WO2013055971A1 - Polymères pour administrer une substance dans une cellule - Google Patents

Polymères pour administrer une substance dans une cellule Download PDF

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WO2013055971A1
WO2013055971A1 PCT/US2012/059828 US2012059828W WO2013055971A1 WO 2013055971 A1 WO2013055971 A1 WO 2013055971A1 US 2012059828 W US2012059828 W US 2012059828W WO 2013055971 A1 WO2013055971 A1 WO 2013055971A1
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polymer
aminoglycoside
diglycidyl ether
diepoxide
polymers
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PCT/US2012/059828
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English (en)
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Kaushal Rege
Thrimoorthy Potta
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Arizona Board Of Regents For And On Behalf Of Arizona State University
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Publication of WO2013055971A1 publication Critical patent/WO2013055971A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/7036—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • 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
    • A61K47/54—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 compound
    • A61K47/55—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 compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A61K47/552—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 compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being an antibiotic
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D303/00—Compounds containing three-membered rings having one oxygen atom as the only ring hetero atom
    • C07D303/02—Compounds containing oxirane rings
    • C07D303/12—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms
    • C07D303/18—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms by etherified hydroxyl radicals
    • C07D303/20—Ethers with hydroxy compounds containing no oxirane rings
    • C07D303/22—Ethers with hydroxy compounds containing no oxirane rings with monohydroxy compounds
    • C07D303/23—Oxiranylmethyl ethers of compounds having one hydroxy group bound to a six-membered aromatic ring, the oxiranylmethyl radical not being further substituted, i.e.
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D303/00—Compounds containing three-membered rings having one oxygen atom as the only ring hetero atom
    • C07D303/02—Compounds containing oxirane rings
    • C07D303/12—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms
    • C07D303/18—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms by etherified hydroxyl radicals
    • C07D303/20—Ethers with hydroxy compounds containing no oxirane rings
    • C07D303/24—Ethers with hydroxy compounds containing no oxirane rings with polyhydroxy compounds
    • C07D303/26—Ethers with hydroxy compounds containing no oxirane rings with polyhydroxy compounds having one or more free hydroxyl radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D303/00—Compounds containing three-membered rings having one oxygen atom as the only ring hetero atom
    • C07D303/02—Compounds containing oxirane rings
    • C07D303/12—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms
    • C07D303/18—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms by etherified hydroxyl radicals
    • C07D303/20—Ethers with hydroxy compounds containing no oxirane rings
    • C07D303/24—Ethers with hydroxy compounds containing no oxirane rings with polyhydroxy compounds
    • C07D303/27—Ethers with hydroxy compounds containing no oxirane rings with polyhydroxy compounds having all hydroxyl radicals etherified with oxirane containing compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D303/00—Compounds containing three-membered rings having one oxygen atom as the only ring hetero atom
    • C07D303/02—Compounds containing oxirane rings
    • C07D303/12—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms
    • C07D303/18—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms by etherified hydroxyl radicals
    • C07D303/28—Ethers with hydroxy compounds containing oxirane rings
    • C07D303/30—Ethers of oxirane-containing polyhydroxy compounds in which all hydroxyl radicals are etherified with oxirane-containing hydroxy compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
    • C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
    • C07D407/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • Viruses are effective at delivering nucleic acids into cells. When used as a vector, viruses raise concerns regarding safety, immunogenicity, repeated dosage, and viral degradation. Manufacturing scale-up issues have motivated the investigation of nonviral approaches for delivering a substance, such as a nucleic acid into a cell.
  • a variety of nonviral delivery vehicles have been used.
  • anionic substances or substances that can be made anionic such as therapeutic agents, peptides, polynucleic acids, and the like
  • cationic polymers are particularly useful.
  • cationic polymers can deliver exogenous DNA to cells and enhance the efficacy of virus-mediated gene transfer.
  • a few examples of known cationic polymers used to deliver genes into cells include poly(L- lysine), poly(ethylene imine), chitosan, polyamidoamine or PAMAM dendrimers and poly(vinyl pyrrolidone).
  • the disclosed subject matter in one aspect, relates to polymers of aminoglycosides that are useful for delivering a substance, such as a nucleic acid, into a cell.
  • the disclosed subject matter relates to pharmaceutical compositions comprising both a polymer and a substance to be delivered into a cell.
  • the disclosed subject matter relates to methods for delivering a substance into a cell using a polymer or pharmaceutical composition.
  • the disclosed subject matter relates to methods for treating a disorder by administering to a subject an effective amount of a polymer and a substance to be delivered into a cell, or by administering to a subject a pharmaceutical composition comprising a polymer and a substance to be delivered into a cell.
  • Figure 1A is a list of the diglycidyl ethers used in making polymers of the present invention.
  • Figures lB-1 through Figure 1B-5 provide a list of the aminoglycosides used in making polymers of the present invention.
  • Figure 2 illustrates an aminoglycoside (Neomycin) and a diepoxide (ethylene glycol diflycidyl ether) and the resulting polymer structure (Neo-EGDE) of the present invention.
  • Figure 3 illustrates the l H NMR spectrum in D2O of an exemplary polymer (Neo- EGDE) of the present invention.
  • the l H- NMR was measured with Varian 400 operating at 400 MHz in the Fourier transform mode.
  • Ten milligrams of polymer were dissolved in D2O mL; proton-decoupled NMR spectrum was measured in D 2 0.
  • Figure 4 illustrates the GPC chromatogram of an exemplary polymer of the present invention.
  • the molecular weights (MWs) of polymers were measured using gel permeation chromatography (GPC) system (Waters 1515) with a refractive index detector (Waters 2414) with ultrahydrogel column- 250 (Waters linear) at a flow rate of 1 mL/min at 35 °C. Water containing 0.1% trifluoroacetic acid and 40% acetonitrile was used as a mobile phase.
  • Poly (2-vinylpyridine) (MW: 3000; 7,000; 12,000; 35,000; 70,000) were used as standards. 5mg of polymer was dissolved in milli Q water, filtered through 0.45 ⁇ filter and injected into the GPC system.
  • the Elution time was 16.545; the retention time was 16545, the adjuste RT (min) was 16.545; the Mn was 3647; the Mw was 4948, the MP was 3557; the Mz+1 was 11231 ; and the Mz/Mw was 1.467039.
  • Figure 5 illustrates the luciferase transgene expression in as a function of polymer chemistry using polymer-mediated pGL3 plasmid DNA delivery to PC3 human prostate cancer cells at a polymer to plasmid ratio (25: 1) in serum-free media.
  • RLU relative luciferase units
  • Figure 6 illustrates the luciferase transgene expression in as a function of polymer chemistry using polymer-mediated pGL3 plasmid DNA delivery to MiaPaCa 2 human pancreatic cancer cell at a polymer to plasmid ratio (25: 1) in serum- free media.
  • RLU relative luciferase units
  • Figure 7A and 7B show the effect of lead aminoglycoside-based polymers and pEI concentration on luciferase transgene expression (RLU/mg) in PC3 cells.
  • Figure 8A and 8B show luciferase transgene expression (RLU/mg) for polymers and pEI in (A) PC3 and (B) 22Rvl human prostate cancer cells, in the presence and absence of serum efficacies of polymers and pEI were investigated at optimal pDNA to polymer ratios, based on dose response study ( Figures 7A & B).
  • * p ⁇ 0.05;
  • * * p ⁇ 0.01.
  • the present invention comprises polymers, compositions and methods for making and using polymers for delivering a substance into a cell.
  • the disclosed polymers are both effective at delivering substances into a cell and generally safe (i.e., not undesirably cytotoxic).
  • Methods of using the polymers comprise delivery of substances into cells.
  • polymers of the present invention are used to deliver nucleic acids into cells, for example, mammalian cells.
  • a polymer of the present invention may comprise at least one aminoglycoside.
  • Polymers of the present invention may be used to enhance or increase the delivery of viruses into cells.
  • polymers are molecules with two or more repeating units.
  • the repeating unit can be any aminoglycoside.
  • the repeating unit can be any one of streptomycin, neomycin, framycetin, paromomycin, ribostamycin, kanamycin, amikacin, arbekacin, bekanamycin, dibekacin, tobramycin, spectinomycin, hygromycin, gentamicin, netilmicin, sisomicin, isepamicin, verdamicin, astromicin, and apramycin.
  • a polymer can be a molecule with two aminoglycosides wherein each aminoglycoside can be the same or different.
  • a polymer can be neomycin-neomycin.
  • the aminoglycosides can be linked together via a linker.
  • the linker can be any linker capable of binding to two or more aminoglycosides.
  • the linker can have at least two electrophilic groups (dielectrophile) that can react with an amine group on an aminoglycoside.
  • electrophilic groups are known in the art and include, but are not limited to, epoxides, carboxylic acids, ketones, and alkyl halidies.
  • suitable linkers include diepoxides, dicarboxylic acids, diketones, dihalides, diacrylates, divinyls, and the like.
  • a "polymer comprising at least one diepoxide” refers to a polymer that includes at least one component that was made from a diepoxide containing compound.
  • compounds and bonds formed from expoxides are contemplated to be included in the phrase "polymer comprising at least one diepoxide.”
  • Figure 2 shows an example of a polymer comprising at least one diepoxide.
  • the ethyleneglycol diglycidyl ether, an epoxide is linked to two neomycin molecules.
  • Such polymer is considered to be a "polymer comprising at least one diepoxide.”
  • polymers at least one dicarboxylic acid wherein the bonds formed via the dielectrophiles are contemplated with such recitation.
  • polymers at least one dielectrophile wherein the bonds formed via the carboxylic acids are contemplated with such recitation.
  • diepoxide and “diepoxide linker” can be used interchangeably.
  • dicarboxylic acid and “dicarboxylic acid linker” can be used interchangeably.
  • the disclosed polymers are polymers of diepoxide and an amine, such as an aminoglycoside.
  • Aminoglycosides can be polymerized via their amine groups using different cross-linkers, for example, diepoxides.
  • Synthesized polymers may be characterized by analytical techniques like NMR, GPC, FT-IR and CFTNS.
  • a library of 50+ aminoglycoside polymers was screened for plasmid DNA delivery and transgene expression in the PC3 prostate cancer and Mia-PaCa-2 pancreatic cancer cell lines. The studies revealed that the polymers have significantly higher transgene expression efficacies and lower toxicities in PC3 and Mia PaCa-2 cells compared to polyethyleneimine, a current standard.
  • Aminoglycoside polymers disclosed herein can be used to deliver nucleic acid, including, but not limited to, plasmid DNA, siRNA, antisense RNA, and plasmid shRNA, mRNA, DNA, nucleic acid genomes, probes, and primers, to mammalian cells. Aminoglycoside polymers, their preparation and use, are described.
  • the polymers may be branched at one or more points on the polymer backbone.
  • the amines of the polymers allow the polymers to be made cationic by subjecting the polymers to acid.
  • the cationic forms are also contemplated.
  • the cationic forms of the polymers can then bind to a variety of substances that can be delivered into a cell.
  • Aminoglycoside polymers are disclosed herein.
  • the aminoglycoside monomers may be linked by a chemical linking moiety that joins two or more aminoglycoside monomers together in an effective manner so that the amoniglycoside polymer functions to deliver nucleic acids or viruses. Diepoxides are disclosed herein as a chemical linking moiety, but the present invention is not to be limited by this disclosure, but comprises other chemical linking moieties.
  • the polymers disclosed are polymers of amines and diepoxides, wherein the amine is represented by the formula:
  • R 1 and R 2 is independently optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, or combinations thereof.
  • R 1 is optionally substituted alkyl or heteroalkyl, such as aminoalkyl.
  • amines showing suitable variations in R 1 , are shown in Figure IB.
  • R 2 is optionally substituted alkyl, optionally substituted heteroalkyl, such as ethylene glycol or polyethylene glycol, optionally substituted cycloalkyl, or optionally substituted heterocycloalkyl.
  • epoxides showing suitable varitations in R 2 , are shown in Figure 1A.
  • Aminoglycosides are a class of small-molecule (typically MW ⁇ 2 kDa) antibiotics which consists of two or more aminosugars joined in glycosidic linkage to a hexose nucleus. These compounds are clinically useful in treating various infections because of their ability to interfere with protein synthesis in microorganism.
  • the bactericidal activity of aminolycosides is attributed to irreversible inhibition of protein synthesis following their binding to the 30S subunit of bacterial ribosome.
  • Aminoglycosides exhibit a positive charge due to the protonable amines; the polycationic nature of aminoglycosides helps in binding to the prokaryotic ribosomal RNA. Aminoglycosides stabilize DNA /RNA based on electrostatic interactions.
  • the present invention comprises a novel class of polymers based on aminoglycosides for efficient delivery of nucleic acids to mammalian cells. Polymers are based on cross linking an aminoglycoside monomer.
  • diepoxides diglycidyl ethers
  • cross-linkers for the aminoglycoside polymer formation, although any molecules that cross-link amines can be used for generating these polymers, and such cross-linking moieties are contemplated by the present invention.
  • a given polymer contains at least one aminoglycoside in its structure.
  • polymers with multiple (>1) aminoglycosides may also be employed for similar purposes.
  • these polymers are also useful for aiding in delivery of viruses to cells.
  • a synthesis protocol comprises the following steps.
  • the sulfate of the aminoglycoside was removed by using amberlite, an aninon exchage resin.
  • Aminoglycosides, free of the sulfate, were allowed to react with digylcidyl ethers (diepoxide cross linker) in 1 :2 molar ratios in a mixture of water and DMF (1.5: 1) for 5 hours at 60 °C.
  • the crude reaction mixture was allowed to cool to room temperature and precipitated using acetone.
  • Precipitated product was washed twice with acetone to remove the unreacted diglycidyl ethers, and dried.
  • Dried product was further purified by the dialysis using 3500 MW cut-off membrane to remove unreacted aminoglycosides.
  • the dialyzed product was freeze-dried to obtain the product (aminoglycoside polymer).
  • the polymers of the present invention may be characterized by methods that include NMR, IR, GPC and others.
  • the present invention further provides methods of determining the transgene efficacy of the synthesized polymer library.
  • Aminoglycoside polymers of the present invention may be used in methods for introducing a nucleic acid and/or a gene product into a cell, thereby providing a method for administering the nucleic acid to cells for variety of purposes
  • the polymers are prepared from monomers having two or more reactive functionalities, namely epoxides and aminoglycosides, and thus ultimately have a variety structures that are generally branched, have one or more amines, including secondary and tertiary amines in the polymer backbone and primary amines as end groups, one or more secondary alcohols (from the ring-opening of the epoxide), and/or one or more epoxides as endgroups.
  • Aminoglycoside polymers may be made with multiple aminoglycoside moieties and multiple chemical linking moieties, such as diepoxides, and the polymers may comprise block polymers and/or branching polymers.
  • the polymers are prepared by reaction between one mole of an aminoglycoside with two moles of a diglycidyl ether molecule.
  • a polymer of aminoglycoside-diepoxide repeating units is formed.
  • an amine end group in an aminoglycoside reacts with a monomeric diglycidyl either and that structure can react with a second aminoglycoside.
  • Further reactions between diepoxides and aminoglycosides may result in polymers, not shown.
  • the ratio of aminoglycosides, glycidyl ethers, and alcohols in the backbone can be modulated by the stoichiometric ratio of the monomers.
  • the molecular weight and structure of the polymer can be likewise modulated by not only the monomer ratio but also by polymerization conditions, such as temperature or duration.
  • the polymers can be made by reacting the monomers in solution or neat.
  • acid can be added to the polymers to protonate one or more amines of the polymer. If the polymer is in solution, only a slight pH modification is needed. Lowering the pH to about 7.4, for example, from a more basic starting point, using acid will suffice to protonate a sufficient number of amines.
  • polymers include polymers produced from any combination of monomers 1 through 8 and monomers A-T, including polymers comprising two monomers of A-T, or copolymers, 1A, IB, 1C, ID, IE, IF, 1G, 1H, II, 1J, IK, 1L, 1M, IN, 10, IP, 1Q, 1R, I S, IT; 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 21, 2J; 2K, 2L, 2M, 2N, 20, 2P, 2Q, 2R, 2S, 2T,3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 31, 3J, 3K, 3L, 3M, 3N, 30, 3P, 3Q, 3R, 3S, 3T; 4A, 4B, 4C, 4D, 4E, 4F, 4G, 4H, 41, 4J, 4K, 4L,
  • Aminoglycosides that may be polymerized in the current invention include, but are not limited to, streptomycin, neomycin, framycetin, paromomycin, ribostamycin, kanamycin, amikacin, arbekacin, bekanamycin, dibekacin, tobramycin, spectinomycin, hygromycin, gentamicin, netilmicin, sisomicin, isepamicin, verdamicin, astromicin, and apramycin.
  • the polymer comprising at least one dielectrophile linker and at least one aminoglycoside comprises streptomycin. In another aspect, the polymer comprising at least one dielectrophile linker and at least one aminoglycoside comprises neomycin. In another aspect, the polymer comprising at least one dielectrophile linker and at least one aminoglycoside comprises framycetin. In another aspect, the polymer comprising at least one dielectrophile linker and at least one aminoglycoside comprises paromomycin. In another aspect, the polymer comprising at least one dielectrophile linker and at least one aminoglycoside comprises ribostamycin.
  • the polymer comprising at least one dielectrophile linker and at least one aminoglycoside comprises kanamycin. In another aspect, the polymer comprising at least one dielectrophile linker and at least one aminoglycoside comprises amikacin. In another aspect, the polymer comprising at least one dielectrophile linker and at least one aminoglycoside comprises arbekacin. In another aspect, the polymer comprising at least one dielectrophile linker and at least one aminoglycoside comprises bekanamycin. In another aspect, the polymer comprising at least one dielectrophile linker and at least one aminoglycoside comprises dibekacin.
  • the polymer comprising at least one dielectrophile linker and at least one aminoglycoside comprises tobramycin. In another aspect, the polymer comprising at least one dielectrophile linker and at least one aminoglycoside comprises spectinomycin. In another aspect, the polymer comprising at least one dielectrophile linker and at least one aminoglycoside comprises hygromycin. In another aspect, the polymer comprising at least one dielectrophile linker and at least one aminoglycoside comprises gentamicin. In another aspect, the polymer comprising at least one dielectrophile linker and at least one aminoglycoside comprises netilmicin.
  • the polymer comprising at least one dielectrophile linker and at least one aminoglycoside comprises sisomicin. In another aspect, the polymer comprising at least one dielectrophile linker and at least one aminoglycoside comprises isepamicin. In another aspect, the polymer comprising at least one dielectrophile linker and at least one aminoglycoside comprises verdamicin. In another aspect, the polymer comprising at least one dielectrophile linker and at least one aminoglycoside comprises astromicin. In another aspect, the polymer comprising at least one dielectrophile linker and at least one aminoglycoside comprises apramycin.
  • the polymer comprising at least one dielectrophile linker and at least one aminoglycoside comprises a dihalide linker. In one aspect, the polymer comprising at least one diacrylate linker and at least one aminoglycoside comprises a dicarboxylic acid linker. In one aspect, the polymer comprising at least one dielectrophile linker and at least one aminoglycoside comprises a divinyl linker. In one aspect, the polymer comprising at least one dielectrophile linker and at least one aminoglycoside comprises a diketone linker. In one aspect, the polymer comprising at least one dielectrophile linker and at least one aminoglycoside comprises a dicarboxylic acid linker.
  • a dicarboxylic acid linker can have the structure HOOC-R 3 -COOH, wherein R 3 comprises an alkyl, alkenyl, alkynyl, alkoxyl, aryl, heteroaryl, cycloalkyl, herterocyclyl, or polyethylene glycol.
  • Suitable dicarboxylic acid linkers include oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azeliaic acid, sebacic acid, phthalic acid, isophthalic acid, terephthalic acid, malic acid, fumaric acid, glutaconic acid, traumatic acid, or muconic acid.
  • Other suitable dicarboxylic acid linkers include dicarboxylic acid polyethylene glycol (PEG).
  • the dicarboxylic acid - PEG are known in the art can have a molecular weight of 250 to 50,000.
  • the polymer comprising at least one dielectrophile linker and at least one aminoglycoside comprises a diepoxide linker.
  • a diepoxide linker can have the structure , wherein R 4 comprises an alkyl, alkenyl, alkynyl, alkoxyl, aryl, heteroaryl, cycloalkyl, herterocyclyl, polyethylene glycol (PEG).
  • Suitable diepoxide linkers include but are not limited to 1,4 butanediol diglycidyl ether (1,4 B); 1,4- cyclohexanedimethanol diglycidyl ether (1,4 C); 4-vinylcyclohexene diepoxide (4VCD); ethyleneglycol diglycidyl ether (EDGE); glycerol diglycidyl ether (GDE); neopentylglycol diglycidyl ether (NPDGE); poly(ethyleneglycol) diglycidyl ether (PEGDE); poly(propyleneglycol) diglycidyl ether (PPGDE); or resorcinol diglycidyl ether (RDE).
  • 4VCD 4-vinylcyclohexene diepoxide diepoxide
  • EDGE ethyleneglycol diglycidyl ether
  • GDE glycerol diglycidyl ether
  • NPDGE ne
  • the polymer comprising at least one diepoxide and at least one aminoglycoside comprises 1,4 butanediol diglycidyl ether. In another aspect, the polymer comprising at least one diepoxide and at least one aminoglycoside comprises 1,4- cyclohexanedimethanol diglycidyl ether. In another aspect, the polymer comprising at least one diepoxide and at least one aminoglycoside comprises 4-vinylcyclohexene diepoxide. In another aspect, the polymer comprising at least one diepoxide and at least one aminoglycoside comprises ethyleneglycol diglycidyl ether.
  • the polymer comprising at least one diepoxide and at least one aminoglycoside comprises glycerol diglycidyl ether. In another aspect, the polymer comprising at least one diepoxide and at least one aminoglycoside comprises neopentylglycol diglycidyl ether. In another aspect, the polymer comprising at least one diepoxide and at least one aminoglycoside comprises poly(ethyleneglycol) diglycidyl ether. In another aspect, the polymer comprising at least one diepoxide and at least one aminoglycoside comprises poly(propyleneglycol) diglycidyl ether. In another aspect, the polymer comprising at least one diepoxide and at least one aminoglycoside comprises resorcinol diglycidyl ether.
  • the polymer comprising at least one diepoxide and at least one aminoglycoside comprises streptomycin. In another aspect, the polymer comprising at least one diepoxide and at least one aminoglycoside comprises neomycin. In another aspect, the polymer comprising at least one diepoxide and at least one aminoglycoside comprises framycetin. In another aspect, the polymer comprising at least one diepoxide and at least one aminoglycoside comprises paromomycin. In another aspect, the polymer comprising at least one diepoxide and at least one aminoglycoside comprises ribostamycin. In another aspect, the polymer comprising at least one diepoxide and at least one aminoglycoside comprises kanamycin.
  • the polymer comprising at least one diepoxide and at least one aminoglycoside comprises amikacin. In another aspect, the polymer comprising at least one diepoxide and at least one aminoglycoside comprises arbekacin. In another aspect, the polymer comprising at least one diepoxide and at least one aminoglycoside comprises bekanamycin. In another aspect, the polymer comprising at least one diepoxide and at least one aminoglycoside comprises dibekacin. In another aspect, the polymer comprising at least one diepoxide and at least one aminoglycoside comprises tobramycin. In another aspect, the polymer comprising at least one diepoxide and at least one aminoglycoside comprises spectinomycin.
  • the polymer comprising at least one diepoxide and at least one aminoglycoside comprises hygromycin. In another aspect, the polymer comprising at least one diepoxide and at least one aminoglycoside comprises gentamicin. In another aspect, the polymer comprising at least one diepoxide and at least one aminoglycoside comprises netilmicin. In another aspect, the polymer comprising at least one diepoxide and at least one aminoglycoside comprises sisomicin. In another aspect, the polymer comprising at least one diepoxide and at least one aminoglycoside comprises isepamicin. In another aspect, the polymer comprising at least one diepoxide and at least one aminoglycoside comprises verdamicin. In another aspect, the polymer comprising at least one diepoxide and at least one aminoglycoside comprises astromicin. In another aspect, the polymer comprising at least one diepoxide and at least one aminoglycoside comprises apramycin.
  • the polymer comprising at least one dicarboxylic acid linker and at least one aminoglycoside comprises streptomycin. In another aspect, the polymer comprising at least one dicarboxylic acid linker and at least one aminoglycoside comprises neomycin. In another aspect, the polymer comprising at least one dicarboxylic acid linker and at least one aminoglycoside comprises framycetin. In another aspect, the polymer comprising at least one dicarboxylic acid linker and at least one aminoglycoside comprises paromomycin. In another aspect, the polymer comprising at least one dicarboxylic acid linker and at least one aminoglycoside comprises ribostamycin.
  • the polymer comprising at least one dicarboxylic acid linker and at least one aminoglycoside comprises kanamycin. In another aspect, the polymer comprising at least one dicarboxylic acid linker and at least one aminoglycoside comprises amikacin. In another aspect, the polymer comprising at least one dicarboxylic acid linker and at least one aminoglycoside comprises arbekacin. In another aspect, the polymer comprising at least one dicarboxylic acid linker and at least one aminoglycoside comprises bekanamycin. In another aspect, the polymer comprising at least one dicarboxylic acid linker and at least one aminoglycoside comprises dibekacin.
  • the polymer comprising at least one dicarboxylic acid linker and at least one aminoglycoside comprises tobramycin. In another aspect, the polymer comprising at least one dicarboxylic acid linker and at least one aminoglycoside comprises spectinomycin. In another aspect, the polymer comprising at least one dicarboxylic acid linker and at least one aminoglycoside comprises hygromycin. In another aspect, the polymer comprising at least one dicarboxylic acid linker and at least one aminoglycoside comprises gentamicin. In another aspect, the polymer comprising at least one dicarboxylic acid linker and at least one aminoglycoside comprises netilmicin.
  • the polymer comprising at least one dicarboxylic acid linker and at least one aminoglycoside comprises sisomicin. In another aspect, the polymer comprising at least one dicarboxylic acid linker and at least one aminoglycoside comprises isepamicin. In another aspect, the polymer comprising at least one dicarboxylic acid linker and at least one aminoglycoside comprises verdamicin. In another aspect, the polymer comprising at least one dicarboxylic acid linker and at least one aminoglycoside comprises astromicin. In another aspect, the polymer comprising at least one dicarboxylic acid linker and at least one aminoglycoside comprises apramycin.
  • the polymers can be tested to evaluate their usefulness as delivery agents for substances to be delivered into a cell using screening techniques known in the art. For example, to evaluate the usefulness of the polymers in delivering an anionic polynucleic acid, such as DNA, into a cell, DNA-binding efficacies of the polymers can be determined using for example, transfection efficacy assays. In vitro and/or in vivo evaluation of the polymers can be evaluated by any method known in the art. Methods of Using the Polymers
  • Contemplated uses of the polymers include delivering a variety of substances into a cell.
  • the disclosed polymers and their cationic forms can form an association with a substance that has an affinity therefor, and as such can function as a delivery vehicle for delivering the substance into a cell.
  • a variety of drugs, bioactive agents, biomolecules, such as peptides, proteins, nucleic acids, polynucleic acids, polynucleotides, among others, which associate or can be caused to associate with a disclosed cationic polymer, can be delivered into a cell.
  • Peptides and proteins include any polymer of at least two residues of a natural of non-natural amino acid.
  • the disclosed polymers can be used in transfection procedures. Accordingly, the disclosed polymers can be used to facilitate the intercellular delivery of nucleic acids, for example, DNA or RNA sequences, whether or not the nucleic acid codes for polypeptides or not.
  • the disclosed polymers can also be used to deliver a plasmid DNA, plasmid shRNA, antisense RNA, siRNA, shRNA, primers, probes, promoters, TAG, or other known types of nucleic acids to a cell.
  • disclosed polymers can be similarly used for the delivery of a polypeptide or protein.
  • polymer-mediated delivery of DNA and RNA polynucleotides or proteins can provide therapy for diseases by supplying deficient or absent gene products to treat any disease in which a defective gene or its product has been identified.
  • Polymer-mediated intracellular delivery can also provide immunizing polypeptides to the cell, either by delivering a polynucleotide coding for the immunogen, or by delivering the immunogen itself.
  • Polymers of the present invention may aid in the delivery of viral particles, viral DNA or RNA, or viral components into cells, or may aid or enhance viral infection of cells.
  • Nucleic acids contemplated by the present invention may comprise naturally occurring nucleotides or modified nucleotides.
  • oligonucleotides including antisense polynucleotide sequences, useful in eliminating or reducing the production of a gene product, as described by Tso, P. et al. Annals New York Acad. Sci. 570:220-241 (1987). Also disclosed is the delivery, by means of the polymer, of ribozymes, or catalytic RNA species, for example, the "hairpin” type as described by Hampel et al. Nucleic Acids Research 18(2):299-304 (1990; or the "Hammerhead” type described by Cech. T. and Bass, B. Annual Rev. Biochem. 55:599-629 (1986).
  • the DNA sequences delivered can be those sequences that do not integrate into the genome of the host cell or those that do integrate into the genome of the host. These can be non-replicating DNA sequences, or specific replicating sequences genetically engineered to lack the genome-integration ability.
  • the nucleic acid to be delivered is mRNA
  • it can be readily prepared from the corresponding DNA in vitro.
  • conventional techniques utilize phage RNA polymerases SP6, T3, or T7 to prepare mRNA from DNA templates in the presence of the individual ribonucleoside triphosphates.
  • An appropriate phage promoter, such as T7 origin of replication site is placed in the template DNA immediately upstream of the gene to be transcribed.
  • Systems utilizing T7 in this manner are well known, and are described in the literature, e.g., in Current Protocols in Molecular Biology, ⁇ 3.8 (vol. 1, 1988).
  • RNA that is chemically blocked at the 5 and/or 3 end to prevent access by RNase (this enzyme is an exonuclease and therefore does not cleave RNA in the middle of the chain).
  • RNase this enzyme is an exonuclease and therefore does not cleave RNA in the middle of the chain.
  • nucleoside or nucleotide analogues having an antiviral effect such as dideoxynucleotides, didehydronucleotides, nucleoside or nucleotide analogues having halo-substituted purine or pyrimidine rings such as 5-trifluoromethyl-2'-deoxyuridine or 5-flurouracil; nucleoside or nucleotide analogues having halo- and azido-substituted ribose moieties, such as 3 -azido- 3 deoxythymidine (AZT), nucleoside analogues having carbon substituted for oxygen in the ribose moiety (carbocyclic nucleosides), or nucleotide analogues having an acyclic pentose such as acyclovir or gancyclovir (DHPG).
  • nucleoside or nucleotide analogues having an antiviral effect such as dideoxy
  • nucleoside analogues comprise phosphatidyl 2', 3 - dideoxynucleosides, 2', 3'-didehydronucleosides, 3 '-azido-2'-deoxynucleosides, 3 - fluorodeosynucleosides and 3'-fluorodideoxynucleosides, 9- -D-arabinofuranosyladenine (araA), ⁇ - ⁇ -D-arabinofuranosylcytidine (araC), nucleosides such as acyclovir and gancyclovir having an acyclic ribose group, or the same nucleoside analogues as diphosphate diglyceride derivatives.
  • peptides comprising physiologic species such as interleukin-2, tumor necrosis factor, tissue plasminogen activator, factor VIII, erythropoietin, growth factors such as epidermal growth factor, growth hormone releasing factor, neural growth factor, and hormones such as tissue insulin, calcitonin, and human growth hormone as well as toxic peptides such as ricin, diphtheria toxin, or cobra venom factor, capable of eliminating diseased or malignant cells.
  • physiologic species such as interleukin-2, tumor necrosis factor, tissue plasminogen activator, factor VIII, erythropoietin
  • growth factors such as epidermal growth factor, growth hormone releasing factor, neural growth factor, and hormones such as tissue insulin, calcitonin, and human growth hormone as well as toxic peptides such as ricin, diphtheria toxin, or cobra venom factor, capable of eliminating diseased or malignant cells.
  • polymers are also contemplated for the intra-cellular delivery of various other agents according to methods known to those skilled in the art, for example as described in Duzgunes, N., Subcellular Biochemistry 1 1 : 195-286 (1985).
  • Materials to be delivered can be proteins or polypeptides, as discussed above, or other negatively charged molecules, monoclonal antibodies, RNA-stabilizing factors and other transcription and translation regulating factors, antisense oligonucleotides, ribozymes, and any molecule possessing intracellular activity that can also associate with or be caused to associate with a disclosed cationic polymer.
  • Polymer-mediated delivery further protects the described agents from non-productive sequestration by substances of the extracellular environment.
  • a polymer and a substance to be delivered into a cell can be administered directly into a subject, as will be discussed below.
  • a cell can be treated with a polymer and a substance to be delivered into the cell, followed by introducing the treated cell into a subject to thereby treat a disorder.
  • a cell of a living organism can be removed from the organism, treated with a polymer and a substance to be delivered into the cell, followed by reintroduction of the treated cell into the organism to thereby treat a disorder.
  • the polymer and/or the substance to be delivered into the cell can be present in a pharmaceutical composition.
  • Local or systemic delivery of the substance can be achieved by administration comprising application or insertion of the pharmaceutical composition into body cavities, inhalation or insufflation of an aerosol, or by parenteral introduction, comprising intramuscular, intravenous, intradermal, peritoneal, subcutaneous and topical administration.
  • the nucleic acids can be delivered to the interstitial space of tissues of the animal body, including those of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue.
  • Interstitial space of the tissues comprises the intercellular, fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels.
  • the effect of the polymers in these pharmaceutical compositions is to enhance the potency and efficiency of the therapeutic agent contained therein by facilitating its intracellular delivery.
  • an effective DNA or mRNA dosage will generally be in the range of from about 0.02 ⁇ g/kg to about 100 mg/kg, usually about 0.005-5 mg/kg. However, as will be appreciated, this dosage will vary in a manner apparent to those of skill in the art according to, e.g., the activity of the peptide coded for by the nucleic acid.
  • Topical formulations are those advantageously applied to the skin or mucosa.
  • Target mucosa can be that of the gastrointestinal tract, comprising the mouth, naso-pharynx and stomach.
  • Other target tissues can be the accessible surfaces and canal of the ear and the ocular tissues.
  • Polymers present in topical formulations can act to facilitate introduction of bioactive molecules into the target tissue, such as the stratum corneum of the skin, by perturbing the barrier properties of the protective membrane, or by introducing perturbing agents or penetration enhancers such as AzoneTM or by promoting the activity of these penetration enhancers. They can also be delivered into muscle or skin using a vaccine gun.
  • compositions comprising the polymers are preparations comprising topical antibiotics such as clindamycin, tobramycin, neomycin, gentamycin, tetracycline, erythromycin; oxidants such as benzoyl peroxide, antifungal agents, such as clotrimazole, miconazole, nystatin, lactoconzole, econazole, and tolnaftate; retinoic acid for the treatment of herpes simplex and comprising antiviral nucleoside analogues such as acyclovir and gancyclovir.
  • topical antibiotics such as clindamycin, tobramycin, neomycin, gentamycin, tetracycline, erythromycin
  • oxidants such as benzoyl peroxide
  • antifungal agents such as clotrimazole, miconazole, nystatin, lactoconzole, econazole, and tolnaftate
  • compositions comprising the disclosed polymers are topical preparations containing an anesthetic or cytostatic agent, immunomodulators, bioactive peptides or oligonucleotides, sunscreens or cosmetics.
  • Preparations for topical use are conveniently prepared with hydrophilic and hydrophobic bases in the form of creams, lotions, ointments or gels; alternatively, the preparation can be in the form of a liquid that is sprayed on the skin.
  • the effect of the cationic polymers is to facilitate the penetration of the active antiviral agent through the stratum corneum of the dermis.
  • Similar preparations for ophthalmic use are those in which the pharmacologically effective agent is timolol, betaxolol, levobunaloa, pilocarpine, and the antibiotics and corticosteriods disclosed for topical applications.
  • composition and form of pharmaceutical preparations comprising the polymers disclosed, in combination with a drug or other therapeutic agents, can vary according to the intended route of administration.
  • Orally administered preparations can be in the form of solids, liquids, emulsions, suspensions, or gels, or preferably in dosage unit form, for example as tablets or capsules. Tablets can be compounded in combination with other ingredients customarily used, such as tale, vegetable oils, polyols, gums, gelatin, starch, and other carriers.
  • the cationic polymers can be dispersed in or combined with a suitable liquid carrier in solutions, suspensions, or emulsions.
  • compositions intended for injection can be prepared as liquids or solid forms for solution in liquid prior to injection, or as emulsions. Such preparations are sterile, and liquids to be injected intravenously should be isotonic. Suitable excipients are, for example, water, dextrose, saline, and glycerol.
  • salts of the substances described herein can be prepared from pharmaceutically acceptable non-toxic bases including organic bases and inorganic bases.
  • Salts derived from inorganic bases include sodium, potassium, lithium, ammonium, calcium, magnesium, and the like.
  • Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, basic amino acids, and the like.
  • Substances for injection can be prepared in unit dosage form in ampules, or in multidose containers.
  • the substances to be delivered can be present in such forms as suspensions, solutions, or emulsions in oily or preferably aqueous vehicles.
  • the salt of the substance can be in lyophilized form for reconstitution, at the time of delivery, with a suitable vehicle, such as sterile pyrogen-free water.
  • a suitable vehicle such as sterile pyrogen-free water.
  • Both liquids as well as lyophilized forms that are to be reconstituted will comprise agents, preferably buffers, in amounts necessary to suitably adjust the pH of the injected solution.
  • the total concentration of solutes should be controlled to make the preparation isotonic, hypotonic, or weakly hypertonic.
  • Nonionic materials such as sugars, are preferred for adjusting tonicity, and sucrose is particularly preferred. Any of these forms can further comprise suitable formulatory agents, such as starch or sugar, glycerol or saline.
  • suitable formulatory agents such as starch or sugar, glycerol or saline.
  • the compositions per unit dosage, whether liquid or solid, can contain from 0.1% to 99% of polynucleotide material.
  • kits comprising the polymers and the substance to be delivered into the cell.
  • the kits can comprise one or more packaged unit doses of a composition comprising the polymer and the substance to be delivered into the cell.
  • the units dosage ampules or multidose containers, in which the polymer and the substance to be delivered are packaged prior to use, can comprise an hermetically sealed container enclosing an amount of polynucleotide or solution containing a substance suitable for a pharmaceutically effective dose thereof, or multiples of an effective dose.
  • the polymer and substance can be packaged as a sterile formulation, and the hermetically sealed container is designed to preserve sterility of the formulation until use.
  • the disclosed polymers can also be present in liquids, emulsions, or suspensions for delivery of active therapeutic agents in aerosol form to cavities of the body such as the nose, throat, or bronchial passages.
  • the ratio of active ingredient to the polymer and the other compounding agents in these preparations will vary as the dosage form requires.
  • the pharmaceutical compositions can be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, lotions, creams, gels, or the like, preferably in unit dosage form suitable for single administration of a precise dosage.
  • the compositions will include, as noted above, an effective amount of the selected lipocomplex in combination with a pharmaceutically acceptable carrier and, in addition, can include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc.
  • conventional nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc., an active compound as described herein and optional pharmaceutical adjuvants in an excipient, such as, for example, water, saline aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension.
  • the pharmaceutical composition to be administered can also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc.
  • Parental administration if used, is generally characterized by injection.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • a more recently revised approach for parental administration involves use of a slow release or sustained release system, such that a constant level of dosage is maintained. See, e.g., U.S. Patent No. 3,710,795, which is incorporated by reference herein.
  • the subject can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian.
  • the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent.
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.
  • a patient refers to a subject afflicted with a disease or disorder.
  • patient includes human and veterinary subjects.
  • a cationic polymer includes mixtures of two or more such cationic polymers
  • reference to “a substance” includes mixtures of two or more such substances
  • reference to “a composition” includes mixtures of two or more such compositions, and the like.
  • Ranges can be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10" is also disclosed.
  • references in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed.
  • X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.
  • a weight percent (wt. %) of a component is based on the total weight of the formulation or composition in which the component is included.
  • the term "substituted" is contemplated to include all permissible substituents of organic compounds.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds.
  • Illustrative substituents include, for example, those described below.
  • the permissible substituents can be one or more and the same or different for appropriate organic compounds.
  • the heteroatoms, such as nitrogen can have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms.
  • substitution or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
  • a 1 ,” “A 2 ,” “A 3 ,” and “A 4 " are used herein as generic symbols to represent various specific substituents. These symbols can be any substituent, not limited to those disclosed herein, and when they are defined to be certain substituents in one instance, they can, in another instance, be defined as some other substituents.
  • alkyl as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like.
  • the alkyl group can also be substituted or unsubstituted.
  • the alkyl group can be substituted with one or more groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below.
  • groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below
  • alkyl is generally used to refer to both unsubstituted alkyl groups and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the specific substituent(s) on the alkyl group.
  • halogenated alkyl specifically refers to an alkyl group that is substituted with one or more halide, e.g., fluorine, chlorine, bromine, or iodine.
  • alkoxyalkyl specifically refers to an alkyl group that is substituted with one or more alkoxy groups, as described below.
  • alkylamino specifically refers to an alkyl group that is substituted with one or more amino groups, as described below, and the like.
  • alkyl is used in one instance and a specific term such as “alkylalcohol” is used in another, it is not meant to imply that the term “alkyl” does not also refer to specific terms such as “alkylalcohol” and the like.
  • cycloalkyl refers to both unsubstituted and substituted cycloalkyl moieties
  • the substituted moieties can, in addition, be specifically identified herein; for example, a particular substituted cycloalkyl can be referred to as, e.g., an "alkylcycloalkyl.”
  • a substituted alkoxy can be specifically referred to as, e.g., a "halogenated alkoxy”
  • a particular substituted alkenyl can be, e.g., an "alkenylalcohol,” and the like.
  • alkoxy as used herein is an alkyl group bound through a single, terminal ether linkage; that is, an “alkoxy” group can be defined as— OA 1 where A 1 is alkyl as defined above.
  • alkoxylalkyl as used herein is an alkyl group that contains an alkoxy substituent and can be defined as— A ⁇ O-A 2 , where A 1 and A 2 are alkyl groups.
  • alkenyl as used herein is a hydrocarbon group of from 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon double bond.
  • the alkenyl group can be substituted with one or more groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below.
  • groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described
  • alkynyl is a hydrocarbon group of 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon triple bond.
  • the alkynyl group can be substituted with one or more groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below.
  • aryl as used herein is a group that contains any carbon-based aromatic group including, but not limited to, benzene, naphthalene, phenyl, biphenyl, phenoxybenzene, and the like.
  • aryl also includes "heteroaryl,” which is defined as a group that contains an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus.
  • non-heteroaryl which is also included in the term “aryl,” defines a group that contains an aromatic group that does not contain a heteroatom. The aryl group can be substituted or unsubstituted.
  • the aryl group can be substituted with one or more groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein.
  • the term "biaryl” is a specific type of aryl group and is included in the definition of aryl. Biaryl refers to two aryl groups that are bound together via a fused ring structure, as in naphthalene, or are attached via one or more carbon-carbon bonds, as in biphenyl.
  • cycloalkyl as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms.
  • examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.
  • heterocycloalkyl is a cycloalkyl group as defined above where at least one of the carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus.
  • the cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted.
  • the cycloalkyl group and heterocycloalkyl group can be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein.
  • Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the like.
  • heterocycloalkenyl is a type of cycloalkenyl group as defined above, and is included within the meaning of the term “cycloalkenyl,” where at least one of the carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus.
  • the cycloalkenyl group and heterocycloalkenyl group can be substituted or unsubstituted.
  • the cycloalkenyl group and heterocycloalkenyl group can be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein.
  • cyclic group is used herein to refer to either aryl groups, non-aryl groups (i.e., cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl groups), or both. Cyclic groups have one or more ring systems that can be substituted or unsubstituted. A cyclic group can contain one or more aryl groups, one or more non-aryl groups, or one or more aryl groups and one or more non-aryl groups.
  • amine or “amino” as used herein are represented by the formula NA A A , where A , A , and A J can be, independently, hydrogen, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
  • carboxylic acid as used herein is represented by the formula -C(0)OH.
  • a “carboxylate” as used herein is represented by the formula -C(0)0 " .
  • esters as used herein is represented by the formula -OC(0)A 1 or - C(0)OA 1 , where A 1 can be an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
  • ether as used herein is represented by the formula A x OA 2 , where A 1 and A 2 can be, independently, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
  • ketone as used herein is represented by the formula A 1 C(0)A 2 , where A 1 and A 2 can be, independently, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
  • halide refers to the halogens fluorine, chlorine, bromine, and iodine.
  • hydroxyl as used herein is represented by the formula -OH.
  • nitro as used herein is represented by the formula -NO 2 .
  • sil as used herein is represented by the formula -SiA ⁇ A 3 , where A 1 , A 2 , and A 3 can be, independently, hydrogen, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
  • sulfo-oxo is represented by the formulas -S(0)A 1 , - S(0) 2 A 1 , -OS(0) 2 A 1 , or -OS(0) 2 OA 1 , where A 1 can be hydrogen, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
  • sulfonyl is used herein to refer to the sulfo-oxo group represented by the formula -S(0) 2 A 1 , where A 1 can be hydrogen, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
  • sulfonylamino or "sulfonamide” as used herein is represented by the formula -S(0) 2 NH-.
  • a 1 S(0) 2 A 2 is represented by the formula A 1 S(0) 2 A 2 , where A 1 and A 2 can be, independently, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
  • sulfoxide as used herein is represented by the formula A 1 S(0)A 2 , where A 1 and A 2 can be, independently, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
  • thiol as used herein is represented by the formula -SH.
  • R 1 ,” “R 2 ,” “R 3 ,” “R”,” where n is an integer, as used herein can, independently, possess one or more of the groups listed above.
  • R 1 is a straight chain alkyl group
  • one of the hydrogen atoms of the alkyl group can optionally be substituted with a hydroxyl group, an alkoxy group, an alkyl group, a halide, and the like.
  • a first group can be incorporated within second group or, alternatively, the first group can be pendant (i.e., attached) to the second group.
  • an alkyl group comprising an amino group the amino group can be incorporated within the backbone of the alkyl group.
  • the amino group can be attached to the backbone of the alkyl group. The nature of the group(s) that is (are) selected will determine if the first group is embedded or attached to the second group.
  • nucleic acid refers to a polymer comprising at least two residues of a nucleotide, which can include any N-glycoside or C-glycoside of a purine or pyrimidine base or of a modified purine or pyrimidine base, which includes those bases that do not occur naturally.
  • Specific examples of such polymers include without limitation any form of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), genomic DNA, messenger RNA (mRNA), complementary DNA (cDNA), antisense RNA (aRNA), a synthetic nucleic acid polymer, or a mixture thereof, among others.
  • Certain materials, compounds, compositions, and components disclosed herein can be obtained commercially or readily synthesized using techniques generally known to those of skill in the art.
  • the starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), Acros Organics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St.
  • 4VCD 4-vinylcyclohexene diepoxide diepoxide
  • EDGE ethyleneglycol diglycidyl ether
  • GDE glycerol diglycidyl ether
  • NPDGE n
  • the sulfate of the aminoglycoside was removed by using amberlite, an aninon exchage resin. Forty (40) gm of amberlite IRA-400 (chloride form) was packed in a separating column. The resin was thoroughly washed with 5 column volumes of milliQ water (250ml) to remove any resin-bound impurities. After resin purification, 1.5 g of each aminoglycoside, were dissolved in 10 mL of milliQ water and allowed to pass through the resin bed to remove the sulfate associated with the molecule structure. The collected fraction was passed through again the same bed to remove trace amounts of sulfate. This procedure was repeated for four more times, after which, the collected fraction was freeze- dried to obtain the sulfate-free version of the aminoglycoside.
  • Each sulfate-free aminoglycoside was dissolved in 1.5 mL milli Q water, followed by the addition of 1 mL DMF.
  • the digylcidyl ether (diepoxide cross linker) was then added in a 1 :2 molar ratio to the aminoglycoside.
  • the reraction mixture was allowed to stir for 5 hours at 60 °C.
  • the crude reaction mixture was allowed to cool to room temperature and precipitated using acetone. Precipitated product was washed twice with acetone to remove the unreacted diglycidyl ethers, and dried.
  • MWCO molecular weight cut off
  • Figure 2 shows an example of an aminoglycoside polymer made from neomycin (aminoglycoside) and ethylene glycol diclycidyl ether (diepoxide). Similar reactions were used for the generation of the library of over fifty cationic polymers.
  • Figures 1A and B shows monomers used in developing the combinatorial matrix of the polymer library.
  • Figure 3 shows the NMR spectrum for the polymer shown in Figure 2.
  • Figure 4 shows the GPC data and chromatogram of the polymer shown in Figure 2.
  • the pGL3 control vector (Promega Corp., Madison, WI, U.S.A.), which encodes for the modified firefly luciferase protein under the control of an SV40 promoter, was used in transfection experiments.
  • the PC3- human prostate cancer cell line was provided by Dr. Michel Sadelain of the Memorial Sloan Cancer Center, New York, NY, U.S.A. The cells were cultured in a 5% CO 2 incubator at 37 °C using RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) and 1% antibiotics (10,000 units /mL penicillin G/ 10,000 ⁇ g/mL streptomycin).
  • FBS heat-inactivated fetal bovine serum
  • MiaPaCa 2 cells were cultured in a 5% CO 2 incubator in Dulbecco's Modified Eagle's Medium (DMEM; BioWhittaker®) containing 4.5 g/L glucose and L-glutamine, supplemented with 10% fetal bovine serum (Invitrogen, CA, U.S.A.) and 1% penicillin / streptomycin (Invitrogen, CA, U.S.A.).
  • DMEM Dulbecco's Modified Eagle's Medium
  • PC3 and MiaPaCa 2 cells were seeded in 24-well plates at a density of 50,000 cells / well and allowed to attach overnight.
  • Polymer:pGL3 control plasmid at weight ratios of 25: 1 polymer concentration 10 ng/ ⁇ and 200 ng pGL3 plasmid in each well
  • serum (10% FBS) serum-containing medium was added to the cells.
  • Figure 5 and 6 show the transfection of PC3 cells and MiaPaca caells with the pGL3 plasmid using different aminoglycoside polymers, respectively. Transfections were carried out with the pGL3 control vector which codes for luciferase protein. A polymer to plasmid ratio of 25: 1 was employed in order to evaluate the transfection efficacies of the selected polymers. The use of nitrogen: phosphorus (N:P) ratio is common in comparing cationic lipid and cationic polymer meditated gene delivery. However, a w/w ratio was used, which has been previously employed for evaluating polymeric transfection agents.
  • N:P nitrogen: phosphorus
  • luminescence relative luminescence units or RLU
  • RLU relative luminescence units
  • both PC3 and 22Rvl cells were transfected with a plasmid encoding enhanced green fluorescent protein (EGFP); the plamid is termed pEGFP.
  • EGFP enhanced green fluorescent protein
  • Transgene expression efficacies were visualized using fluorescence microscopy, and quantified using flow cytometry analysis.
  • the fluorescence microscopy images of PC3 cells transfected with pEGFP both, in the presence and absence of serum were taken. Images of 22Rvl cells were taken under similar conditions. The fluorescence microscopy images were obtained 48 hours following transfection with polymer :pEGFP polyplexes.
  • Figure 9 shows flow cytometry analysis of PC3 cells expressing EGFP in presence and absence of serum. Approximately 15% to 23% cells expressed EGFP with aminoglycoside lead polymers, while transfection with pEI resulted in EGFP expression in only 1.7% cells. The percentage of cells expressing EGFP decreased in serum-containing media for all polymers, including pEI. Polymers exhibited EGFP expression in 10% to 13% cells in the presence of serum, while pEI demonstrated EGFP expression in only 1% cells. It is commonly acknowledged in the literature that decrease of transfection in the presence of serum is likely due to the interference of serum proteins with polyplexes. Finally, we also found that the amount of plasmid DNA delivered did not significantly enhance transgene (EGFP) expression.
  • EGFP transgene
  • the polymer Paromomycin-GDE amount was kept constant at 5 ⁇ g, while the pEGFP amount was varied from 100 ng to 10 ⁇ g.
  • Transgene expression remained largely invariant from 100 - 1000 ng pDNA.
  • EGFP expression was significantly lower for higher pEGFP amounts, presumably due to reduction in the polymenpEGFP ratio, and reduced availability of the polymer for interactions with cells.
  • luciferease and EGFP transgene expression data indicate that aminoglycoside-based polymers perform significantly better than the currently available pEI for different plasmids in several cell lines.

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Abstract

Selon l'invention, des polymères d'aminoglycoside sont formés par liaison de monomères d'aminoglycoside. Les monomères peuvent être liés par un fragment de liaison chimique, tel qu'un diépoxyde, qui relie au moins deux monomères d'aminoglycoside. Les polymères d'aminoglycoside obtenus peuvent être utilisés pour administrer des acides nucléiques ou des virus dans une cellule. Par exemple, les polymères sont utilisés pour administrer des acides nucléiques ou des virus dans des cellules mammifères.
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US8822663B2 (en) 2010-08-06 2014-09-02 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0224987A2 (fr) * 1985-11-29 1987-06-10 Biomatrix, Inc. Systèmes à base de l'hyaluronanne, de ses dérivés et de ses sels pour la libération de médicaments et leur procédé de fabrication
US20020130082A1 (en) * 2001-03-14 2002-09-19 Chisso Corporation Endotoxin adsorbent, and a method of removing endotoxin by using the same
US20070029806A1 (en) * 2004-12-03 2007-02-08 Coman John H Floating electricity production unit
US20080207535A1 (en) * 2007-02-12 2008-08-28 University Of Southern Mississippi Method of attaching drug compounds to non-reactive polymer surfaces
US20100143487A1 (en) * 2007-12-26 2010-06-10 Gel-Del Technologies, Inc. Biocompatible protein-based particles and methods thereof
WO2010132876A1 (fr) * 2009-05-15 2010-11-18 Arizona Board Of Regents For And On Behalf Of Arizona State University Polymères pour distribuer une substance dans une cellule

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0224987A2 (fr) * 1985-11-29 1987-06-10 Biomatrix, Inc. Systèmes à base de l'hyaluronanne, de ses dérivés et de ses sels pour la libération de médicaments et leur procédé de fabrication
US20020130082A1 (en) * 2001-03-14 2002-09-19 Chisso Corporation Endotoxin adsorbent, and a method of removing endotoxin by using the same
US20070029806A1 (en) * 2004-12-03 2007-02-08 Coman John H Floating electricity production unit
US20080207535A1 (en) * 2007-02-12 2008-08-28 University Of Southern Mississippi Method of attaching drug compounds to non-reactive polymer surfaces
US20100143487A1 (en) * 2007-12-26 2010-06-10 Gel-Del Technologies, Inc. Biocompatible protein-based particles and methods thereof
WO2010132876A1 (fr) * 2009-05-15 2010-11-18 Arizona Board Of Regents For And On Behalf Of Arizona State University Polymères pour distribuer une substance dans une cellule

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