CATIONIC MOLECULE AND METHOD OF USE

BACKGROUND

The delivery of a therapeutic compound to a subject is important for its therapeutic effects and usually it can be impeded by limited ability of the compound to reach to targeted cells and tissues. Improvement of such compounds to enter the targeted cells of tissues by a variety of the means of delivery is crucial. The present invention relates the novel lipids, a compositions and method for preparation that facilitate the targeted intracellular delivery of biological active molecules.

Examples of biologically active molecules for which effective targeting to a patients' tissues is often not achieved include: (1) numerous proteins including immunoglobulin proteins, (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 medical practitioners is that a number of different types of nucleic acids are currently being developed as therapeutics for the treatment of a number of diseases. These nucleic acids include DNA in gene therapy, plasmids small interfering nucleic acids (iNA) for use in RNA interference (RNAi), antisense molecules, ribozymes, antagomirs, microRNA and aptamers. As these nucleic are being developed, there is a need to produce lipid formulations that are easy to make and can be readily delivered to a target tissue.

A preferred method for delivering complex biological molecules, including siRNA, to subjects involves the use of liposomes. Lipid carriers increase the rate of transport of compounds into cells. Liposomal drug carriers can protect a drug molecule from degradation while improving its uptake by cells. Also, liposomal drug carriers can encapsulate or bind certain compounds by electrostatic and other interactions, and may interact with negatively charged cell membranes to initiate transport across a membrane.

Technical challenges remain. Liposomal systems involve assembly of multimolecular complexes. The methods for producing liposomal carriers are expensive and complicated, and their variability is substantial, even by highly trained professionals. Even the most robust liposomal systems involve a significant range of particle size, which are highly disperse. The average particle size is an intrinsic property of the liposome, and is not easily controlled by the designer.

The liposomal systems do not permit stable long term storage or even short term stability to allow ease of shipment.

There exists a long-felt need to develop delivery systems that can effectively target cells in a subject's body, while avoiding some or all of the limitations of the liposomal systems that are described above. A key to successful development of novel delivery systems involves identifying molecular components that will supply a solution to these technical demands.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : This shows ApoB siRNA knocking down ApoB expression in mouse liver. CD1 mice were administered intravenously with a single dose of formulated ApoB siRNA at dose of 2 mg/kg in 0.2 ml dose volume. Two days later, the liver was harvested for analyzing gene expression with real-time RT-PCR method (GAPDH as reference gene). Each data point represents the mean + SEM (n=6). The expression level of ApoB mRNA was almost 80% lower than control and untreated groups. The formulations containing the cationic lipid shown in Formula I, below.

Figure 2. This shows lamin A/C siRNA knocking down lamin A/C expression in mouse liver. CD1 mice were administered intravenously with a single dose of formulated lamin A/C siRNA at dose of 2 mg/kg in 0.2 ml dose volume. Two days later, the liver was harvested for analyzing gene expression with real-time RT-PCR method (GAPDH as reference gene). Each data point represents the mean + SEM (n=6). The expression level of lamin A/C mRNA was almost 75% lower than control and untreated groups. The formulations contain the cationic lipid shown in Formula II, below.

Figure 3. This shows a dose-response curve of liver (ApoB) siRNA delivery formulations. Balb/C mice were administered a single dose of 0.2 ml formulated ApoB siRNA intravenously at the indicated dose level. The liver was harvested two days post injection for analyzing gene expression with real time RT-PCR method (GAPDH as reference gene). Serum was also harvested for analyzing total cholesterol level. Each data point represents the mean + SEM (n=6). The ApoB gene knockdown in mouse liver are correlated to serum cholesterol level changes Figure 4. This shows a time course study on ApoB gene expression and corresponding cholesterol reduction Balb/C mice were administered with a single dose of formulated siRNA intravenously at dosing volume of 0.2 ml/mouse and 2 mg/kg. At the specified time point, the liver tissue was harvested for analyzing gene expression as described above. The gene knockdown in liver and cholesterol change in serum almost last three weeks.

Figure 5. Balb/C mice were administered 0.2 ml intravenously of a single dose of formulated siRNA at 2 mg/kg. Two days later, the tumor and liver were harvested for analyzing gene expression with real-time RT-PCR method (GAPDH as reference gene). The tumor model is produced by injecting EMT6 cells (mouse mammary sarcoma original from Balb/C mice) into Glisson capsule of the left lower hepatic lobe. Each data point represents the mean + SEM (n=6).

Figure 6. 129Sl/svImJ mice were administered with a single dose of siRNA intravenously in Agave's proprietary lung selective delivery formulation at dosing volume of 0.2 ml (4 mg/kg siRNA). Three days later, the lung and liver were harvested for analyzing gene expression with realtime RT-PCR method (GAPDH and beta-actin as reference gene). Each data point represents the mean + SEM (n=5). The expression level of the targeted genes was significantly knocked down in lung, but not in liver. Speca-11 and Speca-12 are the siRNAs targeting different genes which are selected for Agave's RNAi therapeutic programs.

Figure 7. 129Sl/svImJ mice were administered a single dose of 0.2 ml formulated siRNA intravenously at the indicated dose level. Three days later, the lung and liver were harvested for analyzing gene expression with real-time RT-PCR method (GAPDH and beta-actin as reference gene). Each data point represents the mean + SEM (n=5). The expression level of the targeted genes was knocked down in lung in dose-responsive manner.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

This disclosure provides novel compounds, compositions and formulations for intracellular and in vivo delivery of drug agents for use, ultimately, as a therapeutic. The compounds and compositions of this disclosure are useful for delivery of drug agents to selected cells, tissues, organs or compartments in order to alter a disease state or a phenotype.

In some aspects, this disclosure provides compounds, compositions and methods to deliver RNA structures to cells to produce the response of RNA interference, antisense effects, or the regulation of genomic expression.

This invention provides a range of novel cationic lipids for use in delivery and

administration of drug agents and in drug delivery systems. The cationic lipids of this disclosure are molecules containing a polyamine molecule and one or more lipophilic tails.

In general, as used herein, general chemical terms refer to all groups of a specified type, including groups having any number and type of atoms, unless otherwise specified. For example "alkenyl" refers broadly to alkyls having 2 to 22 carbon atoms, as defined below, while

(C18:l)alkenyl refers to alkenyls having 18 carbon atoms and one double bond.

The term "alkyl" as used herein refers to a saturated, branched or unbranched, substituted or unsubstituted aliphatic group containing from 1-22 carbon atoms. This definition applies to the alkyl portion of other groups such as, for example, alkoxy, alkanoyl, aralkyl, and other groups defined below. The term "cycloalkyl" as used herein refers to a saturated, substituted or unsubstituted cyclic alkyl ring containing from 3 to 12 carbon atoms. As used herein, the term "C(l -5)alkyl," for example, includes C(l)alkyl, C(2)alkyl, C(3)alkyl, C(4)alkyl, and C(5)alkyl. Likewise, the term "C(3-22)alkyl," for example, includes C(l)alkyl, C(2)alkyl, C(3)alkyl, C(4)alkyl, C(5)alkyl, C(6)alkyl, C(7)alkyl, C(8)alkyl, C(9)alkyl, C(10)alkyl, C(l l)alkyl, C(12)alkyl, C(13)alkyl,

C(14)alkyl, C(15)alkyl, C(16)alkyl, C(17)alkyl, C(18)alkyl, C(19)alkyl, C(20)alkyl, C(21)alkyl, and C(22)alkyl.

The term "alkenyl" as used herein refers to an unsaturated, branched or unbranched, substituted or unsubstituted alkyl or cycloalkyl having 2 to 22 carbon atoms and at least one carbon- carbon double bond. The term "alkynyl" as used herein refers to an unsaturated, branched or unbranched, substituted or unsubstituted alkyl or cycloalkyl having 2 to 22 carbon atoms and at least one carbon-carbon triple bond. The term "alkoxy" as used herein refers to an alkyl, cycloalkyl, alkenyl, or alkynyl group covalently bonded to an oxygen atom. The term "alkanoyl" as used herein refers to ~C(=0)-alkyl, which may alternatively be referred to as "acyl." The term "alkanoyloxy" as used herein refers to— 0~C(=0)-alkyl groups. The term "alkylamino" as used herein refers to the group --NRR', where R and R' are each either hydrogen or alkyl, and at least one of R and R' is alkyl. Alkylamino includes groups such as piperidino wherein R and R' form a ring. The term "alkylaminoalkyl" refers to -alkyl- NRR ^* .

The term "aryl" as used herein refers to any stable monocyclic, bicyclic, or polycyclic carbon ring system of from 4 to 12 atoms in each ring, wherein at least one ring is aromatic. Some examples of an aryl include phenyl, naphthyl, tetrahydro-naphthyl, indanyl, and biphenyl. Where an aryl substituent is bicyclic and one ring is non-aromatic, it is understood that attachment is to the aromatic ring. An aryl may be substituted or unsubstituted.

The term "heteroaryl" as used herein refers to any stable monocyclic, bicyclic, or polycyclic carbon ring system of from 4 to 12 atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfur. Some examples of a heteroaryl include acridinyl, quinoxalinyl, pyrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, and tetrahydroquinolinyl. A heteroaryl includes the N-oxide derivative of a nitrogen-containing heteroaryl.

The term "heterocycle" or "heterocyclyl" as used herein refers to an aromatic or nonaromatic ring system of from five to twenty-two atoms, wherein from 1 to 4 of the ring atoms are heteroatoms selected from oxygen, nitrogen, and sulfur. Thus, a heterocycle may be a heteroaryl or a dihydro or tetrahydro version thereof.

The term "aroyl" as used herein refers to an aryl radical derived from an aromatic carboxylic acid, such as a substituted benzoic acid. The term "aralkyl" as used herein refers to an aryl group bonded to an alkyl group, for example, a benzyl group.

The term "carboxyl" as used herein represents a group of the formula— C(=0)OH or— C(=0)0. The terms "carbonyl" and "acyl" as used herein refer to a group in which an oxygen atom is double-bonded to a carbon atom>C=0. The term "hydroxyl" as used herein refers to OH or O. The term "nitrile" or "cyano" as used herein refers to CN. The term "halogen" or "halo" refers to fluoro (F), chloro (CI), bromo (Br), and iodo (I). The term "substituted" as used herein refers to an atom having one or more substitutions or substituents which can be the same or different and may include a hydrogen substituent. Thus, the terms alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkanoyloxy, alkylamino,

alkylaminoalkyl, aryl, heteroaryl, heterocycle, aroyl, and aralkyl as used herein refer to groups which include substituted variations. Substituted variations include linear, branched, and cyclic variations, and groups having a substituent or substituents replacing one or more hydrogens attached to any carbon atom of the group. Substituents that may be attached to a carbon atom of the group include alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkanoyloxy, alkylamino,

alkylaminoalkyl, aryl, heteroaryl, heterocycle, aroyl, aralkyl, acyl, hydroxyl, cyano, halo, haloalkyl, amino, aminoacyl, alkylaminoacyl, acyloxy, aryloxy, aryloxyalkyl, mercapto, nitro, carbamyl, carbamoyl, and heterocycle. For example, the term ethyl includes without limitation— CH ₂ CH ₃ ,— CHFCH ₃ , -CF ₂ CH ₃ , -CHFCH ₂ F, -CHFCHF ₂ , -CHFCF ₃ , -CF ₂ CH ₂ F, -CF ₂ CHF ₂ , --CF ₂ CF ₃ , and other variations as described above. In general, substituents may be further substituted with any atom or group of atoms.

DEFINITIONS

Definitions of technical terms provided herein should be construed to include without recitation those meanings associated with these terms known to those skilled in the art, and are not intended to limit the scope of the disclosure.

The use herein of the terms "a," "an," "the," and similar terms in describing the disclosure, and in the claims, are to be construed to include both the singular and the plural. The terms

"comprising," "having," "including," and "containing" are to be construed as open-ended terms which mean, for example, "including, but not limited to." Recitation of a range of values herein refers individually to each and any separate value falling within the range as if it were individually recited herein, whether or not some of the values within the range are expressly recited. Specific values employed herein will be understood as exemplary and not to limit the scope of the disclosure.

The term "cationic lipid" refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH (e.g., pH of about 7.0). A number of cationic lipids and related analogs, which are also useful in the present disclosure, have been described in U.S. Patent Publication No. 20060083780; U.S. Pat. Nos. 5,208,036; 5,264,618; 5,279,833;

5,283,185; 5,753,613; U.S. Pat. No. 5,767,099 and 5,785,992; and PCT Publication No. WO

96/10390. Examples of cationic lipids include, but are not limited to, N,N-dioleyl-N,N- dimethylammonium chloride (DODAC), dioctadecyldimethylammonium (DODMA), distearyldimethylammonium (DSDMA), N-(l -(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l-(2,3- dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'- dimethylaminoethane)-carbamoyl)cholesterol (DC-Choi), N-(l ,2-dimyristyloxyprop-3-yl)-N,N- dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), l,2-dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), l,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 06, N ⁴ -spermine cholesteryl carbamate (GL-67), N ⁴ -spermidine cholestryl carbamate (GL-53), 1- (N ⁴ -spermind)-2,3-dilaurylglycerol carbamate (GL-89) and mixtures thereof. As a non-limiting example, cationic lipids that have a positive charge below physiological pH include, but are not limited to, 1 ,2-dimyristoyl-3 -dimethylammonium propane (DODAP), DODMA, and DSDMA. In some cases, the cationic lipids comprise a protonatable tertiary amine head group, CI 8 alkyl chains, ether linkages between the head group and alkyl chains, and 0 to 3 double bonds. Such lipids include, e.g., DSDMA, DLinDMA, DLenDMA, and DODMA. The cationic lipids may also comprise ether linkages and pH titratable head groups. Such lipids include, e.g., DODMA. The cationic lipid may be, e.g., DODAC, DDAB, DOTAP, DOTMA, DODMA, DLinDMA, DLenDMA, or mixtures thereof. Cationic lipids that are useful in the present disclosure can be any of a number of lipid species that carry a net positive charge at physiological pH. Such lipids include, but are not limited to, DODAC, DODMA, DSDMA, DOTMA, DDAB, DOTAP, DOSPA, DOGS, DC -Choi, DMRIE, and mixtures thereof. Additionally, a number of commercial preparations of cationic lipids are available and can be used in the present disclosure.

An emulsion is a mixture of two immiscible (unblendable) substances. One substance (the dispersed phase) is dispersed in the other (the continuous phase). Emulsions are unstable and thus do not form spontaneously. Energy input through shaking, stirring, homogenizers, or spray processes are needed to form an emulsion.

A self-emulsifying lipid/nucleic acid complex is a complex of lipids and nucleic acid that forms an emulsion in an aqueous environment without the input of substantial energy such as sonication, homogenization etc.

As used herein, the term interfering nucleic acid (iNA) refers to a nucleic acid duplexes having a sense and antisense strand, which when entered into a RISC complex induces enzymatic degradation of mRNA. Generally each strand contains predominantly RNA nucleotides but the strands can contain RNA analogs, RNA and RNA analogs, RNA and DNA, RNA analogs and DNA, or one strand that is completely DNA and one strand that is RNA as long as the iNA construct induces enzymatic degradation of a homologous mRNA.

Polar aprotic solvents are solvents that share ion-dissolving power with protic solvents but lack acidic hydrogen. These solvents generally have high dielectric constants and high polarity. Examples of polar aprotic solvents are dimethyl sulfoxide, dimethylformamide, dioxane and hexamethylphosphorotriamide, acetone, acetonitrile, N, N-dimethylformamide (DMF), N- methylpyrrolidone (NMP), dimethylacetamide, dimethyl sulfoxide (DMSO), sulfolane, acetonitrile, hexamethylphosphoric triamide (HMPA), pyridine, tetramethylurea (TMU), urea analogs, N,N- dimethylformamide HCON(CH3)2, Ν,Ν-dimethylacetamide (DMA) CH3CON(CH3)2, and tetramethylurea, (CH3)2NCON(CH3)2, 1,3 -Dimethyl -2-imidazolidinone (DMI), and 1,3-Dimethyl- 3,4,5,6-tetrahydro-2(lH)-pyrimidinone (DMPU). It is possible to substitute the relatively toxic hexamethylphosphoramide (HMPA) with DMPU.

The term surfactant is a blend of "surface acting agent". Surfactants are usually organic compounds that are amphiphilic meaning they contain both hydrophobic groups (their "tails") and hydrophilic groups (their "heads"). Therefore, they are soluble in both organic solvents and water. Surfactants reduce the surface tension of water by adsorbing

http://en.wikipedia.org/wiki/Adsorptionat the liquid-gas interface. They also reduce the interfacial tension between oil and water by adsorbing at the liquid-liquid interface. Many surfactants can also assemble in the bulk solution into aggregates.

The term transfection is used herein generally to mean the delivery and introduction of biologically functional nucleic acid into a cell, i.e., a eukaryotic cell, in such a way that the nucleic acid retains its function within the cell. The term transfection includes the more specific meaning of delivery and introduction of expressible nucleic acid into a cell such that the cell is rendered capable of expressing that nucleic acid. The term expression means any manifestation of the functional presence of the nucleic acid within a cell, including both transient expression and stable expression. Nucleic acids include both DNA and RNA without size limits from any source comprising natural and non-natural bases. Nucleic acids can have a variety of biological functions. They may encode proteins, comprise regulatory regions, and function as inhibitors of gene or RNA expression (e.g., antisense DNA or RNA), function as inhibitors of proteins, function to inhibit cell growth or kill cells, catalyze reactions or function in a diagnostic or other analytical assay. As used herein, the terms "aptamer" or "nucleic acid aptamer" encompass a nucleic acid molecule that binds specifically to a target molecule, wherein the nucleic acid molecule contains a sequence that is recognized by the target molecule in its natural setting. Alternately, an aptamer can be a nucleic acid molecule that binds to a target molecule where the target molecule does not naturally bind to a nucleic acid. For example, the aptamer can be used to bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein.

By "antisense nucleic acid", it is meant a non-enzymatic nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid) interactions and alters the activity of the target RNA. Typically, antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule. However, in certain embodiments, an antisense molecule can bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule can bind such that the antisense molecule forms a loop. Thus, the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence or both. In addition, antisense DNA can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex. The antisense oligonucleotides can comprise one or more RNAse H activating region, which is capable of activating RNAse H cleavage of a target RNA. Antisense DNA can be synthesized chemically or expressed via the use of a single stranded DNA expression vector or equivalent thereof. "Antisense RNA" is an RNA strand having a sequence complementary to a target gene mRNA, that can induce RNAi by binding to the target gene mRNA. Antisense RNA" is an RNA strand having a sequence complementary to a target gene mRNA, and thought to induce RNAi by binding to the target gene mRNA. "Sense RNA" has a sequence complementary to the antisense RNA, and annealed to its complementary antisense RNA to form iNA. These antisense and sense RNAs have been conventionally synthesized with an RNA synthesizer.

Antagomirs are one of a novel class of chemically engineered antisense oligonucleotides. Antagomirs are used in the silencing of endogenous microRNA.

MicroRNAs (miRNA) are single-stranded RNA molecules of about 21-23 nucleotides in length, which regulate gene expression miRNAs are encoded by genes that are transcribed from DNA but not translated into protein (Non-Coding RNA); instead they are processed from primary transcripts known as pri-miRNA to short stem-loop structures called pre-miRNA and finally to functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, and their main function is to downregulate gene expression

"Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral- methyl phosphonates, 2'-0-methyl ribonucleotides, peptide -nucleic acids (PNAs).

By "RNA" is meant a molecule comprising at least one ribonucleotide residue. By

"ribonucleotide" is meant a nucleotide with a hydroxyl group at the 2' position of a .BD-ribo- furanose moiety. The terms include double-stranded RNA, single-stranded RNA, and isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the iNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the instant disclosure can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally occurring RNA. As used herein, the terms "ribonucleic acid" and "RNA" refer to a molecule containing at least one ribonucleotide residue. A ribonucleotide is a nucleotide with a hydroxyl group at the 2' position of a B-D-ribo-furanose moiety. These terms include double- stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified and altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, modification, and/or alteration of one or more nucleotides. Alterations of an RNA can include addition of non-nucleotide material, such as to the end(s) of a iNA or internally, for example at one or more nucleotides of an RNA nucleotides in an RNA molecule include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs. By "nucleotide" as used herein is as recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the Γ position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, nonstandard nucleotides and other;. There are several examples of modified nucleic acid bases known in the art. Some of the non-limiting examples of base modifications that can be introduced into nucleic acid molecules include inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6- trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6- azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others. By "modified bases" in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at the position or their equivalents.

As used herein complementary nucleotide bases are a pair of nucleotide bases that form hydrogen bonds with each other. Adenine (A) pairs with thymine (T) or with uracil (U) in RNA, and guanine (G) pairs with cytosine (C). Complementary segments or strands of nucleic acid that hybridize (join by hydrogen bonding) with each other. By "complementarity" is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence either by traditional Watson - Crick or by other non-traditional modes of binding.

Examples of preservatives include phenol, methyl paraben, paraben, m-cresol, thiomersal, benzylalkonium chloride, and mixtures thereof.

Structure of the cationic lipid

The cationic lipids of the invention include the molecules, as follows:

or

wherein

p, q, r, s and t ; are each independently chosen from 0 to 16;

X and Y are each independently chosen from the group consisting of H, Ac, Boc, or Piv, or from a linker group consisting of C, amide, carbamate, succinamide, maleimide, epoxide, and urethane; and Pvi, P 2, P 3, P 4, R ₅ , R ₆ , R7, and Rs are each independently chosen from a group consisting of a CI - C26 alkane or alkene, a polyunsaturated lipid, a steroid, a PUFA, guanidine or arginine, in any combination; and wherein the steroid is selected from the group consisting of lanosterol, ergosterol, desmosterol, a plant phytosterol, such as stigmasterol, or a bile salt or bile salt derivative such as cholic acid, deoxycholic acid, hydrodeoxycholic acid or dehydrocholic acid.

Preferred embodiments are provided in the following list (Groups α, β, and γ; Formulas I- LXVIII), wherein Ri is H; R ₂ is Me or tert-butoxycarbonyl (Boc); R _{l s} and R ₂ are guanidinyl or N- CNNH); or Ri is H and R ₂ is arginine via an amide bond formation. The cholesterol moiety may be replaced by lanosterol, ergosterol, desmosterol, a plant phytosterol such as stigmasterol, or a bile salt or bile salt derivative such as cholic acid, deoxycholic acid, hyodeoxycholic acid and dehydrocholic acid.

Group a lipids:

- 14- regio-isomers of the same Di-Chol derivative

XII

- 18 -

XXIII

XXVII

XXVIII

XXXII Group γ lipids XXXIII XXXV XXXVI XXXVII XXXVIII

Other examples of molecules derived from three basic polyamine molecules are as follows: XLVII XLVIII XLIX

-27-

-28 -

-30-

LVIII

-33 -

-35 - Synthesis

The following methods illustrate production of certain of the cationic lipids of the invention. Those skilled in the art will recognize other methods to produce these compounds, and to produce also the other compounds of the invention.

The following methods are designed to produce materials with high regioisomeric purity. Methods to produce mixtures of the mono, di and tri substituted cholesterol carbamate analogs of Tetrakis(3-aminopropyl)-l,3-propanediaminecholesterol carbamate consist of simple direct addition of the cholesterol carbamate to a cold stirring solution of desired Tetrakis(3-aminopropyl)-l,3- propanediamine in chloroform, triethylamine mixtures. No protection/deprotection methodology is required. It should be obvious to those skilled in the art that mixtures of a high degree of

compositional accuracy can be obtained by adding the higher order carbamoyl cholesterol compounds back into the pure monocarbamoyl cholesterol substituted polyamines.

A: Synthesis of N 6 -Tetrakis(3-aminopropyl)-l,3-propanediamine cholesteryl carbamate This step in synthesis applies to synthesis of Formulas I -VIII, above.

Tetrakis(3-aminopropyl)-l,3-propanediamine cholesterol carbamate is synthesized according to the following procedure. This procedure gives a molar ratio of monosubsituted to disubstituted cholesterol carbamate polyamine in a 85/15 ratio. It is used as is after flash chromatography purification using methanol chloroform gradient to 15% methanol in the presence of 2% TEA.

Tetrakis(3-aminopropyl)-l,3-propanediamine (25 g, 60.5 mmol) and triethylamine (25 ml, 178 mmol) are dissolved in 500 ml of anhydrous chloroform, cooled to 0-4° C. and stirred under N 2 . Cholesteryl chloro formate (30 g, 67 mmol) is dissolved in 250 ml of chloroform and added to the reaction over a 20 minute period. A white precipitate formed upon addition. After the addition is complete, the reaction is stirred at 0-4° C. for 10 minutes and then at room temperature for 1.5 hr. At this point, the white precipitate completely dissolved. The reaction is followed by TLC with hexane/ethyl acetate 6/4 as eluent (product Rf=0.25). To this reaction mixture is added 500 ml of chloroform and 500 ml of water. The layers are then allowed to separate. The organic layer is dried over MgSO 4 and filtered. The filtrate is concentrated in vacuo to give an oil. Vacuum drying is then carried out overnight. This crude product is a gummy tar. The crude product is purified by column chromatography (2 kg silica gel, eluent-chloroform/methanol 9/1) to give 46.8 g of the Tetrakis(3- aminopropyl)-l,3-propanediamine)carbamoyl cholesterol in 90% yield. B: Synthesis of N 1 ,N 8 -Bis(Arginine carboxamide)-N 6 -Tetrakis(3-aminopropyl)-l,3- propanediamine cholesteryl carbamate.

This step applies to synthesis of Formulas IX -XII, above.

N 1 ,N 8 -Bis(Arginine carboxamide)-N 6 -Tetrakis(3-aminopropyl)-l,3-propanediamine cholesteryl carbamate is prepared as follows.

To N (a),N (e),N (e) (alpha, epsilon, epsilon) -tricarbobenzoxyArginine in CH 2 CI 2 (25 mL) is added N-hydroxysuccinimide (100 mg, 0.89 mmol) and dicyclohexylcarbodiimide (240 mg, 0.89 mmol). The mixture is stirred under N 2 at room temperature for 2.5 hours. N 6 - Tetrakis(3- aminopropyl)-l,3-propanediamine Cholesteryl Carbamate (250 mg, 0.448 mmol) and Et 3 N (0.25 mL, 1.8 mmol) is added and the reaction stirred at room temperature under N 2 for 72 h. The reaction is filtered and the precipitate is washed with CH 2 CI 2 (20 mL). The filtrate is washed with H 2 O (20 mL). The separated organic layer is dried over MgSO 4 and filtered. The filtrate is concentrated in vacuo and the residue is purified by chromatography on 70 g of silica gel (eluent— CHCl 3 /MeOH 95/5). The purified material is concentrated in vacuo and then vacuum dried to give 533 mg (71%) of N 1 , N 3 -Bis (N (a),N (e),N (e) -tricarbobenzoxyArginine carboxamide)-N 6 - Tetrakis(3-aminopropyl)-l,3-propanediamine cholesteryl carbamate. The carbobenzoxy group are removed from N 1 , N 3 -Bis (N (a),N (e),N (e) -tricarbobenzoxyArginine carboxamide)-N 6 - Tetrakis(3-aminopropyl)-l,3-propanediamine cholesteryl carbamate as described in the preparation of N-(N 6 -3-aminopropylTetrakis(3-aminopropyl)-l,3-propanediamine) cholesteryl carbamate. The product, N 1 , N 3 -Bis(Arginine carboxamide)-N 6 -Tetrakis(3-aminopropyl)-l,3-propanediamine cholesteryl carbamate is obtained in 27% yield.

C: 1-Synthesis of (N 6 -Tetrakis(3-aminopropyl)-l,3-propanediamine)-2,3- diarachidonylglycerol carbamate

This step applies in the synthesis of Formulas XIII -XVI, above.

1-(N 6 -Tetrakis(3-aminopropyl)-l,3-propanediamine)-2,3-diarachidon ylglycerol carbamate is prepared as follows. A solution of 3-benzyloxy-l,2-propanediol (1.00 g, 5.49 mmol) in THF (20 mL) is 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) is added and the reaction is refluxed for another two days. After cooling to room temperature the reaction is filtered through a bed of Celite, rinsing with THF. The filtrate is reduced in vacuo to a yellow oil which is redissolved in diethyl ether (100 mL). The ether solution is washed with 0.1 N NaOH (30 mL) and dH20 (2x30 mL). The organic layer is dried over magnesium sulfate, filtered and reduced in vacuo to a red-brown oil. The crude material is purified by flash column chromatography (300 g silica gel) eluting with 3% ethyl acetate/hexanes. The desired product is isolated as a pale yellow oil and characterized by 1 H NMR as 3-OBn-l,2-diarachidonylglycerol (1.70 g, 60%). 3-OBn-l,2-diarachidonylglycerol (1.70 g, 3.28 mmol) in ethanol (100 mL) is stirred with 10% Pd/C (250 mg, 15 wt %) under a hydrogen atmosphere for 24 hours. The reaction is flushed with nitrogen and filtered through Celite, rinsing with ethanol, to remove the catalyst. The filtrate is reduced in vacuo to a solid. The crude material is purified by flash column chromatography (140 g silica gel) eluting with 10% ethyl acetate/hexanes. The desired product is isolated as a white solid and characterized by 1 H NMR as 1,2- diarachidonylglycerol (1.23 g, 88%).

A 1.93 M solution of phosgene in toluene (0.77 mL, 1.49 mmol) is added to a solution of 1 ,2-diarachidonylglycerol (580 mg, 1.35 mmol) and N,N-diisopropylethylamine (0.26 mL, 1.49 mmol) in chloroform (10 mL) and stirred overnight. A solution of N 1 ,N 3 -di-CBz-Tetrakis(3- aminopropyl)-l,3-propanediaminee.2HCl (734 mg, 1.35 mmol) iN 6 0: 25: 4

chloroform/methanol/water (80 mL) is added. After 3 hours another equivalent of N,N- diisopropylethylamine (0.26 mL, 1.49 mmol) is added. An additional 0.5 equivalents of N,N- diisopropylethylamine (0.13 mL, 0.75 mmol) is added three hours later and the reaction is allowed to stir overnight under nitrogen at ambient temperature. The reaction is washed with 1M NaOH (20 mL) and dH20 (15 mL). The organic layer is separated, dried over magnesium sulfate, filtered and reduced in vacuo to a white solid. The crude material is purified by flash column chromatography (125 g silica gel) eluting with 90: 10: 0.5 chloroform/methanol/ammonium hydroxide. The desired product is isolated as an oil and characterized by 1 H NMR as 1 -(N 6 -(N 1 ,N 3 -di-CBz- Tetrakis(3-aminopropyl)-l,3-propanediamine))-2,3-diarachidon ylglycerol carbamate (188 mg, 15%).

The 1-(N 6 -(N 1 ,N 3 -di-CBz-Tetrakis(3-aminopropyl)-l,3-propanediamine))-2,3- diarachidonyl glycerol carbamate (188 mg, 0.203 mmol) is dissolved in glacial acetic acid (10 mL) and stirred with 10% Pd/C (45 mg, 24 wt %) under a hydrogen atmosphere for 5 hours. The catalyst is removed by vacuum filtration rinsing with 10% acetic acid/ethyl acetate (10 mL) The filtrate is reduced to an oil by rotary evaporation. The resulting oil is dissolved in 10% methanol/chloroform (85 mL) and is washed with 1M NaOH (15 mL) and dH20 (10 mL). The organic layer is separated, dried over magnesium sulfate, filtered and reduced in vacuo to an oil. The product is characterized by 1 H NMR as 1-(N 6 -Tetrakis(3-aminopropyl)-l,3-propanediamine)-2,3-diarachidon ylglycerol carbamate (125 mg, 94%).

Other amphiphiles of the invention may be prepared according to procedures that are within the knowledge of those skilled in art.

D: Introduction of the BOC group using DMAP and BOC anhydride.

This step applies to synthesis of Formulas I-IV, above.

Di-tert-butyl dicarbonate (3.60 g, 13.2 mmol) and 4-(dimethylamino)pyridine (84 mg, 0.687 mmol) are added to a solution of amine 1 [2] (1.12 g, 13.2 mmol) in acetonitrile (14 mL) under nitrogen at room temperature and stirred for 21 h. The yellow liquid is concentrated under reduced pressure to generate a yellow oil that is purified by flash chromatography using (MeOH in DCM, 5% TEA) as eluent to afford the title compound 2 (1.77 g, 73%) as a colourless oil.

E: Introduction of guanidine head groups using lH-Pyrazole-l-carboxamidin Hydrochloride This step applies to synthsis of Formulas V-VIII, above.

This procedure is useful for guanylation of sterically unhindered primary and secondary aliphatic amines that are DMF soluble. To the amine, lH-Pyrazole-l-carboxamidin Hydrochloride, and DIEA (2.0 mmol each), is added DMF sufficient to produce a final concentration of

approximately 2 M reactants. The reaction mixture is stirred at room temperature under nitrogen while being monitored by TLC. After a few h, ether (10-15 mL) is added to complete aggregation of the crude product which is collected, washed with ether, and dried. The crude product is

recrystallized from an appropriate solvent system and dried in vacuo.

It can be appreciated that these steps of synthesis can be applied equally by one of ordinary skill to the synthesis of Formulas XXXII -XL VI, above, by substituting starting materials. .

F: Lysine 3-N-dihydrocholesteryl carbamate (These structures are not drawn.) Lysine 3-N-dihydrocholesteryl carbamate was prepared according to the following procedure.

To a solution of dihydrocholesterol (5.0 g, 12.9 mmol, Aldrich), phthalimide (2.0 g, 13.6 mmol, Aldrich), and triphenylphosphine (3.8 g, 13.6 mmol, Aldrich) in THF (20 ml, Aldrich) stirred at 0° C. under a nitrogen atmosphere is added dropwise diethylazodicarboxylate (2.3 ml, 14.5 mmol, Aldrich). Upon the completion of addition the reaction mixture is allowed to warm to ambient temperature and stirred overnight. The reaction mixture is concentrated in vacuo to a residue. This residue is dissolved in 50 ml hexane/ethyl acetate 95/5 and a precipitate formed. The mixture is filtered. The filtrate is concentrated to dryness in vacuo, dissolved in 25 ml of hexane/ethyl acetate 95/5 and chromatographed on 200 g silica gel (eluent 2 L hexane/ethyl acetate 95/5 then 1 L hexane/ethyl acetate 90/10). A 76% yield of the desired 3-phthalimidocholestane (5.43 g) is obtained.

The 3-phthalimidocholestane (5.40 g, 9.75 mmol) is dissolved iN 6 0 mL of methanol and anhydrous hydrazine (3.1 ml, 99 mmol) is added. The reaction mixture is stirred and heated at reflux under a nitrogen atmosphere for 4 hr. This mixture is then cooled to room temperature, 3.1 mL of concentrated HCl is added and the resulting mixture is heated at reflux overnight. Upon cooling to ambient temperature, 100 ml of diethyl ether and 50 ml of 1 N NaOH are added (final pH of 10.1) and the layers are separated. The aqueous layer is extracted with 50 ml of diethyl ether and the combined organic fractions are filtered. The filtrate is concentrated in vacuo and the residue is purified by silica gel chromatography (chloroform/methanol 90/10) to give 2.24 g of 3- aminocholestane in 59% yield.

L-Na,Ne-diBOClysine N-hydroxysuccinimide ester (286 mg, 0.644 mmol, Sigma) and 3- aminocholestane (250 mg, 0.644 mmol) are dissolved in 5 mL of chloroform, 0.1 mL of

triethylamine is added and the resulting solution is stirred under a nitrogen atmosphere at ambient temperature overnight. To the reaction mixture is added 10 mL of water and 25 mL of chloroform and the layers are separated. The aqueous layer is extracted with 25 mL of chloroform and the combined organic fractions are dried over MgSO 4 and filtered. The filtrate is concentrated in vacuo and the residue is purified by chromatography on 25 g of silica gel (eluent—hexane/ethyl acetate 6/4, sample applied in hexane/ethyl acetate 9/1). The purified material is dissolved in 25 mL of chloroform and HCl gas is bubbled through the solution for 2 hr. followed by nitrogen for 10 min. The solution is concentrated in vacuo to give 299 mg of the desired product in 79% yield as the dihydrochloride salt.

G: Synthesis of Tetra-N-Dodecyl-epoxide Tetrakis(3-aminopropyl)-l ,3-propanediamine This step applies to synthesis of Formulas XLVIII-LIV, above

1 ,2-epoxydodecane (2.10 g, 1 1.4 mmol, 4.5 equiv) is added to a 20 mL vial containing amine dried in vacuo overnight (300 mg), 5mL of anhydrous DMF and a magnetic stir bar. The vial is sealed and warmed to 80 °C. The reaction mixture is stirred for 2 days, whereupon analysis of the reaction mixture by TLC showed the reaction is almost complete. The crude mixture is purified by chromatography on silica gel using gradient elution from 5% MeOH in DCM to 15% MeOH in DCM plus 5% TEA. The polydodecylated amine is isolated as a pale yellow viscous oil 50%> yield. This procedure is used for the 1 ,2-epoxytradecane and the Dodecylglycidyl epoxide derivatives. Fully substituted materials are synthesized by using 1.5 excess equivalents over the amine molar ratio or 12 eq. epoxide for the 3-aminopropyl)-l ,3-propanediamine and 3-aminopropyl)-l ,4- butanediamine materials.

H: Synthesis of Tetra-N-Tetradecyl-epoxide Cholesteryl-Tetrakis-(3-aminopropyl)- ammonium chloride

This step applies to synthesis of Formulas LV-LIX, above.

The amine is dried in vacuo overnight and dissolved in anhydrous chloroform containing 5 eq. of anhydrous TEA. Cholesterol chloroformate (l . leq, ) is dissolved in chloroform (5mL) and added dropwise to the cold stirring solution of amine and TEA in chloroform over a 20 minute period. The flask is warmed to 20°C and allowed to react for an additional 1 hour. TLC showed the presence of a cholesterol polyamine product using ninhydrin spray reagent and charring. The crude mixture is purified by chromatography on silica gel using gradient elution from 5% MeOH in DCM to 15% MeOH in DCM plus 5% TEA to yield 75% of the desired cholesteryl polyamine lipid. The material is dried in vacuo before use or further chemical reactions.

The derivatives containing multiple cholesterols are prepared similarly by using 2 eq of cholesterol chloroformate and isolating the more lipophilic materials using silica gel

chromatography and retaining the higher Rf spots. 1 ,2-epoxydodecane (2.10 g, 11.4 mmol, 4.5 equiv) is added to a 20 mL vial containing Cholesteryl derivatised amine dried in vacuo overnight (300 mg), 5mL of anhydrous DMF and a magnetic stir bar. The vial is sealed and warmed to 80 °C. The reaction mixture is stirred for 2 days, whereupon analysis of the reaction mixture by TLC showed the reaction is almost complete. The crude mixture is purified by chromatography on silica gel using gradient elution from 5% MeOH in DCM to 15% MeOH in DCM plus 5% TEA. The polydodecylated amine is isolated as a pale yellow viscous oil 50% yield. This procedure is used for the 1,2-epoxytradecane and the Dodecylglycidyl epoxide derivatives. Fully substituted materials are synthesized by using 1.5 excess equivalents over the amine molar ratio or 12 eq. epoxide for the 3-aminopropyl)-l,3-propanediamine and 3- aminopropyl)-l ,4-butanediamine materials.

I : Tetra-N-Dodecylated-Tetrakis(3 -aminopropyl)- 1 ,3 -propanediamine

This step applies to synthesis of Formulas LX-LXIII, above.

The amine is dried in vacuo overnight and dissolved in anhydrous chloroform containing 5 eq. of anhydrous TEA. The Dodecyl chloro formate (4eq., ) is dissolved in chloroform (5mL) and added dropwise to the cold stirring solution of amine and TEA in chloroform over a 20 minute period. The flask is warmed to 20°C and allowed to react for an additional 1 hour. TLC shows the presence of a cholesterol polyamine product using ninhydrin spray reagent and charring. The crude mixture is purified by chromatography on silica gel using gradient elution from 5% MeOH in DCM to 15%) MeOH in DCM plus 5% TEA to yield 75% of the desired tetradodecyl polyamine lipid. The material is dried in vacuo before use or further chemical reactions.

The derivatives containing fully substituted dodecylated polyamines are prepared similarly by using 12 eq of Dodecyl chloro formate and isolating the more lipophilic materials using silica gel chromatography and retaining the higher Rf spot Tetradecyl chloroformate is also used to prepare the analogous series of compounds. Other acyl lipid chloro formates are also similarly created using this procedure

J: Dodecyl succinamide Tetrakis(3-aminopropyl)-l,3-propanediamine This step applies to synthesis of Formulas XLV-XLVIII, above

Tetrakis(3-aminopropyl)-l,3-propanediamine (2.5 g, 6.1 mmol) is dissolved in lOOmL anhydrous chloroform and dimethylaminopyridine catalyst is added (DMAP, lOOmg). The alkenyl succinic anhydride (ASA, dodecenyl succinic anhydride, 6 eq, 11 g) is added with stirring and 3 A molecular sieve is added after addition of the ASA is complete. The reaction is stirred overnight at room temperature under nitrogen blanket and checked for completion by TLC. The material is washed in aqueous sodium carbonate (2% w/v) and recovered from the chloroform layer after separation. The oily residue is recovered from the organic phase by reduced pressure rotary evaporation and dried overnight in vacuo. The desired material is purified from contaminants by application to a silica gel chromatography bed and eluted using a methanol DCM gradient (5% MeOH to 15% MeOH plus 5% TEA) and visualized using phosphomolybdic acid spray on glass backed F254 TLC plates. 65% yield at 95% percent purity is achieved.

Formulations

The cationic lipid are readily combined with nucleic acid (NA) salts in a solid and stable form. In particular, cationic lipid in combination with interfering nucleic acid salts are produced by bringing into contact a solution of a cationic lipid with an aqueous solution of nucleic acid under conditions wherein the cationic lipid molecules complex with the nucleic acid to form cationic lipid nucleic acid salt aggregates. Aggregation occurs for cationic organic molecules with a carbon number greater than six and a sufficient degree of hydrophobicity to render the nucleic acid insoluble in water. The resulting aggregates contains an amount of the cationic lipid molecular positive charge in a preferable one to one molar concentration with the number of negative charge in nucleotides present in the nucleic acid. The nucleic acid salt organic cationic lipid aggregates can be recovered from the aqueous liquors using filtration, centrifugation and other methods available to those skilled in the art of chemical process. The precipitated cationic lipid salt can be dried and subjected to numerous mechanical treatments to render it suitable for incorporation into solid and liquid dosage forms of NA drugs.

The cationic organic salt aggregates of nucleic acid can be readily solvated in many common organic solvents including solvents that are of the polar aprotic class (dimethylacetamide, dimethylformamide, N-methyl pyrrolidine, diglyme and other ether glycols, chloroform, methylene chloride, some alcohols and other halogenated organic solvents, tetrahydrofuran and other cyclic ether solvents), these solvents can be employed under anhydrous conditions and are of industrial value for the use in chemical transformation and reaction of reagents into new chemical forms. Contacting the nucleic acid solution with the solution of cationic lipids is accomplished by mixing together a first solution of nucleic acids, which is typically an aqueous solution, with a solution of the cationic lipids. The cationic lipid can be in solution in either an organic or aqueous solvent. The ratio of cationic lipid to nucleotides present in nucleic should be preferably 2- 3 to 1 by weight. One of skill in the art will understand that this mixing can take place by any number of methods, for example by mechanical means such as by using vortex, mixers or injection pumps and stirred reactors.

The formulations are produced by mixing the cationic lipid nucleic acid salt with one or more lipids, which include a cationic lipid as described herein, a second cationic lipid, a

phospholipid, a hydrophobic lipid, preferably a sterol or a PEG-linked sterol, most preferably cholesterol or PEG-cholesterol.

The formulation is preferably formed by putting the cationic lipid / nucleic acid salt in solution in an organic solvent, in particular in an aprotic polar solvent and mixed with a solution of one or more lipids mentioned above. The lipids are in solution in an organic solvent, preferably an aprotic polar solvent.

Examples of the second cationic lipids that can be used in creating the cationic lipid nucleic acid salts include any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH (e.g., pH of about 7.0). As used herein, physiological pH refers to the pH of a biological fluid such as blood or lymph as well as the pH of a cellular compartment such as an endosome, an acidic endosome, or a lysosome). Such lipids include, but are not limited to, N,N- dioleyl-N,N-dimethylammonium chloride ("DODAC"); N-(l-(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride ("DOTMA); N,N-dimethyl-(2,3-dioleyloxy)propylamine

("DODMA"); N,N-distearyl-N,N- dimethylammonium bromide ("DDAB"); N-(l-(2,3- dioleoyloxy)propyl)-N,N,N- trimethylammonium chloride ("DOTAP"); 3-(N-(N',N'- dimethylaminoethane)- carbamoyl)cholesterol ("DC-Choi"); N-(l,2-dimyristyloxyprop-3-yl)-N,N- dimethyl-N- hydroxyethyl ammonium bromide ("DMRIE"); l,2-Dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA); and l,2-Dilinolenyloxy-N,N-dimethylaminopropane

(DLenDMA). The following lipids are cationic and have a positive charge at below physiological pH: l,2-dimyristoyl-3 -dimethylammonium propane (DODAP), DODMA, DMDMA and the like. These lipids and related analogs have been described in copending U.S. Ser. No. 08/316,399; U.S. Pat. Nos. 5,208,036, 5,264,618, 5,279,833 and 5,283,185. Additionally, a number of commercial preparations of cationic lipids are available and can be used in the present disclosure.

As a non-limiting example, cationic lipids that have a positive charge below physiological pH include, but are not limited to, l,2-dimyristoyl-3-dimethylammonium propane (DODAP), DODMA, and DSDMA. In some cases, the cationic lipids comprise a protonatable tertiary amine head group, C18 alkyl chains, ether linkages between the head group and alkyl chains, and 0 to 3 double bonds. Such lipids include, e.g., DSDMA, DLinDMA, DLenDMA, and DODMA. The cationic lipids may also comprise ether linkages and pH titratable head groups. Such lipids include, e.g., DODMA. The cationic lipid may be, e.g., DODAC, DDAB, DOTAP, DOTMA, DODMA, DLinDMA, DLenDMA, or mixtures thereof. Cationic lipids that are useful in the present disclosure can be any of a number of lipid species that carry a net positive charge at physiological pH. Such lipids include, but are not limited to, DODAC, DODMA, DSDMA, DOTMA, DDAB, DOTAP, DOSPA, DOGS, DC-Chol, DMRIE, and mixtures thereof. Additionally, a number of commercial preparations of cationic lipids are available and can be used in the present disclosure.

Examples of polyvalent cationic lipids other than those described in detail above, including Formulas I-LXVIII, are polyamines such as the polyvalent cations disclosed herein, and

lipospermines that can be used include N ⁴ -spermine cholesteryl carbamate (GL-67), N ⁴ -spermidine cholestryl carbamate (GL-53), l-(N ⁴ -spermine)-2,3-dilaurylglycerol carbamate (GL-89),

(dipalmitoylphosphatidylethanolamylspermine, DPPES) dioctadecylamido glycylspermine

(Transfectam, DOGS) 2,3 -dioleyloxy-N-[2(sperminecarboxamido)ethyl] -N,N-dimethyl-l - propanaminiu m trifluoroacetate. Lipospermines and lipospermidines are bifunctional molecules consisting of one or more hydrophobic chains covalently linked to a cationic grouping that has three or more amide hydrogens which can complex with a phosphate oxygen of a nucleic acid chain forming an ionic charge complex.

Examples of phospholipids include but are not limited to phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl inositol and diphosphatidyl glycerol, . such as 1,2-Dilauroyl-sn- glycerol (DLG); 1,2-Dimyristoyl-sn-glycerol (DMG); 1 ,2-Dipalmitoyl-sn-glycerol (DPG); 1,2- Distearoyl-sn-glycerol (DSG); l,2-Dilauroyl-sn-glycero-3-phosphatidic acid (sodium salt; DLPA); l,2-Dimyristoyl-sn-glycero-3-phosphatidic acid (sodium salt; DMPA); 1,2-Dipalmitoyl-sn-glycero- 3-phosphatidic acid (sodium salt; DPP A); l,2-Distearoyl-sn-glycero-3-phosphatidic acid (sodium salt; DSPA); l,2-Diarachidoyl-sn-glycero-3-phosphocholine (DAPC); l,2-Dilauroyl-sn-glycero-3- phosphocholine (DLPC); l,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC); 1,2-Dipalmitoyl- sn-glycero-O-ethyl-3-phosphocholine (chloride or triflate; DPePC); l,2-Dipalmitoyl-sn-glycero-3- phosphocholine (DPPC); l,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2-Dilauroyl-sn- glycero-3-phosphoethanolamine (DLPE); 1 ,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE); l,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE); l,2-Distearoyl-sn-glycero-3- phosphoethanolamine (DSPE); l,2-Dilauroyl-sn-glycero-3-phosphoglycerol (sodium salt; DLPG); l,2-Dimyristoyl-sn-glycero-3-phosphoglycerol (sodium salt; DMPG); 1,2-Dimyristoyl-sn-glycero- 3-phospho-sn-l -glycerol (ammonium salt; DMP-sn-l-G); l,2-Dipalmitoyl-sn-glycero-3- phosphoglycerol (sodium salt; DPPG); l,2-Distearoyl-sn-glycero-3-phosphoglycero (sodium salt; DSPG); l,2-Distearoyl-sn-glycero-3-phospho-sn-l -glycerol (sodium salt; DSP-sn-l-G); 1,2- Dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium salt; DPPS); 1 -Palmitoyl -2 -linoleoyl-sn- glycero-3 -phosphocholine (PLinoPC); 1 -Palmitoyl -2 -oleoyl-sn-glycero-3 -phosphocholine (POPC); l-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (sodium salt; POPG); 1 -Palmitoyl -2 -oleoyl-sn- glycero-3 -phosphoglycerol (sodium salt; POPG); l-Palmitoyl-2-oleoyl-sn-glycero-3- phosphoglycerol (ammonium salt; POPG); 1 -Palmitoyl -2 -4-o-sn-glycero-3 -phosphocholine (P-lyso- PC); 1 -Stearoyl -2 -lyso-sn-glycero-3 -phosphocholine (S-lyso-PC); and mixtures thereof

Hydrophobic lipids include molecules such as sterols, zoosterols such as cholesterol, dioleoylphosphatidylethanolamine, phytosterols such as campesterol, sitosterol, stigmasterol and other hydrophobic lipids known in the art. Examples of non-cationic lipids include cholesterols, sterols, and steroids such as gonanes, estranes, androstanes, pregnanes, cholanes, cholestanes, ergostanes, campestanes, poriferastanes, stigmastanes, gorgostanes, lanostanes, cycloartanes, as well as sterol or zoosterol derivatives of any of the foregoing, and their biological intermediates and precursors, which may include, for example, cholesterol, lanosterol, stigmastanol, dihydrolanosterol, zymosterol, zymostenol, desmosterol, 7-dehydrocholesterol, and mixtures and derivatives thereof.

Examples of non-cationic lipids include pegylated cholesterols, and cholestane 3-oxo(Cl- 22acyl) derivatives such as cholesteryl acetate, cholesteryl arachidonate, cholesteryl butyrate, cholesteryl hexanoate, cholesteryl caprylate, cholesteryl n-decanoate, cholesteryl dodecanoate, cholesteryl myristate, cholesteryl palmitate, cholesteryl behenate, cholesteryl stearate, cholesteryl nervonate, cholesteryl pelargonate, cholesteryl n-valerate, cholesteryl oleate, cholesteryl elaidate, cholesteryl erucate, cholesteryl heptanoate, cholesteryl linolelaidate, cholesteryl linoleate, and mixtures and derivatives thereof. Examples of non-cationic lipids derived from steroids include glucocorticoids, Cortisol, hydrocortisone, corticosterone, DELTA ⁵ -pregnenolone, progesterone, deoxycorticosterone, 17-OH- pregnenolone, 17-OH-progesterone, 11-dioxycortisol, dehydroepiandrosterone,

dehydroepiandrosterone sulfate, androstenedione, aldosterone, 18-hydroxycorticosterone, tetrahydrocortisol, tetrahydrocortisone, cortisone, prednisone, 6a-methylpredisone, 9a -fluoro-16a - hydroxyprednisolone, 9a -fluoro-16a -methylprednisolone, 9a -fluorocortisol, adrogens,

testosterone, dihydrotestosterone, androstenediol and mixtures and derivatives thereof. A

polyethylene glycol (PEG) - linked sterol includes a PEG-linked cholesterol and PEG-linked derivatives of cholesterol and PEG-linked phytosterols such as PEG-campesterol, PEG-sitosterol and PEG-stigmasterol. Examples of non-cationic lipids also include polymeric compounds and polymer-lipid conjugates or polymeric lipids, such as pegylated lipids having PEG regions of 300, 500, 1000, 1500, 2000, 3500, or 5000 molecular weight, including polyethyleneglycols, N- (carbonyl-methoxypolyethyleneglycol-2000)-l,2-dimyristoyl-sn -glycero-3-phosphoethanolamine (sodium salt; DMPE-MPEG-2000); N-(carbonyl-methoxypolyethyleneglycol-5000)-l,2- dimyristoyl-sn-glycero-3-phosphoethanolamine (sodium salt; DMPE-MPEG-5000); N-(carbonyl- methoxypolyethyleneglycol 2000)-l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (sodium salt; DPPE-MPEG-2000); N-(carbonyl-methoxypolyethyleneglycol 5000)-l ,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine (sodium salt; DPPE-MPEG-5000); N-(carbonyl-methoxypolyethyleneglycol 750)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine (sodium salt; DSPE-MPEG-750); N- (carbonyl-methoxypolyethyleneglycol 2000)- 1 ,2-distearoyl-sn-glycero-3 -phosphoethanolamine (sodium salt; DSPE-MPEG-2000); N-(carbonyl-methoxypolyethyleneglycol 5000)-l,2-distearoyl- sn-glycero-3-phosphoethanolamine (sodium salt; DSPE-MPEG-5000); sodium cholesteryl sulfate (SCS); pharmaceutically acceptable salts thereof, and mixtures thereof.

Examples of non-cationic lipids include polymeric lipids such as DOPE-PEG, DLPE-PEG, DDPE-PEG DLinPE-PEG, and diacylglycerol-PEG-2000 or -5000, polymeric lipids such as multi- branched pegylated compounds, for example DSPE-PTE020 and DSPE-AM0530K, polymeric lipids such as DSPE-PG8G polyglycerine lipids, dioleoylphosphatidylethanolamine (DOPE),

diphytanoylphosphatidylethanolamine (DPhPE), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine

(DOPC), and l,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC).

Examples of anionic lipids include phosphatidylserine, phosphatidic acid,

phosphatidylcholine, platelet-activation factor (PAF), phosphatidylethanolamine, phosphatidyl -DL- glycerol, phosphatidylinositol, phosphatidylinositol (pi(4)p, pi(4,5)p2), cardiolipin (sodium salt), lysophosphatides, hydrogenated phospholipids, sphingoplipids, gangliosides, phytosphingosine, sphinganines, pharmaceutically acceptable salts thereof, and mixtures thereof.

Examples of non-cationic lipids include lipids having tails ranging from C10:0 to C22:6, for example, DDPE (C10:0) (CAS 253685-27-7), DLPE (C12:0) (CAS 59752-57-7), DSPE (CI 8:0) (CAS 1069-79-0), DOPE (C18:l) (CAS 4004-05-1), DLinPE (CI 8:2) (CAS 20707-71-5), DLenPE (C18:3) (CAS 34813-40-6), DARAPE (C20:4) (CAS 5634-86-6), DDHAPE (C22:6) (CAS 123284- 81-1), DPhPE (16:0-[(CH.sub.3).sub.4]) (CAS 201036-16-0), PEG-linked phospholipids such as DSPC-PEG etc., PEG molecular weight from 200-50000, ceramide-PEG, DSPE-PEG

This disclosure provides pharmaceutically acceptable nucleic acid compositions useful for therapeutic delivery of nucleic acids, plasmids, antisense nucleic acids, ribozymes, aptamers, siRNA, antagomirs, miRNA, gene -silencing iNAs and mixtures thereof. These compositions and methods may be used for prevention and/or treatment of diseases in a mammal.

The lipid/nucleic acid formulations can be made by re -suspending cationic lipid nucleic acid salts in an organic solvent and upon mixing with other lipids produce nucleic acid lipid complexes. After removing the organic solvent by dry or dialysis or any other available skills, the formulation can be administered to an individual for gene therapies using plasmid DNA as the nucleic acid, or for down-regulating a gene using antisense, ribozymes, antagomirs, miRNA, iNA, or to inhibit other conditions using aptamers as the nucleic acid.

Compositions and Formulations for Administration

The nucleic acid-lipid compositions of this disclosure may be administered by various routes, for example, to effect systemic delivery via intravenous, parenteral, intraperitoneal or topical routes. In some embodiments, a siRNA may be delivered intracellularly, for example, in cells of a target tissue such as lung or liver, or in inflamed tissues. In some embodiments, this disclosure provides a method for delivery of siRNA in vivo. A nucleic acid-lipid composition may be administered intravenously, subcutaneously, or intraperitoneally to a subject. In some embodiments, the disclosure provides methods for in vivo delivery of interfering RNA to the lung of a mammalian subject.

In some embodiments, this disclosure provides a method of treating a disease or disorder in a mammalian subject. A therapeutically effective amount of a composition of this disclosure containing a nucleic, a cationic lipid, an amphiphile, a phospholipid, cholesterol and a PEG-linked cholesterol may be administered to a subject having a disease or disorder associated with expression or overexpression of a gene that can be reduced, decreased, downregulated, or silenced by the composition.

The compositions and methods of the disclosure may be administered to subjects by a variety of mucosal administration modes, including by oral, rectal, vaginal, intranasal,

intrapulmonary, or transdermal delivery, or by topical delivery to the eyes, ears, skin or other mucosal surfaces. In some aspects of this disclosure, the mucosal tissue layer includes an epithelial cell layer. The epithelial cell can be pulmonary, tracheal, bronchial, alveolar, nasal, buccal, epidermal, or gastrointestinal. Compositions of this disclosure can be administered using

conventional actuators such as mechanical spray devices, as well as pressurized, electrically activated, or other types of actuators.

Compositions of this disclosure may be administered in an aqueous solution as a nasal or pulmonary spray and may be dispensed in spray form by a variety of methods known to those skilled in the art. Pulmonary delivery of a composition of this disclosure is achieved by

administering the composition in the form of drops, particles, or spray, which can be, for example, aerosolized, atomized, or nebulized. Particles of the composition, spray, or aerosol can be in a either liquid or solid form. Preferred systems for dispensing liquids as a nasal spray are disclosed in U.S. Pat. No. 4,511 ,069. Such formulations may be conveniently prepared by dissolving compositions according to the present disclosure in water to produce an aqueous solution, and rendering said solution sterile. The formulations may be presented in multi-dose containers, for example in the sealed dispensing system disclosed in U.S. Pat. No. 4,511,069. Other suitable nasal spray delivery systems have been described in Transdermal Systemic Medication, Y. W. Chi en ed., Elsevier Publishers, New York, 1985; and in U.S. Pat. No. 4,778,810. Additional aerosol delivery forms may include, e.g., compressed air-, jet-, ultrasonic-, and piezoelectric nebulizers, which deliver the biologically active agent dissolved or suspended in a pharmaceutical solvent, e.g., water, ethanol, or mixtures thereof.

Nasal and pulmonary spray solutions of the present disclosure typically comprise the drug or drug to be delivered, optionally formulated with a surface active agent, such as a nonionic surfactant (e.g., polysorbate-80), and one or more buffers. In some embodiments of the present disclosure, the nasal spray solution further comprises a propellant. The pH of the nasal spray solution may be from about H 6.8 to 7.2. The pharmaceutical solvents employed can also be a slightly acidic aqueous buffer of pH 4-6. Other components may be added to enhance or maintain chemical stability, including preservatives, surfactants, dispersants, or gases.

In some embodiments, this disclosure is a pharmaceutical product which includes a solution containing a composition of this disclosure and an actuator for a pulmonary, mucosal, or intranasal spray or aerosol.

A dosage form of the composition of this disclosure can be liquid, in the form of droplets or an emulsion, or in the form of an aerosol.

A dosage form of the composition of this disclosure can be solid, which can be reconstituted in a liquid prior to administration. The solid can be administered as a powder. The solid can be in the form of a capsule, tablet or gel.

To formulate compositions for pulmonary delivery within the present disclosure, the biologically active agent can be combined with various pharmaceutically acceptable additives, as well as a base or carrier for dispersion of the active agent(s). Examples of additives include pH control agents such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, and mixtures thereof. Other additives include local anesthetics (e.g., benzyl alcohol), isotonizing agents (e.g., sodium chloride, mannitol, sorbitol), adsorption inhibitors (e.g., Tween 80), solubility enhancing agents (e.g., cyclodextrins and derivatives thereof), stabilizers (e.g., serum albumin), and reducing agents (e.g., glutathione). When the composition for mucosal delivery is a liquid, the tonicity of the formulation, as measured with reference to the tonicity of 0.9% (w/v) physiological saline solution taken as unity, is typically adjusted to a value at which no substantial, irreversible tissue damage will be induced in the mucosa at the site of administration. Generally, the tonicity of the solution is adjusted to a value of about 1/3 to 3, more typically 1/2 to 2, and most often 3/4 to 1.7 of the tonicity of 0.9% (w/v) physiological saline solution.

The biologically active agent may be dispersed in a base or vehicle, which may comprise a hydrophilic compound having a capacity to disperse the active agent and any desired additives. The base may be selected from a wide range of suitable carriers, including but not limited to, copolymers of polycarboxylic acids or salts thereof, carboxylic anhydrides (e.g. maleic anhydride) with other monomers (e.g., methyl(meth)acrylate, acrylic acid, etc.), hydrophilic vinyl polymers such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives such as

hydroxymethylcellulose, hydroxypropylcellulose, etc., and natural polymers such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid, and nontoxic metal salts thereof. Often, a biodegradable polymer is selected as a base or carrier, for example, polylactic acid, poly(lactic acid- glycolic acid) copolymer, polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid) copolymer and mixtures thereof. Alternatively or additionally, synthetic fatty acid esters such as polyglycerin fatty acid esters, sucrose fatty acid esters, etc., can be employed as carriers.

Hydrophilic polymers and other carriers can be used alone or in combination, and enhanced structural integrity can be imparted to the carrier by partial crystallization, ionic bonding, crosslinking and the like. The carrier can be provided in a variety of forms, including, fluid or viscous solutions, gels, pastes, powders, microspheres and films for direct application to the nasal mucosa. The use of a selected carrier in this context may result in promotion of absorption of the biologically active agent.

Formulations for mucosal, nasal, or pulmonary delivery may contain a hydrophilic low molecular weight compound as a base or excipient. Such hydrophilic low molecular weight compounds provide a passage medium through which a water-soluble active agent, such as a physiologically active peptide or protein, may diffuse through the base to the body surface where the active agent is absorbed. The hydrophilic low molecular weight compound optionally absorbs moisture from the mucosa or the administration atmosphere and dissolves the water-soluble active peptide. The molecular weight of the hydrophilic low molecular weight compound is generally not more than 10,000 and preferably not more than 3000. Examples of hydrophilic low molecular weight compounds include polyol compounds, such as oligo-, di- and monosaccarides including sucrose, mannitol, lactose, L-arabinose, D-erythrose, D-ribose, D-xylose, D-mannose, D-galactose, lactulose, cellobiose, gentibiose, glycerin, polyethylene glycol, and mixtures thereof. Further examples of hydrophilic low molecular weight compounds include N-methylpyrrolidone, alcohols (e.g., oligovinyl alcohol, ethanol, ethylene glycol, propylene glycol, etc.), and mixtures thereof.

The compositions of this disclosure may alternatively contain as pharmaceutically acceptable carriers substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, and wetting agents, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate,

triethanolamine oleate, and mixtures thereof. For solid compositions, conventional nontoxic pharmaceutically acceptable carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.

In certain embodiments of the disclosure, the biologically active agent may be administered in a time release formulation, for example in a composition which includes a slow release polymer. The active agent can be prepared with carriers that will protect against rapid release, for example a controlled release vehicle such as a polymer, microencapsulated delivery system or bioadhesive gel. Prolonged delivery of the active agent, in various compositions of the disclosure can be brought about by including in the composition agents that delay absorption, for example, aluminum monosterate hydrogels and gelatin.

EXAMPLES

Example 1: Preparation of an Aqueous Solution of the Cationic Lipid DOTMA (N-[l-(2,3- dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) and nucleic acid/DOTMA salts

2 mg of DOTMA was dissolved in 0.1 mL of chloroform in a clean, dry borosilicate clear glass vial and the chloroform was evaporated off by blowing nitrogen gas into the vial and any remaining trace amount of chloroform was removed by drying in vacuo in a vacuum chamber. The vial was removed from the vacuum chamber and 1 mL of sterilized water was added. The vial was sealed with TEFLON® - lined cap and tightly wrapped with a sealing tape. The vial was vortexed or/and sonicated to clarity.

An aqueous solution containing 1 mg siR A was added to the aqueous solution of DOTMA. The addition of the siRNA to the aqueous solution of DOTMA cationic lipid caused immediate aggregation forming a water-insoluble cationic lipid/nucleic acid salt comprised of DOTMA and siRNA.

The water-insoluble cationic lipid/nucleic acid salt in a tube was chilled on ice for at least 10 minutes. The water-insoluble cationic lipid/nucleic acid salt in a tube was then centrifuged in an Eppendorf microcentrifuge at maximum speed for 15 minutes to form a pellet. The aqueous layer was carefully removed and the salt pellet was dried in vacuo to remove additional moisture.

Example 2: Preparation of an Aqueous Solution of the Cationic Lipid DOTMA (N-[l-(2,3- dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) and nucleic acid/DOTMA salts 2 mg of DOTMA was dissolved in 0.1 mL of chloroform in a clean, dry borosilicate clear glass vial and the chloroform was evaporated off by blowing nitrogen gas into the vial and any remaining trace amount of chloroform was removed by drying in vacuo. Sterilized water was added and the vial was vortexed or/and sonicated to clarity.

An aqueous solution containing 1 mg siRNA was added to the aqueous solution of DOTMA. The addition of the siRNA to the aqueous solution of DOTMA cationic lipid caused immediate aggregation forming a water-insoluble cationic lipid/nucleic acid salt comprised of DOTMA and siRNA.

The water-insoluble cationic lipid/nucleic acid salt was centrifuged to form a pellet. The aqueous layer was carefully removed and the salt pellet was dried in vacuo to remove additional moisture.

Example 3: Preparation of an Aqueous Solution of the Cationic Lipid DOTMA (N-[l-(2,3- dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) and nucleic acid/DOTMA salts

2 mg of DOTMA was dissolved in 0.1 mL of chloroform in a clean, dry borosilicate clear glass vial and the chloroform was evaporated off by blowing nitrogen gas into the vial and any remaining trace amount of chloroform was removed by drying in vacuo. Sterilized water was added and the vial was vortexed or/and sonicated to clarity.

The aqueous solution of DOTMA was added to an aqueous solution containing 1 mg siRNA. The addition of the DOTMA cationic lipid to the siRNA solution caused immediate aggregation forming a water-insoluble cationic lipid/nucleic acid salt comprised of DOTMA and siRNA.

The water-insoluble cationic lipid/nucleic acid salt was centrifuged to form a pellet. The aqueous layer was carefully removed and the salt pellet was dried in vacuo to remove additional moisture.

Example 4: Preparation of an Ethanol Solution of the Cationic Lipid DOTMA (N-[l-(2,3- dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) and nucleic acid/DOTMA salts

2 mg of DOTMA was dissolved in 0.1 mL of chloroform in a clean, dry borosilicate clear glass vial and the chloroform was evaporated off by blowing nitrogen gas into the vial and any remaining trace amount of chloroform was removed by drying in vacuox. The vial was removed from the vacuum chamber and 1 mL of ethanol was added. The vial was vortexed or/and sonicated to dissolve the DOTMA.

The solution of DOTMA in ethanol was added to an aqueous solution containing 1 mg siR A. The addition of the DOTMA cationic lipid to the siR A solution caused immediate aggregation forming a water-insoluble cationic lipid/nucleic acid salt comprised of DOTMA and siRNA.

The water-insoluble cationic lipid/nucleic acid salt was centrifuged to form a pellet. The aqueous layer was carefully removed and the pellet was dried in vacuo to remove additional moisture.

Example 5: Preparation of an Aqueous Solution of the Cationic Lipid DC-Chol (cholesteryl 3β-Ν- (dimethylaminoethyl)carbamate hydrochloride) and nucleic acid/DC -Choi salts

2 mg of DC-Chol was dissolved in 0.1 mL of chloroform in a clean, dry borosilicate clear glass vial and the chloroform was evaporated off by blowing nitrogen gas into the vial and any remaining trace amount of chloroform was removed by drying in vacuo. Sterilized water was added and the vial was vortexed or/and sonicated to clarity.

An aqueous solution containing 1 mg siRNA was added to the aqueous solution of DC-Chol. The addition of the siRNA to the aqueous solution of DC-Chol cationic lipid caused immediate aggregation forming a water-insoluble cationic lipid/nucleic acid salt comprised of DC-Chol and siRNA.

The water-insoluble cationic lipid/nucleic acid salt was centrifuged to form a pellet. The aqueous layer was carefully removed and the pellet was dried in vacuo to remove additional moisture.

Example 6: Preparation of an Ethanol Solution of the Cationic Lipid DC-Chol (Cholesteryl 3β-Ν- (dimethylaminoethyl)-carbamate hydrochloride) and nucleic acid/ DC-Chol salts

2 mg of DC-Chol was dissolved in 0.1 mL of chloroform in a clean, dry borosilicate clear glass vial and the chloroform was evaporated off by blowing nitrogen gas into the vial and any remaining trace amount of chloroform was removed by drying in vacuo. 1 mL of ethanol was added the vial was vortexed or/and sonicated to dissolve the DC-Chol. The solution of DC-Chol in ethanol was added to an aqueous solution containing 1 mg siR A. The addition of the DC-Chol cationic lipid to the siR A solution caused immediate aggregation forming a water-insoluble cationic lipid/nucleic acid salt comprised of DC-Chol and siRNA.

The water-insoluble cationic lipid/nucleic acid salt was centrifuged to form a pellet. The aqueous layer was carefully removed and the pellet was dried in vacuo to remove additional moisture.

Example 7: Formulation preparation- 1

After centrifuging and drying the DOTMA/siRNA salt (containing 1 mg siRNA in a complex with DOTMA) which was made as the above examples (Example 1, 2, 3, 4, 5) under vacuum, the salt was re-suspended in 100 of chloroform and mixed with the following chemicals in chloroform: 4.4 mg of N ⁶ -tetrakis(3-aminopropyl)-l,3-propanediamine cholesteryl carbamate, 2.7 mg 20:4 PE (l,2-diarachidonoyl-5/?-glycero-3-phosphoethanolamine), 3.2 mg of Cholesterol, and 14.4 mg of cholesterol-PEG (cholesterol-poly(ethylene glycol)) IK. The mixture was dried under vacuum and stored at 4° C until use. Before injection, 9% sucrose solution was added with shaking to form an isotonic and a self-emulsifying siRNA/lipid suspension of siRNA/lipid formulation.

Example 8: Formulation preparation-2

After centrifuging and drying the DOTMA/siRNA salt (containing 1 mg siRNA in a complex with DOTMA) which was made as the above examples (Example 1, 2, 3, 4, 5) under vacuum, the salt was re-suspended in 100 of chloroform and mixed with the following chemicals in chloroform: 4.4 mg of N ⁶ -tetrakis(3-aminopropyl)-l,3-propanediamine cholesteryl carbamate, 2.7 mg 22:6 PE, 3.2 mg of Cholesterol, and 14.4 mg of cholesterol-PEG (cholesterol-poly(ethylene glycol)) IK. The mixture was dried under vacuum and stored at 4° C until use. Before injection, 9% sucrose solution was added with shaking to form an isotonic and a self-emulsifying siRNA/lipid suspension of siRNA/lipid formulation.

Example 9: Formulation preparation-3 After centrifuging and drying the DOTMA/siRNA salt (containing 1 mg siRNA in a complex with DOTMA) which was made as the above examples (Example 1, 2, 3, 4, 5) under vacuum, the salt was re-suspended in 100 of chloroform and mixed with the following chemicals in chloroform: 4.4 mg of N ⁶ -tetrakis(3-aminopropyl)-l,3-propanediamine cholesteryl carbamate, 2.7 mg DOPE (l,2-dioleoyl-sn-glycero-3-phosphoethanolamine), 3.2 mg of Cholesterol, and 14.4 mg of cholesterol-PEG (cholesterol-poly(ethylene glycol)) IK. The mixture was dried under vacuum and stored at 4° C until use. Before injection, 9% sucrose solution was added with shaking to form an isotonic and a self-emulsifying siRNA/lipid suspension of siRNA/lipid formulation.

Example 10: Formulation preparation-4

After centrifuging and drying the DOTMA/siRNA salt (containing 1 mg siRNA in a complex with DOTMA) which was made as the above examples (Example 1, 2, 3, 4, 5) under vacuum, the salt was re-suspended in 100 of chloroform and mixed with the following chemicals in chloroform: 4.4 mg of N ⁶ -tetrakis(3-aminopropyl)-l,3-propanediamine cholesteryl carbamate, 4.4 mg DLinPE (l,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine), 1.6 mg of Cholesterol, and 14.4 mg of cholesterol-PEG (cholesterol-poly(ethylene glycol)) IK. The mixture was dried under vacuum and stored at 4° C until use. Before injection, 9% sucrose solution was added with shaking to form an isotonic and a self-emulsifying siRNA/lipid suspension of siRNA/lipid formulation.

Example 11: Formulation preparation-5

After centrifuging and drying the DOTMA/siRNA salt (containing 1 mg siRNA in a complex with DOTMA) which was made as the above examples (Example 1, 2, 3, 4, 5) under vacuum, the salt was re-suspended in 100 of chloroform and mixed with the following chemicals in chloroform: 4.4 mg of N ⁶ -tetrakis(3-aminopropyl)-l,3-propanediamine cholesteryl carbamate, 4.4 mg DLinPE, 1.6 mg cholesterol, and 14.4 mg cholesterol-PEG (cholesterol-poly(ethylene glycol)) 660. The mixture was dried under vacuum and stored at 4° C until use. Before injection, 9% sucrose solution was added with shaking to form an isotonic and a self-emulsifying siRNA lipid suspension of siRNA/lipid formulation.

Example 12: Formulation preparation-6 After centrifuging and drying the DC-Chol/siRNA salt (containing 1 mg siRNA in a complex with DC-Choi) which was made as the above examples (Example 6 and 7) under vacuum, the salt was re-suspended in 100 of chloroform and mixed with the following chemicals in chloroform: 4.4 mg N ⁶ -tetrakis(3-aminopropyl)-l,3-propanediamine cholesteryl carbamate, 4.4 mg DLinPE, 1.6 mg cholesterol, and 14.4 mg cholesterol-PEG (cholesterol-poly(ethylene glycol)) 660. The mixture was dried under vacuum and stored at 4° C until use. Before injection, 9% sucrose solution was added with shaking to form an isotonic and a self-emulsifying siRNA/lipid suspension of siRNA/lipid formulation.

Example 13: Formulation preparation for delivery siRNA into tumor

After centrifuging and drying the DOTMA/siRNA salt (containing 1 mg siRNA in a complex with DOTMA) which was made as the above examples (Example 1, 2, 3, 4, 5) under vacuum, the salt was re-suspended in 100 chloroform and mixed with the following chemicals in chloroform: 4.4 mg N ⁶ -tetrakis(3-aminopropyl)-l,3-propanediamine cholesteryl carbamate, 4.4 mg DLinPE, 4.8 mg cholesterol, and 21.6 mg cholesterol-PEG (cholesterol-poly(ethylene glycol)) IK. The mixture was dried under vacuum and stored at 4° C until use. Before injection, 9% sucrose solution was added with shaking to form an isotonic and a self-emulsifying siRNA/lipid suspension of siRNA lipid formulation. The results are shown in Figure 5.

Example 14: Formulation preparation for delivery siRNA into lung

After centrifuging and drying the DOTMA/siRNA salt (containing 1 mg siRNA in a complex with DOTMA) which was made as the above examples (Example 1, 2, 3, 4, 5) under vacuum, the salt was re-suspended in 100 of chloroform and mixed with the following chemicals in chloroform: 8.8 mg N ⁶ -tetrakis(3-aminopropyl)-l,3-propanediamine cholesteryl carbamate, 2.2 mg DLinPE, 1.6 mg cholesterol, and 14.4 mg cholesterol-PEG (cholesterol-poly(ethylene glycol)) 660. The mixture was dried under vacuum and stored at 4° C until use. Before injection, 9% sucrose solution was added with shaking to form an isotonic and a self-emulsifying siRNA/lipid suspension of siRNA lipid formulation. The results are shown in Figures 6 and 7. Example 15: Formulation preparation for delivery siRNA into lung

After centrifuging and drying the DOTMA/siRNA salt (containing 1 mg siRNA in complexed with DOTMA) which was made as the above examples (Example 1, 2, 3, 4, 5) under vacuum, the salt was re-suspended in 100 of chloroform and mixed with the following chemicals in chloroform: 8.8 mg N ⁶ -tetrakis(3-aminopropyl)-l,3-propanediamine cholesteryl carbamate, 2.2 mg DLinPE, 1.6 mg cholesterol, and 14.4 mg C16 PEG750 Ceramide. The mixture was dried under vacuum and stored at 4° C until use. Before injection, 9% sucrose solution was added with shaking to form an isotonic and a self-emulsifying siRNA/lipid suspension of siRNA/lipid formulation. The results are shown in Figures 6 and 7.

Example 16: Formulation preparation for delivery siRNA into lung

After centrifuging and drying the DOTMA/siRNA salt (containing 1 mg siRNA in complexed with DOTMA) which was made as the above examples (Example 1, 2, 3, 4, 5) under vacuum, the salt was re-suspended in 100 of chloroform and mixed with the following chemicals in chloroform: 8.8 mg N ⁶ -tetrakis(3-aminopropyl)-l,3-propanediamine cholesteryl carbamate, 2.2 mg DLinPE, 1.6 mg cholesterol, and 14.4 mg DSPE-PEG 2000. The mixture was dried under vacuum and stored at 4° C until use. Before injection, 9% sucrose solution was added with shaking to form an isotonic and a self-emulsifying siRNA lipid suspension of siRNA/lipid formulation. The results are shown in Figures 6 and 7.

Example 17: Formulation preparation for delivery siRNA into lung

After centrifuging and drying the DOTMA/siRNA salt (containing 1 mg siRNA in complexed with DOTMA) which was made as the above examples (Example 1, 2, 3, 4, 5) under vacuum, the salt was re-suspended in 100 of chloroform and mixed with the following chemicals in chloroform: 8.8 mg N ⁶ -tetrakis(3-aminopropyl)-l,3-propanediamine cholesteryl carbamate, 2.2 mg DLinPE, 1.6 mg cholesterol, and 14.4 mg DOPE-PEG 2000. The mixture was dried under vacuum and stored at 4° C until use. Before injection, 9% sucrose solution was added with shaking to form an isotonic and a self-emulsifying siRNA/lipid suspension of siRNA/lipid formulation. The results are shown in Figures 6 and 7. Example 18: Formulation preparation for delivery siRNA into lung

After centrifuging and drying the DOTMA/siRNA salt (containing 1 mg siRNA in complexed with DOTMA) which was made as the above examples (Example 1, 2, 3, 4, 5) under vacuum, the salt was re-suspended in 100 of chloroform and mixed with the following chemicals in chloroform: 8.8 mg of N ⁶ -tetrakis(3-aminopropyl)-l,3-propanediamine cholesteryl carbamate, 2.2 mg DLinPE, 1.6 mg cholesterol, 7.2 mg cholesterol-PEG 660, and 7.2 mg DSPE-PEG 2000. The mixture was dried under vacuum and stored at 4° C until use. Before injection, 9% sucrose solution was added with shaking to form an isotonic and a self-emulsifying siRNA/lipid suspension of siRNA/lipid formulation. The results are shown in Figures 6 and 7.

Example 19: In vivo gene knockdown examination

All procedures used in animal studies conducted were approved by the Institutional Animal Care and Use Committee (IACUC) and were consistent with local, state and federal regulations as applicable. CDl mice (Charles River) received siRNA formulations at dose volume of 0.2 ml via tail vein. Mouse liver was harvested 2 days after dosing and mRNA was isolated with Turbocapture kit (Qiagen) for analyzing change of gene expression with real-time RT-PCR method (SensiMix SYBR One-Step Kit, Bioline).