WO2001060415A1 - Methodes et compositions d'administration de genes - Google Patents

Methodes et compositions d'administration de genes Download PDF

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Publication number
WO2001060415A1
WO2001060415A1 PCT/US2001/005234 US0105234W WO0160415A1 WO 2001060415 A1 WO2001060415 A1 WO 2001060415A1 US 0105234 W US0105234 W US 0105234W WO 0160415 A1 WO0160415 A1 WO 0160415A1
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WIPO (PCT)
Prior art keywords
polyplex
group
poly
acid
nucleic acid
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PCT/US2001/005234
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English (en)
Inventor
Charles Peter Lollo
Mariusz Banaszczyk
Henry C. Chiou
Dongpei Wu
Patricia M. Mullein
Alison T. Carlo
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The Immune Response Corporation
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Priority to AU2001238485A priority Critical patent/AU2001238485A1/en
Publication of WO2001060415A1 publication Critical patent/WO2001060415A1/fr
Priority to US10/211,214 priority patent/US20030134420A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric

Definitions

  • Nonviral gene delivery systems currently under development are naked DNA, cationic liposomes, cationic polymers, and combinations of both; cationic lipids with cationic polymers (Hickman, et al. Hum Gene Ther 1994, 5, 1477-1483; Wolff, J. A.; et al. Science 1990, 247, 1465-1468; Feigner, P. L. et al. Hum Gene Ther 1997, 8, 511-512; Feigner, et al. Ann NYAcadSci 1995, 772, 126-139; Mahato, R. I. et al. Pharm Res 1997, 14, 853-859; Nicolau, C. et al.
  • complexes of DNA with either cationic lipids or cationic polymers must protect DNA from degradation in extracellular (vascular) compartment, and advantageously should remain intact.
  • both cationic lipid and cationic polymer DNA complexes when challenged with negatively charged molecules (such as those which are typically present in extracellular space) will, to a varying extent, release DNA.
  • These complexes are, generally, unstable and labile. The premature DNA release from labile complexes can result in rapid DNA degradation and poor transfection efficiency.
  • the complex lability and colloidal instability is a challenge for designers of effective gene delivery methods and compositions.
  • the present invention provides novel compositions and formulations for delivering anionic compounds, particularly polynucleotides (DNA and RNA), across cellular boundaries (e.g., cellular membranes) either in vivo or in vitro.
  • anionic compounds particularly polynucleotides (DNA and RNA)
  • cellular boundaries e.g., cellular membranes
  • the invention provides novel molecular complexes, referred to as "polyplexes," containing an anionic compound, such as a nucleic acid, associated with one or, more typically, multiple co-polymer domains, including a cationic domain, a transitional domain, and/or a surface domain.
  • the co-polymer domains function as "delivery enhancers" to facilitate delivery of the anionic compound across cellular boundaries by interacting with or "encapsulating" the anionic compound.
  • the surface domain of the polyplexes optionally also can include cellular ligands which target polyplexes to cells.
  • the invention provides formulants or "penetration enhancers" which can be combined with polyplexes of the invention, or with free (“naked") nucleic acids, to further enhance the ability of these compositions to traverse cellular membranes (i.e., be taken up by cells).
  • Suitable penetration enhancers include, for example, DHPC, bile salts, surfactants and combinations thereof.
  • Other techniques, such as sonification, also can be used in conjunction with the present invention to enhance cellular uptake of polyplexes.
  • Polyplex compositions and formulations of the present invention can be used to enhance delivery and uptake of a wide variety of therapeutic agents agents by cells, particularly in applications of gene therapy.
  • the invention pertains, at least in part, to a method of delivering an anionic agent through a lipid membrane.
  • the method includes contacting the anionic agent with a delivery enhancing formulation, allowing a polyplex to form; and contacting the lipid membrane with a penetration enhancer, such that upon contact of the polyplex with the lipid membrane, the anionic agent is delivered through the • membrane.
  • the delivery enhancing formulation contains one or more components selected from a cationic backbone moiety, a hydrophobic moiety, and a hydrophilic moiety. Typically, the formulation contains all three components.
  • the invention in another embodiment, pertains to a method for enhancing expression of a nucleic acid in a cell.
  • the method includes contacting the nucleic acid with a delivery enhancing formulation (as described above), allowing a polyplex to form, and contacting the membrane of the cell with a penetration enhancer, such that upon contact of the polyplex with the membrane of the cell, the nucleic acid is internalized into the cell and expression of said nucleic acid is enhanced.
  • the invention pertains to a method for treating a subject by administering an effective amount of a penetration enhancer and a polyplex of the present invention (e.g., comprising a nucleic acid, a cationic backbone moiety, a hydrophobic moiety, and a hydrophilic moiety), such that said subject is treated.
  • a penetration enhancer can be administered before, after or concurrently with the polyplex.
  • the invention also pertains, at least in part, to polyplexes of the invention, comprising copolymers as described herein and anionic agents (e.g., nucleic acids, etc.).
  • anionic agents e.g., nucleic acids, etc.
  • pharmaceutical compositions comprising such polyplexes along with an effective amount of a penetration enhancer, combined in a pharmaceutically acceptable carrier to form a therapeutic composition.
  • the invention to pertains to a method for enhancing expression of a nucleic acid in a cell by contacting the cell with a free nucleic acid (i.e., not in the form of a polyplex) and a penetration enhancer, such that the expression of the nucleic acid is enhanced.
  • a free nucleic acid i.e., not in the form of a polyplex
  • a penetration enhancer such that the expression of the nucleic acid is enhanced.
  • Figure 1 shows a polynucleotide carrier complex in Cartesian coordinates.
  • Figure 2 is a drawing of the polynucleotide carrier complex of Figure 1.
  • Figure 3 shows the interaction between a ligand on the surface domain of a polyplex interacting with a cellular receptor.
  • Figure 4 shows polyplex and lipid membrane in equilibrium with a formulant.
  • Figure 5 shows polyplex containing polymers comprising two hydrophobic domains linked with hydrophilic polymer.
  • Figure 5 A depicts polyplex with polymers with one hydrophobic domain each.
  • Figure 5B depicts polyplex formed with conjugates containing two hydrophobic domains, one of which is shown in its unbound state.
  • Figure 5C shows polyplex with conjugates with two hydrophobic domains in the bound state.
  • Figure 6 shows polyplex fusion with cellular membrane facilitated by residues in the second hydrophobic domain of the conjugates.
  • Figure 7 is a representation of n-block co-polymer.
  • Figure 8 is a representation of the compounds described in Table 1.
  • Figure 9 shows the structure of (a) ' randomly grafted hydrophilic PEG chains and randomly grafted hydrophobic chains on a cationic domain and (b) randomly grafted hydrophobic-hydrophilic element on a cationic domain.
  • Figure 10 shows the structure of grafted polymers with one hydrophobic domain per PEG chain.
  • Figure 10a shows a hydrophobic domain between a cationic domain and hydrophilic domains.
  • Figure 10b shows a hydrophobic domain positioned at the terminus of hydrophilic domain that is then grafted on a cationic domain.
  • Figure 11 shows the structure of grafted polymers with two hydrophobic domains per PEG chain.
  • Figure 11a shows a hydrophobic domain between the cationic domain and the surface domain.
  • Figure l ib shows a hydrophobic domain positioned at the terminus of a surface (e.g., hydrophilic) domain, and between the surface (e.g., hydrophilic) and cationic domains.
  • Figure 12 shows the equilibrium between the polynucleotide earner complex (B) with unincorporated formulant (A) and the polynucleotide carrier complex with incorporated formulant (C).
  • Figure 13 is a bar graph showing the effect of a penetration enhancer on the expression of luciferase encapsulated in a polyplex of the invention.
  • the first bar of the graph (white) represents a polyplex formed from copolymers comprised of random grafts of PEG5k on PLL 10k chain
  • the second bar (white) represents a polyplex comprised of copolymers of the formula PLL10k-g-( ⁇ -NH-PEG5k) 14.
  • the remaining bars on the graph represent polyplexes comprised of PLL10k-g-( ⁇ -NH-C 1 o- O-PEG2k) 9 with different penetration enhancing formulants.
  • the third bar (grey) represents no additional formulation enhancer.
  • the fourth bar represents the result with added 0.19% Brij 35 formulant.
  • the fifth bar represents the result with added 0.41% OGP formulant.
  • the sixth bar represents the result with added 0.5% TCDC formulant.
  • the seventh bar represents the results with added 0.4% DHPC formulant.
  • Figure 14 is a graph showing the effects of polyplexes comprised of different copolymers on luciferase expression when administer with the formulant, DHPC.
  • '•' represents PLL9.4k-g-( ⁇ -NH-"Chenodeoxycholic Acid") 16
  • ' ⁇ ' represents the copolymer PLL9.4k-g-( ⁇ -NH-"Cholic Acid")io
  • ' ⁇ ' represents the copolymer PEG5k- b-(Cys-S-C18) 10 -b-(Lys) 5 -g-( ⁇ -NH-Chenodeoxycholic Acid) 10
  • ' ⁇ ' represents the copolymer PEG5k-g-(Cys-S-C 18) 10 -b-(Lys) 120 -g-( ⁇ -NH-"Chenodeoxycholic Acid") 15
  • ' ⁇ ' represents the copolymer PLL9.4k-g-( ⁇ -NH-
  • Figure 15 is a graph showing the effects of the addition of the formulant DHPC on expression of luciferase, when administered with polyplex of the invention.
  • the symbol '•' represents a polyplex formed from the copolymer, PLL9.4k-g-( ⁇ -NH- CO-"Trigalactose”)].6.h the symbol ' ⁇ ' represents a polyplex formed from the copolymer, PLL9.4k-g-( ⁇ -NH-C12-PEG5k) 4 . 7 -g-( ⁇ -NH-' rigalactose") 9 ), the symbol i r ' represents a polyplex formed from the copolymer, PLL9.4k-g-( ⁇ -NH-CO-
  • Figure 16 is a graph showing the expression of luciferase in mice when the gene is administered with a variety of co-polymer polyplexes that were formulated with DHPC.
  • the symbol '•' represents a polyplex which were formed using copolymers constructed from random grafts of hydrophobe (-CH CONHCH 2 CH 2 CH 2 -O- ⁇ - Cholesterol ether) and PEG, PLL9.4k-g-( ⁇ -NH-PEG5k) 12 . 8 -g-( ⁇ -NH- CH 2 CONHCH 2 CH 2 CH 2 -O- ⁇ -Cholesterol ether) 26 .
  • the symbol ' ⁇ ' represents polyplexes comprised of the block co-polymer (PEG5k-b-(Cys-S-Cl 8) 10 -b-(Lys) 5 ); and the symbol ' ⁇ ' represents polyplexes comprised of the block co-polymer (PEG5k-b- (Phe) 14 -b-(Lys) 51 ).
  • Polyplexes comprised of polymers consisting of random grafts of PEG-coupled-hydrophobe with and without Trigalactose ligand, include PLL9.4k-g-( ⁇ - NH-PEG4.4k-C18) 2.8 represented by the symbol ' ⁇ ', PLL10k-g-( ⁇ -NH-C 10 -PEG4.4k- C18) 6 . 6 represented by the symbol ' ⁇ ', PLL9.4k-g-( ⁇ -NH-C 12 -PEG5k) 4 . 7 -g-( ⁇ -NH- CH2CO-"Trigal")9 represented by the symbol' 0 ' .
  • Figures 17A and 17B are bar graphs which show the biodistribution of I25 I- pCMV ⁇ Gal when free (light grey), free with TCDC (medium light grey), encapsulated in a polyplex comprised of block co-polymer (PEG5k-b-(Cys-S-C18) 10 -b-(Lys) 5 ) (BP- A) (dark grey) and encapsulated in a polyplex comprised of block co-polymer (PEG5k- b-(Cys-S-C 18) 10 -b-(Lys) 45 ) (BP-A) with TCDC (black).
  • the biodistribution is determined at 5 minutes ( Figure 17A) and one hour ( Figure 17B).
  • the present invention provides, in one aspect, molecular complexes referred to as "polyplexes" for delivering anionic agents (e.g., anionic polymers or negatively charged therapeutic agents, such as DNA, RNA, proteins, and small molecules) through lipid membranes (e.g., cellular boundaries, e.g., cellular membranes, nuclear membranes, endosomal membranes, etc.).
  • anionic agents e.g., anionic polymers or negatively charged therapeutic agents, such as DNA, RNA, proteins, and small molecules
  • lipid membranes e.g., cellular boundaries, e.g., cellular membranes, nuclear membranes, endosomal membranes, etc.
  • polyplexes of the present invention are made up of multiple co-polymer domains. These domains are organized by the type of functional groups present on the co-polymer making up the domain.
  • the center domain (Zone I of Figure 1) contains the anionic agent. Examples of anionic agents include nucleic acids, negatively charged drugs and other small molecules capable of being delivered via a polyplex through a cellular boundary or lipid membrane.
  • the cationic domain (Zone II of Figure 1) is designed to interact, e.g., electrostatically, with the anionic domain/agent.
  • the cationic domain is comprised of one or more cationic backbone moieties of copolymers, which are described in greater detail below.
  • the transitional domain (Zone III of Figure 1) links the cationic domain with the surface domain, typically via linear or branched co-polymers.
  • the transitional domain may be hydrophobic in nature and may be comprised, at least in part, of hydrophobic moieties of copolymers. When the transitional domain is comprised at least in part of hydrophobic moieties, it is generally referred to as the "hydrophobic domain.”
  • the surface domain defines the polyplex surface by way of, for example, branching elements which allow the introduction of multiple molecules or other polymers on the polyplex surface. Such moieties modify the surface properties of the polyplex so as to enhance overall delivery of the anionic agent.
  • the surface domain may be comprised, at least in part, of hydrophilic moieties of copolymers, as well as other ligands and other surface moieties which allow the polyplex to perfonn its intended function.
  • polyplexes of the invention essentially consist of multiple co-polymer domains which interact (e.g., as a carrier) with an anionic agent which is delivered across a cell boundary or lipid membrane.
  • the functional moieties of the polyplexes can first be attached to a single grafting element which, in turn, can then be grafted onto a desired cationic domain.
  • a hydrophobic moiety is coupled to PEG (a hydrophilic moiety) and then grafted on to a cationic domain.
  • anionic agents to cells or cellular compartments using polyplexes of the invention can, in certain embodiments, be further enhanced using ligand-receptor interactions, endosome disruptive residues, and nuclear localizing sequences.
  • These surface moieties may also aid in polyplex delivery by protecting the polyplex from deleterious interactions in, for example, vascular compartments.
  • Further enhancement can be achieved by attaching additional hydrophobic moieties to the cationic, transition and/or surface domains, such as lather releasing molecules that change permeability of membrane baniers, and as a result, increase overall uptake and expression.
  • other pentration enhancers can also be used to enhance the permeability of the membrane barriers.
  • Polyplexes of the invention can be formed, in one embodiment, with polymers containing one hydrophobic moiety on a grafted cationic backbone moiety.
  • the hydrophobic moiety aids with DNA condensation as evidenced by fluorescent quenching assay.
  • Additional hydrophobic moieties grafted on to the cationic backbone moiety can be used to increase the hydrophobicity of the polyplex.
  • the hydrophobic moieties, through the process of self association, micellization-like processes, and co- micellization processes, can interact with formulant or penetration enhancer molecules which may enhance delivery of the anionic agent through the lipid membrane.
  • polyplexes of the invention can be formulated with permeation enhancers and other delivery formulants which are co- administered with the polyplex.
  • delivery formulants of the invention also can be used to enhance delivery of free DNA.
  • Figure 3 shows that specific cellular entry (e.g., via ligand interactions) can be further enhanced by the coadministration of permeation enhancers.
  • Suitable cellular ligands for incorporation into polyplexes of the invention can include, for example, any natural or synthetic ligand which is capable of binding a cell surface receptor.
  • the ligand can be a protein, polypeptide, glycoprotein, glycopeptide or glycolipid which has functional groups that are exposed sufficiently to be recognized by the cell surface component. It can also be a component of a biological organism such as a virus, cells (e.g., mammalian, bacterial, protozoan).
  • the ligand can comprise an antibody, antibody fragment (e.g., an F(ab') 2 fragment) or analogues thereof (e.g., single chain antibodies) which binds the cell surface component (see e.g., Chen et al. (1994) FEBS Letters 338:167-169, Ferkol et al. (1993) J. Clin. Invest. 92:2394-2400, and Rojanasakul et al. (1994) Pharmaceutical Res. U_(12): 1731-1736).
  • antibody e.g., an F(ab') 2 fragment
  • analogues thereof e.g., single chain antibodies
  • ligands will vary according to the particular cell to be targeted.
  • proteins and polypeptides containing galactose-terminal carbohydrates such as carbohydrate trees obtained from natural glycoproteins, can be used.
  • natural glycoproteins that either contain terminal galactose residues or can be enzymatically treated to expose terminal galactose residues (e.g., by chemical or enzymatic desialylation) can be used.
  • the ligand is an asialoglycoprotein, such as asialoorosomucoid, asialofetuin or desialylated vesicular stomatitis virus.
  • suitable ligands for targeting hepatocytes can be prepared by chemically coupling galactose-terminal carbohydrates (e.g., galactose, mannose, lactose, arabinogalactan etc.) to nongalactose-bearing proteins or polypeptides (e.g., polycations) by, for example, reductive lactosamination.
  • galactose-terminal carbohydrates e.g., galactose, mannose, lactose, arabinogalactan etc.
  • polypeptides e.g., polycations
  • the surface domain of the polyplex can comprise other types of ligands.
  • mannose can be used to target macrophages (lymphoma) and Kupffer cells
  • mannose 6-phosphate glycoproteins can be used to target fibroblasts (fibro- sarcoma), intrinsic factor- vitamin B12 and bile acids (See Kramer et al. (1992) J. Biol. Chem.
  • Apolipoprotein E can be used to target nerve cells
  • pulmonary surfactants such as Protein A
  • Protein A can be used to target epithelial cells
  • ligands include, but are not limited to, Br(CH 2 ) 10 CO-NH- ⁇ - lactosyl amide, N 1 -(bromoacetamide)-N 13 -(chenodeoxycholic acid amide)-4,7,10-trioxo- 1,13- tridecanediamine; 1,1,1 -fris- [(O 6 - ⁇ -D-galactopyranoside)-7, 10,13,16-tetraoxo-5- orie-4-aza-hexadecanyl]- 1 - [ 1 -aza- 11 -amino-2-one-undecanyl] -methane, 1 , 1 , 1 -fris- [(0 16 - ⁇ -D-galactopyranoside)-7, 10,13,16-tefraoxo-5-one-4-aza-hexadecanyl]- 1 -[1,11 -diaza-
  • polyplexes containing co-polymer domains having one or more hydrophobic moieties are able to interact, e.g., bind, with particular formulants and fuse with cellular membranes.
  • Such polyplexes formed with copolymers comprising one or more hydrophobic moieties
  • Figures 5 a and b are shown in Figures 5 a and b, respectively.
  • the equilibrium of the hydrophobic moieties in the second domain between a free state ( Figure 5b) and a bound state ( Figure 5 c) is depicted.
  • This mechanism of equilibration between free and bound states may permit some population of free state form to enhance a docking and fusing step that may be required for cellular entry (Figure 6).
  • This equilibrium can be modulated by relative strength of hydrophobic moieties within the hydrophobic domains of the polyplexes.
  • the "cationic moiety” or “cationic backbone moiety” of the copolymers which make up the cationic domain of the polyplex can include any moiety capable of electrostatically interacting with the anionic agent (e.g., negatively charged polynucleotides).
  • Prefened cationic moieties for use in the carrier include non-peptidic and peptidic polycations, such as polylysine (e.g., poly-L-lysine), polyarginine, polyornithine, spermine, basic proteins such as histones (Chen et al., supra.), avidin, protamines (see e.g., Wagner et al., supra.), modified albumin (i.e., N-acylurea albumin) (see e.g., Huckett et al., supra.) and polyamidoamine cascade polymers (see e.g., Haensler et al. (1993) Bioconjugate Chem. 4: 372-379).
  • polylysine e.g., poly-L-lysine
  • polyarginine e.g., polyarginine
  • polyornithine spermine
  • basic proteins such as histones (Chen et al., supra
  • a preferred polycation is polylysine (e.g., ranging from about 2,000 to about 80,000 daltons, from about 3,800 to about 60,000 daltons, or from about 5,000 to about 50,000 daltons).
  • non- peptidic cationic backbone moities include peptoids (e.g., polymers comprised of modified amino acids or other peptide like polymers) and polyalkylenimines, such as polyethylenimine and polypropylenimine.
  • the cationic backbone moiety comprises polylysine having a molecular weight of about 17,000 daltons (purchased as the hydrogen bromide salt having a MW of a 26,000 daltons), conesponding to a chain length of approximately 100-120 lysine residues.
  • the cationic backbone moiety comprises a polycation having a molecular weight of about 2,600 daltons (purchased as the hydrogen bromide salt having a MW of a 4,000 daltons), corresponding to a chain length of approximately 15-10 lysine residues.
  • hydrophobic moiety includes moieties which make up the hydrophobic domain of the polyplex. Hydrophobic moieties may be selected based on their fusogenic properties or their interactions with components of cellular membranes, such as lectins and lipid head groups. In one embodiment, the hydrophobic moiety comprises linear or branched polymers, linear branched or cyclic, aliphatic, alkenyl, alkynyl groups, aromatic groups or combinations thereof.
  • the hydrophobic moiety may comprise one or more heteroatoms heterocyclic groups, peptides, peptoids, natural products, synthetic compounds, steroids, and steroid derivatives (e.g., hydrophobic moieties which comprise a steroidal nucleus, e.g., a cholesterol ring system) and/or other hydrophobic moieties known in the art which enable the polyplex to perform its function, e.g., deliver an anionic agent across a cell membrane. Delivery of polyplexes also may be further enhanced using penneation enhancers.
  • the hydrophobic moiety contains from about 4 to 40 carbon atoms.
  • hydrophobic groups may be, for example, charged, neutral, ligand bearing, polymeric, polypeptidic, peptoidic, or polypeptoidic.
  • hydrophobic moieties include poly-(C18-S-Cys) and poly (Phe).
  • the hydrophobic domain may be absent.
  • hydrophilic domain or “hydrophilic moieties” may be selected such that the polyplex is capable of performing its intended function, e.g., deliver anionic agents through lipid membranes.
  • hydrophilic moieties which comprise the hydrophilic domains of the polyplexes include polymers such as, for example, polyethers, such as poly(oxyalkylene glycol) (e.g., poly(oxy ethyl ene glycol) (PEG), or poly(oxypropylene glycol), etc.).
  • Other examples of hydrophilic moieties include polyheterocyclic polymers, such as poly(ethyloxazoline) and poly(methyloxazoline).
  • the mass ratio of the hydrophilic moieties to the cationic backbone moiety is from about 1:1 to about 40:1. Other hydrophilic moieties are described in greater detail below.
  • Polyplexes of the invention can be formed using a variety of co-polymers arranged and combined to form several different "architectures" suitable for cell delivery.
  • co-polymers include, for example, block co-polymers and random graft co-polymers, and may also include other chemical or biological constructs which are useful for cell delivery (e.g., peptides or other cellular ligands as described in the previous subsection).
  • Polyplexes can be formed using block co-polymers of the formula (I):
  • A is a hydrophilic moiety
  • B is a hydrophobic moiety
  • C is a cationic backbone moiety.
  • the block copolymers may also comprise one or more additional hydrophobic and/or hydrophilic moieties.
  • the polyplex of the invention is comprised of one or more copolymers of the formula (I).
  • the cationic backbone moiety of one or more copolymers of formula (I) interact with an anionic agent, as described above, to form the cationic domain of the polyplex.
  • the hydrophobic moieties of the copolymer(s) interact to form the transitional or hydrophobic domain and the hydrophilic moieties of the copolymers interact to form the hydrophilic (e.g., suface domain) of the polyplex.
  • the invention uses hydrophilic PEG chains grafted onto through hydrophobic moieties to cationic backbone moieties which evade the reticuloendothelial system.
  • the hydrophilic PEG polymer moieties also minimize serum effects and extend circulation.
  • hydrophobic moiety of the block co- polymer also generates a protective "hydrophobic shell" around the anionic agent (e.g., DNA) during polyplex formation.
  • co-polymers posed the disadvantage of having various chains grafted onto a cationic domain (e.g., poly-L-lysine) via a lysine ⁇ -amino group. These grafted chains introduced steric hindrance to DNA binding and limit the grafted co-polymer architecture.
  • polyplexes of the present invention are formed with co-polymers which reduce the amount of steric hindrance of the anionic agent by using block co-polymers having unmodified cationic domains (except, in certain embodiments, at the two terminal ends) which, thus, can be added or built onto (e.g., other blocks (domains, moieties) can be added on).
  • block architecture of copolymers.
  • Each block is synthesized by a sequential polymerization of appropriate monomers.
  • the initiation step involves the first block (block A) that has a functional group ready to start a polymerization of a monomer B for the second block (block B).
  • block A first block
  • block B second block
  • the second monomer C can be added and polymerization continued until completion.
  • the entire stepwise polymerization can be repeated any number of times until desired composition of block co-polymer is achieved.
  • Each block can then be modified by substituents to further modulate properties of polymers.
  • each individual block (domain), as designated by either a number or a letter, may have additional substituents as shown (Ri through Rn). These substituents may or may not be equal to each other R ⁇ . ⁇ R 2 - ⁇ R 3 ⁇ ... ⁇ Rn) in each individual domain.
  • block co-polymers to form polyplexes of the invention is shown in Figure 8.
  • the constituent chains of the block co-polymer can span the cationic, transitional (e.g., hydrophobic), and/or surface (e.g., hydrophilic) domains.
  • the block copolymers also can be designed in such a way as to create interactions, such as hydrophobic interactions, between the domains that may promote a "closed shell" upon polyplex formation with an anionic agent, such as DNA.
  • interactions such as hydrophobic interactions
  • Other chemical interactions that may be used to close the shell upon polyplex formation are electrostatic interactions, hydrogen bonding, Nan der Waals interactions, ionic interactions, and metal ion complexation.
  • Such interactions can stabilize the interactions between the cationic domain and the anionic agent, such that the cationic moieties assemble near the anionic agent due to the cooperative nature of interactions of closing the shell, and thereby forming the polyplex.
  • the properties of hydrophobic interactions may be modulated in this design by varying the ratios of hydrophobic monomer to initiator during polymerization. This design allows for selection of monomers with stronger or weaker hydrophobes.
  • block copolymers of the invention include those given in Table 1 above as well as block copolymers of the formula PEGl-20k-b/ ⁇ c&-(CysC 18 ) 8-12 -b/ ⁇ c£- (Lys) 10-140 , such as PEG5k-b/oc£-(CysC 18 ) 1 o-b/oc&-(Lys) 45 and PEG5k-bZoc ⁇ -(CysC 18 ) 1 o- b/oc£-(Lys) 1 0 .
  • PEGl-20k-b/ ⁇ c&-(CysC 18 ) 8-12 -b/ ⁇ c£- (Lys) 10-140 such as PEG5k-b/oc£-(CysC 18 ) 1 o-b/oc&-(Lys) 45 and PEG5k-bZoc ⁇ -(CysC 18 ) 1 o- b/oc£-(Lys
  • Polyplexes of the invention can also comprise copolymers which have been formed by the random graft method.
  • the copolymers synthesized by the random graft method are of the formula:
  • A is a hydrophilic moiety
  • B is a hydrophobic moiety
  • C is a cationic backbone moiety
  • n and x are values which can be selected such that the resulting polyplex is capable of performing its intended function (e.g., values of x and n may each range independently from 0 to 1000).
  • polymeric chains are grafted to amino groups on proteins, cationic polymers, or more specifically poly-L-lysine (e.g., 'C above) using activated esters.
  • activated esters The reaction of an activated ester produces an amide bond linked conjugate and, in effect, causes a net loss of charge on the conjugate. Random loss of positive charge can significantly weaken interactions with anionic agents, such as DNA.
  • anionic agents such as DNA.
  • chemistry that leads to charge preservation on the cationic domain may have a minimal impact on interactions with anionic agents, although the interaction will be affected by also by steric hindrance of grafted chains.
  • synthetic chemistries are selected which preserve charges on the cationic domain and produce secondary and tertiary amines, as well as potentially quaternary ammonium salts.
  • These amine species can bear a positive charge at physiological pH and, as a result, bind to anionic agents, e.g., polynucleotides, e.g., DNA.
  • anionic agents e.g., polynucleotides, e.g., DNA.
  • the impact of steric hindrance of grafted chains on polymer-DNA interactions can then be monitored by a fluorescence quenching assay.
  • the design of random graft polymers for use in polyplexes of the present invention is based, in part, on two principles. One is to preserve charge within the cationic domain. The second is to introduce one or more hydrophobic domains into the polyplex to stabilize the polyplex and to allow for interaction with hydrophobic formulants (e.g., penetration enhancers) which interact with these domains through hydrophobic interactions.
  • Hydrophobic formulants e.g., penetration enhancers
  • Ligand-mediated cell targeting also can be used by hydrophobic association of the ligand with the hydrophobic domain and/or a conjugate with an engineered hydrophobic domain.
  • partially hydrophobic conjugates also may be used since they possess moieties that preserve sufficient water solubility (since purely hydrophobic molecules are water insoluble).
  • These conjugates can be made up of two different types of grafts, hydrophilic moieties to maintain adequate water solubility ('A'), and hydrophobic moieties ('B') to introduce a domain with binding and micelle formation properties.
  • the polymer is designed by grafting two or more of these elements onto a cationic backbone moiety (e.g., a cationic polymer, 'C').
  • a suitable grafting element, or hydrophilic moiety for this approach is PEG, which promotes solubility and steric shielding.
  • Another suitable grafting element is any hydrophobic moiety, as described above, which may form domains with binding capabilities. These two or more types of grafting elements can then be randomly distributed along a cationic backbone moiety during the grafting step. As shown in Formula III below, these grafting elements can be simple or complex, and may have additional functionalities.
  • M and K are functional groups for attachment of polymer functional domains.
  • N is a terminal group.
  • Y is functional group for ligand attachment or, alternatively a terminal group.
  • the number of oxyethylene (EO) units in the hydrophilic domain is represented by c; b is represents the number of hydrophobic units in hydrophobic chain; x and n are number of hydrophilic and hydrophobic moieties attached to the cationic backbone moiety.
  • EO oxyethylene
  • polymers have been synthesized with multiple domains. However, as previously discussed, such polymers suffer (due to a large percent of substitution of amino groups on the cationic domain) from increased steric hindrance for DNA binding.
  • One novel strategy of the present invention reduces the percent modification by half and results in stronger polyplexes. It also allows one to vary the position of the hydrophobic moieties with respect to the cationic and hydrophilic moieties ( Figures 10a and 10b).
  • Such hydrophobic domains can be engineered onto cationic backbone moieties using several different methods. First, the hydrophobic moieties may be positioned between the cationic backbone and the hydrophilic moieties ( Figure 10a). Alternatively, they may be attached at the terminus of the hydrophilic domain, which then may be grafted onto a cationic backbone moiety ( Figure 10b).
  • any of these hydrophobic moieties can be made "more hydrophobic" by increasing the number of hydrophobic moieties per individual grafting element ( Figure 1 la vs b).
  • Such hydrophobic moieties also may incorporate natural and synthetic polymers, substituted and unsubstituted linear, branched, aliphatic, alkenyl, and alkynyl groups.
  • the hydrophobic moieties may also include heterocyclic and carbocyclic groups, as well as combinations of groups.
  • the hydrophobic moiety can be any moiety which allows the polyplex to perform its intended function.
  • the overall hydrophobicity of these conjugates can be modulated by changes in grafting densities as well as the substitution and chemical makeup of the hydrophobic moieties.
  • the polyplexes of the invention comprise copolymers formed by the graft method.
  • the copolymers synthesized by the graft method are represented by formula IV, below: wherein each A is an independently selected hydrophobic moiety, each B is an independently selected hydrophilic moiety, C is a cationic backbone moiety and m, n, s, v, and x which are selected such that the resulting polyplex is capable of performing its intended function (e.g., values of m, n, v, s and x may each range independently from 0 to 1000).
  • cationic backbone moieties examples include poly-L-lysine (PLL) polyethylenimine.
  • hydrophobic moieties include alkyl groups having from about 2 to about 80 carbon atoms, alkyl groups having from about 4 to about 40 carbon atoms, etc, cholesterol derivatives, hydroxybenzyl-amidine, biphenyl, cholic acid derivative Trigal-NH(CO)CH 2 Br lactose-(CO)-C 12 -Br picolyl-Cl, or chenodeoxy cholic acid-Br.
  • hydrophilic moieties ('A') include -( ⁇ -NH-PEG2-8k) 10-2 o, ( ⁇ -NH-C10-Igepal-CO-990) 2-10 , ( ⁇ -NH-Brij98) 7-20 , nd (- ⁇ -NH-CH 2 CH(OH)CH 2 O(PO) 30- so(EO) 80 - 15 oOCH 3 ) 5-3 o, TritonX-405-C 10 -Br PEG5k-C 12 -Br, Igepal-C 10 -Br, PEG0.75k- C 10 -Br C 18 -PEG4.4k-Br C 18 -PEG5k-C ⁇ 0 -Br N-(C ⁇ 0 -PEG2k)-N-(C 12 )-N-(COCH 2 I) PEG2k-C 10 -Br, and PEG-Epoxide.
  • polymers which the polyplexes of the invention may be comprised of include poly-L-Lysine-gr ⁇ t-( ⁇ -NH-C 10-PEG2k) 5-15 , poly-L-Lysine-gr ⁇ t ( ⁇ -NH-ClO-Triton X-405) 5-15 , PLL-gr ⁇ /t-( ⁇ -NH-C10-Igepal-CO-990) 2 .
  • the polymers of which the polyplexes of the invention are comprised are PLL10k-gra t-( ⁇ -NH-C10-PEG2k) 9 ; PLL10k-gr ⁇ t-( ⁇ -NH-C10-Triton X-405) 9 ; PLL9.4k-gr ⁇ t-( ⁇ -NH-Cl 0-Igepal-CO-990) 3 . 2 ; PLL9.4k-gra/t-( ⁇ -NH- Brij700) 2.8 ; PLL9.4k-gr t-( ⁇ -NH-C10-Brij700) 6 .
  • the invention provides various penetration enhancers, such as formulants and surfactants, which can be used in combination with polyplexes of the invention, or in combination with free (i.e., uncomplexed) anionic agents (e.g., free DNA), to deliver the anionic agents across lipid membranes and cellular boundaries.
  • penetration enhancers can be used in concert with nucleic acid, alone or with a polyplex formulation, to enhance expression of the nucleic acid.
  • penetration enhancer In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.
  • penetration enhancer The term “penetration enhancer,” “fonnulant” and “surfactant” are used interchangeably herein and refer to reagents which enhance delivery of anionic agents across cellular boundaries, alone or in conjunction with a polyplex of the invention.
  • the invention employs DHPC as a penetration enhancer.
  • penetration enhancers may interact with polyplex.
  • An equilibrium between the interacting formulant and the free formulant is established and is represented by the equilibrium constant, Ki .
  • Ki The equilibrium constant can be modulated, for example, by modifying the strength of hydrophobic domain present in the polyplex or by modifying the hydrophobic domain of the penetration enhancer molecule itself (if applicable).
  • the equilibrium also can be shifted depending on the structure of the pentration enhancer and the architecture of the polyplex and its constituent co-polymers and their hydrophobic moieties.
  • the formulation process may also be accomplished in a stepwise manner.
  • a penetration enhancer may be equilibrated with a copolymer followed by the addition of anionic agent. Stronger penetration enhancers may form stronger co-micelles with conjugate verses polyplex and may in effect release DNA from the polyplex. The DNA release is both conjugate and formulant dependent and can be monitored by a fluorescence DNA release assay.
  • the penetration enhancer will be released and will equilibrate between its polyplex bound form, free form, and cell surface (membrane) bound form, as shown in Figure 12.
  • the lipid bilayer membrane permeability changes, resulting in enhanced internalization, i.e., cellular uptake.
  • nucleic acid e.g., DNA
  • enhanced cellular uptake correlates with enhanced levels of expression.
  • the invention provides methods and compositions for enhancing delivery of anionic agents, e.g., polynucleotides, through cellular membranes, by combining the anionic agent, either in the form of a polyplex or in free form (e.g., free DNA), with a formulant, surfactant, or other penetration enhancer and contacting the resulting composition with the membrane.
  • anionic agents e.g., polynucleotides
  • free form e.g., free DNA
  • Suitable formulants or penetration enhancers for in vitro, ex vivo or in vivo administration of anionic agents (e.g., DNA) to a subject, such as an animal or human include, for example, non-ionic, ethyleneoxide/propyleneoxide formulants; fluorinated type formulants; non-ionic carbohydrate and polyol formulants; ionic negatively charged formulants; bile acids and their derivatives and salts; ionic, cationic and zwitterionic formulants; lipid derivatives; hydrophobes; and other formulants.
  • suitable non-ionic, ethyleneoxide/propyleneoxide type formulants or penetration enhancers include: Brij surfactants (e.g., Brij 30, Brij 35 (C12EO23), Brij 36, Brij 52, Brij 56, Brij 58, Brij 72, Brij 76, Brij 78, Brij 92, Brij 96, Brij 97 (C18-1- EO10), Brij 98, Brij 98/99 (C18-1-EO20), Brij 700 (C18E 100), Brij 721 (C18EO21), 18-1-EO20), Brij 97 (C18-1-EO10 etc.
  • Brij surfactants e.g., Brij 30, Brij 35 (C12EO23), Brij 36, Brij 52, Brij 56, Brij 58, Brij 72, Brij 76, Brij 78, Brij 92, Brij 96, Brij 97 (C18-1- EO10), Brij 98, Brij 98/99 (C
  • fluorinated type formulants examples include Zonyl FSN 100, Zonyl FSA, and mixtures thereof.
  • non-ionic, carbohydrate or polyol type formulants examples include D- glucopyranosides (such as n-decyl- ⁇ -, n-dodecyl- ⁇ -, n-heptyl- ⁇ -, n-octyl- ⁇ -, phenyl- ⁇ -, n-hexyl- ⁇ -, methyI-6-O-N-heptylcarbonyI- ⁇ -, n-octyl- ⁇ -, n-octyl- ⁇ -, n-octyl-racemic mixture, phenyl- ⁇ -), D-1-thioglucopyranosides (such as n-decyl- ⁇ -, n-dodecyl- ⁇ -, n- heptyl- ⁇ -, n-hexyl- ⁇ -, n-octyl- ⁇ -), D-galactopyranosides (such as n-dode
  • Examples of ionic (negatively charged or anionic) type formulants include: N- lauryl sarcosine salt, linolic acid salt, cholesteryl hydrogen succinate, DSPE-PEG, bile acids (e.g., natural and synthetic bile acids, conjugated bile acids, mixtures, and salts), hydrotropes (e.g., 8-(5-carboxy-4-hexyl-cyclohex-2-enyl)-octanoic acid), embonic acid, hydroxy cholic acid sodium salt, linoleic acid sodium salt, N-lauryl sarcosine sodium salt, oleic acid sodium salt, sodium lauryl sulfate and mixtures thereof.
  • bile acids e.g., natural and synthetic bile acids, conjugated bile acids, mixtures, and salts
  • hydrotropes e.g., 8-(5-carboxy-4-hexyl-cyclohe
  • bile acids include, but are not limited to natural and synthetic bile acids, salts, and derivatives thereof.
  • examples of bile acids also include lithocholate, deoxycholate, glycodeoxycholate, taurodeoxycholate, chenodeoxycholate, glycochenodeoxycholate, taurochenodeoxycholate, ursodeoxycholate, glycoursodeoxycholate, tauroursodeoxycholate, cholate, glycocholate, taurocholate, ursocholate, glycoursocholate, or tauroursocholate.
  • Examples of ionic, cationic or zwitterionic type formulants include cetyl pyridinium chloride monohydride, cetyltrimethylammonium bromide, DOCUSATE, N,N-dimethylheptylamine-N-oxide, N,N-dimethylnonylamine-N-oxide, N,N- dimethyloctadecylamine-N-oxide, 2-heptadecylimidazole, 2-undecylimidazole, and mixtures thereof.
  • lipid derivatives useful as permeation enhancers include, for example, l,2-diheptanoyl-s «-glycero-3-phosphocholine, and 1,2-dioctanoyl- ⁇ -glycero- 3-phosphocholine, and mixtures thereof.
  • alcohols include, but are not limited to, aliphatic alcohols such as ethanol, N-propanol, isopropanol, butyl alcohol, and acetyl alcohol.
  • glycols include, but are not limited to, glycerine, propyleneglycol, polyethyleneglycol and other low molecular weight glycols such as glycerol and thioglycerol.
  • Acetates include, for example, acetic acid, gluconol acetate, and sodium acetate.
  • Hypertonic salt solutions include sodium chloride solutions and other pharmaceutically acceptable salt solutions.
  • Heparin-antagonists include quaternary amines, such as prolamine sulfate.
  • Cyclooxygenase inhibitors such as sodium salicylate, salicyclic acid, and non-steroidal anti-inflammatory drugs (NSAIDS) such as indomethacin,. naproxin, diclofenac are also included as penetration enhancers.
  • NSAIDS
  • substances useful for use as permeation enhancers include: ⁇ - carotene, chloroquine diphosphate,, N-decanoyl-N-methylglucamine, DSPE-PEG, menthol, ny statin, N-octanoyl-N-methylglucamide, natural and synthetic saponins.
  • Still other suitable formulants (penetration enhancers) for use in the invention include include alcohols, glycols, heparin antagonists, cyclooxygenase inhibitors, hypertonic salt solutions, and acetates.
  • Such penetration enhancers, formulants and detergents can be administered in conjunction with the anionic agent to be delivered (e.g., in the form of a polyplex of the invention or in free form), before the anionic agent, or after the anionic agent.
  • Advantageous penetration enhancers include N 1 -(cholic acid amide)-4,7,10- trioxo- 1,13 -tridecanediamine, ⁇ -(chenodeoxycholic acid amide)-4,7,10-trioxo-l,13- tridecanediamine, and N-Chenodeoxycholyl-2-aminoethyl-phosphonic acid monopotassium salt.
  • Surfactants are chemical agents which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of anionic agents or polyplexes of the invention interacting with such surfactants through cellular membranes is enhanced.
  • cell membrane permeability is significantly increased.
  • cellular uptake of, e.g., polyplexes can be increased. The increased cellular uptake can be observed by fluorescence histology as described in the Examples herein.
  • Suitable surfactants for use in the invention include, for example, bile salts and fatty acids.
  • Other suitable surfactants include sodium lauryl sulfate, polyoxyethylene-9- lauryl ether and polyoxyethylene-20-cetyl ether (see, Lee et al. Crit. Rev. Ther. Drug Carrier Systems, 1991, p. 91); and perfluorochemical emulsions, such as FC-43 (Takahashi, et al. J. Pharm. Pharmacol. 1988 40:252).
  • surfactants include, for example, sodium dodecyl sulfate (SDS), lysolecithin, polysorbate 80, nonylphenoxypolyoxyethylene, lysophosphatidyl choline, polyethyleneglycol 400, polysorbate 80, polyoxyethylene ethers, polyglycol ether surfactants and DMSO.
  • SDS sodium dodecyl sulfate
  • lysolecithin polysorbate 80
  • nonylphenoxypolyoxyethylene lysophosphatidyl choline
  • polyethyleneglycol 400 polysorbate 80
  • polyoxyethylene ethers polyglycol ether surfactants
  • DMSO DMSO
  • Still other suitable surfactants include ZWITTERGENT 3-14 detergent, CHAPS (3-[(3-Cholamidopropyl)dimethylammonio] 1 -propanesulfonate hydrate), Big CHAP, Deoxy Big CHAP, TRITON-X-100 detergent, C12E8, Octyl-B-D-Glucopyranoside, PLURONIC-F68 detergent, TWEEN 20 detergent, and TWEEN 80 detergent.
  • Suitable fatty acids and their derivatives which can be used as penetration enhancers according to the present invention include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid) myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaproate, tricaproate, monoolein (1-monooleolyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaparate, l-dodecylazacycloheptan-2- one, acylcartinines, acycl cholines, Cj.io alkyl esters thereof (e.g., methyl, isopropyl, and t-butyl), and mono- and di- glycerides thereof (e.g., oleate, laurate, caproate, myristate, palmiate, stea
  • bile salts includes any of the naturally occurring components of bile as well as any synthetic derivatives thereof.
  • examples of bile salts include, for example, cholic acid (or its pharmaceutically acceptable salts, e.g., sodium cholate), dehydrocholic acid, sodium dehydrocholate, deoxycholic acid, sodium deoxycholate, glucholic acid, sodium glucholate, glycholic acid, sodium glycocholate, glycodeoxycholic acid, sodium glycodeoxycholate, taurocholic acid, sodium taurocholate, taurodeoxycholic acid, sodium taurodeoxycholate, chenodeoxycholic acid, sodium chenodeoxycholate, ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydrofusidate (STDHF), sodium glycodihydrof sidate and polyoxyethylene-9-lauryl ether (PO
  • Suitable bile acids and derivatives include natural and synthetic bile acids, their organic and inorganic salts, and conjugated bile acids and their organic and inorganic salts. Still further examples include bigchap, chaps, chapso, chenodeoxycholic acid, cholic acid methyl ester, cholesteryl hydrogen succinate, cholesteryl sulfate potassium salt, dehydrocholic acid, dehydrocholic acid sodium salt, deoxycholic acid (sodium deoxycholate), deoxy-bigchap, fusidic acid, glucholic acid(sodium glucholate), glycholic acid (sodium glycholate), glycodeoxycholic acid(sodium glycodeoxycholate), lithocholic acid, sodium tauro-24, 25-dihydro- fasidate(STDHF), sodium glycodihydrofusidate, taurocholic acid sodium salt, taurodeoxycholic acid(sodium taurodeoxycholate), taurolit
  • Chelating agents which can be used in the present invention include compounds which remove metallic ions from solution by forming complexes with the metallic ions, resulting in absorption of the anionic agent, e.g., polynucleotides or polyplexes, through cellular membranes. Chelating agents also provide the advantage of serving as DNAase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr. 1993, 613, 315).
  • Suitable chelating agents for use in the invention include, for example, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5- methoxysalicytate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N- amino acyl derivatives of ⁇ -diketones (enamines).
  • EDTA disodium ethylenediaminetetraacetate
  • citric acid e.g., citric acid
  • salicylates e.g., sodium salicylate, 5- methoxysalicytate and homovanilate
  • N-acyl derivatives of collagen e.g., laureth-9 and N- amino acyl derivatives of ⁇ -diketones (enamines).
  • Non-chelating, non-surfactant, formulants which can be used in the present invention include compounds which have insignificant activity as chelating agents or as surfactants, but nonetheless enhance absorption of oligonucleotides through membranes (Muranishi, Crit. Rev. in Therapeutic Drug Carrier Systems 1990, 7:1).
  • This class of formulants includes, for example, unsaturated cyclic ureas, 1- alkyl and 1-alkenylazacyclo-alkanone derivatives and non-steroidal anti-inflammatory agents such as dichlofenac sodium, indomethacin, and phenylbutazone.
  • agents which can be used as formulants to enhance uptake of polynucleotides or polyplexes at the cellular level include cationic lipids (such as lipofectin, U.S. 5,705,188), cationic glycerol derivatives and polycationic molecules, such as polylysine (WO 97/30731).
  • cationic lipids such as lipofectin, U.S. 5,705,188
  • cationic glycerol derivatives such as polycationic molecules, such as polylysine (WO 97/30731).
  • n is an integer from 1 to 10
  • Xi is a cholic acid group, a deoxycholic acid group, or an analog or derivative thereof
  • X 2 and X 3 are each independently selected from the group consisting of a cholic acid group, a deoxycholic acid group, and a saccharide group. At least one of X 2 and X 3 is a saccharide group.
  • saccharide groups include, for example, pentose monosaccharide groups, hexose monosaccharide groups, pentose-pentose disaccharide groups, hexose-hexose disaccharide groups, pentose-hexose disaccharide groups, and hexose-pentose disaccharide groups.
  • the penetration enhancer has the following formula:
  • Xi and X 2 are selected from the group consisting of a cholic acid group and a deoxycholic acid group and X 3 is a saccharide group.
  • WO 98/35554 includes other penetration enhancers of the invention.
  • Non-Chemical Penetration Enhancers Sound waves also can be employed in conjunction with the invention to facilitate uptake of polynucleotides and polyplexes by cells.
  • Polyplex and DNA formulations of the present invention can be administered to cells in vitro or in vivo (i.e., to a subject, such as a mammal) using a variety of suitable techniques known in the art, such as injection, oral administration and, in some cases, topical delivery.
  • the invention pertains, at least in part, to a method of delivering an anionic agent through a lipid membrane.
  • the method includes contacting the anionic agent with a delivery enhancing formulation, allowing a polyplex to form; and contacting the lipid membrane with a penetration enhancer, such that upon contact of the polyplex with the lipid membrane, the anionic agent is delivered through the membrane.
  • the formulation comprises a cationic backbone moiety, a hydrophobic moiety, and a hydrophilic moiety;
  • the invention also pertains to a method for enhancing expression of a nucleic acid in a cell.
  • the method includes contacting the nucleic acid with a delivery enhancing formulation (as described above), allowing a polyplex to form, and contacting the membrane of the cell with a penetration enhancer, such that upon contact of the polyplex with the membrane of the cell, the nucleic acid is internalized into the cell and expression of said nucleic acid is enhanced.
  • a delivery enhancing formulation as described above
  • “enhanced” includes any expression of the nucleic acid that is greater than that observed by administering the DNA to a subject or a culture of cells with out any a polyplex or penetration assistance.
  • the delivery enhancing formulation is generally comprised of copolymers, as described above, with a variety of architectures which are allow the polyplex to perform its intended function, e.g., deliver an anionic agent across a lipid membrane, e.g., a cellular boundary, e.g., a cellular membrane, a nuclear membrane, an endosomal membrane, etc.
  • the delivery enhancing formulation may be a copolymer which comprises a cationic backbone moiety, a hydrophobic moiety, and a hydrophilic moiety.
  • the polyplex comprises a polymer which includes a polylysine back bone moiety, a hydrophobic moiety, and a poly(oxyethylene glycol) hydrophilic moiety.
  • cultured cells can be incubated with the the compositions of the invention in an appropriate medium under conditions conducive to uptake of the compositions by the cells.
  • the compositions also can be delivered ex vivo to cells or tissues which have been removed from an organism, incubated the compositions of the invention, and then returned to the organism.
  • compositions of the invention can be administered to a subject in a pharmaceutically acceptable vehicle.
  • pharmaceutically acceptable carrier is intended to include any physiologically acceptable carrier for stabilizing the compositions invention for administration in vivo, including, for example, saline and aqueous buffer solutions, solvents, dispersion media, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • compositions of the invention may be administered in vivo by any suitable route of administration.
  • the appropriate dosage may vary according to the selected route of administration.
  • the compositions are preferably injected intravenously in the form of a solution.
  • Other suitable routes of administration include intravascular, subcutaneous (including slow-release implants), topical and oral. Appropriate dosages may be determined empirically, as is routinely practiced in the art.
  • subject include organisms and cells which can be advantageously treated or altered through interaction with the polyplexes or anionic agents of the the invention.
  • the term “subject” includes protists,birds, reptiles, monera, bacteria, and preferrably,mammals, such as dogs, cats, horses, pigs, bears, cows, sheep, goats, rats, mice, hamsters, and, primates, such as chimpanzees, gorillas, and humans.
  • the subject is suffering from a genetic or an acquired disorder. Examples of disorders which the subject may be suffering from include, but are not limited to, 68.
  • the method of claim 57 wherein the subject is treated for a disorder selected from the group consisting of hepatitis, inflammatory diseases, hemophilia, metabolic deficiencies, metabolic disorders, immune rejection of transplanted tissue, infections by invading pathogens, tissue trauma, ischemia, lipid metabolism disorders, cholesterolimia, hypercholesterolimia, peripheral and central nervous system disorders and regeneration, obesity, allergies, allergic rhinitis, asthma, Gaucher's disease, epilepsy, Parkinson's disease, ocular diseases, elevated intraocular pressure, cancer, skin disorders, and alopecia.
  • a disorder selected from the group consisting of hepatitis, inflammatory diseases, hemophilia, metabolic deficiencies, metabolic disorders, immune rejection of transplanted tissue, infections by invading pathogens, tissue trauma, ischemia, lipid metabolism disorders, cholesterolimia, hypercholesterolimia, peripheral and central nervous system disorders and regeneration, obesity, allergies, allergic rhinitis, asthma, Gaucher's disease, epilepsy, Parkinson's disease
  • treatment includes the diminishment or alleviation of at least one symptom associated or caused by the state, disorder or disease being treated.
  • treatment can be diminishment of one or several symptoms of a disorder or complete eradication of a disorder.
  • composition includes preparations suitable for administration to mammals, e.g., humans.
  • pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
  • phrases "pharmaceutically acceptable carrier” is art recognized and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present invention to mammals.
  • the carriers include liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in canying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each earner must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, ⁇ -tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin
  • Formulations of the present invention include those suitable for oral, nasal, topical, transdermal, buccal, sublingual, rectal, vaginal and/or parenteral administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a earner material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.
  • Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in- water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient.
  • a compound of the present invention may also be administered as a bolus, electuary or paste.
  • the active ingredient is mixed with one or more pharmaceutically acceptable caniers, such as sodium citrate or dicalcium phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pynolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol and glycerol mono
  • compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres.
  • compositions may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions which can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluent commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluent commonly used in the art, such as, for example, water or other solvents, solubilizing agents and e
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, macrocrystalline cellulose, aluminum metahydroxide, bentonite, agar- agar and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, macrocrystalline cellulose, aluminum metahydroxide, bentonite, agar- agar and tragacanth, and mixtures thereof.
  • Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable noninitating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • suitable noninitating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
  • Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
  • the ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the active compound in a polymer matrix or gel.
  • compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous earners examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride
  • the absorption of the drug in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drag in an oil vehicle.
  • Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drag in liposomes or microemulsions which are compatible with body tissue. The preparations of the present invention may be given orally, parenterally, topically, or rectally. They are of course given by forms suitable for each administration route.
  • compositions or capsule form are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories.
  • Systemic administration is prefened.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, infraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • systemic administration means the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.
  • the anionic agent, or cationic polymeric agent of the present invention which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular anionic agent or cationic polymeric agent of the present invention employed, the polyplex or penetration enhancing agent used, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses anionic agent of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable daily dose of anionic agent of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect.
  • Such an effective dose will generally depend upon the factors described above.
  • the effective daily dose of the polyplex of the invention may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • a compound of the present invention While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical composition.
  • compositions of the invention may comprise compounds which may contain a basic functional group, such as amino or alkylamino, and are, thus, capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids.
  • pharmaceutically acceptable salts is art recognized and includes relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed.
  • Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and Iaurylsulphonate salts and the like. (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J Pharm. Sci. 66:1-19).
  • the compounds which can be incorporated into the compositions of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases.
  • pharmaceutically acceptable salts in these instances includes relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine.
  • Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like.
  • Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.
  • pharmaceutically acceptable esters refers to the relatively non-toxic, esterified products of compounds, such as, for example, penetration enhancers of the present invention. These esters can be prepared in situ during the isolation and purification, or by separately reacting the compound in its free acid form or hydroxyl with a suitable esterifying agent. Carboxylic acids can be converted into esters via treatment with an alcohol in the presence of a catalyst.
  • Hydroxyls can be converted into esters via treatment with an esterifying agent such as alkanoyl halides.
  • esterifying agent such as alkanoyl halides.
  • the term also includes lower hydrocarbon groups capable of being solvated under physiological conditions, e.g., alkyl esters, methyl, ethyl and propyl esters. (See, for example, Berge et al., supra.) This invention is illustrated further by the following examples which should not be construed as further limiting the subject invention.
  • the contents of all references and published patent applications cited throughout this application are hereby incorporated by reference.
  • compositions (e.g., polyplexes) of the present invention can be used to deliver a variety of nucleic acids to cells, e.g., to be expressed.
  • Polyplexes can contain more than one copy of the same polynucleotide or one or more different polynucleotides.
  • polynucleotide is intended to include any single or double-stranded DNA or RNA molecule, or any analogue thereof.
  • the polynucleotide is a gene encoding a desired therapeutic protein (e.g., a blood clotting factor, growth factor, enzyme, antagonist, immunogen, cell surface receptor or any other beneficial protein).
  • a desired therapeutic protein e.g., a blood clotting factor, growth factor, enzyme, antagonist, immunogen, cell surface receptor or any other beneficial protein.
  • the gene is generally in a form suitable for expression, processing and secretion by the target cell.
  • the gene must be operably linked to appropriate genetic regulatory elements which are functional in the target cell.
  • Such regulatory sequences include, for example, promoter sequences which drive transcription of the gene.
  • Suitable promoters include a broad variety of viral promoters, such as SV40 and CMV promoters.
  • the gene may also include appropriate signal sequences which provide for trafficking of the encoded protein to intracellular destinations and/or extracellular secretion.
  • the signal sequence may be a natural sequence of the protein or an exogenous sequence.
  • regulatory sequences required for gene expression, processing and secretion are art-recognized and are selected to direct expression of the desired protein in an appropriate cell.
  • regulatory sequence includes promoters, enhancers and other expression control elements. Such regulatory sequences are known and discussed in Goeddel, Gene expression Technology: Methods in Enzymology, p. 185, Academic Press, San Diego, CA (1990).
  • the gene can be contained in an expression vector such as a plasmid or a transposable genetic element along with the genetic regulatory elements necessary for expression of the gene and secretion of the gene-encoded product.
  • the polynucleotide is an antisense polynucleotide (DNA or RNA), or is a gene which is transcribed into an antisense RNA (e.g., a ribozyme).
  • Antisense polynucleotides can be chemically synthesized using standard techniques well known in the art. For example, various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See e.g., Itakura et al, U.S. Patent No. 4,598,049; Caruthers et al, U.S. PatentNo. 4,458,066; and Itakura, U.S. Patent Nos. 4,401,796 and 4,373,071, incorporated by reference herein).
  • polynucleotides When administered in vivo, synthetic and natural polynucleotides are subject to degradation by exo- and endonucleases in a manner equivalent to any cellular nucleic acid. Accordingly, these polynucleotides can be chemically modified to provide substantial nuclease resistance.
  • chemically modified polynucleotides include, for example, phosphorothioate polynucleotides, in which one of the phosphate oxygens is replaced by a sulfur atom (See e.g., U.S. Patent No: 5,262,530, the teachings of which are incorporated by reference herein).
  • Phosphorothioates may be synthesized using automated techniques employing either phosphoramidite or phosphonate chemistries.
  • modified polynucleotides with increased stability include, for example, nonionic DNA analogs, such as alkyl- or arylphosphonates, in which the charged phosphate oxygen is replaced by an alkyl or aryl group (see e.g., U.S. Patent No: 4,469,863, the teachings of which are incorporated by reference herein), and alkylphosphotriesters, in which the charged oxygen moiety is alkylated (See e.g., U.S. Patent No: 5,023,243 and European Patent No: 092,574, the teachings of which are incorporated by reference herein). Both of these DNA analogs can be prepared by automated solid-phase synthesis using commercially available reagents.
  • nonionic DNA analogs such as alkyl- or arylphosphonates, in which the charged phosphate oxygen is replaced by an alkyl or aryl group
  • alkylphosphotriesters in which the charged oxygen moiety is alkylated
  • genetic markers such as, luciferase gene, ⁇ -galactosidase gene, hygromycin resistance, neomycin resistance, green fluorescent protein (GFP) or chloramphenicol acetyl transferase.
  • nucleic acids include sequences which encode proteins such as low density lipoprotein receptors, coagulation factors, suppressors of tumors, cytokines, angiogenesis factors, tumor antigens, immune modulators, major histocompatibility proteins, antioncogenes, pl6, p53, thymidine kinase, IL2, IL4, IL10, or TNF ⁇ . Still other examples include nucleic acids which encode for viral proteins, bacterial proteins, cell surface markers, HIV antigens, HIV p24 antigens, HSVgD antigens, HBVS antigens.
  • the nucleic acid incorporated into the polyplex of the invention also can be RNA, for example, a sense RNA, an antisense RNA, or a ribozyme.
  • Tris (2-carboxyethyl) phosphine hydrochloride (TCEP»HCL) was purchased from Pierce Chemical Co. (Rockford, IL).
  • CM hydrophobic interaction column
  • Polyethylene glycol (PEG) amino 5k (MW5254; Substitution: 98% (1H NMR), 98.2% (titration)) and Polyethylene glycol (PEG) epoxides 2K (M n 1554; M w /M n 1.044 (GPC)), 3K (M n 2696; M w /M n 1.035 (GPC)), and 5K (M n 5231; M w /M n 1.017 (GPC)) were purchased from Shearwater Polymers, Inc. (Huntsville, AL). The PEG amino 5k was dried in vacuo at 40°C. Acrylonitrile was purchased from Avacado Research Chemicals, Ltd., Lancaster, England.
  • L-cysteine, 1- bromooctadecane, and LiOH «H2 ⁇ were purchased from Aldrich Chemical Co. (Milwaukee, WI). Potassium Hydroxide and tetrahydrofuran (THF) were purchased from VWR Scientific Products, West Chester, PA. and double distilled from sodium benzophenyl ketal. Bis(trichloromethyl)carbonate (friphosgene) and N- ⁇ -Z-L-lysine were purchased from Fluka Chemical Corp., Milwaukee, WI.
  • Plasmid DNA pCMVb, Clontech, Palo Alto, CA and pCMV-Luciferase was prepared by BIO 101 (San Diego, CA). Plasmid DNA preparation contained more than 90% covalently closed circular DNA as determined by agarose gel electrophoresis.
  • Brij 700 (C18EO100), Brij 721 (C18EO21), Brij35 (C12EO23), Brij 98/99 (C18-1-EO20), Brij 97 (C18-1-EO10), Polyoxyethylene Ether W-l, Triton X-405, Triton X-100, N-Lauryl Sarcosine, sodium salt, n-Dodecyl- ⁇ -D-glucopyranoside, n-Heptyl- ⁇ -D-1-thioglucopyranoside, n-octyl- ⁇ - D-galactopyranoside, n-Decyl- ⁇ -D-maltopyranoside, Taurocholic Acid sodium salt, Saponin (from Quilaja Bark), Nystatin, and Chloroquine, diphosphate were purchased from Sigma Chemical Co.
  • N-octyl-N-methyl glucamide, n-Octyl- ⁇ -D- glucopyranoside, n-Octyl-D-glucopyranoside, n-Octyl- ⁇ -D-1-thioglucopyranoside, N- Decanoyl-N-methyl glucamine, N,N-Dimethylnonylamine-N-oxide, N,N- Dimethyloctadecylamine-N-oxide, ⁇ -Carotene, and Cholesterol PEG-900 were purchased from Fluka Chemical Co. (Milwaukee, WI).
  • Igepal CO-990, 2- Undecylimidazole, and Ethylenediamine tetrakis(propoxylate-b/ ⁇ c&-ethoxylate tetrol) were purchased from Aidrich Chemical Co. Inc. (Milwaukee, WI).
  • l,2-Diheptanoyl-,y «- glycero-3-phophocholine and l,2-Dioctanoyl-s «-glycero-3-phosphocholine were purchased from Avanti Polar-Lipids, Inc. (Alabaster, AL).
  • Zonyl FSN 100 and Zonyl FSA were purchased from Dupont Corp. (Wilmington, DE).
  • Phenyl- ⁇ -D- glucopyranosidesides and n-Octyl- ⁇ -D-glucopyranosides were purchased from Pfanstiehl Laboratories, Inc., Waukegan, II.
  • N-Heptyl- ⁇ -D-glucopyranosides were purchased from Calbiochem (La Jolla, CA).
  • N-Hexyl- ⁇ -D-glucopyranosides, n-Hexyl- ⁇ -D-1-thioglucopyranosides were purchased from Toronto Research Inc. (Ontario, Canada).
  • Linolic Acid, sodium salt and 2-Heptadecylimidazole were purchased from TCI America, Inc. (Portland, OR).
  • Cholesteryl Hydrogen Succinate was purchased from ICN Biomedicals, Inc. (Aurora, OH).
  • DSPE-PEG was purchased from Shearwater Polymers Inc. (Huntsville, AL).
  • Tetronic 908 was purchased from BASF (Mount Olive, NJ).
  • bile acids were purchased from the following suppliers: Lithocholic acid [434-13-9], Chenodeoxycholic Acid, Glycochenodeoxy cholic acid, sodium salt [16564-43-5], Deoxycholic acid [88-44-3], and Taurochenodeoxycholic acid, sodium salt [6009-98-9] were purchased from Sigma Chemical Co (St. Louis, MO). Glycodeoxycholic acid, sodium salt, Ursodeoxycholic acid [128-13-2], and Ursocholate were purchased from Fluka Chemical Corp. (Milwaukee, WI). Taurocholic acid, sodium salt [145-42-6], CHAPS [75621-03-3], and CHAPSO [82473-24-3] were purchased from Aldrich Chemical Co., Inc. (Milwaukee, WI). Cholic Acid [81-25-4] was purchased from Avacado Research Chemicals, Ltd. (Lancanster, England).
  • Polynucleotide ca ier complexes were prepared by rapidly adding an equal volume of plasmid DNA to a volume of the copolymer.
  • DNA (2x) was prepared in water and copolymers were dissolved in the 2x diluent before mixing.
  • Polynucleotide carrier complex concentrations are reported by DNA content and were 10 ⁇ g/ml unless otherwise noted.
  • Polynucleotide canier complexes were fonnulated at room temperature by rapidly mixing 500 ⁇ L of DNA (2x) and 500 ⁇ L of copolymer stock solution. Final DNA concentration was 50 ⁇ g/mL at a charge ratio of 1.0 (+/-) in 150 mM NaCl. Each polynucleotide carrier complex solution was divided into five 200 ⁇ L aliquots and incubated at room temperature for 30 minutes. Anionic molecules were added to the polyplex aliquots in increasing amounts (charge ratio 1, 4, 7, 10, and 100 per phosphate group). The samples were then incubated for 20 hours and analyzed on agarose gel (0.6%).
  • Tris-borate EDTA urea gels were obtained through Novex (San Diego, CA). The gels were run in IX TBE buffer. The samples were mixed with an equal volume of sample buffer containing 40% sucrose, 0.1% methyl green dye (Sigma Chemical Co., St. Louis, MO) 7.2 M urea in 1 x TBE. The gels were run at 180 volts with polarity reversed for approximately 2 hours, stained with coomassie brilliant blue, and photographed.
  • Carbon-coated copper grids with formvar support film (Tel Pella, Inc., Redding, CA) were glow-discharged for 30 seconds just prior to sample preparation.
  • Samples were negatively stained with uranyl acetate by one of the following methods: 1) the grid was floated on a 15 ⁇ L droplet containing polyplexes (at 10 ⁇ g/ml unless otherwise stated) for three minutes, then wicked to filter paper. The grid was washed 2x by placing on a distilled water droplet for 15 seconds followed by thoroughly removing liquid by wicking to filter. 2) The grid was floated on a droplet containing equal volumes of sample and 1.5% uranyl acid stain (1 minute) followed by two washes with water. The grids were examined under a Zeiss EM 10b microscope at 10,000x and 40,000x magnification.
  • Synthesis, purification, and characterization of grafting elements can be performed as follows.
  • the synthesis of grafting elements with a cholesterol nucleus is shown in Scheme 1. The synthesis was accomplished in three steps starting with cyanoethylation of cholesterol, followed by catalytic reduction of cyanoethyl derivative, and finally bromoacetylation of 3 -aminopropyl- ⁇ -cholesterol ether.
  • 2-Cyanoethyl-O- ⁇ -cholesterol ether (2.02 g, 4.43 mmol) and slurry of Raney- Nickel catalyst (0.65 g) were added to a 250 mL glass hydrogenation vessel containing a solution of NaOH (20 mg) in 80 mL of 95% ethanol.
  • the mixture was placed in a Parr Hydrogenation apparatus under hydrogen (50 psi) and shaken overnight with gentle heating.
  • the catalyst was removed by vacuum filtration and rinsed with 95% ethanol.
  • the solvent was evaporated in vacuo, and the residue dissolved in water.
  • the aqueous layer was extracted with CH2C1 2 .
  • 1,10-Dibromodecane (6.0 g, 20 mmol) was dissolved in acetonitrile (10 mL) followed by addition of pyridine (0.36 g, 4.55 mmol). The resulting solution was refluxed for 5 hours. After evaporation of the solvent in vacuo, the residue was purified by flash chromatography (silica gel, EtOAc/HAc/MeOH/H 2 O, 12:3:4:4, v/v/v/v) to give 1.64 g (95.2%) of a pink solid.
  • the N-(l l-bromoundecanoyl)- ⁇ -lactosylamide was synthesized from ⁇ -Lactose in two steps.
  • the first step involved formation of ⁇ -lactosyl amine that was converted to 11-bromoundecanoyl amide in the second step.
  • the final product was used to prepare co-polymers containing terminal galactose as a potential ligand.
  • Lactosylamine ⁇ -Lactose monohydrate (3.60 g, 10 mmol) was dissolved in 25 mL of concentrated aqueous ammonium hydroxide (16 M) to form a 0.4 M of ⁇ -Lactose solution.
  • ammonium bicarbonate was added to form a 0.4 M solution.
  • the resulting solution was heated at 33 °C for 3 days. Then the solvent was removed in vacuo.
  • the crude product was repeatedly dissolved in water and water was evaporated in vacuo. The entire process was repeated eight times (8 x 50 mL) to remove remaining ammonium salts.
  • Lactosylamine (0.99 g, 2.9 mmol) and Br(CH 2 ) 10 COONHS (1.05 g, 2.9 mmol) were added to DMF (20 mL). The resulting suspension was stined at room temperature for 3 days. Then the solvent was removed in vacuo and the yellow solid was recrystalized from MeOH/H 2 O (10:1, v/v) to give 0.44 g (26%) of a white product.
  • triantennary galactose ligand was synthesized and its structure is shown below. This ligand-amine was further converted to bromoacetyl derivative that was used for synthesis of grafted co-polymers.
  • FAB-MS calculated for M+l, 1443.7; found for CHNO, M+l, 1443.
  • ESP-MS M+l, 1443.2.
  • trigalactose-ligand-amine (0.36 g, 0.25 mmol) was lyophihzed from H 2 O, then dissolved in 20 mL of MeOH, and combined with BrCH 2 COONHS (0.624 g, 2.63 mmol). Triethylamine (37.6 ⁇ l, 0.27 mmol) was added and the solution was stined overnight. The solvent was removed in vacuo and the residue purified by Sephadex G-25 column with 0.05 M Acetic Acid in 30%
  • Bile acids are transported into hepatocytes via system of protein receptors/transporters that are distinct from the ASGPr. Bile acids enter hepatocytes via a non-endocytic pathway, and therefore, can serve as a possible ligand for targeted delivery to liver. Derivatives of bile acids that can be grafted on cationic polymers were then prepared and are described below in Scheme 3.
  • the grafting element containing cholic acid was prepared in two steps starting with methyl cholate as shown in Scheme 3.
  • the final product, iodoacetamide derivative was used for grafting without further purification.
  • the polymeric products obtained in such procedure were purified by standard procedures and are described in later sections.
  • Scheme 3 depicts the synthesis of grafting elements containing bile acid
  • Scheme 3A depicts the synthesis of N -(Iodoacetamide)-N -(Cholic Acid Amide)-4,7, 10-trioxo- 1,13 -tridecanediamine)
  • Scheme 3B depicts the synthesis of N 1 - (Bromoacetamide)-N -(Chenodeoxy Cholic Acid Amide)-4,7, 10-trioxo- 1,13- tridecanediamine.
  • Iodoacetamide was prepared as described earlier for bromoacetamide derivative.
  • Iodoacetamide was prepared as described for iodoacetamide of chenodeoxy acid derivative above. Yield 860 mg (94 %). The product was used for a next step without further purification.
  • N-Hydroxysuccinimide (2.93 g, 25.5 mmol) was added to the solution of Chenodeoxycholic acid (5 g, 12.7 mmol) in 200 mL of freshly distilled THF and stirred for 2.5 hours at room temperature. After 30 minutes of stining white precipitate of DCU was formed and later removed by filtration. The solution was evaporated in vacuo to give the crude active ester as a white solid. The crude active ester was dissolved in 150 mL of CHC1 3 and washed with saline/brine solution (0.1 N Na 2 CO 3 , 5 M NaCl, 3X 150 mL). The organic layer was separated and dried over anhydrous Na 2 SO 4 .
  • 2-Aminoethylphosphonic acid (0.30 g, 2.4 mmol) was dissolved in 5 mL of aqueous solution of potassium hydroxide (0.27g, 4.8 mmol) and lyophihzed to obtain 2- aminoethylphosphonic acid dipotassium salt as a colorless glass.
  • This dipotassium salt was dissolved in 20 mL of MeOH and added to the 20 mL of methanolic solution of Chenodeoxycholic Acid N-Hydroxysuccinimidyl ester (1.41 g, 2.88 mmol). The resulting clear solution was stined at room temperature overnight.
  • TLC indicated that this solid still contained some N-Hydroxysuccinimide.
  • the solid (0.87 g) was divided into four batches of approximately equal weight (-200 mg) and each batch was further purified with prepacked Amprep C18 column (500 mg sorbent per column). Each batch of solid dissolved in 0.5 mL of water was loaded on the column. The column was eluted first with 4 mL of water then with 4 mL of ethanol. The fractions containing the product were combined. The solvents were removed in vacuo and the final product was freeze-dried from water to give a white solid. Overall yield of the desired product was 0.24 g (18.6%).
  • poly-L-lysine-gr ⁇ t-copolymers A variety of poly-L-lysine-gr ⁇ t-copolymers was successfully synthesized through epoxide, tosyl, vinyl sulfone and haloacetamido chemistries. These chemistries were selected over typical activated ester approach to preserve charges on polycation and minimize impact on conjugate-DNA binding. These copolymers, poly-L-lysine- gr /t-R 1 -gr t-R 2 -gr t-R 3 co-polymers, could have a variety of molecules grafted on amino groups of cationic poly-L-lysine in a stepwise synthesis.
  • PEG molecules are grafted first (R ⁇ ), followed by introduction of other molecules (R 2 ), and finally fluorescent tags or ligand molecules (R 3 ), are covalently attached to some copolymers.
  • Poly-L-lysine-gr ⁇ /t-PEG polymers were prepared by reacting a PEG-electrophile with ⁇ -NH 2 lysine groups under basic conditions. For individual co-polymers, the ratios of PEG-electrophile to poly-L-lysine, PEG-electrophile size, and poly-L-lysine size were varied. The conditions of the syntheses are summarized in Table 2 and the general procedure is described for PEG-epoxide below.
  • Poly-L-lysine 10K (600 mg, 0.06 mmol) and lithium hydroxide monohydrate (41 mg, 2.9 mmol) were dissolved in water (2 ml) and methanol (6 ml) in a siliconized glass flask. Solid PEG5K-epoxide (600 mg, 0.12 mmol) was added, the flask was then sealed, and the solution incubated at 65° C for 48 h. After incubation, the solvent was removed in vacuo. The product was redissolved in a loading buffer (0.1 M sodium phosphate pH 6 in 10% MeOH (v/v)) and loaded on cation exchange column (Amersham Pharmacia SP Sepharose FF resin) followed by extensive washing step (up to 10 column volumes).
  • a loading buffer 0.1 M sodium phosphate pH 6 in 10% MeOH (v/v)
  • cation exchange column (Amersham Pharmacia SP Sepharose FF resin) followed by extensive washing step (up to 10 column volumes).
  • the product then was eluted with 0.1 N NaOH in 10% MeOH solution.
  • the macromolecular fractions containing the product were combined and the solvent removed in vacuo.
  • the product containing inorganic salts was re-dissolved in minimum amount of 0.05 M acetic acid in 30% MeOH solution and eluted over a G-25 column (Amersham Pharmacia Sephadex G-25 fine resin) with the same acetic acid solution.
  • the macromolecular fractions were pooled and lyophihzed.
  • the ratio of PEG chains to poly-L-lysine chains was determined by ! H NMR.
  • hydrophobically modified series of poly-L-lysine-gr t-R 1 -gr ⁇ -R 2 co- polymers was synthesized tlirough epoxide, bromoalkyl and amidine chemistry.
  • the products of such syntheses are listed in Tables 2 and 3.
  • the exemplary synthesis is described below.
  • Lithium hydroxide monohydrate (30.6 mg, 0.73 mmol) dissolved in water (1 mL) was added to a solution of poly-L-Lysine 26K (257.3 mg, 0.0099 mmol) in 10 mL of MeOH.
  • PEG5K-epoxide (495 mg, 0.099 mmol) was then added.
  • the flask was sealed and the solution incubated at 65 °C for 8 hours.
  • Br(CH 2 ) 10 -N-pyridinuim bromide 300 mg, 0.79 mmol was added and the resulting solution was incubated at 65 °C for 3 days. After incubation, the solvent was removed in vacuo and the product was re-dissolved in a minimum amount of water.
  • the pH of the solution was adjusted to pH 4 with glacial acetic acid.
  • the resulting solution was eluted over a G-25 column (Amersham Phannacia Sephadex G-25 fine resin) with 0.1 M of acetic acid.
  • the macromolecular fractions were pooled and lyophihzed yielding 486 mg.
  • grafting elements were attached to cationic polymer by amidine functionality. Such grafting elements were prepared starting with cyano derivatives that were transformed into imino methyl esters in a presence of HCl and anhydrous methanol in CH 2 C1 2 and as shown on Scheme 4 and 5. The polymeric products are described in Table 4.
  • Diphenylacetoimino methyl ester hydrochloride Diphenylacetonitrile (9.65 g, 49.9 mmol) was dissolved under argon in dichloromethane (60 mL), methanol (6.52 mL, 165 mmol) was added, and the reaction mixture was cooled to 0°C. The reaction mixture was saturated with HCl gas and maintained at 0°C overnight. The final product was then precipitated in ethyl ether to yield 9.0 g (70%) as a white solid. The product was used for the next step without further purification.
  • PL 10k-gr ⁇ -(PEG5k) 7 , 9 -gr ⁇ /t-(NH-C( NH)-benzyl) 5 .
  • PL10k-gr ⁇ /t-( ⁇ -NH-(CH 2 ) 10 PEG2k) 4 (PL-E) (115 mg, 0.0062 mmol) and 4- Picolyl Chloride (49 mg, 0.301 mmol) were dissolved in methanol (8 ml). Lithium Hydroxide (22 mg, 0.54 mmol) was added as a solution in methanol (1 mL). The reaction was incubated at 65°C for 6 days. The reaction mixture was then evaporated to dryness. It was re-dissolved in 0.05 N acetic acid in 30% methanol, and chromatographed over G-25 column. The macromolecular fraction was collected, and evaporated to dryness to yield 84 mg of red-brown solid.
  • Tris (2-carboxy ethyl) phosphine hydrochloride (TCEP»HCL) was purchased from Pierce Chemical Co. (Rockford, IL).
  • PD 10 Sepadex G-25M (pre-packed) and phenyl sepharose high performance (hydrophobic interaction column [HIC]) columns and G-25M resin were purchased from Pharmacia Biotech Inc. (Piscataway, NJ).
  • the CM/M Poros column (CM) was purchased from PerSeptive Biosystems, Inc. (Farmington, MA).
  • Synthetic polylysine, (Lys) 48 Cys was purchased from Dr. Schwabe (Protein Chemistry Facility at the Medical University of South Carolina).
  • Polyethylene glycol (PEG) epoxides 2K (M n 1554; M w /M n 1.044 (GPC)), 3K ( perhaps 2696; M w /M n 1.035 (GPC)), and 5K (Mlois 5231; M w /M n 1.017 (GPC)) were purchased from Shear Water Polymers, Inc. (Huntsville, AL). LiOH»H 2 ⁇ was purchased from Aldrich Chemical Co. (Milwaukee, WI). Plasmid DNA (pCMV ⁇ , Clontech, Palo Alto, CA and pCMV-Luciferase was prepared by BIO 101 (San Diego, CA). Plasmid DNA preparation contained more than 90% covalently closed circular DNA as determined by agarose gel electrophoresis.
  • the resulting solution was stined at room temperature under Argon and in the dark for two days, and then, it was briefly refluxed for 2 hours resulting in a cloudy solution.
  • the solvent was evaporated in vacuo and the yellow solid was dissolved in a minimum amount of chloroform and precipitated into anhydrous ethyl ether.
  • the solid was collected by centrifugation and rinsed three times with ethyl ether.
  • the desired product was dried in vacuum oven overnight and obtained as a white solid (17.05 g, 3.31 mmol, 65.5%).
  • 3-Chloroperoxybenzoic acid Prior to use, commercially available 3-Chloroperoxybenzoic acid (0.60 g, 60%, 2.1 mmol) was dissolved in toluene (100 mL) and dried over anhydrous sodium sulfate for two hours. The polymer from the previous step (5g, 0.97 mmol) was added to 3-Chloroperoxybenzoic acid (80 mL of toluene solution) and stirred for two days. The solvent was removed in vacuo producing a white solid. The crade product was dissolved in CH 2 C1 2 and precipitated into anhydrous ethyl ether cooled in a dry ice- acetone bath.
  • 3-Chloroperoxybenzoic acid Prior to use, commercially available 3-Chloroperoxybenzoic acid (11.7 g, 57%, 38.7 mmol) was dissolved in toluene (300 mL) and dried over anhydrous sodium sulfate overnight. Brij98 (25 g, 21.7 mmol) was added to 3-Chloroperoxybenzoic acid (280 mL of toluene solution) and stirred overnight. The solvent was removed in vacuo producing a yellow oil. The crade product was dissolved in CH 2 C1 2 and precipitated into anhydrous ethyl ether cooled in a dry ice-acetone bath.
  • 3-Chloroperbenzoic Acid (2.9 g, 10.08 mmol) was added to Toluene (80 mL, 0.75 mmol) and dried over anhydrous Na 2 SO overnight. Solution became clear and yellow once it was dry. This solution was added to 20 g of allyl ether- O(PO) 6 i(EO) n3 OCH 3 ; MeO(EO) ⁇ 3 (PO) 6 iOCH 2 CHCH 2 (1373-079) and stined over weekend. Solvent was then reduced in vacuo to 40 mL and product was precipitated from 600 mL ether that was chilled to -70 °C.
  • Poly-L-lysine-gr ⁇ t-(PEG-Hydrophobe) polymers were prepared by reacting a PEG-hydrophobe-electrophile with ⁇ -NH 2 lysine groups under basic conditions. For individual co-polymers, the ratios of PEG-hydrophobe-electrophile to poly-L-lysine, PEG-hydrophobe-electrophile size, and poly-L-lysine size were varied. The conditions of the syntheses are summarized in Table 8 and the general procedure is described for Triton X-405-C10-Br and PEG-C 10 -Br below.
  • Lithium hydroxide (18.1 mg, 0.43 mmol) dissolved in water (0.5 ml) was added to a solution of MeOPEG2k-C 10 -Br (1.4 g, 0.63 mmol) and Poly-L-Lysine 10k (150 mg, 0.016 mmol) in methanol (8 ml). The flask was sealed and incubated at 65°C overnight. After 18 h, additional PEG2k-C 10 -Br (160 mg. 0.072 mmol) and lithium hydroxide (2.6 mg, 0.062 mmol ) were added, and the flask was sealed and incubated at 65°C.
  • PL 1 Ok-graft-f ⁇ -NH-C 10-Triton X-405) Lithium hydroxide (12.4 mg, 0.28 mmol) dissolved in water (1 ml) was added to a solution of PLIOk (100 mg, 0.01 mmol) and Triton X-405-C10-Br (2.04 g, 0.44 mmol) in methanol (8 mL). The flask was sealed and incubated at 65°C for 48 h. Then additional lithium hydroxide (2.6 mg) and Triton X-405-C10-Br (255 mg) were added, and the reaction mixture incubated at 65°C for 48 h. The solvents were evaporated in vacuo and the residue was re-dissolved in 0.05 M Acetic Acid.
  • the solid product was first dissolved into 150 mL of a solution of 0.1 M of sodium phosphate buffer pH 6 containing 10 % of methanol v/v and then loaded on SP Sepharose FF Cation-Exchange Column. After 10 column volume washes to remove excess unreacted PEG-hydrophobe starting material, the final product was eluted with 0.1 M NaOH containing 10% of methanol v/v. The ninhydrin positive fractions were combined and pH was adjusted to pH 4 - 5 by dropwise addition of acetic acid. The solvent was removed in vacuo and the residue re-dissolved in 0.05 M Acetic Acid in 30% methanol v/v.
  • the product was purified by Sephadex G-25 column eluted with 0.05 M HAc in 30% methanol v/v. The ninhydrin positive fractions were combined and lyophihzed to give the product as a white solid.
  • the purity of the final conjugate was established by two analytical methods. Gel electrophoresis was performed to exclude contamination by poly-L-lysine and TLC to exclude free PEG-hydrophobe contamination. Typically, the final product did not contain unreacted poly-L-lysine or PEG-hydrophobe starting material.
  • Poly-L-aspartic acid (P(Asp)) sodium salt 10K [DP (Vis) 76, Mw 10,400 (Vis); DP (LALLS) 57, Mw (LALLS) 7,800] and ethidium bromide were purchased from Sigma Chemical Co., St. Louis, MO.
  • Plasmid DNA (pCMVb, Clontech, Palo Alto, CA and pCMV -Luciferase was prepared by BIO 101 (San Diego, CA). Plasmid DNA preparation contained more than 90% covalently closed circular DNA as determined by agarose gel electrophoresis. Tetrahydrofurnan (THF) was purchased from VWR and doubly distilled from sodium benzophenyl ketal.
  • Polyethylene glycol (PEG) amino 5k (MW 5254; Substitution: 98% ⁇ HNMR), 98.2% (titration)) purchased from Shearwater Polymers, Inc. (Huntsville, AL) was dried in vacuo at 40°C. All other reagents were used without further purification.
  • L-cysteine and 1-bromooctadecane were purchased from the Aldrich Chemical Co.
  • Bis(trichloromethyl)carbonate (friphosgene) and N ⁇ -Z- L-Lysine were purchased from Fluka Chemika. Potassium Hydroxide was obtained from VWR Scientific.
  • N-Carboxyanhydride of L-Cys-S-C 18 Synthesis of N-Carboxyanhydride of L-Octadecylcysteine was carried out by the Fuchs-Farthing method using friphosgene.
  • L-octadecylcysteine (6.0 g, 0.014 mol) was suspended in dry THF (30 ml).
  • Bis(trichloromethyl) carbonate 2.2 g, 0.007 mol
  • the reaction mixture was filtered over fritted glass filter (M porosity), and the filtrate was poured into hexanes (300 ml) and stored at -20°C overnight. The precipitate was filtered, washed with cold hexane (3 x 50 ml) and dried in vacuo. The white solid was recrystallized from THF/hexanes three times until the melting point remained constant. (m.p. 83-86°C). The product was characterized by 1 HNMR (CDC1 3 ) and IR.
  • N-Carboxyanhydrides of e-(Benzyloxycarbonyl)-L-lysine and of L-phenylalanine were synthesized and characterized as previously reported.
  • NCA-LysZ (9.2 g, 0.03 mol) was suspended in 100 mL THF and PEG5k— b/oc£-(CysC 18 ) 10 -NH 2 ( 33 ml > 0.0002 mol) was added.
  • the solution was stined at 40°C for 72 hours, and became very viscous after 24 hours. The solution was monitored by
  • PEG5k-b/oc&-(CysC 18 ) 10 -b/ocA:-(LysZ) 45 was prepared as described above. The removal of ⁇ -N-carboxy benzyl protecting group was performed as previously described.
  • Polyplexes were prepared by rapidly adding an equal volume of plasmid DNA to a volume of the copolymer.
  • DNA (2x) was prepared in water and copolymers were dissolved in the 2x diluent before mixing. Polyplex concentrations are reported by DNA content and were 10 ⁇ g/ml unless otherwise noted.
  • Polyplexes were formulated at room temperature by rapidly mixing 500 ⁇ L of
  • DNA (2x) and 500 ⁇ L of copolymer stock solution. Final DNA concentration was 50 ⁇ g/mL at a charge ratio of 1.0 (+/-) in 150 mM NaCl. Each polyplex solution was divided into five 200 ⁇ L aliquots and incubated at room temperature for 30 minutes.
  • Anionic molecules were added to the polyplex aliquots in increasing amounts (charge ratio 1, 4, 7, 10, and 100 per phosphate group). The samples were then incubated for 20 hours and analyzed on agarose gel (0.6%).
  • mice were anesthetized with a 80 ⁇ l intramuscular injection of a cocktail prepared from 20 ml isotonic saline, 7.5 ml ketamine (100 mg/ml), 3.8 ml xylazine (20 mg/ml) and 0.75 ml acepromazine (10 mg/ml) prior to treatment.
  • a cocktail prepared from 20 ml isotonic saline, 7.5 ml ketamine (100 mg/ml), 3.8 ml xylazine (20 mg/ml) and 0.75 ml acepromazine (10 mg/ml) prior to treatment.
  • 500 ⁇ l to as low as 200 ⁇ l of isotonic saline containing 15 - 20 ⁇ g of pDNA formulated with conjugate and any formulant was injected.
  • animals were sacrificed 24 hours post- injection by asphyxiation with CO 2 .
  • Organs were excised and rinsed twice with phosphate buffered saline (PBS). Organ weight was determined gravimetrically and recorded. Organs were dounce homogenized in ten volumes of cell lysis buffer (100 mmol/L potassium phosphate pH 7.8, 0.2 % Triton X-100). The resultant cell lysate was centrifuged for 5 min at maximum speed in a clinical centrifuge tube. The clear aqueous phase was collected from between the lipid layer on top and the cell pellet on the bottom of the tube. This clear lysate was further clarified by an additional 5 minute centrifugation at high speed in a microcentrifuge.
  • PBS phosphate buffered saline
  • the luciferase assay was performed on 0.1-100 ⁇ l of the final supernatant.
  • the luciferase activity of aliquots of tissue homogenate was measured with an Analytical Luminescence 2010 Luminometer. Background measurement was subtracted and the relative light units were converted to picograms of protein as calculated from standard curves based on purified luciferase protein standards (Analytical Luminescence Laboratories, San Diego, CA).
  • polyplexes were administered to anesthetized Buffalo, SHR, or Lewis rats by tail vein injection of 5 ml of the polyplex. Serum samples for interferon measurements were obtained at various time points by retro-orbital puncture and stored at -70° C prior to assay.
  • Serum concentrations of IFN- ⁇ 2b were measured using an ELISA kit (Endogen Inc., Cambridge, MA) according to the manufacturer's protocol. The ELISA is specific for human IFN- ⁇ 2b and does not cross react with murine IFN. Non-specific signal was accounted for by subtracting 3X background level from each value. Animal data are reported as mean with standard deviation.
  • 0.5 ml of fluorescent CY5 polyplex was injected into the tail vein of 12 week-old Balb/C mice. Five minutes after injection, the animals were killed by cervical dislocation and the livers excised and rinsed in PBS. Liver tissue was cut into 2 mm by 2 mm squares and fixed in 4 % paraformaldehyde for 4 hours. Tissue was infused in 0.5 molar sucrose overnight and then frozen in liquid nitrogen chilled isopentane. Frozen tissue was cut on a Leica cryostat at 10 mm and allowed to air dry for tissue attachment to slides.
  • Liver sections were counterstained with the nuclear stain DAPI, mounted with immunomount (Shandon Lipshaw, Pittsburgh, PA), and viewed on an Olympus BH2 microscope equipped with filter cubes designed for emission wavelengths of 461 nm (DAPI) and 670 nm (Cy5). Images were captured and superimposed on one another using a CCD camera and Metamorph software.
  • Table 9 shows the results of in vivo studies. The mice were injected with a 200 Cl dose, which contained 15 ⁇ g of DNA per injection. The structure of the steroidal fomulant is given below the table.
  • Figure 13 shows the effects of varying polyplexes and using a formulant to enhance luciferase expression.
  • the mice were injected with a 0.5 mL dose which contained 15 ⁇ g/mL of DNA (pCMVLuc).
  • pCMVLuc DNA
  • the polyplex which was constructed from the copolymer PLL9.4k-g-( ⁇ -NH-C 1 o-PEG2k) 14 when administered with DHPC, TCDC, OGP, Brij 35 resulted in enhanced expression of the gene luciferase.
  • enhancement was also found, to a lesser degree, when the DNA was administered without a penetration enhancer in a polyplex of the invention.
  • Figure 14 shows the effects of varying polyplexes and using a formulant to enhance luciferase expression.
  • the mice were injected with a 0.5 mL dose which contained 15 ⁇ g/mL of DNA (pCMVLuc). It was found that the polyplex which was constructed from the copolymer PLL9.4k-g-( ⁇ -NH-C10-PEG2k) 1 when administered with DHPC resulted in enhanced expression of the gene luciferase, as compared to the other formulations tested in this trial.
  • Figure 15 shows that the addition of the formulant, DHPC, greatly enhances the expression of luciferase.
  • mice were injected a dose of 200 ⁇ L which contained 15 ⁇ g/mL of DNA (pCMVLuc). Both (PLL9.4k-g-(e-NH-CO-"Trigal") 16. and (PLL9.4k-g-(e-NH-C12-PEG5K) 4 . 7 -g-(e-NH-"Trigal") (LG-E) advantageously allowed for enhanced expression of luciferase in vivo with and without the addition of formulant.
  • Figure 16 shows that when mice were injected with a dose of 200 ⁇ l containing 15 ⁇ g/mL of DNA (pCMVLuc), expression of luciferase was dependent on the architecture of the conjugate used. It was found that the conjugate comprised of random grafts of PEG and the hydrophobe Cholesterol ( 10KPL-5KPEG-cholesterol) had a wide range of luciferase expression when administered with the formulant, DHPC (represented by '•'). The range of luciferase expression ranged from below 0.1 pg Luc per gram of liver to over 1000 g of Luc per gram of liver.
  • Polyplexes comprised of block co-polymer (PEG5k-b-(Cys-S-C18) 10 -b-(Lys) 45 ) and (PEG5k-b-(Phe) 1 -b-(Lys) 51 ) (represented by ' ⁇ ' and ' ⁇ ', respectively), resulted in some luciferase expression.
  • Polyplexes comprised of polymers consisting of random grafts of PEG-coupled-hydrophobe with and without Trigalactose ligand included PLL9.4k-g-( ⁇ -NH-PEG4.4k-C18) 2 .

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Abstract

La présente invention concerne de nouvelles compositions et formulations d'administration de composés anioniques, en particulier des polynucléotides (ADN et ARN) à travers les limites cellulaires (par exemple, les membranes cellulaires) soit in vivo soit in vitro.
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