WO2008070141A2 - Compositions pour administrer des agents thérapeutiques - Google Patents

Compositions pour administrer des agents thérapeutiques Download PDF

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Publication number
WO2008070141A2
WO2008070141A2 PCT/US2007/024966 US2007024966W WO2008070141A2 WO 2008070141 A2 WO2008070141 A2 WO 2008070141A2 US 2007024966 W US2007024966 W US 2007024966W WO 2008070141 A2 WO2008070141 A2 WO 2008070141A2
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poly
polymer
units
chains
ethylene glycol
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WO2008070141A3 (fr
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May Pang XIONG
Glen S. Kwon
Younsoo Bae
Kazunori Kataoka
Shigeto Fukushima
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Wisconsin Alumni Research Foundation
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Wisconsin Alumni Research Foundation
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • 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
    • A61K47/56—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • 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
    • A61K47/6455—Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
    • 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
    • A61K47/66—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 the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • A61K47/67—Enzyme prodrug therapy, e.g. gene directed enzyme drug therapy [GDEPT] or VDEPT
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
    • C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
    • C08G69/10—Alpha-amino-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
    • C08G73/06—Polycondensates having nitrogen-containing heterocyclic rings in the main chain of the macromolecule
    • C08G73/10—Polyimides; Polyester-imides; Polyamide-imides; Polyamide acids or similar polyimide precursors
    • C08G73/1092—Polysuccinimides

Definitions

  • GDEPT Gene Directed Enzyme Prodrug Therapy
  • Viral gene delivery methods which usually involve transfecting with lentivirus vectors containing a gene of interest are viewed with reservation because of potential problems with immunogenicity and other safety risks.
  • the first block can include aspartic acid or glutamic acid units.
  • the second block can include lysine or arginine units.
  • the aspartic acid or glutamic acid units can be covalently linked to poly(ethylene glycol) chains.
  • the molecular weight of the poly(ethylene glycol) chains can be about 500 to about 50,000.
  • a plurality of aspartic acid or glutamic acid units can be covalently linked to poly(ethylene glycol) chains and the poly(ethylene glycol) chains terminate in methoxy, amino, or acetamide groups.
  • the ratio of aspartic acid or glutamic acid units to polyethylene(glycol) chains can be about 1 : 1 to about 20: 1.
  • the polymer can include poly(lysine) units wherein one or more of the lysine side chain amino group is protected with nitrogen protecting groups, such as trifluoroacetate groups or carbonyl-benzyloxy (CBz) groups.
  • the first block is poly(aspartic acid) comprising about 20 to about 60 aspartic acid units.
  • the second block is poly(lysine) comprising about 30 to about 80 lysine units.
  • the first block is poly(glutamic acid) comprising about 20 to about 60 arginine units.
  • the second block is poly(arginine) comprising about 30 to about 80 arginine units.
  • embodiments of the invention include replacing one or more units of a 'lysine' block with arginine units, aspartate units, aspartate unit derivatives such as aspartate with a diethyl triamine side chain (an N-(2-aminoethyl)-2-aminoethyl group in the side chain), or an imidazole acetic acid group on a side chain. Additionally, embodiments also include replacing any aspartic acid unit with a glutamic acid unit, optionally in combination with a replacement of one or more lysine units.
  • a typical polymer of the invention will include at least about 60 repeating units. In other embodiments, the polymers of the invention may include up to about 150 repeating units.
  • the invention also provides a polyamide block polymer comprising at least one block of poly(aspartic acid) units and at least one block of poly(lysine) units; wherein at least one side chain of an aspartic acid unit is covalently linked to a poly(ethylene glycol) chain through a hydrazide moiety, and wherein the hydrazide moiety is linked to the poly(ethylene glycol) chain at the N* nitrogen of the hydrazide through a hydrazone bond.
  • a plurality of aspartic acid units can be covalently linked to poly(ethylene glycol) chains and the molecular weight of the poly(ethylene glycol) chains can be about 500 to about 10,000.
  • a plurality of aspartic acid units can be covalently linked to poly(ethylene glycol) chains and the poly(ethylene glycol) chains can terminate in various organic groups, such as methoxy, amino, or acetamide groups, or combinations thereof.
  • the ratio of aspartic acid units to polyethylene(glycol) chains can be about 1:1 to about 20:1, or any ratio in between.
  • the polymer can include poly(lysine) units wherein the side chain amino group is protected with a nitrogen protecting group, for example, a trifluoroacetate group or a carbonylbenzyloxy group. Protecting groups can optionally be removed before the polymer is used to prepare a micelle that incorporates various cargo.
  • the poly(aspartic acid) block can include about 20 to about 60 aspartic acid units.
  • the poly(lysine) block can include about 30 to about 80 lysine units.
  • the invention further provides a micelle composition that includes a polymer as described herein and a therapeutic agent, wherein the polymer substantially encapsulates the therapeutic agent and the poly(ethylene glycol) chains of the polymer align toward the outside surface of the micelle.
  • the therapeutic agent can include a nucleic acid, a gene, a drug, a prodrug, or a combination thereof.
  • the nucleic acid can be plasmid DNA encoding gene products or RNAi.
  • polyamide polymer comprising amino acid units with side chains that can have positive, neutral, or negative charges; wherein at least one side chain of an amino acid of the polyamide is covalently linked to a poly(ethylene glycol) chain through a hydrazide moiety, and wherein the hydrazide moiety is linked to the poly(ethylene glycol) chain at the N 1 nitrogen of the hydrazide through a hydrazone bond.
  • the amino acid units can include a variety of amino acids, including aspartic acid units, lysine units, or both.
  • a plurality of aspartic acid units can be covalently linked to poly(ethylene glycol) chains.
  • the molecular weight of the poly(ethylene glycol) chains can be about 500 to about 10,000.
  • a plurality of aspartic acid units can be covalently linked to poly(ethylene glycol) chains and the poly(ethylene glycol) chains can terminate in PEG-capping groups, for example, amino or acetamide groups.
  • the ratio of aspartic acid units to polyethylene(glycol) chains can be about 1 : 1 to about 20: 1, or any ratio in between.
  • One or more of the lysine side chain amino group can be protected with a nitrogen protecting group, such as trifluoroacetate groups or carbonylbenzyloxy groups.
  • the polymer can include about 5 to about 100 amino acid units in the first block.
  • the polymer can include about 10 to about 150 amino acid units in the second block.
  • the amino acids can be aspartic acid or glutamic acid in the first block, and lysine or arginine in the second block.
  • the polymer can also include about 5 to about 60 units, about 20 to about 100 units, about 10 to about 80 units, or about 20 to about 60 units, in the first block.
  • the polymer can also include about 10 to about 80 units, about 20 to about 150 units, about 20 to about 120 units, about 30 to about 100 units, or about 30 to about 80 units, in the second block.
  • the invention further provides a polymer comprising formula I:
  • the group L can be a direct bond from NH to R 1 , or L can be a carbonyl or sulfonyl group.
  • R 1 can be H, or together with the carbonyl to which it is attached, can be a nitrogen protecting group, for example, trifluoroacetate or carbonylbenzyloxy.
  • R 2 can be hydrogen, hydroxy, (Ci-Ci 2 )alkyl, alkoxy, -NHR 3 wherein R 3 is hydrogen,an amino protecting group, or any PEG-capping group; and X can be a direct bond, an organic linking group, or a group of about 1 to about 100 amino acids; or a salt thereof.
  • m can be about 30 to about 50
  • n can be about 40 to about 60
  • p can be selected such that the molecular weight of the poly(ethylene glycol) chain is about 2,000 to about 10,000.
  • R 2 can be -NHR 3 wherein R 3 is an acetyl group.
  • the invention also provides a polymer comprising formula II:
  • a method of activating a therapeutic prodrug that includes contacting a micelle composition described herein and a cell, wherein the micelle composition contains a nucleic acid, gene, or vector that encodes an enzyme that converts the prodrug to an active drug.
  • the method can further include introducing the prodrug to the vicinity of the cell after the cell has synthesized the enzyme so that the enzyme activates the prodrug to the active drug.
  • the introduction of the prodrug to the vicinity of the cell can be via systemic delivery, such as by IV or other injection.
  • the formulation for injection can include a pharmaceutically acceptable carrier or diluent, for example, saline that optionally includes a buffer.
  • a method of delivering a nucleic acid to a cell that includes encapsulating nucleic acid in a micelle comprising a polymer described herein; and contacting a cell with the micelle.
  • the nucleic acid can be a gene, a vector, or a specific type of RNA, such as siRNA, or a plasmid that codes for RNAi.
  • the invention additionally provides a method of treatment for a disease or a condition in a human or an animal by administering a micelle composition that includes a polymer disclosed herein and a therapeutic agent, such as a nucleic acid or a drug.
  • a therapeutic agent such as a nucleic acid or a drug.
  • the nucleic acid can encode for a therapeutic protein, such as an enzyme.
  • a prodrug can then be administered to a patient, whereby cells that produce the enzyme then activate the prodrug to a drug, and then the drug takes its effect on the cell.
  • One aspect of the invention involves the preparation of a pH-sensitive polyamide copolymer, such as a poly(Aspartate-Hydrazide-PEG)-b-poly(L- Lysine) block-copolymer.
  • the poly(L-Lysine) segment of the polyamide can be used to condense an agent within an association of polyamide molecules, such as in a micelle core or polyamide particle core.
  • the agent can be a therapeutic agent, such as a drug or a gene therapy agent, for example, a pDNA, or the agent can be a diagnostic agent or an imaging agent.
  • Aldehyde-PEG groups can be attached, for example, through the formation of hydrazone bonds to hydrazide groups, forming a variable brush layer in the particle shell, composed of acid labile PEG moieties. At a pH of less than approximately 7.0, these acid labile PEG moieties can be released from the polymer, leaving behind hydrazide groups that can enhance the escape of pDNA from endolysosomes via membrane disruptions. The rapid release of the PEG brush layer can further enhance agent unpacking, for example, pDNA unpackaging.
  • compositions and methods thus provide non-viral transfection vehicles with reduced toxicity compared to standard poly(lysine) delivery vehicles.
  • the compositions and methods also improved in vivo stability, wherein the rate of release of encapsulated agents can be tuned by varying the amount of PEG groups on the cationic polymers.
  • the invention thus provides novel polymers, polymer compositions, including micelles, and methods of making and using the polymers and polymer compositions.
  • the polymers and compositions can be used to treat diseases or disorders of mammals, for example, using GDEPT.
  • the polymers and compositions can also be used to prepare a medicament to treat diseases in mammals, for example, cancer in a human. Also provided are useful intermediates for the preparation of the polymers disclosed herein.
  • Figure 1 illustrates a block-copolymer polyplex according to an embodiment of the invention.
  • the first structure illustrates hydrazine groups exposed at the surface of the polyplex particle.
  • the second structure illustrates post-PEGylation of the polyplexes, which include a brush layer of PEG.
  • the second arrow indicates pH-triggered release and degradation of the cationic polymer in a cell's reductive environment.
  • Figure 2(a) illustrates a GPC (10 raM LiCl in DMF) of p(Asp-Hyd) 36 - PLL(TF A)s3 reaction with aldehyde-PEG7k at a 1:1 molar ratio after 2 and 5 days incubation at RT. Peak a is un-reacted PEG7k, peak b is un-reacted p(Asp- Hyd) 36 -PLL(TFA) 53 block-copolymer and peak c is the multimodal distribution of pre-PEGylated block-copolymers.
  • Figure 2(b) illustrates a GPC (10 mM LiCl in DMF) of p(As ⁇ -Hyd) 36 - PLL(TFA) 50 reaction with aldehyde-PEG7k at a 1 :0.5 molar ratio after 2 and 5 days incubation at RT. Peak a is un-reacted PEG7k, peak b is un-reacted p(Asp-Hyd) 36 -PLL(TFA) 53 block-copolymer and peak c is the unimodal distribution of pre-PEGylated block-copolymers.
  • Figure 2(c) illustrates a GPC (10 mM LiCl in DMF) of p(Asp-Hyd) 36 - PLL(TFA) 50 reaction with aldehyde-PEG7k at a 1 :0.1 molar ratio after 2 and 5 days incubation at RT. Peak a is un-reacted PEG7k, peak b is un-reacted p(Asp- Hyd) 36 -PLL(TFA) 53 block-copolymer and peak c is perhaps pre-PEGylated block-copolymers.
  • Figure 3 illustrates transfection efficiency of PEI25, BC, and PBC at N/P 70-100 in MDA-MB-231 cells.
  • Figure 5 illustrates the acid sensitivity of p(Asp-Hyd-PEG)-poly(L- lysine(TFA)) , according to an embodiment of the invention. Release of PEG from the block-copolymer was verified by GPC analysis by the addition of various amounts of TFA and incubating overnight. At a pH of 7.4, very little PEG was released. At a pH ⁇ 7.0, the release of PEG was immediate and pH- dependent, with over 50% PEG release within 20 minutes of incubation at pH 3.0.
  • Figure 6 illustrates a SEC example of a block copolymer aggregation after PEG following 20 minutes incubation at pH 3, 5.5, and 7.4, according to an embodiment.
  • Figure 7 illustrates (A) successful ligation of the yCD (-500 bp) into the
  • Figure 8 illustrates (A) expression of yCD as shown by (A): a Coomassie-stained gel of the crude lysate after induction of yCD; and (B) a Western blot of crude protein extracts probed with Tetra-His conjugate.
  • Figure 9 illustrates transfection activity at increasing equivalent N/P ratios and toxicity profiles of the free polymers in serum media for (A) C2C12; and (B) MDA-MB 231.
  • FIG 10 illustrates a Gene Directed Enzyme Prodrug Therapy (GDEPT) concept according to an embodiment of the invention.
  • GDEPT Gene Directed Enzyme Prodrug Therapy
  • Figure 11 illustrates a pH-sensitive poly(Aspartate-Hydrazide-PEG)-b- poly(L-Lysine) block-copolymer particle and its release of PEG groups, according to an embodiment of the invention.
  • Figure 12 illustrates the formation of polyplex micelles through the electrostatic interaction between block catiomers and plasmid DNA, according to an embodiment of the invention.
  • Figure 13 illustrates structures of the block copolymers: a) poly(aspartate-hydrazide)- ⁇ /oc ⁇ >poly(L-lysine) (BC); and b) pH-sensitive poly(aspartate-hydrazide-PEG)- ⁇ /ocA>poly(L-lysine) (pH-PBC), and covalent poly(aspartate-hydrazide-PEG)-6/oc ⁇ :-poly(L-lysine) (cov-PBC).
  • Figure 14 illustrates a) cationic polyplexes formed with PLL (or PEI) are cytotoxic to cells; in contrast, neutral shielded particles [parts b), c), and d)] cause minimal membrane damage during endocytosis: following a decrease in pH, hydrazide amino groups protonate b) and/or acid-labile PEG chains are released c); this increases endosomal membrane disturbances with the resulting charged particle and enhances escape into the cytosol (pH 7.4), whereupon sensing the new pH, hydrazide amino groups deprotonate again, once more imparting neutral properties to the PIC and minimizing intracellular toxicity; d) cov-PBC particles cannot release PEG chains following a decrease in pH, and this can minimize particle interactions with the endosomal membrane.
  • FIG 15 illustrates protonation profiles comparing branched PEI, BC, and PLL.
  • PEI can buffer over a relatively wide pH range.
  • BC carries little buffering capacity, similar to PLL (note: error bars omitted to simplify profile).
  • Figure 17 illustrates a) size and b) ⁇ profiles for BC (white) and pH-PBC (gray) at the indicated pH values, according to an embodiment of the invention.
  • RI refractive index
  • Figure 19 illustrates membrane toxicity of polymers at concentrations of 20 and 40 ⁇ g mL "1 toward MDA-MB-231 (human breast cancer) cells in DMEM serum-free media; polymers were incubated both in free form [parts a) and c)] and also pre-complexed to 1 ⁇ g pGL3 [parts b) and d)].
  • Figure 20 illustrates metabolic toxicity of polymers incubated for 4 hours at various concentrations toward MDA-MB-231 (human breast cancer) cells in DMEM serum-containing media.
  • Polymers were incubated in both free form and pre-complexed to 1 ⁇ g pGL3; a) free polymers with cationic components are toxic to cells at higher concentrations, with PEI being more toxic than BC (*p ⁇ 0.05 using two-way ANOVA); and b) when complexed, BC results in negligible toxicity to the cell, similar to pH-PBC particles (*p>0.05 using two-way ANOVA).
  • the invention provides polymers, particularly block co-polymers, that can have refined properties that make them "tunable” in response to various environmental conditions, addition of ligands, excipients, etc.
  • Block copolymers that include PEG groups are of interest because PEG is unique in its ability to facilitate transfer of appended agents across cell membranes. PEG is both water soluble and membrane permeable.
  • the inclusion of multiple PEG residues in the polymers disclosed herein allows for the covalent yet labile attachment of targeting ligands and tenability or modulation of the release of agents, such as DNA packages, into the polycationic core.
  • the contents of the particle can be released upon hydrolytic removal of PEG residues in response to slightly lower than physiological pH, such as the pH found in cancerous cells.
  • One embodiment of the invention comprises a poly(PEG)-poly(lysine) copolymer that encapsulates a vector encoding the non-mammalian enzyme yeast cytosine deaminase (yCD), which has shown potential in activating the prodrug 5-fluorocytosine (5FC) into cytotoxic 5-fluorouracil (5FU) in several tumor models.
  • Cytosine deaminase can be obtained both from S. Cerevisiae (yeast) and E. CoIi (bacteria). Both the prodrug and the gene encoding the enzyme can be preferentially delivered to tumor cells because the slightly more acidic extracellular environment, combined with the leaky vasculature of tumor cells, favors delivery of the DNA vector and prodrug to tumor cells.
  • Variations of this embodiment can be considered a platform for the creation of a library of tunable poly(PEG)-polycation copolymers with modulatable gene and therapeutic agent delivery properties.
  • the invention adds to the compendium of non-viral vehicles for tunable, specific gene and prodrug delivery into cells.
  • the invention also provides nonviral gene vectors from synthetic catiomers (polyplexes) as alternatives to viral vectors.
  • polyplexes synthetic catiomers
  • One embodiment employs the use of poly(ethylene glycol) ("PEG")-linked block catiomers, which form a nanoscaled core-shell polyplex with biocompatible PEG palisades.
  • PEG poly(ethylene glycol)
  • the catiomers can spontaneously associate with pDNA to form polyplex micelles at the sub- 100-nm scale with a dense and hydrophilic PEG palisade surrounding the polyplex core.
  • These polyplex micelles with PEG palisades can display high colloidal stability under physiological conditions and can afford appreciable levels of reporter-gene expression to various cell lines even after preincubation in a serum-containing medium.
  • the polyplex micelles can demonstrat longevity in blood circulation, offering the possibility of their use in systemic gene delivery.
  • PEG-linked block catiomers with various amine functionalities have been prepared by a simple and affordable synthetic procedure based on aminolysis reactions. These PEG-linked block catiomers can be used as gene carriers.
  • the catiomers with various side chains are capable of efficient and low toxicity transfection even toward primary cells, highlighting important structural factors of the cationic units in the construction of polyplex- type gene vectors.
  • the polyplex micelle for transfection with cells for example, primary osteoblasts, allows for therapeutic methods, including bone regeneration in vivo, mediated by nonviral gene transfection.
  • references in the specification to "one embodiment”, “an embodiment”, “an example embodiment”, etc., indicate that the embodiment described may include a particular feature, structure, or characteristic, but every embodiment may not necessarily include that particular feature, structure, or characteristic. Moreover, such phrases are not necessarily referring to the same embodiment. Further, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is submitted that it is within the knowledge of one skilled in the art to affect such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described.
  • block copolymer refers to a polymer with repeating units of one type adjacent to each other in a linear manner to form a block, which is linked, for example, through a covalent bond to a second block made up of repeating units of a second type, which are adjacent to one another in a linear manner to form a second block of the block copolymer.
  • the first block of the polyamide block polymer includes amino acid units derived from amino acid units with side chains that can have neutral or negative charges and the second block of the polyamide block polymer includes amino acid units derived from amino acid units with side chains that can have positive charges.
  • amino acid units derived from amino acid units with side chains that can have negative charges refers to amino acid units (repeat segments of a polyamide) whose side chains possess a group that can form an anion, for example, at a slightly basic pH, such as under mammalian physiological conditions. In some embodiments, the group can form an anion at intracellular pH. The group can also be uncharged at a neutral pH. Examples of suitable amino acids include aspartic acid, glutamic acid, and derivatives thereof.
  • the amino acid units of the first block can undergo a variety of reactions to provide multifunctional polyamides. Some side chain groups may remain as the initial unreacted side chains. Others can be converted to hydrazide groups.
  • more than 90% of the first block side chains will be converted to hydrazide groups. Many of these groups can be further reacted to provide hydrazone groups that link the amino acid unit to PEG groups, for example, by a condensation reaction between the N 1 nitrogen and an aldehyde group of a PEG chain.
  • amino acid units derived from amino acid units with side chains that can have positive charges refers to amino acid units (repeat segments of a polyamide) whose side chains possess a group that can form a cation, for example, at a slightly acidic pH, such as under certain mammalian physiological conditions.
  • the group can form a cation at certain intracellular pH ranges, such as at a pH less than 7.
  • suitable amino acids include lysine, arginine, glutamine, histadine, and derivatives thereof.
  • hydrazone refers to an N-alkylidene derivative of a hydrocarbyl derivative of a hydrazine.
  • hydrazide refers to a hydrazine that has an acyl substituent.
  • therapeutic agent refers to biologically active agents, prodrugs, or drugs, including, for example, any organic or inorganic small molecule compound (e.g., a molecule with a molecular weight of less than about 700, or less and about 500), polymeric species (including nucleic acids (D ⁇ A, such as pD ⁇ A, and R ⁇ A), proteins, peptides, hormones, carbohydrates, and derivatives thereof), lipids and mixtures thereof, wherein said drug or agent can be administered in vivo (in humans or animals) for the treatment of a disease, condition, or disorder.
  • the micelle cargo can be lactate dehydrogenase (LKD).
  • the micelle cargo can be a plasmid that encodes for an enzyme that can activate a prodrug to a drug.
  • the therapeutic agent when it is pD ⁇ A, it can either be used for expression of a prodrug activating protein, or the pD ⁇ A can include sequences that allow for transposition of a gene into a host genome. Such a method can be used for long term protein expression of, for example, a deficient gene, for treating a genetic disorder.
  • a method can be used for long term protein expression of, for example, a deficient gene, for treating a genetic disorder.
  • One significant advantage of non- viral vehicles over viral vehicles is that the micelle polyplexes described herein can encapsulate very large sized agents, such as a plasmid with about 5K or about 6K base pairs. For example, pGL3 plasmid (5280 bp) (Promega Corp.) was used as a reporter assay in various experiments.
  • the ⁇ /P 1 for pGL3 was determined by running agarose gels, defining a suitable ratio as the lowest amount of polymer it takes to retard 1 ⁇ g of pGL3 (pDNA). This ratio will of course change as other forms of DNA are selected for use in the micelles.
  • RNAi-based drugs currently in pre-clinical development include those targeting respiratory syncytial virus, hepatitis C, HIV, Huntington's disease and several other neurodegenerative disorders (Check (2005) Na/ Med 11(3): 243-244; Schmidt Knockout Punch: The Promise of RNAi. bio.com, New & Features 2005 Jun 28).
  • prodrug is a pharmacological substance that can be administered in an inactive (or significantly less active) form that the active compound that the substance is metabolized in vivo.
  • Some specific examples of prodrugs include, but are not limited to, enalapril, valaciclovir, levodopa, chloramphenicol, codeine, and gamma-butyrolactone (GBL).
  • GBL gamma-butyrolactone
  • the term “micelle” refers to a supermolecular structure having a core- shell form. Micelle formation is entropy driven and water molecules are typically excluded into the bulk phase. When above critical micelle concentration (CMC), amphiphilic portions of the polymer employed aggregate into structured micelles.
  • CMC critical micelle concentration
  • Polymeric micelles are typically spherical and can have nanoscopic dimensions in the range of about 1 to about 250 nm, typically in the 20-100 nm range. This is advantageous because circulating particles less than about 200 nm can avoid filtering by interendothelial cell slits at the spleen. Additionally, delivery vehicles of less than about 150 nm are much more efficiently taken up by cells. Polymeric micelles have been shown to circulate in the blood for prolonged periods and capable of targeted delivery of therapeutic agents, for example, nucleic acids or poorly water-soluble compounds. Upon disassociation, micelle unimers are typically ⁇ 50,000 g/mol, permitting elimination by the kidneys. These properties allow for prolonged circulation with little or no buildup of micelle components in the liver that could lead to storage diseases.
  • a "micelle composition" as used herein is any composition that includes a micelle, for example, a novel micelle as described herein.
  • PEG refers to poly(ethylene glycol) and derivatives thereof.
  • the molecular weight of the PEG chain can be about 500 to about 20,000.
  • the PEG group can have a molecular weight of about 1,000 to about 20,000, about 2,000 to about 15,000,about 3,500 to about 12,000, or about 3,000 to about 9,000.
  • the PEG groups can have a molecular weight of about 4,000 or about 7,000.
  • PEG groups can terminate in any variety of groups including hydroxy, alkyl, alkoxy, aryl, arylalkyl, amino, and the like, referred to herein as PEG-capping groups.
  • PEG-capping group refers to a group at the end of a PEG chain.
  • the PEG chain can terminate in any variety of groups including hydroxy, alkyl, alkoxy, aryl, arylalkyl, amino, and the like.
  • Other PEG-capping groups that can be used include acyl groups such as acetyl, acryloyl, and benzoyl, various silane protecting groups, benzyl groups, or heterocyclic groups, or the terminal hydroxy group of the PEG can be oxidize to an aldehyde or a carboxylic acid (i.e., the CH 2 CH 2 -R 2 group of formula I is oxidized to CH 2 CO 2 H).
  • PEG groups can also have an amino acid capping group, such as a group of 1 to about 30 amino acids.
  • the PEG capping group is melittin, a 26 amino acid membrane disrupting agent.
  • the melittin can be attached directly to a amino acid unit of the polymer backbone, allowing it to be exposed when the PEG groups of the polymer are hydrolyzed from side chain to which they are linked.
  • the amount of PEG groups relative to the number of amino acid units in a particular block can be effectively adjusted by linking branched PEG groups to the appropriate linking functional groups of the amino acid side chains.
  • organic linking group refers to a group that includes at least one carbon atom that links two other groups together, for example, two polymer blocks.
  • protecting group refers to any group which, when bound to a hydroxyl, nitrogen, or other heteroatom prevents undesired reactions from occurring at this group and which can be removed by conventional chemical or enzymatic steps to reestablish the 'unprotected' hydroxyl, nitrogen, or other heteroatom group.
  • the particular removable group employed is often interchangeable with other groups in various synthetic routes.
  • Certain removable protecting groups include conventional substituents such as, for example, allyl, benzyl, acetyl, chloroacetyl, thiobenzyl, benzylidine, phenacyl, methyl methoxy, silyl ethers (e.g., trimethylsilyl (TMS), f-butyl-diphenylsilyl (TBDPS), or f-butyldimethylsilyl (TBS)) and any other group that can be introduced chemically onto a hydroxyl functionality and later selectively removed either by chemical or enzymatic methods in mild conditions compatible with the nature of the product.
  • TMS trimethylsilyl
  • TDPS f-butyl-diphenylsilyl
  • TBS f-butyldimethylsilyl
  • These protecting groups can likewise be used to protect a nitrogen atom.
  • Typical nitrogen protecting groups described in Greene include benzyl ethers, silyl ethers, esters including sulfonic acid esters, carbonates, sulfates, and sulfonates.
  • suitable nitrogen protecting groups include substituted methyl ethers; substituted ethyl ethers; /?-chlorophenyl, /?-methoxyphenyl, 2,4-dinitrophenyl, benzyl; substituted benzyl ethers (p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, /7-halobenzyl, 2,6-dichlorobenzyl, /?-cyanobenzyl, /?-phenylbenzyl, 2- and 4-picolyl, diphenylmethyl, 5-dibenzosuberyl, triphenylmethyl.p-methoxy- phenyldiphenylmethyl,
  • GDEPT Gene Directed Enzyme Prodrug Therapy
  • GDEPT Gene Directed Enzyme Prodrug Therapy
  • a gene coding for a prodrug activating enzyme e.g. yeast cytosine deaminase
  • a specific delivery vector for example, a polyplex particle that includes a p(Asp-Hyd-PEG)-poly(L- lysine(TFA)) coating.
  • the long circulating nanoparticles extravasate across leaky tumor blood vessels into the cancer.
  • New proteins are synthesized by the nucleus that express the incorporated gene coding, e.g., the encoded enzymes.
  • a non-toxic prodrug such as 5-fluorocytosine is injected intravenously. Only cells expressing the enzyme can convert prodrugs into toxic drug molecules (e.g. 5-fluorouracil), thereby minimizing negative side effects to healthy tissues.
  • a pH-sensitive poly(Aspartate-Hydrazide-PEG)-b-poly(L-Lysine) block- copolymer particle and its release of PEG groups, according to an embodiment of the invention, is illustrated in Figure 11.
  • Poly(L-Lysine) can be used to condense pDNA in a polyplex core.
  • PEG groups are attached through the formation of hydrazone bonds to hydrazide groups, forming a variable brush layer in the shell.
  • pH ⁇ 7.0 acid labile PEG is released from the polymer, leaving behind hydrazide groups that enhance the escape of pDNA from endolysosomes via membrane disruptions.
  • the rapid release of the PEG brush layer may further enhance pDNA unpackaging.
  • the variables m and n can be about 2 to about 200, and in various embodiments, about 20 to about 100, or about 40 to about 60, depending on reaction conditions and the desired properties of the polyplex polymers.
  • the non-viral delivery vehicles described herein can furthermore deliver pDNA to cancer cells. Upon deliver of the pDNA to the cell interior, the cancer cells can then code for antigens and cytokines to illicit or enhance the body's immune response toward cancer cells.
  • Today nearly 40% of all gene therapy clinical trials have used genes coding for antigens and cytokines (using mostly viral vehicles) with about 70% of trials targeting cancer diseases.
  • Gene therapy agents that can be used in conjunction with the micelles described herein include those being studied in worldwide clinical trials.
  • RNA interference can be obtained from Ambion, Austin, TX, and from their technical resources and technical notes found at www.ambion.com.
  • Polyplex micelle (or "poly-ion complexes") can be prepared by various methods, including cosolvent evaporation methods.
  • Micelles can be prepared by mixing solutions of the cationic polymers disclosed herein and pDNA in various ratios of N/P, for which N is the total number of amine groups in the block catiomer and P represents the number of phosphate units in the pDNA.
  • N is the total number of amine groups in the block catiomer
  • P represents the number of phosphate units in the pDNA.
  • a polymer and an agent can be dissolved in a water miscible solvent system.
  • the solution can be slowly added to a vigorously stirred aqueous solution, followed by solvent evaporation.
  • the resulting composition can be nanof ⁇ ltered and/or centrifuged to remove unincorporated agent.
  • Other useful techniques for preparing micelles have been reported by Kwon and coworkers, Pharm. Res. 2004, 27, 1184-1191.
  • the ratio of the solvent phase to the aqueous phase can be adjusted to adjust the size of the resulting micelles. For example, in one embodiment, using a lower proportion of the solvent phase results in smaller micelles.
  • the ratio of solvent phase to aqueous phase can be in the range of about 1 :1 to about 1 : 10, or about 1 : 2 to about 1 :6.
  • micelle carriers can be employed for the delivery of therapeutic agents without chemically modifying the agent.
  • the structure of the polymers described herein can be tailored in order to enhance the properties of the micelles for therapeutic agent delivery. Such tailoring includes varying the amount and nature of amino acid side chain modifications, such as those described in the Examples below.
  • Micelles formed from the polymers disclosed herein allow for the PEG side-groups of the polymers to concentrated at outer portions of the micelles, referred to as the micelle corona.
  • the micelle corona is therefore hydrophilic and allows for its incorporation into cells.
  • micelle compositions include their ease of storage and delivery. Micelle compositions can be lyophilized and reconstituted before intravenous administration. This allows for a lower risk of agent precipitation, which can in some cases lead to embolism formation. Micelle compositions are capable of long blood circulation, low mononuclear phagocyte uptake, and low levels of renal excretion. Also, micelle compositions have enhanced permeability and retention (EPR) to increase the likelihood of their encapsulated therapeutics reaching their targets, for example, tumors. Tumors typically have high vascular density, as well as defective vasculature. Accordingly, high extravasation occurs and there may be impaired lymphatic clearance.
  • EPR permeability and retention
  • Polyplex micelles of various diameters can be prepared.
  • the unloaded or empty micelles can be prepared.
  • the resultant micelles can have average diameters of less than about 200 nm, or less than about 100 nm.
  • the micelles can have an average diameter of between about 55 nm and about 90 nm.
  • cumulant diameters of polyplex micelles can be about 70 nm to about 90 nm for polymer/pDNA complexes with N/P ratios of 1 to about 20.
  • the small size of polymeric micelles that have PEG coronas can help the micelle carrier to stay unrecognized, as self, in a biological system.
  • Other advantages associated with nanoscopic dimensions of polymeric micelles include the ease of sterilization via filtration and safety of administration.
  • the core of the micelles can take up, protect and retain biologically active agents, leading to improved solubility and stability of the agents in vivo, their controlled release, and overall reduced toxicity and attenuated pharmacokinetic interaction with other treatment agents.
  • Micelles can be suitably formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo.
  • a pharmaceutical composition is provided that includes micelles as described herein, in admixture with a suitable diluent or carrier.
  • the compositions containing micelles can be prepared by known methods for the preparation of pharmaceutically acceptable compositions that can be administered to subjects, such that an effective quantity of the therapeutic agent within the micelles is combined in a mixture with a pharmaceutically acceptable vehicle.
  • compositions include, albeit not exclusively, solutions of the micelles in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
  • pharmaceutical compositions can be used to enhance biodistribution and drug delivery of therapeutic agents, such as drugs.
  • the described micelles may be administered to a subject in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art.
  • the micelles of the invention may be administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump, or transdermal administration and the pharmaceutical compositions formulated accordingly.
  • Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal and topical modes of administration. Parenteral administration may be by continuous infusion over a selected period of time.
  • a micelle may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet.
  • the micelle of the invention may be incorporated with excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • a micelle may also be administered parenterally. Solutions of a micelle can be prepared in water suitably mixed with suitable excipients. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersion and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form should be sterile and should be fluid to the extent that easy syringability exists.
  • compositions for nasal administration may conveniently be formulated as aerosols, drops, gels and powders.
  • Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomizing device.
  • the sealed container may be a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after use.
  • the dosage form comprises an aerosol dispenser, it will contain a propellant which can be a compressed gas such as compressed air or an organic propellant such as fluorochlorohydrocarbon.
  • compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, wherein the active ingredient is formulated with a carrier such as sugar, acacia, tragacanth, or gelatin' and glycerine.
  • Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base such as cocoa butter.
  • the compositions described herein can be administered to an animal alone or in combination with pharmaceutically acceptable carriers, as noted above, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration and standard pharmaceutical practice.
  • the pharmaceutical compositions are administered in a convenient manner such as by direct application to the infected site, e. g. by injection (subcutaneous, intravenous, etc. ).
  • injection subcutaneous, intravenous, etc.
  • the pharmaceutical compositions or micelles or biologically active agents in the micelles of the invention may be coated in a material to protect the micelles or agents from the action of enzymes, acids, and other natural conditions that may inactivate certain properties of the composition or its encapsulated agent.
  • compositions for non- pharmaceutical purposes are also included within the scope of the present invention, such as for diagnostic or research tools.
  • the biologically active agents or micelles comprising such agents can be labeled with labels known in the art, such as florescent or radio-labels or the like.
  • the present invention includes a delivery system that can be used to deliver biologically active agents or formulations or pharmaceutical compositions.
  • the invention includes the delivery of nucleic acids.
  • the invention includes delivery of biologically active agents by loading them into micelles comprising a amphiphilic core and a hydrophilic outer surface, thus improving their delivery in aqueous mediums, such as blood and body fluids.
  • the invention includes the delivery of biologically active agents while reducing their toxicity profile.
  • the invention also includes a method for reducing aggregation of the micelle delivery vesicles of the invention. As such, it provides for better biodistribution of biologically active agents resulting in decreased toxicity and/or improved therapeutic efficacy.
  • Another aspect of the invention includes a method of delivering biologically active agents to treat a disease, condition, or disorder in a subject in need thereof comprising administering an effect amount of an agent-loaded micelle to a subject.
  • the disease, condition or disorder is cancer or drug resistant cancers, infectious disease or an autoimmune disease.
  • the dosage of the micelles of the invention can vary depending on many factors such as the pharmacodynamic properties of the micelle, the biologically active agent, the rate of release of the agent from the micelles, the mode of administration, the age, health and weight of the recipient, the nature and extent of the symptoms, the frequency of the treatment and the type of concurrent treatment, if any, and the clearance rate of the agent and/or micelle in the subject to be treated.
  • the micelles may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response. For ex vivo treatment of cells over a short period, for example for 30 minutes to 1 hour or longer, higher doses of micelles may be used than for long term in vivo therapy.
  • the micelles can be used alone or in combination with other agents that treat the same and/or another condition, disease or disorder.
  • the micelle or biologically active agent is labeled, one can conduct in vivo or in vitro studies for determining optimal dose ranges, drug loading concentrations and size of micelles and targeted drug delivery for a variety of diseases.
  • polymers and compositions described herein can be prepared by any of the applicable techniques of organic synthesis and the related arts. Many such techniques are well known to the skilled artisan. Accordingly, many of the known techniques are elaborated in, for example, Compendium of Organic
  • a polyamide chain can be prepared by methods known to those of skill in the art. These and other methods, such as those provided in the Examples below, provide for the efficient synthesis of various polyamides.
  • the amino acid side chains of the polyamides can then be modified, for example, by attaching hydrophilic groups, such as PEG chains, and PEG chain derivatives.
  • Various linkers can be used to prepare polyamides with side groups that degrade under certain physiological conditions.
  • hydrazone linkers can be used to link PEG chains to the polyamide backbone. Hydrazone linkers provide the advantage of molecular stability at neutral pH, while allowing for the hydrolysis of PEG groups via hydrazone bonds in an acidic environment.
  • the polyamide polymers of the invention are di-block copolymers.
  • this concept can be varied by attaching additional blocks to one end or the other of this di-block, for example, to form a triblock polymer.
  • the third block can be the same as one of the di-block groups, or it can be a different block, such as a physiologically acceptable poly(amino acid) of about 5 to about 150 repeating units.
  • the two blocks of the di-block polymers described herein can be separated by an organic linking group by methods well known to those of skill in the art.
  • one of the blocks of the polymer can be replaced with a octa-poly(arginine) block, or one or more octa-poly(arginine) units can be conjugated to the polymer through a hydrazide group to provide additional cell-penetrating peptide properties to the polymer.
  • a hydrazide group to provide additional cell-penetrating peptide properties to the polymer.
  • PEG Poly(ethylene glycol)
  • 1"5 In vivo studies in mice have demonstrated that PEGylated-PEI25 complexes are taken into RES organs less than PEI25. However, PEG minimizes interactions with cells and can reduce transfection efficiency. 7 Wagner et al. have recently shown already that pH-triggered deshielding of PEG from PLL resulted in better in vivo gene transfer in mice compared to polyplexes with covalent linkages of PEG.
  • the second block containing hydrazine groups will remain exposed to the outer-particle environment at this pH; thus polymers containing PEG "brushes" for pre- and post-PEGylation of polyplexes can be prepared.
  • This approach will allow for the formation of more stable polyplexes for in vivo delivery because pre-PEGylation may hinder the formation of well- packed polyplexes for pH-sensitive release of PEG from polyplexes after endocytosis of the complexes.
  • the pH-triggered release of PEG from complexes in endosomal compartments will aid in unpackaging the pDNA from the polymer and facilitate therapeutic agent, e.g., pDNA, escape into the cytoplasm. See for example, the schematic diagram shown in Figure 1.
  • BLA-NCA PBLA ⁇ -Benzyl L-aspartate N-carboxyanhydride (“BLA- ⁇ CA”) was synthesized by the Fuchs-Farthing method.
  • Three grams of BLA- ⁇ CA (12 mM, MW 249 g/mol) was dissolved in an anhydrous cocktail mixture of 1:10 DMF to CH 2 Cl 2 (40.5 mL CH 2 Cl 2 , 4.5 mL DMF) under vacuum.
  • the flask was quickly moved to a 35 0 C water bath with a stir bar to react for 24 hours.
  • PBLA poly( ⁇ -benzyl L-aspartate), or "PBLA”
  • PBLA poly( ⁇ -benzyl L-aspartate)
  • the final product poly( ⁇ -benzyl L-aspartate), or "PBLA” was precipitated from cold ether and the pellet of fine crystals was collected by centrifugation (10,000 RPM at 4 0 C, 10 minutes). After carefully pouring out the supernatant, a small amount Of CH 2 Cl 2 was added to the white crystal pellet to aid dissolution and the solution was transferred to a new flask where 100 mL of benzene was added for overnight freeze-drying. Degree of polymerization of PBLA was checked by 1 H-NMR measurement in DMSO-ds at 80 0 C with respect to the methyl peak of butylamine (CH 3 -: ⁇ 0.8). 2.2.
  • R CF 3 or OBn p(Asp-Hyd-PEG)-PLL(TFA/Z)
  • R CF 3 or OBn Varying molar ratios of 1:1, 1:0.5 and 1:0.1 p(Asp-Hyd)-PLL(TFA) to CHO-PEG7K-COCH 3 were reacted together in anhydrous DMSO. Assuming 100% hydrazine substitution of PBLA-PLL(TFA), 240 ⁇ L of p(Asp-Hyd)- PLL(TFA) (stock concentration 10 mg/mL in DMSO) was respectively reacted with 672 ⁇ L, 336 ⁇ L and 68 ⁇ L of activated PEG7k (stock concentration 50 mg/mL in DMSO).
  • PBLA Poly( ⁇ -benzyl L-aspartate), or "PBLA”
  • BLA-NCA Poly( ⁇ -benzyl L-aspartate), or "PBLA”
  • the ratio of butylamine initiator to use was in proportion to the desired chain length of PBLA. In this case, theoretical calculations were made for a chain length of 35 and hence a 1:35 molar ratio of initiator to BLA-NCA was used.
  • BLA-NCA is quite sensitive to moisture and it is important to seal the system under inert argon gas and/or store under vacuum to ensure good polymerization results.
  • NCA's are hydrophobic molecules and the best solvent for solubilizing various types of NCA's will vary but in the case of BLA-NCA, a suitable solvent for the reactions was anhydrous CH 2 Cl 2 .
  • the actual degree of polymerization obtained for PBLA was calculated by analyzing its 1 H-NMR spectra (DMSOd 6 at 80 0 C) and integrating peaks with respect to the methyl group present on PBLA that originated from butylamine (CH 3 -: ⁇ 0.8). From this integration, the peak intensity ratio of the phenyl protons on PBLA ( ⁇ 7.2) revealed the degree of polymerization of PBLA to be 36. This allowed calculation of an exact molecular weight of 7,453 g/mol for PBLA. From 3 grams of starting BLA-NCA, 2.204 grams of PBLA 36 were obtained, compared to a calculated theoretical yield of 2.4 grams (92% yield).
  • PBLA 36 -PLL(Z) Poly( ⁇ -benzyl L-aspartate)-poly(l-lysine(Z))
  • PBLA 36 -PLL(TFA) Poly( ⁇ -benzyl L-aspartate)-poly(l-lysine(TFA))
  • PBLA 36 -PLL(TFA) Poly( ⁇ -benzyl L-aspartate)-poly(l-lysine(TFA))
  • hydrazine The pKa of hydrazine is approximately 3-4 versus that of lysine, which is closer to 10.
  • pH near endosomal pH of 4-5
  • both hydrazine and lysine terminal -NH 2 would be protonated and formation of a Schiff base with activated aldehyde-PEG would be hindered.
  • hydrazine moieties would being to deprotonate and only PLL should carry a charge on its terminal amine. This is ideal because it is desireable for PLL to condense DNA at physiologic pH, exposing the hydrazine groups.
  • two types of PBLA 36 -PLL(X) block-copolymers were prepared to investigate effects of deprotection on hydrazine reactivity to aldehyde-PEG.
  • PBLA was used to initiate the ring opening polymerization of NCA- Lys(Z) and NCA-Lys(TFA).
  • a target degree of polymerization of 55 was attempted for both types of PLL chains and appropriate PBLA as initiator was used.
  • the reaction was allowed to proceed for 20 hours at which point an IR spectra was taken to confirm that polymerization had ended.
  • the degree of polymerization was determined by integration with respect to the methyl group of butylamine (CH 3 -: ⁇ 0.79), followed by analysis of the peak intensity ratio of the three lysine methylene groups (-CH 2 -: ⁇ 1.32, 1.92, 2.45).
  • CH 3 -: ⁇ 0.79 the degree of polymerization was 49.
  • a PDI of 1.038 was obtained and for Ace-PEG7K-NH 2 a PDI of 1.027 was obtained.
  • a 1 H-NMR spectra of each Ace-PEG4k/PEG7k-NH2 was taken in D 2 O at 25 0 C to determine actual molecular weight. PEG4k was found to be about 4K and PEG7k was found to be about 7K.
  • the ⁇ -terminal amine of heterobifunctional poly(ethylene glycol) was acetylated and the acetal group was deprotected into an aldehyde to yield activated aldehyde PEG. Because the activated PEG is quite reactive, there is possibility of polymer coupling to form dimers. GPC of activated PEG4k and PEG7k, however, revealed almost unimodal curves. Mobile phase was 10 mM LiCl in DMF at a flowrate of 0.8 mL/min. From analysis of GPC runs of the activated PEG4k, about 4.8% of the reaction formed dimers and for activated PEG7k, about 2.1% formed dimers.
  • Poly(Asp-Hyd)-PLL(TFA) was pre-PEGylated at three different molar ratios of hydrazines to PEG7k. Assuming that all 36 monomers of PBLA reacted with hydrazine, equivalent molar ratios of PEG7k for 1 : 1 , 1 :0.5 and 1 :0.1 were calculated.
  • organic solvents CH 2 Cl 2 , DMF, AcN, DMSO, MeOH
  • the reactions were allowed to proceed with shaking at RT for up to 5 days.
  • a GPC of the reactions was taken at day 2 and day 5 to monitor extent of pre-PEGylation over time.
  • the GPC solvent used was 10 mM LiCl in DMF, which was suitable but a more different solvent or solvent system may solubilizing these hydrophobic block-copolymers in a more optimal manner.
  • poly(Asp-Hyd)-poly(L-lysine(TFA)) and p(Asp-Hyd)-poly(L- lysine(Z)) have been successfully synthesized. It has been shown, using p(Asp- Hyd)-poly(L-lysine(TFA)), that hydrazide groups on a block-copolymer can interact with PEG-aldehydes to form an acid-sensitive Schiff bases (pre- PEGy lation strategy for forming polyplexes).
  • yeast cytosine deaminase has shown potential in activating the prodrug 5-fiuorocytosine (5FC) into cytotoxic 5- fluorouracil (5FU) in several tumor models.
  • yCD's instability limits its bolus delivery in enzyme-prodrug type applications.
  • a degradable poly(amino acid) block-copolymer poly(aspartatehydrazide)-poly(L-lysine) with acid-labile PEG attached through the formation of Schiff bases to hydrazide groups was synthesized. PEG provides 'stealth properties' to the complex at physiologic pH, minimizing immunogenic responses and prolonging systemic circulation.
  • PEG release is pH-triggered from the polymer under acidic conditions (pH ⁇ 7).
  • the yCD gene was subcloned into the Hermes HRIgfp vector for regulation by a transactivator (RetroTet RTAb(+)) that binds to its promoter only in the presence of tetracycline.
  • the results from this study provide valuable data supporting the low toxicity effects to cells in the presence of PEG and the importance of PEG release inside the cells for greater gene expression.
  • yeast cytosine deaminase has shown potential in activating the therapeutic prodrug 5-fluorocytosine (5FC) into cytotoxic 5-fluorouracil (5FU) in several tumor models.
  • yCD's instability limits its bolus delivery in prodrug-type applications. Therefore, this Example is directed to enhancing the delivery of the corresponding nucleic acid to the tumor site through a novel non-viral gene carrier system.
  • the design of a pH-sensitive poly(aspartatehydrazide-PEG)-poly(L-lysine) block-copolymer addresses these issues.
  • enzyme-prodrug therapy A promising two-step therapy approach for tackling the shortcomings of chemotherapy was revived in 1987 by Bagshawe et al. and is generally known as enzyme-prodrug therapy (see Br. J. Cancer 1987, 56:531-532).
  • the core idea behind enzyme-prodrug therapy is to deliver a specific enzyme that will synergistically activate an explicit prodrug in tumor cells.
  • the nontoxic prodrug is systemically injected only after sufficient time has been allowed for the enzyme to localize at the tumor site. Only prodrug molecules in the tumor vicinity are activated, hence minimizing normal cell exposure to toxic drugs.
  • GDEPT gene-directed enzyme prodrug therapy
  • the S. cerevisiae yeast cytosine deaminase (yCD) enzyme was cloned and expressed.
  • the resulting 474 bp gene was subcloned into pET-15b to facilitate purification of the protein via a fused histidine tag and transformed into competent BL21(DE3) cells for protein expression.
  • An estimate of about 25-30 mg of pure yCD-His was collected from a 1 -liter culture.
  • Thrombin cleavage of the histidine tag on yCD-His was achieved by incubating the protein for 2 hours at 23 0 C in thrombin cleavage buffer.
  • Enzyme activity characteristics for yCD and yCD-His was investigated using both the prodrug 5FC and the enzyme's natural substrate, the DNA base cytosine. Five-FC turnover rates remained within 10 5 M *1 • s "1 (Table 1 ) for both proteins (see E. Kievit et al. Cancer Res. 2000, 60:6649-6655). The diffusion controlled limit for extremely efficient enzymes and their substrates in solution is in the range 10 8 -10 M " • s " , therefore it was concluded that yCD was a very efficient pretargeting enzyme for catalyzing 5FC to 5FU conversion, especially applicable to GDEPT. Table 1 shows enzyme activity of yCD and His-yCD, with average values ⁇ SE, with the number of experiments shown in parentheses.
  • Cytosine (4) 3.75 ⁇ 0.69 741 1.98 X 10 5 a Amount of enzyme used was 10 uL drawn from a stock of 4.52 ug/mL b Amount of enzyme used was 10 uL drawn from a stock of 4.23 ug/mL
  • PEG block-copolymer e.g., poly(Asp-Hyd-PEG)-poly(L- lysine) of Example 1, successfully mediates gene delivery at about the same order of magnitude as the BC but shows dramatically less toxicity in C2C12 and MDA-MB-231 cell lines, especially evident at higher N/P ratios 70 to 100.
  • Figure 3 illustrates the transfection efficiency of PEI25, BC, and PBC at N/P ratios of 70-100 in MDA-MB-231 cells.
  • the yCD gene was subcloned into the Hermes HRIgfp vector for regulation by a transactivator (RetroTet RTAb(+) or RetroTet RtAb(-)) that will bind to its promoter only in the presence or absence of tetracycline (see A.M. Kringstein et al. Proc. Natl. Acad. ScL USA 1998, 95, 13670-13675). This allows controlled studies of yCD expression in cells when looking at 5FC or 5FU toxicities.
  • Figure 8 illustrates (A) expression of yCD: Coomassie-stained gel of the crude lysate after induction of yCD at 28°C with 0.4 mM IPTG showing, from left to right, crude protein extract, 1, 1.5, and 2.5 hrs after induction; and (B) a Western blot of crude protein extracts probed with Tetra-His conjugate at 1 : 1500 dilution followed by horse-radish peroxidase.
  • Figure 9 illustrates transfection activity of PEI-25, the block copolymer (BC), and PLL at increasing equivalent N/P ratios and toxicity profiles of the free polymers in serum media for (A) C2C12; and (B) MDA-MB 231.
  • Example 3 Poly(Asp-Hyd-PEG)-b-PLL Purification An ion exchange column was used to purify the poly(Asp-Hyd-PEG)-b-
  • the ionic strength of the wash was kept low (50 mM phosphate, pH 8.0) with the flowthrough (unreacted PEG) being collected while the cationic polymer with PEG remains remained on the column.
  • the polymer was collected by using a high ionic strength buffer (50 mM phosphate, 2 M NaCl, pH 8.0) and was dialyzed against 50 mM phosphate (pH 8.0) to remove the salt, before lyophilizing. NMR was used to estimate the average degree of PEG substitution on each polymer chain.
  • Example 4 Polyplex Formation with p(Asp-Hyd)-poly(L-lysine(TFA/Z)) Post-PEGylation can be carried out using variously protected block- copolymers or by deprotecting the block-copolymer first followed by formation of a polyplex.
  • the block-copolymers may be suitably soluble in an aqueous environment once deprotected. Suitable solubility is advantageous because polyplexes form via electrostatic interactions with various therapeutic agents, e.g., DNA, which does not occur in an optimal manner in many organic solvent systems.
  • the intelligent design and characterization of dynamic cationic nanoparticles formed from the self-association of a pH-responsive block copolymer is described in this Example.
  • the platform allows pH-sensitive multilevel PEGylation and imparts nanoparticles with the ability to transition from neutral to charged at a pH of about 5.
  • the block copolymer has negligible toxicity elsewhere to the cell.
  • the poly(L-lysine) segment with a high pK a value of ⁇ 9.4, preferentially forms a poly-ion complex with the negative phosphate groups of pDNA, whereas the pH-responsive poly(aspartate-hydrazide) segment, with the comparatively lower pK a ⁇ 5.0, is characterized by a substantial fraction of unprotonated amino groups at physiological pH.
  • complexation between such a polymer and pDNA leads to the formation of a two-layered nanoparticle.
  • the nanoparticle possesses an unprotonated pH-responsive segment to serve as both a scaffold for acid-labile linkages of various moieties, such as aldehyde-PEG, and to transition from neutral to charged for disrupting endosomal membranes, and safely enhancing gene expression.
  • This system supports an endosomal escape mechanism based on charge interactions rather than the proton-sponge effect, providing a new classes of nonviral vectors.
  • Figure 13 shows the chemical structure of the dual-cationic block copolymer, poly(aspartate-hydrazide)-W ⁇ c&-poly(L-lysine) (abbreviated BC), based on functional pKa differences between respective cationic blocks.
  • the poly(L-Lys) (PLL) segment with a high pK a value of ⁇ 9.4, preferentially forms a poly-ion complex (PIC) with the negative phosphate groups of pDNA, whereas the pH-responsive poly(Asp-Hyd) segment, with a comparatively lower pK ⁇ value of -5.0, is characterized by a substantial fraction of unprotonated amino groups at physiological pH (Figure 13a).
  • Asp-Hyd residues are coupled to aldehyde-PEG chains (ALD-PEG) through acid-labile hydrazone linkages to impart favorable stealth properties to the PIC ( Figure 13b).
  • ALD-PEG aldehyde-PEG chains
  • Acid-labile hydrazone linkages to impart favorable stealth properties to the PIC ( Figure 13b).
  • the complexation between such a polymer and pDNA may lead to the formation of a two-layered particle possessing an unprotonated pH-responsive poly(Asp-Hyd) segment that functions as both a scaffold for acid-labile linkages of various moieties, and that has the ability to undergo a transition from neutral to charged for disrupting endosomal membranes, as illustrated in Figure 14.
  • the PIC described herein is formed from associations of such block copolymer chains: a) a pDNA condensing core made of PLL chains (known for not contributing to the PSE), and b) a shell composed of segments with repeating Asp-Hyd residues.
  • the backbone of the block copolymer is derived from poly( ⁇ -benzyl-L- aspartate)-6/oc ⁇ :-poly(L-lysine) (PBLA-6-PLL; see Example 1).
  • PBLA was prepared by the ring-opening polymerization of ⁇ -benzyl-L-aspartate N- carboxyanhydride (BLA-NCA), initiated by the terminal -NH 2 group of butylamine to yield a polymer with narrow distribution and degree of polymerization (DP) of 36.
  • the final PEGylated block copolymers [poly(Asp-Hyd-PEG)-6-PLL] had either about five ALD-PEG chains conjugated to each poly-(Asp-Hyd) block to form the acid-labile hydrazone linkage (pH-PBC), or about six COOH-PEG chains conjugated covalently by amide linkages to hydrazide groups to generate the non-hydrolysable control (cov-PBC), as shown in Figure 13. There are substantial unconjugated hydrazide groups that remain on each block copolymer chain. 2.2. Physical characterization
  • Ri-NH 2 R 2 -NH R 3 -N commercial PEI 25,000 323 3U ⁇ 29 ⁇ 7 p(Asp-Hyd) 36 -6-PLL 4 9 11,000 100 n/a n/a commercial poly(L-lysine) 9,200 100 n/a n/a
  • PEG-shielded polymers exhibited low metabolic toxicity.
  • PICs with concealed cationic PLL cores BC, pH-PBC, and cov-PBC
  • gave little evidence of metabolic toxicity to the cell, in contrast to PEI-formulated particles Figure 20b).
  • the pDNA encoding firefly luciferase ( ⁇ GL3, 5.3 kb) was obtained from Promega (Madison, WI, USA), transformed into electrocompetent DH5 ⁇ cells, propagated in LB broth (1 L) supplemented with ampicillin (100 ⁇ g mL "1 ), and purified with a plasmid Maxiprep kit (BioRad, Hercules, CA, USA). All pDNA had purity levels of 1.8 or greater by UV/Vis (A 2 ⁇ o/A 28 o).
  • Dulbecco's modified Eagle's medium DMEM
  • RPMI 1640 phosphate buffered saline
  • FBS fetal bovine serum
  • trypsin-EDTA 0.25% trypsin, 2.21 mm EDTA in Hank's balanced salt solution (HBSS)
  • penicillin / streptomycin purchased from Cellgro (Mediatech, Herndon, VA, USA).
  • Metabolic toxicity was assessed through the resazurin dye (Sigma-Aldrich, Milwaukee, WI, USA) by incubating the respective polymers (free or complexed with 1 ⁇ g pGL3) in serum- containing media and monitoring the toxic effects of the polymers on the metabolic rates of cells through reduction of the dye. Briefly, 96-well plates were seeded at 20000 cellswell "1 and incubated for 24 hours. Free polymer at increasing concentrations of 0-50 ⁇ g mL 1 was added to wells and incubated in serum-containing media for 4 hours before refreshing the media.
  • MDA-MB-231 human breast cancer
  • C2C12 murine myoblast
  • MCF-7 human breast cancer
  • Protein content was obtained using the DC protein assay kit from BioRad (Hercules, CA, USA), absorbance was measured with the Spectramax 190 microplate reader (Molecular Devices, Sunnyvale, CA, USA), and results were fit to a known protein calibration curve.
  • Cells were seeded at either 300000 cells well "1 in 6-well plates or 150000 cells well "1 in 12-well plates and cultured for 24 hours. The next day, wells were aspirated, washed with PBS (2 x 1 mL), and appropriate medium for each cell line was added (supplemented with 10% FBS and 1 % penicillin/streptomycin).
  • RLUs Luciferase relative light units
  • the degree of polymerization (DP) of PBLA was checked with 1 H-NMR in DMSO-d 6 at 80 0 C by integrating the phenyl protons (-CH 2 C 6 H 5 -: ⁇ 7.3 ppm) with respect to the methyl peak of butylamine (CH 3 -: ⁇ 0.8 ppm) and found to be 36 with a polydispersity index (PDI) of 1.20 (TSK-gel G3000PWXL and TSK-gel G4000PWXL; 10 mM LiCl in DMF; 0.8 mL/min).
  • PDI polydispersity index
  • the terminal amine of PBLA was used to initiate the ring opening polymerization of NCA-Lys(TFA).
  • a target DP of 55 was attempted for the PLL chain, and appropriate amounts of PBLA as initiator was used.
  • the final composition of the block-copolymer was checked with IR, GPC and TLC. 1 H- NMR was taken in DMSO-d ⁇ at 80 0 C by integrating with respect to the methyl group of butylamine (CH 3 -: ⁇ 0.8 ppm) and then looking at the peak intensity ratio of the three lysine methylene groups (-CH 2 -: ⁇ 1.26, 1.56, 1.95 ppm).
  • CH 3 -: ⁇ 0.8 ppm the peak intensity ratio of the three lysine methylene groups
  • the deprotection method was done in MeOHZH 2 O (20: 1) using K 2 CO 3 as a base catalyst.
  • the polymer was slightly insoluble in MeOH/H 2 O due to the hydrophobic PLL(TFA) and free hydrazide groups (despite presence of PEG) and DMSO was added as needed to improve the solubility of the block-copolymer.
  • the reaction proceeded for 3 hrs under reflux at 60 0 C.
  • the expected product would have a molecular weight of 51 ,500 g/mol, hence it was filtered against water 3x using Millipore Amicon Ultra centrifugal filter devices (MWCO 30,000 g/mol) and the filtrate containing the deprotected pH-sensitive polymer was lyophilized.
  • This Example provides several combinations of enzyme/prodrug systems that can be used for GDEPT (Table 3). It is worthwhile to note that most clinical trials of GDEPT have utilized viral vectors as gene delivery vehicles, but the success of these enzyme/prodrug combinations could very well translate to other carriers, especially as progress is being made in the non- viral field of gene delivery.
  • HSV-tk/ganciclovir The most common gene used for GDEPT is the Herpes Simplex Virus type-1 gene which codes for the thymidine kinase enzyme (HSV-tk) [21] . When the gene is transfected into tumor cells, it renders them sensitive to the prodrug ganciclovir (GCV). This is the only GDEPT system that has reached Phase III clinical testing f22] and is commonly investigated for treating malignant gliomas [23 ' 24 ⁇ Fusion proteins of GFP and HSV-tk reveal that the enzyme is localized mostly in the nuclei [25] .
  • GCV prodrug ganciclovir
  • Cells expressing HSV-tk are capable of monophosphorylating GCV, which in turn gets triphosphorylated by cellular kinases, and induces cell death by incorporating into the DNA. It was found that the bystander effect of GCV was not due to passive diffusion but due to transfer through cellular gap junctions between cells [26] . GCV appeared to work better during S phase of mitosis when the cells were actively dividing, therefore this property could be a drawback in slower dividing hypoxic regions of a tumor [22) .
  • CD/5FC Cytosine deaminase
  • CD Cytosine deaminase
  • 5FC non-toxic antifungal agent 5-fluorocytosine
  • 5FU toxic 5-fluorouracil
  • the drug inhibits synthesis of DNA or RNA when its metabolite is incorporated into each respective chain, and the drug can also irreversibly bind to thymidylate synthase (an enzyme critical for DNA synthesis).
  • Fungal CD has superior turnover rates both for the DNA base cytosine and 5FC, compared to bacterial CD [16] , but is reportedly less stable [27] .
  • 5FC and 5FU are small non-polar molecules, they can therefore freely diffuse across cell membranes, generating a good bystander effect.
  • the toxicity of 5FU is not cell-cycle specific and the antitumor effect of CD/5FC has been demonstrated in a variety of tumor systems [22] .
  • a colon cancer model revealed that when 4% of cells expressed CD, it was sufficient to give a 60% cure rate in tumor-bearing animals, compared to the HSV-tk/GCV system where 50% of the cells had to be expressing to give the same cure rate [28] .
  • CE/CPT-11 Carboxylesterase (CE) can hydrolyze esters into acids and carbamates into alcohols.
  • the enzyme is endogenously expressed as two major isoforms in the human liver, and is also present in tumor tissues [30] .
  • the rabbit liver analogue of CE is reported to be up to 1000-fold more efficient than human CE at converting the prodrug irinotecan (7-ethyl- 10[4-( 1 -piperidino)- 1 - piperidno] carbonyloxy- camptothecin, CPT-11) into the potent topoisomerase I inhibitor SN-38 (7-ethyl- 10-hydroxycamptothecin) [31'33] .
  • the SN-38 inhibitor binds to the topoisomerase I enzyme (which cleaves, unwinds and ligates DNA strands) and prevents the DNA from re-ligating after being unwound.
  • CPT-11 demonstrates good bystander effects [34 ⁇ , which makes it suitable for GDEPT.
  • the CE/CPT-11 conversion efficiency was evaluated in 59 patients undergoing surgery for treatment of liver or colorectal cancers in France to monitor prodrug conversion in normal vs. tumor tissues [24J . In 12 out of 53 patients, a ratio > 5 for prodrug conversion in the tumor with respect to normal tissue was observed. This system shows promise in GDEPT since successful delivery and expression of rabbit CE may further increase prodrug/drug conversion at the tumor site [33] .
  • bGlu/HMR-1826 This is an enzyme found in prokaryotes and eukaryotes.
  • beta-glucuronidase (bGlu) is also expressed at higher levels in tumor lysosomes [15> 36) .
  • the enzyme can hydrolyze beta-glucuronic linkages from glycosaminoglycans and is highly specific for the glucuronyl residue t37] , hence the linker is useful in the design of various prodrugs such as doxorubicin (HMR- 1826), paclitaxel or 5FU [33> " "40 I Most prodrugs tend to be cell permeable for conversion into the active drug inside the cell, where the enzyme is being generated. However, HMR- 1826 is hydrophilic and therefore cell impermeable.
  • NTR/CB1954 Most of the GDEPT studies with this enzyme have used the NfsB gene coding for nitroreductase (NTR) from E. CoIi [42] .
  • the enzyme requires a flavin mononucleotide (a cofactor derived from vitamin B2) to efficiently reduce quinones and aromatic nitrogen groups into hydroxylamines [43] .
  • NTR has broad substrate specificity for nitrogen mustards and carbamates [44] , making this enzyme attractive for the design and activation of a variety of prodrugs.
  • CB 1954 is a prodrug that is efficiently reduced by NTR into the corresponding hydroxylamine, which is further metabolized by cellular kinases into the toxic agent capable of crosslinking DNA.
  • CB 1954 demonstrates good bystander effect [45] .
  • the first clinical trial for this system was reported in 2004 when 18 patients with liver or colorectal cancers were treated by direct intratumoral administration of a replicant-deficient adenovirus vector coding for NTR l46] .
  • the study revealed that high expression of NTR was well-tolerated by patients.
  • GDEPT in cancer therapy has been clinically tested over the years using a variety of enzyme/prodrug combinations. Certain combinations appear to sufficiently kill cancer cells, and each has its advantages. However, they all aim to improve the therapeutic efficacy of a lethal drug by minimizing non-specific tissue toxicities.
  • the micelles of the invention overcome these obstacles to provide a significantly improved method of delivering these drugs than current methods.
  • HSV-tk thymydine kinase
  • CD cytosine deaminase
  • CE carboxylesterase
  • bGlu beta-glucuronidase
  • N77? nitroreductase
  • Enzyme/prodrug gene therapy comparison of cytosine deaminase/5-fluorocytosine versus thymidine kinase/ganciclovir enzyme/prodrug systems in a human colorectal carcinoma cell line. Cancer research 55(21 ), 4808-4812.
  • Gene-directed enzyme prodrug therapy quantitative bystander cytotoxicity and DNA damage induced by CB 1954 in cells expressing bacterial nitroreductase. Gene Ther 5(1), 105-112.
  • Glucuronidase from Escherichia coli as a gene-fusion marker.

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Abstract

L'invention concerne des composés et des compositions de polyamide, ainsi que des procédés pour leur préparation et des procédés pour leur utilisation. L'invention fournit également des compositions de micelle qui comprennent des agents thérapeutiques encapsulés, par exemple des acides nucléiques ou des promédicaments, et des procédés pour leur préparation. L'invention fournit en outre des procédés d'administration d'agents thérapeutiques à des cellules, et des procédés d'activation de promédicaments thérapeutiques.
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Cited By (8)

* Cited by examiner, † Cited by third party
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WO2010006194A3 (fr) * 2008-07-10 2010-08-05 Baxter International Inc. Modification non covalente de microparticules et leur procédé de préparation
WO2010131015A1 (fr) * 2009-05-15 2010-11-18 Almac Sciences (Scotland) Limited Procédé d'étiquetage d'interférons par glycol polyéthylénique (peg)
US20110142886A1 (en) * 2009-12-01 2011-06-16 Intezyne Technologies, Incorporated Pegylated polyplexes for polynucleotide delivery
US20120148631A1 (en) * 2009-12-01 2012-06-14 Intezyne Technologies, Incorporated Pegylated polyplexes for polynucleotide delivery
US8383136B2 (en) 2009-09-25 2013-02-26 Wisconsin Alumni Research Foundation Micelle encapsulation of therapeutic agents
EP3252068A2 (fr) 2009-10-12 2017-12-06 Larry J. Smith Procédés et compositions permettant de moduler l'expression génique à l'aide de médicaments à base d'oligonucléotides administrés in vivo ou in vitro
JP2018188642A (ja) * 2008-11-25 2018-11-29 エコール ポリテクニク フェデラル ド ローザンヌ（エーペーエフエル） ブロックコポリマーおよびその使用
EP3505555A4 (fr) * 2016-08-23 2020-04-29 Kawasaki Institute of Industrial Promotion Polymère, procédé de production de polymère et conjugué de médicament

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JP2002515932A (ja) * 1997-04-18 2002-05-28 カリフォルニア インスティチュート オブ テクノロジー 多機能性ポリマー性組織コーティング
JP2007526220A (ja) * 2003-06-05 2007-09-13 ジェネンテック・インコーポレーテッド Ｂ細胞疾患の併用療法
MX2007012157A (es) * 2005-04-01 2008-03-14 Intezyne Technologies Llc Micelas polimericas para suministro de farmacos.

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010006194A3 (fr) * 2008-07-10 2010-08-05 Baxter International Inc. Modification non covalente de microparticules et leur procédé de préparation
JP2018188642A (ja) * 2008-11-25 2018-11-29 エコール ポリテクニク フェデラル ド ローザンヌ（エーペーエフエル） ブロックコポリマーおよびその使用
WO2010131015A1 (fr) * 2009-05-15 2010-11-18 Almac Sciences (Scotland) Limited Procédé d'étiquetage d'interférons par glycol polyéthylénique (peg)
US8383136B2 (en) 2009-09-25 2013-02-26 Wisconsin Alumni Research Foundation Micelle encapsulation of therapeutic agents
US8529917B2 (en) 2009-09-25 2013-09-10 Wisconsin Alumni Research Foundation Micelle encapsulation of a combination of therapeutic agents
US8858965B2 (en) 2009-09-25 2014-10-14 Wisconsin Alumni Research Foundation Micelle encapsulation of a combination of therapeutic agents
EP3252068A2 (fr) 2009-10-12 2017-12-06 Larry J. Smith Procédés et compositions permettant de moduler l'expression génique à l'aide de médicaments à base d'oligonucléotides administrés in vivo ou in vitro
EP4089169A1 (fr) 2009-10-12 2022-11-16 Larry J. Smith Procédés et compositions permettant de moduler l'expression génique à l'aide de médicaments à base d'oligonucléotides administrés in vivo ou in vitro
US20110142886A1 (en) * 2009-12-01 2011-06-16 Intezyne Technologies, Incorporated Pegylated polyplexes for polynucleotide delivery
US20120148631A1 (en) * 2009-12-01 2012-06-14 Intezyne Technologies, Incorporated Pegylated polyplexes for polynucleotide delivery
EP3505555A4 (fr) * 2016-08-23 2020-04-29 Kawasaki Institute of Industrial Promotion Polymère, procédé de production de polymère et conjugué de médicament
EP3971227A1 (fr) * 2016-08-23 2022-03-23 Kawasaki Institute of Industrial Promotion Polymère, procédé de production de polymère et conjugué de médicament

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