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  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
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  • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
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  • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
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  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
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  • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
  • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
• • A—HUMAN NECESSITIES
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  • 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
• • A—HUMAN NECESSITIES
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  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
  • A61K9/1273—Polymersomes; Liposomes with polymerisable or polymerised bilayer-forming substances
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  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/51—Nanocapsules; Nanoparticles
  • A61K9/5107—Excipients; Inactive ingredients
  • A61K9/513—Organic macromolecular compounds; Dendrimers
  • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
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  • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
  • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
  • C08G65/32—Polymers modified by chemical after-treatment
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  • C08G65/32—Polymers modified by chemical after-treatment
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  • C08G75/02—Polythioethers
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  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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Definitions

• Block copolymers containing charged blocks or chemical moieties sensitive to oxidation or hydrolysis have been developed. We describe the use of such block copolymers in supramolecular structures, e.g., micelles or vesicles, and pharmaceutical compositions and in methods of preparing the supramolecular structures and pharmaceutical compositions.
• the invention is particularly useful for the delivery of pharmaceutical agents, e.g., nucleic acids, to cells. Accordingly, in one aspect, the invention features a block copolymer including a hydrophilic block and a hydrophobic block wherein at least one of the blocks is interrupted with a hydrolysable or oxidation-sensitive chemical moiety.
• the pharmaceutical agent is selected from dexamethasone, paclitaxel, cyclosporine A, sirolimus, everolimus, tacrolimus, amphotericin B, or adriamycin.
• the pharmaceutical agent is a polypeptide, such as a protein or an antibody or an antigen-binding fragment of an antibody.
• the pharmaceutical agent is encapsulated in the supramolecular structure at an efficiency greater than 5%, 25%, 50%, 75%, 90%, or even 95%.
• the supramolecular structure may be included in a pharmaceutical composition with a pharmaceutically acceptable diluent.
• the invention features a method of encapsulating a pharmaceutical agent in a supramolecular structure, e.g., a micelle or a vesicle.
• the method includes contacting the pharmaceutical agent with an excipient and a block copolymer containing a hydrophilic block and a hydrophobic block, applying heat to homogenize the mixture of the pharmaceutical agent, excipient, and block copolymer, and diluting the homogenized mixture in an aqueous solution.
• the excipient is DBU or PEG of molecular weight between 400 and 800.
• the invention further features a method of making a vesicle including forming micelles from a block copolymer containing a hydrophilic block and a hydrophobic block, wherein a vesicle formed by the block copolymer is thermodynamically more stable than a micelle formed by the block copolymer, and heating the micelles to form the vesicle.
• the vesicles formed by the method are 70 to 800 nm in diameter.
• the micelles are suspended in a solution containing a pharmaceutical agent, and the pharmaceutical agent is encapsulated in the vesicles upon heating the solution.
• the hydrophilic block of the block copolymer contains PEG, and the hydrophobic block of the block copolymer contains PPS.
• the invention also provides a vesicle prepared by this method.
• the invention features a dry formulation containing micelles of a block copolymer having a hydrophilic block and a hydrophobic block, wherein the water content of the formulation is less than 5%, e.g., less than 2%.
• the dry formulation may further contain a pharmaceutical agent.
• the hydrophilic block of the block copolymer contains PEG, and the hydrophobic block of the block copolymer contains PPS.
• the invention further features a supramolecular structure containing a block copolymer containing a positively charged block and a nucleic acid, wherein the supramolecular structure has a maximal diameter of less than 60 nm.
• the block copolymer may further include a hydrophilic block, e.g., PEG, and a hydrophobic block, e.g., PPS.
• the block copolymer contains PPS, PEG, and polyethylene imine (PEI).
• the nucleic acid is a single-stranded oligonucleotide, a short interfering RNA, an aptamer, or plasmid DNA.
• the maximal diameter of the supramolecular structure is less than 40 nm.
• the invention features a method of transfecting a cell with a nucleic acid including contacting the cell with a supramolecular structure containing a block copolymer containing a positively charged block and the nucleic acid.
• the invention features a block copolymer containing PPS, PEG, and PEL
• the PPS block and the PEI block are attached via a bond that is labile in an endosome, e.g., a disulfide bond, vinyl ether, orthoester, acyl hydrazone, or a -N-PO 3 group.
• the block copolymer includes a nucleic acid that is bound to the PEI block.
• the block copolymer may be included in a pharmaceutical composition containing a pharmaceutical agent and a pharmaceutically acceptable diluent.
• the invention further features a micelle between 10 and 60 nm in diameter containing two block copolymers, the first of which contains a hydrophilic block and a hydrophobic block, the second of which contains a hydrophilic block, a hydrophobic block, and a positively charged block.
• the first block copolymer contains PEG and PPS
• the second block copolymer contains PEG, PPS, and PEL
• the micelle has a maximal diameter between 20 and 50 nm.
• the micelle may be included in a pharmaceutical composition containing a pharmaceutical agent and a pharmaceutically acceptable diluent.
• the invention features a supramolecular structure containing block copolymers containing a hydrophilic block, e.g., PEG, and a hydrophobic block, e.g., PPS, wherein 5-25% of the repeating units in the block copolymer have a charged chemical moiety disposed at the outer surface of the supramolecular structure.
• the charged chemical moiety is carboxylic acid, sulfate, or sulfone.
• the supramolecular structure may be included in a pharmaceutical composition containing a pharmaceutical agent and a pharmaceutically acceptable diluent.
• the hydrophilic block may contain poly(ethylene glycol), poly(ethylene oxide)-co-poly(propylene oxide) di- or multiblock copolymers, poly(ethylene oxide), polyvinyl alcohol), poly(ethylene-co-vinyl alcohol), poly(N-vinyl pyrrolidone), poly(acrylic acid), poly(ethyloxazoline), poly(alkylacrylates), poly(acrylamide), poly(N- alkylacrylamides), polypeptide, polysaccharide, poly(N,N-dialkylacrylamides), hyaluronic acid, or poly (N-acryloylmorpholine).
• the hydrophobic block may contain poly(propylene sulfide), poly(propylene glycol), esteril ⁇ ed poly(acrylic acid), esterified poly(glutamic acid), esterified poly(aspartic acid), or a polypeptide.
• the charged block is PEI, a polypeptide, poly(amidoamine), poly(sodium l-(iV-acryloylpiperazin-l-yl)diazen-l-ium- 1 ,2-diolate), poly(sodium 1 -(N-acryloylhomopiperazin- 1 -yl)diazen- 1 -ium- 1,2- diolate) or poly(sodium l-(N-acryloyl-2,5-dimethylpiperazin-l-yl)diazen-l- ium-l,2-diolate).
• PEI a polypeptide
• poly(amidoamine) poly(sodium l-(iV-acryloylpiperazin-l-yl)diazen-l-ium- 1 ,2-diolate)
• a block that is "interrupted with" a hydrolysable chemical moiety is meant a block of the same repeating unit that includes within it the hydrolysable chemical moiety so that, when the chemical moiety is hydrolyzed, the number of repeating units in the block decreases. Upon hydrolysis, the block may decrease in size by at least, e.g., 2, 4, 10, 15, 20, 30, 50, 75, 100, or 115 repeating units.
• Hydrolysable moieties include, e.g., esters, amides, thioesters, anhydrides, and ketals.
• An exemplary block that is interrupted with a hydrolysable chemical moiety is PEG 46 esterified to PEG 4 .
• a “hydrolysable chemical moiety” is meant a chemical moiety that is cleaved in aqueous solution with a half life of 1 year or less at pH 7.4 and 37 0 C.
• the half life of the moiety at pH 7.4 and 37 0 C is one month or less.
• nucleic acid is meant any nucleobase oligomer.
• modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone are also considered to be nucleobase oligomers.
• nucleic acids are antisense oligonucleotides, small interfering RNAs (siRNAs), aptamers. and plasmid DNA.
• Fig. IA Conversion of poly (ethylene glycol) monomethyl ether (PEG MME) to PEG thioacetate (PEG TAc).
• PEG MME was dried using a Dean Stark trap, and 2.5 eq of thionyl bromide was added. The reaction was then re fluxed for 4 h at 140 0 C. The toluene was evaporated, and the polymer was dissolved in dichloromethane (DCM) and precipitated in cold diethyl ether. The final conversion was accomplished by dissolving the PEG-Br in dimethylformamide (DMF) with 5 eq K 2 CO 3 and 5 eq thiolacetic acid, and stirring overnight. The product was filtered, and the DMF was evaporated and dissolved in DCM. The PEG-TAc was purified over activated charcoal and precipitated in cold diethyl ether.
• DMF dichloromethane
• Fig. 1C The final polymer was synthesized by forming the PPS block initiated from benzenethiol, and using the PEG-acrylate monomer as an end- capping reagent. This was accomplished by dissolving DBU base into THF and degassing. The benzenethiol was added under argon flow, and propylene sulfide monomer was added through a gastight septum. After 1 hr, the PEG- ester-EG 4 -ester-acrylate was added, dissolved in THF, and degassed (0.5 eq). The reaction was allowed to stir overnight. The final product was purified by dissolving the dried mixture in toluene and filtering. After evaporating the toluene, the polymer was dissolved in DCM and precipitated in cold diethyl ether.
• Fig. 4 Dynamic scanning calorimetry of PEG-PPS blended with DBU- HCl. The polymer and salt melted together creating a homogeneous blend of the two materials.
• Fig. 8 Degradation study analyzed using proton NMR. Unheated, heated, and heated with salt preparations using the polymer EG 46 -PSi 2 were compared. The polymer and salt with polymer samples were heated at 95°C for three hours prior to extraction in THF and precipitation in diethyl ether. The purified fractions were measured via proton-NMR in chloroform-D containing 0.1% TMS as an internal standard. Fig. 9. Dynamic scanning calorimetry of the constituents separately and mixed together. The mixture was prepared by mixing EG 46 -PS 12 50/50 wt/wt with DBU-HCl and heating at 95 0 C for 60 min. Samples were mixed thoroughly and measured on the DSC. For comparison, the polymer and DBU- HCl salt were also measured separately. The salt and polymer dissolved into a molten state upon heating.
• Fig. 10 Imaging vesicles formed via direct hydration using cryogenic TEM. The formed lamellar phases were vitrified on a holey carbon grid prior to imaging. Aggregates were extruded prior to analysis using cryo-TEM.
• Fig. HB Encapsulation efficiency of dexamethasone and paclitaxel in PEG formulations.
• Dexamethasone or paclitaxel were incubated with the indicated block copolymers and the indicated PEG or control formulations according to the methods of the invention.
• Bovine serum albumin and ovalbumin were prepared at 50 mg/mL in distilled water.
• the formulations were prepared as follows. Ten milligrams of PEG-PPS was heated with lOmg of PEG500 dimethyl ether and heated at 95 0 C for 15 minutes. The melt was mixed and allowed to cool to room temperature. After, a volume of protein solution (5 ⁇ L or lO ⁇ L) was added and slowly diluted with distilled water up to 1 mL volume with mixing. To calculate the encapsulation efficiencies, standard curves were generated for both BSA (using fluorescamine) or ovalbumin (using FITC-ovalbumin). The dispersed vesicle solutions were centrifuged for 10 minutes at 10,00Og to sediment the vesicles, and we measured the free protein in the supernatant.
• Fig. 12 Optical density change over time with heating of the polymer at 95°C.
• PEG-PPS EG 46 -PS 64
• OD optical density
• DLS dynamic light scattering
• Fig. 13 The samples for optical density (50 ⁇ l) were dispersed into 550 ⁇ l of double distilled water and measured using DLS. Both the OD and DLS data clearly display a trend towards larger particle size, and changing morphology.
• Fig. 14 Cryogenic Transmission Electron Microscopy of the thermal transition of PEG-PPS micelles into vesicles.
• the micelle sample (left) displays small aggregates of PEG-PPS in good agreement with the DLS results from Fig 2.
• the 30 min sample (right) shows the vesicles (polymersomes) created during heating.
• Fig. 15 The aggregation of PEG-PPS micelles during heating captured using negative staining TEM.
• 3 min sample from the heating experiment was added to a 400 mesh carbon coated copper grid which had been prepared by glow discharge.
• the sample was then blotted off after 60 sec, and stained 30 sec with 2% uranyl acetate.
• the image above was taken at 75,000x.
• the 2-D surface vesicles form slowly into short worm like micelles.
• Fig. 16A Particle size distribution by dynamic light scattering of PEG44-PPS20 cyclosporine A-loaded polymer micelles.
• Fig. 16B Particle size distribution of the micelles of Fig. 16A after drying and rehydration.
• Fig. 17A Stability OfPEG 44 -PPS 20 after exposure to gastric pH, as measured by gel permeation chromatography.
• Fig. 17B Stability OfPEG 44 -PPS 20 after exposure to gastric pH, as measured by dynamic light scattering.
• Fig. 18 A synthetic route to PEG-PPS-PEI. A disulfide link between the PPS block and the PEI block allows destabilization of the polymer after endocytosis. Fig. 19. PEG-PPS-PEI was demonstrated to condense plasmid DNA into nanoparticles of size distribution for transfection.
• Fig. 20 PEG-PPS-PEI was demonstrated to transfect cells very efficiently, even difficult-to-transfect cells such as 3T3 fibroblasts, shown here. Other cells were transfected at even higher transfection efficiency, including 239T cells at 96% with PEG2kDa-PPS 27 -PEI 96 .
• Fig. 21 Cytotoxicity with PEG-PPS-PEI was much lower than that with linear PEI of the same molecular weight at the same PEI concentration.
• Fig. 25 Gene complexes formed with a 10:1 ratio of PEG-PPS- PEI:PEG-PPS efficiently transfect cells.
• PEG-PPS was used to reduce the size of PEG-PPS-PEI micelles resulting in smaller complexes with gene-based pharmaceutical agents.
• cells were incubated with 30 nm complexes containing the green fluorescent protein (GFP) gene sequence.
• GFP green fluorescent protein
• carbohydrate polymers such as hyaluronic acid (HA) may swell to about 1000 times their volume and are used in nature to prevent protein absorption.
• carbohydrate polymer or molecule candidates are found in nature.
• Exemplary hydrophilic blocks include poly(ethylene glycol), poly(ethylene oxide)-co-poly(propylene oxide) di- or multiblock copolymers, poly(ethylene oxide), poly(vinyl alcohol), poly(ethylene-co-vinyl alcohol), poly(N- vinyl pyrrolidone), poly(acrylic acid), poly(ethyloxazoline), poly(alkylacrylates), poly(acrylamide), poly(N- alkylacrylamides), polypeptides, polysaccharides, poly(N-acryloylmorpholine), or poly(N,N-dialkylacrylamides), potentially bearing polar, ionic, or ionizable groups in the aliphatic chains.
• Hydrophilic blocks having molecular weights between 500 and 10,000 Da are practical and convenient, although higher molecular weight hydrophilic blocks may be employed.
• hydrophilic blocks a number of repeating units between about 10 and about 250 is preferable because of the ease with which these materials may be eliminated from the body by renal filtration.
• a PEG hydrophilic block is preferably between 750 and 5500 Da, e.g., between 2 and 5 kDa (e.g., a block containing 115 units).
• Hydrophilic blocks with a larger number of repeating units may also be cleared by the kidney but at slower rates than hydrophilic polymers of lower number of repeating units, which may place limits on doses that can be applied. Hydrophobic blocks.
• Nucleobase oligomers having modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
• morpholino linkages formed in part from the sugar portion of a nucleoside
• siloxane backbones sulfide, sulfoxide and sulfone backbones
• formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
• alkene containing backbones sulfamate backbones
• sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH 2 components.
• PNA Peptide Nucleic Acid
• the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
• the nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
• PNAs include, but are not limited to, U.S. Patent Nos.: 5,539,082; 5,714,331 ; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.
• the nucleobase oligomers have phosphorothioate backbones and nucleosides with heteroatom backbones, and in particular -CH 2 -NH-O-CH 2 -, -CH 2 -N(CH 3 )-O-CH 2 - (known as a methylene (methylimino) or MMI backbone), -CH 2 -O-N(CH 3 )-CH 2 -, -CH 2 - N(CH 3 )-N(CH 3 )-CH 2 -, and -O-N(CH 3 )-CH 2 -CH 2 -.
• the oligonucleotides have morpholino backbone structures described in U.S. Patent No. 5,034,506.
• nucleobase oligomers include one of the following at the 2' position: C 1 to C 10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of a nucleobase oligomer, a group for improving the pharmacodynamic properties of a nucleobase oligomer, or other substituents having similar properties.
• Preferred modifications are 2'-O-methyl and 2'- methoxyethoxy (2'-0-CH 2 CH 2 OCH 3 , also known as 2'-O-(2-methoxyethyl) or 2'-MOE).
• Another desirable modification is 2'-dimethylaminooxyethoxy (i.e., O(CH 2 ) 2 ON(CH 3 ) 2 ), also known as 2'-DMAOE.
• Other modifications include, 2'-aminopropoxy (2'-OCH 2 CH 2 CH 2 NH 2 ) and 2'-fluoro (2'-F).
• nucleobase oligomers may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
• Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos.
• nucleobases are particularly useful for increasing the binding affinity of an antisense or (partially) complementary oligonucleotide of the invention to a target nucleic acid.
• These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5- propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 0 C. (Sanghvi, Y. S., Crooke, S. T.
• nucleobase oligomer of the invention involves chemically linking to the nucleobase oligomer one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide.
• moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 86:6553-6556, 1989), cholic acid (Manoharan et al., Bioorg. Med. Chem.
• a thioether e.g., hexyl-S-tritylthiol
• Manoharan et al. Ann. N.Y. Acad. Sci., 660:306-309, 1992Manoharan et al., Bioorg. Med. Chem. Let., 3:2765-2770, 1993
• a thiocholesterol Olet al., Nucl.
• Acids Res., 18:3777-3783, 1990 a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 14:969-973, 1995), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 36:3651-3654, 1995), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1264:229- 237, 1995), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp.
• Chimeric nucleobase oligomers may be formed as composite structures of two or more nucleobase oligomers as described above. Such nucleobase oligomers, when oligonucleotides, have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include U.S. Patent Nos. 5,013,830, 5,149,797, 5,220,007, 5,256,775, 5,366,878, 5,403,71 I 5 5,491,133, 5,565,350, 5,623,065, 5,652,355, 5,652,356, and 5,700,922, each of which is herein incorporated by reference in its entirety.
• ANAs can also be employed in the present invention.
• ANAs are nucleobase oligomers based on D-arabinose sugars instead of the natural D-2'-deoxyribose sugars.
• Underivatized ANA analogs have similar binding affinity for RNA as do phosphorothioates.
• fluorine 2' F-ANA
• an enhancement in binding affinity results, and selective hydrolysis of bound RNA occurs efficiently in the resulting ANA/RNA and F-ANA/RNA duplexes.
• These analogs can be made stable in cellular media by a derivatization at their termini with simple L sugars.
• the use of ANAs in therapy is discussed, for example, in Damha et al., Nucleosides Nucleotides & Nucleic Acids 20: 429-440, 2001.
• the copolymers may utilize biological pathways for both delivery and therapeutic action.
• a block copolymer that self-assembles in aqueous environments into nanoscale micelles or vesicles may be employed for the delivery of pharmaceutical agents, such as siRNA or other nucleic acids.
• a block copolymer of the invention can exploit changing intracellular environments, e.g., the reductive environment of the endosome, for efficient delivery of the pharmaceutical agent and a biological pathway for therapeutic action, e.g., the activation of the RNAi pathway for gene silencing.
• a biological pathway for therapeutic action e.g., the activation of the RNAi pathway for gene silencing.
• colloidal particles such as nanospheres, liposomes, and micelles have been studied extensively for site-specific pharmaceutical agent delivery. Unless the reticuloendothelial system (RES) is a target, the particles must escape capture by the RES of the liver and the filtration activity of the lungs. Prolonged survival of colloidal systems in the blood has been obtained by the use of PEG- containing amphiphiles (Lasic et al., Ed. Stealth Liposomes; CRC Press: Boca Raton, FL, 1995). By virtue of marked reduction of opsonization by plasma proteins, the macrophages clearance of PEG-based liposomes has been drastically reduced (Torchilin et al., Biochim Biophys Acta 1994, 1195, 11-20).
• RES reticuloendothelial system
• internalization agents i.e., compounds or species that enhance the internalization of the copolymers of the invention, such as antibodies, growth factors, cytokines, adhesion factors, oligonucleotide sequences and nuclear localization sequences has served to enhance the delivery capabilities of PEG-coated liposomes, and it has been demonstrated that the maximal activity is shown by ligands tethered to the distal end of PEG chains (Blume et al., Biochim. Biophys. Acta 1993, 1149, 180-184; Zalipsky et al., Bioconjugate Chem. 1995, 6, 705-708; Zalipsky, J. Controlled Release 1996, 39, 153-161; Gabizon, Bioconjugate Chem.
• the copolymers of the present invention are useful for any application in the controlled release, e.g., in the cytosol or nucleus, of a pharmaceutical agent, e.g., nucleic acid.
• the release of the contents, e.g., the nucleic acid, of the self-assembled aggregate, such as a micelle or vesicle, may be achieved through sensitivity of the aggregate to the environment, such as triggering a release based on the lowering of pH, increase in the extent of oxidation, and increase in the concentration of proteases during the process of intracellular trafficking from the endosome to the lysosome.
• Excipients may also be incorporated along with the pharmaceutical agent to help it in reaching its final biological target, such as incorporation of agents that assist in destabilizing or permeabilizing biological membranes, such as the endosomal or lysosomal membranes, to enhance transport of the nucleic acid into the cytoplasm or ultimately into the nucleus.
• the block copolymers of the invention may be used to deliver nucleic acids for the up- or down-regulation of genes.
• nucleic acids include siRNA, ODN (antisense), and pDNA, including pDNA encoding therapeutic proteins.
• the internalization of DNA/positively charged polymer complexes can be enhanced by the covalent attachment of ligands, such as transferrin, folate, lectins, epidermal growth factor (EGF), RGD peptides, and mannose- containing species such as mannose-containing glycopeptides to bind to the mannose receptor (Kircheis, R., et al., Gene Ther, 1997. 4(5): p.
• the method may be used to treat infection by a pathogen or to treat a nonpathogenic disease, e.g., cancer, postsurgical adhesions, scar formation, or restenosis after removal of arterial block (e.g., via balloon angioplasty or stenting).
• a nucleic acid internalized in the cell specifically reduces or inhibits the expression of a target gene, e.g., one associated with the disease (e.g., all or a region of a gene, a gene promoter, or a portion of a gene and its promoter).
• exemplary pathogens include bacteria, protozoan, yeast, and fungi.
• the nucleic acid or other molecule inhibits the expression of an endogenous gene in a vertebrate cell or a pathogen cell (e.g., a bacterial, a yeast cell, or a fungal cell), or inhibits the expression of a pathogen gene in a cell infected with the pathogen (e.g., a plant or animal cell).
• the nucleic acid or other molecule may also reduce or inhibit the expression of an endogenous gene, e.g., in a cancer cell or in cells that produce undesirable effects, e.g., restenosis, scar formation, and postsurgical adhesions.
• the target gene is a gene associated with cancer, such as an oncogene, or a gene encoding a protein associated with a disease, such as a mutant protein, a dominant negative protein, or an overexpressed protein.
• the nucleic acid or other pharmaceutical agent delivered may increase the expression of a gene.
• the copolymer of the invention may be used to deliver a plasmid or other gene vector to the nucleus where one or more genes contained on the plasmid may be expressed.
• Such a system may be employed to enable expression of gene products that are not expressed endogenously, to increase expression of endogenous gene products, and to replace gene products that are mutated or otherwise non- functional.
• compositions of the inventions may be used to treat a disease, .e.g., cancer, in an animal, e.g., a human.
• a disease .e.g., cancer
• Exemplary cancers that can be treated using the methods described herein include prostate cancers, breast cancers, ovarian cancers, pancreatic cancers, gastric cancers, bladder cancers, salivary gland carcinomas, gastrointestinal cancers, lung cancers, colon cancers, melanomas, brain tumors, leukemias, lymphomas, and carcinomas. Benign tumors may also be treated or prevented using the methods of the present invention.
• Other cancers and cancer related genes that may be targeted are known in the art.
• Exemplary endogenous proteins that may be associated with disease include ANA (anti-nuclear antibody) found in SLE (systemic lupus erythematosis), abnormal immunoglobulins including IgG and IgA, Bence Jones protein associated with various multiple myelomas, and abnormal amyloid proteins in various amyloidoses including hereditary amyloidosis and Alzheimer's disease.
• ANA anti-nuclear antibody
• SLE systemic lupus erythematosis
• abnormal immunoglobulins including IgG and IgA
• Bence Jones protein associated with various multiple myelomas
• abnormal amyloid proteins in various amyloidoses including hereditary amyloidosis and Alzheimer's disease.
• HD a genetic abnormality in the HD (huntingtin) gene results in an expanded tract of repeated glutamine residues.
• HD patients have a copy of chromosome 4 which has a normal sized CAG repeat.
• methods of the invention can be used
• Exemplary diseases that may be treated with the methods include infection by pathogens, such as a virus, a bacterium, a yeast, a fungus, a protozoan, or a parasite.
• the nucleic acid may be delivered to the pathogen or to a cell infected with the pathogen.
• the pathogen may be an intracellular or extracellular pathogen.
• the target nucleic acid sequence is, for example, a gene of the pathogen that is necessary for replication and/or pathogenesis, or a gene encoding a cellular receptor necessary for a cell to be infected with the pathogen.
• Such methods may be employed prior to, concurrent with, or following the administration of the in-dwelling device to a patient to prevent infections.
• In-dwelling devices include, but are not limited to, surgical implants, prosthetic devices, and catheters, i.e., devices that are introduced to the body of an individual and remain in position for an extended time.
• Such devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, and continuous ambulatory peritoneal dialysis (CAPD) catheters.
• devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, and continuous ambulatory peritoneal dialysis (CAPD) catheters.
• CAPD continuous ambulatory peritoneal dialysis
• a bacterial infection may be due to one or more of the following bacteria: Chlamydophila pneumoniae, C. psittaci, C. abortus, Chlamydia trachomatis, Simkania negevensis, Parachlamydia acanthamoebae, Pseudomonas aeruginosa, P. alcaligenes, P. chlororaphis, P. fluorescens, P. luteola, P. mendocina, P. monteilii, P. oryzihabitans, P. pertocinogena, P. pseudalcaligenes, P. putida, P.
• a viral infection may be due to one or more of the following viruses: Hepatitis B, Hepatitis C, picornarirus, polio, HIV, coxsacchie, herpes simplex virus Type I and 2, St. Louis encephalitis, Epstein-Barr, myxoviruses, JC, coxsakieviruses B, togaviruses, measles, paramyxoviruses, echoviruses, bunyaviruses, cytomegaloviruses, varicella-zoster, mumps, equine encephalitis, lymphocytic choriomeningitis, rhabodoviruses including rabies, simian virus 40, human polyoma virus, parvoviruses, papilloma viruses, primate adenoviruses, coronaviruses, retroviruses, Dengue, yellow fever, Japanese encephalitis virus, BK, Retrovirus, Herpesvirus, Hepad
• the target viral nucleic acid sequence is, for example, necessary for replication and/or pathogenesis of the virus in an infected cell.
• viral target genes are necessary for the propagation of the virus and include, e.g., the HIV gag, env, and pol genes, the HPV6 LI and E2 genes, the HPV 1 1 LI and E2 genes, the HPV 16 E6 and E7 genes, the BPV 18 E6 and E7 genes, the HBV surface antigens, the HBV core antigen, HBV reverse transcriptase, the HSV gD gene, the HSVvp 16 gene, the HSV gC, gH, gL and gB genes, the HSV ICPO, ICP4 and ICP6 genes, Varicella zoster gB, gC and gH genes, and the BCR-abl chromosomal sequences, and non-coding viral polynucleotide sequences which provide regulatory functions necessary for transfer of the infection from cell to cell, e.g
• the copolymers of the invention can be used to treat subjects already infected with a virus, such as HIV, in order to shut down or inhibit a viral gene function essential to virus replication and/or pathogenesis, such as HIV gag.
• a virus such as HIV
• this method can be employed to inhibit the functions of viruses, which exist in mammals as latent viruses, e.g., Varicella zoster virus, the causative agent of shingles.
• the nucleic acid or other molecule is administered in an amount sufficient to treat the disease or condition, e.g., to prevent, stabilize, or inhibit the growth of the pathogen or to kill the pathogen or to increase or decrease the expression of an endogenous gene whose under- or ovcrexprcssion results in a disease.
• PPS serves as a useful hydrophobic block in micelle and vesicle formation, as it can be oxidized to form the hydrophilic sulfone and sulfoxide products, after which it can presumably be excreted from the body by renal filtration. It is possible to build into the block copolymeric amphiphile other degradable segments, for example hydrolyzable chemical moieties. Such moieties may be placed within the hydrophilic domain of the block copolymer, so that moieties' accessibility to water does not become a limiting factor in hydrolysis rate and thus release rate. One route by which to accomplish this is shown in Figure 1.
• Excipien ⁇ s can be used to enhance encapsulation efficiency of hydrophobic pharmaceutical agents in micelles.
• Samples used in Table 2 were prepared as follows. 10 mg of PEG-PPS was added to a 1.5 mL centrifuge tube with either 90 mg of PEG600 or DBU- HCl, and 2 mg of either paclitaxel or dexamethasone. This was heated at 95°C for 15 min and mixed thoroughly. After cooling to RT, the blend was slowly diluted to 1 mL with distilled water. The free pharmaceutical agent was pelleted via centrifugation for 10 minutes at 10.000 g, and the pellet and supernatant were separately freeze dried, and analyzed in THF via gel permeation chromatography. Dexamethasone and paclitaxel results were quantified using a standard curve.
• Dexamethasone and amphotericin B were more efficiently encapsulated via solvent dispersion than via the method of the invention.
• paclitaxel was more efficiently encapsulated via the method of the invention.
• Other pharmaceutical agents such as sirolimus and everolimus were also efficiently encapsulated using the methods of the invention.
• the encapsulation efficiency depends on the structure of the pharmaceutical agent. The flexibility of the PEG-PPS system to encapsulate pharmaceutical agents is very large because the system accommodates a variety of techniques to encapsulate most pharmaceutical agents at high efficiencies.
• Example 3 Excipients can be used to enhance encapsulation efficiency of hydrophilic pharmaceutical agents in vesicles.
• Polymeric vesicles represent very powerful tools for protection and delivery of hydrophilic pharmaceutical agents, such as peptides, proteins, nucleic acids, and genes; however, they are difficult to load.
• hydrophilic pharmaceutical agents such as peptides, proteins, nucleic acids, and genes; however, they are difficult to load.
• One method is to dissolve in the polymer an excipient that is soluble both in the polymer and in water, such as DBU or PEGs, as illustrated above in the formation of polymer micelles.
• An aqueous solution of the pharmaceutical agent to be encapsulated is added to the polymer mixture with the excipient (the so-called direct hydration method). Typical results are illustrated in Table 3 and Figures 5-11.
• Example 4 Thermal transitions can induce vesicle formation from micelles.
• vesicles are powerful tools with which to encapsulate hydrophilic pharmaceutical agents, to modulate their release, to target their release, and to protect them from biological clearance and degradation mechanisms.
• the micelles can be metastable and can be concentrated to a high degree.
• Application of heat to the metastable micelles induces spontaneous formation of vesicles, which can be very small and homogeneous in size distribution.
• Pharmaceutical agent incorporated in the micelle suspension will be loaded within the vesicles during their formation. The approach is illustrated in Figures 12-15.
• the ultrasmall size of the polymer vesicles formed by this method may be particularly useful in some applications. For example, in targeting tumors from the bloodstream via the enhanced permeation and retention effect, smaller particles are more effective than larger particles in penetration of the fenestrated endothelium in the tumor microcirculation. Smaller particles are more effective than larger ones in penetration of the arterial wall under physiological pressure or mild overpressure, in penetration of mucosal surfaces and targeting cells beneath, such as dendritic cells, in permeation of the interstitium to target lymph nodes draining the tissue site, and in targeting the lymphatics in the gut.
• Example 5 PEG-PPS vesicle formulations can be stable upon drying and rehydration.
• Formulations that can solubilize hydrophobic pharmaceutical agents and can be administered in dry form are useful in a number of pharmaceutical applications.
• PEG-PPS micelles can be dried into a tablet and subsequently resuspended rapidly, to the same size distribution, without loss of encapsulated pharmaceutical agent.
• PEG 44 -PPS 2O micelles were formed with size mean of 21 nm ( Figure 16A), loaded with cyclosporine A. The suspension was dried, and then the dried sample was placed in water to allow brief rehydration. The measured size distribution showed a mean of 20.3 nm (Figure 16B). Throughout the process, high encapsulation efficiency was maintained (Table 7). The particles, being primarily sensitive to oxidation, are stable at gastric pH ( Figure 17).
• PEG-PPS-PEI copolymers can efficiently deliver gene-based pharmaceutical agents.
• PEG-PPS- polycation triblock copolymers including the case where the polycation blocks were based on peptides.
• Example 7 Blends of PEG-PPS and PPS-PEI can form very small complexes with gene-based pharmaceutical agents.
• nanoparticles with gene- based pharmaceutical agents that are very small. This was possible by using mixed micelles of PEG-PPS and PPS-PEI to obtain very small complexes.
• PEG-PPS nanoparticles are biospecific, as the control macromolecule dextran was not well transported, and the transport was blocked at cold temperatures.
• PPS nanoparticles formed with a terminal hydroxyl group were about 10-fold better transported than analogous particles with terminal methoxy groups.
• PPS nanoparticles with 90% -OCH 3 and 10% COOH are actively transported across LECs (5x better than others).
• PPS nanoparticles with 90% -OH and 10% COOH are actively transported across Caco-2 cells (1Ox better than others).

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• 2024
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