z--~vhere z is other than 0; (b)--[[C'H3(CH2)y]N] -(CH2)z--where z is other than 0; and (c~--[[CH3(CH2)X][C'H3(cH2)y]lN -(CH2)z - where z is other than 0.
With respect to the circumstance where one or both Of R1 and R2 are tertiary amines, such as is represented in Structure (c) above, it is understood that a hydrogen atom corresponding to either R3 or R4, as appropriate, may or may ll CA 02260034 l999-01-ll not be present since such hydrogen atorns correspond to the N:H(+) structure whose level of protonation will vary according to pH.
The terrn "polyalkylamine" as referred to herein defines a polymeric structure in which at least two alkylamines are joined. The alkylamine units that 5 are so joined may be primary or secondary, and the polyalkylamines that resultmay contain primary, secondary, or tertiary N-atoms. The alkylamine (sub)units may be saturated or unsaturated, and therefore the term "alkylamine"
encompasses alkenylamines in the description of the invention.
Representative resultant polyalkylarnines include: (d)--INH-(cH2)(z)]q 10 --, where z is other than 0, and q is 2 or higher; (e)--[NH-(CH2)(y)]p--[NH-(CH2) (z) ]q--, where y and z are each other than 0, and p and q are each other than 0; (fl - LNH-(CH2)(x)]n--[NH-(CH2)(y)]p--[NH-(CH2)(z)]q--, where x, y, and z are each other than 0, and n, p and q are each other than 0; (g)--[NH-(cH2)(w)lm - [NH-(CH2)(x)]n--[NH-(CH2)(y)]p--[NH-(CH2)(z)]q--, where 15 w, x, y, and z are each other than 0, and m, n, p, and q are each other than 0; (h) - [NH-(CH2)(~ m--[NH-(cH2)(x)]n -[[cH3(cH2)y]N] -(CH2)z--, where x, n and z are each other than 0; (i) --[NH-(cH2)(w)~p - [[CH3(CH2)x]N]-(CH2)y -[~H-(CH2)(z)]q --, ~~here w, p, y, z, and p are each other than 0; and (j) - [NH-(CH2) (v) ] 1--[NH-(CH2) (W) ]m--[NH-(CH2) (X)] n -[NH-(CH2) (y) ]p -20 [NH-(CH2) (z) ]q --, where v, w, x, y, and z are each other than 0, and l, m, n, p, and q are each other than 0.
As mentioned above R1 and R~, independently, can be -- NH--, an alkylamine, or a polyalkylamine, and can be the same or different from each other, except that both R1 and R2 cannot be --NH-- in order to (1) preserve the 25 "T- shape" of the resultant compound, and (2) to provide for the stability thereof. It is preferred that - in combination- the combined backbone length of R3R1 (or of R4R2) be less than about 40 atoms of nitrogen and carbon, more 2~

CA 02260034 l999-01-ll WO 98/02190 PCTtUS97/12105 preferrablv less than about 30 atoms of nitrogen and carbon.
In the case where the R1 group adjacent to R3 (or R2 adjacent to R4) includes a terminal nitrogen atom that defines a tertiary center, then a quatemary amine is formed (at that nitrogen atom of R1) if R3 is an aliphatic group, and a5 tertiary arnine remains (at that nitrogen atom of R1) if R3 is H. Accordingly, with respect to such resultant R3R1 or R4R2 structures, representative respective formulas are:
(k~ H-(CH2)(w)--~[cH3(cH2)x][cH3(cH2)y]N] -(CH2)z--, where w and z are each other than zero; and (l) H--[[cH3(cH2)x][cH3(cH2)y]N]-(cH2)z--, 10 where z is other than zero.
In connection with inte~le~g the structural diagrams described herein, it is intended tl~.at the attachrnent of R3R1--(or R4R~ ) structures to atom "X"is through the right hand side (as depicted) of the R3R1--, that is, through a CH2--moiety. The coefficents (i.e. v, w, x, y, and z and l, m, n, p, and q) as 1~ depicted herein represent whole numbers. For the purposes of the invention, "whole number" means O and the natural nLunbers 1,2,3,4,5,6.. and up, unless specifically restricted.
With respect to the amphiphiles of the invention including those represented by forrnulas (a) to (l), it is noted that there are certain preferences 2~ concerning the design of such groups depending on whether atorn 'X" as it is shown according to structure (I) above, is a nitrogen atom or a carbon atom. If "X" is nitrogen then amphiphiles containing R3-R1 ( or R4-R2 ) groups that end in an N atom [ i.e formula (e) where z equals O and q=1; formula (h) where z equals 0~ are not preferred, since the resultant N-N linkage involving pcsition X
2~ results in an armphiphile that may be unstable and/or difficult to prepare. An additional group of structures that are difficult to prepare and/or are unstable is represented, for example, by the R sequence (whether in R1, or bridging R1 and CA 02260034 l999-Ol-ll R3)--NH- CH2-NH-CH2--. Accordingly, use of such structures [ i.e. formula (a) where Z equals 1, formula (e) where one or both of y and z equals 1]in the practice of the invention is not preferred.
With respect to the design of structures (such as those depicted above) for 5 inclusion in cationic amphiphiles, the following further considerations are ofnote. Any combination of altemating amine and aLl~yl moieties ~eates an R
structure within the scope of the invention. A polyalkylamine may be represented, for example, by the formulas above, although many more structures (such structures being within the scope of the invention) can be depicted by 10 extending the number of, or types or combinations of, alkylamine subunits within the amphiphile structure. That further such variations can be made is apparent to those skilled in the art.
It is noted that a polyalkylamine group (or resultant R3R1 group) that is very long may interfere, for example, with the solubility of the resultant 15 amphiphile, or interfere with its ability to stably interact with the biologically active molecule selected for intracellular delivery In this regard, polyalkylamines (or resultant R3R1 groups) having a backbone length of about 40 nitrogen and carbon atoms, or more, may not be suitable for inclusion in amphiphiles. However, for each such proposed structure, its properties may be 20 determined by experimentation, and its use is nonetheless within the practice of the int~ention.

Accordingly, specific alkylamine and polyalkylamine structures result as follows:
Table 1 and/or R2 (1) ~
(2) -NH-(CH2) (2)-(3) -NH-(CH2) (3) ~
(4) -NH-(CH2) (4)~
(5) -NH-(cH2) (6) ~
(6) -NH-(CH2)(3)- NH-(cH2)(4) (7) -NH-(CH2)(2)- NH-(cH2)(2)-(8) -NH-(CH2) (4)- NH-(CH2) (3) -(9) -NH-(CH2) (y)- NH-(CH2) (z)-(10) -NH-(CH2) (x)-NH-(cH2) (y) -NH-(CH2) (Z) -(11) -NH-(cH2) (w) -NH-(cH2) (x)-NH-(cH2) (y) -NH-(CH2) (z) -(12) -NH-(CH2) (v) - NH-(cH2) (w) -NH-(cH2) (x)-NH-(cH2) (y)-NH-(cH2) (z) -(13) -[NH-(CH2)(w)]m - [NH-(cH2)(x)]ll--[lcH3(cH2)y]N] -(CH2)z-(14) -[NH-(CH2)(x)]n -[[cH3(cH2)y]N] -(CH2)z -(15) -[NH-(cH2) (w) ]m--[NH-(cH2) (x)] n--[[cH3(cH2) y]N] -(CH2) z -(16) - [[CH3(CH2)x]~CH3(CH2)ylN]-(CH2)z -(17) -NH-(CH2)(z)- NH-(18) -NH-(CH2) (y) NH-(CH2) (z)-NH -(19) -NH-(CH2)(y) CH=CH-(CH2)z--(20) --[NH-(cH2) ~ w) ]p--~[cH3(cH2) x]N] -(cH2) y - [NH-(CH2) (z) ]q -CA 02260034 l999-Ol-ll For R3 and/or R4 (1) H--(2) CH3--(3) CH3-(CH2)2-(4) CH3-(CH2)4-(5) CH3-(CH2)z-(6) CH3-[cH3-(cH2)z]cH--(7) CH3-~CH3-(CH2)2]CH--(8) CH3-[[CH3-(CH2)y][CH3-(CH2)z3]C--(9) CH3-(CH2)z-CH=CH-CH2-(10) CH3-[CH3-(CH2)y-CH=CH-(CH2)z]CH--(11) CH3-[[CH3-(CH2)~CH=CH-(CH2)Xl[CH3-(cH2)y-cH=cH-(cH2)zllcH--(12) CH3-[CH3-(CH2)y]CH-(CH2)z-Group II Amphiphiles Additionally there are provided cationic amphiphiles of Group II (see Figure 5) capable of facilitating transport of biologically active molecules into cells said amphiphiles having the structure (II), (R3) (R1) \
(~ (Y) (~

(R4)--(R~) (II) wherein:
Z is a steroid;
10 X is a carbon atom or a nitrogen atom;
Y is a lin~ing group or Y is absent;
R3 is an amino a~id, a derivatized am~no acid, H or alkyl;
R1 is --NH--, an alkylamine, or a polyalkylamine;
R4 is an amino a id, a derivatized amino acid, H or alkyl;
15 R2 is -NH--, an. alkylamine, or a polyalkylamine;
and wherein R1 :is the same or is different from R2, except that both R1 and R2 cannot be --NH-.
Representative amphiphiles provided according to Group II include amphiphiles 87, 91, 93, 9S, 97, 99,100, and 103. With respect to the structural 20 features of these amphiphiles, and the other amphiphiles of Group II, the following should be considered.

CA 02260034 l999-Ol-ll WO 98/02190 PCT/US97tl210S

The steroid group may be selected according to the criteria defined above for the Group I amphiphiles. Accordingly, ~lefeLL~d amphiphiles include those selected from 3- sterols, wherein the sterol molecule is linked by the 3-O- group thereof, or by N in replacement thereof, to '~'.
The linlcing group Y of the Group II amphiphiles consists of an N-acylamino acid (or a derivative thereof), or consists of a group (such as > C=O or > C=S) wherein no more than one atom of said group forms a bond with both "X" and "Z". Optionally, group Y may be absent. Representative N-acylarnino groups include an N-Acyl serine ( No. 87), an N-Acyl glycine (No. 91), and an N-Acyl aspartic acid ( No. 103). With respect to the use of N-Acyl aspartic acid in amphiphile No. 103, it is noted that, as provided, the gamma carboxyl thereof isfurther derivatized to an additional alkylarnine moiety.
The crtiteria for selection of Rl and R2 are as set forth for the Group I
amphiphiles. R3 and R4 represent H or alkyl, or may be natural or artificial amino acids including derivatives of either. Representative examples of R3 or R4amino acid groups include those derived from tryptophan ( No. 97) and from arginine ( No. 9~).

CA 02260034 lsss-ol-ll wo 98/02190 PCT/US97/12105 Group III Amphiphiles Additionally there are provided cationic amphiphiles of Group m (see Figure 6) capable of facilitating transport of bioiogically active molecules into cells said amphiphiles having the structure (m), (R3 \
//

(R4)--(R2) (m) wherein:
Z is an alkylamine or a dialkylamine, linked by the N-atom thereof, to Y, or directly to X if Y is absent, wherein if Z is a dialkylamine, the aLIcyl groups 10 thereof can be the same or different;
~ is a carbon atom or a nitrogen atom;
Y is a short linking group, or Y is absent;
R3 is H, or a sahlrated or unsaturated aliphatic group;
R1 is--NH--, an alkylamine, or a polyalkylamine;
1~ R~ is H, or a saturated or unsaturated aliphatic group;
R~ is--NH--, an alkylamine, or a polyalkylamine;
and wherein R1 is the same or is different from R2, except that both Rl and R2 cannot be --NEI--.
Representative cationic amphiphiles according to the practice of the 20 invention that contain an alkyl amine or dialkylamine as lipophilic group include, for example, N,N-dioctadecyllysineamide; N1, Nl-dioctadecyl-1,2,h-triaminohexane; N,N-didodecyllysineamide; N,N- didecyllysineamide;
spermidine- N,N- dioctadecyl urea; N-myristyllysineamide; and N-WO 98tO2190 PCTIUS97/12105 (dioctyldecylaminoethyl)-lysineamide . Representative amphiphiles are depicted (Figure 6) as arnphiphiles 43, 47, 56, 60, and 73. With respect to the structural features of these amphiphiles, and the other amphiphiles of Group m, the following should be considered.
With respect to the selection of the lipophilic alkylamine or diaL~cylaIIune group "Z", Table 2 below provides representative structures.
Table 2 For "Z"
(1) CH3-(CH2)13-NH--(2) CH3-(CH2)z-NH--(3) [[CH3(CH2) 17][CH3(CH2) 17]]N-(4) ~[C~3(CH2) 11][CH3(CH2) 11]]N--(5) [[CH3(CH2)9][CH3(CH2)9]]N--(6) [ [CH3(CH2) x] ~CH3(CH2) y]]N--1~ (7) [[cH3(cH2)x][cH3(cH2)ycH=cH(cH2)z]]N--(8) [[CH3(CH2)W][CH3(cH2)xcH=cH(cH2)ycH=cH(cH2)z]]N--In connection with the selection of suitable alkylamine or dialkylamine groups for inclusion at position Z in the amphiphiles of the invention, an alkylchain(s) of the group should not be so large in molecular weight that it interferes with the solubility of the amphiphile, or interferes with its ability to interact with plasmid DNA. Additionally, an alkyl chain of an alkylamine or dialkylamine may include one or more points of unsaturation.
The selection of R groups R1, R2, R3, and R4 follows that disclosed for the Group I amphiphiles, and these R groups may be selected, for example, from 2~ Table I Linking group Y may be seleected as for the Group I amphiphiles, and preferred examples thereof include--CH2--, and > C=O.

. ,_ . _ _ . __, . , ,,,.,, _ __ . _ CA 02260034 lsss-ol-ll wo 98/02190 PCTIUS97/12105 Group IV Amphiphiles Additionally there are provided cationic amphiphiles of Group IV (see Figure 7) capabl~ of facilitating transport of biologically active molecules into cells said amphiphiles having the structure (rV), (A)--¦~ (D)l (E)~
5 (B) (R2)--(R4) (R ) (R6) (IV) wherein:
A and B are independently O, N or S;
10 R~ and R6 are independently alkyl or acyl groups and may be sahlrated or contain sites of ~msahuration;
C is selected from the group consisting of -CH2-, >C=O, and >C=S;
E (analogous to "X" in struchures I, II, m) is a carbon atom or a nitrogen atom;D is a lin~;ing group such as -NH(C=O)- or -O(C=O)-, or D is absent;
15 R3 is H, or a sahlrated or unsaturated aliphatic group;
R1 is --NH--, an alkylamine, or a polyalkylamine;
R4 is H, or a sah1rated or unsahurated aliphatic group;
R2 is --NH--, an alkylamine, or a polyalkylamine;
and ~~herein Rl is the same or is different from R2, except that both R1 and R2 20 car not be--NH--Representative amphiphiles of Group rv include Nos. 64, 76, 85, 89, 9g, 98, 102, 10~, 110, and 111 With respect to the structural features of these amphiphiles, and the other amphiphiles of Group IV, the following should be considered.
With respect to the selection of R1, R2, ~3, and R4, the teachings provided for Group I, ~, and m amphiphiles are applicable. As aforementioned, group "E" represents a carbon atom or a nitrogen atom.
R5 and R6 are independently aLI~yl or acyl groups, ~left:lldbly containing about 8 to about 30 carbon atoms, and such groups may contain one or more points of unsaturation.
With respect to the selection of Group D, linlcers such as -NH(C=O)- or -O(C=O)- are ylere~.ed, and are depicted such that the left side thereof in intended to bond to "C" and the right side thereof is intended to bond to "E".
Optionally, group D may be absent (amphiphile No.94). Additional linkers may be selected based on the teachings provided with respect to Groups I, II, and m above, and based upon the in vivo test date derived ( Figure 15), it is ~'efer~ed that the linker D be short or absent.
Co-lipids Representative cc~lipids that are useful according to the practice of the in~ention for mixing with one or more cationic amphiphiles include dioleoylphosphatidylethanolarnine ("DOPE"), diphytanoylphosphatidyl-ethanolamine, lyso-phosphatidylethanolamines other phosphatidyl-ethanolamines, phosphatidylcholines, lyso-phosphatidylcholines and cholesterol.
~ Typically, a preferred rnolar ratio of cationic amphiphile to colipid is about 1:1.
However, it is within the practice of the invention to vary this ratio (see Example 3 below), including also over a considerable range.
It is generally believed in the art that preparing cationic amphiphiles as complexes with co-lipids (particularly neutral co-lipids) enhances the capability of the amphiphile to facilitate transfections. Although colipid-enhanced . _ . ... . ~

W O 98/02190 PCT~US97/12105 performance has been observed for numerous of the amphiphiles of the invention, the arnphiphiles of the invention are active as transfectants without co-lipid. Accordingly, the practice of the present invention is neither to be considered limited by theories as to co-lipid partiapation in intracellular delivery 5 mechanisms, no r to require the involvement of co-lipids.
Transacylation Reactions Although heretofore unrecognized in the art, it has been deternuned also that certain co-lipids may react ch~ lly with certain types of cationic amphiphiles under conditions of co-storage, there resulting new molecular 10 species. Generalion of such new species is believed to occur via mechanisms such as transacylation. In this regard, see Figure 4 which depicts a transacylation reaction involviI~g sperrnine cholesterol carbamate(No.67) and DOPE, there resulting lyso PE species and multiple forrns of particular acyl- cationic amphiphile ( designated No. 80~.
lS With respect to such reactions, the following remarks are of interest.
With respect to use of amphiphile No.67, it has been obser~ed that a mixture of amphiphile and DOPE, in chloroform solvent, does not appear to participate in such reactions. However, preparing the amphiphile and co-lipid in an aqueous solution where bilayer-containing structures such as liposomes can form will permit transacylation. Additionally, if amphiphile and co-lipid are dried down to a thin film, such as from chloroform (thereby placing the 2 species in intimate contact), then trcmsacylation also occurs, possibly as a result of entropic effects. It is expected that these phenomena would also apply to Iyophilized amphiphile/DOPE preparations.
Accordingly, it is highly preferred to maintain such amphiphile /DOPE
preparations at very cold temperatures, such as -70 degrees C. Preparation of CA 02260034 l999-01-ll amphiphile No. 67 as a mono, di, or tri acetate salt has also been determined toslow transacylations.
It is to be understood that therapeutically-effective pharmaceutical compositions of the present invention may or may not contain such 5 transacylation byproducts, or other byproducts, and that the presence of such byproducts does not prevent the therapeutic use of the compositions containing them. Rather use of such compositions is within the practice of the invention, and such compositions and the novel molecular species thereof are therefore specifically claimed.
Preparation of Pharmaceutical Compositions and Administration Thereof The present invention provides for pharmaceutical compositions that facilitate intracellular delivery of therapeutically effective amounts of biologically active molecules. Pharmaceutical compositions of the invention facilitate entry of biologically active molecules into tissues and organs such as the gastric mucosa, 15 heart, lung, and solid tumors. Additionally, compositions of the invention facilitate entry of biologically active molecules into cells that are maintained in vitro , such as in tissue culture. The amphiphilic nature of the compounds of the invention enables them to associate with the lipids of cell membranes, other cell surface molecules, and tissue surfaces, and to fuse or to attach thereto. One type 20 of structure that can be formed by amphiphiles is the liposome, a vesicle formed into a more or less spherical bilayer, that is stable in biological fluids and can entrap biological molecules targeted for intracellular delivery. By fusing with cell membranes, such liposomal compositions permit biologically active molecules carried therewith to gain access to the interior of a cell through one or 25 more cell processes including endocytosis and pinocytosis. However, unlike the case for many classes of amphiphiles or other lipid-like molecules that have been proposed for use in therapeutic compositions, the cationic amphiphiles of the CA 02260034 lsss-ol-ll invention need not forrn highly organized vesicles in order to be effective, and in fact can assume (with the biologically active molecules to which they bind) a wide variety of ]oosely organized structures. Any of such structures can be present in pharrnaceutical preparatior s of the invention and can contnbute to 5 the ef~Liv~-esss thereof.
BiologicaLly active molec~ that can be provided intracellularly in therapeutic amounts using the amphiphiles of the invention include:
(a) polynucleotides such as genomic DNA, cDNA, and mRNA that encode for therapeutically useful proteins as are known in the art, 10 (b) ribosomal RNA;
(c) antisense polynucleotides, whether RNA or DNA, that are useful to inactivatetranscription products of genes and which are useful, for example, as therapies to regulate the gro wth of malignant cells; and (d) ribozymes.
In general, and owing to the potential for leakage of contents theref~ "
vesicles or other structures formed from numerous of the cationic amphiphiles are not preferred by those skilled in the art in order to deliver lo w molecularweight biologically active molecules. Although not a preferred ernbodiment of the present invention, it is nonetheless within the practice of the invention to20 deliver such low molecular weight molecules intracellularly. Representative of the types of low molecular weight biologically active molecules that can be delivered includ e hormones and antibiotics.
Cationic amphiphile species of the invention may be blended so that two or more species thereof are used, in combination, to facilitate entry of 25 biologically acti ve molecules into target cells and/or into subcellular compartments tnereof. Cationic amphiphiles of the invention can also be blended for such use with amphiphiles that are known in the art.

Dosages of the pharmaceutical compositions of the invention will vary, depending on factors such as half-life of the biologically-active molecule, potency of the biologically-active mo}ecule, half-life of the amphiphile(s), any potential adverse effects of the amphiphile(s) or of degradation products thereof, the route 5 of administration, the condition of the patient, and the like. Such factors are capable of determination by those skilled in the art.
A variety of methods of adrninistration may be used to provide highly accurate dosages of the pharmaceutical compositions of the invention. Such preparations can be administered orally, parenterally, topically, transmucosally, 10 or by injection of a preparation into a body cavity of the patient, or by using a sustained-release formulation containing a biodegradable material, or by onsite delivery using additional micelles, gels and liposomes. Neb~ 7ing devices, powder inhalers, and aerosoli7ed solutions are representative of methods that may be used to administer such preparations to the respiratory tract.
Additionally, the therapeutic compositions of the invention can in general be formulated with excipients (such as the carbohydrates lactose, trehalose, sucrose, mannitol, maltose or galactose) and may also be lyophilized (and then rehydrated) in the presence of such excipients prior to use. Conditions of optimized forrnulation for each amphiphile of the invention are capable of 20 determination by those skilled in the pharmaceutical art. By way of example, for spermidine cholesterol carbamate (amphiphile No. 53), it has been determined that use of sucrose is preferred over mannitol in order to prevent formation of amphiphile/DNA aggregates, particularly as the concentration of DNA is increased therein. Addition of such excipients maintains the consistency of 25 lyophilized ~harmaceutical compositions during storage, and prevent difficulties such as aggregation, or insolubity, that may likely occur upon rehydration from the lyophilized state.

..

CA 02260034 lsss-ol-ll Accordin~,ly, a principal aspect of the invention involves providing a composition that comprises a biologically active molecule (for example, a polynucleotide) and one or more cationic amphiphiles (including optionally one or more co-lipids), and then maintaining said composition in ~e presence of one 5 ore more excipients as aforementioned, said resu~tant composition being in liquid or solid (preferably lyophilized) form, so that: (1) the therapeutic activity of the biologically active molecules is substantially preserved; (2) the transfection-enh~mcing nature of the amphiphile( or of amphiphile/ DNA
complex) is maintained. Without being limited as to theory, it is believed that 10 the excipients stabilize the interaction (complexes)of the amphiphile and biologically active molecl~le through one or more effects including:
(1) minirnizing interactions with container surfaces, (2) preventing irreversible aggregation of the complexes, and (3) maintaining amphiphile/DNA complexes in a chemically-stable state, i.e., preventing o~idation and/or hydrolysis.
Although the presence of excipients in the pharmaceutical compositions of the invention stabilizes the compositions and faciliates storage and manipulation thereof, it has also been determined that moderate concentrations of numerous excipients may interfere with the transfection-enhancing capability of ~0 pharmaceutical f~rmulations containing them. In this regard, an additional and valuable characteristic of the amphiphiles of the invention is that any such potentially adverse effect can be minimized owing to the greatly enhanced in vivo activity of the amphiphiles of the invention in comparison with amphiphilic compounds known in the art. Without being limited as to theory, it '75 is believed that osmotic stress ( at low total solute concentration) may contribute positively to the successful transfection of polynucleotides into cells in vivo .
Such a stress may occur when the pharmaceutical composition, provided in ll unbuffered water, contacts the target cells. Use of such otherwise ~lefe-,ed compositions may therefore be incompatible with treating target tissues that already are stressed, such as has damaged lung tissue of a cystic fibrosis patient.
According}y, and using sucrose as an example, selection of concentrations of this 5 excipient that range from about 15 rnM to about 200 mM provide a coll,~rc,~Lise betweeen the goals of (1) stabilizing the pharmaceutical composition to storage and (2) mimizing any effects that high concentrations of solutes in the composition may have on transfection ~e~ulll~ance Selection of optimum concentrations of particular excipients for particular 10 formulations is subject to experimentation, but can be determined by those skilled in the art for each such formulation.
An additional aspect of the invention concerns the protonation state of the cationic amphiphiles of the invention prior to their contac~ng plasmid DNA in order to form a therapeutic composition. It is within the practice of the invention 15 to utilize fully protonated, partially protonated, or free base forms of the amphiphiles in order to form such therapeutic compositions. With respect to amphiphile No. 67 (spermine cholesterol carbamate), it has been observed that when providing this amphiphile for a transfecting composition with DOPE (itself provided as a zwitterion), transgene expression was best for the free base, but 20 decreased if the amphiphile was prepared as an acetate salt. Activity decreased step-wise through the mono and di acetate salts and was minimal for the tri-acetate salt. Under the circumstances described, the plasmid DNA provided for contacting with the amphiphile was prepared (without buffer) as a sodium salt inwater.
25 T~ansfection of the Vascular Svstem A further aspect of the invention involves transfection of the vascular system. By transfection of the vascular system is meant that the .

therapeutic composition (comprising one or more cationic amphiphiles, a therapeutic polynucleotide, and optionally, one or more co-lipids) is placed in a blood vessel of a. patient through which it will be distributed to blood vessel cells.
Blood vessels suitable for application in the practice of the invention include 5 those of the arterial, venous, or capillary systerns. Blood vessel cells that may be transfected acco;rding to the practice of the invention also include those of the arterial, venous, or capillary systems.
It is also ~vithin the practice of the invention to transfect cells of identifiable vess~els of the lymphatic system.
The catiol~ic amphiphiles of the invention (including those defined herein by Groups I, II, III and IV) can be formulated with co-lipids and polynucleotides for such therapeutic application.
The vasc~Llar system of a patient is contacted with a composition that comprises a cationic amphiphile and a polynucleotide that encodes a protein 15 having therapeutic properties, such that cells of the vascular system are transfected thert by, and express said protein from said polynucleotide. In a preferred aspect. the protein is one norrnally secreted from cells, and the encoding polynucieotide includes, for example, sequences for pre- or pro-peptides, or for amino acids that are to be glycosylated, such that the encoded 20 protein is secreted into the vascular circulation of a patient, after which the secreted protein provides therapeutic benefit at a site remote or adjacent to the transfected blood vessel cells. Examples of therapeutic proteins that can be expressed in patients according to this aspect of the invention include adenosine deaminease, glucocerebidase, and further include numerous of the protein 25 hormones such as growth hormones, insulin and the like. Efficient expression and secretion of such a protein is demonstrated in Example 11.

CA 02260034 l999-Ol-ll Methods of Syntheses The following methods illustrate production of certain of the cationic amphiphiles of the invention. Those skilled in the art will recogr~ize other methods to produce these compounds, and to produce also the other compounds 5 of the invention.
Group I amphiphiles (A) N~Spermidine cholestervl carbamate Spermidine cholesterol carbarnate (Figure 1, No. 53) was synthesi~ed according to the following procedure which is outlined in Figure 8.
10 Synthesis of Nl N8-DiCBZ-N4-Sperrnidine Cholesterol Carbamate N1, N8 - dicarbobenzoxyspermidine (61% yield, m.p. 104 - 105~ C) was prepared according to the procedure of S. K. Sharma, M. J. Miller, and S. M.
Payne, J. Med. Chem, 1989, 32, 357-367. The N1, N8-dicarbobenzoxyspermidine (25 g, 60.5 rrunol) and triethylamine (25 ml, 178 15 mmol) were dissolved in 625 ml of anhydrous methylene chloride, cooled to 0 -4~C and stirred under N2. Cholesteryl chloroformate (27.2 g, 60.6 mmol) was dissolved in 250 ml of methylene chloride and added to the reaction over a 20 minute period. A white precipitate formed upon addition. After the addition was complete, the reaction was stirred at 0 - 4~C for 10 minutes and then at room 20 temperature for 1.5 hr. At this point, the white precipitate completely dissolved.
The reaction was followed by TLC with hexane / ethyl acetate 6 / 4 as eluent (product Rf = 0.25). To this reaction mixture was added 625 ml of methylene chloride and 625 ml of water. The layers were then allowed to separate. The organic layer was dried over MgS04 and filtered. The filtrate was concentrated 25 in vacuo to give an oil. Vacuum drying was then carried out overnight. This crude product had a glue-li~e consistency. The crude product was purified by column chromatography (2 ~g silica gel, eluent - hexane / ethyl acetate 6 / 4) to ~, CA 02260034 l999-Ol-ll WO 98/02190 PCT/US97/1210~;

give 46.8 g of the 3~ N~(Nl,N8-dicarbobenzoxysperrnidine)carbamoyl~
cholesterol (also described herein as N1, N~ diCBZ-N4- spermidine cholesterol carbamate) in 9;3% yield.
Final Svnthesis of Sperrnidine Cholesterol Carbamate To 6.0 grams of 10% palladium on activated carbon under N2 was added a solution of 30 grams of 3-~-[N4-(N1,N8-dicarbobenzoxys~ idine)carbamoyl]
cholesterol in 1 liter of ethanol, see Figure 13. The reaction rnixture was purged with N2 and stirred under H2 (atrnospheric pressure) for 18 hr. The rnixture wasagain purged with N2 and filtered through a 10 g bed of celite. The filter cake was washed with 2 liters of 10% triethylamine in ethanol and the combined filtrates were ccncentrated in ~7acuo to a gel. The product was then dried undervacuum overnight to a sticky solid. This crude product was purified by column chromatography (2 kg of silica gel, eluent - 4 L of chloroforrn / methanol 95 / 5 followed by 30 lL of chloroforrn / methanol / iso-propylamine 95 / 5 / 5, Rf =
0.24) to obtain 13.1 g of the desired spermidine cholesterol carbamate in 64%
yield. HPLC (C-18 reversed phase column, linear gradient elution profile -methanol / iso-propanol / water / trifluoroacetic acid 60 / 20 / 20 / 0.1 to methanol / iso-propanol / trifluoroacetic acid 70 / 30 / 0.1 to methanol / iso-propanol / chloroforrn / trifluoroacetic acid 60 / 20 / 20 / 0.1) analysis of this material showed it to be 99.2% pure with the 7-dehydrocholesterol analog present at a level of 0.8%.
In connection with this example and those that follow, it is noted that all TLC plates were visualized with phosphomolybdic acid.
(B) N4-Spermine cholestervl carbamate Spermine cholesterol carbamate (Figure 1, No. 67) was prepared according to the followin~ procedure which is outlined in Figure 9.
Nl N 12 -di CBZ -spermine .

ll CA 02260034 l999-01-ll Benzylchloroformate (1.76g, 1.5 ml, 10.36 mmol) was dissolved in methylene chloride (5 rnl) and placed in a three neclc flask under a nitrogen atrnosphere. Imidazole (1.4 g, 20.6 mrnol) was dissolved in methylene chloride (20 rnl) and placed in an addition funnel. The three neck flask was cooled to 0~C
and the irnidazole solution was added gradually over 20 rr~in. The mixture was stirred at room temperatllre for 1 hour and then methylene chloride ( 25 mL) andcitric acid (10%, 25 ml) were added. The layers were separated and the organic fraction was washed with citric acid (10%, 25 rnl). The organic component was dried over magnesium suLfate and concentrated in vacuo. The residue was dried urlder high vacuurn for 1 hour at ambient temperature.
To the residue was added dimethylaminopyridine (35 mg), methylene chloride (25 ml) and the mLxture was cooled to 0~C, under a nitrogen atmosphere. To an addition funnel was added a solution of spermine (lg, 4.94 rnrnol) in methylene chloride (25 ml). The sperrnine solution was added gradually over 15 min. The reaction mixture was stirred overnight at ambient temperature and then concentrated in vacuo. The residue was dissolved in ethyl acetate (SO ml) and washed three times with water (15 ml). The organics were dried over magnesium sulfate, filtered and concentrated in vacuo to give a crudewhite solid. The material was purified by flash chromatography (65g silica gel, 100:100:10 CHCl3: MeOH: NH40H, product ~f.=0.33), to give after drying under high vacuum 1.01g (2.146 mmol, 43 % yield) of product.
NlN12-diCBZ- N4- spermine cholestryl carbamate Cholesteryl chloroformate (964 mg, 2.15 rnmol) was dissolved in chloroform (10 ml) and added dropwise to a cooled (0~C) solution of N1,N12-diCBZ spermine (1.Olg, 2.15 mmol), triethylamine (1 ml) in chloroform (10 ml).
The reaction was allowed to warm to room temperature and stirred for 2 hours.
To the reaction solution was added water (25 ml) and chloroform (25 ml). The WO 98/02190 PCTrUS97/12105 layers were separated and the organic fraction dried over magnesium sulfate.
The solution was concentrated in vacuo to give a crude material that was purified by flash chromatography (68g silica gel, MeO~ / CHCl3 1/4, product Rf. =0.36) to give 1.23 g (1.39 mmol, 65% yield) of product.
final synthesis of N4-Spermine Cholesteryl Carbamate N1 ,N 12-cLiCBZ-N4-spermine cholesteryl carbarnate (262 mg, 0.300 rnmol) was dissolved in 5 ml of acetic acid and 45 mg of 10% Pd on C was added. The solution was purged with nitrogen and stirred under hydrogen at atmospheric pressure. The hydrogenolysis was allowed to proceed for 7 hours. The reaction mixture was filtered and the catalyst was washed with 40 rnl of ethyl acetate /
acetic acid 9 / 1 and the filtrate will be concentrated in v~cuo to give a residue.
The crude product was dissolved in 35 mL of lN NaOH and extracted three times with 40 m~ of chloroform / methanol 9 / 1. The combined organic fractions were w ashed with 20 mL of water and dried over Na2SO4. The solution was filtered, concentrated in vac~o and dried under vacuum to give 125 mg of the desired product in 67% yield.
In connection with the above procedure, it is noted that the hydrogenolysis ,hould be carried out under acidic conditions, in order to minimize the poisoning of the catalyst.
Urea ana]ogs - such as sperrnine or spermidine cholestamine urea - can be prepared by a sequence of reactions well known to those versed in the art of organic synthesis. For example an arnine can be treated with an equal molar amount of carbcnyldiimidazole followed by the addition of a second amine to give the desired urea.
(C) N N Bis t3-aminopropvl)-O-cholestery~ -3-carbamate N,N Bis (3-aminopropyl)-O-cholesteryl-3-carbamate (Figure 1, No. 69) was prepared according to the following procedure.

W O 98102190 PCT~US97/12105 Bis (3-CBZ aminopropyl) amine was prepared usmg the method described above for Nl,N12 -diCBZ-sperrnine, except that N-(3-arninopropyl)1,3-propanediamine was substituted for sperrnine as reactant. The pure product was isolated in 34 %yield by silica gel flash chromatography using as solvent CHCl3/ MeOH/
NH40H 80/20/0.5.
The Bis (3-CBZ aminopropyl) arnine so prepared was then reacted with cholesteryl chloroformate according to the method described above for the synthesis of N1, N8-DiCBZ -N4 spermidine cholesteryl carbamate. The pure product (N,N Bis ( 3-CBZ aminopropyl)-~cholesteryl-3-carbamate) was obtained in 73% yield.
Synthesis of N,N Bis(3-aminopropyl)~cholesteryl-3-carbamate was completed by hydrogenolysis of the CBZ groups from N,N Bis(3-CBZ
aminopropyl)-O-cholesteryl-3-carbamate following the procedure described above in relation to the synthesis of N4-spermidine cholesteryl carbamate. The product was obtained in 23% yield without silica gel chromatography purification.
(D) N.N Bis (6-aminohexvl~-O-cholestervl -3- carbamate.
N,N Bis (6-aminohexyl)-O-cholesteryl-3-carbamate (Figure 1, No. 70) was prepared according to the following procedure.
First, Bis (6-CBZ aminohexyl) amine was prepared using the method described above for N1,N 12 -diCBZ-spermine, except that Bis(hexamethylene)triamirle was substituted for spermine as reactant. Pure product was isolated in 24% yield by recrystallization from toluene.
Bis (6-CBZ aminohexyl) amine was then reacted with cholesteryl chloroformate according to the method described above for the synthesis of N1, N8-DiCBZ -N4-spermidine cholesteryl carbamate. Product N,N Bis(6-CBZ

CA 02260034 l999-01-ll W O 98/02190 PCTrUS97/12105 aminohexyl)-C~cholesteryl-3-carbamate was isolated in 40% yield by silica gel flash chromatography using hexanes/ethyl acetate 7/3 .
~ Lysine 3-N- dihydrocholestervl carbamate Lysine 3-N- dihydrocholesteryl carbamate (Figure 1, panel C) was 5 prepared accorcling to the following procedure.
To a solution of dihydrocholesterol (5.0 g, 12.9 rnrnol, Aldrich), phthal~nide (2.() g, 13.6 mmol, Aldrich), and triphenylphosphine (3.8 g, 13.6 mmol, Aldrich) in THF (20 ml, Aldrich) stirred at 0~ C under a nitrogen atmosphere was added dropwise diethylazodicarboxylate (2.3 ml, 14.5 rnmol, 10 Aldrich). Upon the completion of arl~ition the reaction mixture was allowed to warm to ambient temperature and stirred overnight. The reaction rnixture was concentrated in vacuo to a residue. This residue was dissolved in 50 ml hexane /ethyl acetate 95 / 5 and a precipitate formed. The mixture was filtered. The filtrate was concentrated to dryness in vacuo, dissolved in 25 rnl of hexane / ethyl acetate 95 / 5 and chromatographed on 2C0 g silica gel (eluent 2 L hexane / ethyl acetate 95 / 5 then 1 L hexane / ethyl acetate 90 / 10). A 76% yield of the desired 3-phthalimidocholestane (5.43 g) was obtained.
The 3-phthalimidocholestane (5.40 g, 9.75 mmol) was dissolved in 60 mL
of methanol and anhydrous hydrazine (3.1 ml, 99 mmol) was added. The 20 reaction mixture was stirred and heated at reflw~ under a nitrogen atmosphere for 4 hr. This mixture was then cooled to room temperature, 3.1 mL of concentrated HC l was added and the resulting mixture was heated at reflux overnight. Upon cooling to ambient temperature, 100 ml of diethyl ether and 50 ml of 1 N NaOH were added (final pH of 10.1) and the layers were separated.
25 lhe aqueous layer was extracted with 50 ml of diethyl ether and the combined organic fractions were filtered. The filtrate was concentrated in v~cuo and the CA 02260034 l999-01-ll residue was purified by silica gel chromatography (chloroform / methanol 90 /
10) to give 2.24 g of 3-aminocholestane in 59 % yield.
L-N~,N~-diBOClysine N-hydroxysuccinimide ester (286 mg, 0.644 mmol, Sigma) and 3-aminocholestane (250 mg, 0.644 mmol) were dissolved in 5 mL of methylene chloride, 0.1 mL of triethylarnine was added and the resulting solution was stirred under a nitrogen atmosphere at arnbient temperature overnight. To the reaction rnixture was added 10 mL of water and 25 mL of methylene chloride and the layers were separated. The aqueous layer was extracted with 25 mL of methylene chloride and the combined organic fractions were dried over MgSO4 and fi}tered. The filtrate was concentrated in vacuo and the residue was purified by chromatography on 25 g of silica gel (eluent - hexane / ethyl acetate 6 / 4, sarnple applied in hexane / ethyl acetate 9 / 1). The purified material was dissolved in 25 mL of chloroforrn and HCl gas was bubbled through the solution for 2 hr. followed by nitrogen for 10 min. The solution was concentrated in vacuo to give 299 mg of the desired product in 79%
yield as the dihydrochloride salt.
(F) N_N--Bis(3-aminopropvl)-N4-sperrnidine cholestervl carbamate N1,N8-Bis(3-aminopropyl)-N4-spermidine cholesteryl carbamate (Figure 1, No. 75) was prepared according to the following procedure.
N4-Spermidine cholesteryl carbamate (1.14g, 2.04 mmol) was dissolved in MeOH (5 mL). Freshly distilled acrylonitrile (0.28 mL, 4.29 mmol) was added and the solution was stirred at room temperature for 18 h. The solvent was concentrated in vacuo to give an oil. Vacuum drying was then carried out overnight. The crude product was purified by column chromatography (125 g silica gel, eluent- CHC13 MeOH 1/9) to give 1.15 g (85 %) of the N1,N8-Bis (cyanoethyl) N~Spermidine cholesteryl carbamate.

W O 98/02190 PCT~US97/12105 Raney Nickel 50% slurry (1.2 g, Aldrich) was placed in a Parr Bomb with lM
NaOH in 95% F.tOH (50 mL). The Nl,N8-Bis (cyanoethyl) N4-Spermidine cholesteryl carbamate. was dissolved in EtOH (35 mL) and added to the bomb.
The vesicle was evacuated and placed under Argon pressure (80-100 psi), three times and then evacuated and placed under Hydrogen pressure (100 psi), three times. The reaction was stirred under hydrogen pressure (100 psi) at room temperature fo:r 72h. The vesicle was evacuated and placed under argon pressure. The catalyst was removed by filtration. The filtrate was concentrated in v~cuo . The res~;~ting oil was dissolved in 2:1 CH2a2: MeOH (100 mL) and washed with H20 (35 and 25 mL). The organic layer was dried over Na2S04 and filtered. The filtrate was conce~ aLed in vacuo and the residue was purified by chromatography on 100 g of silica gel (eluent - CHC13/MeOH/conc. NH40H
40/25/10, sample applied in CHC13/MeOH 40/25). The purified material was concentrated in vacuo with iPrOH (3 X 50 mL) and CH2a2(3X50 mL) and then vacuurn dried to give 986 mg (85%) of N1,N8-Bis(3-aminopropyl)-N ' spermidine cholesteryl carbamate.
(G) N(N4-3-aminopropvl-spermidine) cholesteryl carbamate N(N4-3-aminopropyl-spermidine) cholesteryl carbamate (Figure 1, No. 78) was prepared as follows:
N1, N8-dicarbobenzoxyspermidine (1.0 g, 2.4 mmol) was dissolved in MeOH
(10 mL). Fresh]y distilled acrylonitrile (0.3 mL, 4.5 mmol) was added and the reaction was stirred at room temperature for 18 h. The solvent was concentrated in vacuo to give an oil. The crude product was purified by column chromatography (100 g silica gel, eluent - CHCI3/MeOH 1/19) to give 1.10 g (97 %) of N4-2-Cyanoethyl-N1, N8 - dicarbobenzoxyspermidine.
Il~e N4-2-Cyanoethyl-N1, N8-dicarbobenzoxyspermidine (0.5 g, 1.07 mmol) ~,vas dissolved in MeOH (5 mL) and CoC12 (280 mg, 2.15 mmol, Aldrich) was WO 98/02190 PCT/US9711210~

added. The blue solution was cooled in an ice ~ath and NaBH4 (405 mg, 10.7 mmol, Aldrich) was added in portions over 15 mir~. The resu~ting black solution was stirred at room temperature for 1 h. The black solution turned blue over this period. To the reaction was added CH2a2/MeOH 2/1 (30 mL). A black ppt formed. To this was added H20 (20mL) and the mixture was filtered. The resulting layers were separated and the organic layer dried with MgSO4. The drying agent was filtered and the filtrate concentrated in vacuo to give an oil.The crude product was purified by colwnn chromatography (50 g silica gel, eluent - CHCl3/MeOH/conc NH40H 100/100/5) to give 309 mg (62 %) of the N4 3-aminopropyl-N1, N8 - dicarbobenzoxysperrnidine.
To the N4 3-aminopropyl-Nl, N8 - dicarbobenzoxyspermidine (300 mg, 0.66 mmol) dissolved in CH2Cl2 was added Et3N under N2. Cholesteryl chloro formate (326 mg, 0.726 rnmol, Aldrich) was dissolved in CH2a2 and added to the reaction dropwise. The mixture was stirred for 2h at room temperature. Afteradding CH2Cl2 (25 mL) and H2O (10 mL), the layers were separated. The organic layer was dried with MgSO4 and filtered. The filtrate was concentrated in vacuo to give 640 mg of crude product. The residue was purified by chromatography on 80 g of silica gel (eluent - CHC13 / MeOH 90 /10, sample applied in CHC13 / MeOH 90/10). The purified material was concentrated in vacuo and then vacuum dried to give 329 mg (57%) of N-(N4-3-aminopropyl-N1, N8 - dicarbobenzoxyspermidine) cholesteryl carbamate.
To 10% Pd on carbon (65 mg, Aldrich) was added a solution of N-(N4-3-aminopropyl-N1, N8 - dicarbobenzoxyspermidine) cholesteryl carbamate (300 mg) in acetic acid (25 mL). The reaction was placed under H2 and stirred at roomtemperature overnight. After being placed under N2, the reaction was filtered.
~Ihe catalyst was washed with 10 % acetic acid in EtOAc (50 mL). The filtrate was concentrated in vacuo to give an oil. The oil was dissolved in 2/1 CH2Cl2/MeO~:[ (35 mL) and washed with 1 M NaOH (15 mL). The organic layer was dried with MgSO4 and filtered. The filtrate was concentrated in vacuo and vacuum dried to give 196 mg (93%) of N-(N4-3-aminopropylsperrnidine) cholesteryl carbamate.
n N-~N-N- N8~Tris (3-arninopropyl) spermidinel cholesteryl carbamate N-[N1,N4,N8~Tris (3-aminopropyl) spermidine] cholesteryl carbamate (Figure 1, No. 96) was prepared by reacting N-(N4-3-aminopropylsperrnidine) cholesteryl carbamate with acrylonitrile (90% yield) and subsequent reduction ofthe di adduct with Raney nickel (75 % yield) as described for the preparation ofN1,N8Bis(3-aminopropyl)-N~spermidine cholesteryl carbamate.
(n N N-Bis(4-aminobutvl~ cholesteryl carbamate N,N-Bis(~aminobutyl) cholesteryl carbamate (Figure 1, No. 82) was prepared as follows.
To a mixture of Benzylamine (2.0 g, 18.6 mmol, Aldrich), Na2CO3 (4.4g, 42 rnmol) and KI (].. 4 g, 9.5 mmol) in n-butanol (50 mL) was added 4-Chlorobutyronitrile (4.0 mL, 95 mmol) under nitrogen. The reaction was stirred at reflux of 48 h under nitrogen. After cooling to room temperature, diethyl ether (50 mL) was added and the precipitate filtered off. The filtrate was concentrated in vacuo to an oil. Toluene (100 mL) was added and the solution was 20 concentrated in vacuo . Chloroforrn (100 mL) was added and again the solutionwas concentrated in vacuo and then vacuum dried for 18 h. The resulting oil was dissolved in Ch]oroform (100 mL) filtered and concentrated in vacuo . The crude product was purified by column chromatography (250 g silica gel, eluent -hexanes/EtOAc 60/40) to give 3.75g (97 %) of N,N-Bis (3-cyanopropyl) 2~ benzylamine.
The N,N-Bis (3-cyanopropyl) benzylamine (3.7 g, 17.8 mmol) was dissolved in EtOH (150 mL) and Acetic acid (4 mL) was added. This solution was added to 10% Pd on carbon (400 mg) under N2. The mixture was placed under H2 and the reaction stirred for 18 h at room temperature. The reaction was placed under N2.The catalyst was filtered off and washed with EtOH (150 mL). The filtrate was concentrated in vacuo, chloroforrn (50 mL) was added and again concentrated in 5 vacuo . The resulting oil was vacuum dried for 0.5 h and used directly in the next reaction. To this oil dissolved in CH2C12 (lOOmL) was added Et3N (5 mL, 35 mmol) under N2 and the solution cooled in an ice bath. Cholesteryl chloro formate (6.2 g, 13.87 mmol) was dissolved in CH2Cl2 (100 mL) and this solution was added to the reaction dropwise over 10 min. The cooling bath was removed and the reaction stirred at room temperature for 18 h under N2. CH2a2 ~100 mL) and H2O (100 mL) was added and the resulting layers were separated. The organic layer was dried with MgSO4 and filtered. The filtrate was concentrated in vacuo and vacuum dried for 1 h. The crude product was purified by colurnn chromatography (600 g silica gel, eluent - hexanes/EtOAc 60/40) to give 1.05g (10 %) of N,N-Bis (3-cyanopropyl) cholesteryl carbamate.
Raney Nickel 50% slurry (1.2 g) was placed in a Parr Bomb with lM NaOH in 95% EtOH (50 mL). The N,N-Bis (3-cyanopropyl) cholesteryl carbamate (1.0 g, 1.77 rrLmol was dissolved in EtOH (100 mL) and added to the bomb. The vesicle was evacuated and placed under Argon pressure (80-100 psi), three times and then evacuated and placed under Hydrogen pressure (100 psi), three times. The reaction was stirred under hydrogen pressure (100 psi) at room temperature for four days. The vesicle was evacuated and placed under argon pressure. The catalyst was removed by filtration. The filtrate was concentrated in vacuo . Theresulting oil was dissolved in 2:1 CH2a2: MeOH (250 mL) and washed twice with H~O (75 and 50 mL). The organic layer was dried over Na2SO4 and filtered.
The filtrate was concentrated in VRCI~O and the residue was purified by chromato, ,raphy on 110 g of silica ~el (eluent - CHCI3/MeOH/iPrNH2 95/5/5, sample applied in CHCl3/MeOH 95/5). The purified material was concentrated in vacuo and then vacuum dried to give 900 mg (85%) of N,N~Bis(~arninobutyl) cholesteryl carbamate.
m N.N-Bi~(N'-3-aminopropvl~-aminobutyl~ chol~teryl carbamate S N,N-Bis(N'-3-arninopropyl~aminobutyl) cholesteryl carbamate (Figure 1, No. 83) was prepared by reacting N,N~Bis(~arninobutyl) cholesteryl carbarnate with acrylonitrile (82% yield) and subsequent reduction of the di acrylonitrile adduct with Raney nickel (81 % yield) as described for the ~ tion of Nl,N8-Bis(3-arninopropyl)-N~spermidine cholesteryl carbamate.
~Q N4 Spermidine cholesteryl carboxamide N4 Spern Lidine cholesteryl carboxarnide ( Figure 1, No. 90) was prepared as follows.
A solution of cholesteryl chloride (5.0 g, 12.3 rnmol) in THF (50 mL) was added dropwise over 0.5 h under reflux to Magnesium turnings (390 mg) in THF
1~ (25 mL). Initially a pinch of Iodine and three drops of Iodomethane were added to initiate the reaction. After refluxing for 3 h. the reaction was cooled to room temperature. I~is mixture was poured onto Dry ice (10 g) and then stirred for lh.
This solution WclS cooled in an ice bath and added to ice cold 1 M H2SO4 (100 mL). After stirring for 5 min., sodium chloride (1 g) and diethyl ether (100 mL)was added. The layers were separated and the aqueous layer was extracted with diethyl ether (100 mL). The combined organic layers were washed with a solution of Sodium thiosulfate pentahydrate(120 mg) in H20 (30 mL). The organic layer WclS concentrated in vac~o and vacuum dried for 18 h. The crude solid was titrated with hexanes (25 mL). After filtration the solid was washed with ice cold he:canes (10 mL). The solid was vacuum dried for lh. The cholesteryl carboxylic acid obtained (3.0 g, 59 %) was ca. 90 % pure and used without further purification.

CA 02260034 l999-Ol-ll Cholesteryl carboxylic acid (500 mg, 1.2 mmol) and N-hydroxysuccinimide (140 mg, 1.2 mmol) was dissolved in CH2C12 . To this solution was added Dicyclohexylcarbodiimide (275 mg, 1.32 mmol) was added and the reaction was stirred under N2 for 2h. Nl, N8-dicarbobenzoxyspermidine (474 mg, 1.2 rnmol) and Et3N (1.0 mL, 7.1 rnmol) was added and the reaction was stirred under N2 for 72 h. The reaction was filtered and the precipitate was washed with CH2Cl2 (50 mL). The filtrate was washed with H20 (25mL). The separated organic layer was dried over MgS04 and filtered. The filtrate was concentrated in vacuo and the residue was purified by chromatography on 150 g of silica gel (eluent - hexanes / EtOAc 1/ 1). lhe purified material was concentrated in vacuo and then vacuum dried to give 680 mg (70%) of Nl,N8-dicarbobenzoxy-N4-spermidine cholesteryl carboxamide.
The carbobenzoxy group were removed from N1,N8-dicarbobenzoxy-N4-spermidine cholesteryl carboxamide as described in the preparation of N4-spermidine cholesteryl carbamate. The purified product, N4 Spermidine cholesteryl carboxamide, was obtained in 53 % yield.
Group ll Amphiphiles (A~ N- N8-Bis(Arginine carboxamide~-N~spermidine cholestervl carbamate N1, N8-Bis(Arginine carboxamide)-N4-spermidine cholesteryl carbamate (Figure 5, No. 95) was prepared as follows.
To N (a) ,N (e) ,N (e) (alpha, epsilon, epsilon) -tricarbobenzoxyArginine in CH2Cl2 (25 mL) was added N-hydroxysuccinimide (100 mg, 0.8g rnmol) and dicyclohexylcarbodiimide (240 mg, 0.89 mmol). The mixture was stirred under N2 at room temperature for 2.5 hours. N4- Sperrnidine Cholesteryl Carbamate (250 mg, 0.448 mmol) and Et3N ( 0.25 mL, 1.8 mmol) was added and the reaction stirred at room temperature under N2 for 72 h. The reaction was filtered and theprecipitate was washed with CH2CI2 (20 mL). The filtrate was washed with H20 CA 02260034 l999-01-ll WO 98/02190 PCT/11S97/1210!;

(20 mL). The separated orgaruc layer was dried over MgS04 and filtered. The filtrate was concentrated in vacuo and the residue was purified by chromatographv on 70 g of silica gel (eluent - CHC13 / MeOH 95/ 5). The purified materia l was concentrated in v~cuo and then vacuu3rn dried to give 5335 mg (71%) of Nl, N8-Bis (N(a),N(e),N(e)-tricarboberlzoxyArgiinine carboxamide)- N4 spermidine cholesteryl carbamate.
The carbobenzoxy group were removed from N1, N8-Bis (N(a),N(e),N(e)-tricarbobenzoxyArginine carboxamide)-N4 spermidine cholesteryl carbamate as described in the preparation of N~(N4-3-aminopropylspermidine) cholesteryl 10 carbamate. The product,Nl,N8-Bis(Argininecarboxamide)-N4-sp~rm~ ne cholesteryl carb; mate was obtained in 27 % yield.
Group III Amphiphiles (A) N N-Dioctadecvllvsineamide N,N- dio.tadecyllysineamide( Figure 6, No.73) was prepared according to the following procedure. N,N-dioctadecylamine (1.35 g, 2.58 mmol, Fluka) and L-Na,N~-diBOC lysine N-hydroxysuccinirnide ester ( 1.00 g, 2.58 mrnol, Sigma) were combined in 15 ml of methylene chloride and 2 ml triethyla3rnine was added. The reaction mixture was heated briefly to effect complete dissolution and then stirred at ambient temperature overnight. Water (20 ml) and methylene 20 chloride (50 ml) were added to the reaction mixture and the layers were separated. The aqueous 3fraction was extracted a second time with 50 ml methylene chloride. The combined organic fractions were dried over MgS04, filtered and concentrated in v~cuo. The residue was purified by column chromatography (150 g silica gel, eluent - hexane/ethyl acetate 8/2). The 25 purified material, N,N-dioctadecyl-Na,NE-diBOC lysineamide(1.59 g) was dissolved in 25 mi of chioroform and stirred for 2 hr. while HCI gas was bubbledthrough the soll ~tion. This solution was purged with N2 gas and concentrated in CA 02260034 l999-Ol-ll WO 98/02190 PCl:'/US97/12105 vacuo. N,N -dioctadecyllysineamide (1.34 g) was obtained in 68% yield as the di HCl salt.
rB) Nl NLDioctadecyl-1,2,6-triaminohexane Nl,N 1-Dioctadecyl-1,2,~triaminohexane (Figure 6, No. 47) was prepared 5 as follows. To N,N-Dioctadecyl-Na,N~-diBOClysinearnide (760 mg, 0.823 rnrnol) in 30 ml anhydrous T~ stirred at ambient temperature was added LiAlH4 ~185 mg, 4.87 mmol) in portions. The reaction mixture was stirred at ambient temperature overnight under a nitrogen atmosphere. The reaction was quenched by the dropwise addition of 2 ml water and the resulting solution was 10 concentrated in vacuo. To this residue was added in order 10 mL of 1 M HCl, 50 ml of methylene chloride, and 10 rnl of lM NaOH (final pH 10). The layers were separated and the aqueous fraction was extracted a second time with 50 ml of methylene chloride. The combined organic layers were dried over MgSO4 and filtered. The filter cake was washed with 50 ml of methylene chloride. The 15 combined filtIates were concentrated in vacuo to give 700 mg of crude product.
The crude product was purified by column chromatography (80 g silica gel, eluent - hexane/ethyl acetate 7/3). The fractions containing the purified product were combined and concentrated in vacuo to obtain 490 mg of the product protected as the diBOC derivative. To 200 mg of this diBOC derivative was 20 added 4 ml of chloroform and 1 ml of TFA. The resulting reaction mixture was stirred at ambient temperature for 2 hr and concentrated in vacuo . The residue was dissolved in 25 ml of water and 25 mL of methylene chloride and adjusted to pH 10 with approximately 2 ml of concentrated ammonium hydroxide. The layers were separated and the aqueous layer was extracted a second tirne with 2525 ml of methylene chloride. The organic fractions were combined, dried over Na2SO4 and concentrated in vacuo . The resulting residue was dissolved in 10 ml of diethyl ether, ~ICl gas was bubbled through the solution for 2 minutes and the CA 02260034 lggg-ol-ll WO 98/02l90 PCT/US97/l2l05 solution was cooled at 4~ C overnight. The precipitated product was collected byfiltration, washed with cold (4~ C) diethyl ether, and dried under vacuum to obtain 160 mg of the desired product in 67% yield.
Group IV AmF~hiphiles (~O 1-rN~spermine~-2 3-dilaurylglycerol carbamate 1-(N~spermine)-2,3-dilaurylglycerol carbamate (Figure 7, No. 89) was prepared as fol Lows. A solution of 3-benzyloxy-1,2-propanediol (1.00 g, 5.49 mmol) in l~IF (20 mL) was added to a suspension of sodium hydride (60% w/w in oil, 550 mg, 13.725 mmol) in THF (30 mL) and allowed to reflux overnight under dry nitrogen. A solution of dodecyl methane sulfonate (3.39 g, 12.078 mrnol) in THF (20 mL) was added and the reaction was refluxed for another two days. After cooling to room temperature the reaction was filtered through a bed of Celite, rinsing with THF. The filtrate was reduced in vacrlo to a yellow oil which was redissolved in diethyl ether (100 mL). The ether solution was washed wit,n 0.1 N NaOH (30 mL) and dH20 (2 x 30 mL). The organic layer was dried over magnesium sulfate, filtered and reduced in vacuo to a red-brown oil. The crude material was purified by flash column chromatography (300 g silica gel) elutinc, with 3% ethyl acetate/ hexanes. The desired product was isolated as a pale yellow oil and characterized by lH NMR as 3-OBn-1,2-dilaurylglycerol (1.70 g, 60%). 3-OBn-1,2-dilaurylglycerol (1.70 g, 3.Z8 mmol) in ethanol (100 mL) was stirred with 10~~, Pd/C (250 mg, 15 wt%) under a hydrogen atrnosphere for 24 hours. The reaction was flushed with nitrogen and filtered through Celite, rinsing with ethanol, to remove the catalyst. The filtrate was reduced in vacuo to a solid. The crude material was purified by flash column chromatography (140 g silica gel) eluting with 10% ethyl acetate/ hexanes. The desired product was isolated as a white solid and characterized by lH NM~ as 1,2-dilaurylglycerol (1.23g, 8S%).

CA 02260034 l999-Ol-ll A 1.93 M solution of phosgene in toluene (0.77 mL, 1.49 mmol) was added to a solution of 1,2-dilaurylglycerol (580 mg, 1.35 mmol) and N,N-diisopropylethylamine (0.26 mL, 1.49 mmol) in methylene chloride (10 mL) and stirred overnight. A solution of N1,N12-di-CBz-spermine-2HCl (734 mg, 1.35 5 mmol) in 60: 25: 4 chlo.o~ / methanol/ water (80 mL) was added. After 3 hours another equivalent of N,N-diisopropylethylarnine (0.26 mL, 1.49 mmol) was added. An additional 0.5 equivalents of N,N-diisopropylethylarnine (0.13 mL, 0.75 mmol) was added three hours later and the reaction was allowed to stir overnight under nitrogen at ambient temperature. The reaction was washed 10 wiTh lM NaOH (20 mL) and dH20 (15 mL). The organic layer was separated, dried over magnesium sulfate, filtered and reduced in VQCUO to a white solid Thecrude material was purified by flash colurnn chromatography (125 g silica gel) eluting with 90: 10: 0.5 chloroform/ methanol/ arnmonium hydroxide. The desired product was isolated as an oil and characterized by lH NMR as 1-(N4 ~T 1~ 12-di-CBz-spermine))-2,3-dilaurylglycerol carbamate (188 mg, 15%).
The 1-(N4-(N l,N 17 di-CBz-spermine))-2,3-dilaurylglycerol carbamate (188 mg, 0.203 mmol) was dissolved in glacial acetic acid (10 mL) and stirred with 10% Pd/C (45 mg, 24 wt %) under a hydrogen atmosphere for 5 hours. The catalyst was removed by vacuum filtration rinsing with 10% acetic acid/ ethyl acetate (10 mL) The filtrate was reduced to an oil by rotary evaporation. The resultmg oil was dissolved in 10% methanol/ chloroform (85 mL) and was washed with lM NaOH (15 mL) and dH20 (10 mL). The organic layer was separated, dried over magnesium sulfate, filtered and reduced in vncuo to an oil.
The product was characterized by lH NMR as 1-(N~spermine)-2,3-dilaurylglycerol carbamate (125 mg, 94%).
Other amphiphiles of the invention may be prepared according to procedures that are within the knowledge of those skilled in art r ~ I - ~~--~~~ ' lr CA 02260034 lggg-ol-ll ~Uo 98/02190 PCT/US97/12105 Examples The following Examples are representative of the practice of the invention.
Example 1 - Ce]l Transfection Assay Separate 3.35 ~Lmole samples of spermidine cholesterol carbamate (amphiphile No. 53) and the neutral lipid dioleoylphosphatidylethanolamine ("DOPE") were each dissolved in chloiofo,l-L as stock preparations. Following combination of the solutions, a thin film was produced by removing chloroform from the mixture by evaporation under reduced pressure (20 mm Hg). The film was further dried under vacuum (1 mm Hg) for 24 hours. As aforementioned, some of the arnphiphiles of the invention participate in transacylation reactions with co-lipids s uch as I ~OPE, or are subject to other reactions which may cause decomposition thereof. Accordingly, it is ylefelled that amphiphile/co-lipid compositions be stored at low temperature, such as -70 degrees C, until use.
To produce a dispersed suspension, the lipid film was then hydrated with steri}e deionized water (1 ml) for 10 minutes, and then vortexed for 1 minute ( sonication for 11~ to 20 seconds in a bath sonicator may also be used, and sonication has proved useful for other amphiphiles such as DC-chol). The resulting suspel~sion was then diluted with 4 ml of water to yield a solution that is 670uM in cationic amphiphile and 670!1M in neutral colipid.
Experiments were also performed using spermine cholesterol carbamate (amphiphile No. 67) and other amphiphiles of the invention. With respect to spermine cholesterol carbamate, the optimum molar ratio of amphiphile to DOPE under the conditions tested was deterrnined to be 1:2, not 1:1. Optimized ratios for many of the amphiphiles of the invention are reported in Figures 13,14 and 15, and are readily determined by those skilled in the art.
For preparation of the transfecting solution, DNA encoding for B-galactosidase (pCMV~, ClonTech., Palo Alto, CA) was dissolved in OptiMEM

CA 02260034 l999-01-ll WO 98/02190 PCT/US97/1210~

culture medium (Gibco/ BRL No. 31885-013). The resulting solution had a DNA
concentration of 960 ~LM (assuming an average molecular weight of 330 daltons for nucleotides in the encoding DNA).
The following procedure was used to test a 1:1 molar mixture of the 5 cationic amphiphile spermidine cholesterol carbamate in combination with DOPE. A 165 111 aliquot of spermidine cholesterol carbamate (670 ~lM) containingalso the colipid ( at 670 ~LM ) was pipetted into 8 separate wells in a 96-well plate containing OptiMEM (165~1l) in each well. The resulting 335 IlM solutions were then serially diluted 7 times to generate 8 separate amphiphile~ontaining solutions having concentrations ranging from 335 ~M to 2.63 ~lM, with each resultant solution having a volume of 165 ~Ll. Thus, 64 solutions were prepared in all, there being 8 wells each of 8 different concentrations of amphiphile/DOPE.
Independently, DNA solutions (165~Ll, 96011M) were pipetted into 8 wells containing OptiMEM (165 ~l), and the resulting 480~M solutions were then 1~ serially diluted 7 times to generate 8 separate 165 ~Ll solutions from each well, with the concentrations of DNA in the wells ranging from 480 IlM to 3.75 IlM.
The 64 test solutions (cationic amphiphile: neutral lipid) were then combined with the 64 DNA solutions to give separate mixtures in 64 wells, each having a volume of 330~Llr with DNA concentrations ranging from 240 tlM to 1.875 ~M along one axis, and lipid concentrations ranging from 167 ~lM to 1.32 ,LM along the other axis. Thus 64 solutions were prepared in all, each having a different amphiphile: DNA ratio and/or concentration. The solutions of DNA
and amphiphile were allowed to stand for 15 to 30 minutes in order to allow complex formation.
A CFr-1 cell line (human cystic fibrosis bronchial epithelial cells immortalized with papillomavirus) provided by Dr. James Yankaskas, University of North Carolina, Chapel Hill, was used for the in vifro assay. The cells are homozygous far a mutant allele (deletion of phenylalanine at position 508, hereinafter A F508 ) of the gene encoding for cystic fibrosis transmembrane conductance regulator ("CFIF~") protein. C~-l~ is a cAMP-regulated chloride (a-) channel protein. Mutation of the CFTR gene results typically in complete 5 loss ( or at least substantial impairment) of Cl- charmel activity across, for example, cell membranes of affected epithelial tissues.
The ~ F508 mutation is the most cornmon mutation associated with cystic fibrosis disease. For a discussion of the properties of the A F508 mutation and the genetics of cystic fibrosis disease see, in particular, Cheng et al., Cell. 63, 827-834 (1990). See alsc, Riordan et al., Science, 245l 1066-1073 (1989); published European Patent Application No. 91301819.8 of Gregory et al., bearing publication number O 446 017 A1; and Gregory et al., Nature, 347, 382-385 (1990).
The cells were cultured in Harns F12 nutrient media (Gibco/ BRL No.
31765-027) supplemented with 2% fetal bovine serum ("FBS", Irvine Scientific, No. 3000) and i' additional supplements. Cells were then plated into 96-well tissue culture plates at a density of approximately 7,500 cells/well. Before being used in the assay, cells were allowed to grow for periods of 5-7 days until a confluent pattem had been achieved.
Following the allotted time period, three 96-well plates with CFT-1 cells were aspirated in order to remove the growth medium. The various concentrations of DNA-lipid complex (in 100 ~Ll aliquots) were transferred to each of three 9~well plates bringing the DNA-lipid complexes in contact with the cells. DNA-only/cell and lipid-only/cell control wells were also prepared onone of the three plates.
The 100 Ill solutions of DNA-lipid complex were maintained over the cells for 6 hours, after which 50 Ill of 30% FBS (in OptiMEM) was added to each well.
After a further 20-hour incubation period, an additional 100 ~ll of 10% FBS in 6~

OptiMEM was also added. Following a further 24-hour incubation period, cells were assayed for ~ ures~ion of protein and B-galactosidase.
For the assays, the resultant medium was removed from the plates and the cells washed with phosphate buffered saline. Lysis buffer (50 yl, 250 mM Tris-HCl, pH 8.0, 0.15% Triton X-100) was then added, and the cells were lysed for 30minutes. The 96-well plates were carefully vortexed for 10 seconds to dislodge the cells and cell debris, and 5 ~Ll volurnes of lysate from each well were transferred to a plate containing 100111 volumes of Coomassie Plus(~) protein assay reagent (Pierce Company, No. 23236). The protein assay plates were read by a Bio-Rad Model 450 plate-reader containing a 595nm filter, with a protein standard curve included in every assay.
The level of B-galactosidase activity in each well was measured by adding phosphate buffered saline (50 ~11) to the remaining lysates, followed by addition of a buffered solution consisting of chlorophenol red galactopyranoside (100 ~Ll, 1 mg per ml, Calbiochem No. 220~88), 60 mM disodium hydrogen phosphate pH
8.0, 1 mM magnesium sulfate, 10 mM potassium chloride, and 50 mM 2-mercaptoethanol. The chlorophenol red galactopyranoside, following enzymatic ( B-galactosidase) hydrolysis, gave a red color which was detected by a plate-reader containing a 570 nm filter. A B-galactosidase (Sigma No. G6512) standard curve was included to calibrate every assay.
Following subtraction of background readings, optical data determined by the plate-reader allowed determination of B-galactosidase activity and protein content. In comparison to the amount of B-galactosidase expressed by known transfectants, for example, DMRIE (1,2-dimyristyloxypropyl-~dimethyl-hydroxyethyl ammonium bromide), compounds of the invention are particularly effective in transfecting airway epithelial cells and inducing therein B-galactosidase expression. Relative to DMRIE:DOPE (1:1), the spermidine ,~ , , , .. . lr WO 98/02190 PCT/I~S97/12105 cholesterol carbamate: DOPE mixture (also 1:1~ demonstrated transfection efficiency improved by a factor of about 5 (see, for example, Figures 13, 14 and15).
Example 2 - Transfection of the Gene Encoding for Human Cystic Fibrosis 5 Transmembrane Conductance Regulator Protein The ability of the cationic amphiphiles of the invention to transfect cells and to induce therein biochemical corrections was demonstrated with a separate in vitro assay. immorta~i7ed human cystic fibrosis airway cells (CFT-1, as above) were used.
In preparation for the assay, the cells were grown on glass coverslips until approximately ~0% confluent. The cells were then transfected with a complex of spermidine cholesterol carbamate:DOPE (1:1) and a plasmid(pCMV- CFrR) containing a cDNA that encodes wild type hurnan CFTR. pCMV-CFTR plasrnid is a construct contairung the encoding sequence for CFTR and the following regulatory elem ents, a CMV promoter and enhancer, and an SV40 polyadenylation signal. Additional constructs suitable for the practice of this example include pMT-CFTR, Cheng et al., ~L 63, S27-834 (1990). The complex used was 10.5 llmolar of spermidine cholesterol carbamate (also of DOPE) and 30 ~nolar of pCMV-CFTR based on nucleotide.
48 hours after amphiphile-mediated transfection, cells were tested for cAMP-stimulated Cl~ channel activity using the 6-methoxy-N-~3-sulfopropyl)quinolinium ("SPQ") assay. See S. Cheng et al., ~ 66,1027-1036 (1991) for further information concerning assay methodology. In the assay, cAMP-dependent Cl~ channel activity was assessed using "SPQ" (from ~5 Molecular Probes, Eugene, Oregon), a halide-sensitive fluorophore. Increases in halide permeability results in a more rapid increase in SPQ fluorescence, and the rate of change (rather than the absolute change in fluorescence) is the important W O 98/02190 PCTrUS97112105 variable in assessing Cl- permeability. See also Rich et al., Nature, 347, 358-363 (1990) for baclcground information.
~ luorescence of the SPQ molecule in individual cells was measured using an inverted microscope, Nikon,, a digital imaging system from Universal 5 Imaging, and an ICCD camera, Hamamatsu, Inc.. Cells were selected for analysiswithout prior knowledge of their expected rate-of-change- in-fluorescence characteristics .
In each experiment, up to five microscope fields of between 90 and 100 cells were exarnined on a given day, and studies under each condition were 10 repeated on at least 3 different days. Since e,~lession of ( :~ l K is heterogenous (i.e. cells do not produce identical amounts of CFTR), the data presented were for the 20% of cells in each field exhibiting the greatest response.
As expected, cells that were mock transfected failed to exhibit any measurable increase in cAMP-stimulated halide fluorescence. In contrast, cells 15 that had been transfected with the wild type CFIR cDNA displayed a rapid increase in SPQ fluorescence upon stirnulation with cAMP agonist, indicating increased permeability to anions. Approximately 60% of the cells assayed exhibited measurable cA~IP-stimulated Cl~ channel acti~it~. Accordingly, spermidine cholesterol carbamate, and other cationic amphiphiles of the 20 invention similarly tested, are effective in transferring CFTR-encoding plasmid into immortalized CF airway cells.
Example 3 - CAT Assav part A
This assay was used to assess the ability of the cationic amphiphiles of the '~5 invention to transfect cells in vivo from live specimens. In the assay, the lungs of balb/c mice were instilled intra-nasally (the procedure can also be performed trans-tracheally) with 100 ul of cationic amphiphile:DNA complex, which was . _ CA 02260034 l999-01-ll allowed to form during a 1~minute period prior to administration according to the following p rocedure. The amphiphile (premixed with co-lipid, see below) was hydrated iII water for 10 minutes, a period sufficient to yield a suspension at twice the final concentration required. This was vortexed for two rninutes and 5 aliquoted to provide 55 microliter quantities for each mouse to be instilled.
Similarly, DNA encoding the reporter (CAT) gene was diluted with water to a concentration t~ice the required final concentration, and then aliquoted at 55 microliters for each mouse to be instilled. The lipid was gently combined with the DNA (in a polysLyl~le tube), and the complex allowed to forrn for 15 rrunutes 10 before the rnice were instilled therewith.
The plas]~ud used (pCMVHI-CAT, see Example 4) provides an encoding DNA for chloramphenicol transferase enzyme. Specifics on the arnphiphile:DNA
complexes are provided below.
Two days following transfection, mice were sacrificed, and the lungs and 1~ trachea removed, weighed, and homogenized in a buffer solution (250 rnM Tris,pH 7.S, 5mM El)TA). The homogenate was clarified by centrifugation, and the deacetylases therein were inactivated by heat treatment at 70 ~C for 10 minutes.Lysate was incubated overnight with acetyl coenzyme A and C14--chloramphenicol. CAT enzyme activity was then visualized by thin layer 20 chromatography ("TLC") following an ethyl acetate extraction. Enzyme activity was quantitated by comparison with a CAT standard curve.
The presence of the enzyme CAT will cause an acetyl group to be transferred frorm acetylcoenzyme A to C14 -chloramphenicol. The acetylated/radiolabeled chloramphenicol migrates faster on a TLC plate and 2~ thus its preseno~ can be detected. The amount of CAT that had been necessary to generate the dei ermined amount of acetylated chloramphenicol can then be calculated from standards.

The activity of spermidine cholesterol carbamate (amphiphile No.53) was determined in the CAT assay in relation to the recognized transfection reagents DMRIE and DC-Chol. Figure 10 demonstrates dramatically (as ng CAT activity per 100 mg tissue) the enhanced ability of spermidine cholesterol carbamate (amphiphile No. 53) to transfect cells in vivo, which enhancement is about 20-fold, or greater, in this assay. In the assay, activity was measured as ng CAT
erlzyme per 100 mg lung tissue. As a comparison, it is generally observed that DMRIE, a well known transfectant, when ~aled as a 1:1 molar rni~cture with DOPE and then complexed with plasmid DNA (1!7 rnM DMRIE, 1.7 mM DOPE, 1.2 mM plasmid DNA measured as nucleotide) gives about 1 to 2 ng activity per 100 mg lung tissue in this assay.
With respect to the comparison provided by Figure 10, the following conditions are of note. The transfection solution for spermidine cholesterol carbamate contained 6mM DNA measured as concentration of nucleotide, and 1.5 mM of cationic amphiphile. Following generally the procedure of Example 1, each amphiphile had also been premixed with DOPE, in this case at 1:1 molar ratio. For transfection with DC-chol, the molar ratio of DC-chol to DOPE was 3:2, and the concentrations of cationic amphiphile and of DNA (as nucleotide) were 1.3 mM and 0.9 mM, respectively. For transfection with DMRIE, the molar ratio of DMRIE to DOPE was 1:1 and the concentrations of cationic amphiphile and of DNA were 1.7 mM and 1.2 rnM, respectively. These concentrations (and concentration ratios) for each amphiphile, and colipid and DNA, had been determined to be optimal for transfection for that respective amphiphile, and accordingly were used as the basis for the comparison presented herein.
For spermidine cholesterol carbamate (amphiphile No. 53), optimization experiments were also performed to determine preferred concentrations of piasmid for a particular amphiphile concentration (see Figure 11), and also to W O 98/02190 PCTrUS97/12105 determine ~lef~:L~ed concentrations of the same amphiphile in relation to a particular plasnud concentration (see Figure 12). Transfection efficiency was optimal at an arnphiphile concentration of 1.5 rnM (DOPE also being present at 1.5 rnM), and albout 6 mM (by nucleotide) of plasmid, or about at a ratio of 1:4. It was noted, however, that concentrations of about 0.75 rnM of arnphiphile, and 3.0 mM of plasmid were less toxic to the target cells.
Intra-nasal transfection with pCMV~-CAT vector was also performed in rnice using spermidine cholesterol carbamate as cationic amphiphile but with cholesterol as co-lipid. In this experiment, the concentrations of sperrnidine cholesterol carbamate tested were between 1.0 and 1.5 mM (cholesterol being present at a 1:1 molar ratio in each case, with the mixing of amphiphile and co-lipid being performed as above). The DNA concentration ( measured as nucleotide concentration) was between 4.0 and 6.0 mM. Transfection efficiency (again measured as ng CAT/100 mg tissue) was less effective than with DOPE as co-lipid; however, the transfections were substantially more effective than those achieved using DC-Chol/DOPE.
part B
Additional experiments were performed to compare in vivo the transfection efficiency of cationic amphiphiles depicted in Figures 1, 5 and 7.
Results therefor are reported in Figures 13,14 and 15 respectively. The compounds were administered intra-nasally using between 12 and 1~ mice per compound. As in part A above, ng CAT activity was measured per 100 mg of tissue. However, improved vectors (pCF1/CAT and its near equivalent pCF2/CAT) were used. In part resulting from improved vector performance, incubations of Iysate with acetyl coenzyme A and C14-chloramphenicol were conducted for only 30 minutes. Construction of pCF1 /CAT and pCF2/CAT is described below in Example 4.

CA 02260034 l999-Ol-ll The in vivo data reported in Figures 13,14 and 15 were compiled generally as follows. As aforementioned, Figures 10 and 11 report data from the complete in vivo optirnization of amphiphile No. 53. Amphiphile No. 67 was sub~ected to a similar partial optimization. With respect to all of the other 5 cationic amphiphiles reported on, and taking advantage of numerous structural similari*es, op*~i7e~l compositions for in vivo testing were extrapolated from in vitro results. This facilitated the screening of large numbers of arnphiphiles and produced broadly, if not precisely, comparable data. For all amphiphiles other than Nos. 53 and 67, the ratio, for in vivo testing, of amphiphile concentration to 10 DOPE concentration was taken from the in vitro experiments, as was the optimized ratio of amphiphile concentration to DNA concentration (see Example 1). Accordingly, for such arnphiphiles the in vivo test concentration was fixed at lrnM, thereby fixing also the co-lipid concentration. [Broadly, the molar ratio of the amphiphile to co-lipid DOPE ranged from 1:2 (for example, spermine 1~ cholesterol carbamate, No. 67) through 1:1 (for example, spermidine cholesterol carbamate, No. 53~ to about 2:1 (for example, amphiphile No. 75)]. The concentration of plasmid DNA varied for each amphiphile species tested in order to duplicate the optimized amphiphile/DNA ratio that had been determined in vitro.
20 part C
That the novel amphiphiles of the invention are an important contribution to the art is irr~nediately seen by comparing their performance - as in vivo transfection enhancers - to that of closely related cationic amphiphiles that lack the novel T-shape. It has been determined that spermidine cholesterol carbamate 2~ provides a much greater level of enhancement than N1-spermidine cholesteryl carbamate which contains the same number of carbon and nitrogen atoms in its cationic alkylamine component but which is linear and not "T-shaped".

t I I '" I'r CA 02260034 lsss-ol-ll Following generally the procedures of Example 3, part B, and using respectively 6mM (as nucleotide), 1.5 mM, and 1.5 mM concentrations of DNA, amphiphiIe and of co-lipid, the transfection enhancement provided by spermidine chiolesterol carbamate (amphiphile No.53), in relation to Nl-sperrnidine cholesteryl carbaimate, was determined to be about 30 fold.
Also following the procedures of Example 3, part B, and using respectively 4m~i (as nucleotide), lmM, and 2 mM concentrations of DNA, amphiphile and co-lipid, the transfection enhancement provided by spermine cholesterol carba.mate (amph;iphile No. 67)--in relation to Nl-thermospermine cholesteryl carbamate and Nl-spermine cholesteryl carbamate to whichi sperrr~inecholesterol carba.mate is similarly related--is at least about 30 fold.
Example ~ Construction of vectors As aforernentioned, numerous types of biologically active molecules can be transported ir-to cells in thierapeutic compositions that comprise one or more of the cationic arnphiphi}es of the invention. In an important embodiment of theinvention, the biologically active macromolecule is an encoding DNA. There follo~ ~s a description of novel vectors (plasmids) that are preferred in order to facilitate expression of such encoding DNAs in target cells.
part A--pCMVHI-CAT
pCMVHI CAT is representative of plasmid constructs useful in the practice of the invention. Although the plasmid is provided in a form carrying areporter gene (see Example 3), transgenes having therapeutic utility may also beincluded therein.
The pCMVHI-CAT vector is based on the commercially available vector pChlV~ (Clontech). The pCMV~ construct has a pUC19 backbone a Vieira, et al., Gene ,19, 25'3-268, 1982) that includes a procaryotic origin of replicationderived original~y from pBR322. Basic features of the pCMVHI-CAT plasmid (as WO 98/02190 rCT/US97/12105 constructed to include a nucleotide sequence coding for CAT) are as follows.
Proceeding clockwise--the human cytomegalovirus imrnediate early gene promoter and enhancer, a fused tripartite leader from adenovirus and a hybrid intron, a linker sequence, the CAT cDNA, an additional linker sequence, the late5 SV40 polyadenylation signal, and the pUC origin of replication and backbone that includes the gene for ampicillin le~isL~Ice.
The human cytomegalovirus irnsne~ te early gene promoter and enhancer spans the region from nucleotides 1-639. This corresponds to the region from -522 to +72 relative to the transcriptional start site (+1) and includes alrnost the entire enhancer region from -524 to -118 as originally defined by Boshart et al, Cell 41:521-530, 1985. The CAAT box is located at nucleotides 487-491 and the TATA box is at nucleotides 522-526 in pCMVHI-CAT. The CAT
transcript is predicted to initiate at nucleotide 549, which is the kanscriptional start site of the CMV promoter. The tripartite leader-hybrid intron is composed of a fused tri-partite leader from adenovirus containing a 5' splice donor signal, and a 3' splice acceptor signal derived from an IgG Ooene. The elements in the intron are as follows: the first leader, the second leader, part of the third leader, the splice donor sequence and inkon region from the first leader, and the mouse imrnunoglobulin gene splice donor sequence. The length of the inkon is 230 nucleotides. The CAT coding region comprises nucleotides 1257-1913. The SV40 poly A signal extends from nucleotide 2020 to 2249.
Accordingly, construction of the pCMVHI-CAT plasrnid proceeded as follows. The vector pCMV~ (Clontech, Palo Alto, CA) was digested with Not } to excise the ~-galactosidase gene. The vector fragment lacking the ~-galactosidasegene was isolated and ligated to form pCMV.
The hybrid intron (Figure 17) was obtained from the plasrrLid pAD~
(Clontech) The hybrid intron had been isolated from a 695 base pair XhoI-EcoRI

~ . I ..

CA 02260034 l999-Ol-ll fragment of p9~ 023(B), see Wong et al., Science, 228, 810-815 ~1985). The hybrid intron contains the fused Llipal lile leader from adenovirus, the donor site from the first segment of the tripartite leader, and the acceptor site from an IgG gene, and has a length of 230 bp.
pAD~ w,as digested with Prnl 1 and Not I, and the -500 base-pair (bp) fragment was isolated, and then ligated into the Not I site of pBluescript~ KS(-) (Stratagene, La Jolla, CA) to form pBlueII-HI.
- pBlueII-~ was digested with XhoI and Not~ to excise the hybrid intron fragment. This fragment was ligated into the XhoI and NotI sites of pCMV, replacing the S~140 intron to form pCMV~.
The CAI gene was obtained from the Chloramphenicol Acetyltransferase GenBlock (Pharmacia, Piscataway, NJ). This 792 bp Hind m fragment was blunted with the Klenow fragrnent of DNA Polymerase I, then Not I linkers (New England 13iolabs) were ligated to each end. After digestion with Not I to 1~ expose the Not [ sticky ends, the fragment was subcloned into the Not I site of pCMV to forrn pC~I-CAT. pCMV-CAT was digested with Not I to excise the CAT fragment. The CAT fragrnent was ligated into pCMVHI to form pCMVHI-CAT which is depicted in Figure 16.
part B--pCF1 ~md pCF2 Although pCMVHI is suitable for therapeutic transfections, further performance enhancements (including increased expression of transgenes) are provided by the pCF} and pCF2 plasmids. A map of pCF1/CAT is shown in Figure 18, panel A, and a map of pCF2/CAT is shown in panel B.
Briefly, pCF1 contains the enhancer/promoter region from the immediate early gene of cytomegalovirus (CMV) . A hybrid intron is located between the promoter and the transgene cDNA. The polyadenylation signal of the bovine growth hormone gene was selected for placement downstream from the CA 02260034 l999-01-ll transgene. The vector also contains a drug-resistance marker that encodes the arninoglycosidase 3'-phosphotransferase gene (derived from the transposon Tn903, A. Oka et al., Journal of Molecular Biology, 147, 217-226, 1981) thereby conferring resistance to kanamycin. Further details of pCF1 structure are 5 provided directly below, including description of placement therein of a cDNA
sequence encoding for cystic fibrosis transmembrane conductance regulator (CFTR) protein.
The pCF1 vector is based on the commercially available vector pCh~VB
(Clontech). The pCMV~ construct has a pUC19 backbone a Vieira, et al., Gene, 19, 259-268, 1982) that includes a procaryotic origin of replication derived originally from pBR322.
Basic features of the pCF1-plasmid (as constructed to include a nucleotide sequence coding for CFTR) are as follows. Proceeding clockwise--the human cytomegalovirus immediate early gene promoter and enhancer, a fused tripartite 15 leader from adenovirus and a hybrid intron, a linker sequence, the CFTR cDNA,an additional linker sequence, the bovine growth hormone polyadenylation signal, pUC origin of replication and backbone, and the kanamycin resistance gene. The pCF1-CFTR plasmid has been completely sequenced on both strands.
The human cytomegalovirus immediate early gene promoter and 20 enhancer spans the region from nucleotides 1-639. This corresponds to the region from -522 to +72 relative to the transcriptional start site (+1) and includes almost the entire enhancer region from -524 to -118 as originally defined by Boshart et al., Cell 41, 521-530 (1985). The CAAT box is located at nucleotides 486-490 and the TATA box is at nucleotides 521-525 in pCF1-CFTR. The CFTR
25 transcript is predicted to initiate at nucleotide 548, which is the transcriptional start site of the CMV promoter.

~ , , _ rr The hybrid intron is composed of a fused tri-partite leader from adenovirus containing a 5' splice donor signal, and a 3' splice acceptor signal derived from an IgG gene. The elements in the intron are as follows: the first leader (nucleotiides 705-7g5), the second leader (nucleotides 746-816), the third leader (partial, nucleotides 817-877), the splice donor sequence and intron region from the first leader (nucleotides 878-1042), and the mouse immunoglobulin gene splice donor sequence (nucleotides 104~1138). The donor site (G I ~E) is at nucleotides 887-888, the acceptor site (AG I G) is at nucleotides 1128-1129, andthe length of the intron is 230 nucleotides. The CFl~ coding region comprises nucleotides 1183-5622.
Within the CFI'R-encoding cDNA of pCF1-C~ , there are two differences from the originally-published predicted cDNA sequence a ~iordan et al., Science, 245, 1066-1073, 1989); (1) an A to C change at position 1990 of the CFTR cDNA which corrects an error in the original published sequence, and (2) a T to C change introduced at position 936. The change at position 936 was introduced by site-directed mutagenesis and is silent but greatly increases the stability of the cDNA when propagated in bacterial plasmids (R. J. Gregor,v et al.
et al., Nature,347, 382-386,1990). The 3' untranslated region of the predicted CFI R transcript comprises 51 nucleotides of the 3' untranslated region of the 2b CFIR cDNA, 2] nucleotides of lin}~er sequence and 114 nucleotides of the BGH
poly A signal.
The BGH poly A signal contains 90 nucleotides of flanking sequence 5' to the conserved AAUAAA and 129 nucleotides of flanking sequence 3' to the AAUAAA motif. The primary CFTR transcript is predicted to be cleaved ~5 downstream of the BGH polyadenylation signal at nucleotide 5808. There is adeletion in pCFl-CFI'R at position +46 relative to the cleavage site, but the deletion is not predicted to effect either polyadenylation efficiency or cleavage CA 02260034 l999-01-ll site accuracy, based on the studies of E.C. Goodwin et al., J. Biol. Chem., 267,16330-16334 (1992). After the addition of a poly A tail, the size of the resulting transcript is approximately 5.1 kb.
pCF2 plasmid, Figure 18 (B), contains a second CMV enhancer, in tandem 5 with the first. En'hanced expression of transgenes from pCF1 or pCF2 results from the combination of a strong promoter, the presence of a highly efficient polyadenylation signal, a leader se~uence that enhances translation, and an intron to increase message stability.
Example 5- Correction of Chloride Ion Transport Defect in Nasal Polyp 10 Epithelial Cells of a Cystic Fibrosis Patient by Cationic Amphiphile-Mediated Gene Transfer Prirnary (non-immortalized) nasal polyp cells from an adult male cystic fibrosis patient (homozygous for the a F508mutation) were grown on collagen-coated permeable filter supports (Millic~ ) to form a polarized and confluent 15 epithelial monolayer. Once the monolayer was electrically tight (about 5 to 7days post seeding, and as indicated by the development of resistance across the cell sheet), the apical surface can be exposed to formulations of cationic amphiphile: DNA complex.
In this case, the amphiphile (spermidine cholesterol carbarnate ) was 20 provided as a 1:1 (by mole) mixture with DOPE, and this mixture was then complexed with pCMV-CFTR plasmid vector (a construct encoding wild type human cystic fibrosis transmembrane conductance regulator protein, see above).
Concentrations in the final mixture were 4~ llmolar of spermidine cholesterol carbamate(and also of DOPE) and 60 llmolar (based on molarity in nucleotides) 25 of the plasmid expression vector.
Expression of CFrR was determined by measuring cAMP-stimulated transepithelial chloride secretion in a modified Ussing chamber, Zabner et al., CA 02260034 l999-Ol-ll l~ah~re Genetics ,6, 75-~3 (1984). The mucosal side of the epithelium was bathedin Ringer's bicarbonate solution bubbled with 95% ~2 and 5% CO2. The composition of l:he submucosal solution was similar to the mucosal solution withthe exception that sodiurn gluconate replaced sodium chloride. Transepithelial 5 voltage was clamped to 0 mV and short circuit current was recorded. Amiloride (10 IlM) was applied into the apical bath, followed by the mu~osal addition of forskolin and IBMX (at 100 IlM each). ~nitro-2-(3-phenylpropylamino) benzoic acid ("NPPB"), an inhibitor of C~-lK chloride channels, was then added to the mucosal solutiorl at 10 to 30 ,uM.
Chloride secretion (i.e. movement of chloride from the epithelial cells to the mucosal solution) is shown as an upward deflection (see Figure 19A). The same plasmid vector, but containing a reporter ~ene~ was used as a negative control (~ ure 19B) . A cAMP stimulaled current (O.S to 2.5 ~ampere/cm2) was observed in monolayers transfected witll wild type Cl;TR gene. Current was not detected with the 15 pCMB-,B-galactosidase control.
Fxample 6- Correction of Chloride lon Transport Defect in Ain~ray Epithelial Cells of a Cystic Fibrosis Patient bv Cationic Amphiphile-Mediated Gene Transfer A recommended procedure for formulating and using the pharmaceutical 20 compositions of the invention to treat cystic fibrosis in human patients is as fol~ows.
Following generally the procedures described in Example 1, a thin film (evaporated from chloroform) is produced wherein spermine cholesterol carbamate (amphiphile No. 67) and WPE are present in the molar ratio of 1: 2.
25 The amphiphile-containing film is rehydated in water-for-injection with gentle vortexing to a resultant amphiphile concentration of about 3mM. However, in order to increase the amount of amphiphile/DNA complex that may be stably CA 02260034 l999-Ol-ll delivered by aerosol as a homogeneous phase (for example, using a Puritan Bennett Raindrop nebulizer from Lenexa Medical Division, Lenexa, KS, or the PARI LC JetTM nebulizer from PARI Res~ildLoly Equipment, Inc., Richrnond, VA), it may be advantageous to prepare the amphiphile-containing film to 5 include also one or more further ingredients that act to sta~lize the final amphiphile/DNA composition. Accordingly, it is ~iesently ~re~lled to prepare the amphiphile-containing film as a 1: 2: 0.05 molar mixture of amphiphile No.
67, DOPE, and PEG~5000)-DMpE. [A suitable source of PE~D~E, polyethylene glycol 5000 - dimyristoylphoshatidyl ethanolarr~ine, is Catalog No.10 880210 from Avanti Polar Lipids, Alabaster, AL]. Additional fatty acid species of PEG-PE may be used in replacernent therefor.
Without being limited as to theory, PEG(5000)-DMpE is believed to stablize the therapeutric compositions by preventing further agrregation of formed amphiphile/DNA complexes. Additionally it is noted that PEG(200o)-15 DMPE was found to be less effective in the practice of the invention.
pCF1-CFTR plasmid (containing an encoding sequence for human cystic fibrosis transmembrane conductance regulator, see Example 4) is provided in water-for-injection at a concentration, measured as nucleotide, of 4 mM.
Complexing of the plasmid and amphiphile is then allowed to proceed by gentle 20 contacting of the two solutions for a period of 10 minutes.
It is presently preferred to deliver aerosolized DNA to the lung at a concentration thereof of between about 2 and about 12 mM (as nucleotide). A
sample of about 10 to about 40 ml is generally sufficient for one aerosol administration to the lung of an adult patient who is homozygous for the ~ F508 25 mutation in the CFTR-encoding gene.
It is expected that this procedure (using a freshly prepared sample of amphiphile/DNA) will need to be repeated at time intervals of about two weeks, T . I ' ' ' - '' ' lr CA 02260034 lsss-ol-ll but depending considerably upon the response of the patient, duration of expression from the transfected DNA, and the appearance of any potential adverse effects such as inflammation, all of which can be determined for each individual patie~tt and taken into account by the patient's physicians.
One irnportant advantage of the cationic amphiphiles of the present invention is that they are substantially more effective--in vivo--than other presently available amphiphiles, and thus may be used at substantially lower concentrations than known cationic arnphiphiles. There results the opportunity to substantially minimize side effects (such as amphiphile toxicity, inflamrnatory response) that would otherwise affect adversely the success of the gene therapy.A further rparticular advantage associated with use of many of the amphiphiles of the invention should again be mentioned. Many of the amphiphiles of the invention were designed so that the metabolism thereof would rapidly proceed toward relatively harrnless biologically-compatib}e components. In this regard, highly active amphiphiles 53, 67, and 75 are of particular note.
Alternate Procedure to Prepare an Amphiphile/Co-lipid Composition In order ta formulate material that is suitable for clinical administration, it may be preferable to avoid use of chloroform when the cationic amphiphile and the co-lipid are p:repared together. An altemate method to produce such compositions is suggested using formulation of amphiphile 67 (N4- spermine cholestryl carbarnate, Figure lA) as the example.
The cationic amphiphile, the neutral co-lipid DOPE, and PEG(5000)-DMpE
are weighed into vials, and each is dissolved in t-butanol:water 9:1 with vortexing, followed by transfer to a single volumetric flask. An appropriate amount of each lipid is selected to obtain a molar ratio of cationic amphiphile to DOPE to DMPE-I'EG of 1: 2: 0.05. The resultant solution is vortexed, and , . . _ .. _. ...

CA 02260034 l999-01-ll WO g8/02190 PCT/US97/12105 further diluted as needed with t-butanol:water 9:1, to obtain the desired concentration. The solution is then filtered using a sterile filter (0.2 nucron,nylon).
One mL of the resultant filtered 1: 2: 0.05 solution is then pipetted into individual vials. The vials are partially stoppered with 2-leg butyl stoppers and placed on a tray for lyophili~ation. The t-butanol:water 9:1 solution is removedby freeze drying over 2 to 4 days at a temperature of approximately -5~C. The lyophilizer is then backfilled with argon that is passed through a sterile 0.2 rnicron filter. The stoppers are then fully inserted into the vials, and the vials are 10 then c~iInped shut with an alurninum crimp-top. The vials are then maintained at -70~C until use .

Example 7- Further ErLhancements in Plasmid Design for Gene Therapv:
Replicating Episomal Plasmids Although the above design features substantially enhance th performance of available plasmids, further modifications are desirable in order that therapeutic compositions comprising such plasmids and cationic amphiphiles have optimal performance for gene therapy.
It is desirable that plasmids for gene therapy also be able to replicate in the 20 cells of patients, since continued presense of the plasmid will provide correction of the genetic defect (in the case of cystic fibrosis, lack of functioning C~
protein in the cell membrane of lung epithelial cells or other cells) over an extended period of time. There is concem that plasmids representative of the current art (that is, those that cannot replicate in the targeted cells of a patient) may be degraded after only a relatively short period of maintenance in the patient, thus requiring excessive repeat administrations.

lr CA 02260034 l999-01-ll W O 98/02190 PCT~US97/12105 Long term correction could perhaps be achieved using a vector designed to integrate into chromosomes in the patient's targeted cells (for example, vectors patterned on retrovirus). Such a strategy, however, involves risks including (1)that the vector 1~.rill integrate into an essential region of a chromosome, or (2) that 5 the vector will integrate adjacent to an oncogene and activate it.
Accordingly, it would be desirable to provide for con~inlle~ maintenance of gene therapy vectors (plasmids) in target cells by other means. One such strategy is to construct a plasmid capable of being maintained separately in thenucleus of a tsrget cell, and that is also able to replicate there (i.e. an episome).
Plasmidc, provided according to this aspect of the invention can be constructed as follows. It has been ~let~m ined (C. McWhinney et al., Nucleic Acids Research. 18, 1233-1242,1990) that the 2.4 kb Hindm-XhoI fragment that is present immediately 5' to exon 1 of the human c-myc gene contains an origin of replication. The fragment was then cloned into a plasmid that if transfected into 15 HeLa cells was ;hown to persist therein for more than 300 generations under drug selection. Replication was shown to be semiconservative (C. McWhinney et al.). Although approximately 5% of the plasmid population was lost per cell generation without drug selection in those experiments, this result nonetheless demonstrates substantial stabilization would be of benefit with respect to the 20 design of therapeutic plasmids for gene therapy.
Accordingly, in one example of a replicating episomal vector, a variant of pCF1-CFTR (or pCF1-CAT) can be constructed in which a copy of the 2.4 kb Hindm-XhoI fragment is placed just 5' to the CMV enhancer/promoter region of the pCF1 backbone. Alternatively, between 2 and about 4 - in tandem - copies of 25 the 2.4 kb fragrnent may be similarly positioned. The increase in plasmid size that results from insertion of the 2.4 kb fragment (or multiple copies thereof) is CA 02260034 lsss-ol-ll wo 98/02190 PCT/US97tl2105 predicted to provide an additional benefit, that is, to facilitate plasmid unwinding, thus facilitating the activity of DNA polymerase.
Use of this origin of replication, or multiple copies thereof, allows the resultant plasrnid to replicate efficiently in human cells. Other DNA sequences 5 containing other origins of replication may also be used (for example, as found in the hu~nan ~-globin gene, or the mouse DH~;R gene.
A plasrnid that can be constructed according to this aspect of the invention and containing the cytomegalovirus promoter and enhancer, an intron, the CFTR
cDNA, the bovine growth hormone polyadenylation signal, the kanamycin resistance transposon Tn903, and 4 copies of the 2.4 kb 5' flanking region of the human c-myc gene is shown in Figure 20.
Example 8- Further Enhancements in Plasmid Design for Gene Therapv:
Use of Cvtokine Promoters to Modulate Expression of Transgenes in Gene Therapv Chronic inflammation is associated with numerous of the disease states that can be treated by gene therapy. Representative of such disease states are cystic fibrosis (using CFTR), bronchitis, adult respiratory distress s~ndrome 20 (using alpha-1 antitrypsin), and metastatic cancers (through upregulation of p~3, llMP-l, and T~MP-2). Inflammatory conditions typically involve many interrelated processes (for example, involvement by many types of immune system cells and liver proteins), whereby the body attempts to heal a damaged orinfected tissue. ~Iowever, chronic inflammation w hich persists as a result of an 25 unresolved condition may lead to perrnanent tissue damage, as is the case with respect to lung tissue affected by cystic fibrosis and associated and unresolvedlung infections. In fact, permanent damage to the ~ung tissue of cystic fibrosispatients is a leading cause of their mortality. It would be desirable to provide ~4 CA 02260034 lsss-ol-ll gene therapy in such a manner as to treat inflamrnatory conditions associated with the targeted disease state.
Accordingly, a further aspect of the present invention involves construction of gene therapy vectors in which the therapeutic transgene is placed 5 under control of an RNA polymerase promoter from a cytokine gene (or a gene that encodes aIIother similar regulatory protein) such as, for example, the promoter for any of interleukin 2, interleukin 8, interleukin I, interleukin 11,interleukin 6, endothelin -1, monocyte chemoattractant protein -1, IL-lra (receptor agoni.st), or for GM-CSF.
Cytokines may be defined as hormone-like intercellular signal proteins that are involved in regulation of cell proliferation, differentiation, and function, such as conceming haematopoiesis and lymphopoiesis. The interleulcins are a particular group of cytokines having promoters that are useful in the practice of the invention. The interleukins are proteins, typically of unrelated origin, which 15 act as intercellular signals mediating reactions between i~ununoreactive cells.
However, it is understood that many "interleukins" have effects upon additional cell types including endothelial cells, epithelial cells, and fibroblasts.
Since the concentration of many cytokines is upregulated at an affected site in response to the level of inflammation that is present, gene therapy vectors 20 can be designed wherein the level of therapeutic transgene expressed thele is determined, in part, by the level of inflammation present. There follows hereafter description of how such vectors are designed using primarily properties of the interleu}~in 8 gene as an example.
It has been determined that numerous biologically active molecules are 25 present in tissues at concentrations thereof that increase with the severity of an inflammatory c ondition (for example, turnor necrosis factor "TNF" and potentially transcription factors such as NF-kB, AP-1, NF-IL6 and octamer binding protein).

CA 02260034 lggg-ol-ll It has also been determined that interleukin 8, a polypeptide of 8,500 MW, is upregulated by irlflammation and acts as a potent chemoattractant for T
Iymphocytes and neutrophil cells that are themselves involved in the inflarnmation response. The interleukin 8 gene is regulated primarily at the 5 transcriptional level, and it has also been determined (H. Naka~nura et al., Joumal of Biological Chemistry, 266, 19611-19617, 1991) that TNF can increase interleukin 8 transcription by more than 30 -fold in vitro in bronchial epithelial cells. Accordingly, there follows description of gene therapy vectors which takeadvantage of the above.
10A plasmid can be constructed that is substantially sirnilar to pCF1, that is, derived from a pUC plasrnid containing a bacterial-derived origin of replicationand a gene conferring resistance to kanarnycin. The resultant plasmid contains also, in se~uence, a C~V enhancer, a promoter, a hybrid intron, a cDNA
sequence encoding CFll~, and the bovine growth hormone polyadenylation 15signal. As RNA polymerase promoter there is selected the -335 to ~ 54 region of the interleukin 8 promoter. 'Ihis region gave the highest ratio in terms of promoter activity plus TNF over minus TNF (Nakamura, 1991) Such a plasmid has particularly valuable performance attributes. As inflammation increases in a cystic fibrosis-affected lung (and therefore the need 20 to treat the lung with gene therapy also increases), the concentration of various inflammation-related molecules ( such as TNF) will increase. By placing the CFrR-encoding cD~JA of the therapeutic plasmid under the control of a transcriptional promoter (that of interleukin 8, for example) that is itself sensitive to the concentration of inflammation-related substances in contact with the cell, ~a the promoter will function as a natural gene switch such that the amount of beneficial CFIR transcription will be tailored to the amount of inflammation. As r I I lr CA 02260034 lsss-ol-ll aforementioned, RNA polymerase promoter sequences derived from the other aforementioned genes are also useful in the practice of the invention.
Example 9 Intravenous Delivery of Transgenes For some disease states, such as cystic fibrosis, it is desirable to 5 deliver transgenes to the lung. Delivery by aerosol is the most direct approach to achieve this goal. However, given the ~ifficl-lti~c inherent with the delivery of an aerosol together with the potential need to target organs other than the lung (for example, the pancreas for cystic fibrosis), it is important to evaluate the feasibility of lung delivery using non-aerosol 10 delivery formats Accordingly, il~L~dvellous delivery of a reporter transgene was performed using a mouse model and the feasibility of intravenous organ targeting was assessed. A comparison was made of feasibility of deli very to the lung and the heart.
The reporl er plasmid pCF-1 CAT (~:xample 4) was used and was 1~ purified to mirLimize endotoxin (<1 EU/mg pDNA), and also chromosomal DNA contamination (< 2%). Amphiphile No. 53 (1:1 with DOPE) / DNA
complex was prepared according to the procedures of Example 3. The amphiphile was provided as the free base, the plasmid was prepared as a sodium salt in water, and the DOPE was provided in zwitterionic form.
The animal model was the BALB/c mouse. Females ~ weeks old weighing 16-18 g were injected intravenously using the tail vein, using 5 animals per group. The volume of lipid:pDNA complex used was 100 ~l in all experiments. Unless noted otherwise, mice were sacrificed 48 h following adminctration of the complex. Organs were frozen immediately ~5 on dry ice to store for subse~uent analysis.
Expressior of chloramphenicol acetyl transferase (CAT) was quantitated using a radiochemical assay for CAT enzymatic activity.

.. . .

CA 02260034 l999-01-ll Organs were weighed and homogenized on ice in a lysis buffer containing protease inhibitors. The lysate was freeze-thawed 3X, centrifuged, and heated to 65~C to inactivate deacetylases before adding it to a reaction mixture contairung 14C-chloramphenicol. After an incubation at 37~C, the 5 rnixture was extracted with ethyl acetate, concentrated, spotted onto TLC
plates and eluted with CHC13/MeOH. Spots corresponding to the acylated reaction products were quantitated (Betagen) and converted to ng CAT
activity using authentic CAT standards.
It was surprisingly dete~..Lined that targting to the heart could be 10 substantially ~ ed by altering the molar ratio (at a constant DNA
concentration of 0.9 mM, measured as nucleotide) of arnphiphile/DNA in the therapeutic composition. This information is of value in connection with gene therapy for the heart, such as for coronary disease. However, targeLing to the lung remained relatively constant over a range of 15 amphiphile/DNA ratios, all at constant DNA concentration (Figure 21).
At molar ratios of less than about 0.5, the organ distribution was found to be strongly weighted toward the lung. At this molar ratio, the zeta potential of the complex is negative (about -30 mV) due, in part, to excess negative charge from the DNA relative to the amphiphile. At an 20 amphiphile/DNA ratio of 1.25, however, where the complex has a positive zeta potential (about +30mV), organ distribution was remarkably altered and substantial expression was found in the heart (Figure 21).
Zeta potentials of the samples can be measured (using typically 5 measurements per sample) employing a Malvern Zetasizer 4 (Malvern 25 Instruments, Southborough, MA.) and a zeta cell (AZ-104 cell, Malvem Instruments Co.). Dried lipid films containing the cationic lipid and DOPE
are hydrated in distilled water (dH2O). DNA typically should be diluted to T I I ' ' ' ~r CA 02260034 l999-01-ll a concentration of about 300 ~LM in dH20. The DNA solution (1.5 mL) can then be added to an equal volume of cationic lipid vesicles and incubated at room temperature for 10 min. Enough NaCl (for example, 4 rnM stock) may be added to result in a final concentration of 1 mM NaCl. If necessary, the 5 sample can be diluted furtlner with 1 mM NaCl (to maintain a photomultiplier signal below 4000 counts per second), and distilled water can be used in place of the NaCl solutions.
According to this aspect of the invention, amphiphiles No. 53 and No. 67 are among those ~efe,led for use in intravenous talgeLil~g of the 10 heart, as are many other amphiphiles selected from Groups I and r[.
Example 10- Additional Experimental Procedures (A) Additional synthesis procedure for N~spermine cholestervl carbamate, amphiphile No. 67 (S~mthesis of N1,N12 -diCbz-sperrnine di-HCl salt) Benzylchloroforrnate (15 mL, 105 rnmol) was dissolved in methylene chloride (335 ml ) and placed in a three neck flask under a nitrogen atmosphere.Imidazole (14 g, 206 mmol) was dissolved in methylene chloride (200 mL). The three neck flask was cooled to 0-2 ~C using an ice-water bath and the irnidazolesolution was added gradually over 30 min. The cooling bath was removed and the mixture stirIed at room temperature for 1 hour. Methylene chloride ( 250 mL)and aqueous cib-ic acid (10%, 250 mL) were added to the mixture. The layers were separated imd the organic layer was washed with aqueous citric acid (10%, 2~0 mL). The orgar ic fraction was dried over magnesium sulfate and concentrated in uacl~o. The resulting oil was vacuum dried for 2 hours at ambient temperature. Tc) the oil was added dimethylaminopyridine (530 mg, 4.3 mmol) and methylene chloride (250 mL). The mixture was cooled to 0-2 ~C and kept under a nitrogen atmosphere. A solution of spermine (lOg, 49 mmol) in CA 02260034 l999-01-ll W O 98/02190 PCT~US97/12105 methylene chloride (250 mL) was added gradually over 15 minutes, maintaining a reaction temperature of 0-2 ~C. The reaction mixture was stirred overnight at ambient temperature and then concentrated in vacuo. To the resulting material was added lM hydrochloric acid (67 mL) and methanol (400 mL). The solution 5 was cooled overnight at 4 ~C yielding a white precipitate. The precipitate wasisolated by vacuum filtration using Whatrnan #1 filter paper. The Nl,N 12-diCbz-spermine di HCl salt (13.38g, 24.7 mmol, 50% yield) thus obtained was dried under vacuum at ambient temperature for 17 hours.
(Synthesis of Nl,N12-diCbz-N4-spermine cholesteryl carbamate) N1,N12-diCbz-spermine di HCl salt (13.38g, 24.7 rnrnol) was dissolved in a chloroform, methanol and water rnixture in the ratio 65:25:4 (940 mL). The solution was stirred at room temperature and cholesteryl chloroformate (llg, 24.5 mmol) was added. The solution was stirred at ambient temperature for 1.5 hours and then diluted with lM sodium hydroxide solution (165 mL). The organic and 15 aqueous layers were separated and the organic layer containing the product was washed with water (110 mL). The organic fraction was dried over sodium sulfate, concentrated i~ VQCUO and vacuum dried. The crude oil was purified by chromatography usin3 silica gel (60A, 1 Kg) . The silica was pacl;ed in 10% MeOH/ CHCl3 and the column was eluted with 25% MeOH / CHCl3. Fractions of 900 20 mL were collected and analyzed by thin layer chromatography. Fractions containing the product (Rf. = 0.5 in 20% MeOH / CHCl3) were combined and concentrated in vac~o. The resulting oil was dried under vacuurn for 17 hours togive 8.5g (9.67 mmol, 39% yield) of product.

r ~ ! ' '''' 1~

CA 02260034 lsss-ol-ll (Synthesis of N4-spermine cholesteryl carbamate) N~ diCbz-N4-sperrnine cholesteryl carbamate (8.5g, 9.67 rMnol) was dissolved in 200 mL of acetic acid and 1.66 g of 10% Pd on carbon was added.
The solution was purged with nitrogen and stirred under hydrogen at atmospheric pressure. The hydrogen was supplied to the reaction flask using a balloon filled with hydrogen gas. The hydrogenolysis was al~owed to proceed for 3 hours. The reaction mixture was filtered through Whatman #1 filter paper and the catalyst was washed with 250 mL of 10% acetic acid in ethyl acetate. The filtrate was concentrated in vacuo to give a residue, coevaporation with chloroform aids removal of the acetic acid. To the crude product was added lM
sodium hydroxide solution (400 mL) and the solution was extracted three times with 10% MeOH / CHC13 (700 mL). The combined organic ~ractions were washed with water (600 mL) and dried over sodium sulfate. The solution was filtered, concentrated in vacuo and vacuum dried at ambient temperature for 48 hours. The crude material was purified by chromatography on silica gel (500 g).
The column was packed in 40:25 MeOH: CHC13 and eluted with 40:25 MeOH:
CHC13 and then 40:25:10 MeOH: CHC13: NH40H. The fractions collected were analyzed by thin layer chromatography and the product containing fractions were combined and concentrated in vacuo (the evaporation was assisted by the addition of iso-propanol in order to azeotrope the residual water). The materialwas vacuum dried at ambient temperature for 48 hours to give N4-spermine cholesteryl carbamate (4g, 6.5 mmol, 67% yield).
rB) N~-(N'-cholestervl carbamate glycineamide)-spermine (amphiphile No. 91) N-t-BC~C-glycine-N-hydroxysuccinimide ester (0.5 g, 1.83 mmol) was added to a solution of diCbz-spermine-2HCl (1.0 g, 1.94 rnmol) and N,N-diisopropylethy]amine (0.3 mL, 1.72 mmol) in 65/ 25/ 4 chloroform/ methanol/
water (50 mL). 1~he solution was stirred ovemight at room temperature.

CA 02260034 lsss-ol-ll wo 98/02190 PCT/US97/12105 Analysis of the reaction by TLC (20% methanol/ chioroform) indicated the presence of a new spot. The reaction was washed first with lM NaOH (10 mL) then with H2O (10 mL). The organic layer was separated, dried over sodium sulfate, vacuum filtered, and reduced in vacuo to an oil. The crude material was5 purified by flash column chromatography (85 g silica gel) eluting with 20%
methanol/ chloroform. The desired product was isolated and characterized by lH NM~ as Nl,NL~diCbz-N4 (N'-t-BOC-glycineamide)-spermine (402 mg, 0.65 mmol, 35%).
Benzyl chloroformate (100 mg, 0.58 mmol) was added to a solution of N1,N12-diCbz-N4-(N'-t-BOC-glycineamide)-spermine (220 mg, 0.354 mmol) and triethylamine (4 drops) in methylene chloride (20 mL). The reaction was stirred overnight at room temperature. Analysis of the reaction by TLC (20% methanol/
ch~oroform) indicated the presence of a new, higher running spot. The reaction was quenched by the addition of lM HCl (5 mL). The organic layer was isolated, washed with H20 (5 mL), dried over sodium sulfate, filtered, and reduced in vacuo .
The resulting crude material was dissolved in chloroform (30 mL) and anhydrous HCI gas was bubbled through the solution for 2 hours. Analysis of the reaction by TLC (10% methanol/ chloroform) indicated the complete disappearance of the starting material. The reaction was purged with dry nitrogen, and washed with lM NaOH (2 x 10 mL) and dH20 (l0 mL). The organic layer was isolated, dried over sodium sulfate, filtered, and reduced in vacuo to give N1,N9,N1~triCbz-N4-glycineamide-spermine (219 mg, 0.33 mmol, 93% yield for two steps).
Cholesteryl chloroformate (148 mg, 0.33 mmol) w as added to a solution of N1,N9,N12-triCbz-N4-glycineamide-spermine (219 mg, 0.33 mmol) and triethylamine (0.3 mL, 2.15 mmol) in methylene chloride (30 mL). The reaction W O 98/02190 PCT~US97/12105 was stirred at r oom temperature for 3 hours. The reaction was washed with H2O
(10 mL). The organic layer was separated, dried over sodium sulfate, filtered, and reduced iti~ vacuo . The crude material was purified by flash column chromatography (30 g silica gel) eluting with 65% ethyl acetate/ hexanes. The 5 desired product was isolated and char~ct~ri7e~l by lH NI~ as Nl,N9,N~
tri0z-N~(N'-cholesteryl carbamate glyriT~ irle)-sperrnine (221 mg, 0.2 mmol, 62% yield).
N1,N9,Nl;~tri-Cbz-N~(N'-cholesteryl carbamate glycineamide)-spermine (221 mg, 0.2 mmol) was stirred with 10% Pd/C (50 mg) in glacial acetic acid (10 10 mL) under a hydrogen atmosphere for 2.5 hours. Analysis of the reaction by TLC (65% ethyl acetate/ hexanes) indicated the complete disappearance of the starting material. The flask was purged with nitrogen and the catalyst was removed by vacuum filtration through filter paper rinsing with 10% acetic acid/
ethyl acetate (2~ mL). The filtrate was reduced in vacuo to an oil which was dissolved in 10~/o methanoll chloroform (100 mL) and washed with lM NaOH
(20 mL) and H~O (15 mL). The organic layer was separated, dried over sodium sulfate, filtered, and reduced in v~cuo. The isolated product was characterized by lH NMR as N4-(N'-cholesteryl carbamate glycineamide)-spermine (128 mg, 0.19 mrnol, 95% yie]d).
20 (C) Svnthesis of N--spermidine-2.3-dilaurvloxypropvlamine. amphiphile No. 94.2,3 Dimyristoylglycerol (600 mg, 1.4 mmol) was dissolved in pyridine and the solution cooled to 0~C. The solution was stirred under a nitrogen atmosphere and p-toluenesulfonyl chloride (300 mg, 1.57 rrunol) was added. The solution was al'loweti to warrn to room temperature and was then stirred 25 ovemight at ambient temperature. To the solution was added hydrochloric acid (2.5M, 20 mL) and the solution was extracted three times with methylene chloride (25 mL,). The combined organic extracts were dried over sodium sulfate, .. ..... .

CA 02260034 l999-01-ll WO 98tO2190 PCT/US97/12105 filtered and concentrated in vacuo to give a crude oil. The oil was purified by flash chromatography (50g of silica gel, 60~) eluting with 5% ethyl acetate /
hexane. The oil obtained by flash chromatography was dried under high vacuum at ambient temperature to give 2,3-Dirnyristoylglycerol-tosylate(630 mg, 77% yield).
2,3-Dimyristoylglycerol-tosylate (300 mg, 0.51 rnrnol) and Nl,N8-diCbz-spermidine (1.5g, 3.6 rnmol) were dissolved in toluene (15 mL). The solution wasstirred under a nitrogen atmosphere and heated at reflux (110~C). The reaction was heated for ~ days at reflux temperature. The reaction was cooled to room temperature and then filtered through Whatman #1 filter paper. The filtrate was concentrated in vacuo . The residue was dissolved in chloroforrn (50 mL) and washed with sodium hydroxide solution (1 M, 10 mL) and water (10 mL). The organic fraction was dried over sodium sulfate, fi}tered and concentrated in vacuo . The crude material was purified ~y flash chromatography (30g si~ica gel, 60A) eluting with 5% methanol / chloroform. The product containing fractions were concentrated in vacuo. The material was purified by a second flash chromatography column (20 g silica, 60A) eluting with 50% ethyl acetate /
hexane. Chromatography gave, after drying the product under high vacuum at ambient temperature, N~(Nl,N8)-diCbz-spermidine-2,~
dilauryloxypropylamine, as an oil (142 mg, 35% yield).
N4-(Nl,N8)-diCbz-sperrnidine-2,3-dilauryloxypropylamine (142 mg, 0.18 mmol) in glacial acetic acid (5 mL) was stirred with 10% Pd/C (50 mg) under a hydrogen atmosphere, for 2 hours. The catalyst was removed by vacuum filtration through Whatman #l filter paper. The catalyst was washed with ethyl / acetate hexane (10%,10 mL). The filtrate was concentrated in vacuo and dried for 2 hours under high vacuum. To the residue was added sodium hydroxide solution (1 M, 8 mL) and the solution was extracted three times with methanol /

t I I n CA 02260034 1999-01~11 chloroform (lO~o, 20 mL). The combined organic extracts were dried over sodium sulfate, filtered and concentrated in vacuo to give after drying under high vacuum N4 spermidine-2,3-dilauryloxypropylamine (52 mg, 52% yield).
~ Synthesis of N4-spermine-~3-dilaurylox,vpropvlamine, amphiphile No. 102 Nl,N 12-diCbz-spermine (0.87g, 1.85 mmol) and 2,3 dimyristoylglycerol-tosylate (280mg, 0.48 mmol) were dissolved in toluene t25 rnL) and heated at reflux temperature (110~C) for 3 days. The solution was concentrated in VQCUO
and the resulting material was purified by flash chromatography (30g silica gel,60A) eluting with 10% methanol /chloroform. The material isolated was dissolved in methanol / chloroform (10%, 85 mL) and washed twice with sodium hydroxide solution (1 M, 15 mL) and water (10 mL). The organic fraction was dried over sodium sulfate, filtered and concentrated in vacuo . ~he material wasdried under high vacuum overnight, at ambient temperature, to yield N4-~ 2-diCbz-spermine)-2,3-dilaurylo~y~ro~ylamine (180 mg, 43 % yield).
N4-~N1,I~Jl~diCbz-spermine)-2,3-dilauryloxypropylmine (180 mg, 0.2 mmol) in glacial acetic acid (10 mL) was stirred with 10% Pd/C (50 mg) under a hydrogen atmosphere, for 3 hours. The catalyst was removed by vacuum filtration through Whatrnan .tl filter paper. The catalyst was washed with ethyl/ acetate hexane (10%, 30 mL). The filtrate was concentrated in vacuo and dried for 2 hours under high vacuum. To the residue was added methanol /
chloroform (10~~" 85 mL) and the organic layer was washed twice with sodium hydroxide solution (1 M, 15 mL) and water (lO mL). The organic fraction was dried over sodium sulfate, filtered and concentrated in vacuo to give after drying under hi,gh vacuum N4-spermine-2,3-dilauryloxypropylamine (50 mg, 40% yield).

Example 11- Expression of a Secreted Protein from Vascular Tissue . ~

CA 02260034 l999-Ol-ll W O 98/02190 PCTrUS97/12105 Human secreted alkaline phosphatase (SEAP) was detected in the serum of BALB/c mice following intravenous administration of a plasmid containing an encoding cDNA. Following generally the procedures of Examples 1 and 3, a cationic amphiphile plasmid composition was prepared. A cDNA encoding S sequence for human SEAP was placed in pCF1 plasmid (see Example 4). rhe transfecting composition was prepared to contain 0.75 rnM of amphiph e No. 67, 1.5 mM DOPE, and 2mM (as nucleotide) of pCF1 plasmid. Thus the amphiphile/DNA ratio was 1:4 thereby providing a negative zeta potential.
As demonstrated in Figure 22, substantial expression of SEAP protein was 10 detected in the serurn of BALB/c rnice following tail vein administration of the amphiphile/DNA complex. pCF1 plasmid with the encoding cDNA was used as control and error bars are shown. Similar results were achieved using amphiphile No. 53 (see Fxample 3 for ~efel~ed procedures). The amphiphile/DNA complex was provided as 0.5 mM of amphiphile No. 53, 0.5 15 mM DOPE, and 2mM (as nucleotide) of pCF1 plasmid.

The above descriptions of preferred embodiments of the invention have been presented to illustrate the invention to those skilled in the art. They are not 20 intended to limit the invention to the precise forrns disclosed.

n

Claims

Claims
1. A method of transfecting a blood vessel in vivo comprising first providing a therapeutic composition, itself comprising a (1) a DNA molecule that includes an encoding sequence for a therapeutic protein that is secreted from cells, and (2) a cationic amphiphile selected from Groups I, II; III, or IV of amphiphiles, wherein:
Z is a steroid;
X is a carbon atom or a nitrogen atom;
Y is a short linking group, or Y is absent;
R3 is H, or a saturated or unsaturated aliphatic group;
R1 is -NH-, an alkylamine, or a polyalkylamine;
R4 is H, or a saturated or unsaturated aliphatic group;
R2 is -NH-, an alkylamine, or a polyalkylamine;
and wherein R1 is the same or is different from R2, except that both R1 and R2 cannot be -NH-;

wherein:
Z is a steroid;
X is a carbon atom or a nitrogen atom;
Y is a linking group or Y is absent;
R3 is an amino acid, a derivatized amino acid, H or alkyl;
R1 is -NH-, an alkylamine, or a polyalkylamine;
R4 is an amino acid, a derivatized amino acid, H or alkyl;
R2 is -NH-, an alkylamine, or a polyalkylamine;
and wherein R1 is the same or is different from R2, except that both R1 and R2 cannot be -NH-;

wherein:

Z is an alkylamine or a dialkylamine, linked by the N-atom thereof, to Y (or directly to X, if Y is absent), wherein if Z is a dialkylamine, the alkyl groupsthereof can be the same or different;
X is a carbon atom or a nitrogen atom;
Y is a short linking group, or Y is absent;
R3 is H, or a saturated or unsaturated aliphatic group;
R1 is -NH-, an alkylamine, or a polyalkylamine;
R4 is H, or a saturated or unsaturated aliphatic group;
R2 is -NH-, an alkylamine, or a polyalkylamine;
and wherein R1 is the same or is different from R2, except that both R1 and R2 cannot be -NH-; and wherein:
A and B are independently O, N or S;
R5 and R6 are independently alkyl or acyl groups and may be saturated or contain sites of unsaturation;
C is selected from the group consisting of --CH2--, >C=O, and >C=S;
E is a carbon atom or a nitrogen atom;
D is a linking group such as -NH(C=O)- or -O(C=O)-, or D is absent;
R3 is H, or a saturated or unsaturated aliphatic group;

R1 is -NH-, an alkylamine, or a polyalkylamine;
R4 is H, or a saturated or unsaturated aliphatic group;
R2 is -NH-, an alkylamine, or a polyalkylamine;
and wherein R1 is the same or is different from R2, except that both R1 and R2 cannot be -NH-;

and, second, administering said composition into the vascular or lymphatic system of a patient.
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