Title:  Compositions and methods for drug delivery using amphiphile binding molecules

United States Patent:  6,740,643

Issued:  May 25, 2004

Inventors:  Wolff; Jon A. (Madison, WI); Hagstrom; James E. (Madison, WI); Monahan; Sean D. (Madison, WI); Budker; Vladimir (Middleton, WI); Rozema; David B. (Madison, WI); Slattum; Paul M. (Madison, WI)

Assignee:  Mirus Corporation (Madison, WI)

Appl. No.:  726792

Filed:  November 29, 2000

Abstract

The present invention relates to the delivery of desired compounds (e.g., nucleic acids) into cells using noncovalent delivery systems which include complexing nucleic acids, amphipathic binding agents, and amphiphiles.

SUMMARY OF THE INVENTION

The invention relates to noncovalent amphiphile binding systems for use in biologic systems. More particularly, amphiphile-binding agents and polymers of amphiphile-binding agents are utilized in the delivery of molecules, polymers, nucleic acids and genes to cells.

Described in a preferred embodiment is a process for obtaining an expression product by delivering a polynucleotide to a cell, comprising the step of associating an amphiphile binding agent, an amphiphile, and a polynucleotide to form a complex. Then, delivering the complex to the cell and expressing the polynucleotide in the cell.

In another preferred embodiment, a complex is described for delivering and expressing DNA in a mammal, comprising an amphiphile binding agent, an amphiphile, and DNA in complex

Another preferred embodiment is a process for obtaining an expression product in vivo, comprising forming a complex with a cyclodextrin, an amphiphile and a polynucleotide. Then, delivering the complex to a cell in a mammal which expresses the polynucleotide.

DETAILED DESCRIPTION OF THE INVENTION

The following description provides exemplary embodiments of the systems, compositions, and methods of the present invention. These embodiments include a variety of systems that have been demonstrated as effective delivery systems. The invention is not limited to these particular embodiments.

Cyclodextrin Structure and Binding Properties

Cyclodextrins are naturally occurring cyclic oligomers of glucose in 1-4 .alpha. linkages.

Cyclodextrin composed of six glucose units (N=6) is called .alpha.-cyclodextrin, 7 units is called .beta.-cyclodextrin, and 8 is called .gamma.-cyclodextrin. The cyclic structure is torroidal in shape with the center of the torroid relatively nonpolar compared to water. For this reason, cyclodextrins will bind to nonpolar sections of amphipathic compounds,also known as amphiphilic compounds or amphiphiles, in water. Amphiphiles are compounds that contain both hydrophilic and hydrophobic functional groups. Examples include lipids, acyl-glycerol, sterols, polyethyleneglycol, and amino acids. Hydrophilic groups indicate in qualitative terms that the chemical moiety is water-preferring. Typically, such chemical groups are water soluble, and are hydrogen bond donors or acceptors with water. Examples of hydrophilic groups include compounds with the following chemical moieties; carbohydrates, polyoxyethylene, peptides, oligonucleotides and groups containing amines, amides, alkoxy amides, carboxylic acids, sulfirs, or hydroxyls. Hydrophobic groups indicate in qualitative terms that the chemical moiety is water-avoiding. Typically, such chemical groups are not water soluble, and tend not to hydrogen bonds. Hydrocarbons are hydrophobic groups. Amphipathic compounds bound by cyclodextrins include hydrophobic amino acids (e.g. leucine and phenylalanine), surfactants (e.g. sodium dodecylsulfate and Triton X-100), and lipids (e.g.palmitic acid). The strength of the interaction between cyclodextrin and an amphiphilic compound depends on the size of both the hydrophobic part of the amphiphile and the cyclodextrin. For example, .alpha.-cyclodextrin will bind linear alkyl chains, but not branched tertiary alkyl groups, which are bound by .beta.-cyclodextrin (Stella, V. J., Rajewsci, R. A. Pharm. Res. 1997, 14, 556. Stella, V. J., Rao, V. M. Zannov, E. A., Zia, V. Adv. Drug Del. Rev. 1999, 36, 3.).

Nucleic Acid Delivery by Polvcations and Cationic Lipids

There are many nonviaal nucleic acid complexes that have been shown to aid in delivery of DNA into cells. Nucleic acid includes DNA (plasmid DNA, anti-sense, oligonucleotides) and RNA (ribozymes, oligonucleotides, artificial messenger RNA). In general, these nonviral complexes may be grouped into two classes: cationic lipid complexes (lipoplexes) and cationic polymer (polyplexes) complexes. In either case, the polyanionic DNA is complexed with a cation. In lipoplexes, the cations are associated noncovalently by hydrophobic lipid-lipid interactions to form a polycation. In polymer complexes, the positive charges are attached covalently to form a polycation. Nucleic acids are delivered to cells for the purpose of gene therapy and anti-sense therapy.

Nucleic Acids Complexes Containing Cyclodextrins

As mentioned previously, cyclodextrins form complexes with amphipathic molecules that may be positively or negatively charged. Therefore, a polymer composed of cyclodextrins will become a polyion, a noncovalent amphiphilic electrolyte, when associated with a charged amphiphile. For example, association between a polymer composed of cyclodextrins and a cationic amphiphile will result in a polycation that may interact with DNA. In a preferred embodiment, a cyclodextrin-contaming polymers are constructed by reacting cyclodextrin with epichlorohydrin under alkaline conditions to produce cyclodextrin-epichlorohydrin copolymer. This cyclodextrin-epichlorohydrin copolymer, compacts pDNA upon addition of cations such as 1adamantanamine or 1-dodecylamine. The complex of cyclodextrin-epichlorohydrin copolymer and 1-adamanta amine or 1-dodecylamine is a cationic noncovalent amphiphilic polyelectrolyte, which is capable of condensing DNA. In addition, cationic amphiphiles that are polymers that are bound to monomeric or polymeric amphiphile binding agents may be used to compact DNA. Such DNA-containing complexes may be used for taansfection of cells.

Amphiphile binding agents may also be used to create anionic noncovalent amphiphilic polyclectrolytes. Association between a polymer composed of cyclodextrins and an anionic amphiphile will result in a polyanion that will interact with a positively-charged DNA-polycation complex, i.e. "recharge" the DNA complex. In a preferred embodiment, the complex between cyclodextrin-epichlorohydrin copolymer and 4-t-butylbenzoic acid, to form an anionic noncovalent amphiphilic polyelectrolyte, was added to particles of DNA and poly-L-lysine. The resulting particles were found to transfect cells in vitro. In addition, anionic amphiphiles that are polymers that are bound to monomeric or polymeric amphiphile binding agents may be used to "recharge" DNA particles. For example, succinyloleoylpoly-L-lysine is an anionic polymeric amphiphile which complexes with the amphiphile binding agent .beta.-cyclodextrin and interacts ("recharges") a poly-L-lysine condensed DNA particle. The addition of the cyclodextrin increased the transfection of the recharged particle 33 fold over recharged particle in the absence of cyclodextrin.

Not only is the cycbodextrin the basis for the DNA-polyion interaction, but cyclodextrinbased polyions may have properties (e.g. surface charge and stability) different from standard polyions. In contrast to standard polyions, the polyions derived from cyclodextrin-containing polymers and charged amphiphiles are reversible. The existence of the polyion is dependent upon the concentration of the cyclodextrin-containing polymer and the charged amphiphile, such that the disruption of the polyion maybe trigger by simple dilution of either cyclodextrin or charged amphiphile.

Monomeric cyclodextrins may also be incorporated into nucleic acid complexes by association with amphiphile molecules in a DNA complex. In this case, the cyclodcxtxins are not the basis for the DNA-electrolyte interactions, but may be used to change the properties of the DNA-electrolyte complex, e.g. stability or surface charge. The addition of cyclodextrin into a DNA particle also adds hydrophilic, but not charged, moieties to the particle. Hydrophilic molecules (e.g. PEG) have been shown to increase solubility of DNA particles, decrease the surface charge and thereby increase their stability. Cyclodextrins have the ability to bind to other nonionic hydrophilic molecules such as PEG. Therefore, addition of PEG to a cyclodextrin-containing DNA particle will result in PEG-particle interactions, which may confer the particle with added stability. Unlike other examples of PEG stabilization of DNA particles, the interaction between DNA particle and PEG is transient and may release under dilute, delivery conditions. The rate at which the PEG may be released may be altered by the number of PEG molecules incorporated, the number of cyclodextrins, and the incorporation of PEG derivatives with strong cyclodextrin binding regions (e.g. t-octylphenyl group of Triton X-100). In a preferred embodiment, addition of the PEG-derived detergent Triton X-100 to particles of DNA and poly-L-lysine-succinyl-.beta.-cyclodextrin resulted in particles that were more stable than particles without addition of the Triton X-100.

Likewise, cell targeting ligands aid in transport to a cell but may not be necessary, and may inhibit, transport into a cell. In all of these cases, the reversible attachment of the interaction modifier, through a labile bond, would be beneficial.

The present invention provides for the transfer of polynucleotides, and other biologically active compounds into cells in culture (also known as "in vitro"). Compounds or kits for the tansfection of cells in culture is commonly sold as "transfection reagents" or "transfection kits". The present invention also provides for the transfer of polynucleotides, and biologically active compounds into cells within tissues in situ and in vivo, and delivered intravascurary (U.S. patent application Ser. No. 08/571,536), intrarterially, intravenous, orally, intraduodenaly, via the jejunum (or ileum or colon), rectally, transdermally, subcutaneously, intramuscularly, intaaperitoraally, intraparenterally, via direct injections into tissues such as the liver, lung, heart, muscle, spleen, pancreas, brain (including intraventricular), spinal cord, ganglion, lymph nodes, lymphatic system, adipose tissues, thryoid tissue, adrenal glands, kidneys, prostate, blood cells, bone marrow cells, cancer cells, tumors, eye retina, via the bile duct, or via mucosal membranes such as in the mouth, nose, throat, vagina or rectum or into ducts of the salivary or other exocrine glands. Compounds for the transfection of cells in vivo in a whole organism can be sold as "in vivo transfection reagents" or "in vivo transfection kits" or as a pharmaceutical for gene therapy.

EXAMPLES

The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

Example 1

Synthesis of Succinyl-.beta.-cyclodextrin

.beta.-Cyclodextrin (0.5 gm, 0.38 mmol) and succinic anhydride (0.5 gm, 5 mmol) were dissolved in anhydrous pyridine (10 mL) for 4 h. The succinyl-.beta.-cyclodextrin was then precipitated by addition of 40 mL isopropyl alcohol. The precipitate was then washed 3 times with 10 mL isopropyl alcohol.

Example 2

Synthesis of Poly-L-lysine-succinyl-.beta.-cyclodextrin

Succinyl-.beta.-cyclodextrin (75 mg, 0.05 mmol) and poly-L-lysine (2 mg, MW 52,000, 0.01 mmol amines) were dissolved in 1 mL water. To this mixture was added N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (40 mg, 0.2 mnmol) and the reaction was allowed to proceed overnight. The reaction mixture was then placed into a dialysis bag (12,000 molecular weight cutoff) and dialyzed against 3x1 L water for 72 hr. Lyophilization resulted in 6.7 mg of poly-L-lysine-succinyl-.beta.-cyclodextrin, which is 35% modification of the amine residues. The polymer was then dissolved in 0.2 mL of water.

Example 3

Synthesis of Oleoyl poly-L-lysine

Poly-L-lysine (5 mg, 0.02 mmol amines) was dissolved in 0.5 mL water, to this solution was added oleoyl chloride (0.5 mg, 0.002 mmol) in 20 .mu.L of acetonitrile.

Example 3:

Synthesis of Succinyloleoylpoly-L-lysine

To a solution of poly-L-lysine-oleoyl amide (2 mg) in 200 mL water was added succinic anhydride (20 mg, 0.2 mmol) and potassium carbonate (100 mg 0.7 mmol). After 5 minutes, the succinylpoly-L-lysine-oleoyl amide was precipitated by addition of 1 mL isopropyl alcohol.

Example 3

Synthesis of Epichlorohydrin-.beta.-cyclodextrin Copolymer

.beta.-Cyclodextrin (0.5 gm, 0.38 mmol) and sodium hydroxide (0.18 gm, 4.5 mmol) were dissolved in water (0.8 mL) and heated to 30^oC. To this solution was added epichlorohydrin (0.345 mL, 4.4 mmol) and the immiscible solutions were stirred at 30^oC. for 3.5 h, during which time the epichlorohydrin dissolved in the aqueous solution. The epichlorohydrin-.beta.-cyclodextrin copolymer was then precipitated by the addition of acetone (10 mL). The acetone was decanted and the precipitate was dissolved in water (20 mL) and dialyzed in 14,000 molecular weight cutoff tubing against 2x1L water for 48 h. The polymer was then isolated by lyophilization to yield 0.3 gm of polymer.

Example 4

Characterization of Particles Formed by Poly-L-lysine, Epichlorohydrin-.beta.-cyclodextrin Copoyvmer, and 4-t-butylbenzoic Acid

To a solution of epichlorohydrin-.beta.-cyclodextrin copolymer (100 .mu.g/mL) and poly-L-lysine (100 .mu.g/mL) was added 4-t-butylbenzoic acid (3 mM). The size of the particle formed by the three agents was 100 nm, measured by a Brookhaven ZetaPlus Particle Sizer. Particle formation is observed only in the presence of all three components and is independent of the order of addition of each component.

Examnple 5

Characterization of Particles Formed by Plasmid DNA, Epichlorohydrin-.beta.-cyclodextrin Copolymer, and Oleoylamine

To a solution of epichlorohydrin-.beta.-cyclodextrin copolymer (50 .mu.g/mL) and plasmid DNA (10 .mu.g/mL) was added oleoylamine (0.1 mM). The size of the particle formed by the three agents was 78 nm, measured by a Brookhaven ZetaPlus Particle Sizer. Particle formation is observed only in the presence of all three components and is independent of the order of addition of each component.

Example 6

Characterization of Particles Formed Between Plasmid DNA and Poly-L-lysine-succinyl-.beta.-cyclodextrin

To a solution of plasmid DNA (10 .mu.g/mL) was added poly-L-lysine-succinyl-.beta.-cyclodextrin (30 .mu.g/mL). The size of the particle formed was 88 run and its charge was 11+7 mV, measured by a Brookhaven ZetaPlus Particle Sizer. To these particle was added Triton X-100 (0.2 mg/mL) resulting in a particle that was 140 nm in size with a charge of 22+4 mV. Addition of sodium chloride (100 mM) to these particles resulted in particles that were 115 nm in size with a charge of 7+2 mV. If Triton x-100 is not added to the particles prior to the addition of sodium chloride the particles become large, >200 nm.

Example 7

In Vitro Transfection with DNA-poly-L-lysine-succinylpoly-L-lysine-oleoyl Amide Particles in the Presence of .beta.-Cyclodextrin

To plasmid DNA pCIluc (10 .mu.g/mL, 2.6 .mu.g/.mu.L pCIluc; prepared according to Danko I, Williams P, Herweijer H, Zhang G, Latendresse J S, Bock I, Wolff J A Hum. Mol. Genet. 1997, 6, 1435) in 0.5 mL of 0 or 3 mM aqueous .beta.-cyclodextrin was added poly-Llysine (30 .mu.g/mL). Subsequently, 0.15 mg/mL of succinyloleoylpoly-L-lysine was added. The DNA complexes were then added (200 .mu.L) to a well containing 3T3 mouse embryonic fibroblast cells in 290 mM glucose and 5 mM HEPES buffer pH 7.5. After 1.5 h, the glucose solution was replaced with Dubelco's modified Eagle Media and the cells were allowed to incubate for 48 h. The cells were then harvested and then assayed for luciferase activity. Luciferase activity in the presence of .beta.-cyclodextrin was 33-fold higher (324,305 relative light units) than in the absence of .beta.-cyclodextrin (RLU=9,924).

Example 8

In Vitro Transfection with DNA-poly-L-lysine-epichlorohydrin-.beta.-cyclodextrin Copolymer in the Presence of p-t-butyl-benzoic Acid

To plasmid DNA pCIluc (10 .mu.g/mL, 2.6 .mu.g/.mu.L pCIluc) in 0.5 mL of aqueous 0 or 3 mM 4-t-butylbenzoic acid was added poly-L-lysine (30 .mu.g/mL). Subsequently, 0.15 mg/mL of succinylated poly-L-lysine or epichlorohydrin-.beta.-cyclodextrin copolymer was added. The DNA complexes were then added (200 .mu.L) to a well containing 3T3 mouse embryonic fibroblast cells in Dubelco's modified Eagle Media. After 1.5 h, the media was changed and the cells were allowed to incubate for 48 h. The cells were then harvested and then assay for luciferase activity. Luciferase activity for the particles composed of epichlorohydrin-.beta.-cyclodextrin copolymer was 81-fold higher (314166 relative light units(RLU)) than those particles composed of succinylated poly-L-lysine (3868 RLU).

Example 9

Characterization of Complexes of Plasmid DNA, Dodecylamine, and .beta.-cyclodextrin-epichlorohydrin Copolymer

To a solution of plasmid DNA (10 .mu.g/mL) and .beta.-cyclodextrin-epichlorohydrin copolymer (50 .mu.g/mL) was added dodecylamine (100 .mu.M). The size of the particle formed was 181 nm as measured by a Brookhaven ZetaPlus Particle Sizer. Prior to the addition of dodecylamine there were no particles formed and solutions of .beta.-cyclodextrin epichlorohydrin copolymer and dodecyl amine do no not form particles.

Example 10

Characterization of Complexes of Plasmid DNA. 1-adamantamine, and .beta.-cyclodextrin-epichlorohydrin Copolymer

To a solution of plasind DNA (10 .mu.g/mL) and .beta.-cyclodextrin-epichlorohydrin copolymer (50 .mu.mL) was added various amounts of 1-adamantanamine (100-600 .mu.M). The size of the particle formed was 181 nm as measured by a Brookhaven ZetaPlus Particle Sizer. Prior to the addition of dodecylamine there were no particles formed and solutions of .beta.-cyclodextrin epichlorohydrin copolymer and dodecyl amine do no not form particles.

Example 11

In Vivo Expression of Complexes of Plasmid DNA, 1-adamantamine, and .beta.-cyclodextrin-epichlorohydrin Copolymer

A complex of pCI Luc (50 .mu.g/mL), 250 .mu.g/mL .beta.-cyclodextrin-epichlorohydrin copolymer, and 6 mM aantamine in 0.2 mL were diluted to 2.5 mL in Ringers solution. Tail vein injections of 2.5 mL of the complex were performed as previously described (Zhang, G., Budker, V., Wolff, J. A. Hum. Gene Ther. 1999, 10, 1735.) Luciferase expression was determined as previously reported (Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsadi, G., Jani, A. and Felgner, P. L. Direct gene transfer into mouse muscle in vivo. Science, 1465-1468,1990.). A Lumat LB 9507 (EG&G Berthold, BadWildbad, Germany) luminometer was used.

Example 12

In Vivo Expression of Complexes of Digoxin-labeled Plasmid DNA and .gamma.-cyclodextrin

Plasmid DNA was labeled with Mirus' LabelIt.RTM. digoxin labeling kit according to protocol. A complex of digoxin-labeled pCI Luc (2 .mu.g) and .gamma.-cylodextrin (17 mg) were formulated in 2.5 mL in Ringers solution. Tail vein injections of the complex were performed as previously described (Zhang, G., Budker, V., Wolff, J. A. Hum. Gene Ther. 1999, 10, 1735.) Luciferase expression was determined as previously reported (Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsad, G., Jani, A. and Felgner, P. L. Direct gene transfer into mouse muscle in vivo. Science, 1465-468, 1990.). A Lumat LB 9507 (EG&G Berthold, Bad-Wildbad, Germany) luminometer was used.

Claim 1 of 15 Claims

We claim:

1. A process for obtaining an expression product by delivering a polynucleotide to a cell, comprising:

a) associating a noncovalent amphiphilic polyelectrolyte a cyclodextrin, and a polynucleotide thereby forming a complex, wherein the noncovalent amphiphilic polyelectrolyte consists of an polymeric amphiphile binding agent and charged amphiphiles;

b) delivering the complex to the cell; and,

c) expressing the polynucleotide.

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