WO2001042200A1 - Cationic amphiphiles for intracellular delivery of therapeutic molecules - Google Patents

Abstract

Novel cationic amphiphiles are provided that facilitate transport of biologically active (therapeutic) molecules into cells. There are provided also therapeutic compositions prepared typically bycontacting 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.

Description

Cationic Amphiphiles For Intracellular Delivery of Therapeutic Molecules

Background of the Invention

The present invention relates to novel cationic amphiphilic compounds that facilitate

the intracellular delivery of biologically active (therapeutic) molecules. The present

invention relates also to pharmaceutical compositions that comprise such cationic

amphiphiles, and that are useful to deliver into the cells of patients therapeutically effective

amounts of biologically active molecules. The novel cationic amphiphilic compounds of

the invention are particularly useful in relation to gene therapy.

Effective therapeutic use of many types of biologically active molecules has not

been achieved 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 therapeutic benefit. Efficient delivery of therapeutically

sufficient amounts of such molecules into cells has often proved difficult 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 structures in the cell, such as lyposomal

compartments, 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 limited.

Although such selectivity is generally necessary in order that proper cell function

can be maintained, it comes with the disadvantage that many therapeutically valuable

substances (or therapeutically effective amounts thereof) are excluded. Additionally, the

complex structure, behavior, and environment presented by an intact tissue targeted for intracellular delivery of biologically active molecules often interferes substantially with

such delivery, in comparison with the case presented by populations of cells cultured in

vitro.

Examples of biologically active molecules for which effective targeting to a patients'

tissues is often not achieved include: (1) numerous proteins such as immunoglobins, (2)

polynucleotides such as genomic DNA, cDNA, or mRNA (3) antisense polynucleotides; and

(4) many low molecular weight compounds, whether synthetic or naturally occurring, such

as the peptide hormones and antibiotics.

One of the fundamental challenges now facing the medical practitioner is that

although the defective genes that are associated with numerous inherited diseases (or that

represent disease risk factors including for various cancers) have been isolated and

characterized, methods to correct the disease states themselves by providing patients with

normal copies of such genes (the technique of gene therapy) are substantially lacking.

Accordingly, the development of improved 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 cystic fibrosis, Gaucher's disease, Fabry's disease, and muscular

dystrophy. Representative of acquired disorders that can be treated are: (1) for cancers -

multiple myeloma, leukemias. melanomas, ovarian carcinoma and small cell lung cancer;

(2) for cardiovascular conditions - progressive heart failure, restenosis, and hemophilias;

and (3) for neurological conditions - traumatic brain injury.

Gene therapy requires successful transfection of target cells in a patient.

Transfection may generally be defined as the process of introducing an expressible polynucleotide (for example a gene, a cDNA, or an mRNA patterned thereon) into a cell.

Successful expression of the encoding polynucleotide leads 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 ("CFTR"), and which regulates the movement of ions (and therefore

fluid) across the cell membrane of epithelial cells, including lung epithelial cells. Abnormal

ion transport in airway cells leads to abnormal mucous secretion, inflammation and

infection, tissue 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 inherited diseases. There remains however a critical need to develop compounds that

facilitate entry of functional genes into cells, 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 intracellular delivery (whether for in vitro or in vivo application) meet this

definition. One particularly important class of such amphiphiles is the cationic amphiphiles.

In general, cationic amphiphiles 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) molecules including, for example, negatively charged polynucleotides

such as DNA.

Examples of cationic amphiphilic compounds that have both polar and non-polar

domains and that are stated to be useful in relation to intracellular delivery of biologically

active molecules are found, for example, in the following references, which contain also

useful discussion 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. Feigner, et al., Proc. Natl. Acad. Sci. USA 84: 7413-

7417 (1987) disclose use of positively-charged synthetic cationic lipids including N-[l(2,3-

dioleoyloxy)propyll-N,N,N-trimethylammonium chloride ("DOTMA"), to form lipid/DNA

complexes suitable for transfections. See also Feigner et al., The Journal of Biological

Chemistry 269(4): 2550-2561 (1994). Behr et al., Proc. Natl. Acad. Sci. USA 86: 6982-

6986 (1989) disclose numerous amphiphiles including dioctadecylamidologlycylspermine

("DOGS"). U.S. Patent 5,283,185 to Epand et al. describes additional classes and species of amphiphiles including 39 [N-(N1,N1 - dimethylaminoethane)carbamoyl] cholesterol, termed

"DC-chol." Additional compounds that facilitate transport of biologically active molecules

into cells are disclosed in U.S. Patent No. 5,264,618 to Feigner et al. See also Feigner et

al., The Journal Of Biological Chemistrv 269(4): 2550-2561 (1994) for disclosure therein of

further compounds including "DMRIE" 1,2- dimyristyloxypropyl-3-dimethyl-hydroxyethyl

ammonium bromide, which is discussed below. Reference to amphiphiles suitable for

intracellular delivery of biologically active molecules is also found in U.S. Patent No.

5,334,761 to Gebeyehu et al., and in Feigner et al., Methods in Enzvmology 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 vitro) the entry of

biologically active molecules into cells, it is believed that the uptake efficiencies provided

thereby are insufficient to support numerous therapeutic applications, particularly gene

therapy. Additionally, since the above-identified compounds are understood to have only

modest activity, substantial quantities thereof must be used leading to concerns about the

toxicity of such compounds or of the metabolites thereof.

Moreover, several barriers to the systemic use of cationic lipid:pDNA complexes

have been identified. These include low, non-therapeutic levels of biologically active

molecule expression, aggregation and opsonization of the complexes and also abnormal

blood and liver parameters indicating acute toxicity.

Accordingly there is a need to develop a "second generation" of cationic amphiphiles

whose activity is so sufficient that successful therapies can be achieved therewith. Summary of the Invention

The present invention provides for cationic amphiphiles that are particularly

effective to facilitate transport of biologically active molecules into cells. In a preferred

embodiment, these cationic amphiphiles are particularly effective for intravenous delivery

of biologically active molecules into cells. Representative amphiphiles are provided

according to the following structure:

wherein:

(A) and (B) are independently O, N, S or -O(C=0);

(C) is chosen from -CH₂-, >C=O, and >C=S;

(D) is a linking group, which may be chosen from but is not limited to: -NH(C=O)-

-O(C=O)-; -(CH₂)₂-; -(CH₂)₃-; -(CH₂)-(C=O)-; and -NH-(C=O)-O- or (D) is absent;

(E) is -(CH₂)- or -NH- or is absent;

R¹ is -NH-, an alkylamine, or a polyalkylamine; R² is -NH-, an alkylamine, or a polyalkylamine and wherein R¹ is the same or is

different from R², except that both R' and R² cannot be NH;

R³ is H, or a saturated or unsaturated aliphatic group;

R⁴ is H, or a saturated or unsaturated aliphatic group; and

R⁵ and R⁶ are independently alkyl or acyl groups and may be saturated or contain

sites of unsaturation.

Representative amphiphiles may also be dimers of formula I which are provided

according to the following structure:

wherein:

(A) and (B) are independently O, N, S or -O(C=0);

(C) is chosen from -CH₂-, >C=O, and >C=S;

(D) is a linking group, which may be chosen from but is not limited to: -NH(C=O)-;

-O(C=O)-; -(CH₂)₂-; -(CH₂)₃-; -(CH₂)-(C=O)-; and -NH-(C=O)-O- or (D) is absent;

(E) is -(CH₂)- or -NH- or is absent;

R¹ is -N-, an alkylamine, or a polyalkylamine; R² is -N-, an alkylamine, or a polyalkylamine and wherein R¹ is the same or is

different from R², except that both R¹ and R² cannot be N;

R³ is H, or a saturated or unsaturated aliphatic group;

R⁴ is H, or a saturated or unsaturated aliphatic group; and

R⁵ and R⁶ are independently alkyl or acyl groups and may be saturated or contain

sites of unsaturation.

The invention provides also for pharmaceutical compositions that comprise one or

more cationic amphiphiles, and one or more biologically active molecules, wherein said

compositions facilitate intracellular delivery in the tissues of patients of therapeutically

effective amounts of the biologically active molecules. The pharmaceutical compositions of

the invention may be formulated to contain one or more additional physiologically

acceptable substances that stabilize the compositions for storage and/or contribute to the

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 dispersion with a biologically active

molecule to form a complex between said amphiphile 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 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 including those known as "DC-chol", and those disclosed by Behr et al. Proc. Natl. Acad.

Sci. USA 86: 6982-6986 (1989), a representative structure of which is dioctadecylamidolo-

glycylspermine ("DOGS"), may be included in the formulations of the present invention.

In a still further embodiment of the invention, particular organs or tissues may be

targeted for gene therapy by intravenous administration of amphiphile/biologically active

molecule complexes and by adjusting the ratio of amphiphile to DNA in such complexes,

and/or by adjusting the apparent charge or zeta potential thereof.

Yet another embodiment of the invention, is improved expression and toxicity

profiles by slow infusion of the amphiphile/biologically active molecule complexes rather

than by administering the complex as a bolus.

Cationic amphiphiles of the invention representative of formula (I) provided above

are as follows:

(

Brief Description of the Drawings

FIGURES 1 and 2 depict representative cationic amphiphiles.

FIGURE 3 provides a map of the pCF2/CAT plasmid.

Detailed Description of the Invention

This invention provides for cationic amphiphile compounds, and compositions

containing them, that are useful to facilitate transport of biologically active molecules 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 practice of the invention possess several novel

features. These features may be seen in comparison with, for example, cationic amphiphile

structures such as those disclosed in U. S. Patent No. 5,283,185 to Epand et al., a

representative structure of which is 3β [N-(N',N' - dimethylaminoethane)-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-glycylspermine ("DOGS").

In connection with the practice of the present invention, it is noted that "cationic"

means that the R' and R² as defined herein, may have one or more positive charges in a

solution that is at or near physiological pH. Such cationic character may enhance

interaction of the amphiphile with therapeutic molecules (such as nucleic acids) or with cell

structures (such as plasma membrane glycoproteins) thereby contributing to successful entry

of the therapeutic molecules into cells, or processing within subcompartments (such as the

nucleus or an endosomal vesicle) 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. It should be noted that in vitro and in vivo efficacy has also been determined for cationic amphiphiles of the invention when such amphiphiles (either in fully deprotonated

"free base" form or partially protonaled form) are formulated with a co-lipid.

Cationic amphiphiles of the present invention are provided according to the

following formula:

wherein:

(A) and (B) are independently O, N, S or -O(C=0);

(C) is chosen from -CH₂-, >C=O, and >C=S;

(D) is a linking group, which may be chosen from but is not limited to: -NH(C=O)-

-O(C=O)-; -(CH₂)₂-; -(CH₂)₃-; -(CH₂)-(C=O)-; and -NH-(C=O)-O- or (D) is absent;

(E) is -(CH₂)- or -NH- or is absent;

R¹ is -NH-, an alkylamine, or a polyalkylamine;

R² is -NH-, an alkylamine, or a polyalkylamine and wherein R' is the same or is

different from R², except that both R¹ and R² cannot be NH;

R³ is H, or a saturated or unsaturated aliphatic group;

R⁴ is H, or a saturated or unsaturated aliphatic group; and R⁵ and R⁶ are independently alkyl or acyl groups and may be saturated or contain

sites of unsaturation.

Cationic amphiphiles of the present invention may also be dimers of formula I which

are provided according to formula II:

wherein:

(A) and (B) are independently O, N, S or -O(C=0);

(C) is chosen from -CH₂-, >C=O, and >C=S;

(D) is a linking group, which may be chosen from but is not limited to: -NH(C=O)-

-O(C=O)-; -(CH₂)₂-; -(CH₂)₃-; -(CH₂)-(C=O)-; and -NH-(C=O)-O- or (D) is absent;

(E) is -(CH₂)- or -NH- or is absent;

R' is -N-, an alkylamine, or a polyalkylamine;

R² is -N-, an alkylamine, or a polyalkylamine and wherein R¹ is the same or is

different from R², except that both R¹ and R² cannot be N;

R³ is H, or a saturated or unsaturated aliphatic group;

R⁴ is H, or a saturated or unsaturated aliphatic group; and R⁵ and R⁶ are independently alkyl or acyl groups and may be saturated or contain

sites of unsaturation.

Preferred compounds representative of formula (I) include:

GL 194

Biological molecules for which transport into cells can be facilitated according to the

practice of the invention include, for example, genomic DNA, 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 pharmaceutical

compositions of the invention.

In an important embodiment of the invention, the biologically active 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 ("CFTR") to allow possession of the

biological property of epithelial cell anion channel regulation.

In connection with the design of cationic amphiphiles of the invention capable of

facilitating transport of biologically active molecules into cells, the following considerations

are of note.

With respect to the selection of R . 1¹, D R2²,

and R⁴ preferred groups are set forth in

Table 1

Table 1

For R¹ and R²

1) -NH-

2) -NH-(CH₂)

3) -NH-(CH₂)

4) -NH-(CH₂)

5) -NH-(CH₂)

6) -NH-(CH₂) NH-(CH₂)₍₄₎-

7) -NH-(CH₂), NH-(CH₂)₍₂₎-

8) -NH-(CH₂), NH-(CH₂)₍₃₎-

9) -NH-(CH₂) 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)_(V)-NH-(CH₂)_(Z)- 13) -[NH-(CH₂)_(w)]_m-[NH-(CH₂)_(x)]_n-[[CH₃(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)_(V)]N]-(CH₂)_(Z) - 17) -NH-(CH₂)_(Z)-NH-

18) -NH-(CH,)_(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- For R³ and 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),]CH- (8) CH₃-[[CH₃-(CH₂)_(y)][CH₃-(CH₂)_(z)]]C-

(9) CH₃-(CH₂)_(z)-CH=CH-CH₂-

(10) CH₃-[CH₃-(CH,)_(y)-CH=CH-(CH₂)_(z)]CH-

(l l) CH₃-[[CH₃-(CH₂)_(w)-CH=CH-(CH₂)_(x)][CH₃-(CH₂)_(y)-CH=CH-(CH₂)_(z)]]CH- (12) CH₃-[CH₃-(CH₂)_(y)]CH-(CH₂)_(z) -

R⁵ and R⁶ are independently alkyl or acyl groups, preferably containing about 8 to 30

carbon atoms, and such groups may contain one or more points of unsaturation. For the

dimers of formula (II), R' may be attached to R² through any carbon or nitrogen atom of R'

and R², while R³ and R⁴ may be attached to R¹ and R² through and carbon or nitrogen atom

of R¹ and R².

It is noted that a polyalkylamine group that is very long may interfere, for example,

with the solubility of the resultant amphiphile, or interfere with its ability to stably interact

with the biologically active molecule selected for intracellular delivery. In this regard,

polyalkylamines having a backbone length of about 40 nitrogen and carbon atoms, or more,

may not be suitable for inclusion in amphiphiles. It is preferred that said backbone length

be about 30 nitrogen and carbon atoms in length, or less. However, for each such proposed

structure, its properties may be determined by experimentation, and its use is nonetheless

within the practice of the invention. The Linking Group

Preferably the linking group (D) that connects the lipophilic group to the cationic

group is relatively short. It is generally preferred that within linking group (D) are

contained no more than about three or four atoms that themselves form a bridge of covalent

bonds between (E) and the lipophilic group.

Examples of (D) groups include -NH(C=O)-; -O(C=O)-; -(CH₂)₂-; -(CH₂)₃-;

-(CH₂)-(C=O)-; and -NH-(C=O)-O-. Additional linking groups useful in the practice of the

invention are those patterned on small amino acids such as glycinyl, alanyl, beta-alanyl,

serinyl, threoninyl, and the like.

In certain preferred embodiments of the invention, (D) is a linking group wherein no

more than one atom of this group forms a bond with both (E) and the lipophilic group.

Alternatively, the linking group (D) may be absent entirely.

Co-lipids

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 performance has been observed for

numerous of the amphiphiles of the invention, the amphiphiles 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 participation in intracellular delivery

mechanisms, nor to require the involvement of co-lipids.

Representative co-lipids that are useful according to the practice of the invention for

mixing with one or more cationic amphiphiles include dioleoylphosphatidylethanolamine ("DOPE"), diphytanoylphosphatidylethanolamine, lyso-phosphatidylethanolamines other

phosphatidylethanolamines, phosphatidylcholines, lyso-phosphatidylcholines and

cholesterol. Typically, a preferred molar ratio of cationic amphiphile to co-lipid is about

1 :1. However, it is within the practice of the invention to vary this ratio, including also over

a considerable range.

Transacylation Reactions

Although heretofore unrecognized in the art, it has been determined also that certain

co-lipids may react chemically with certain types of cationic amphiphiles under conditions

of co-storage, resulting in new molecular species. Generation of such new species is

believed to occur via mechanisms such as transacylation. For a further discussion thereof,

see international patent publication WO 96/18372 at pages 43-44, and also Figure 4 thereof.

It is to be understood that therapeutically effective pharmaceutical compositions of

the present invention may or may not contain such 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, 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 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 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 invention need not form 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 loosely organized structures. Any of such structures can be present in

pharmaceutical preparations of the invention and can contribute to the effectiveness thereof.

Biologically active molecules 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, (b) ribosomal RNA; (c) antisense polynucleotides, whether RNA or DNA,

that are useful to inactivate transcription products of genes and which are useful, for

example, as therapies to regulate the growth of malignant cells; and (d) ribozymes. In general, and owing to the potential for leakage of contents therefrom, vesicles or

other structures formed from many cationic amphiphiles are not preferred by those skilled in

the art in order to deliver low molecular weight biologically active molecules. Although not

a preferred embodiment of the present invention, it is nonetheless within the practice of the

invention to deliver such low molecular weight molecules intracellularly. Representative of

the types of low molecular weight biologically active molecules that can be delivered

include 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 biologically active molecules

into target cells and/or into subcellular compartments thereof. 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 molecule, half-life of the amphiphile(s), any potential adverse effects of the

amphiphile(s) or of degradation products thereof, the route 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 administration may be used to provide highly accurate

dosages of the pharmaceutical compositions of the invention. Such preparations can be

administered orally, parenterally, topically, transmucosally, intravenously, 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. Nebulizing devices, powder inhalers, and aerosolized 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 inorganic or organic salts) and may also be lyophilized (and then

rehydrated) in the presence of such excipients prior to use. Conditions of optimized

formulation for each amphiphile of the invention are capable of determination by those

skilled in the pharmaceutical art.

Accordingly, 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 the presence of one or more excipients as aforementioned,

said resultant 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-enhancing nature of the amphiphile (or of amphiphile/ DNA complex) is

maintained. Without being limited as to theory, it is believed that the excipients stabilize

the interaction of the amphiphile and biologically active molecule through one or more

effects including: (1) minimizing 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 oxidation and/or hydrolysis.

Although the presence of excipients in the pharmaceutical compositions of the

invention stabilizes the compositions and facilitates storage and manipulation thereof, it has

also been determined that moderate concentrations of numerous excipients may interfere with the transfection enhancing capability of pharmaceutical formulations 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 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 unbuffered water, contacts the target cells. Use of such otherwise

preferred compositions may therefore be incompatible with treating target tissues that

already are stressed, such as has damaged lung tissue of a cystic fibrosis patient.

Accordingly, and using sucrose as an example, selection of concentrations of this excipient

that range from about 15 mM to about 200 mM provide a compromise between the goals of

(1) stabilizing the pharmaceutical composition to storage and (2) minimizing any effects

that high concentrations of solutes in the composition may have on transfection

performance.

Selection of optimum concentrations of particular excipients for particular

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 contacting plasmid DNA in order to form a

therapeutic composition, or prior to the time when said therapeutic composition contacts a

biological fluid. It is within the practice of the invention to provide fully protonated, partially protonated, or free base forms of the amphiphiles in order to form, or maintain,

such therapeutic compositions.

Another aspect of this invention concerns more effective gene transduction

following intravenous delivery of cationic lipid:pDNA complexes, using the novel series of

cationic lipids.

The invention will be further clarified by the following examples, which are

intended to be illustrative of the invention, but not limiting thereof.

-2.4- Examples

Example 1 - Methods of Synthesis

The cationic amphiphiles of the present invention preferably result from coupling a

polycationic headgroup to the lipophilic moiety through a primary amine. The following

methods illustrate production of certain of the cationic amphiphiles of the invention. Those

skilled in the art will recognize other methods to produce these compounds, and to produce

also other compounds of the invention.

Synthesis of 2,3-dilaurylglycerol N 4 Spermine Carbamate (Lipid 202)

3-OBn-glycerol (1. 00 g, 5. 49 mmol) in THF (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 mmol) 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 vacuo to a yellow oil which was redissolved in

diethyl ether (100 mL). The ether solution was washed with 0. 1 N NaOH (30 mL) and

dH2O (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) eluting with 3% ethyl acetate/hexanes. The desired

product was isolated as a pale yellow oil and characterized by IH NMR as 3-OBn-l,2-

dilaurylglycerol (1. 70 g, 60%).

3-OBn-l,2-dilaurylglycerol (1. 70 g, 3. 28 mmol) in ethanol (100 mL) was stirred

with 10% Pd/C (250 mg, 15 wt%) under a hydrogen atmosphere 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 'H NMR as 1,2-

dilaurylglycerol (1. 23g, 88%).

4-Nitrophenylchloroformate (3. 02 g, 14. 98 mmol) was added to a chilled (0 °C) solution

of 1 ,2-dilaurylglycerol (4. 58 g, 10. 68 mmol) in methylene chloride (40 mL). The reaction

was stirred for 10 minutes at 0 °C and brought to room temperature and stirred for an

additional 30-45 minutes. Pyridine (1. 2 mL 14. 84 mmol) was added dropwise and the

reaction was allowed to stir. After 3 hours, the reaction was diluted with chloroform (150

mL). The chloroform solution was washed with saturated sodium bicarbonate (3 x 70 mL)

and saturated sodium chloride solution (100 mL). The organic layer was dried over

magnesium sulfate, filtered, and reduced in vacuo to a colorless oil. The colorless oil was

further dried under high vacuum for several hours and characterized by 'H NMR as the 4-

nitrophenylcarbonate of 1.2-dilaurylglycerol.

The carbonate was redissolved in a solution of DMF (40 mL ) and methylene

chloride (30 mL) and was added dropwise to a cooled (0 °C) solution of spermine (19. 42 g,

95. 97 mmol) and diisopropylethylamine ( 2 mL, 1 1. 48 mmol) in DMF (70 mL) and

methylene chloride (40 mL). The reaction was brought to room temperature and stirred for

2 hours. The DMF was removed in vacuo. The resultant oil was redissolved in 2:1

methylene chloride: methanol. A yellow precipitate was filtered out and washed several

times with additional 2:1. The filtrate was collected. Total volume of filtrate was

approximately 150 mL. This solution was washed with IM NaOH (3 x70 mL) and dH2O (2

x 70 mL). The organic layer was dried over sodium sulfate, filtered and reduced to an oil in vacuo. The crude material was purified by flash column chromatography (500g silica gel)

eluting with 40:25:5, chloroform: methanol: ammonium hydroxide. The desired product

was isolated as a clear oil (4. 52 g, 60% ) and characterized by 'H NMR as Lipid 202, 2,3-

dilaurylglycerol N 4-spermine carbamate.

Synthesis of 3-(l '-sperminecarboxyl)-l ,2-dilauryIaminocarboxyIglycerol (Lipid

194)

3-OBn-glycerol (0. 25 gl. 37 mmol) and dodecyl isocyanate (1. 1 g, 4. 8 mmol) in

toluene (25 mL) was refluxed under Nitrogen for 18h. After cooling to room temperature

the solvent was removed in vacuo . The crude material was purified by flash column

chromatography (150 g silica gel) eluting with 20% ethyl acetate/ hexanes. The desired

product was isolated as a colorless oil and characterized by Η NMR as 3-OBn-l,2-

dilaurylaminocarboxylglycerol (0. 71 g, 86%).

3-OBn-l,2-dilaurylaminocarboxylglycerol (0. 71 g, 1. 17 mmol) in ethanol (35 mL)

was stirred with 10% Pd on Carbon (180 mg) under a hydrogen atmosphere for 48 hours.

The reaction was flushed with nitrogen, filtered and rinsed with ethanol to remove the

catalyst. The filtrate was reduced in vacuo to a gray solid. The crude material was

purified by flash column chromatography (70 g silica gel) eluting with 35% ethyl acetate/

hexanes. The desired product was isolated as a white solid and characterized by 'H NMR as

1,2-dilaurylaminocarboxylglycerol (0. 54g , 90%).

A solution of phosgene in toluene (1. 94M, 1. 6 mL) was added to the 1,2-

dilaurylaminocarboxylglycerol (0. 32 g, 0. 62 mmol) in methylene chloride (40 mL) and the

solution was stirred under nitrogen at room temperature for 18 h. A solution of imidazole

(0. 445 g, 6. 51 mmol) in methylene chloride (7. 5 mL) was added. After stirring for 2 h at room temperature, methylene chloride (50 mL) was added and the organic layer was

washed with 15 % citric acid solution. The organic layer was dried with sodium sulfate and

the solvent removed in vacuo. The crude imidazole formate was dissolved in methylene

chloride (50 mL). Spermine (0. 63 g, 3. 1 mmol) and dimethylaminopyridine (10 mg, 0. 09

mmol) was added. The solution was stirred at room temperature for 18 h. Methylene

chloride (50 mL) was added and the organic layer was washed with water (25 mL). The

organic layer was dried with sodium sulfate and the solvent removed in vacuo. The crude

material was purified by flash column chromatography (45 g silica gel) eluting in succession

with 40:25 chloroform: methanol, 40:25:2 chloroform: methanol: cone, ammonium

hydroxide, 40:25:5 chloroform: methanol: cone, ammonium hydroxide, and finally

40:25:10 chloroform: methanol: cone, ammonium hydroxide. The desired product was

isolated and characterized by 'H NMR as Lipid 194, 3-(l'-sperminecarboxyl)-l,2-

dilaurylaminocarboxylglycerol (0. 26g, 56%) .

Example 2 - Construction of vectors

As aforementioned, numerous types of biologically active molecules can be

transported into cells in therapeutic compositions that comprise one or more of the cationic

amphiphiles of the invention. In an important embodiment of the invention, the biologically

active macromolecule is an encoding DNA. There follows a description of novel vectors

(plasmids) that are preferred in order to facilitate expression of such encoding DNAs in

target cells. Construction of 12CF1

A map of pCFl /CAT is shown in Figure 3. Briefly, pCFl contains the enhancer/

promoter region from the immediate early gene of cytomegalovirus (CMV) . A hybrid

intron is located between the promoter and the biologically active cDNA. The

polyadenylation signal of the bovine growth hormone gene was selected for placement

downstream from the biologically active. The vector also contains a drug-resistance marker

that encodes the aminoglycosidase X-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 pCFl structure are provided directly below,

including description of placement therein of a cDNA sequence encoding for cystic fibrosis

transmembrane conductance regulator (CFTR) protein.

The pCFl vector is based on the commercially available vector pCMVβ (Clontech).

The pCMVβ construct has a pUC 19 backbone (J. Vieira, et al., Gene 19: 259-268 (1982))

that includes a prokaryotic origin of replication derived originally from pBR322.

Basic features of the pCFl -plasmid (as constructed to include a nucleotide sequence

coding for CFTR) are as follows. Proceeding clockwise, the basic features are: the human

cytomegalovirus immediate early gene promoter and enhancer, a fused tripartite 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 pCFl -CFTR plasmid has been

completely sequenced on both strands.

The human cytomegalovirus immediate 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 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 pCFlCFTR.

The CFTR transcript is predicted to initiate at nucleotide 548, which is the transcriptional

start site of the CMV promoter.

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 (nucleotides, 705-745), 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 1043-1 138). The donor site (G |

QT is at nucleotides 887-888, the acceptor site (AG | G) is at nucleotides 1 128-1129 , and

the length of the intron is 230 nucleotides. The CFTR coding region comprises nucleotides

1 183-5622.

Within the CFTR-encoding cDNA of pCFI-CFTR, there are two differences from

the originally-published predicted cDNA sequence (J. Riordan 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. See R. J.

Gregory et al., Nature 347: 382-386 (1990). The 3' untranslated region of the predicted

CFTR transcript comprises 51 nucleotides of the 3' untranslated region of the CFTR cDNA,

21 nucleotides of linker 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 downstream of the BGH

polyadenylation signal at nucleotide 5808. There is a deletion in pCFl-CFTR at position

+46 relative to the cleavage site, but the deletion is not predicted to effect either

polyadenylation efficiency or cleavage 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.

Example 3 - Methods of Using A Pharmaceutical Composition According to the

Invention

Cell Transfection

Cell transfection assays are well known in the art. Examples of such assays are

present in U. S. Patent No. 5,840,710, herein incorporated by reference. The cationic

amphiphiles of the present invention facilitate the intracellular delivery of biologically

active molecules by the use of such cell transfection assays.

CAT Assay

Assays to assess the ability of the cationic amphiphiles of the invention to transfect

cells in vivo from live specimens are also well known in the art. One example of such an

assay is a CAT assay, as described in U. S. Patent 5,840,710.

Optimized compositions for in vivo testing are extrapolated from in vitro results.

This facilitates the screening of large numbers of amphiphiles and produces broadly, if not

precisely, comparable data. Thus, the ratio, for in vivo testing, of amphiphile concentration to DOPE concentration, is taken from the in vitro experiments, as is the optimized ratio of

amphiphile concentration to DNA concentration.

Choosing a Cationic Amphiphile Formulation

In developing a non-viral gene therapy vector for treating a disease state, the

following set of rules governing the systemic delivery of cationic lipid:pDNA complexes for

optimal expression should be considered to provide effective gene transduction following

intravenous delivery of cationic lipid:pDNA complexes. It is preferred to couple the

polycationic headgroup to the lipophilic moiety through a primary amine group rather than

through a secondary amine. Such a coupling results in a linear molecule rather than a "T"

shaped molecule, and can result in a ten-fold increase in expression.

It is also preferred to formulate the cationic amines as free bases rather than

protonated. Such a formulation can result in a ten-fold increase in transduction.

Optimizing the 'neutral' co-lipid is also preferred. A preferred molar ratio of cationic

lipid to the neutral co-lipid is 1 :2 molar ratio. Also, substituting diphytanoylphosphatidyl

ethanolamine for the commonly used DOPE results in a significant increase in expression.

In fact, including a low (0.05) mole percentage DMPE-PEGjo_oo in the lipid formulation has

been found to inhibit aggregation of the cationic lipid:pDNA complexes at the high

concentrations required for the most effective transduction.

In view of the foregoing, the following experiments were performed. A complex

was prepared by mixing equal volumes of the plasmid DNA and the cationic amphiphile of

the invention, both in water, and leaving them to complex for 15 minutes. The complex was

lOOμl of cationic lipid:pDNA at a ratio of 1 :lmM. BALB/c mice in groups of four to five

were injected with complex via their tail vein. The mice were then sacrificed 48 hours later and their lungs were harvested for assay of the CAT transgene. The absolute level of CAT

is determined and reported or presented as a proportion of a control group where

appropriate.

Comparative experiments were performed as described above except the cationic

amphiphile was "T" shaped. In other words, a series of matched lipids, differing only in the

orientation of the headgroup, were tested, i.e., the lipid anchor groups in these matched

lipids were identical. The anchor groups were dilauryl anchors, and cholesterol lipid

anchors.

These experiments demonstrated a good correlation between the orientation of

polycationic headgroups and transfection efficiency in the lung. Those cationic amphiphiles

that were coupled through a secondary amine, i.e., forming a "T"-shape, were generally

more effective intranasally than if coupled through a primary amine, i.e., forming a linear

shape. However, the linear shape yielded a higher level of expression in intravenous

delivery.

In addition, these experiments demonstrated that an alkyl lipid anchor provides

improved delivery as compared to a cholesterol lipid anchor. Moreover, a C,₂ alkyl anchor

provides improved delivery as compared to a C_uι, C,₄ alkyl lipid anchor, or an alkyl lipid

anchor group having more than fourteen carbons.

Formulating the Pharmaceutical Compositions

A recommended procedure for formulating the pharmaceutical compositions of the

invention is as follows. A thin film can be produced wherein the amphiphile and DOPE are

present in a molar ratio of 1 : 1 , generally as follows. Separate samples of an amphiphile and the neutral lipid dioleoylphosphatidyl

ethanolamine ("DOPE") are dissolved in chloroform as stock preparations. Following

combination of the solutions (as a 1 : 1 molar composition), a thin film is further dried under

vacuum (1 mm Hg) for 24 hours. Some of the amphiphiles of the invention participate in

transacylation reactions with co-lipids such as DOPE, or are subject to other reactions which

may cause decomposition thereof. Accordingly, it is preferred that amphiphile/co-lipid

compositions be stored at low temperature, such as -70 degrees C under inert gas, until use.

The amphiphile-containing film is then rehydrated 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 delivered 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 Respiratory

Equipment, Inc., Richmond, VA), it may be advantageous to prepare the amphiphile-

containing film to include also one or more further ingredients that act to stabilize the final

amphiphile/DNA composition. Accordingly, it may be preferred to prepare the amphiphile

containing film using an additional ingredient, PEG(5000)-DMPE. [A suitable source of

PEG-DMPE, polyethylene glycol 5000 - dimyristoylphoshatidylethanolamine, is Catalog

No. 880210 from Avanti Polar Lipids, Alabaster, AL]. Additional fatty acid species of

PEG-PE or other PEG derivatives may be used in replacement therefor.

Without being limited as to theory, PEG derivatives, such as PEG(5000)-DMPE is

believed to stabilize the therapeutic compositions by preventing further aggregation of

formed amphiphile/DNA complexes. Additional discussion of the use of these ingredients

in found in aforementioned WO 96/18372 at, for example, page 87. pCFl-CFTR plasmid (containing an encoding sequence for human cystic fibrosis

transmembrane conductance regulator) 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 contacting of the two solutions for a period of 10 minutes.

Using the Pharmaceutical Compositions

One method of delivering the DNA to the lung is by aerosolization. The aerosolized

DNA is delivered 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 A F508

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, 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 patient and taken into account by the

patient's physicians.

It is presently preferred to deliver solutions intravenously. Moreover, it is preferred

to deliver the amphiphile/biologically active molecule complex by slow infusion. Such

slow infusion improves expression and toxicity profiles as compared to delivery as a bolus.

For some disease states, such as cystic fibrosis, it is desirable to deliver biologically

active molecules to the lung. While delivery by aerosol is the most direct approach to

achieve this goal, there are difficulties inherent with such delivery. Moreover, 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

delivery formats.

One method of intravenous delivery of the DNA complex can be performed as

follows. The reporter plasmid pCF-I CAT is purified to minimize endotoxin (<1

EU/mg pDNA), and also chromosomal DNA contamination (< 2%). An amphiphile

according to the invention (1 :1 with DOPE) / DNA complex was prepared according to the

procedures set forth above. Preferably, the amphiphile is provided as the free base. The

plasmid is prepared as a sodium salt in water, and the DOPE is provided in zwitterionic

form.

A female BALB/c mouse is injected intravenously using the tail vein. Preferably 5

animals per group are used. The volume of Hpid:pDNA complex used is 100 μl. The mice

are sacrificed 48 h following administration of the complex. Organs can be frozen

immediately on dry ice to store for subsequent analysis.

Expression of chloramphenicol acetyl transferase (CAT) is quantitated using a

radiochemical assay for CAT enzymatic activity. Organs are weighed and homogenized on

ice in a lysis buffer containing protease inhibitors. The lysate is freeze-thawed 3X,

centrifuged, and heated to 65°C to inactivate deacetylases before adding it to a reaction

mixture containing C_]4-chloramphenicol. After an incubation at 37°C, the mixture is

extracted with ethyl acetate, concentrated, spotted onto TLC plates and eluted with

CHC13/MeOH. Spots corresponding to the acylated reaction products are then quantitated

(Betagen) and converted to ng CAT activity using authentic CAT standards.

One important 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

amphiphiles. There results the opportunity to substantially minimize side effects (such as

amphiphile toxicity, inflammatory response) that would otherwise affect adversely the

success of the gene therapy.

A further particular 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 harmless

biologically-compatible components.

Example 4 - Alternate Procedure to Prepare an Amphiphile/Co-lipid Composition

In order to 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

prepared together. An alternate method to produce such compositions may be as follows.

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-PEG of 1 :2:0.05. The

resultant solution is then vortexed, and 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

micron, 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 lyophilization. The t-butanol: water 9:1 solution is removed by 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 micron filter. The stoppers are then fully inserted into the

vials, and the vials are then crimped shut with an aluminum crimp-top. The vials are then

maintained at -70 °C until use.

The specification is most thoroughly understood in light of the teachings of the references cited within the specification which are hereby incorporated by reference. The embodiments within the specification provide an illustration of embodiments of the

invention and should not be construed to limit the scope of the invention. The skilled

artisan readily recognizes that many other embodiments are encompassed by the invention.

Claims

We Claim:

1. A cationic amphiphile effective for facilitating transport of at least one

biologically active molecule into a cell, said cationic amphiphile having the structure:

wherein:

(A) and (B) are independently O, N, S or -O(C=0);

(C) is -CH₂-, >C=O, or >C=S;

(D) is a linking group or is absent;

(E) is -(CH₂)- or -NH- or is absent;

R¹ is -NH-, an alkylamine, or a polyalkylamine;

R² is -NH-, an alkylamine, or a polyalkylamine and wherein said R¹ is the same or is

different from said R², except that both said R¹ and R² cannot be NH;

R³ is H, or a saturated or unsaturated aliphatic group;

R⁴ is H, or a saturated or unsaturated aliphatic group; and R⁵ and R⁶ are independently alkyl or acyl groups and may be saturated or contain

sites of unsaturation.

2. A cationic amphiphile according to claim 1 , wherein said linking group, (D),

is -NH(C=O)-; -O(C=O)-; -(CH₂)₂-; -(CH₂)₃-; -(CH₂)-(C=O)-; or -NH-(C=O)-O-.

3. A composition effective for facilitating transport of at least one biologically

active molecule into a cell, said composition comprising:

said cationic amphiphile according to claim 1 ; and

a biologically active molecule.

4. A composition according to claim 3, further comprising a co-lipid.

5. A composition according to claim 4, wherein said co-lipid is

diphytanoylphosphatidylethanolamine.

6. A composition according to claim 3, further comprising a PEG derivative.

7. A cationic amphiphile according to claim 1, wherein said cationic

amphiphile is

GL 194

8. A cationic amphip ile according to claim 1, wherein said cationic

amphiphile is

9. A cationic amphiphile effective for facilitating transport of at least one

biologically active molecule into a cell, said cationic amphiphile having the structure:

wherein:

(A) and (B) are independently O, N, S or -O(C=0);

(C) is -CH₂-, >C=O, or >C=S; (D) is a linking group or is absent;

(E) is -(CH₂)- or -NH- or is absent;

R¹ is -N-, an alkylamine, or a polyalkylamine;

R² is -N-, an alkylamine, or a polyalkylamine and wherein said R¹ is the same or is

different from said R², except that both said R¹ and R² cannot be N;

R³ is H, or a saturated or unsaturated aliphatic group;

R⁴ is H, or a saturated or unsaturated aliphatic group; and

R⁵ and R⁶ are independently alkyl or acyl groups and may be saturated or contain

sites of unsaturation.

10. A cationic amphiphile according to claim 9, wherein said linking group (D)

is -NH(C=O)-; -O(C=O)-; -(CH₂)₂-; -(CH₂)₃-; -(CH₂)-(C=O)-; or -NH-(C=O)-O-.

11. A composition effective for facilitating transport of at least one biologically

active molecule into a cell, said composition comprising:

said cationic amphiphile according to claim 9; and

a biologically active molecule.

12. A composition according to claim 11 , further comprising a co-lipid.

13. A composition according to claim 12, wherein said co-lipid is

diphytanoylphosphatidylethanolamine.

14. A composition according to claim 11 , further comprising a PEG derivative.