AU721245B2 - Emulsion and micellar formulations for the delivery of biologically active substances to cells - Google Patents

Description

WO 97/11682 PCT/US96/15388 1 Title of Invention Emulsion and Micellar Formulations for the Delivery of Biologically Active Substances to Cells Field of the Invention The present invention relates to the use of lipid dispersions to deliver biologically active substances to cells. In particular, the present invention relates to emulsion and micellar formulations and to the ability of these formulations to form stable complexes with biologically active substances and thereby facilitate the delivery of these substances to cells.

Background of the Invention Cationic liposomes are of interest as a non-viral vehicle for the delivery of biologically active substances such as drugs, hormones, enzymes, nucleic acids and antigens, including viruses, to cells both in vitro and in vivo. Indeed, cationic liposomes have been demonstrated to deliver genes in vivo (Nabel, et al. (1990) Science, 249: 1285-1288), (Brigham, et al. (1989) Am. J. Respir. Cell Mol. Biol., 195-200, Stribling, et al. (1992) Proc. Natl. Acad. Sci. 89: 11277- 11281), (Plautz, et al. (1993) Proc. Natl. Acad.

Sci. 90: 4645-4649, Stewart, et al. (1992) Hum. Gene Ther., 3: 267-275). However, the inhibition by serum components of the transfer of nucleic acids by cationic liposomes limits the application of liposomes as a vector for nucleic acids in vivo to regional administrations which avoid exposure to serum.

In addition, stability is a major problem limiting the use of liposomes, both in terms of shelf life and after administration in vivo. Thus, it is desirable to explore the use of other types of lipid dispersions as delivery systems utility for biologically active substances.

U.S. Patent 4,610,888 refers to the use as a drug-delivery system of water-in-oil emulsions in which the volume of aqueous phase ranges from about 0.7% to about 10.25% of the volume of the lipid components used.

However, such water-in-oil emulsions are unsuitable for delivering substances in blood or in other aqueous body tissues.

Summary of Invention The present invention relates to novel emulsion and micellar formulations useful for delivering biologically active substances to cells. The emulsion and micellar formulations of this invention are compatible with blood, retain activity in the presence of serum and are stable in storage. The formulations of this invention comprise lipid components, a nucleic acid and an aqueous carrier, where the lipid components comprise a cationic amphiphile component, preferably a cationic lipid, and a nonionic surfactant i' component. The micellar formulations may comprise a cationic amphiphile component a nonionic surfactant component and an aqueous carrier. The 2 lipid components of the emulsion and micellar formulation of the present invention may further comprise a neutral phospholipid component.

"Component" as used throughout the specification and claims is defined as: comprising at least one cationic amphiphile or a mixture of amphiphiles when used in the phrase "amphiphile component"; comprising at least one oil or a mixture of oils when used in the phrase "oil component"; 25 comprising at least one nonionic surfactant or a mixture of nonionic surfactants when used in the phrase "nonionic surfactant component"; comprising at least one neutral phospholipid or a mixture of neutral phospholipids when used in the phrase "neutral phospholipid component".

.The invention further relates to complexes formed by combining biologically active substances and the above-identified emulsion and micellar formulations. These biologically active substance :emulsion and biologically active substance:micelle complexes are stable over time and may have therapeutic and/or prophylactic utility in vivo depending on the activity of the biologically active substance contained in the complex.

This invention also provides a method for delivering a nucleic acid to cells by exposing cells to the formulations of this invention. In one

S

embodiment, a method of exposing cells to a nucleic acid is provided, said method comprising culturing said cells in the presence of a nucleic acid:emulsion complex or a nucleic acid:micelle complex thereby facilitating delivery of the nucleic acid to cells.

The invention further provides a method of delivering a nucleic acid to cells in vivo comprising administering to an animal or human the formulations of this invention. It is to be understood that the formulations used for the delivery of nucleic acids to cells in vitro or in vivo may be freshly prepared by admixture or may be prepared earlier and stored prior to their use.

The invention further relates to a kit containing an emulsion or micellar formulation of the present invention.

The invention also provides a kit containing a formulation formed between a nucleic acid substance and an emulsion or micellar formulation of the present invention.

Methods for producing emulsion and micellar formulations according to the invention are also provided herein.

In one embodiment, a method of producing an oil-in-water emulsion formulation comprises: combining an oil component, a cationic amphiphile component and a nonionic surfactant component; adding aqueous carrier to produce said emulsion formulation; and combining the emulsion formulation with a nucleic acid.

In another embodiment, a method of producing a micellar formulation comprises: combining a cationic amphiphile component and a nonionic surfactant component; adding aqueous carrier to produce said micellar formulation;and combining the micellar formulation with a nucleic acid.

In a further embodiment, a method for producing an emulsion formulation of this invention comprises a) combining an organic solvent with an oil component, a cationic amphiphile component and a nonionic surfactant component; b) removing the organic solvent to leave a lipid film; and c) suspending the lipid film in an aqueous carrier to produce said emulsion formulation miscible in aqueous solution.

4 In an alternative embodiment, the oil may serve as the organic solvent in step such that the method for producing an emulsion formulation of this invention comprises a) combining an oil component, a cationic amphiphile component and a nonionic surfactant component; and b) adding an aqueous carrier to the combination of step When a neutral phospholipid component is to be included in the emulsion, the neutral phospholipid component is combined with the above components in step In yet another embodiment, a method for producing a micellar formulation miscible in aqueous solution comprises: a) combining an organic solvent with a cationic amphiphile component and a nonionic surfactant component; b) removing the organic solvent to leave a lipid film; and c) suspending the lipid film in an aqueous carrier to produce said micellar formulation miscible in aqueous solution.

When a neutral phospholipid component is to be included in the micellar formulation, the neutral phospholipid component is combined with the above components in step In a further embodiment, the present invention provides a method of producing a lipid film having an oil component, a nucleic acid, a cationic amphiphile component and a nonionic surfactant component; said method including: 25 combining an organic solvent with the oil component, the nucleic acid, the amphiphile component and the nonionic surfactant component; and removing the organic solvent to leave said lipid film.

In yet another embodiment, the present invention provides a method of producing a lipid film having a nucleic acid, a cationic amphiphile 30 component and a nonionic surfactant component; said method including: combining an organic solvent with the nucleic acid, the amphiphile component and the nonionic surfactant component; and removing the organic solvent to leave said lipid film.

The present invention also provides a lipid film capable of forming an oil-in-water emulsion upon suspension in an aqueous carrier, said film having an oil component, a nucleic acid, a cationic amphiphile component and a nonionic surfactant component.

Further, the present invention provides a lipid film capable of forming a micelle upon suspension in solution, said film having a nucleic acid, a cationic amphiphile component and a nonionic surfactant component.

In addition, the present invention provides a formulation including lipid components, a nucleic acid and an aqueous carrier, wherein the lipid components include a cationic amphiphile component, a nonionic surfactant component, and optionally, an oil component, and wherein the cationic amphiphile component is selected from the group consisting of 1,2 bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP), dioleoyloxy) propyl]-N, N, N- trimethyl ammonium chloride (DOTMA), 1,2dioleoyl-3-(4' trimethylammonio) butanoyl-sn-glycerol (DOBT), cholesteryl (4' trimethylammonia) butanoate (ChOTB), DL-1, 2-dioleoyl-3dimethylaminopropyl-B-hydroxyethylammonium (DORI), DL-1,2-O-dioleoyl- 3-dimethylaminopropyl-p-hydroxyethylammonium (DORIE), 1,2-dioleoyl-3succinyl-sn-glycerol choline ester (DOSC), cholesteryl hemisuccinate choline ester (ChOSC), doctadecylamidoglycyl-spermine (DOGS), dipalmitoyl phosphatidyesthanolamidospermine (DPPES), cholesteryl-3p-carboxyl-amidoethylenetrimethylammonium iodide, 1 -dimethylamino-3-trimethylammonio- DL-2-propyl-cholesteryl carboxylate iodide, cholesteryl-33carboxyamidoethyleneamine, cholesteryl-3p-oxysuccinamidoethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonio- DL-2-propyl-cholesteryl-3p-oxysuccinate iodide, 2-[(2-trimethylammonio)- 25 ethylmethylamino] ethyl-cholesteryl-3p-oxysuccinate iodide, N'dimethylaminoethane) carbamoyl] cholesterol (DC-Chol), and 3p-[N- (polyethyleneimine)-carbamoyl] cholesterol.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will 30 be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

WO 97/11682 PCT/US96/15388 5 o DESCRIPTION OF FIGURES Figure 1 shows the optimization of transfection of BL6 cells with complexes formed between pCMV-Luc DNA and formulations #21 #27 #28 or (see Table 3 for compositions of formulations) by mixing 6 pl of each formulation (6 Al of each formulation contained 4.5 pg of DC-Chol) with varying amounts of pCMV- Luc DNA as indicated on the horizontal axis.

Figure 2 shows the optimization of transfection of BL6 cells with complexes formed between pCMV-Luc DNA and varying amounts of formulations #21 #27 #28 or #30 as indicated on the horizontal axis (see Table 3 for compositions of formulations). The amount of pCMV-Luc DNA was fixed at 2 Ag for formulations #27 and #30 and at 1.5 Ag pCMV-Luc DNA for formulations #21 and #28 and formulation" on the horizontal axis refers to Lgs of total lipid components present in the amount of formulation combined with pCMV-Luc DNA to form complex.

Figure 3 shows the stability of the complexes formed between DNA and the indicated emulsion or micellar formulations (see Table 3 for composition of formulations). Complex was prepared with 2 Ag of pCMVCAT and 16 pl of the indicated formulations containing the same amount of DC-Chol (12 Ag) in a final volume of 250 i1, except for the DC-Chol/DOPE liposome:DNA complexes which were prepared with 1 Ag pCMVCAT and 6 Ag liposome in a final volume of 250 il.

Figure 4 shows the effect of varying the concentration of Tween 80 in emulsions containing 0.25 mg oil, 0.25 mg DOPE, 0.75 mg DC-Chol and x mg Tween 80 per ml on the average diameter of concentrated and diluted pCMV-Luc DNA/emulsion complexes.

Figures 5A and 5B show the effect of Tween 80 on the WO 97/11682 PCT/US96/15388 6 transfection activity of concentrated (Figure 5A) and diluted (Figure 5B) pCMV-Luc DNA/emulsion complexes in BL6 cells in medium containing either 0 or 20% serum. The emulsion formulations used to produce the diluted and concentrated DNA:emulsion complexes contained 0.25 mg oil, 0.25 mg DOPE, 0.75 mg DC-Chol and varying amounts of Tween per ml. Concentrated DNA/emulsion complex was formed by adding 2 pl of solution containing 8 pg of pCMV-Luc DNA to 72 pl of emulsion and diluted DNA/emulsion complex was formed by combining 2 pg of pCMV-Luc DNA in 125 il with 18 pl of emulsion diluted to 125 pl.

Figure 6 shows CAT reporter gene expression in mice injected via the tail vein with DNA:emulsion or DNA:micelle complexes. The complexes were formed as follows: 200 il each of 4x concentrates of formulations #21 (1100 Ag total lipid components), #28 (1000 Ag total lipid components), #34 (900 ig total lipid components) and #31 (700 pg total lipid components) were mixed with 6 Al of 5M NaCi to a final concentration of NaC1 of 0.15M and then combined with 25 Al of 4 ig/Al pCMV-CAT DNA (100 pg).

The amount of DC-Chol contained in each DNA:emulsion and DNA:micelle complex was 600 ig. After two days, organs were excised and protein was extracted. CAT activity was measured by using 0.1 ICi[ 4 C]chloramphenicol as substrate.

Each bar represents the mean of two mice.

Detailed Description of Invention The present invention relates to emulsion and micellar formulations which form stable complexes with biologically active and thereby facilitate the delivery of the biologically active substances to cells.

The emulsion formulations of this invention are oilin-water emulsions which comprise an aqueous carrier and the following lipid components, an oil component, a cationic amphiphile component, a nonionic surfactant WO 97/11682 PCT/US96/15388 7 component and optionally, a neutral phospholipid component.

Preferably, the total lipid components are present in the emulsion formulation in an amount from about 0.001 to about 20% by weight, more preferably from about 0.01 to about 10% by weight and most preferably from about 0.05 to about 2% by weight, with the remainder of the emulsion by weight being aqueous carrier. Thus, for example, for formulation #1 in Table 1 where 0.625 mg of total lipid components are present in 0.5 ml of PBS, the weight of total lipid components in formulation #1 can be calculated as follows: Assuming 1 ml of PBS, like 1 ml of water, weighs approximately 1000 mg, then 0.5 ml of PBS weighs 500 mg and the weight of total lipid components contained in formulation #1 is 0.625 mg x l00 0.125%.

0.625 mg 500 mg Of the total lipid components present in the emulsion formulations of this invention, preferably, the amphiphile component is present in an amount from about 5 to about weight of the total lipid components in the emulsion formulation; the oil component is present in an amount from about 10 to about 80 weight of the total lipid components; the nonionic surfactant component is present in an amount from about 5 to about 50 weight of the total lipid components, and optionally, the neutral phospholipid component is present in the formulation in an amount from about 5 to about 25 weight of the total lipid component.

More preferably, the oil component is present in an amount from about 10-60 weight of the total lipid components in the emulsion formulation; the amphiphile component is present in an amount from about 20-60 weight of the total lipid components; the nonionic surfactant component is present in an amount from about 10-50 weight S of the total lipid components and optionally, a neutral WO 97/11682 PCT/US96/15388 8 o phospholipid component is present in amount from about weight of the total lipid components.

Most preferably, the emulsion formulation comprises the oil component in amount from about 10-20 weight of the total lipid components; the amphiphile component in an amount from about 40-60 weight of the total lipid components; the nonionic surfactant component in an amount from about 20-50 weight of the total lipid components and optionally, the neutral phospholipid component in an amount from about 10-20 weight of the total lipid components. A particularly preferred emulsion formulation contains an oil, a cationic amphiphile, a nonionic surfactant and a neutral phospholipid in a weight ratio of about 2:6:1:2.

The micellar formulations of this invention are compatible with blood. The micellar formulations comprise an aqueous carrier and the following lipid components: a cationic amphiphile component, a nonionic surfactant component and optionally, a neutral phospholipid component.

Preferably, the total lipid components are present in the micellar formulation in an amount ranging from about 0.0001 to about 70% by weight, more preferably from about 0.001 to about 60% by weight and most preferably from about 0.001 to about 50 by weight, with the remainder by weight of the micellar formulation being aqueous carrier.

Thus, for example, for formulation #15 in Table 2 where 1.25 mg of total lipid components are present in 1 ml of PBS, the weight of total lipid components in formulation can be calculated as follows: Assuming 1 ml of PBS, like 1 ml of water, weighs approximately 1000 mg, then the weight of total lipid components in formulation #15 is 1.25 mg x 100 0.125%.

1.25 mg+1000 mg Of the total lipid components contained in the WO 97/11682 PCT/US96/15388 -9micellar formulations of this invention, preferably, the amphiphile component is present in an amount from about to about 90 weight of the total lipid components in the micellar formulation, the nonionic surfactant component is present in an amount from about 10 to about 90 weight of the total lipid components; and optionally, the neutral phospholipid component is present in an amount from about to about 40 weight of the total lipid components.

More preferably, the amphiphile component is present in an amount from about 30 to about 90 weight of the total lipid components in the micellar formulation, the nonionic surfactant component is present in an amount from about 10 to about 70 weight of the total lipid components and optionally, the neutral phospholipid component is present in an amount from about 5 to about weight of the total lipid components.

Most preferably, the amphiphile component is present in an amount from about 50 to about 90 weight of the total lipid components in the micellar formulation, the nonionic surfactant component is present in an amount from about 10 to about 50 weight of the total lipid components and optionally, the neutral phospholipid component is present in an amount from about 10 to about weight of the total lipid components. A particularly preferred micellar formulation contains a cationic amphiphile, a nonionic surfactant and a neutral phospholipid in a weight ratio of about 6:1:2.

By oil component as used herein is meant any water immiscible component that is conventionally referred to as an oil. It is understood that the oil component may include mixtures of two or more oils. Examples of oils which can be used to produce the emulsion formulations of the present invention include, but are not limited to, natural oils such as almond oil, coconut oil, cod liver oil, corn oil, cottonseed oil, castor oil, olive oil, palm Ie oil, peanut oil, peppermint oil, rose oil, safflower oil, WO 97/11682 PCT/US96/15388 10 S sesame oil, soybean oil, sunflower oil and vegetable oil and synthetic oils such as triethylglycerol and diethylglycerol. A preferred oil is castor oil.

The cationic amphiphile component of the formulations of this invention may be any cationic amphiphile or mixture of amphiphiles which is effective for use in liposomes or for producing lipid complexes capable of delivering a biologically active substance to cells. For example, the amphiphiles described in Bolcsak et al U.S.

Patent 5,100,662, which is incorporated herein by reference, would be suitable for use in this invention.

Additional examples of cationic amphiphiles suitable for formulating the emulsion and micellar formulations of this invention include, but are not limited to, cationic lipids such as 1, 2 bis(oleoyloxy)-3- (trimethylammonio) propane (DOTAP); -(2,3-dioleoyloxy) propyl] N, Ntrimethyl ammonium chloride (DOTMA) or other N-1dialkoxy)-alklyl-N, N, N-trisubstituted ammonium surfactants; 1, 2 dioleoyl-3-(4'-trimethylammonio) butanoyl-sn-glycerol (DOBT) or cholesteryl (4' trimethylammonia) butanoate (ChOTB) where the trimethylammonium group is connected via a butanoyl spacer arm to either the double chain (for DOTB) or cholesteryl group (for ChOTB); DORI (DL-1, 2-dioleoyl-3dimethylaminopropyl-B-hydroxyethylammonium) or DORIE (DL- 1, 2-O-dioleoyl-3-dimethylaminopropyl-0hydroxyethylammonium) (DORIE) or analogs thereof as disclosed in WO 93/03709, incorporated herein by reference; 1, 2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC); cholesteryl hemisuccinate ester (ChOSC); lipopolyamines such as doctadecylamidoglycylspermine (DOGS) and dipalmitoyl phosphatidyesthanolamidospermine (DPPES) or the cationic lipids disclosed in US Patent Number 5,283,185, incorporated herein by reference, S iodide, l-dimethylamino-3-trimethylammonio-DL-2-propyl- WO 97/11682 PCT/US96/15388 11 cholesteryl carboxylate iodide, cholesteryl-33carboxyamidoethyleneamine, ethylenetrimethylammonium iodide, l-dimethylamino-3trimethylammonio-DL-2-propyl-cholesteryl-30-oxysuccinate iodide, 2-[(2-trimethylammonio)-ethylmethylamino] ethylcholesteryl-33-oxysuccinate iodide, N'dimethylaminoethane) carbamoyl] cholesterol (DC-Chol), and 33-[N-(polyethyleneimine)-carbamoyl]cholesterol.

Examples of preferred amphiphiles include iodide, l-dimethylamino-3-trimethylammonio-DL-2-propylcholesteryl carboxylate iodide, cholesteryl-33carboxyamidoethyleneamine, amidoethylenetrimethylammonium iodide, l-dimethylamino-3trimethylammonio-DL-2-propyl-cholesteryl-30-oxysuccinate iodide, 2-[(2-trimethylammonio)ethylmethylamino]ethyl- N'dimethylaminoethane)-carbamoyl]-cholesterol (DC-Chol), and 3 [N- (polyethyleneimine)-carbamoyl]cholesterol.

Since an attribute of the emulsion and micellar formulations of the present invention is their stability when stored alone or as complexes with biologically active substances, it will be understood by those of ordinary skill in the art that preferred cationic amphiphiles are cationic lipids in which bonds between the lipophilic group and the amino group are stable in aqueous solution.

Such bonds include amide bonds, ester bonds, ether bonds and carbamoyl bonds. A more preferred cationic lipid is DC-Chol.

The nonionic surfactant component of the formulations of this invention includes at least one nonionic surfactant of a molecular weight between 200 and 20,000.

In one embodiment, these surfactants may be formed by reacting a hydrophobic hydroxyl-containing compound an alcohol or phenol) with ethylene oxide where the number of ethylene oxide groups may be added to any desired WO 97/11682 PCTIUS96/1 5388 12 extent. However, those of ordinary skill in the art would understand that the ability to stabilize the emulsions or micelles of this invention may depend on the relative amount of ethylene oxide added to a given hydrophobic group. It is further understood that surfactants having branched chain ethylene oxide moieties cover more surface area than surfactants having single chain ethylene oxide moieties and that therefore, the single chain surfactants may have to be used in larger amounts than the branched chain surfactants to produce the emulsion and micellar formulation of this invention.

Examples of nonionic surfactants of this invention include, but are not limited to, polyethylene glycol, derivatives of phosphatidylethanolamine and synthetic detergents commercially available under the brand names Span,

OH

OH

0 R= CHCOO SPAN HO II R-C 31 OO SPAN C CH -0 C R R- CIcOO SPAN \H O SPAN Brij,

CH

3

(CH

2

(OCH

2 CH20)x -OH Brij 72 y=17 x=2 Brij 76 y=17 Brij 78 y=17 Brij 100 y=17 x=100 WO 97/11682 PCTIUS96/1 5388 13 Tween,

(OCHCH

3

OH

HO (CH 2

CH

2

O).W

CH(OCH

2

CHZ),OH

H 2 C- (OOH 2

CH).-R

R-q01COOween4O R- C 17 H65COO Tween R- C 17 H33COO Tween F68 and F127,

CH

3 HO (CI1 2

CH

2 O) (CHCH 2 O) (CH 2

CH

2 O) H pluronic F6B x=75 y=30 Pluronic F127 x=98 y=67 z=98 WO 97/11682 PCT/US96/15388 14 o Triton X-100 and Triton x-114

CH

3 (CHa) 3 CCH2 C O(CH 2 CH3 n 9-10; Triton X-100 n 7-8; Triton X-114 Preferred surfactants are branched chain surfactants such as Tween 20, Tween 40, Tween 60 and Tween When optionally added to the emulsion and micellar formulations of this invention, the neutral phospholipid component may be a single neutral phospholipid or a mixture of neutral phospholipids. Examples of neutral phospholipids which may be optionally added to the formulations of this invention include, but are not limited to, phosphatidylcholine (PC) or phosphatidylethanolamine (PE) or fully saturated or partially hydrogenated phosphatidylcholines (PC) or phosphatidylethanolomines (PE) having aliphatic chains between 6 and 24 atoms in length such as dioleoyl-PC (DOPC) and dioleoyl-PE (DOPE). A preferred neutral phospholipid is DOPE.

Methods for producing the emulsion and micellar WO 97/11682 PCT/US96/15388 15 formulations of the present invention are also provided.

One method for producing emulsion formulations of this invention comprises: combining an organic solvent with an oil component, a cationic amphiphile component, a nonionic surfactant component and optionally, a neutral phospholipid component; removing the organic solvent to leave a lipid film; and suspending the lipid film in an aqueous carrier to produce said emulsion formulation.

An alternative method for producing the emulsion formulations of this invention comprises: combining an oil component, a cationic amphiphile component, a nonionic surfactant component and optionally, a neutral phospholipid component; and adding an aqueous carrier to the combination of components in step to produce said emulsion.

Preferably, average diameters of the emulsion formulations are less than about 1000 nm, more preferably less than 800 nm, and most preferably less than 500 nm.

Preferred components of the emulsions of the present invention include phosphate-buffered saline (PBS) as the aqueous carrier, castor oil as the oil component, DC-Chol as the amphiphile component, Tween 80 as the nonionic surfactant component and optionally, phosphatidylcholine or DOPE as the neutral phospholipid component.

A method for producing the micellar formulations of this invention comprises: combining an organic solvent with a cationic amphiphile component, a nonionic surfactant component and optionally a neutral phospholipid component; removing the organic solvent to leave a lipid film; and WO 97/11682 PCT/US96/15388 16 suspending the lipid film in an aqueous carrier to form said micellar formulation.

Preferably, average diameters of the micellar formulations are less than about 1000 nm, more preferably less than about 800 nm; and most preferably less than about 500 nm.

Preferred components of the micellar formulations of the present invention include phosphate-buffered saline as the aqueous carrier, DC-Chol as the amphiphile component, Tween 80 as the nonionic surfactant component and optionally, PC or DOPE as the neutral phospholipid component.

When an organic solvent is used in the above methods to produce the micellar and emulsion formulations of this invention, any organic solvent which does not leave a toxic residue following removal and which solubilizes the lipid components of the emulsion and micellar formulations of this invention is suitable for use. Examples of suitable solvents include lower alcohols, dimethoxyethane, dioxane, tetrahydrofuran, tetrahydropyran, diethylether, acetone, dimethylsulfoxide (DMSO), dimethylformamides (DMF), and halogenated hydrocarbons, such as chloroform, acetonitrile, or mixtures thereof. A preferred organic solvent is chloroform.

The organic solvent may be removed by drying the combination of step under a suitable gas such as argon or nitrogen and/or under a vacuum. The dried film may then be lyophilized and stored at about -80 to about 37 0

C

or may be resuspended in a suitable aqueous carrier.

Aqueous carriers suitable for use in this invention are non-toxic to cells and may or may not be buffered. When the carriers are buffered, suitable buffers include buffers such as citrate, carbonate, bicarbonate, acetate, Tris, glycinate and maleate. Aqueous carriers which may be used in the formulations of this invention include, but IF are not limited to, distilled water, normal saline WO 97/11682 PCT/US96/15388 17 S solution and phosphate-buffered saline. It is to be understood that a preferred pH range for the emulsion and micellar formulations of this invention is a pH range in which the particular cationic amphiphile component present in a formulation is positively charged. Those of ordinary skill in the art would readily be able to determine such a pH range from the pKa of the cationic amphiphile component present in a particular formulation.

It is further understood that the aqueous carrier in which the lipid film is suspended may include ingredients such as stabilizers, antibiotics, or antifungal and antimycotic agents.

Once formed, the micellar and emulsion formulations may be mixed with biologically active substances to produce complexes which are stable in storage as reflected by a retention of the activity of the biologically activity substance over time or by retention of the diameter of the emulsion or micellar formulation over time.

In one embodiment, the ability of an emulsion or micellar formulation of this invention to deliver a biologically active substance to a cell may be tested by exposing cells to complexes formed between an emulsion or micellar formulation and a plasmid construct containing a reporter gene as the biologically active substance. Such reporter genes are known to those of ordinary skill in the art and include, but are not limited to, the chloramphenicol acetyltransferase gene, the luciferase gene, the 0-galactosidase gene and the human growth hormone gene.

By "biologically active substance" as used throughout the specification and claims is meant a molecule, compound, or composition, which, when present in an effective amount, reacts with and/or affects living cells and organisms. It is to be understood that depending on -F the nature of the active substance, the active substance WO 97/11682 PCT/US96/15388 18 S may either be active at the cell surface or produce its activity, such as with DNA or RNA, after being introduced into the cell.

Examples of biologically active substances include, but are not limited to, nucleic acids such as DNA, cDNA, RNA (full length mRNA, ribozymes, antisense RNA, decoys), oligodeoxynucleotides (phosphodiesters, phosphothioates, phosphoramidites, and all other chemical modifications), oligonucleotide (phosphodiesters, etc.) or linear and closed circular plasmid DNA; carbohydrates; proteins and peptides, including recombinant proteins such as for example cytokines (eg interleukins), trophic and growth or naturation factors (eg NGF, G-CSF, GM-CSF), enzymes, vaccines (eg HBsAg, gpl20); vitamins, prostaglandins, drugs such as local anesthetics procaine), antimalarial agents chloroquine), compounds which need to cross the blood-brain barrier such as antiparkinson agents leva-DOPA), adrenergic receptor antagonists propanolol), anti-neoplastic agents doxorubicin), antihistamines, biogenic amines (e.g.

dopamine), antidepressants desipramine), anticholinergics atropine), antiarrhythmics (e.g.

quinidine), antiemetics chloroprimamine) and analgesics codeine, morphine) or small molecular weight drugs such as cisplatin which enhance transfection activity, or prolong the life time of DNA in and outside the cells.

When the biologically active substance is an antigenic protein or peptide, the complexes formed by the emulsion or micellar formulations of the present invention may be utilized as vaccines. In this embodiment, the presence of oil in the emulsion formulation may enhance an adjuvant effect of the complex.

Preferred biologically active substances are negatively charged substances such as nucleic acids, negatively charged proteins and carbohydrates including WO 97/11682 PCT/US96/15388 19 polysaccharides, or negatively charged drugs.

In a more preferred embodiment the biologically active substances are nucleic acids and in a most preferred embodiment, the nucleic acids are nucleic acids which encode a gene or a gene fragment or which effect transcription and/or translation.

The complexes of the present invention may be utilized to deliver biologically active substances to cells in vitro or in vivo.

When the biologically active substance is a nucleic acid, it is believed that the cationic amphiphile binds to the negatively charged nucleic acid. Preferably, nucleic acid:emulsion complexes of this invention to be used in vitro or in vivo have a weight ratio of nucleic acid: total lipid components in the emulsion of about 1:1 to about 1:50, more preferably a weight ratio of nucleic acid:total lipid components in the emulsion of about 1:1 to about 1:30 and most preferably, a weight ratio of nucleic acid:total lipid components in the emulsion of about 1:1 to about 1:20. Thus for example, in Example 11 where a DNA:emulsion complex was formed by combining 100 pg of DNA with a volume of emulsion formulation #21 containing 1100 pg total lipid components, the weight ratio of DNA:total lipid components of emulsion #21 in the complex was 100 pg/1100 pg or 1:11.

Preferably, nucleic acid:micelle complexes of this invention to be used in vitro or in vivo have a weight ratio of nucleic acid:total lipid components in the micelle of about 1:1 to about 1:50, more preferably a weight ratio of nucleic acid:total lipid components in the micelle about 1:1 to about 1:30 and most preferably a weight of nucleic acid:total lipid components of the micelle ratio of about 1:1 to about 1:20. Thus for example, in Example 11 where a DNA:micelle complex was formed by combining 100 pg of DNA with a volume of micellar formulation #31 containing 700 pg total lipid WO 97/11682 PCT/US96/15388 20 components, the weight ratio of DNA:total lipid components of micelle #31 in the complex was 100 ug/700 pg or 1:7.

It is to be understood that the combining of emulsion or micellar formulations with a nucleic acid to form the nucleic acid:emulsion or nucleic acid:micellar complexes of this invention may be carried out for at least minutes in the presence or absence of serum at a temperature from about 4 0 C to about 37 0 C. The resultant nucleic acid:emulsion and nucleic acid:micelle complexes may then be immediately used in vitro or in vivo or may be stored prior to use.

Preferably, average diameters of nucleic acid:emulsion or nucleic acid:micelle complexes to be used in vitro or in vivo are 100-4000 nm, more preferably 100- 2000 nm and most preferably 100-1000 nm.

In one embodiment, the nucleic acid micelle and nucleic acid:emulsion complexes of this invention may be used to transfect cells with nucleic acid. Cells suitable for transfection in vitro include eukaryotic cells, including all mammalian cell lines suitable for transfection by cationic lipids, cells put into primary culture from a host, or cells resulting from passage of the primary culture.

When, for example, 105 cells are transfected in vitro, transfection is carried out by exposing the cells to preferably from about 0.1 to about 5 pgs of nucleic acid:emulsion complex, more preferably to about 0.5 to from about 2 pgs of nucleic acid:emulsion complex.

When 105 cells are to be transfected with nucleic acid:micelle complex, transfection is carried out by exposing the cells to preferably from about 0.1 to about Ags of nucleic acid:micelle complex; more preferably to about 1 to about 10 pgs of nucleic acid:micelle complex.

As used herein, pg of nucleic acid:emulsion complex or pg of nucleic acid:micelle complex refers to the sum of the Ag amount of nucleic acid and the Ag amount of total WO 97/11682 PCT/US96/15388 21 lipid components in the emulsion or micellar formulation contained in the complex. For example, 5 pg of nucleic acid:emulsion complex might contain 0.5 pg of nucleic acid and 4.5 pg of total lipid components of emulsion formulation.

Those of ordinary skill in the art would readily understand that the total amount of nucleic acid:emulsion or nucleic acid:micelle complex to be used varies directly with the number of cells to be transfected. One advantage of the emulsion and micellar formulations of this invention over prior art cationic lipid vectors is that the emulsions and micelles of the invention, when complexed with nucleic acid, are more effective for transfecting cells cultured in serum-containing medium.

The present invention therefore relates to the use of the nucleic acid:emulsion and nucleic acid:micelle complexes of this invention to deliver nucleic acids to cells in an animal or human in vivo. Thus, the present invention also relates to the use of nucleic acid:emulsion or nucleic acid:micelle complexes as delivery systems in gene therapy.

Suitable routes of administration of the nucleic acid containing complexes of this invention to an animal or human include inoculation or injection by, for example, intravenous, oral, intraperitoneal, intramuscular, subcutaneous, intra-aural, intraarticular or intra-mammary routes, topical application, for example on the skin, scalp, ears or eyes and by absorption through epithelial or mucocutaneous linings, for example, nasal, oral, vaginal, rectal and gastrointestinal among others, and as an aerosol. Those of ordinary skill in the art would readily understand that the mode of administration may determine the sites in the organism to which the biologically active substance will be delivered and may effect the amount of complex to be administered.

Since as shown in Example 11, administration of WO 97/11682 PCT/US96/15388 22 approximately 4 fg of nucleic acid:emulsion or nucleic acid:micelle complex/gram of body weight to a 25 gram mouse produced transfection activity in vivo, those of ordinary skill in the art using this ratio of 4 Ag of complex/gram of mouse body weight could obtain other ratios of g of complex/gram of body weight which are optimized for transfection activity in other animals or humans.

In an alternative embodiment, the emulsion and micellar formulations themselves may bind with biomacromolecules molecules produced by the animal or human) in situ after systematic or topical administrations and behave as a local depot for endogenous bioactive substances.

All articles or patents referenced herein are incorporated by reference. The following examples illustrate various aspects of the invention but are in no way intended to limit the scope thereof.

Examples Material and Methods Materials: DC-Chol cationic lipid was synthesized according to Gao and Huang (Biochem. Biophys. Res. Commun., 179:280- 285, 1991). Tween 80 and castor oil were obtained from Fisher, pluronic co-polymer L63 was obtained from BASF.

Brij, Span, and pluronic F68 and F127 surfactants were purchased from Sigma. Dioleoyl phosphatidylethanolamine (DOPE) and egg phosphatidylcholine (PC) were obtained from Avanti Polar Lipids. LipofectAMINE liposomes (DOSPA (2, 3-dioleyloxy-N-[2(spermine carboxamido)ethyl]-N,N,dimethyl-l- propanaminium) and DOPE) in a weight ratio of 3:1) were obtained from Life Technologies, Inc.

Preparation of emulsions and micelles: Tween 80 diluted in chloroform was combined with DC- Chol (micelles) and, where indicated DOPE or WO 97/11682 PCT/US96/15388 23 S phosphatidylcholine; or with castor oil, DC-Chol and, where indicated, DOPE or phosphatidylcholine (emulsions) at different weight ratios. The organic solvent was then evaporated under a stream of nitrogen gas and the lipid film was vacuum desiccated at 4 0 C overnight to remove residual organic solvent. One ml of phosphate buffered saline (PBS, pH 7.4) was then added and the mixture was allowed to hydrate for 1 h. The lipid suspension was then mixed with a vortex mixer and subsequently homogenized for 3-4 min using a tissue tearer at a speed of about 20,000 rpm. Average diameters of the emulsion or micelle formulations and of the DNA:emulsion or DNA:micelle complexes were measured by laser light scattering using a Coulter N4SD submicron particle sizer.

Preparation of DC-Chol/DOPE liposomes: Unilamellar small liposomes of approximately 100 nm in diameter were prepared by microfluidization of hydrated mixture of DC-Chol and DOPE (weight ratio of 1:1) and filter sterilized. The final lipid concentration of the DC-Chol/DOPE liposomes used in the transfection experiments was 1.2 g/ll of PBS.

Tissue culture: Murine melanoma BL6 cells were cultured in RPMI medium supplemented with 10% fetal bovine serum. Human embryonic kidney 293 cells and F, were cultured in DMEM medium supplemented with 10% fetal bovine serum. CHO cells were cultured in F12 medium supplemented with 10% fetal bovine serum.

Plasmid DNA: A pCDNA 3 plasmid, pCMV-Luc, containing the luciferase gene under the control of cytomegalovirus (CMV) immediate early promoter was used to assess the efficiency of transfection. A similar plasmid, pRSV-Luc, containing the same luciferase gene under the control of a Rous sarcoma virus promoter was also used to assess transfection efficiency. Plasmid pCMV-CAT is a pUC18 based plasmid WO 97/11682 PCT/US96/15388 24 containing the E.coli chloramphenicol acetyltransferase (CAT) gene downstream from the CMV promoter. The preparation and purification of plasmid DNA was carried out according to standard procedure (Sambrook, J., Fritsch, Maniatis, T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Lab Press: Plainview, 1: pp 21-52, 1989.).

Transfection: Cells cultured in 48 well plates (about 70-80% confluent) were used for transfection and 3 wells were transfected with each formulation. The pCMV-Luc or pRSV- Luc plasmid DNAs were diluted in 125Al of serum free CHO- S-SFM medium (Life Technologies, Inc.). The emulsion, micelle, DC-Chol/DOPE liposomes or lipofectAMINE liposomes were diluted in 125

L

1 of Hank's balanced salt solution (HBSS). The diluted DNA and formulations or liposomes were combined, with or without the addition of fetal bovine serum to 20%, and incubated at room temperature for 5-10 min, before being added to the cells. The cells were incubated at 37 0 C for 5 h. Transfection medium was replaced with growth medium containing 10% fetal bovine serum, and cells were then cultured for 2 days before the luciferase assay was performed.

Luciferase assay: Cells were washed twice with PBS and incubated at room temperature for 10 min in the presence of 100 pl lysis buffer (0.1M Tris-HC-1, pH 7.8/0.05% Triton X- 100/2mM EDTA) and then centrifuged at 12,000xg. Ten pl of supernatant was taken for luciferase assay using the luciferase assay system (Promega) in a luminometer (AutoLumat LB953 from EG&G, Berthhold). Luciferase activity is given in relative light units (RLU).

Animal studies: Female CD 1 mice, 5 weeks old, were purchased from Charles River Breeding Laboratories. Animal care was according to the institutional guidelines. 200 pl each of WO 97/11682 PCT/US96/15388 25 4x concentrates (for example, the 4x concentrate of formulation #21 contained 1.0 mg oil, 1.0 mg PC, 0.5 mg Tween 80 and 3.0 mg DC-Chol per ml of PBS) of formulations #21 (1100 ig total lipid components), #28 (1100 Ag total lipid components), #34 (900 pg total lipid components) and #31 (700 jig total lipid components) (600 pg DC-Chol per formulation) were mixed with 5M NaCI to a final concentration of NaC1 of 0.15M and then combined with Al of 4 Mg/Ml pCMV-CAT DNA (100 Ag). The total mixture (231 pl) was injected into mice by tail vein. Two days later, mice were killed and liver, spleen, kidney, lung and heart were excised for CAT assay.

Chloramphenicol acetyltransferase(CAT) assay: The organs excised from animals were homogenized in Tris-HCl, pH 7.5;10 mM EDTA;150 mM NaC1. After homogenization, cells were lysed by three freeze-thaw cycles, and the lysate was heated at 65 0 C for 10 min to inactive deacetylases and centrifuged for 10 min. The protein concentration of the supernatant extracts was measured with a Coomassie blue G 250-assay (Pierce).

Protein was extracted from each organ and 200 Ag of extract was then assayed for the CAT activity using chloramphenicol as a substrate as previously described (Ausubel, Breht, Kingstone, et al.

Current Protocols in Molecular Biology (Wiley, Boston), Vol. 1, pp 962-965, 1991.).

Example 1 Physical stability of emulsion formulations and transfection ability of DNA:emulsion complexes To test which components of the emulsion formulations are important for physical stability and transfection ability, 9 different emulsion formulations containing different amounts of castor oil, egg phosphatidylcholine Tween 80 and DC-Chol were formulated. The average diameter of the formulations was measured as was their ability to transfect 293 cells by combining 6 pl of each WO 97/11682 PCT/US96/15388 26 emulsion (7.5 pg total lipid components) with 0.5 pg RSV- Luc. The resulting data are presented in Table 1.

Table 1 Transfection activity of DNA:emulsion complexes formed by combining DNA with different emulsion formulations Formulation Composition (mg) Average Transfection of formulation Diameter (nm) Activityb Oil PC Tween 80 DC-Chol PBS of formulation (RLU x 6 /well) #1 0.125 0.250 0.125 0.125 0.50 170 118±22 #2 0.250 0.250 0.250 0.750 1.20 151 444±4 #3 0.750 0.250 0.025 0.250 1.02 212 265±46 #4 0.125 0.125 0.025 0.750 0.82 218 332±89 0.250 0.125 0.125 0.250 0.60 181 220±7 #6 0.750 0.125 0.250 0.125 1.00 154 68±1 #7 0.125 0.500 0.250 0.250 0.90 165 65±20 #8 0.250 0.500 0.025 0.125 0.72 201 1±0.6 #9 0.750 0.500 0.125 0.750 1.70 161 261±47 DC-Chol/DOPE 122 445±2 (1:1 ratio by weight) LipofectAMINE (DOSPA and 100 427 DOPE in a 3:1 weight ratio) a: The final concentration of the emulsion was 1.25 pg total lipid components/pl. Lipid concentrations of DC-Chol/DOPE liposome and LipofectAMINE were 1.2 pg/pl and 2.0 pg/pl respectively.

b: 293 cells were transfected with complexes formed by combining 0.5 lg of pRSV-Luc and 6 pl of emulsion (7.5 pg total lipid components) or with complexes formed by combining 0.5 pg of pRSV-Luc and 2.5 pl of DC-Chol/DOPE liposomes (3 ig lipid components) or with complexes formed by combining 0.5 pg of pRSV-Luc and 2.25 pl of lipofectAMINE liposomes (4.5 Ug lipid components). Each well contained approximately 70-80 pg extractable protein.

WO 97/11682 PCT/US96/15388 28 The data show that the emulsions are physically stable with size ranging from 150 to 218 nm in average diameter as measured by laser light scattering. Further, complexes of DNA with those emulsions with high content of DC-Chol (0.750 mg), the only cationic component in the emulsion, showed high transfection activity comparable to that of the cationic DC-Chol/DOPE and LipofectAMINE liposomes.

Example 2 Transfection activity of DNA:emulsion and DNA:micelle complexes Emulsion and micellar formulations which contained high content of the cationic amphiphile DC-Chol (0.75 mg or more) were complexed with pRSV-Luc DNA and examined for transfection activity in BL6 and 293 cells. The data presented in Table 2 Table 2 Transfection activity of DNA:emulsion and DNA:micelle complexes Formulation Composition (mg) Average Transfection Activity' of formulation Diameter (inn) of BL6 cells 293 cells Oil PC Tween 80 DC-Chol DOPE Stearylamine PBS formulation (RLU x 10 3 /well) (RLU x 10 6 /well) 0.250 0.250 0.250 6.750 1.2 129 13±8 530±78 #11 0.125 0.125 0.250 0.375 0.7 143 15±4 546±62 #12 0.125 0.125 0.375 0.375 0.8 127 4±2 239±15 #13 0.125 0.125 0.125 0.500 0.7 153 54±34 708±213 #14 0.125 0.125 0.125 0.750 0.9 152 112±37 880±13 0.250 0.250 0.750 1.0 165 19±2 861±22 #16 0.250 0.250 0.750 1.0 161 78±14 710±47 #17 0.250 0.250 0.750 0.250 1.2 155 23±10 463±90 #18 0.250 0.250 0.250 0.400 0.90 193 3±1 9±1 #19 0.250 0.750 0.8 186 262±104 806±71 0.250 0.250 0.750 1.0 160 19±9 840±20 DC-ChoI/DOPE 0.600 0.600 1.0 122 81±40 221±35 a: The final concentration of the formulations was 1.25 jsg total lipid components/IAL Lipid concentration of DC-CboIIDOPE liposome was 1.2 jig/Ipl.

b: 293 cells were transfected with complexes formed by combining 0.5 ug of pRSV-Luc and 6 IAI of the indicated emulsion or micellar formulation jig total lipid components) or with complexes formed by combining 0.5 jig of pRSV-Luc and 2.5 jil DC-Chol/DOPE liposomes (3.0 pg lipid components). Each well contained approximately 70-80 jug extractable protein except for #18 which contained 50 p~g protein.

WO 97/11682 PCT/US96/15388 30 show that complexes of DNA with the emulsions and the two micellar formulations (#15 and #19) were active. However, the complex formed by combining DNA with formulation #18 that contained stearylamine instead of DC-Chol showed low transfection activity which may be due to the toxicity of stearylamine to cells as evidenced by the fact that the amount of protein extractable from wells transfected with complex containing formulation #18 was less than the amount of protein extractable from wells transfected with complexes containing all other formulations (see Table 2, footnote Of interest, the activities of a micellar and an emulsion DNA complex were high, and indeed more active, than the cationic liposome formulation (Dc-Chol/DOPE) in both cell lines.

Example 3 Transfection activity of more DNA:emulsion and DNA:micelle complexes The physical diameter of additional emulsion and micellar formulations was measured as was the transfection 2 activity of complexes formed between DNA and these formulations. The results are shown in Table 3.

W 0 LIJ 0 Table 3 Transfection activity of more DNA:emulsion and DNA:micelle complexes Composition (mg) l of formulation Formulation Pluronic L63 Oil PC Tween 80 DC-Chol DOPE #21 #22 #23 #24 #26 #27 #28 #29 #31 #32 #33 #34 DC-Chol/DOPE 0.250 0.250 0.250 0.250 0.250 0.250 0.250 A c 0.

0.

0.

0.

0.

0.

250 0.125 250 0.250 250 0.500 250 0.250 250 0.250 250 0.250 0.125 0.125 0.750 0.750 0.750 1.000 1.500 0.750 0.750 0.750 0.750 0.750 0.750 0.750 0.750 0.750 Average Diameter (nm) of formulation 137 218 133 152 152 168 163 169 146 202 179 204 212 200 122 n Icn Transfection Activityb BL6 293 (RLU x 10S/well) (RLU x 10 7 /well)

U

0.250 0.125 0.125 0.250 0.250 0.500 0.250 228±40 235 67 2±0.4 322±76 98 28 450 11 426 83 645±219 117±44 759±65 324 22 150±45 107±32 890±35 1033 204 101± 11±16 104±4 34 ±14 122±40 130±26 34±1 56±4 94±58 188 88±20 82±16 40±13 a: 1 ml of PBS was added to each formulation. The concentration of DC-Chol in each formulation was 0.75 mg/ml except #24 and in which the DC-Chol concentrations were 1.0 mg/ml and 1.5 mg/ml respectively. However, the total lipid concentration or eacn formulation was different.

b: Cells were transfected with complexes formed by combining 0.5 pg of pCMV-Luc and 6 pl of each formulation (4.5 pg DC-Chol per 6 ul of each formulation) or with complexes formed by combining 0.5 pg pCMV-Luc and 2.5 pl of DC-Chol/DOPE liposomes (3.0 g lipid components). Each well contained approximately 70-80 pg extractable protein.

WO 97/11682 PCT/US96/15388 32 The results show that complexes of DNA and micelles containing DC-Chol and Tween 80 (#30 and were again quite active and replacing Tween 80 with pluronic L63 (#32 and #33) did not significantly alter the average diameters of the formulations or their transfection activity in 293 cells. Another micelle containing DC-Chol, Tween 80 and PC (phosphatidylcholine) was also active. The remaining formulations (#22-25, 27-29) were emulsions which contained castor oil. Complexes of DNA and these emulsion formulations were fairly active in transfection.

When complexes of DNA and these micellar and emulsion formulations were tested in another cell line (BL6 mouse melanoma cells), qualitatively similar results were obtained except the activity in this cell line was generally lower than that of the 293 cells. The transfection activities of complexes of DNA and these emulsions and micelles were comparable to that of the cationic liposome formulation (DC-Chol/DOPE) in 293 cells, but somewhat lower in BL6 cells.

Example 4 Optimization of transfection conditions: To optimize the transfection activity of the DNA:emulsion and DNA:micelle complexes, the amount of DC- 2 Chol in each emulsion or micelle formulation was kept constant at 4.5 Ag (6 ul of formulations #s 21, 27, 28 and respectively were used; refer to Table 3 for compositions of formulations) and the amount of pCMV-Luc DNA was varied from 0 to 5 ig. As shown in Figure 1, 3 complexes of DNA and formulations #27 and #30 (refer to Table 3 for compositions) showed a maximal transfection activity in BL6 cells at 2 ig DNA while complexes of DNA and formulations #21 and #28 showed a maximum activity at ig DNA.

Next, the amount of pCMV-Luc DNA was fixed at 2 ig for formulations #27 and #30 and at 1.5 g for for formulations #27 and #30 and at 1.5 yg for WO 97/11682 PCT/US96/15388 33 formulations #21 and #28 and complexes of DNA and varying amounts of emulsion or micelle formulation as indicated on the horizontal axis of Figure 2 (where pg formulation on the horizontal axis refers to pg total lipid components present in the volume of formulation combined with pCMV- Luc DNA to form complex) were produced. The results presented in Figure 2 show that complexes of DNA and formulations #27, #28 and #30 exhibited a relatively broad peak of activity in BL6 cells with the optimal amount of total lipid components present in the volume of formulation combined with DNA to produce complex being about 18pg. However, complexes of DNA and formulation #21 exhibited a narrower peak of activity with the optimal amount of total lipid components present in the volume of formulation combined with DNA to produce complex being about 13 pg.

Example Sensitivity of transfection activity of DNA:emulsion and DNA:micelle complexes to serum All the above described transfection experiments were carried out in a serum-free medium. Therefore, to determine the sensitivity of transfection activity of DNA/emulsion and DNA/micelle complexes to the presence of serum, BL6 cells were transfected in the medium containing 0 or 20% fetal bovine serum with complexes of DNA and different emulsions or micelles (or DC-Chol/DOPE liposomes) as follows.

Different formulations of the compositions shown in Table 4 were prepared in a total volume of 1 ml PBS (pH BL6 cells in a 48 well plate were then transfected with 2 pg of pCMV-Luc and 16 A1 of each formulation (containing 12 pg DC-Chol), or with 2 pg of pCMV-Luc and 16 1l of DC-Chol/DOPE liposomes (1.2 pg/pl), in medium containing either 0 or 20% fetal bovine serum and luciferase activity was detected. Each well contained WO 97/11682 PCTIUS96/1 5388 34 approximately 70-80 gig extractable protein. The results are shown in Table 4 Table 4. Transfection Activity Of Additional DNA:emulsion and DNA:micelle complexes FORMULATION COMPOSITION(mg) 9 LUCIFERASE ACTIVITY (RLU/well x of formulation Oil PC DOPE Tween. 80 DC-Chol -Serum 0.25 0.25 0.125 0.75 14 ±3 49 ±2 #36 0.25 0.125 0.75 32±6 98±6 #37 0.25 0.125 0.75 170 ±7 5 ±3 #38 0.25 0.25 0.75 24 0.8 56 ±13 #39 0.25 0.25 0.125 0 0 0.25 0.25 0.125 0.75 50 ±3 151 ±6 #41 0.25 -0.25 0.75 40±5 85±8 #42 0.25 0.125 0.75 140 ±5 267 ±44 #43 0.125 0.75 156 ±13 298 ±42 #44 0.25 0.75 259± 12 307 ±8 DC-Cho1IDOPE liposomes 0.60 0.60 107 ±21 47 ±9 a The average diameter of each formulation was 100-250 un.

WO 97/11682 PCT/US96/15388 36 demonstrate that the transfection activity of DC-Chol/DOPE liposomes was quite sensitive to serum (only about 33% activity remained in the presence of serum) while of the formulations tested, only complex of DNA and formulation #37 showed serum sensitivity where serum sensitivity is a reduction in transfection activity in the presence of serum relative to the level of activity observed in the absence of serum. In addition, the fact that complex of DNA and formulation #39 showed no activity in the presence or absence of serum demonstrated that the presence of cationic amphiphile is critical to transfection activity. Particularly interesting are complexes of DNA and formulations #35, #36 and (corresponding in composition to formulations 21, #34 and #28 respectively in Table 3) which, of the formulations tested, exhibited the greatest enhancement of transfection activity in the presence of serum.

Example 6 Sensitivity Of Transfection Activity Of Complexes Of DNA and Selected Formulations To Serum In Different Cell Lines To determine if the serum sensitivity observed in BL6 cells in Table 4 was observed in other cell lines, F 0 cells, CHO cells and 293 cells were transfected with 16 Al of selected formulations (each containing 12 pg DC-Chol/16 Al) from Table 4 combined with 2 Ag of pCMV-Luc DNA or, with 16 Al DC-Chol/DOPE liposomes (1.2 ug/tl) combined with 2 ig of pCMV-Luc DNA, in medium containing 0 or serum as in Example 5. The results of these experiments are shown in Table 5. The data show that complexes of DNA and all formulations are active in transfecting cells and are in general, serum-resistant.

Table 5. Transfection Activity of Complexes Of DNA And Selected Formulations in Different Cell Lines.

FORMULATION LUCIFERASE ACTIVITY (RLU/Well x (wiw)

F

0 Cells CHO Cells 293 Cells -Serum Serum -Serum Serum -Serum Serum (Oil/PC/Tw/DC-Chol 8.3 3.7 13.2 ±7.0 8.0 0.7 5.9 ±0.4 33.0 ±7.8 37.0 3.4 (2:2:1:6)1 #37 (Oil/Tw/DC-ChoI) 2.7 0.2 1.6 ±0.9 1.6 1.6 10.4 ±1.3 33.3 ±11.3 44.2 1.2 (2:1:6:rb (Oil/DOPE/Tw/DC-Chol) 8.7 2.1 12.0 ±2.1 0.4 0.0 7.5 ±0.4 27.9 ±7.0 34.7 2.1 (2:2:1 :6)c #44 (TwIDC-Chol) 22.0 ±3.3 13.2 ±1.1 0.4 0.1 1.5 ±0.3 68.0 ±17.0 168.0 17.0 (2:6)d flOPE/DC-Chol litnosomes 2.7 +0.2 0.5 ±0.1 1.7 0.0 2.8 ±0.5 88.0 ±3.9 67.0 3.9 l)C a 2:2:1:6 0.25 mg/0.25 mg/0.125 nig/0.75 mg per ml of solution.

b 2:1:6 =0.25 mg/0.125 mgIO.75 mg per ml of solution.

C 2:2:1:6 0.25 mg/0.25 mgIO.125 mg/0.75 mg per ml of solution.

d 2:6 0.25 mgIO.75 mg per ml of solution.

e 1: 1 0. 6mgfO.6mg permnl of solution.

WO 97/11682 PCT/US96/15388 38 Example 7 Stability of DNA:emulsion and DNA:micelle complexes Five different formulations 26, 27, 28, 29 and 34 of Table 3) were tested for the stability of their complex with DNA. Complex was prepared by combining 2pg pCMV-CAT DNA and 16A1 of the indicated emulsion or micelle formulation (where 16 p1 of each formulation contained the same amount of DC-Chol, 12 pg) or by combining 1 pg pCMV- CAT DNA and 6 pg of DC-Chol/DOPE liposomes. As can be seen in Figure 3, formulations #26 #28 and #29 formed relatively small complexes with DNA; the average diameter of the complex as measured by laser light scattering ranged from 200-300 nm, and remained small even after days at 4 0 C. Formulation #34 and #27, on the other hand, 1 formed larger complexes with DNA with average diameters of 600 and 900 nm, respectively. In contrast, DC-Chol/DOPE liposomes had formed large aggregates (1,800 nm on day 1) which had grown to even larger ones (>4,000 nm) on day 3 and subsequently precipitated out of solution (data not 2 shown). Thus, all new formulations could form complexes with DNA that had physical stability better than that of complexes formed with the DC-Chol/DOPE liposomes.

Example 8 Effect of Different Surfactants on the Transfection Activity of Complexes of DNA and Emulsions Composed of Oil/DOPE/DC-Chol/Surfactant in a Weight Ratio of 2:2:6:x Emulsions containing different surfactants were prepared in 1 ml of PBS containing 0.25 mg of oil, 0.25 mg of DOPE, 0.75 mg of DC-Chol and different amounts of the indicated surfactants where the total amount of surfactant used in each formulation was approximately the same by mole. BL6 cells were then transfected with 2 pg of pCMV- Luc DNA combined with 16 1l of formulation (12 pg DC- Chol/16 Ai of each formulation) and assayed for luciferase activity. The results of this experiment are shown in Table 6.

WO 97/11682 WO 9711682PCTIUS96/1 5388 39 Table 6. Effect Of Different Surfactants On The Transfection Activity Of Complexes Of DNA and Emulsions Composed Of OIL/DOPE/DC- Chol/surfactant (0.25 mg:0.25 mg:0.75 mg:X mg per ml of PBS) Surfactant X (mg) LUCIFERASE ACTIVITY (RLU/Well) x W0 Tween Brij 72 74 76 100 Span 20 80 pluronic F pluronic F 20 68 127 0.117 0.122 0.125 0.034 0.068 0.110 0.446 0.033 0.03 8 0.041 0.041 0.802 1.202 -Serum .9 ±0.4 .9 ±0.3 .2 ±0.3 .0 ±0.7 .4 ±1.6 .4 ±0.8 1.3 ±0.2 .2 ±0.0 .9 ±0.1 .4 ±0.4 .4 ±0.4 .8 ±0.3 1.9 ±0.9 +Serum 4.9 ±1.1 5.3 ±0.7 5.6 10.0 6.5 ±0.3 0.08 ±0.03 0.003 ±0.006 0.1 ±0.0 0.1 ±0.0 0.3 ±0.1 0.3 ±0.1 9.0 ±1.4 9.9 ±1.1 WO 97/11682 PCT/US96/15388 40 Of the surfactants tested, complexes formed from emulsions containing Tween 20, Tween 40, Tween 60, Brij 72, Brij 74, F68 or F127 demonstrated transfection activity that was not sensitive to the presence of serum and complexes formed from formulations containing the Tween series of detergents showed the greatest increase in transfection activity in the presence of serum relative to that observed in the absence of serum.

Example 9 Effect of Tween 80 Concentration in Emulsions On the Average Diameters of Concentrated and Diluted DNA/Emulsion Complexes Concentrated DNA/emulsion complex was formed by adding 2 pl of solution containing 8 pg of DNA (pCMV-Luc) directly to 72 pl of emulsions containing 0.25 mg Oil/0.25 mg DOPE/0.75 mg DC-Chol and varying mg amounts of Tween per ml. As in the prior examples diluted DNA/emulsion complex was formed by combining 2 ig of DNA in 125 pl with 18 pl of the same emulsions used in the concentrated complex but diluted to 125 pl. The average diameters of the concentrated and diluted complexes were measured 1 hour after incubation at room temperature. The results shown in Figure 4 demonstrate that increasing amounts of Tween 80 reduced the size of the concentrated complexes 2 but had no effect on the size of the diluted complexes.

Example Effect of the Amount of Tween 80 in an Emulsion on Transfection Activity of Concentrated and Diluted DNA/Emulsion Complexes in the Presence or Absence of 20% Serum Emulsions and concentrated and diluted pCMV-Luc DNA /emulsion complexes were prepared as in Example 9 and the transfection activity of the complexes was measured in BL6 cells in the presence or absence of 20% serum. The results of these experiments are shown in Figures WO 97/11682 PCT/US96/15388 41 (concentrated complex) and 5B (diluted complex). While the diluted complexes appear to show better activity than the concentrated complexes, the need to keep the volume of complex administered to an animal small may favor the use of more concentrated complexes in vivo.

Example 11 Animal studies with DNA:emulsion and DNA:micelle Complexes Formulations #21, 28, 31 and 34 (refer to Table 3 for compositions) were tested for gene transfer activity in mice. 200 pl each of 4x concentrates of formulations #21 (1100 pg total lipid components), #28 (1000 pg total lipid components), #34 (900 pg total lipid components) and #31 (700 pg total lipid components)) were mixed with 6 Al of NaCl to a final concentration of 0.15M NaC1 and then combined with 4 pg/Ml pCMV-CAT DNA (100 pg). The complexes were then injected i.v. via the tail vein of the mouse (each mouse weighed approximately 25 grams) and CAT activity was measured in major organs two days after injection. The data presented in Figure 6 clearly demonstrates that complexes of DNA and formulations #28 (emulsion), #31 (micelle) or #34 (micelle) could transfect various organs with relatively high activity while complex of DNA and formulation #21 (emulsion), on the other hand, was weak and comparable to that of DC-Chol/DOPE liposomes.

In addition, the activity of complex of DNA and formulation #31 seems to be lung specific, as no other organs were significantly transfected; complex of DNA and formulation #28 could transfect all organs quite well with only weak transfection of the kidney, and complex of DNA and formulation #34 showed a high activity in the heart with very low activity in the kidney.

Claims (32)

1. A formulation including lipid components, a nucleic acid and an aqueous carrier, wherein the lipid components include a cationic amphiphile component, and a nonionic surfactant component.

2. The formulation of claim 1 wherein said formulation is an oil-in-water emulsion formulation including an oil component, a cationic amphiphile component, a nonionic surfactant component and an aqueous carrier.

3. The formulation of claim 1 or claim 2 wherein said formulation is an oil- in-water emulsion formulation including an oil component present in an amount from about 10 to about 80 weight of the total lipid components in the formulation, an amphiphile component present in an amount from about 5 to about 80 weight of the total lipid components and a nonionic surfactant component present in an amount from about 5 to about 50 weight of the total lipid components.

4. The formulation according to any one of claims 1 to 3 further including a neutral phospholipid component. The formulation according to any one of claims 1 to 4, wherein the S. amphiphile component is a cationic lipid selected from the group consisting of 1,2 bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP), 25 dioleoyloxy) propyl]-N, N, N-trimethyl ammonium chloride (DOTMA), 1,2- dioleoyl-3-(4' trimethylammonio) butanoyl-sn-glycerol (DOBT), cholesteryl (4' trimethylammonia) butanoate (ChOTB), DL-1, 2-dioleoyl-3- dimethylaminopropyl-B-hydroxyethylammonium (DORI), DL-1,2-O-dioleoyl-3- dimethylaminopropyl--hydroxyethylammonium (DORIE), 1,2-dioleoyl-3- 30 succinyl-sn-glycerol choline ester (DOSC), cholesteryl hemisuccinate choline ester (ChOSC), doctadecylamidoglycyl-spermine (DOGS), dipalmitoyl phosphatidyesthanolamidospermine (DPPES), cholesteryl-3P-carboxyl-amido- ethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonio-DL- 2-propyl-cholesteryl carboxylate iodide, carboxyamidoethyleneamine, ethylenetrimethylammonium iodide, 1 -dimethylamino-3 -trimethylammonio- DL-2-propyl-cholesteryl-30-oxysuccinate iodide, trimethylammonio)-ethylmethylamino] iodide, N'-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol), and 30-[N-(polyethyleneimine)-carbamoyl] cholesterol.

6. The formulation of claim 1, wherein the formulation is a micellar formulation including a cationic amphiphile component, a nonionic surfactant component and an aqueous carrier.

7. The micellar formulation of claim 6, wherein the lipid components further include a neutral phospholipid component.

8. The micellar formulation of claim 6 or claim 7 wherein the amphiphile component is present in an amount from about 10 to about 90 weight of the total lipid components and the nonionic surfactant component is present in an amount from about 90 to about 10 weight o/o of the total lipid components.

9. The micellar formulation according to any one of claims 6 to 8, further including a neutral phospholipid component present in an amount from about to about 40 weight of the total lipid components. The micellar formulation according to any one of claims 6 to 9, wherein the amphiphile component is a cationic lipid selected from the group consisting of 1,2 bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP), dioleoyloxy) propyl]-N, N, N-trimethyl ammonium chloride (DOTMA), 1,2- dioleoyl-3-(4' trimethylammonio) butanoyl-sn-glycerol (DOBT), cholesteryl (4' trimethylammonia) butanoate (ChOTB), DL-1, 2-dioleoyl-3- S dimethylaminopropyl-B-hydroxyethylammonium (DORI), DL-1,2-O-dioleoyl-3- dimethylaininopropyl-p-hydroxyethylammonium (DORIE), 1,2-dioleoyl-3- succinyl-sn-glycerol choline ester (DOSC), cholesteryl hemisuccinate choline ester (ChOSC), doctadecylamidoglycyl-spermine (DOGS), dipalmitoyl phosphatidyesthanolamidospermine (DPPES), cholesteryl-3P-carboxyl-amido- ethylenetrimethylamnmonium iodide, 1-dimethylamino-3-trimethylammonio-DL- 2-propyl-cholesteryl carboxylate iodide, carboxyamidoethyleneamine, cholesteryl-3 -oxysuccinamido- ethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonio-DL- 2-propyl-cholesteryl-3 -oxysuccinate iodide, 2-[(2-trimethylammonio)- ethylmethylamino] ethyl-cholesteryl-30-oxysuccinate iodide, N'- dimethylaminoethane) carbamoyl] cholesterol (DC-Chol), and 3p-[N- (polyethyleneimine)-carbamoyl] cholesterol.

11. The formulation of claim 2 or claim 6 for facilitating the delivery of the nucleic acid to cells, wherein the cationic amphiphile is N'- dimethylaminoethane) carbamoyl] cholesterol (DC-Chol).

12. The formulation according to any one of claims 2 to 5, wherein the oil component is a natural oil.

13. The formulation according to any one of claims 1 to 12, wherein the weight ratio of nucleic acid to total lipid components in the emulsion formulation is about 1:1 to about 1:50.

14. The formulation of claim 12, wherein the oil component is castor oil.

15. A method for delivering a nucleic acid to cells including exposing the cells to the formulation according to any one of claims 1 to 14 thereby S16. The method of claim 15 wherein the cells are mammalian cells exposed to 25 the formulation in the presence of serum.

17. The method according to claim 15 or claim 16 wherein the cells are exposed to the formulation in vivo by administering the formulation to an animal or human in an amount effective to facilitate the delivery of the nucleic acid to 30 the cells of the animal or human. o 18. The formulation according to any one of claims 1 to 14, wherein the nonionic surfactant is selected from the group consisting of polyethylene glycol, derivatives of phosphatidylethanolamine and synthetic detergents.

19. The method according to claim 15 or claim 16 wherein the cells are exposed to the formulation in vitro. A method of producing an oil-in-water emulsion formulation including: combining an oil component, a cationic amphiphile component and a nonionic surfactant component; adding aqueous carrier to produce said emulsion formulation; and combining the emulsion formulation with a nucleic acid.

21. A method of producing a micellar formulation including: combining a cationic amphiphile component and a nonionic surfactant component; adding aqueous carrier to produce said micellar formulation;and combining the micellar formulation with a nucleic acid.

22. The methods of claim 20 and 21 further including combining the components of step with a neutral phospholipid component.

23. The method according to any one of claims 20 to 22, wherein the components of step are combined in an organic solvent and the solvent is removed to leave a lipid film prior to step

24. A method of producing a lipid film having an oil component, a nucleic acid, a cationic amphiphile component and a nonionic surfactant component; 25 said method including: combining an organic solvent with the oil component, the nucleic acid, the amphiphile component and the nonionic surfactant component; and removing the organic solvent to leave said lipid film. 30 25. A method of producing a lipid film having a nucleic acid, a cationic amphiphile component and a nonionic surfactant component; said method including: combining an organic solvent with the nucleic acid, the amphiphile component and the nonionic surfactant component; and removing the organic solvent to leave said lipid film.

26. A lipid film capable of forming an oil-in-water emulsion upon suspension in an aqueous carrier, said film having an oil component, a nucleic acid, a cationic amphiphile component and a nonionic surfactant component.

27. A lipid film capable of forming a micelle upon suspension in solution, said film having a nucleic acid, a cationic amphiphile component and a nonionic surfactant component.

28. The lipid films of claims 26 and 27, said films further having a neutral phopholipid component.

29. A formulation including lipid components, a nucleic acid and an aqueous carrier, wherein the lipid components include a cationic amphiphile component, a nonionic surfactant component, and optionally, an oil component, and wherein the cationic amphiphile component is selected from the group consisting of 1,2 bis (oleoyloxy)-3-(trimethylammonio)propane (DOTAP), (2,3-dioleoyloxy) propyl]-N, N, N- trimethyl ammonium chloride (DOTMA), 1,2- dioleoyl-3-(4' trimethylammonio) butanoyl-sn-glycerol (DOBT), cholesteryl (4' trimethylammonia) butanoate (ChOTB), DL-1, 2-dioleoyl-3- dimethylaminopropyl-B-hydroxyethylammonium (DORI), DL-1,2-O-dioleoyl-3- dimethylaminopropyl-p-hydroxyethylammonium (DORIE), 1,2-dioleoyl-3- ~succinyl-sn-glycerol choline ester (DOSC), cholesteryl hemisuccinate choline ester (ChOSC), doctadecylamidoglycyl-spermine (DOGS), dipalmitoyl i" phosphatidyesthanolamidospermine (DPPES), S 25 ethylenetrimethylammonium iodide, 1 -dimethylamino-3-trimethylammonio-DL- 2-propyl-cholesteryl carboxylate iodide, carboxyamidoethyleneamine, cholesteryl-3p-oxysuccinamido- ethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonio-DL- 0. 2-propyl-cholesteryl-3 0-oxysuccinate iodide, 2- [(2-trimethylammonio)- ethylmethylamino] ethyl-cholesteryl-3-oxysuccinate iodide, N'- dimethylaminoethane) carbamoyl] cholesterol (DC-Chol), and (polyethyleneimine)-carbamoyl] cholesterol. The formulation of claim 18 wherein the cationic amphiphile component is N'-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol).

31. The formulation according to any one of claims 1 to 14, 18, 29 or wherein the nonionic surfactant is a nonionic surfactant derivative of phosphatidylethanolamine.

32. The formulation according to any one of claims 1 to 14, 18, 29 or wherein the nonionic surfactant is a synthetic detergent.

33. The formulation according to claim 32, wherein the synthetic detergent is selected from the group consisting of sorbitan fatty acid ester, sorbitan polyoxyethylene fatty acid ester, Pluronic and octoxynol surfactants.

34. The formulation according to claim 32, wherein the synthetic detergent is selected from the group consisting of sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, poloxamer 188, poloxamer 407, polyoxyethylene (10) isooctylphenyl ether, and polyoxyethylene isooctylphenyl ether.

35. The formulation according to any one of claims 1 to 14, 18 or 29 to 34, wherein the nucleic acid is selected from the group consisting of DNA and RNA.

36. The method according to claim 17, wherein the formulation is i" administered to the animal or human by an intravenous, oral, intraperitoneal, 25 intramuscular, subcutaneous, intraaural, topical, intraarticular or intramammary route.

37. The formulation according to any one of claims 1 to 14, 18 or 29 to wherein the formulation is suitable for transfection in vitro.

38. The formulation according to any one of claims 1 to 14, 18 or 29 to wherein the formulation is suitable for transfection in vivo.

39. The formulation according to claim 6, wherein the formulation is suitable for administration to a human. 48 Dated this second day of May 2000 UNIVERSITY OF PITTSBURGH Patent Attorneys for the Applicant: FB RICE &CO 4. 9* 9 a 9. 9 9 4 4 *49 9 *999 .4 4 0* 9* 4. 9 9.. 9 9 9*p* 9 9 I. 99 9. 9 .99 4 .9 4 99 09 9